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Sample records for allosteric sites implications

  1. Assessing the structural conservation of protein pockets to study functional and allosteric sites: implications for drug discovery

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    Daura Xavier

    2010-03-01

    Full Text Available Abstract Background With the classical, active-site oriented drug-development approach reaching its limits, protein ligand-binding sites in general and allosteric sites in particular are increasingly attracting the interest of medicinal chemists in the search for new types of targets and strategies to drug development. Given that allostery represents one of the most common and powerful means to regulate protein function, the traditional drug discovery approach of targeting active sites can be extended by targeting allosteric or regulatory protein pockets that may allow the discovery of not only novel drug-like inhibitors, but activators as well. The wealth of available protein structural data can be exploited to further increase our understanding of allosterism, which in turn may have therapeutic applications. A first step in this direction is to identify and characterize putative effector sites that may be present in already available structural data. Results We performed a large-scale study of protein cavities as potential allosteric and functional sites, by integrating publicly available information on protein sequences, structures and active sites for more than a thousand protein families. By identifying common pockets across different structures of the same protein family we developed a method to measure the pocket's structural conservation. The method was first parameterized using known active sites. We characterized the predicted pockets in terms of sequence and structural conservation, backbone flexibility and electrostatic potential. Although these different measures do not tend to correlate, their combination is useful in selecting functional and regulatory sites, as a detailed analysis of a handful of protein families shows. We finally estimated the numbers of potential allosteric or regulatory pockets that may be present in the data set, finding that pockets with putative functional and effector characteristics are widespread across

  2. Exploiting protein flexibility to predict the location of allosteric sites

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    Panjkovich Alejandro

    2012-10-01

    Full Text Available Abstract Background Allostery is one of the most powerful and common ways of regulation of protein activity. However, for most allosteric proteins identified to date the mechanistic details of allosteric modulation are not yet well understood. Uncovering common mechanistic patterns underlying allostery would allow not only a better academic understanding of the phenomena, but it would also streamline the design of novel therapeutic solutions. This relatively unexplored therapeutic potential and the putative advantages of allosteric drugs over classical active-site inhibitors fuel the attention allosteric-drug research is receiving at present. A first step to harness the regulatory potential and versatility of allosteric sites, in the context of drug-discovery and design, would be to detect or predict their presence and location. In this article, we describe a simple computational approach, based on the effect allosteric ligands exert on protein flexibility upon binding, to predict the existence and position of allosteric sites on a given protein structure. Results By querying the literature and a recently available database of allosteric sites, we gathered 213 allosteric proteins with structural information that we further filtered into a non-redundant set of 91 proteins. We performed normal-mode analysis and observed significant changes in protein flexibility upon allosteric-ligand binding in 70% of the cases. These results agree with the current view that allosteric mechanisms are in many cases governed by changes in protein dynamics caused by ligand binding. Furthermore, we implemented an approach that achieves 65% positive predictive value in identifying allosteric sites within the set of predicted cavities of a protein (stricter parameters set, 0.22 sensitivity, by combining the current analysis on dynamics with previous results on structural conservation of allosteric sites. We also analyzed four biological examples in detail, revealing

  3. The allosteric site regulates the voltage sensitivity of muscarinic receptors.

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    Hoppe, Anika; Marti-Solano, Maria; Drabek, Matthäus; Bünemann, Moritz; Kolb, Peter; Rinne, Andreas

    2018-01-01

    Muscarinic receptors (M-Rs) for acetylcholine (ACh) belong to the class A of G protein-coupled receptors. M-Rs are activated by orthosteric agonists that bind to a specific site buried in the M-R transmembrane helix bundle. In the active conformation, receptor function can be modulated either by allosteric modulators, which bind to the extracellular receptor surface or by the membrane potential via an unknown mechanism. Here, we compared the modulation of M 1 -Rs and M 3 -Rs induced by changes in voltage to their allosteric modulation by chemical compounds. We quantified changes in receptor signaling in single HEK 293 cells with a FRET biosensor for the G q protein cycle. In the presence of ACh, M 1 -R signaling was potentiated by voltage, similarly to positive allosteric modulation by benzyl quinolone carboxylic acid. Conversely, signaling of M 3 -R was attenuated by voltage or the negative allosteric modulator gallamine. Because the orthosteric site is highly conserved among M-Rs, but allosteric sites vary, we constructed "allosteric site" M 3 /M 1 -R chimeras and analyzed their voltage dependencies. Exchanging the entire allosteric sites eliminated the voltage sensitivity of ACh responses for both receptors, but did not affect their modulation by allosteric compounds. Furthermore, a point mutation in M 3 -Rs caused functional uncoupling of the allosteric and orthosteric sites and abolished voltage dependence. Molecular dynamics simulations of the receptor variants indicated a subtype-specific crosstalk between both sites, involving the conserved tyrosine lid structure of the orthosteric site. This molecular crosstalk leads to receptor subtype-specific voltage effects. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Identification of the Allosteric Regulatory Site of Insulysin

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    Noinaj, Nicholas; Bhasin, Sonia K.; Song, Eun Suk; Scoggin, Kirsten E.; Juliano, Maria A.; Juliano, Luiz; Hersh, Louis B.; Rodgers, David W. (U. Sao Paulo); (Kentucky)

    2012-05-25

    Insulin degrading enzyme (IDE) is responsible for the metabolism of insulin and plays a role in clearance of the A{beta} peptide associated with Alzheimer's disease. Unlike most proteolytic enzymes, IDE, which consists of four structurally related domains and exists primarily as a dimer, exhibits allosteric kinetics, being activated by both small substrate peptides and polyphosphates such as ATP. The crystal structure of a catalytically compromised mutant of IDE has electron density for peptide ligands bound at the active site in domain 1 and a distal site in domain 2. Mutating residues in the distal site eliminates allosteric kinetics and activation by a small peptide, as well as greatly reducing activation by ATP, demonstrating that this site plays a key role in allostery. Comparison of the peptide bound IDE structure (using a low activity E111F IDE mutant) with unliganded wild type IDE shows a change in the interface between two halves of the clamshell-like molecule, which may enhance enzyme activity by altering the equilibrium between closed and open conformations. In addition, changes in the dimer interface suggest a basis for communication between subunits. Our findings indicate that a region remote from the active site mediates allosteric activation of insulysin by peptides. Activation may involve a small conformational change that weakens the interface between two halves of the enzyme.

  5. Identification of the Allosteric Regulatory Site of Insulysin

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    Noinaj, Nicholas; Bhasin, Sonia K.; Song, Eun Suk; Scoggin, Kirsten E.; Juliano, Maria A.; Juliano, Luiz; Hersh, Louis B.; Rodgers, David W.; Gerrard, Juliet Ann

    2011-06-24

    Background Insulin degrading enzyme (IDE) is responsible for the metabolism of insulin and plays a role in clearance of the Aβ peptide associated with Alzheimer's disease. Unlike most proteolytic enzymes, IDE, which consists of four structurally related domains and exists primarily as a dimer, exhibits allosteric kinetics, being activated by both small substrate peptides and polyphosphates such as ATP. Principal Findings The crystal structure of a catalytically compromised mutant of IDE has electron density for peptide ligands bound at the active site in domain 1 and a distal site in domain 2. Mutating residues in the distal site eliminates allosteric kinetics and activation by a small peptide, as well as greatly reducing activation by ATP, demonstrating that this site plays a key role in allostery. Comparison of the peptide bound IDE structure (using a low activity E111F IDE mutant) with unliganded wild type IDE shows a change in the interface between two halves of the clamshell-like molecule, which may enhance enzyme activity by altering the equilibrium between closed and open conformations. In addition, changes in the dimer interface suggest a basis for communication between subunits. Conclusions/Significance Our findings indicate that a region remote from the active site mediates allosteric activation of insulysin by peptides. Activation may involve a small conformational change that weakens the interface between two halves of the enzyme.

  6. Prediction of allosteric sites on protein surfaces with an elastic-network-model-based thermodynamic method.

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    Su, Ji Guo; Qi, Li Sheng; Li, Chun Hua; Zhu, Yan Ying; Du, Hui Jing; Hou, Yan Xue; Hao, Rui; Wang, Ji Hua

    2014-08-01

    Allostery is a rapid and efficient way in many biological processes to regulate protein functions, where binding of an effector at the allosteric site alters the activity and function at a distant active site. Allosteric regulation of protein biological functions provides a promising strategy for novel drug design. However, how to effectively identify the allosteric sites remains one of the major challenges for allosteric drug design. In the present work, a thermodynamic method based on the elastic network model was proposed to predict the allosteric sites on the protein surface. In our method, the thermodynamic coupling between the allosteric and active sites was considered, and then the allosteric sites were identified as those where the binding of an effector molecule induces a large change in the binding free energy of the protein with its ligand. Using the proposed method, two proteins, i.e., the 70 kD heat shock protein (Hsp70) and GluA2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, were studied and the allosteric sites on the protein surface were successfully identified. The predicted results are consistent with the available experimental data, which indicates that our method is a simple yet effective approach for the identification of allosteric sites on proteins.

  7. In Vivo Investigation of Escitalopram’s Allosteric Site on the Serotonin Transporter

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    Murray, Karen E.; Ressler, Kerry J.; Owens, Michael J.

    2015-01-01

    Escitalopram is a commonly prescribed antidepressant of the selective serotonin reuptake inhibitor class. Clinical evidence and mapping of the serotonin transporter (SERT) identified that escitalopram, in addition to its binding to a primary uptake-blocking site, is capable of binding to the SERT via an allosteric site that is hypothesized to alter escitalopram’s kinetics at the SERT. The studies reported here examined the in vivo role of the SERT allosteric site in escitalopram action. A knockin mouse model that possesses an allosteric-null SERT was developed. Autoradiographic studies indicated that the knockin protein was expressed at a lower density than endogenous mouse SERT (approximately 10–30% of endogenous mouse SERT), but the knockin mice are a viable tool to study the allosteric site. Microdialysis studies in the ventral hippocampus found no measurable decrease in extracellular serotonin response after local escitalopram challenge in mice without the allosteric site compared to mice with the site (p = 0.297). In marble burying assays there was a modest effect of the absence of the allosteric site, with a larger systemic dose of escitalopram (10-fold) necessary for the same effect as in mice with intact SERT (p = 0.023). However, there was no effect of the allosteric site in the tail suspension test. Together these data suggest that there may be a regional specificity in the role of the allosteric site. The lack of a robust effect overall suggests that the role of the allosteric site for escitalopram on the SERT may not produce meaningful in vivo effects. PMID:26621784

  8. Allosteric and orthosteric sites in CC chemokine receptor (CCR5), a chimeric receptor approach

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    Thiele, Stefanie; Steen, Anne; Jensen, Pia C

    2011-01-01

    -allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5...... preserved, the allosteric enhancement of chemokine binding was disrupted. In summary, the Trojan horse chimera revealed that orthosteric and allosteric sites could be structurally separated and still act together with transmission of agonism and antagonism across the different receptor units....

  9. Non-site-specific allosteric effect of oxygen on human hemoglobin under high oxygen partial pressure.

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    Takayanagi, Masayoshi; Kurisaki, Ikuo; Nagaoka, Masataka

    2014-04-08

    Protein allostery is essential for vital activities. Allosteric regulation of human hemoglobin (HbA) with two quaternary states T and R has been a paradigm of allosteric structural regulation of proteins. It is widely accepted that oxygen molecules (O2) act as a "site-specific" homotropic effector, or the successive O2 binding to the heme brings about the quaternary regulation. However, here we show that the site-specific allosteric effect is not necessarily only a unique mechanism of O2 allostery. Our simulation results revealed that the solution environment of high O2 partial pressure enhances the quaternary change from T to R without binding to the heme, suggesting an additional "non-site-specific" allosteric effect of O2. The latter effect should play a complementary role in the quaternary change by affecting the intersubunit contacts. This analysis must become a milestone in comprehensive understanding of the allosteric regulation of HbA from the molecular point of view.

  10. Identification of an allosteric binding site for RORγt inhibition

    NARCIS (Netherlands)

    Scheepstra, M.; Leysen, S.; van Almen, G.; Miller, J.R.; Piesvaux, J.; Kutilek, V.; van Eenennaam, H.; Zhang, H.; Barr, K.; Nagpal, S.; Soisson, S.M.; Kornienko, M.; Wiley, K.; Elsen, N.; Sharma, S.; Correll, C.C.; Trotter, B.W.; Stelt, van der M.; Oubrie, A.; Ottmann, C.; Parthasarathy, G.; Brunsveld, L.

    2015-01-01

    RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of

  11. Allosteric ligands and their binding sites define γ-aminobutyric acid (GABA) type A receptor subtypes.

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    Olsen, Richard W

    2015-01-01

    GABAA receptors (GABA(A)Rs) mediate rapid inhibitory transmission in the brain. GABA(A)Rs are ligand-gated chloride ion channel proteins and exist in about a dozen or more heteropentameric subtypes exhibiting variable age and brain regional localization and thus participation in differing brain functions and diseases. GABA(A)Rs are also subject to modulation by several chemotypes of allosteric ligands that help define structure and function, including subtype definition. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABA(A)Rs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Two classes of pharmacologically important allosteric modulatory ligand binding sites reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site and the high-affinity, relevant to intoxication, ethanol site. The benzodiazepine site is specific for certain GABA(A)R subtypes, mainly synaptic, while the ethanol site is found at a modified benzodiazepine site on different, extrasynaptic, subtypes. In the transmembrane domain are allosteric modulatory ligand sites for diverse chemotypes of general anesthetics: the volatile and intravenous agents, barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are endogenous positive allosteric modulators. X-ray crystal structures of prokaryotic and invertebrate pentameric ligand-gated ion channels, and the mammalian GABA(A)R protein, allow homology modeling of GABA(A)R subtypes with the various ligand sites located to suggest the structure and function of these proteins and their pharmacological modulation. © 2015 Elsevier Inc. All rights reserved.

  12. Molecular sites for the positive allosteric modulation of glycine receptors by endocannabinoids.

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    Gonzalo E Yévenes

    Full Text Available Glycine receptors (GlyRs are transmitter-gated anion channels of the Cys-loop superfamily which mediate synaptic inhibition at spinal and selected supraspinal sites. Although they serve pivotal functions in motor control and sensory processing, they have yet to be exploited as drug targets partly because of hitherto limited possibilities for allosteric control. Endocannabinoids (ECs have recently been characterized as direct allosteric GlyR modulators, but the underlying molecular sites have remained unknown. Here, we show that chemically neutral ECs (e.g. anandamide, AEA are positive modulators of α(1, α(2 and α(3 GlyRs, whereas acidic ECs (e.g. N-arachidonoyl-glycine; NA-Gly potentiate α(1 GlyRs but inhibit α(2 and α(3. This subunit-specificity allowed us to identify the underlying molecular sites through analysis of chimeric and mutant receptors. We found that alanine 52 in extracellular loop 2, glycine 254 in transmembrane (TM region 2 and intracellular lysine 385 determine the positive modulation of α(1 GlyRs by NA-Gly. Successive substitution of non-conserved extracellular and TM residues in α(2 converted NA-Gly-mediated inhibition into potentiation. Conversely, mutation of the conserved lysine within the intracellular loop between TM3 and TM4 attenuated NA-Gly-mediated potentiation of α(1 GlyRs, without affecting inhibition of α(2 and α(3. Notably, this mutation reduced modulation by AEA of all three GlyRs. These results define molecular sites for allosteric control of GlyRs by ECs and reveal an unrecognized function for the TM3-4 intracellular loop in the allosteric modulation of Cys-loop ion channels. The identification of these sites may help to understand the physiological role of this modulation and facilitate the development of novel therapeutic approaches to diseases such as spasticity, startle disease and possibly chronic pain.

  13. Identification of an allosteric binding site for RORγt inhibition

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    Scheepstra, Marcel; Leysen, Seppe; vanAlmen, Geert C.; Miller, J. Richard; Piesvaux, Jennifer; Kutilek, Victoria; van Eenennaam, Hans; Zhang, Hongjun; Barr, Kenneth; Nagpal, Sunil; Soisson, Stephen M.; Kornienko, Maria; Wiley, Kristen; Elsen, Nathaniel; Sharma, Sujata; Correll, Craig C.; Trotter, B. Wesley; van der Stelt, Mario; Oubrie, Arthur; Ottmann, Christian; Parthasarathy, Gopal; Brunsveld, Luc (Merck); (Eindhoven)

    2015-12-07

    RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors.

  14. Structural insight to mutation effects uncover a common allosteric site in class C GPCRs

    DEFF Research Database (Denmark)

    Harpsøe, Kasper; Boesgaard, Michael W; Munk, Christian

    2017-01-01

    MOTIVATION: Class C G protein-coupled receptors (GPCRs) regulate important physiological functions and allosteric modulators binding to the transmembrane domain constitute an attractive and, due to a lack of structural insight, a virtually unexplored potential for therapeutics and the food industry....... Combining pharmacological site-directed mutagenesis data with the recent class C GPCR experimental structures will provide a foundation for rational design of new therapeutics. RESULTS: We uncover one common site for both positive and negative modulators with different amino acid layouts that can...

  15. Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site.

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    Adams, Julian; Chen, Zhi-Ping; Van Denderen, Bryce J W; Morton, Craig J; Parker, Michael W; Witters, Lee A; Stapleton, David; Kemp, Bruce E

    2004-01-01

    AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.

  16. Mutations that silence constitutive signaling activity in the allosteric ligand-binding site of the thyrotropin receptor.

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    Haas, Ann-Karin; Kleinau, Gunnar; Hoyer, Inna; Neumann, Susanne; Furkert, Jens; Rutz, Claudia; Schülein, Ralf; Gershengorn, Marvin C; Krause, Gerd

    2011-01-01

    The thyrotropin receptor (TSHR) exhibits elevated cAMP signaling in the basal state and becomes fully activated by thyrotropin. Previously we presented evidence that small-molecule ligands act allosterically within the transmembrane region in contrast to the orthosteric extracellular hormone-binding sites. Our goal in this study was to identify positions that surround the allosteric pocket and that are sensitive for inactivation of TSHR. Homology modeling combined with site-directed mutagenesis and functional characterization revealed seven mutants located in the allosteric binding site that led to a decrease of basal cAMP signaling activity. The majority of these silencing mutations, which constrain the TSHR in an inactive conformation, are found in two clusters when mapped onto the 3D structural model. We suggest that the amino acid positions identified herein are indicating locations where small-molecule antagonists, both neutral antagonists and inverse agonists, might interfere with active TSHR conformations.

  17. Signaling-sensitive amino acids surround the allosteric ligand binding site of the thyrotropin receptor.

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    Kleinau, Gunnar; Haas, Ann-Karin; Neumann, Susanne; Worth, Catherine L; Hoyer, Inna; Furkert, Jens; Rutz, Claudia; Gershengorn, Marvin C; Schülein, Ralf; Krause, Gerd

    2010-07-01

    The thyrotropin receptor [thyroid-stimulating hormone receptor (TSHR)], a G-protein-coupled receptor (GPCR), is endogenously activated by thyrotropin, which binds to the extracellular region of the receptor. We previously identified a low-molecular-weight (LMW) agonist of the TSHR and predicted its allosteric binding pocket within the receptor's transmembrane domain. Because binding of the LMW agonist probably disrupts interactions or leads to formation of new interactions among amino acid residues surrounding the pocket, we tested whether mutation of residues at these positions would lead to constitutive signaling activity. Guided by molecular modeling, we performed site-directed mutagenesis of 24 amino acids in this spatial region, followed by functional characterization of the mutant receptors in terms of expression and signaling, measured as cAMP accumulation. We found that mutations V421I, Y466A, T501A, L587V, M637C, M637W, S641A, Y643F, L645V, and Y667A located in several helices exhibit constitutive activity. Of note is mutation M637W at position 6.48 in transmembrane helix 6, which has a significant effect on the interaction of the receptor with the LMW agonist. In summary, we found that a high proportion of residues in several helices surrounding the allosteric binding site of LMW ligands in the TSHR when mutated lead to constitutively active receptors. Our findings of signaling-sensitive residues in this region of the transmembrane bundle may be of general importance as this domain appears to be evolutionarily retained among GPCRs.

  18. Role of allosteric switch residue histidine 195 in maintaining active-site asymmetry in presynaptic filaments of bacteriophage T4 UvsX recombinase.

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    Farb, Joshua N; Morrical, Scott W

    2009-01-16

    Recombinases of the highly conserved RecA/Rad51 family play central roles in homologous recombination and DNA double-stranded break repair. RecA/Rad51 enzymes form presynaptic filaments on single-stranded DNA (ssDNA) that are allosterically activated to catalyze ATPase and DNA strand-exchange reactions. Information is conveyed between DNA- and ATP-binding sites, in part, by a highly conserved glutamine residue (Gln194 in Escherichia coli RecA) that acts as an allosteric switch. The T4 UvsX protein is a divergent RecA ortholog and contains histidine (His195) in place of glutamine at the allosteric switch position. UvsX and RecA catalyze similar strand-exchange reactions, but differ in other properties. UvsX produces both ADP and AMP as products of its ssDNA-dependent ATPase activity--a property that is unique among characterized recombinases. Details of the kinetics of ssDNA-dependent ATP hydrolysis reactions indicate that UvsX-ssDNA presynaptic filaments are asymmetric and contain two classes of ATPase active sites: one that generates ADP, and another that generates AMP. Active-site asymmetry is reduced by mutations at the His195 position, since UvsX-H195Q and UvsX-H195A mutants both exhibit stronger ssDNA-dependent ATPase activity, with lower cooperativity and markedly higher ADP/AMP product ratios, than wild-type UvsX. Reduced active-site asymmetry correlates strongly with reduced ssDNA-binding affinity and DNA strand-exchange activity in both H195Q and H195A mutants. These and other results support a model in which allosteric switch residue His195 controls the formation of an asymmetric conformation of UvsX-ssDNA filaments that is active in DNA strand exchange. The implications of our findings for UvsX recombination functions, and for RecA functions in general, are discussed.

  19. Conopeptide ρ-TIA defines a new allosteric site on the extracellular surface of the α1B-adrenoceptor.

    Science.gov (United States)

    Ragnarsson, Lotten; Wang, Ching-I Anderson; Andersson, Åsa; Fajarningsih, Dewi; Monks, Thea; Brust, Andreas; Rosengren, K Johan; Lewis, Richard J

    2013-01-18

    The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α(1B)-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

  20. Overlapping binding site for the endogenous agonist, small-molecule agonists, and ago-allosteric modulators on the ghrelin receptor

    DEFF Research Database (Denmark)

    Holst, Birgitte; Frimurer, Thomas M; Mokrosinski, Jacek

    2008-01-01

    A library of robust ghrelin receptor mutants with single substitutions at 22 positions in the main ligand-binding pocket was employed to map binding sites for six different agonists: two peptides (the 28-amino-acid octanoylated endogenous ligand ghrelin and the hexapeptide growth hormone......, and PheVI:23 on the opposing face of transmembrane domain (TM) VI. Each of the agonists was also affected selectively by specific mutations. The mutational map of the ability of L-692,429 and GHRP-6 to act as allosteric modulators by increasing ghrelin's maximal efficacy overlapped with the common....... It is concluded that although each of the ligands in addition exploits other parts of the receptor, a large, common binding site for both small-molecule agonists--including ago-allosteric modulators--and the endogenous agonist is found on the opposing faces of TM-III and -VI of the ghrelin receptor....

  1. Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB2 Receptors

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    Eva Martínez-Pinilla

    2017-10-01

    Full Text Available The mechanism of action of cannabidiol (CBD, the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB2 receptors (CB2Rs it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs; however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB2R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB2R. Using membrane preparations from CB2R-expressing HEK-293T (human embryonic kidney 293T cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB2R where the synthetic cannabinoid, [3H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB2R-selective compound, CM-157. The effect on binding to CB2R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the KD. CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB2R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

  2. Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB2 Receptors.

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    Martínez-Pinilla, Eva; Varani, Katia; Reyes-Resina, Irene; Angelats, Edgar; Vincenzi, Fabrizio; Ferreiro-Vera, Carlos; Oyarzabal, Julen; Canela, Enric I; Lanciego, José L; Nadal, Xavier; Navarro, Gemma; Borea, Pier Andrea; Franco, Rafael

    2017-01-01

    The mechanism of action of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB 2 receptors (CB 2 Rs) it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs); however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB 2 R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB 2 R. Using membrane preparations from CB 2 R-expressing HEK-293T (human embryonic kidney 293T) cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB 2 R where the synthetic cannabinoid, [ 3 H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB 2 R-selective compound, CM-157. The effect on binding to CB 2 R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the K D . CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB 2 R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

  3. Identification of potential small molecule allosteric modulator sites on IL-1R1 ectodomain using accelerated conformational sampling method.

    Directory of Open Access Journals (Sweden)

    Chao-Yie Yang

    Full Text Available The interleukin-1 receptor (IL-1R is the founding member of the interleukin 1 receptor family which activates innate immune response by its binding to cytokines. Reports showed dysregulation of cytokine production leads to aberrant immune cells activation which contributes to auto-inflammatory disorders and diseases. Current therapeutic strategies focus on utilizing antibodies or chimeric cytokine biologics. The large protein-protein interaction interface between cytokine receptor and cytokine poses a challenge in identifying binding sites for small molecule inhibitor development. Based on the significant conformational change of IL-1R type 1 (IL-1R1 ectodomain upon binding to different ligands observed in crystal structures, we hypothesized that transient small molecule binding sites may exist when IL-1R1 undergoes conformational transition and thus suitable for inhibitor development. Here, we employed accelerated molecular dynamics (MD simulation to efficiently sample conformational space of IL-1R1 ectodomain. Representative IL-1R1 ectodomain conformations determined from the hierarchy cluster analysis were analyzed by the SiteMap program which leads to identify small molecule binding sites at the protein-protein interaction interface and allosteric modulator locations. The cosolvent mapping analysis using phenol as the probe molecule further confirms the allosteric modulator site as a binding hotspot. Eight highest ranked fragment molecules identified from in silico screening at the modulator site were evaluated by MD simulations. Four of them restricted the IL-1R1 dynamical motion to inactive conformational space. The strategy from this study, subject to in vitro experimental validation, can be useful to identify small molecule compounds targeting the allosteric modulator sites of IL-1R and prevent IL-1R from binding to cytokine by trapping IL-1R in inactive conformations.

  4. Molecular modeling study on the allosteric inhibition mechanism of HIV-1 integrase by LEDGF/p75 binding site inhibitors.

    Directory of Open Access Journals (Sweden)

    Weiwei Xue

    Full Text Available HIV-1 integrase (IN is essential for the integration of viral DNA into the host genome and an attractive therapeutic target for developing antiretroviral inhibitors. LEDGINs are a class of allosteric inhibitors targeting LEDGF/p75 binding site of HIV-1 IN. Yet, the detailed binding mode and allosteric inhibition mechanism of LEDGINs to HIV-1 IN is only partially understood, which hinders the structure-based design of more potent anti-HIV agents. A molecular modeling study combining molecular docking, molecular dynamics simulation, and binding free energy calculation were performed to investigate the interaction details of HIV-1 IN catalytic core domain (CCD with two recently discovered LEDGINs BI-1001 and CX14442, as well as the LEDGF/p75 protein. Simulation results demonstrated the hydrophobic domain of BI-1001 and CX14442 engages one subunit of HIV-1 IN CCD dimer through hydrophobic interactions, and the hydrophilic group forms hydrogen bonds with HIV-1 IN CCD residues from other subunit. CX14442 has a larger tert-butyl group than the methyl of BI-1001, and forms better interactions with the highly hydrophobic binding pocket of HIV-1 IN CCD dimer interface, which can explain the stronger affinity of CX14442 than BI-1001. Analysis of the binding mode of LEDGF/p75 with HIV-1 IN CCD reveals that the LEDGF/p75 integrase binding domain residues Ile365, Asp366, Phe406 and Val408 have significant contributions to the binding of the LEDGF/p75 to HIV1-IN. Remarkably, we found that binding of BI-1001 and CX14442 to HIV-1 IN CCD induced the structural rearrangements of the 140 s loop and oration displacements of the side chains of the three conserved catalytic residues Asp64, Asp116, and Glu152 located at the active site. These results we obtained will be valuable not only for understanding the allosteric inhibition mechanism of LEDGINs but also for the rational design of allosteric inhibitors of HIV-1 IN targeting LEDGF/p75 binding site.

  5. Piracetam Defines a New Binding Site for Allosteric Modulators of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors§

    Science.gov (United States)

    Ahmed, Ahmed H.; Oswald, Robert E.

    2010-01-01

    Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to both GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators. PMID:20163115

  6. Piracetam defines a new binding site for allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors.

    Science.gov (United States)

    Ahmed, Ahmed H; Oswald, Robert E

    2010-03-11

    Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators.

  7. A novel polyamine allosteric site of SpeG from Vibrio cholerae is revealed by its dodecameric structure.

    Science.gov (United States)

    Filippova, Ekaterina V; Kuhn, Misty L; Osipiuk, Jerzy; Kiryukhina, Olga; Joachimiak, Andrzej; Ballicora, Miguel A; Anderson, Wayne F

    2015-03-27

    Spermidine N-acetyltransferase, encoded by the gene speG, catalyzes the initial step in the degradation of polyamines and is a critical enzyme for determining the polyamine concentrations in bacteria. In Escherichia coli, studies have shown that SpeG is the enzyme responsible for acetylating spermidine under stress conditions and for preventing spermidine toxicity. Not all bacteria contain speG, and many bacterial pathogens have developed strategies to either acquire or silence it for pathogenesis. Here, we present thorough kinetic analyses combined with structural characterization of the VCA0947 SpeG enzyme from the important human pathogen Vibrio cholerae. Our studies revealed the unexpected presence of a previously unknown allosteric site and an unusual dodecameric structure for a member of the Gcn5-related N-acetyltransferase superfamily. We show that SpeG forms dodecamers in solution and in crystals and describe its three-dimensional structure in several ligand-free and liganded structures. Importantly, these structural data define the first view of a polyamine bound in an allosteric site of an N-acetyltransferase. Kinetic characterization of SpeG from V. cholerae showed that it acetylates spermidine and spermine. The behavior of this enzyme is complex and exhibits sigmoidal curves and substrate inhibition. We performed a detailed non-linear regression kinetic analysis to simultaneously fit families of substrate saturation curves to uncover a simple kinetic mechanism that explains the apparent complexity of this enzyme. Our results provide a fundamental understanding of the bacterial SpeG enzyme, which will be key toward understanding the regulation of polyamine levels in bacteria during pathogenesis. Copyright © 2015. Published by Elsevier Ltd.

  8. Characterization of an allosteric citalopram-binding site at the serotonin transporter

    DEFF Research Database (Denmark)

    Chen, Fenghua; Breum Larsen, Mads; Neubauer, Henrik Amtoft

    2005-01-01

    The serotonin transporter (SERT), which belongs to a family of       sodium/chloride-dependent transporters, is the major pharmacological       target in the treatment of several clinical disorders, including       depression and anxiety. In the present study we show that the dissociation......       rate, of [3H]S-citalopram from human SERT, is retarded by the presence of       serotonin, as well as by several antidepressants, when present in the       dissociation buffer. Dissociation of [3H]S-citalopram from SERT is most       potently inhibited by S-citalopram followed by R......-citalopram, sertraline,       serotonin and paroxetine. EC50 values for S- and R-citalopram are 3.6 +/-       0.4 microm and 19.4 +/- 2.3 microm, respectively. Fluoxetine, venlafaxine       and duloxetine have no significant effect on the dissociation of       [3H]S-citalopram. Allosteric modulation of dissociation...

  9. CavityPlus: a web server for protein cavity detection with pharmacophore modelling, allosteric site identification and covalent ligand binding ability prediction.

    Science.gov (United States)

    Xu, Youjun; Wang, Shiwei; Hu, Qiwan; Gao, Shuaishi; Ma, Xiaomin; Zhang, Weilin; Shen, Yihang; Chen, Fangjin; Lai, Luhua; Pei, Jianfeng

    2018-05-10

    CavityPlus is a web server that offers protein cavity detection and various functional analyses. Using protein three-dimensional structural information as the input, CavityPlus applies CAVITY to detect potential binding sites on the surface of a given protein structure and rank them based on ligandability and druggability scores. These potential binding sites can be further analysed using three submodules, CavPharmer, CorrSite, and CovCys. CavPharmer uses a receptor-based pharmacophore modelling program, Pocket, to automatically extract pharmacophore features within cavities. CorrSite identifies potential allosteric ligand-binding sites based on motion correlation analyses between cavities. CovCys automatically detects druggable cysteine residues, which is especially useful to identify novel binding sites for designing covalent allosteric ligands. Overall, CavityPlus provides an integrated platform for analysing comprehensive properties of protein binding cavities. Such analyses are useful for many aspects of drug design and discovery, including target selection and identification, virtual screening, de novo drug design, and allosteric and covalent-binding drug design. The CavityPlus web server is freely available at http://repharma.pku.edu.cn/cavityplus or http://www.pkumdl.cn/cavityplus.

  10. Conopeptide ρ-TIA Defines a New Allosteric Site on the Extracellular Surface of the α1B-Adrenoceptor*♦

    Science.gov (United States)

    Ragnarsson, Lotten; Wang, Ching-I Anderson; Andersson, Åsa; Fajarningsih, Dewi; Monks, Thea; Brust, Andreas; Rosengren, K. Johan; Lewis, Richard J.

    2013-01-01

    The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β1-adrenergic receptor crystal structure, we modeled the α1B-adrenoceptor (α1B-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α1B-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α1-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs. PMID:23184947

  11. 8-anilino-1-naphthaline sulfonate binds at the hemoglobin allosteric regulatory sites: inhibitory analyses

    International Nuclear Information System (INIS)

    Bokut', S.B.; Parul', D.A.; Yachnik, N.N.; Milyutin, A.A.

    2001-01-01

    The present study focused on the localization at least one of the ANS binding sites in the major form of human hemoglobin HbA. High-resolution docking predict ANS binding to the hemoglobin central cavity. Steady-state fluorescence titration data obtained in the absence/presence of natural effector inositol hexaphosphate (IHP) allowed to conclude that IHP competitively inhibited ANS binding to HbA. Thus, we must conclude that one of the ANS binding sites is central cavity, which makes it possible to monitor changes at this region upon ligation/deligation, effector binding and changes in hemoglobin structure

  12. The Low-Affinity Binding of Second Generation Radiotracers Targeting TSPO is Associated with a Unique Allosteric Binding Site

    Czech Academy of Sciences Publication Activity Database

    Rojas, C.; Stathis, M.; Coughlin, J. M.; Pomper, M.; Slusher, Barbara S.

    2018-01-01

    Roč. 13, č. 1 (2018), s. 1-5 ISSN 1557-1890 Institutional support: RVO:61388963 Keywords : translocator protein 18KDa (TSPO) * allosteric modulation * residence time Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 3.339, year: 2016

  13. An allosteric binding site at the human serotonin transporter mediates the inhibition of escitalopram by R-citalopram: kinetic binding studies with the ALI/VFL-SI/TT mutant.

    Science.gov (United States)

    Zhong, Huailing; Hansen, Kasper B; Boyle, Noel J; Han, Kiho; Muske, Galina; Huang, Xinyan; Egebjerg, Jan; Sánchez, Connie

    2009-10-25

    The human serotonin transporter (hSERT) has primary and allosteric binding sites for escitalopram and R-citalopram. Previous studies have established that the interaction of these two compounds at a low affinity allosteric binding site of hSERT can affect the dissociation of [(3)H]escitalopram from hSERT. The allosteric binding site involves a series of residues in the 10th, 11th, and 12th trans-membrane domains of hSERT. The low affinity allosteric activities of escitalopram and R-citalopram are essentially eliminated in a mutant hSERT with changes in some of these residues, namely A505V, L506F, I507L, S574T, I575T, as measured in dissociation binding studies. We confirm that in association binding experiments, R-citalopram at clinically relevant concentrations reduces the association rate of [(3)H]escitalopram as a ligand to wild type hSERT. We demonstrate that the ability of R-citalopram to reduce the association rate of escitalopram is also abolished in the mutant hSERT (A505V, L506F, I507L, S574T, I575T), along with the expected disruption the low affinity allosteric function on dissociation binding. This suggests that the allosteric binding site mediates both the low affinity and higher affinity interactions between R-citalopram, escitalopram, and hSERT. Our data add an additional structural basis for the different efficacies of escitalopram compared to racemic citalopram reported in animal studies and clinical trials, and substantiate the hypothesis that hSERT has complex allosteric mechanisms underlying the unexplained in vivo activities of its inhibitors.

  14. Allosteric transition: a comparison of two models

    DEFF Research Database (Denmark)

    Bindslev, Niels

    2013-01-01

    Introduction Two recent models are in use for analysis of allosteric drug action at receptor sites remote from orthosteric binding sites. One is an allosteric two-state mechanical model derived in 2000 by David Hall. The other is an extended operational model developed in 2007 by Arthur...... of model both for simulation and analysis of allosteric concentration-responses at equilibrium or steady-state. Conclusions As detailed knowledge of receptors systems becomes available, systems with several pathways and states and/ or more than two binding sites should be analysed by extended forms...

  15. Allosteric enhancers, allosteric agonists and ago-allosteric modulators: where do they bind and how do they act?

    DEFF Research Database (Denmark)

    Schwartz, Thue W; Holst, Birgitte

    2007-01-01

    Many small-molecule agonists also display allosteric properties. Such ago-allosteric modulators act as co-agonists, providing additive efficacy--instead of partial antagonism--and they can affect--and often improve--the potency of the endogenous agonist. Surprisingly, the apparent binding sites...... different binding modes. In another, dimeric, receptor scenario, the endogenous agonist binds to one protomer while the ago-allosteric modulator binds to the other, 'allosteric' protomer. It is suggested that testing for ago-allosteric properties should be an integral part of the agonist drug discovery...... process because a compound that acts with--rather than against--the endogenous agonist could be an optimal agonist drug....

  16. Derivation of the Crick-Wyman equation for allosteric proteins defining the difference between the number of binding sites and the Hill coefficient.

    Science.gov (United States)

    Poitevin, Frédéric; Edelstein, Stuart J

    2013-05-13

    In response to a 100-word footnote in the 1965 article by Monod, Wyman, and Changeux, a detailed manuscript signed by Francis Crick and Jeffries Wyman with 6000 words and 30 equations entitled "A Footnote on Allostery" circulated in 1965 among a limited group of scientists interested in allosteric interactions. This interesting and provocative document is published in this special issue for the first time. An intriguing equation in their text relates the difference between n (the number of ligand binding sites) and n' (the Hill coefficient) to the ratio of the saturation functions Y¯, for oligomers with n-1 and n binding sites. A compact derivation of this equation was not provided by Crick and Wyman, but one is presented here based on a definition of Y¯ involving the binding polynomial and its first derivative. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

    Science.gov (United States)

    Fago, Angela; Malte, Hans; Storz, Jay F.; Gorr, Thomas A.

    2013-01-01

    In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3−), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3− levels and metabolic rates increase. Examination of α- and β-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: β82Lys→Gln, β143His→Val, and β146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3− binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues. PMID:23720132

  18. Identification of a negative allosteric site on human α4β2 and α3β4 neuronal nicotinic acetylcholine receptors.

    Directory of Open Access Journals (Sweden)

    Ryan E Pavlovicz

    Full Text Available Acetylcholine-based neurotransmission is regulated by cationic, ligand-gated ion channels called nicotinic acetylcholine receptors (nAChRs. These receptors have been linked to numerous neurological diseases and disorders such as Alzheimer's disease, Parkinson's disease, and nicotine addiction. Recently, a class of compounds has been discovered that antagonize nAChR function in an allosteric fashion. Models of human α4β2 and α3β4 nicotinic acetylcholine receptor (nAChR extracellular domains have been developed to computationally explore the binding of these compounds, including the dynamics and free energy changes associated with ligand binding. Through a blind docking study to multiple receptor conformations, the models were used to determine a putative binding mode for the negative allosteric modulators. This mode, in close proximity to the agonist binding site, is presented in addition to a hypothetical mode of antagonism that involves obstruction of C loop closure. Molecular dynamics simulations and MM-PBSA free energy of binding calculations were used as computational validation of the predicted binding mode, while functional assays on wild-type and mutated receptors provided experimental support. Based on the proposed binding mode, two residues on the β2 subunit were independently mutated to the corresponding residues found on the β4 subunit. The T58K mutation resulted in an eight-fold decrease in the potency of KAB-18, a compound that exhibits preferential antagonism for human α4β2 over α3β4 nAChRs, while the F118L mutation resulted in a loss of inhibitory activity for KAB-18 at concentrations up to 100 µM. These results demonstrate the selectivity of KAB-18 for human α4β2 nAChRs and validate the methods used for identifying the nAChR modulator binding site. Exploitation of this site may lead to the development of more potent and subtype-selective nAChR antagonists which may be used in the treatment of a number of neurological

  19. Exploring allosteric coupling in the α-subunit of Heterotrimeric G proteins using evolutionary and ensemble-based approaches

    Directory of Open Access Journals (Sweden)

    Hilser Vincent J

    2008-05-01

    Full Text Available Abstract Background Allosteric coupling, which can be defined as propagation of a perturbation at one region of the protein molecule (such as ligand binding to distant sites in the same molecule, constitutes the most general mechanism of regulation of protein function. However, unlike molecular details of ligand binding, structural elements involved in allosteric effects are difficult to diagnose. Here, we identified allosteric linkages in the α-subunits of heterotrimeric G proteins, which were evolved to transmit membrane receptor signals by allosteric mechanisms, by using two different approaches that utilize fundamentally different and independent information. Results We analyzed: 1 correlated mutations in the family of G protein α-subunits, and 2 cooperativity of the native state ensemble of the Gαi1 or transducin. The combination of these approaches not only recovered already-known details such as the switch regions that change conformation upon nucleotide exchange, and those regions that are involved in receptor, effector or Gβγ interactions (indicating that the predictions of the analyses can be viewed with a measure of confidence, but also predicted new sites that are potentially involved in allosteric communication in the Gα protein. A summary of the new sites found in the present analysis, which were not apparent in crystallographic data, is given along with known functional and structural information. Implications of the results are discussed. Conclusion A set of residues and/or structural elements that are potentially involved in allosteric communication in Gα is presented. This information can be used as a guide to structural, spectroscopic, mutational, and theoretical studies on the allosteric network in Gα proteins, which will provide a better understanding of G protein-mediated signal transduction.

  20. Discovery of a Hepatitis C Virus NS5B Replicase Palm Site Allosteric Inhibitor (BMS-929075) Advanced to Phase 1 Clinical Studies

    Energy Technology Data Exchange (ETDEWEB)

    Yeung, Kap-Sun; Beno, Brett R.; Parcella, Kyle; Bender, John A.; Grant-Young, Katherine A.; Nickel, Andrew; Gunaga, Prashantha; Anjanappa, Prakash; Bora, Rajesh Onkardas; Selvakumar, Kumaravel; Rigat, Karen; Wang, Ying-Kai; Liu, Mengping; Lemm, Julie; Mosure, Kathy; Sheriff, Steven; Wan, Changhong; Witmer, Mark; Kish, Kevin; Hanumegowda, Umesh; Zhuo, Xiaoliang; Shu, Yue-Zhong; Parker, Dawn; Haskell, Roy; Ng, Alicia; Gao, Qi; Colston, Elizabeth; Raybon, Joseph; Grasela, Dennis M.; Santone, Kenneth; Gao, Min; Meanwell, Nicholas A.; Sinz, Michael; Soars, Matthew G.; Knipe, Jay O.; Roberts, Susan B.; Kadow, John F.

    2017-05-04

    The hepatitis C virus (HCV) NS5B replicase is a prime target for the development of direct-acting antiviral drugs for the treatment of chronic HCV infection. Inspired by the overlay of bound structures of three structurally distinct NS5B palm site allosteric inhibitors, the high-throughput screening hit anthranilic acid 4, the known benzofuran analogue 5, and the benzothiadiazine derivative 6, an optimization process utilizing the simple benzofuran template 7 as a starting point for a fragment growing approach was pursued. A delicate balance of molecular properties achieved via disciplined lipophilicity changes was essential to achieve both high affinity binding and a stringent targeted absorption, distribution, metabolism, and excretion profile. These efforts led to the discovery of BMS-929075 (37), which maintained ligand efficiency relative to early leads, demonstrated efficacy in a triple combination regimen in HCV replicon cells, and exhibited consistently high oral bioavailability and pharmacokinetic parameters across preclinical animal species. The human PK properties from the Phase I clinical studies of 37 were better than anticipated and suggest promising potential for QD administration.

  1. Allosteric modulation of G-protein coupled receptors

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Spalding, Tracy A

    2004-01-01

    are believed to activate (agonists) or inhibit (competitive antagonists) receptor signalling by binding the receptor at the same site as the endogenous agonist, the orthosteric site. In contrast, allosteric ligands modulate receptor function by binding to different regions in the receptor, allosteric sites....... In recent years, combinatorial chemistry and high throughput screening have helped identify several allosteric GPCR modulators with novel structures, several of which already have become valuable pharmacological tools and may be candidates for clinical testing in the near future. This mini review outlines...... the current status and perspectives of allosteric modulation of GPCR function with emphasis on the pharmacology of endogenous and synthesised modulators, their receptor interactions and the therapeutic prospects of allosteric ligands compared to orthosteric ligands....

  2. Physician-Rating Web Sites: Ethical Implications.

    Science.gov (United States)

    Samora, Julie Balch; Lifchez, Scott D; Blazar, Philip E

    2016-01-01

    To understand the ethical and professional implications of physician behavior changes secondary to online physician-rating Web sites (PRWs). The American Society for Surgery of the Hand (ASSH) Ethics and Professionalism Committee surveyed the ASSH membership regarding PRWs. We sent a 14-item questionnaire to 2,664 active ASSH members who practice in both private and academic settings in the United States. We received 312 responses, a 12% response incidence. More than 65% of the respondents had a slightly or highly unfavorable impression of these Web sites. Only 34% of respondents had ever updated or created a profile for PRWs, although 62% had observed inaccuracies in their profile. Almost 90% of respondents had not made any changes in their practice owing to comments or reviews. One-third of respondents had solicited favorable reviews from patients, and 3% of respondents have paid to improve their ratings. PRWs are going to become more prevalent, and more research is needed to fully understand the implications. There are several ethical implications that PRWs pose to practicing physicians. We contend that it is morally unsound to pay for good reviews. The recourse for physicians when an inaccurate and potentially libelous review has been written is unclear. Some physicians have required patients to sign a waiver preventing them from posting negative comments online. We propose the development of a task force to assess the professional, ethical, and legal implications of PRWs, including working with companies to improve accuracy of information, oversight, and feedback opportunities. It is expected that PRWs will play an increasing role in the future; it is unclear whether there will be a uniform reporting system, or whether these online ratings will influence referral patterns and/or quality improvement. Copyright © 2016 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.

  3. Allosteric regulation of epigenetic modifying enzymes.

    Science.gov (United States)

    Zucconi, Beth E; Cole, Philip A

    2017-08-01

    Epigenetic enzymes including histone modifying enzymes are key regulators of gene expression in normal and disease processes. Many drug development strategies to target histone modifying enzymes have focused on ligands that bind to enzyme active sites, but allosteric pockets offer potentially attractive opportunities for therapeutic development. Recent biochemical studies have revealed roles for small molecule and peptide ligands binding outside of the active sites in modulating the catalytic activities of histone modifying enzymes. Here we highlight several examples of allosteric regulation of epigenetic enzymes and discuss the biological significance of these findings. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Development of allosteric modulators of GPCRs for treatment of CNS disorders.

    Science.gov (United States)

    Nickols, Hilary Highfield; Conn, P Jeffrey

    2014-01-01

    The discovery of allosteric modulators of G protein-coupled receptors (GPCRs) provides a promising new strategy with potential for developing novel treatments for a variety of central nervous system (CNS) disorders. Traditional drug discovery efforts targeting GPCRs have focused on developing ligands for orthosteric sites which bind endogenous ligands. Allosteric modulators target a site separate from the orthosteric site to modulate receptor function. These allosteric agents can either potentiate (positive allosteric modulator, PAM) or inhibit (negative allosteric modulator, NAM) the receptor response and often provide much greater subtype selectivity than orthosteric ligands for the same receptors. Experimental evidence has revealed more nuanced pharmacological modes of action of allosteric modulators, with some PAMs showing allosteric agonism in combination with positive allosteric modulation in response to endogenous ligand (ago-potentiators) as well as "bitopic" ligands that interact with both the allosteric and orthosteric sites. Drugs targeting the allosteric site allow for increased drug selectivity and potentially decreased adverse side effects. Promising evidence has demonstrated potential utility of a number of allosteric modulators of GPCRs in multiple CNS disorders, including neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, as well as psychiatric or neurobehavioral diseases such as anxiety, schizophrenia, and addiction. © 2013.

  5. Orthosteric and allosteric potentiation of heteromeric neuronal nicotinic acetylcholine receptors.

    Science.gov (United States)

    Wang, Jingyi; Lindstrom, Jon

    2018-06-01

    Heteromeric nicotinic ACh receptors (nAChRs) were thought to have two orthodox agonist-binding sites at two α/β subunit interfaces. Highly selective ligands are hard to develop by targeting orthodox agonist sites because of high sequence similarity of this binding pocket among different subunits. Recently, unorthodox ACh-binding sites have been discovered at some α/α and β/α subunit interfaces, such as α4/α4, α5/α4 and β3/α4. Targeting unorthodox sites may yield subtype-selective ligands, such as those for (α4β2) 2 α5, (α4β2) 2 β3 and (α6β2) 2 β3 nAChRs. The unorthodox sites have unique pharmacology. Agonist binding at one unorthodox site is not sufficient to activate nAChRs, but it increases activation from the orthodox sites. NS9283, a selective agonist for the unorthodox α4/α4 site, was initially thought to be a positive allosteric modulator (PAM). NS9283 activates nAChRs with three engineered α4/α4 sites. PAMs, on the other hand, act at allosteric sites where ACh cannot bind. Known PAM sites include the ACh-homologous non-canonical site (e.g. morantel at β/α), the C-terminus (e.g. Br-PBTC and 17β-estradiol), a transmembrane domain (e.g. LY2087101) or extracellular and transmembrane domain interfaces (e.g. NS206). Some of these PAMs, such as Br-PBTC and 17β-estradiol, require only one subunit to potentiate activation of nAChRs. In this review, we will discuss differences between activation from orthosteric and allosteric sites, their selective ligands and clinical implications. These studies have advanced understanding of the structure, assembly and pharmacology of heteromeric neuronal nAChRs. This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc. © 2017 The British Pharmacological Society.

  6. Emerging Computational Methods for the Rational Discovery of Allosteric Drugs.

    Science.gov (United States)

    Wagner, Jeffrey R; Lee, Christopher T; Durrant, Jacob D; Malmstrom, Robert D; Feher, Victoria A; Amaro, Rommie E

    2016-06-08

    Allosteric drug development holds promise for delivering medicines that are more selective and less toxic than those that target orthosteric sites. To date, the discovery of allosteric binding sites and lead compounds has been mostly serendipitous, achieved through high-throughput screening. Over the past decade, structural data has become more readily available for larger protein systems and more membrane protein classes (e.g., GPCRs and ion channels), which are common allosteric drug targets. In parallel, improved simulation methods now provide better atomistic understanding of the protein dynamics and cooperative motions that are critical to allosteric mechanisms. As a result of these advances, the field of predictive allosteric drug development is now on the cusp of a new era of rational structure-based computational methods. Here, we review algorithms that predict allosteric sites based on sequence data and molecular dynamics simulations, describe tools that assess the druggability of these pockets, and discuss how Markov state models and topology analyses provide insight into the relationship between protein dynamics and allosteric drug binding. In each section, we first provide an overview of the various method classes before describing relevant algorithms and software packages.

  7. Allosteric Regulation of Proteins

    Indian Academy of Sciences (India)

    ... Lecture Workshops · Refresher Courses · Symposia · Live Streaming. Home; Journals; Resonance – Journal of Science Education; Volume 22; Issue 1. Allosteric Regulation of Proteins: A Historical Perspective on the Development of Concepts and Techniques. General Article Volume 22 Issue 1 January 2017 pp 37-50 ...

  8. Surface dynamics in allosteric regulation of protein-protein interactions: modulation of calmodulin functions by Ca2+.

    Directory of Open Access Journals (Sweden)

    Yosef Y Kuttner

    2013-04-01

    Full Text Available Knowledge of the structural basis of protein-protein interactions (PPI is of fundamental importance for understanding the organization and functioning of biological networks and advancing the design of therapeutics which target PPI. Allosteric modulators play an important role in regulating such interactions by binding at site(s orthogonal to the complex interface and altering the protein's propensity for complex formation. In this work, we apply an approach recently developed by us for analyzing protein surfaces based on steered molecular dynamics simulation (SMD to the study of the dynamic properties of functionally distinct conformations of a model protein, calmodulin (CaM, whose ability to interact with target proteins is regulated by the presence of the allosteric modulator Ca(2+. Calmodulin is a regulatory protein that acts as an intracellular Ca(2+ sensor to control a wide variety of cellular processes. We demonstrate that SMD analysis is capable of pinpointing CaM surfaces implicated in the recognition of both the allosteric modulator Ca(2+ and target proteins. Our analysis of changes in the dynamic properties of the CaM backbone elicited by Ca(2+ binding yielded new insights into the molecular mechanism of allosteric regulation of CaM-target interactions.

  9. Virtual screening with AutoDock Vina and the common pharmacophore engine of a low diversity library of fragments and hits against the three allosteric sites of HIV integrase: participation in the SAMPL4 protein-ligand binding challenge.

    Science.gov (United States)

    Perryman, Alexander L; Santiago, Daniel N; Forli, Stefano; Martins, Diogo Santos; Olson, Arthur J

    2014-04-01

    To rigorously assess the tools and protocols that can be used to understand and predict macromolecular recognition, and to gain more structural insight into three newly discovered allosteric binding sites on a critical drug target involved in the treatment of HIV infections, the Olson and Levy labs collaborated on the SAMPL4 challenge. This computational blind challenge involved predicting protein-ligand binding against the three allosteric sites of HIV integrase (IN), a viral enzyme for which two drugs (that target the active site) have been approved by the FDA. Positive control cross-docking experiments were utilized to select 13 receptor models out of an initial ensemble of 41 different crystal structures of HIV IN. These 13 models of the targets were selected using our new "Rank Difference Ratio" metric. The first stage of SAMPL4 involved using virtual screens to identify 62 active, allosteric IN inhibitors out of a set of 321 compounds. The second stage involved predicting the binding site(s) and crystallographic binding mode(s) for 57 of these inhibitors. Our team submitted four entries for the first stage that utilized: (1) AutoDock Vina (AD Vina) plus visual inspection; (2) a new common pharmacophore engine; (3) BEDAM replica exchange free energy simulations, and a Consensus approach that combined the predictions of all three strategies. Even with the SAMPL4's very challenging compound library that displayed a significantly lower amount of structural diversity than most libraries that are conventionally employed in prospective virtual screens, these approaches produced hit rates of 24, 25, 34, and 27 %, respectively, on a set with 19 % declared binders. Our only entry for the second stage challenge was based on the results of AD Vina plus visual inspection, and it ranked third place overall according to several different metrics provided by the SAMPL4 organizers. The successful results displayed by these approaches highlight the utility of the computational

  10. Virtual screening with AutoDock Vina and the common pharmacophore engine of a low diversity library of fragments and hits against the three allosteric sites of HIV integrase: participation in the SAMPL4 protein-ligand binding challenge

    Science.gov (United States)

    Perryman, Alexander L.; Santiago, Daniel N.; Forli, Stefano; Santos-Martins, Diogo; Olson, Arthur J.

    2014-04-01

    To rigorously assess the tools and protocols that can be used to understand and predict macromolecular recognition, and to gain more structural insight into three newly discovered allosteric binding sites on a critical drug target involved in the treatment of HIV infections, the Olson and Levy labs collaborated on the SAMPL4 challenge. This computational blind challenge involved predicting protein-ligand binding against the three allosteric sites of HIV integrase (IN), a viral enzyme for which two drugs (that target the active site) have been approved by the FDA. Positive control cross-docking experiments were utilized to select 13 receptor models out of an initial ensemble of 41 different crystal structures of HIV IN. These 13 models of the targets were selected using our new "Rank Difference Ratio" metric. The first stage of SAMPL4 involved using virtual screens to identify 62 active, allosteric IN inhibitors out of a set of 321 compounds. The second stage involved predicting the binding site(s) and crystallographic binding mode(s) for 57 of these inhibitors. Our team submitted four entries for the first stage that utilized: (1) AutoDock Vina (AD Vina) plus visual inspection; (2) a new common pharmacophore engine; (3) BEDAM replica exchange free energy simulations, and a Consensus approach that combined the predictions of all three strategies. Even with the SAMPL4's very challenging compound library that displayed a significantly lower amount of structural diversity than most libraries that are conventionally employed in prospective virtual screens, these approaches produced hit rates of 24, 25, 34, and 27 %, respectively, on a set with 19 % declared binders. Our only entry for the second stage challenge was based on the results of AD Vina plus visual inspection, and it ranked third place overall according to several different metrics provided by the SAMPL4 organizers. The successful results displayed by these approaches highlight the utility of the computational

  11. Implications of NGA for NEHRP site coefficients

    Science.gov (United States)

    Borcherdt, Roger D.

    2012-01-01

    Three proposals are provided to update tables 11.4-1 and 11.4-2 of Minimum Design Loads for Buildings and Other Structures (7-10), by the American Society of Civil Engineers (2010) (ASCE/SEI 7-10), with site coefficients implied directly by NGA (Next Generation Attenuation) ground motion prediction equations (GMPEs). Proposals include a recommendation to use straight-line interpolation to infer site coefficients at intermediate values of ̅vs (average shear velocity). Site coefficients are recommended to ensure consistency with ASCE/SEI 7-10 MCER (Maximum Considered Earthquake) seismic-design maps and simplified site-specific design spectra procedures requiring site classes with associated tabulated site coefficients and a reference site class with unity site coefficients. Recommended site coefficients are confirmed by independent observations of average site amplification coefficients inferred with respect to an average ground condition consistent with that used for the MCER maps. The NGA coefficients recommended for consideration are implied directly by the NGA GMPEs and do not require introduction of additional models.

  12. Supramolecular Allosteric Cofacial Porphyrin Complexes

    International Nuclear Information System (INIS)

    Oliveri, Christopher G.; Gianneschi, Nathan C.; Nguyen, Son Binh T.; Mirkin, Chad A.; Stern, Charlotte L.; Wawrzak, Zdzislaw; Pink, Maren

    2008-01-01

    Nature routinely uses cooperative interactions to regulate cellular activity. For years, chemists have designed synthetic systems that aim toward harnessing the reactivity common to natural biological systems. By learning how to control these interactions in situ, one begins to allow for the preparation of man-made biomimetic systems that can efficiently mimic the interactions found in Nature. To this end, we have designed a synthetic protocol for the preparation of flexible metal-directed supramolecular cofacial porphyrin complexes which are readily obtained in greater than 90% yield through the use of new hemilabile porphyrin ligands with bifunctional ether-phosphine or thioether-phosphine substituents at the 5 and 15 positions on the porphyrin ring. The resulting architectures contain two hemilabile ligand-metal domains (Rh I or Cu I sites) and two cofacially aligned porphyrins (Zn II sites), offering orthogonal functionalities and allowing these multimetallic complexes to exist in two states, 'condensed' or 'open'. Combining the ether-phosphine ligand with the appropriate Rh I or Cu I transition-metal precursors results in 'open' macrocyclic products. In contrast, reacting the thioether-phosphine ligand with RhI or CuI precursors yields condensed structures that can be converted into their 'open' macrocyclic forms via introduction of additional ancillary ligands. The change in cavity size that occurs allows these structures to function as allosteric catalysts for the acyl transfer reaction between X-pyridylcarbinol (where X = 2, 3, or 4) and 1-acetylimidazole. For 3- and 4-pyridylcarbinol, the 'open' macrocycle accelerates the acyl transfer reaction more than the condensed analogue and significantly more than the porphyrin monomer. In contrast, an allosteric effect was not observed for 2-pyridylcarbinol, which is expected to be a weaker binder and is unfavorably constrained inside the macrocyclic cavity.

  13. An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering.

    Science.gov (United States)

    Keedy, Daniel A; Hill, Zachary B; Biel, Justin T; Kang, Emily; Rettenmaier, T Justin; Brandao-Neto, Jose; Pearce, Nicholas M; von Delft, Frank; Wells, James A; Fraser, James S

    2018-06-07

    Allostery is an inherent feature of proteins, but it remains challenging to reveal the mechanisms by which allosteric signals propagate. A clearer understanding of this intrinsic circuitry would afford new opportunities to modulate protein function. Here we have identified allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal 'hidden' low-occupancy conformational states for protein and ligands. Our results converge on allosteric sites that are conformationally coupled to the active-site WPD loop and are hotspots for fragment binding. Targeting one of these sites with covalently tethered molecules or mutations allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure, revealed here by multitemperature crystallography, can elucidate allosteric mechanisms and open new doors for long-range control of protein function. © 2018, Keedy et al.

  14. Selective Negative Allosteric Modulation Of Metabotropic Glutamate Receptors - A Structural Perspective of Ligands and Mutants

    DEFF Research Database (Denmark)

    Harpsøe, Kasper; Isberg, Vignir; Tehan, Benjamin G

    2015-01-01

    modulators. In this analysis, we make the first comprehensive structural comparison of all metabotropic glutamate receptors, placing selective negative allosteric modulators and critical mutants into the detailed context of the receptor binding sites. A better understanding of how the different m......Glu allosteric modulator binding modes relates to selective pharmacological actions will be very valuable for rational design of safer drugs....

  15. Pharmacological characterization and modeling of the binding sites of novel 1,3-bis(pyridinylethynyl)benzenes as metabotropic glutamate receptor 5-selective negative allosteric modulators

    DEFF Research Database (Denmark)

    Mølck, Christina; Harpsøe, Kasper; Gloriam, David E

    2012-01-01

    )pyridine (MPEP)-derived negative allosteric modulators, 2-, 3-, and 4-BisPEB, have been characterized. 2-, 3-, and 4-BisPEB are 1,3-bis(pyridinylethynyl)-benzenes and differ only by the position of the nitrogen atoms in the pyridine rings. Despite their high structural similarity, 2-BisPEB [1,3-bis(pyridin-2......-ylethynyl)-benzene, nitrogen atoms in ortho positions], with an IC(50) value in the nanomolar range, is significantly more potent than the 3- and 4-pyridyl analogs. Mutational analysis, directed by a previously published mGluR5 homology model, was used to determine key residues for the ligand...... that the higher potency of 2-BisPEB is due to hydrogen bonding to Ser809 because the S809A mutation made 2-BisPEB equipotent to 3- and 4-BisPEB (IC(50), 1-2.5 µM). The potency of MPEP was also greatly affected by S809A (52-fold), suggesting that a Ser809-mediated hydrogen bond is also a key interaction between...

  16. Use of allosteric targets in the discovery of safer drugs.

    Science.gov (United States)

    Grover, Ashok Kumar

    2013-01-01

    The need for drugs with fewer side effects cannot be overemphasized. Today, most drugs modify the actions of enzymes, receptors, transporters and other molecules by directly binding to their active (orthosteric) sites. However, orthosteric site configuration is similar in several proteins performing related functions and this leads to a lower specificity of a drug for the desired protein. Consequently, such drugs may have adverse side effects. A new basis of drug discovery is emerging based on the binding of the drug molecules to sites away (allosteric) from the orthosteric sites. It is possible to find allosteric sites which are unique and hence more specific as targets for drug discovery. Of many available examples, two are highlighted here. The first is caloxins - a new class of highly specific inhibitors of plasma membrane Ca²⁺ pumps. The second concerns the modulation of receptors for the neurotransmitter acetylcholine, which binds to 12 types of receptors. Exploitation of allosteric sites has led to the discovery of drugs which can selectively modulate the activation of only 1 (M1 muscarinic) out of the 12 different types of acetylcholine receptors. These drugs are being tested for schizophrenia treatment. It is anticipated that the drug discovery exploiting allosteric sites will lead to more effective therapeutic agents with fewer side effects. Copyright © 2013 S. Karger AG, Basel.

  17. The second extracellular loop of the adenosine A1 receptor mediates activity of allosteric enhancers.

    Science.gov (United States)

    Kennedy, Dylan P; McRobb, Fiona M; Leonhardt, Susan A; Purdy, Michael; Figler, Heidi; Marshall, Melissa A; Chordia, Mahendra; Figler, Robert; Linden, Joel; Abagyan, Ruben; Yeager, Mark

    2014-02-01

    Allosteric enhancers of the adenosine A1 receptor amplify signaling by orthosteric agonists. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied by an agonist, thereby conferring specificity to stressed or injured tissues that produce adenosine. To explore the mechanism of allosteric enhancer activity, we examined their action on several A1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants, and (4) site-specific mutants. These findings were combined with homology modeling of the A1 receptor and in silico screening of an allosteric enhancer library. The binding modes of known docked allosteric enhancers correlated with the known structure-activity relationship, suggesting that these allosteric enhancers bind to a pocket formed by the second extracellular loop, flanked by residues S150 and M162. We propose a model in which this vestibule controls the entry and efflux of agonists from the orthosteric site and agonist binding elicits a conformational change that enables allosteric enhancer binding. This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists.

  18. Steric hindrance mutagenesis in the conserved extracellular vestibule impedes allosteric binding of antidepressants to the serotonin transporter

    DEFF Research Database (Denmark)

    Plenge, Per; Shi, Lei; Beuming, Thijs

    2012-01-01

    be involved in the allosteric binding in the extracellular vestibule located above the central substrate binding (S1) site. Indeed, mutagenesis of selected residues in the vestibule reduces the allosteric potency of (S)-citalopram and clomipramine. The identified site is further supported by the inhibitory...

  19. Differential modulation of thresholds for intracranial self-stimulation by mGlu5 positive and negative allosteric modulators: implications for effects on drug self-administration

    Directory of Open Access Journals (Sweden)

    M. Foster eOlive

    2012-01-01

    Full Text Available Pharmacological manipulation of the type 5 metabotropic glutamate (mGlu5 receptor alters various addiction related behaviors such as drug self-administration and the extinction and reinstatement of drug-seeking behavior. However, the effects of pharmacological modulation of mGlu5 receptors on brain reward function have not been widely investigated. We examined the effects of acute administration of positive and negative allosteric modulators (PAMs and NAMs, respectively on brain reward function by assessing thresholds for intracranial self-stimulation (ICSS. In addition, when acute effects were observed, we examined potential changes in altered ICSS thresholds following repeated administration. Male Sprague-Dawley rats were implanted with bipolar electrodes into the medial forebrain bundle and trained to respond for ICSS, followed by assessment of effects of mGlu5 ligands on ICSS thresholds using a discrete trials current intensity threshold determination procedure. Acute administration of the selective mGlu5 NAMs MTEP (0, 0.3, 1 or 3 mg/kg and fenobam (0, 3, 10, or 30 mg/kg dose-dependently increased ICSS thresholds (~70% at the highest dose tested, suggesting a deficit in brain reward function. Acute administration of the mGlu5 PAMs CDPPB (0, 10, 30 and 60 mg/kg or ADX47273 (0, 10, 30 and 60 mg/kg was without effect at any dose tested. When administered once daily for 5 consecutive days, the development of tolerance to the ability of threshold-elevating doses of MTEP and fenobam to increase ICSS thresholds was observed. We conclude that mGlu5 PAMs and NAMs differentially affect brain reward function, and that tolerance to the ability of mGlu5 NAMs to reduce brain reward function develops with repeated administration. These brain reward deficits should be taken into consideration when interpreting acute effects of mGlu5 NAMs on drug self-administration, and repeated administration may be an effective method to reduce these deficits.

  20. GABAA receptor: Positive and negative allosteric modulators.

    Science.gov (United States)

    Olsen, Richard W

    2018-01-31

    gamma-Aminobutyric acid (GABA)-mediated inhibitory neurotransmission and the gene products involved were discovered during the mid-twentieth century. Historically, myriad existing nervous system drugs act as positive and negative allosteric modulators of these proteins, making GABA a major component of modern neuropharmacology, and suggesting that many potential drugs will be found that share these targets. Although some of these drugs act on proteins involved in synthesis, degradation, and membrane transport of GABA, the GABA receptors Type A (GABA A R) and Type B (GABA B R) are the targets of the great majority of GABAergic drugs. This discovery is due in no small part to Professor Norman Bowery. Whereas the topic of GABA B R is appropriately emphasized in this special issue, Norman Bowery also made many insights into GABA A R pharmacology, the topic of this article. GABA A R are members of the ligand-gated ion channel receptor superfamily, a chloride channel family of a dozen or more heteropentameric subtypes containing 19 possible different subunits. These subtypes show different brain regional and subcellular localization, age-dependent expression, and potential for plastic changes with experience including drug exposure. Not only are GABA A R the targets of agonist depressants and antagonist convulsants, but most GABA A R drugs act at other (allosteric) binding sites on the GABA A R proteins. Some anxiolytic and sedative drugs, like benzodiazepine and related drugs, act on GABA A R subtype-dependent extracellular domain sites. General anesthetics including alcohols and neurosteroids act at GABA A R subunit-interface trans-membrane sites. Ethanol at high anesthetic doses acts on GABA A R subtype-dependent trans-membrane domain sites. Ethanol at low intoxicating doses acts at GABA A R subtype-dependent extracellular domain sites. Thus GABA A R subtypes possess pharmacologically specific receptor binding sites for a large group of different chemical classes of

  1. The origins of enhanced activity in factor VIIa analogs and the interplay between key allosteric sites revealed by hydrogen exchange mass spectrometry

    DEFF Research Database (Denmark)

    Rand, Kasper D; Andersen, Mette D; Olsen, Ole H

    2008-01-01

    Factor VIIa (FVIIa) circulates in the blood in a zymogen-like state. Only upon association with membrane-bound tissue factor (TF) at the site of vascular injury does FVIIa become active and able to initiate blood coagulation. Here we used hydrogen exchange monitored by mass spectrometry to invest......Factor VIIa (FVIIa) circulates in the blood in a zymogen-like state. Only upon association with membrane-bound tissue factor (TF) at the site of vascular injury does FVIIa become active and able to initiate blood coagulation. Here we used hydrogen exchange monitored by mass spectrometry...... to investigate the conformational effects of site-directed mutagenesis at key positions in FVIIa and the origins of enhanced intrinsic activity of FVIIa analogs. The differences in hydrogen exchange of two highly active variants, FVIIa(DVQ) and FVIIa(VEAY), imply that enhanced catalytic efficiency was attained...

  2. HIV integration sites and implications for maintenance of the reservoir.

    Science.gov (United States)

    Symons, Jori; Cameron, Paul U; Lewin, Sharon R

    2018-03-01

    To provide an overview of recent research of how HIV integration relates to productive and latent infection and implications for cure strategies. How and where HIV integrates provides new insights into how HIV persists on antiretroviral therapy (ART). Clonal expansion of infected cells with the same integration site demonstrates that T-cell proliferation is an important factor in HIV persistence, however, the driver of proliferation remains unclear. Clones with identical integration sites harbouring defective provirus can accumulate in HIV-infected individuals on ART and defective proviruses can express RNA and produce protein. HIV integration sites differ in clonally expanded and nonexpanded cells and in latently and productively infected cells and this influences basal and inducible transcription. There is a growing number of cellular proteins that can alter the pattern of integration to favour latency. Understanding these pathways may identify new interventions to eliminate latently infected cells. Using advances in analysing HIV integration sites, T-cell proliferation of latently infected cells is thought to play a major role in HIV persistence. Clonal expansion has been demonstrated with both defective and intact viruses. Production of viral RNA and protein from defective viruses may play a role in driving chronic immune activation. The site of integration may determine the likelihood of proliferation and the degree of basal and induced transcription. Finally, host factors and gene expression at the time of infection may determine the integration site. Together these new insights may lead to novel approaches to elimination of latently infected cells.

  3. SB265610 is an allosteric, inverse agonist at the human CXCR2 receptor

    Science.gov (United States)

    Bradley, ME; Bond, ME; Manini, J; Brown, Z; Charlton, SJ

    2009-01-01

    Background and purpose: In several previous studies, the C-X-C chemokine receptor (CXCR)2 antagonist 1-(2-bromo-phenyl)-3-(7-cyano-3H-benzotriazol-4-yl)-urea (SB265610) has been described as binding competitively with the endogenous agonist. This is in contrast to many other chemokine receptor antagonists, where the mechanism of antagonism has been described as allosteric. Experimental approach: To determine whether it displays a unique mechanism among the chemokine receptor antagonists, the mode of action of SB265610 was investigated at the CXCR2 receptor using radioligand and [35S]-GTPγS binding approaches in addition to chemotaxis of human neutrophils. Key results: In equilibrium saturation binding studies, SB265610 depressed the maximal binding of [125I]-interleukin-8 ([125I]-IL-8) without affecting the Kd. In contrast, IL-8 was unable to prevent binding of [3H]-SB265610. Kinetic binding experiments demonstrated that this was not an artefact of irreversible or slowly reversible binding. In functional experiments, SB265610 caused a rightward shift of the concentration-response curves to IL-8 and growth-related oncogene α, but also a reduction in maximal response elicited by each agonist. Fitting these data to an operational allosteric ternary complex model suggested that, once bound, SB265610 completely blocks receptor activation. SB265610 also inhibited basal [35S]-GTPγS binding in this preparation. Conclusions and implications: Taken together, these data suggest that SB265610 behaves as an allosteric inverse agonist at the CXCR2 receptor, binding at a region distinct from the agonist binding site to prevent receptor activation, possibly by interfering with G protein coupling. PMID:19422399

  4. The allosteric communication pathways in KIX domain of CBP

    Science.gov (United States)

    Palazzesi, Ferruccio; Barducci, Alessandro; Tollinger, Martin; Parrinello, Michele

    2013-01-01

    Allosteric regulation plays an important role in a myriad of biomacromolecular processes. Specifically, in a protein, the process of allostery refers to the transmission of a local perturbation, such as ligand binding, to a distant site. Decades after the discovery of this phenomenon, models built on static images of proteins are being reconsidered with the knowledge that protein dynamics plays an important role in its function. Molecular dynamics simulations are a valuable tool for studying complex biomolecular systems, providing an atomistic description of their structure and dynamics. Unfortunately, their predictive power has been limited by the complexity of the biomolecule free-energy surface and by the length of the allosteric timescale (in the order of milliseconds). In this work, we are able to probe the origins of the allosteric changes that transcription factor mixed lineage leukemia (MLL) causes to the interactions of KIX domain of CREB-binding protein (CBP) with phosphorylated kinase inducible domain (pKID), by combing all-atom molecular dynamics with enhanced sampling methods recently developed in our group. We discuss our results in relation to previous NMR studies. We also develop a general simulations protocol to study allosteric phenomena and many other biological processes that occur in the micro/milliseconds timescale. PMID:23940332

  5. Change in Allosteric Network Affects Binding Affinities of PDZ Domains: Analysis through Perturbation Response Scanning

    Science.gov (United States)

    Gerek, Z. Nevin; Ozkan, S. Banu

    2011-01-01

    The allosteric mechanism plays a key role in cellular functions of several PDZ domain proteins (PDZs) and is directly linked to pharmaceutical applications; however, it is a challenge to elaborate the nature and extent of these allosteric interactions. One solution to this problem is to explore the dynamics of PDZs, which may provide insights about how intramolecular communication occurs within a single domain. Here, we develop an advancement of perturbation response scanning (PRS) that couples elastic network models with linear response theory (LRT) to predict key residues in allosteric transitions of the two most studied PDZs (PSD-95 PDZ3 domain and hPTP1E PDZ2 domain). With PRS, we first identify the residues that give the highest mean square fluctuation response upon perturbing the binding sites. Strikingly, we observe that the residues with the highest mean square fluctuation response agree with experimentally determined residues involved in allosteric transitions. Second, we construct the allosteric pathways by linking the residues giving the same directional response upon perturbation of the binding sites. The predicted intramolecular communication pathways reveal that PSD-95 and hPTP1E have different pathways through the dynamic coupling of different residue pairs. Moreover, our analysis provides a molecular understanding of experimentally observed hidden allostery of PSD-95. We show that removing the distal third alpha helix from the binding site alters the allosteric pathway and decreases the binding affinity. Overall, these results indicate that (i) dynamics plays a key role in allosteric regulations of PDZs, (ii) the local changes in the residue interactions can lead to significant changes in the dynamics of allosteric regulations, and (iii) this might be the mechanism that each PDZ uses to tailor their binding specificities regulation. PMID:21998559

  6. Effects of the dopamine D2 allosteric modulator, PAOPA, on the expression of GRK2, arrestin-3, ERK1/2, and on receptor internalization.

    Directory of Open Access Journals (Sweden)

    Dipannita Basu

    Full Text Available The activity of G protein-coupled receptors (GPCRs is intricately regulated by a range of intracellular proteins, including G protein-coupled kinases (GRKs and arrestins. Understanding the effects of ligands on these signaling pathways could provide insights into disease pathophysiologies and treatment. The dopamine D2 receptor is a GPCR strongly implicated in the pathophysiology of a range of neurological and neuropsychiatric disorders, particularly schizophrenia. Previous studies from our lab have shown the preclinical efficacy of a novel allosteric drug, 3(R-[(2(S-pyrrolidinylcarbonylamino]-2-oxo-1-pyrrolidineacetamide (PAOPA, in attenuating schizophrenia-like behavioural abnormalities in rodent models of the disease. As an allosteric modulator, PAOPA binds to a site on the D2 receptor, which is distinct from the endogenous ligand-binding site, in order to modulate the binding of the D2 receptor ligand, dopamine. The exact signaling pathways affected by this allosteric modulator are currently unknown. The objectives of this study were to decipher the in vivo effects, in rats, of chronic PAOPA administration on D2 receptor regulatory and downstream molecules, including GRK2, arrestin-3 and extracellular receptor kinase (ERK 1/2. Additionally, an in vitro cellular model was also used to study PAOPA's effects on D2 receptor internalization. Results from western immunoblots showed that chronic PAOPA treatment increased the striatal expression of GRK2 by 41%, arrestin-3 by 34%, phospho-ERK1 by 51% and phospho-ERK2 by 36%. Results also showed that the addition of PAOPA to agonist treatment in cells increased D2 receptor internalization by 33%. This study provides the foundational evidence of putative signaling pathways, and changes in receptor localization, affected by treatment with PAOPA. It improves our understanding on the diverse mechanisms of action of allosteric modulators, while advancing PAOPA's development into a novel drug for the

  7. An allosteric conduit facilitates dynamic multisite substrate recognition by the SCFCdc4 ubiquitin ligase

    Science.gov (United States)

    Csizmok, Veronika; Orlicky, Stephen; Cheng, Jing; Song, Jianhui; Bah, Alaji; Delgoshaie, Neda; Lin, Hong; Mittag, Tanja; Sicheri, Frank; Chan, Hue Sun; Tyers, Mike; Forman-Kay, Julie D.

    2017-01-01

    The ubiquitin ligase SCFCdc4 mediates phosphorylation-dependent elimination of numerous substrates by binding one or more Cdc4 phosphodegrons (CPDs). Methyl-based NMR analysis of the Cdc4 WD40 domain demonstrates that Cyclin E, Sic1 and Ash1 degrons have variable effects on the primary Cdc4WD40 binding pocket. Unexpectedly, a Sic1-derived multi-CPD substrate (pSic1) perturbs methyls around a previously documented allosteric binding site for the chemical inhibitor SCF-I2. NMR cross-saturation experiments confirm direct contact between pSic1 and the allosteric pocket. Phosphopeptide affinity measurements reveal negative allosteric communication between the primary CPD and allosteric pockets. Mathematical modelling indicates that the allosteric pocket may enhance ultrasensitivity by tethering pSic1 to Cdc4. These results suggest negative allosteric interaction between two distinct binding pockets on the Cdc4WD40 domain may facilitate dynamic exchange of multiple CPD sites to confer ultrasensitive dependence on substrate phosphorylation.

  8. Fatty Acid-Mediated Inhibition of Metal Binding to the Multi-Metal Site on Serum Albumin: Implications for Cardiovascular Disease.

    Science.gov (United States)

    Blindauer, Claudia A; Khazaipoul, Siavash; Yu, Ruitao; Stewart, Alan J

    2016-01-01

    Human serum albumin (HSA) is the major protein in blood plasma and is responsible for circulatory transport of a range of small molecules including fatty acids, metal ions and drugs. We previously identified the major plasma Zn2+ transport site on HSA and revealed that fatty-acid binding (at a distinct site called the FA2 site) and Zn2+ binding are interdependent via an allosteric mechanism. Since binding affinities of long-chain fatty acids exceed those of plasma Zn2+, this means that under certain circumstances the binding of fatty acid molecules to HSA is likely to diminish HSA Zn2+-binding, and hence affects the control of circulatory and cellular Zn2+ dynamics. This relationship between circulatory fatty acid and Zn2+ dynamics is likely to have important physiological and pathological implications, especially since it has been recognised that Zn2+ acts as a signalling agent in many cell types. Fatty acid levels in the blood are dynamic, but most importantly, chronic elevation of plasma fatty acid levels is associated with some metabolic disorders and disease states - including myocardial infarction and other cardiovascular diseases. In this article, we briefly review the metal-binding properties of albumin and highlight the importance of their interplay with fatty acid binding. We also consider the impact of this dynamic link upon levels and speciation of plasma Zn2+, its effect upon cellular Zn2+ homeostasis and its relevance to cardiovascular and circulatory processes in health and disease.

  9. Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Yi; Qin, Ling; Ortiz Zacarías, Natalia V.; de Vries, Henk; Han, Gye Won; Gustavsson, Martin; Dabros, Marta; Zhao, Chunxia; Cherney, Robert J.; Carter, Percy; Stamos, Dean; Abagyan, Ruben; Cherezov, Vadim; Stevens, Raymond C.; IJzerman, Adriaan P.; Heitman, Laura H.; Tebben, Andrew; Kufareva, Irina; Handel , Tracy M. (Vertex Pharm); (Leiden-MC); (USC); (BMS); (UCSD)

    2016-12-07

    CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases2 including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer3. These disease associations have motivated numerous preclinical studies and clinical trials4 (see http://www.clinicaltrials.gov) in search of therapies that target the CCR2–chemokine axis. To aid drug discovery efforts5, here we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]7) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein–protein interactions, receptor–chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.

  10. Structural and kinetic studies of the allosteric transition in Sulfolobus solfataricus uracil phosphoribosyltransferase: Permanent activation by engineering of the C-terminus

    DEFF Research Database (Denmark)

    Christoffersen, Stig; Kadziola, Anders; Johansson, Eva

    2009-01-01

    and PPi, in the other sites. Combined with three existing structures of uracil phosphoribosyltransferase in complex with UMP and the allosteric inhibitor cytidine triphosphate (CTP), these structures provide valuable insight into the mechanism of allosteric transition from inhibited to active enzyme...

  11. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins

    Science.gov (United States)

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-01-01

    Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel’s ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators. PMID:27384555

  12. Evolution of allosteric regulation in chorismate mutases from early plants

    Energy Technology Data Exchange (ETDEWEB)

    Kroll, Kourtney; Holland, Cynthia K.; Starks, Courtney M.; Jez, Joseph M.

    2017-09-28

    Plants, fungi, and bacteria synthesize the aromatic amino acids: l-phenylalanine, l-tyrosine, and l-tryptophan. Chorismate mutase catalyzes the branch point reaction of phenylalanine and tyrosine biosynthesis to generate prephenate. In Arabidopsis thaliana, there are two plastid-localized chorismate mutases that are allosterically regulated (AtCM1 and AtCM3) and one cytosolic isoform (AtCM2) that is unregulated. Previous analysis of plant chorismate mutases suggested that the enzymes from early plants (i.e. bryophytes/moss, lycophytes, and basal angiosperms) formed a clade distinct from the isoforms found in flowering plants; however, no biochemical information on these enzymes is available. To understand the evolution of allosteric regulation in plant chorismate mutases, we analyzed a basal lineage of plant enzymes homologous to AtCM1 based on sequence similarity. The chorismate mutases from the moss/bryophyte Physcomitrella patens (PpCM1 and PpCM2), the lycophyte Selaginella moellendorffii (SmCM), and the basal angiosperm Amborella trichopoda (AmtCM1 and AmtCM2) were characterized biochemically. Tryptophan was a positive effector for each of the five enzymes examined. Histidine was a weak positive effector for PpCM1 and AmtCM1. Neither tyrosine nor phenylalanine altered the activity of SmCM; however, tyrosine was a negative regulator of the other four enzymes. Phenylalanine down-regulates both moss enzymes and AmtCM2. The 2.0 Å X-ray crystal structure of PpCM1 in complex with the tryptophan identified the allosteric effector site and reveals structural differences between the R- (more active) and T-state (less active) forms of plant chorismate mutases. Molecular insight into the basal plant chorismate mutases guides our understanding of the evolution of allosteric regulation in these enzymes.

  13. Heat shock protein 70 inhibitors. 2. 2,5'-thiodipyrimidines, 5-(phenylthio)pyrimidines, 2-(pyridin-3-ylthio)pyrimidines, and 3-(phenylthio)pyridines as reversible binders to an allosteric site on heat shock protein 70.

    Science.gov (United States)

    Taldone, Tony; Kang, Yanlong; Patel, Hardik J; Patel, Maulik R; Patel, Pallav D; Rodina, Anna; Patel, Yogita; Gozman, Alexander; Maharaj, Ronnie; Clement, Cristina C; Lu, Alvin; Young, Jason C; Chiosis, Gabriela

    2014-02-27

    The discovery and development of heat shock protein 70 (Hsp70) inhibitors is currently a hot topic in cancer. In the preceding paper in this issue ( 10.1021/jm401551n ), we have described structure-activity relationship studies in the first Hsp70 inhibitor class rationally designed to bind to a novel allosteric pocket located in the N-terminal domain of the protein. These ligands contained an acrylamide to take advantage of an active cysteine embedded in the allosteric pocket and acted as covalent protein modifiers upon binding. Here, we perform chemical modifications around the irreversible inhibitor scaffold to demonstrate that covalent modification is not a requirement for activity within this class of compounds. The study identifies derivative 27c, which mimics the biological effects of the irreversible inhibitors at comparable concentrations. Collectively, the back-to-back manuscripts describe the first pharmacophores that favorably and selectively interact with a never explored pocket in Hsp70 and provide a novel blueprint for a cancer-oriented development of Hsp70-directed ligands.

  14. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    Science.gov (United States)

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-11-01

    Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

  15. A dynamically coupled allosteric network underlies binding cooperativity in Src kinase.

    Science.gov (United States)

    Foda, Zachariah H; Shan, Yibing; Kim, Eric T; Shaw, David E; Seeliger, Markus A

    2015-01-20

    Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity.

  16. Allosteric regulation of rhomboid intramembrane proteolysis.

    Science.gov (United States)

    Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

    2014-09-01

    Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. © 2014 The Authors.

  17. Allosteric inhibitors of Coxsackie virus A24 RNA polymerase.

    Science.gov (United States)

    Schein, Catherine H; Rowold, Diane; Choi, Kyung H

    2016-02-15

    Coxsackie virus A24 (CVA24), a causative agent of acute hemorrhagic conjunctivitis, is a prototype of enterovirus (EV) species C. The RNA polymerase (3D(pol)) of CVA24 can uridylylate the viral peptide linked to the genome (VPg) from distantly related EV and is thus, a good model for studying this reaction. Once UMP is bound, VPgpU primes RNA elongation. Structural and mutation data have identified a conserved binding surface for VPg on the RNA polymerase (3D(pol)), located about 20Å from the active site. Here, computational docking of over 60,000 small compounds was used to select those with the lowest (best) specific binding energies (BE) for this allosteric site. Compounds with varying structures and low BE were assayed for their effect on formation of VPgU by CVA24-3D(pol). Two compounds with the lowest specific BE for the site inhibited both uridylylation and formation of VPgpolyU at 10-20μM. These small molecules can be used to probe the role of this allosteric site in polymerase function, and may be the basis for novel antiviral compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. RET Functions as a Dual-Specificity Kinase that Requires Allosteric Inputs from Juxtamembrane Elements

    Directory of Open Access Journals (Sweden)

    Iván Plaza-Menacho

    2016-12-01

    Full Text Available Receptor tyrosine kinases exhibit a variety of activation mechanisms despite highly homologous catalytic domains. Such diversity arises through coupling of extracellular ligand-binding portions with highly variable intracellular sequences flanking the tyrosine kinase domain and specific patterns of autophosphorylation sites. Here, we show that the juxtamembrane (JM segment enhances RET catalytic domain activity through Y687. This phospho-site is also required by the JM region to rescue an otherwise catalytically deficient RET activation-loop mutant lacking tyrosines. Structure-function analyses identified interactions between the JM hinge, αC helix, and an unconventional activation-loop serine phosphorylation site that engages the HRD motif and promotes phospho-tyrosine conformational accessibility and regulatory spine assembly. We demonstrate that this phospho-S909 arises from an intrinsic RET dual-specificity kinase activity and show that an equivalent serine is required for RET signaling in Drosophila. Our findings reveal dual-specificity and allosteric components for the mechanism of RET activation and signaling with direct implications for drug discovery.

  19. The different ways through which specificity works in orthosteric and allosteric drugs.

    Science.gov (United States)

    Nussinov, Ruth; Tsai, Chung-Jung

    2012-01-01

    Currently, there are two types of drugs on the market: orthosteric, which bind at the active site; and allosteric, which bind elsewhere on the protein surface, and allosterically change the conformation of the protein binding site. In this perspective we argue that the different mechanisms through which the two drug types affect protein activity and their potential pitfalls call for different considerations in drug design. The key problem facing orthosteric drugs is side effects which can occur by drug binding to homologous proteins sharing a similar binding site. Hence, orthosteric drugs should have very high affinity to the target; this would allow a low dosage to selectively achieve the goal of target-only binding. By contrast, allosteric drugs work by shifting the free energy landscape. Their binding to the protein surface perturbs the protein surface atoms, and the perturbation propagates like waves, finally reaching the binding site. Effective drugs should have atoms in good contact with the 'right' protein atoms; that is, the contacts should elicit propagation waves optimally reaching the protein binding site target. While affinity is important, the design should consider the protein conformational ensemble and the preferred propagation states. We provide examples from functional in vivo scenarios for both types of cases, and suggest how high potency can be achieved in allosteric drug development.

  20. Orthosteric and Allosteric Regulation in Trypsin-Like Peptidases

    DEFF Research Database (Denmark)

    Kromann-Tofting, Tobias

    Trypsin-like serine peptidases play an important role in many physiological and pathophysiological processes, the latter including cardiovascular diseases and cancer. Binding of natural ligands to functional sites on the peptidase surface balances the level of activity and substrate specificity......-ray crystallography to determine crystal structures of active and inactive conformations of muPA, combined with biochemical analysis, elucidated an allosteric regulatory mechanism, which is now believed to be highly conserved in the trypsin-like serine peptidases. Targeting zymogen activation represents an attractive...

  1. Yield and Depth of Burial Hydrodynamic Calculations in Granodiorite: Implications for the North Korean Test Site

    Science.gov (United States)

    2011-09-01

    the existence of a test site body wave magnitude (mb) bias between U. S. and the former Soviet Union test sites in Nevada and Semipalatinsk . The use...YIELD AND DEPTH OF BURIAL HYDRODYNAMIC CALCULATIONS IN GRANODIORITE:IMPLICATIONS FOR THE NORTH KOREAN TEST SITE Esteban Rougier, Christopher R...Korean test site and the May 2009 test . When compared to the Denny and Johnson (1991) and to the Heard and Ackerman (1967) cavity radius scaling models

  2. Allosteric regulation and communication between subunits in uracil phosphoribosyltransferase from Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Arent, Susan; Harris, Pernille; Jensen, Kaj Frank

    2005-01-01

    organisms. To understand the allosteric regulation, crystal structures were determined for S. solfataricus UPRTase in complex with UMP and with UMP and the allosteric inhibitor CTP. Also, a structure with UMP bound in half of the active sites was determined. All three complexes form tetramers but reveal...... to rearrangements in the quaternary structure imply that this residue plays a major role in regulation of the enzyme and in communication between subunits. The ribose ring of UMP adopts alternative conformations in the cis and trans subunits of the UPRTase-UMP tetramer with associated differences...

  3. Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins.

    Science.gov (United States)

    Yao, Xin-Qiu; Malik, Rabia U; Griggs, Nicholas W; Skjærven, Lars; Traynor, John R; Sivaramakrishnan, Sivaraj; Grant, Barry J

    2016-02-26

    G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gαi. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Allosteric cross-talk in chromatin can mediate drug-drug synergy

    Science.gov (United States)

    Adhireksan, Zenita; Palermo, Giulia; Riedel, Tina; Ma, Zhujun; Muhammad, Reyhan; Rothlisberger, Ursula; Dyson, Paul J.; Davey, Curt A.

    2017-03-01

    Exploitation of drug-drug synergism and allostery could yield superior therapies by capitalizing on the immensely diverse, but highly specific, potential associated with the biological macromolecular landscape. Here we describe a drug-drug synergy mediated by allosteric cross-talk in chromatin, whereby the binding of one drug alters the activity of the second. We found two unrelated drugs, RAPTA-T and auranofin, that yield a synergistic activity in killing cancer cells, which coincides with a substantially greater number of chromatin adducts formed by one of the compounds when adducts from the other agent are also present. We show that this occurs through an allosteric mechanism within the nucleosome, whereby defined histone adducts of one drug promote reaction of the other drug at a distant, specific histone site. This opens up possibilities for epigenetic targeting and suggests that allosteric modulation in nucleosomes may have biological relevance and potential for therapeutic interventions.

  5. Molecular Basis for Allosteric Inhibition of Acid-Sensing Ion Channel 1a by Ibuprofen

    DEFF Research Database (Denmark)

    Lynagh, Timothy; Romero-Rojo, José Luis; Lund, Camilla

    2017-01-01

    -clamp fluorometry. Our results show that ibuprofen is an allosteric inhibitor of ASIC1a, which binds to a crucial site in the agonist transduction pathway and causes conformational changes that oppose channel activation. Ibuprofen inhibits several ASIC subtypes, but certain ibuprofen derivatives show some...

  6. Molecular dynamics simulation study of PTP1B with allosteric inhibitor and its application in receptor based pharmacophore modeling

    Science.gov (United States)

    Bharatham, Kavitha; Bharatham, Nagakumar; Kwon, Yong Jung; Lee, Keun Woo

    2008-12-01

    Allosteric inhibition of protein tyrosine phosphatase 1B (PTP1B), has paved a new path to design specific inhibitors for PTP1B, which is an important drug target for the treatment of type II diabetes and obesity. The PTP1B1-282-allosteric inhibitor complex crystal structure lacks α7 (287-298) and moreover there is no available 3D structure of PTP1B1-298 in open form. As the interaction between α7 and α6-α3 helices plays a crucial role in allosteric inhibition, α7 was modeled to the PTP1B1-282 in open form complexed with an allosteric inhibitor (compound-2) and a 5 ns MD simulation was performed to investigate the relative orientation of the α7-α6-α3 helices. The simulation conformational space was statistically sampled by clustering analyses. This approach was helpful to reveal certain clues on PTP1B allosteric inhibition. The simulation was also utilized in the generation of receptor based pharmacophore models to include the conformational flexibility of the protein-inhibitor complex. Three cluster representative structures of the highly populated clusters were selected for pharmacophore model generation. The three pharmacophore models were subsequently utilized for screening databases to retrieve molecules containing the features that complement the allosteric site. The retrieved hits were filtered based on certain drug-like properties and molecular docking simulations were performed in two different conformations of protein. Thus, performing MD simulation with α7 to investigate the changes at the allosteric site, then developing receptor based pharmacophore models and finally docking the retrieved hits into two distinct conformations will be a reliable methodology in identifying PTP1B allosteric inhibitors.

  7. 2013 Philip S. Portoghese Medicinal Chemistry Lectureship: Drug Discovery Targeting Allosteric Sites†

    Science.gov (United States)

    2015-01-01

    The identification of sites on receptors topographically distinct from the orthosteric sites, so-called allosteric sites, has heralded novel approaches and modes of pharmacology for target modulation. Over the past 20 years, our understanding of allosteric modulation has grown significantly, and numerous advantages, as well as caveats (e.g., flat structure–activity relationships, species differences, “molecular switches”), have been identified. For multiple receptors and proteins, numerous examples have been described where unprecedented levels of selectivity are achieved along with improved physiochemical properties. While not a panacea, these novel approaches represent exciting opportunities for tool compound development to probe the pharmacology and therapeutic potential of discrete molecular targets, as well as new medicines. In this Perspective, in commemoration of the 2013 Philip S. Portoghese Medicinal Chemistry Lectureship (LindsleyC. W.Adventures in allosteric drug discovery. Presented at the 246th National Meeting of the American Chemical Society, Indianapolis, IN, September 10, 2013; The 2013 Portoghese Lectureship), several vignettes of drug discovery campaigns targeting novel allosteric mechanisms will be recounted, along with lessons learned and guidelines that have emerged for successful lead optimization. PMID:25180768

  8. Allosteric Binding in the Serotonin Transporter - Pharmacology, Structure, Function and Potential Use as a Novel Drug Target

    DEFF Research Database (Denmark)

    Loland, Claus J.; Sanchez, Connie; Plenge, Per

    2017-01-01

    The serotonin transporter (SERT) is an important drug target and the majority of currently used antidepressants are potent inhibitors of SERT, binding primarily to the substrate binding site. However, even though the existence of an allosteric modulator site was realized more than 30 years ago......, the research into this mechanism is still in its early days. The current knowledge about the allosteric site with respect to pharmacology, structure and function, and pharmacological tool compounds, is reviewed and a perspective is given on its potential as a drug target....

  9. Strontium-90 at the Hanford Site and its ecological implications

    International Nuclear Information System (INIS)

    RE Peterson; TM Poston

    2000-01-01

    Strontium-90, a radioactive contaminant from historical operations at the U.S. Department of Energy (DOE) Hanford Site, enters the Columbia River at several locations associated with former plutonium production reactors at the Site. Strontium-90 is of concern to humans and the environment because of its moderately long half-life (29.1 years), its potential for concentrating in bone tissue, and its relatively high energy of beta decay. Although strontium-90 in the environment is not a new issue for the Hanford Site, recent studies of near-river vegetation along the shoreline near the 100 Areas raised public concern about the possibility of strontium-90-contaminated groundwater reaching the riverbed and fall chinook salmon redds. To address these concerns, DOE asked Pacific Northwest National Laboratory (PNNL) to prepare this report on strontium-90, its distribution in groundwater, how and where it enters the river, and its potential ecological impacts, particularly with respect to fall chinook salmon. The purpose of the report is to characterize groundwater contaminants in the near-shore environment and to assess the potential for ecological impact using salmon embryos, one of the most sensitive ecological indicators for aquatic organisms. Section 2.0 of the report provides background information on strontium-90 at the Hanford Site related to historical operations. Public access to information on strontium-90 also is described. Section 3.0 focuses on key issues associated with strontium-90 contamination in groundwater that discharges in the Hanford Reach. The occurrence and distribution of fall chinook salmon redds in the Hanford Reach and characteristics of salmon spawning are described in Section 4.0. Section 5.0 describes the regulatory standards and criteria used to set action levels for strontium-90. Recommendations for initiating additional monitoring and remedial action associated with strontium-90 contamination at the Hanford Site are presented in Section 6

  10. Strontium-90 at the Hanford Site and its ecological implications

    Energy Technology Data Exchange (ETDEWEB)

    RE Peterson; TM Poston

    2000-05-22

    Strontium-90, a radioactive contaminant from historical operations at the U.S. Department of Energy (DOE) Hanford Site, enters the Columbia River at several locations associated with former plutonium production reactors at the Site. Strontium-90 is of concern to humans and the environment because of its moderately long half-life (29.1 years), its potential for concentrating in bone tissue, and its relatively high energy of beta decay. Although strontium-90 in the environment is not a new issue for the Hanford Site, recent studies of near-river vegetation along the shoreline near the 100 Areas raised public concern about the possibility of strontium-90-contaminated groundwater reaching the riverbed and fall chinook salmon redds. To address these concerns, DOE asked Pacific Northwest National Laboratory (PNNL) to prepare this report on strontium-90, its distribution in groundwater, how and where it enters the river, and its potential ecological impacts, particularly with respect to fall chinook salmon. The purpose of the report is to characterize groundwater contaminants in the near-shore environment and to assess the potential for ecological impact using salmon embryos, one of the most sensitive ecological indicators for aquatic organisms. Section 2.0 of the report provides background information on strontium-90 at the Hanford Site related to historical operations. Public access to information on strontium-90 also is described. Section 3.0 focuses on key issues associated with strontium-90 contamination in groundwater that discharges in the Hanford Reach. The occurrence and distribution of fall chinook salmon redds in the Hanford Reach and characteristics of salmon spawning are described in Section 4.0. Section 5.0 describes the regulatory standards and criteria used to set action levels for strontium-90. Recommendations for initiating additional monitoring and remedial action associated with strontium-90 contamination at the Hanford Site are presented in Section 6

  11. Heat Capacity Changes and Disorder-to-Order Transitions in Allosteric Activation.

    Science.gov (United States)

    Cressman, William J; Beckett, Dorothy

    2016-01-19

    Allosteric coupling in proteins is ubiquitous but incompletely understood, particularly in systems characterized by coupling over large distances. Binding of the allosteric effector, bio-5'-AMP, to the Escherichia coli biotin protein ligase, BirA, enhances the protein's dimerization free energy by -4 kcal/mol. Previous studies revealed that disorder-to-order transitions at the effector binding and dimerization sites, which are separated by 33 Å, are integral to functional coupling. Perturbations to the transition at the ligand binding site alter both ligand binding and coupled dimerization. Alanine substitutions in four loops on the dimerization surface yield a range of energetic effects on dimerization. A glycine to alanine substitution at position 142 in one of these loops results in a complete loss of allosteric coupling, disruption of the disorder-to-order transitions at both functional sites, and a decreased affinity for the effector. In this work, allosteric communication between the effector binding and dimerization surfaces in BirA was further investigated by performing isothermal titration calorimetry measurements on nine proteins with alanine substitutions in three dimerization surface loops. In contrast to BirAG142A, at 20 °C all variants bind to bio-5'-AMP with free energies indistinguishable from that measured for wild-type BirA. However, the majority of the variants exhibit altered heat capacity changes for effector binding. Moreover, the ΔCp values correlate with the dimerization free energies of the effector-bound proteins. These thermodynamic results, combined with structural information, indicate that allosteric activation of the BirA monomer involves formation of a network of intramolecular interactions on the dimerization surface in response to bio-5'-AMP binding at the distant effector binding site.

  12. Mars: Periglacial Morphology and Implications for Future Landing Sites

    Science.gov (United States)

    Heldmann, Jennifer L.; Schurmeier, Lauren; McKay, Christopher; Davila, Alfonso; Stoker, Carol; Marinova, Margarita; Wilhelm, Mary Beth

    2015-01-01

    At the Mars Phoenix landing site and in much of the Martian northern plains, there is ice-cemented ground beneath a layer of dry permafrost. Unlike most permafrost on Earth, though, this ice is not liquid at any time of year. However, in past epochs at higher obliquity the surface conditions during summer may have resulted in warmer conditions and possible melting. This situation indicates that the ice-cemented ground in the north polar plains is likely to be a candidate for the most recently habitable place on Mars as near-surface ice likely provided adequate water activity approximately 5 Myr ago. The high elevation Dry Valleys of Antarctica provide the best analog on Earth of Martian ground ice. These locations are the only places on Earth where ice-cemented ground is found beneath dry permafrost. The Dry Valleys are a hyper-arid polar desert environment and in locations above 1500 m elevation, such as University Valley, air temperatures do not exceed 0 C. Thus, similarly to Mars, liquid water is largely absent here and instead the hydrologic cycle is dominated by frozen ice and vapor phase processes such as sublimation. These conditions make the high elevation Dry Valleys a key Mars analog location where periglacial processes and geomorphic features can be studied in situ. This talk will focus on studies of University Valley as a Mars analog for periglacial morphology and ice stability. We will review a landing site selection study encompassing this information gleaned from the Antarctic terrestrial analog studies plus Mars spacecraft data analysis to identify candidate landing sites for a future mission to search for life on Mars.

  13. Implications of generator siting for CO2 pipeline infrastructure

    International Nuclear Information System (INIS)

    Newcomer, Adam; Apt, Jay

    2008-01-01

    The location of a new electric power generation system with carbon capture and sequestration (CCS) affects the profitability of the facility and determines the amount of infrastructure required to connect the plant to the larger world. Using a probabilistic analysis, we examine where a profit-maximizing power producer would locate a new generator with carbon capture in relation to a fuel source, electric load, and CO 2 sequestration site. Based on models of costs for transmission lines, CO 2 pipelines, and fuel transportation, we find that it is always preferable to locate a CCS power facility nearest the electric load, reducing the losses and costs of bulk electricity transmission. This result suggests that a power system with significant amounts of CCS requires a very large CO 2 pipeline infrastructure

  14. Allosteric small-molecule kinase inhibitors

    DEFF Research Database (Denmark)

    Wu, Peng; Clausen, Mads Hartvig; Nielsen, Thomas E.

    2015-01-01

    current barriers of kinase inhibitors, including poor selectivity and emergence of drug resistance. In spite of the small number of identified allosteric inhibitors in comparison with that of inhibitors targeting the ATP pocket, encouraging results, such as the FDA-approval of the first small...

  15. Allosteric Modulation of Muscarinic Acetylcholine Receptors

    Czech Academy of Sciences Publication Activity Database

    Jakubík, Jan; El-Fakahany, E. E.

    2010-01-01

    Roč. 3, č. 9 (2010), s. 2838-2860 ISSN 1424-8247 R&D Projects: GA ČR GA305/09/0681 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic acetylcholine receptors * allosteric modulation * Alzheimer´s disease Subject RIV: CE - Biochemistry

  16. The allosteric switching mechanism in bacteriophage MS2

    Energy Technology Data Exchange (ETDEWEB)

    Perkett, Matthew R.; Mirijanian, Dina T.; Hagan, Michael F., E-mail: hagan@brandeis.edu [Martin Fisher School of Physics, Brandeis University, Waltham, Massachusetts 02474 (United States)

    2016-07-21

    We use all-atom simulations to elucidate the mechanisms underlying conformational switching and allostery within the coat protein of the bacteriophage MS2. Assembly of most icosahedral virus capsids requires that the capsid protein adopts different conformations at precise locations within the capsid. It has been shown that a 19 nucleotide stem loop (TR) from the MS2 genome acts as an allosteric effector, guiding conformational switching of the coat protein during capsid assembly. Since the principal conformational changes occur far from the TR binding site, it is important to understand the molecular mechanism underlying this allosteric communication. To this end, we use all-atom simulations with explicit water combined with a path sampling technique to sample the MS2 coat protein conformational transition, in the presence and absence of TR-binding. The calculations find that TR binding strongly alters the transition free energy profile, leading to a switch in the favored conformation. We discuss changes in molecular interactions responsible for this shift. We then identify networks of amino acids with correlated motions to reveal the mechanism by which effects of TR binding span the protein. We find that TR binding strongly affects residues located at the 5-fold and quasi-sixfold interfaces in the assembled capsid, suggesting a mechanism by which the TR binding could direct formation of the native capsid geometry. The analysis predicts amino acids whose substitution by mutagenesis could alter populations of the conformational substates or their transition rates.

  17. Allosteric substrate switching in a voltage-sensing lipid phosphatase.

    Science.gov (United States)

    Grimm, Sasha S; Isacoff, Ehud Y

    2016-04-01

    Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We found that the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), has not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage-sensing domain (VSD). Using fast fluorescence resonance energy transfer (FRET) reporters of PIPs to monitor enzyme activity and voltage-clamp fluorometry to monitor conformational changes in the VSD, we found that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage-sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This two-step allosteric control over a dual-specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility, endocytosis and exocytosis.

  18. Allosteric substrate switching in a voltage sensing lipid phosphatase

    Science.gov (United States)

    Grimm, Sasha S.; Isacoff, Ehud Y.

    2016-01-01

    Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We find the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), to have not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage sensing domain (VSD). Using fast FRET reporters of PIPs to monitor enzyme activity and voltage clamp fluorometry to monitor conformational changes in the VSD, we find that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This novel 2-step allosteric control over a dual specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility and endo/exocytosis. PMID:26878552

  19. Studies on allosteric phenomena in glycogen phosphorylase b.

    Science.gov (United States)

    Madsen, N B; Avramovic-Zikic, O; Lue, P F; Honikel, K O

    1976-03-26

    This article attempts to trace, from a personal point of view, the history of discoveries of allosteric phenomena in phosphorylase b and the later development of systematic attempts to fit the data into comprehensive theoretical models. Work from our own laboratory is emphasized, but we try to integrate this into the results from other investigators and show their contributions to our ideas and experiments. Finally, some recent unpublished data is presented together with some conclusions and predictions from a new hypothesis. The discoveries by Carl and Gerty Cori of the activation of phosphorylase by AMP, the inhibition of glucose and the enzymatic interconversion of two forms fo the enzyme with different control properties helped lay the foundations of our present understanding of allosteric mechanisms. The later discovery of the oligomeric nature of phosphorylase and its relationship to AMP binding served as a basis for many years of research into the structure-function relationships of phosphorylase and other enzymes. Data showing that AMP lowers the entropy of activation is discussed with respect to the role of the nucleotide and its binding close to the active site. The discovery of the control of phosphorylase b by common metabolites and the impetus this gave to the intensive kinetic studies of the last ten years, wherein fitting to theoretical models has been a common feature, is reviewed.

  20. Allosteric Inhibition of Macrophage Migration Inhibitory Factor Revealed by Ibudilast

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Y.; Crichlow, G; Vermeire, J; Leng, L; Du, X; Hodsdon, M; Bucala, R; Cappello, M; Gross, M; et al.

    2010-01-01

    AV411 (ibudilast; 3-isobutyryl-2-isopropylpyrazolo-[1,5-a]pyridine) is an antiinflammatory drug that was initially developed for the treatment of bronchial asthma but which also has been used for cerebrovascular and ocular indications. It is a nonselective inhibitor of various phosphodiesterases (PDEs) and has varied antiinflammatory activity. More recently, AV411 has been studied as a possible therapeutic for the treatment of neuropathic pain and opioid withdrawal through its actions on glial cells. As described herein, the PDE inhibitor AV411 and its PDE-inhibition-compromised analog AV1013 inhibit the catalytic and chemotactic functions of the proinflammatory protein, macrophage migration inhibitory factor (MIF). Enzymatic analysis indicates that these compounds are noncompetitive inhibitors of the p-hydroxyphenylpyruvate (HPP) tautomerase activity of MIF and an allosteric binding site of AV411 and AV1013 is detected by NMR. The allosteric inhibition mechanism is further elucidated by X-ray crystallography based on the MIF/AV1013 binary and MIF/AV1013/HPP ternary complexes. In addition, our antibody experiments directed against MIF receptors indicate that CXCR2 is the major receptor for MIF-mediated chemotaxis of peripheral blood mononuclear cells.

  1. SH2-catalytic domain linker heterogeneity influences allosteric coupling across the SFK family.

    Science.gov (United States)

    Register, A C; Leonard, Stephen E; Maly, Dustin J

    2014-11-11

    Src-family kinases (SFKs) make up a family of nine homologous multidomain tyrosine kinases whose misregulation is responsible for human disease (cancer, diabetes, inflammation, etc.). Despite overall sequence homology and identical domain architecture, differences in SH3 and SH2 regulatory domain accessibility and ability to allosterically autoinhibit the ATP-binding site have been observed for the prototypical SFKs Src and Hck. Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix. As a relatively unconserved region between SFK family members, SH2-CD linker sequence variability across the SFK family is likely a source of nonredundant cellular functions between individual SFKs via its effect on the availability of SH3 and SH2 domains for intermolecular interactions and post-translational modification. Using a combination of SFKs engineered with enhanced or weakened regulatory domain intramolecular interactions and conformation-selective inhibitors that report αC helix conformation, this study explores how SH2-CD sequence heterogeneity affects allosteric coupling across the SFK family by examining Lyn, Fyn1, and Fyn2. Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases. Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

  2. Students' Participation in Social Networking Sites: Implications for Social Work Education

    Science.gov (United States)

    Mukherjee, Dhrubodhi; Clark, Janet

    2012-01-01

    Social work students have few guidelines to help them evaluate the implication of their posted information on Internet-based social networking sites (SNSs). There is a national trend among employers of human services to cross-check publicly available online information on applicants. Based on data from a survey of 105 baccalaureate and master's…

  3. Labeling by ( sup 3 H)1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

    Energy Technology Data Exchange (ETDEWEB)

    Rothman, R.B.; Reid, A.; Mahboubi, A.; Kim, C.H.; De Costa, B.R.; Jacobson, A.E.; Rice, K.C. (National Institute of Mental Health, Bethesda, MD (USA))

    1991-02-01

    Equilibrium binding studies with the sigma receptor ligand ({sup 3}H)1,3-di(2-tolyl)guanidine (({sup 3}H)DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. (Life Sci. 45:1721-1732 (1989)). Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and low affinity for most other sigma ligands. Kinetic experiments demonstrated that ({sup 3}H)DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of ({sup 3}H)DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of ({sup 3}H)DTG from site 2, suggesting an association of this binding site with calcium channels.

  4. Labeling by [3H]1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

    International Nuclear Information System (INIS)

    Rothman, R.B.; Reid, A.; Mahboubi, A.; Kim, C.H.; De Costa, B.R.; Jacobson, A.E.; Rice, K.C.

    1991-01-01

    Equilibrium binding studies with the sigma receptor ligand [ 3 H]1,3-di(2-tolyl)guanidine ([ 3 H]DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. [Life Sci. 45:1721-1732 (1989)]. Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and low affinity for most other sigma ligands. Kinetic experiments demonstrated that [ 3 H]DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of [ 3 H]DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of [ 3 H]DTG from site 2, suggesting an association of this binding site with calcium channels

  5. The future of type 1 cannabinoid receptor allosteric ligands.

    Science.gov (United States)

    Alaverdashvili, Mariam; Laprairie, Robert B

    2018-02-01

    Allosteric modulation of the type 1 cannabinoid receptor (CB1R) holds great therapeutic potential. This is because allosteric modulators do not possess intrinsic efficacy, but instead augment (positive allosteric modulation) or diminish (negative allosteric modulation) the receptor's response to endogenous ligand. Consequently, CB1R allosteric modulators have an effect ceiling which allows for the tempering of CB1R signaling without the desensitization, tolerance, dependence, and psychoactivity associated with orthosteric compounds. Pain, movement disorders, epilepsy, obesity are all potential therapeutic targets for CB1R allosteric modulation. Several challenges exist for the development of CB1R allosteric modulators, such as receptor subtype specificity, translation to in vivo systems, and mixed allosteric/agonist/inverse agonist activity. Despite these challenges, elucidation of crystal structures of CB1R and compound design based on structure-activity relationships will advance the field. In this review, we will cover recent progress for CB1R allosteric modulators and discuss the future promise of this research.

  6. Allosteric modulation of endogenous metabolites as an avenue for drug discovery.

    Science.gov (United States)

    Wootten, Denise; Savage, Emilia E; Valant, Celine; May, Lauren T; Sloop, Kyle W; Ficorilli, James; Showalter, Aaron D; Willard, Francis S; Christopoulos, Arthur; Sexton, Patrick M

    2012-08-01

    G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and a key drug target class. Recently, allosteric drugs that can co-bind with and modulate the activity of the endogenous ligand(s) for the receptor have become a major focus of the pharmaceutical and biotechnology industry for the development of novel GPCR therapeutic agents. This class of drugs has distinct properties compared with drugs targeting the endogenous (orthosteric) ligand-binding site that include the ability to sculpt cellular signaling and to respond differently in the presence of discrete orthosteric ligands, a behavior termed "probe dependence." Here, using cell signaling assays combined with ex vivo and in vivo studies of insulin secretion, we demonstrate that allosteric ligands can cause marked potentiation of previously "inert" metabolic products of neurotransmitters and peptide hormones, a novel consequence of the phenomenon of probe dependence. Indeed, at the muscarinic M(2) receptor and glucagon-like peptide 1 (GLP-1) receptor, allosteric potentiation of the metabolites, choline and GLP-1(9-36)NH(2), respectively, was ~100-fold and up to 200-fold greater than that seen with the physiological signaling molecules acetylcholine and GLP-1(7-36)NH(2). Modulation of GLP-1(9-36)NH(2) was also demonstrated in ex vivo and in vivo assays of insulin secretion. This work opens up new avenues for allosteric drug discovery by directly targeting modulation of metabolites, but it also identifies a behavior that could contribute to unexpected clinical outcomes if interaction of allosteric drugs with metabolites is not part of their preclinical assessment.

  7. The risk implications of approaches to setting soil remediation goals at hazardous waste contaminated sites

    Energy Technology Data Exchange (ETDEWEB)

    Labieniec, Paula Ann [Carnegie Mellon Univ., Pittsburgh, PA (United States)

    1994-08-01

    An integrated exposure and carcinogenic risk assessment model for organic contamination in soil, SoilRisk, was developed and used for evaluating the risk implications of both site-specific and uniform-concentration approaches to setting soil remediation goals at hazardous-waste-contaminated sites. SoilRisk was applied to evaluate the uncertainty in the risk estimate due to uncertainty in site conditions at a representative site. It was also used to evaluate the variability in risk across a region of sites that can occur due to differences in site characteristics that affect contaminant transport and fate when a uniform concentration approach is used. In evaluating regional variability, Ross County, Ohio and the State of Ohio were used as examples. All analyses performed considered four contaminants (benzene, trichloroethylene (TCE), chlordane, and benzo[a]pyrene (BAP)) and four exposure scenarios (commercial, recreational and on- and offsite residential). Regardless of whether uncertainty in risk at a single site or variability in risk across sites was evaluated, the exposure scenario specified and the properties of the target contaminant had more influence than variance in site parameters on the resulting variance and magnitude of the risk estimate. In general, variance in risk was found to be greater for the relatively less degradable and more mobile of the chemicals studied (TCE and chlordane) than for benzene which is highly degradable and BAP which is very immobile in the subsurface.

  8. The risk implications of approaches to setting soil remediation goals at hazardous waste contaminated sites

    International Nuclear Information System (INIS)

    Labieniec, P.A.

    1994-08-01

    An integrated exposure and carcinogenic risk assessment model for organic contamination in soil, SoilRisk, was developed and used for evaluating the risk implications of both site-specific and uniform-concentration approaches to setting soil remediation goals at hazardous-waste-contaminated sites. SoilRisk was applied to evaluate the uncertainty in the risk estimate due to uncertainty in site conditions at a representative site. It was also used to evaluate the variability in risk across a region of sites that can occur due to differences in site characteristics that affect contaminant transport and fate when a uniform concentration approach is used. In evaluating regional variability, Ross County, Ohio and the State of Ohio were used as examples. All analyses performed considered four contaminants (benzene, trichloroethylene (TCE), chlordane, and benzo[a]pyrene (BAP)) and four exposure scenarios (commercial, recreational and on- and offsite residential). Regardless of whether uncertainty in risk at a single site or variability in risk across sites was evaluated, the exposure scenario specified and the properties of the target contaminant had more influence than variance in site parameters on the resulting variance and magnitude of the risk estimate. In general, variance in risk was found to be greater for the relatively less degradable and more mobile of the chemicals studied (TCE and chlordane) than for benzene which is highly degradable and BAP which is very immobile in the subsurface

  9. Targeting S-adenosylmethionine biosynthesis with a novel allosteric inhibitor of Mat2A

    Energy Technology Data Exchange (ETDEWEB)

    Quinlan, Casey L.; Kaiser, Stephen E.; Bolaños, Ben; Nowlin, Dawn; Grantner, Rita; Karlicek-Bryant, Shannon; Feng, Jun Li; Jenkinson, Stephen; Freeman-Cook, Kevin; Dann, Stephen G.; Wang, Xiaoli; Wells, Peter A.; Fantin, Valeria R.; Stewart, Al E.; Grant, Stephan K. (Pfizer)

    2017-05-29

    S-Adenosyl-L-methionine (SAM) is an enzyme cofactor used in methyl transfer reactions and polyamine biosynthesis. The biosynthesis of SAM from ATP and L-methionine is performed by the methionine adenosyltransferase enzyme family (Mat; EC 2.5.1.6). Human methionine adenosyltransferase 2A (Mat2A), the extrahepatic isoform, is often deregulated in cancer. We identified a Mat2A inhibitor, PF-9366, that binds an allosteric site on Mat2A that overlaps with the binding site for the Mat2A regulator, Mat2B. Studies exploiting PF-9366 suggested a general mode of Mat2A allosteric regulation. Allosteric binding of PF-9366 or Mat2B altered the Mat2A active site, resulting in increased substrate affinity and decreased enzyme turnover. These data support a model whereby Mat2B functions as an inhibitor of Mat2A activity when methionine or SAM levels are high, yet functions as an activator of Mat2A when methionine or SAM levels are low. The ramification of Mat2A activity modulation in cancer cells is also described.

  10. Computational modeling of allosteric regulation in the hsp90 chaperones: a statistical ensemble analysis of protein structure networks and allosteric communications.

    Directory of Open Access Journals (Sweden)

    Kristin Blacklock

    2014-06-01

    Full Text Available A fundamental role of the Hsp90 chaperone in regulating functional activity of diverse protein clients is essential for the integrity of signaling networks. In this work we have combined biophysical simulations of the Hsp90 crystal structures with the protein structure network analysis to characterize the statistical ensemble of allosteric interaction networks and communication pathways in the Hsp90 chaperones. We have found that principal structurally stable communities could be preserved during dynamic changes in the conformational ensemble. The dominant contribution of the inter-domain rigidity to the interaction networks has emerged as a common factor responsible for the thermodynamic stability of the active chaperone form during the ATPase cycle. Structural stability analysis using force constant profiling of the inter-residue fluctuation distances has identified a network of conserved structurally rigid residues that could serve as global mediating sites of allosteric communication. Mapping of the conformational landscape with the network centrality parameters has demonstrated that stable communities and mediating residues may act concertedly with the shifts in the conformational equilibrium and could describe the majority of functionally significant chaperone residues. The network analysis has revealed a relationship between structural stability, global centrality and functional significance of hotspot residues involved in chaperone regulation. We have found that allosteric interactions in the Hsp90 chaperone may be mediated by modules of structurally stable residues that display high betweenness in the global interaction network. The results of this study have suggested that allosteric interactions in the Hsp90 chaperone may operate via a mechanism that combines rapid and efficient communication by a single optimal pathway of structurally rigid residues and more robust signal transmission using an ensemble of suboptimal multiple

  11. Diacylglycerol Acyltransferase 1 Is Regulated by Its N-Terminal Domain in Response to Allosteric Effectors.

    Science.gov (United States)

    Caldo, Kristian Mark P; Acedo, Jeella Z; Panigrahi, Rashmi; Vederas, John C; Weselake, Randall J; Lemieux, M Joanne

    2017-10-01

    Diacylglycerol acyltransferase 1 (DGAT1) is an integral membrane enzyme catalyzing the final and committed step in the acyl-coenzyme A (CoA)-dependent biosynthesis of triacylglycerol (TAG). The biochemical regulation of TAG assembly remains one of the least understood areas of primary metabolism to date. Here, we report that the hydrophilic N-terminal domain of Brassica napus DGAT1 (BnaDGAT1 1-113 ) regulates activity based on acyl-CoA/CoA levels. The N-terminal domain is not necessary for acyltransferase activity and is composed of an intrinsically disordered region and a folded segment. We show that the disordered region has an autoinhibitory function and a dimerization interface, which appears to mediate positive cooperativity, whereas the folded segment of the cytosolic region was found to have an allosteric site for acyl-CoA/CoA. Under increasing acyl-CoA levels, the binding of acyl-CoA with this noncatalytic site facilitates homotropic allosteric activation. Enzyme activation, on the other hand, is prevented under limiting acyl-CoA conditions (low acyl-CoA-to-CoA ratio), whereby CoA acts as a noncompetitive feedback inhibitor through interaction with the same folded segment. The three-dimensional NMR solution structure of the allosteric site revealed an α-helix with a loop connecting a coil fragment. The conserved amino acid residues in the loop interacting with CoA were identified, revealing details of this important regulatory element for allosteric regulation. Based on these results, a model is proposed illustrating the role of the N-terminal domain of BnaDGAT1 as a positive and negative modulator of TAG biosynthesis. © 2017 American Society of Plant Biologists. All Rights Reserved.

  12. Allosteric regulation by oleamide of the binding properties of 5-hydroxytryptamine7 receptors.

    Science.gov (United States)

    Hedlund, P B; Carson, M J; Sutcliffe, J G; Thomas, E A

    1999-12-01

    Oleamide belongs to a family of amidated lipids with diverse biological activities, including sleep induction and signaling modulation of several 5-hydroxytryptamine (5-HT) receptor subtypes, including 5-HT1A, 5-HT2A/2C, and 5-HT7. The 5-HT7 receptor, predominantly localized in the hypothalamus, hippocampus, and frontal cortex, stimulates cyclic AMP formation and is thought to be involved in the regulation of sleep-wake cycles. Recently, it was proposed that oleamide acts at an allosteric site on the 5-HT7 receptor to regulate cyclic AMP formation. We have further investigated the interaction between oleamide and 5-HT7 receptors by performing radioligand binding assays with HeLa cells transfected with the 5-HT7 receptor. Methiothepin, clozapine, and 5-HT all displaced specific [3H]5-HT (100 nM) binding, with pK(D) values of 7.55, 7.85, and 8.39, respectively. Oleamide also displaced [3H]5-HT binding, but the maximum inhibition was only 40% of the binding. Taking allosteric (see below) cooperativity into account, a K(D) of 2.69 nM was calculated for oleamide. In saturation binding experiments, oleamide caused a 3-fold decrease in the affinity of [3H]5-HT for the 5-HT7 receptor, without affecting the number of binding sites. A Schild analysis showed that the induced shift in affinity of [3H]5-HT reached a plateau, unlike that of a competitive inhibitor, illustrating the allosteric nature of the interaction between oleamide and the 5-HT7 receptor. Oleic acid, the product of oleamide hydrolysis, had a similar effect on [3H]5-HT binding, whereas structural analogs of oleamide, trans-9,10-octadecenamide, cis-8,9-octadecenamide, and erucamide, did not alter [3H]5-HT binding significantly. The findings support the hypothesis that oleamide acts via an allosteric site on the 5-HT7 receptor regulating receptor affinity.

  13. Convergent transmission of RNAi guide-target mismatch information across Argonaute internal allosteric network.

    Science.gov (United States)

    Joseph, Thomas T; Osman, Roman

    2012-01-01

    In RNA interference, a guide strand derived from a short dsRNA such as a microRNA (miRNA) is loaded into Argonaute, the central protein in the RNA Induced Silencing Complex (RISC) that silences messenger RNAs on a sequence-specific basis. The positions of any mismatched base pairs in an miRNA determine which Argonaute subtype is used. Subsequently, the Argonaute-guide complex binds and silences complementary target mRNAs; certain Argonautes cleave the target. Mismatches between guide strand and the target mRNA decrease cleavage efficiency. Thus, loading and silencing both require that signals about the presence of a mismatched base pair are communicated from the mismatch site to effector sites. These effector sites include the active site, to prevent target cleavage; the binding groove, to modify nucleic acid binding affinity; and surface allosteric sites, to control recruitment of additional proteins to form the RISC. To examine how such signals may be propagated, we analyzed the network of internal allosteric pathways in Argonaute exhibited through correlations of residue-residue interactions. The emerging network can be described as a set of pathways emanating from the core of the protein near the active site, distributed into the bulk of the protein, and converging upon a distributed cluster of surface residues. Nucleotides in the guide strand "seed region" have a stronger relationship with the protein than other nucleotides, concordant with their importance in sequence selectivity. Finally, any of several seed region guide-target mismatches cause certain Argonaute residues to have modified correlations with the rest of the protein. This arises from the aggregation of relatively small interaction correlation changes distributed across a large subset of residues. These residues are in effector sites: the active site, binding groove, and surface, implying that direct functional consequences of guide-target mismatches are mediated through the cumulative effects of

  14. Convergent transmission of RNAi guide-target mismatch information across Argonaute internal allosteric network.

    Directory of Open Access Journals (Sweden)

    Thomas T Joseph

    Full Text Available In RNA interference, a guide strand derived from a short dsRNA such as a microRNA (miRNA is loaded into Argonaute, the central protein in the RNA Induced Silencing Complex (RISC that silences messenger RNAs on a sequence-specific basis. The positions of any mismatched base pairs in an miRNA determine which Argonaute subtype is used. Subsequently, the Argonaute-guide complex binds and silences complementary target mRNAs; certain Argonautes cleave the target. Mismatches between guide strand and the target mRNA decrease cleavage efficiency. Thus, loading and silencing both require that signals about the presence of a mismatched base pair are communicated from the mismatch site to effector sites. These effector sites include the active site, to prevent target cleavage; the binding groove, to modify nucleic acid binding affinity; and surface allosteric sites, to control recruitment of additional proteins to form the RISC. To examine how such signals may be propagated, we analyzed the network of internal allosteric pathways in Argonaute exhibited through correlations of residue-residue interactions. The emerging network can be described as a set of pathways emanating from the core of the protein near the active site, distributed into the bulk of the protein, and converging upon a distributed cluster of surface residues. Nucleotides in the guide strand "seed region" have a stronger relationship with the protein than other nucleotides, concordant with their importance in sequence selectivity. Finally, any of several seed region guide-target mismatches cause certain Argonaute residues to have modified correlations with the rest of the protein. This arises from the aggregation of relatively small interaction correlation changes distributed across a large subset of residues. These residues are in effector sites: the active site, binding groove, and surface, implying that direct functional consequences of guide-target mismatches are mediated through the

  15. Activation of Hsp90 Enzymatic Activity and Conformational Dynamics through Rationally Designed Allosteric Ligands.

    Science.gov (United States)

    Sattin, Sara; Tao, Jiahui; Vettoretti, Gerolamo; Moroni, Elisabetta; Pennati, Marzia; Lopergolo, Alessia; Morelli, Laura; Bugatti, Antonella; Zuehlke, Abbey; Moses, Mike; Prince, Thomas; Kijima, Toshiki; Beebe, Kristin; Rusnati, Marco; Neckers, Len; Zaffaroni, Nadia; Agard, David A; Bernardi, Anna; Colombo, Giorgio

    2015-09-21

    Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP-regulated internal dynamics are critical for its function and current pharmacological approaches block the chaperone with ATP-competitive inhibitors. Herein, a general approach to perturb Hsp90 through design of new allosteric ligands aimed at modulating its functional dynamics is proposed. Based on the characterization of a first set of 2-phenylbenzofurans showing stimulatory effects on Hsp90 ATPase and conformational dynamics, new ligands were developed that activate Hsp90 by targeting an allosteric site, located 65 Å from the active site. Specifically, analysis of protein responses to first-generation activators was exploited to guide the design of novel derivatives with improved ability to stimulate ATP hydrolysis. The molecules' effects on Hsp90 enzymatic, conformational, co-chaperone and client-binding properties were characterized through biochemical, biophysical and cellular approaches. These designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Bitopic Ligands and Metastable Binding Sites

    DEFF Research Database (Denmark)

    Fronik, Philipp; Gaiser, Birgit I; Sejer Pedersen, Daniel

    2017-01-01

    of orthosteric binding sites. Bitopic ligands have been employed to address the selectivity problem by combining (linking) an orthosteric ligand with an allosteric modulator, theoretically leading to high-affinity subtype selective ligands. However, it remains a challenge to identify suitable allosteric binding...... that have been reported to date, this type of bitopic ligands would be composed of two identical pharmacophores. Herein, we outline the concept of bitopic ligands, review metastable binding sites, and discuss their potential as a new source of allosteric binding sites....

  17. Sparse networks of directly coupled, polymorphic, and functional side chains in allosteric proteins.

    Science.gov (United States)

    Soltan Ghoraie, Laleh; Burkowski, Forbes; Zhu, Mu

    2015-03-01

    Recent studies have highlighted the role of coupled side-chain fluctuations alone in the allosteric behavior of proteins. Moreover, examination of X-ray crystallography data has recently revealed new information about the prevalence of alternate side-chain conformations (conformational polymorphism), and attempts have been made to uncover the hidden alternate conformations from X-ray data. Hence, new computational approaches are required that consider the polymorphic nature of the side chains, and incorporate the effects of this phenomenon in the study of information transmission and functional interactions of residues in a molecule. These studies can provide a more accurate understanding of the allosteric behavior. In this article, we first present a novel approach to generate an ensemble of conformations and an efficient computational method to extract direct couplings of side chains in allosteric proteins, and provide sparse network representations of the couplings. We take the side-chain conformational polymorphism into account, and show that by studying the intrinsic dynamics of an inactive structure, we are able to construct a network of functionally crucial residues. Second, we show that the proposed method is capable of providing a magnified view of the coupled and conformationally polymorphic residues. This model reveals couplings between the alternate conformations of a coupled residue pair. To the best of our knowledge, this is the first computational method for extracting networks of side chains' alternate conformations. Such networks help in providing a detailed image of side-chain dynamics in functionally important and conformationally polymorphic sites, such as binding and/or allosteric sites. © 2014 Wiley Periodicals, Inc.

  18. Allosteric mechanisms within the adenosine A2A-dopamine D2 receptor heterotetramer

    Science.gov (United States)

    Ferré, Sergi; Bonaventura, Jordi; Tomasi, Dardo; Navarro, Gemma; Moreno, Estefanía; Cortés, Antonio; Lluís, Carme; Casadó, Vicent; Volkow, Nora D.

    2017-01-01

    The structure constituted by a G protein coupled receptor (GPCR) homodimer and a G protein provides a main functional unit and oligomeric entities can be viewed as multiples of dimers. For GPCR heteromers, experimental evidence supports a tetrameric structure, comprised of two different homodimers, each able to signal with its preferred G protein. GPCR homomers and heteromers can act as the conduit of allosteric interactions between orthosteric ligands. The well-known agonist/agonist allosteric interaction in the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer, by which A2AR agonists decrease the affinity of D2R agonists, gave the first rationale for the use of A2AR antagonists in Parkinson’s disease. We review new pharmacological findings that can be explained in the frame of a tetrameric structure of the A2AR-D2R heteromer: first, ligand-independent allosteric modulations by the D2R that result in changes of the binding properties of A2AR ligands; second, differential modulation of the intrinsic efficacy of D2R ligands for G protein-dependent and independent signaling; third, the canonical antagonistic Gs-Gi interaction within the frame of the heteromer; and fourth, the ability of A2AR antagonists, including caffeine, to also exert the same allosteric modulations of D2R ligands than A2AR agonists, while A2AR agonists and antagonists counteract each other’s effects. These findings can have important clinical implications when evaluating the use of A2AR antagonists. They also call for the need of monitoring caffeine intake when evaluating the effect of D2R ligands, when used as therapeutic agents in neuropsychiatric disorders or as probes in imaging studies. PMID:26051403

  19. Allosteric modulation of Ras and the PI3K/AKT/mTOR pathway: emerging therapeutic opportunities

    Science.gov (United States)

    Hubbard, Paul A.; Moody, Colleen L.; Murali, Ramachandran

    2014-01-01

    GTPases and kinases are two predominant signaling modules that regulate cell fate. Dysregulation of Ras, a GTPase, and the three eponymous kinases that form key nodes of the associated phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)/AKT/mTOR pathway have been implicated in many cancers, including pancreatic cancer, a disease noted for its current lack of effective therapeutics. The K-Ras isoform of Ras is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC) and there is growing evidence linking aberrant PI3K/AKT/mTOR pathway activity to PDAC. Although these observations suggest that targeting one of these nodes might lead to more effective treatment options for patients with pancreatic and other cancers, the complex regulatory mechanisms and the number of sequence-conserved isoforms of these proteins have been viewed as significant barriers in drug development. Emerging insights into the allosteric regulatory mechanisms of these proteins suggest novel opportunities for development of selective allosteric inhibitors with fragment-based drug discovery (FBDD) helping make significant inroads. The fact that allosteric inhibitors of Ras and AKT are currently in pre-clinical development lends support to this approach. In this article, we will focus on the recent advances and merits of developing allosteric drugs targeting these two inter-related signaling pathways. PMID:25566081

  20. Substrate specificity changes for human reticulocyte and epithelial 15-lipoxygenases reveal allosteric product regulation.

    Science.gov (United States)

    Wecksler, Aaron T; Kenyon, Victor; Deschamps, Joshua D; Holman, Theodore R

    2008-07-15

    Human reticulocyte 15-lipoxygenase (15-hLO-1) and epithelial 15-lipoxygenase (15-hLO-2) have been implicated in a number of human diseases, with differences in their substrate specificity potentially playing a central role. In this paper, we present a novel method for accurately measuring the substrate specificity of the two 15-hLO isozymes and demonstrate that both cholate and specific LO products affect substrate specificity. The linoleic acid (LA) product, 13-hydroperoxyoctadienoic acid (13-HPODE), changes the ( k cat/ K m) (AA)/( k cat/ K m) (LA) ratio more than 5-fold for 15-hLO-1 and 3-fold for 15-hLO-2, while the arachidonic acid (AA) product, 12-( S)-hydroperoxyeicosatetraenoic acid (12-HPETE), affects only the ratio of 15-hLO-1 (more than 5-fold). In addition, the reduced products, 13-( S)-hydroxyoctadecadienoic acid (13-HODE) and 12-( S)-hydroxyeicosatetraenoic acid (12-HETE), also affect substrate specificity, indicating that iron oxidation is not responsible for the change in the ( k cat/ K m) (AA)/( k cat/ K m) (LA) ratio. These results, coupled with the dependence of the 15-hLO-1 k cat/ K m kinetic isotope effect ( (D) k cat/ K m) on the presence of 12-HPETE and 12-HETE, indicate that the allosteric site, previously identified in 15-hLO-1 [Mogul, R., Johansen, E., and Holman, T. R. (1999) Biochemistry 39, 4801-4807], is responsible for the change in substrate specificity. The ability of LO products to regulate substrate specificity may be relevant with respect to cancer progression and warrants further investigation into the role of this product-feedback loop in the cell.

  1. The transport implications of siting policies for the disposal of low-level radioactive wastes

    International Nuclear Information System (INIS)

    James, I.A.

    1986-01-01

    This report has been produced to be complementary to the previously issued report ''The Transport Implications of Regional Policies for The Disposal of Intermediate Level Radioactive Wastes''. The same combinations of disposal facilities have been used so that direct comparison with intermediate waste results can be made. Low level wastes and short-lived intermediate level wastes for near-surface disposal are assumed to share a common infrastructure on the rail system and hence a methodology of separating total costs between these two waste types has been derived. Two transport modes, road and rail have been analysed. Hybrid transport, a combination of road and rail systems, has not been examined since no site is considered to produce sufficient waste to justify a dedicated rail service. Sellafield, has not been included in this examination since it is assumed to be served by its own disposal site at Drigg. (author)

  2. Discovery of a novel allosteric modulator of 5-HT3 receptor

    DEFF Research Database (Denmark)

    Trattnig, Sarah M; Harpsøe, Kasper; Thygesen, Sarah B

    2012-01-01

    The ligand-gated ion channels in the Cysloop receptor superfamily mediate the effects of neurotransmitters acetylcholine, serotonin, GABA and glycine. Cysloop receptor signaling is susceptible to modulation by ligands acting through numerous allosteric sites. Here we report the discovery of a novel...... receptor guided by a homology model, PU02 is demonstrated to act through a transmembrane intersubunit site situated in the upper three helical turns of TM2 and TM3 in the (+)subunit and TM1 and TM2 in the (minus)subunit. The Ser248, Leu288, Ile290, Thr294 and Gly306 residues are identified as important...

  3. Allosteric Mutant IDH1 Inhibitors Reveal Mechanisms for IDH1 Mutant and Isoform Selectivity

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Xiaoling; Baird, Daniel; Bowen, Kimberly; Capka, Vladimir; Chen, Jinyun; Chenail, Gregg; Cho, YoungShin; Dooley, Julia; Farsidjani, Ali; Fortin, Pascal; Kohls, Darcy; Kulathila, Raviraj; Lin, Fallon; McKay, Daniel; Rodrigues, Lindsey; Sage, David; Touré, B. Barry; van der Plas, Simon; Wright, Kirk; Xu, Ming; Yin, Hong; Levell, Julian; Pagliarini, Raymond A. (Novartis)

    2017-03-01

    Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 “regulatory segment,” which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

  4. Coarse-grained molecular simulations of allosteric cooperativity

    Energy Technology Data Exchange (ETDEWEB)

    Nandigrami, Prithviraj; Portman, John J. [Department of Physics, Kent State University, Kent, Ohio 44242 (United States)

    2016-03-14

    Interactions between a protein and a ligand are often accompanied by a redistribution of the population of thermally accessible conformations. This dynamic response of the protein’s functional energy landscape enables a protein to modulate binding affinities and control binding sensitivity to ligand concentration. In this paper, we investigate the structural origins of binding affinity and allosteric cooperativity of binding two Ca{sup 2+} ions to each domain of Calmodulin (CaM) through simulations of a simple coarse-grained model. In this model, the protein’s conformational transitions between open and closed conformational ensembles are simulated explicitly and ligand binding and unbinding are treated implicitly within the grand canonical ensemble. Ligand binding is cooperative because the binding sites are coupled through a shift in the dominant conformational ensemble upon binding. The classic Monod-Wyman-Changeux model of allostery with appropriate binding free energies to the open and closed ensembles accurately describes the simulated binding thermodynamics. The simulations predict that the two domains of CaM have distinct binding affinity and cooperativity. In particular, the C-terminal domain binds Ca{sup 2+} with higher affinity and greater cooperativity than the N-terminal domain. From a structural point of view, the affinity of an individual binding loop depends sensitively on the loop’s structural compatibility with the ligand in the bound ensemble, as well as the conformational flexibility of the binding site in the unbound ensemble.

  5. Computational Analysis of Residue Interaction Networks and Coevolutionary Relationships in the Hsp70 Chaperones: A Community-Hopping Model of Allosteric Regulation and Communication.

    Directory of Open Access Journals (Sweden)

    Gabrielle Stetz

    2017-01-01

    Full Text Available Allosteric interactions in the Hsp70 proteins are linked with their regulatory mechanisms and cellular functions. Despite significant progress in structural and functional characterization of the Hsp70 proteins fundamental questions concerning modularity of the allosteric interaction networks and hierarchy of signaling pathways in the Hsp70 chaperones remained largely unexplored and poorly understood. In this work, we proposed an integrated computational strategy that combined atomistic and coarse-grained simulations with coevolutionary analysis and network modeling of the residue interactions. A novel aspect of this work is the incorporation of dynamic residue correlations and coevolutionary residue dependencies in the construction of allosteric interaction networks and signaling pathways. We found that functional sites involved in allosteric regulation of Hsp70 may be characterized by structural stability, proximity to global hinge centers and local structural environment that is enriched by highly coevolving flexible residues. These specific characteristics may be necessary for regulation of allosteric structural transitions and could distinguish regulatory sites from nonfunctional conserved residues. The observed confluence of dynamics correlations and coevolutionary residue couplings with global networking features may determine modular organization of allosteric interactions and dictate localization of key mediating sites. Community analysis of the residue interaction networks revealed that concerted rearrangements of local interacting modules at the inter-domain interface may be responsible for global structural changes and a population shift in the DnaK chaperone. The inter-domain communities in the Hsp70 structures harbor the majority of regulatory residues involved in allosteric signaling, suggesting that these sites could be integral to the network organization and coordination of structural changes. Using a network-based formalism of

  6. Modulation of global low-frequency motions underlies allosteric regulation: demonstration in CRP/FNR family transcription factors.

    Science.gov (United States)

    Rodgers, Thomas L; Townsend, Philip D; Burnell, David; Jones, Matthew L; Richards, Shane A; McLeish, Tom C B; Pohl, Ehmke; Wilson, Mark R; Cann, Martin J

    2013-09-01

    Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distinct site. There is growing evidence that allosteric cooperativity can be communicated by modulation of protein dynamics without conformational change. The mechanisms, however, for communicating dynamic fluctuations between sites are debated. We provide a foundational theory for how allostery can occur as a function of low-frequency dynamics without a change in structure. We have generated coarse-grained models that describe the protein backbone motions of the CRP/FNR family transcription factors, CAP of Escherichia coli and GlxR of Corynebacterium glutamicum. The latter we demonstrate as a new exemplar for allostery without conformation change. We observe that binding the first molecule of cAMP ligand is correlated with modulation of the global normal modes and negative cooperativity for binding the second cAMP ligand without a change in mean structure. The theory makes key experimental predictions that are tested through an analysis of variant proteins by structural biology and isothermal calorimetry. Quantifying allostery as a free energy landscape revealed a protein "design space" that identified the inter- and intramolecular regulatory parameters that frame CRP/FNR family allostery. Furthermore, through analyzing CAP variants from diverse species, we demonstrate an evolutionary selection pressure to conserve residues crucial for allosteric control. This finding provides a link between the position of CRP/FNR transcription factors within the allosteric free energy landscapes and evolutionary selection pressures. Our study therefore reveals significant features of the mechanistic basis for allostery. Changes in low-frequency dynamics correlate with allosteric effects on ligand binding without the requirement for a defined spatial pathway. In addition to evolving suitable three-dimensional structures, CRP/FNR family transcription factors have been selected to

  7. Modulation of global low-frequency motions underlies allosteric regulation: demonstration in CRP/FNR family transcription factors.

    Directory of Open Access Journals (Sweden)

    Thomas L Rodgers

    2013-09-01

    Full Text Available Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distinct site. There is growing evidence that allosteric cooperativity can be communicated by modulation of protein dynamics without conformational change. The mechanisms, however, for communicating dynamic fluctuations between sites are debated. We provide a foundational theory for how allostery can occur as a function of low-frequency dynamics without a change in structure. We have generated coarse-grained models that describe the protein backbone motions of the CRP/FNR family transcription factors, CAP of Escherichia coli and GlxR of Corynebacterium glutamicum. The latter we demonstrate as a new exemplar for allostery without conformation change. We observe that binding the first molecule of cAMP ligand is correlated with modulation of the global normal modes and negative cooperativity for binding the second cAMP ligand without a change in mean structure. The theory makes key experimental predictions that are tested through an analysis of variant proteins by structural biology and isothermal calorimetry. Quantifying allostery as a free energy landscape revealed a protein "design space" that identified the inter- and intramolecular regulatory parameters that frame CRP/FNR family allostery. Furthermore, through analyzing CAP variants from diverse species, we demonstrate an evolutionary selection pressure to conserve residues crucial for allosteric control. This finding provides a link between the position of CRP/FNR transcription factors within the allosteric free energy landscapes and evolutionary selection pressures. Our study therefore reveals significant features of the mechanistic basis for allostery. Changes in low-frequency dynamics correlate with allosteric effects on ligand binding without the requirement for a defined spatial pathway. In addition to evolving suitable three-dimensional structures, CRP/FNR family transcription factors have

  8. An evolution-based strategy for engineering allosteric regulation

    Science.gov (United States)

    Pincus, David; Resnekov, Orna; Reynolds, Kimberly A.

    2017-04-01

    Allosteric regulation provides a way to control protein activity at the time scale of milliseconds to seconds inside the cell. An ability to engineer synthetic allosteric systems would be of practical utility for the development of novel biosensors, creation of synthetic cell signaling pathways, and design of small molecule pharmaceuticals with regulatory impact. To this end, we outline a general approach—termed rational engineering of allostery at conserved hotspots (REACH)—to introduce novel regulation into a protein of interest by exploiting latent allostery that has been hard-wired by evolution into its structure. REACH entails the use of statistical coupling analysis (SCA) to identify ‘allosteric hotspots’ on protein surfaces, the development and implementation of experimental assays to test hotspots for functionality, and a toolkit of allosteric modulators to impinge on endogenous cellular circuitry. REACH can be broadly applied to rewire cellular processes to respond to novel inputs.

  9. A Non-Competitive Inhibitor of VCP/p97 and VPS4 Reveals Conserved Allosteric Circuits in Type I and II AAA ATPases.

    Science.gov (United States)

    Pöhler, Robert; Krahn, Jan H; van den Boom, Johannes; Dobrynin, Grzegorz; Kaschani, Farnusch; Eggenweiler, Hans-Michael; Zenke, Frank T; Kaiser, Markus; Meyer, Hemmo

    2018-02-05

    AAA ATPases have pivotal functions in diverse cellular processes essential for survival and proliferation. Revealing strategies for chemical inhibition of this class of enzymes is therefore of great interest for the development of novel chemotherapies or chemical tools. Here, we characterize the compound MSC1094308 as a reversible, allosteric inhibitor of the type II AAA ATPase human ubiquitin-directed unfoldase (VCP)/p97 and the type I AAA ATPase VPS4B. Subsequent proteomic, genetic and biochemical studies indicate that MSC1094308 binds to a previously characterized drugable hotspot of p97, thereby inhibiting the D2 ATPase activity. Our results furthermore indicate that a similar allosteric site exists in VPS4B, suggesting conserved allosteric circuits and drugable sites in both type I and II AAA ATPases. Our results may thus guide future chemical tool and drug discovery efforts for the biomedically relevant AAA ATPases. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. A3 Adenosine Receptor Allosteric Modulator Induces an Anti-Inflammatory Effect: In Vivo Studies and Molecular Mechanism of Action

    Directory of Open Access Journals (Sweden)

    Shira Cohen

    2014-01-01

    Full Text Available The A3 adenosine receptor (A3AR is overexpressed in inflammatory cells and in the peripheral blood mononuclear cells of individuals with inflammatory conditions. Agonists to the A3AR are known to induce specific anti-inflammatory effects upon chronic treatment. LUF6000 is an allosteric compound known to modulate the A3AR and render the endogenous ligand adenosine to bind to the receptor with higher affinity. The advantage of allosteric modulators is their capability to target specifically areas where adenosine levels are increased such as inflammatory and tumor sites, whereas normal body cells and tissues are refractory to the allosteric modulators due to low adenosine levels. LUF6000 administration induced anti-inflammatory effect in 3 experimental animal models of rat adjuvant induced arthritis, monoiodoacetate induced osteoarthritis, and concanavalin A induced liver inflammation in mice. The molecular mechanism of action points to deregulation of signaling proteins including PI3K, IKK, IκB, Jak-2, and STAT-1, resulting in decreased levels of NF-κB, known to mediate inflammatory effects. Moreover, LUF6000 induced a slight stimulatory effect on the number of normal white blood cells and neutrophils. The anti-inflammatory effect of LUF6000, mechanism of action, and the differential effects on inflammatory and normal cells position this allosteric modulator as an attractive and unique drug candidate.

  11. Phloem development in nematode-induced feeding sites: The implications of auxin and cytokinin

    Directory of Open Access Journals (Sweden)

    Birgit eAbsmanner

    2013-07-01

    Full Text Available Sedentary plant parasitic nematodes such as root-knot nematodes and cyst nematodes induce giant cells or syncytia, respectively, in their host plant’s roots. These highly specialized structures serve as feeding sites from which exclusively the nematodes withdraw nutrients. While giant cells are symplastically isolated and obtain assimilates by transporter-mediated processes syncytia are massively connected to the phloem by plasmodesmata. To support the feeding sites and the nematode during their development, phloem is induced around syncytia and giant cells. In the case of syncytia the unloading phloem consists of sieve elements and companion cells and in the case of root knots it consists exclusively of sieve elements. We applied immunohistochemistry to identify the cells within the developing phloem that responded to auxin and cytokinin. Both feeding sites themselves did not respond to either hormone. We were able to show that in root knots an auxin response precedes the differentiation of these auxin responsive cells into phloem elements. This process appears to be independent of B-type Arabidopsis response regulators. Using additional markers for tissue identity we provide evidence that around giant cells protophloem is formed and proliferates dramatically. In contrast, the phloem around syncytia responded to both hormones. The presence of companion cells as well as hormone-responsive sieve elements suggests that metaphloem development occurs. The implication of auxin and cytokinin in the further development of the metaphloem is discussed.

  12. Allosteric conformational barcodes direct signaling in the cell.

    Science.gov (United States)

    Nussinov, Ruth; Ma, Buyong; Tsai, Chung-Jung; Csermely, Peter

    2013-09-03

    The cellular network is highly interconnected. Pathways merge and diverge. They proceed through shared proteins and may change directions. How are cellular pathways controlled and their directions decided, coded, and read? These questions become particularly acute when we consider that a small number of pathways, such as signaling pathways that regulate cell fates, cell proliferation, and cell death in development, are extensively exploited. This review focuses on these signaling questions from the structural standpoint and discusses the literature in this light. All co-occurring allosteric events (including posttranslational modifications, pathogen binding, and gain-of-function mutations) collectively tag the protein functional site with a unique barcode. The barcode shape is read by an interacting molecule, which transmits the signal. A conformational barcode provides an intracellular address label, which selectively favors binding to one partner and quenches binding to others, and, in this way, determines the pathway direction, and, eventually, the cell's response and fate. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Cell survival following alpha particle irradiation: critical sites and implications for carcinogenesis

    International Nuclear Information System (INIS)

    Lloyd, E.L.; Gemmell, M.A.; Henning, C.B.; Gemmell, D.S.; Zabransky, B.J.

    1976-01-01

    In experiments in which mammalian cells were irradiated with 5.6 MeV alpha particles from a Tandem Van de Graaff machine we have confirmed the finding of others that the mean lethal dose (D 0 ) is about 100 rad, but by measurements of the area of the cell nuclei as irradiated we found that this mean lethal dose corresponds not to 1, as expected, but to about 27 alpha particles per cell nucleus. (The exact number appears to change slightly with cell passage number.) This allows for the possibility that the direct action of alpha particles on the nucleus may be the important event in carcinogenesis, a theory which was previously difficult to accept if a single particle hitting the nucleus anywhere was considered to be lethal. Evidence is presented to implicate the nucleolus as a possible critical site for the inhibition of reproductive integrity of the cell

  14. Allosteric Inhibition of Factor XIIIa. Non-Saccharide Glycosaminoglycan Mimetics, but Not Glycosaminoglycans, Exhibit Promising Inhibition Profile.

    Directory of Open Access Journals (Sweden)

    Rami A Al-Horani

    Full Text Available Factor XIIIa (FXIIIa is a transglutaminase that catalyzes the last step in the coagulation process. Orthostery is the only approach that has been exploited to design FXIIIa inhibitors. Yet, allosteric inhibition of FXIIIa is a paradigm that may offer a key advantage of controlled inhibition over orthosteric inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We reasoned that targeting a collection of basic amino acid residues distant from FXIIIa's active site by using sulfated glycosaminoglycans (GAGs or non-saccharide GAG mimetics (NSGMs would lead to the discovery of the first allosteric FXIIIa inhibitors. We tested a library of 22 variably sulfated GAGs and NSGMs against human FXIIIa to discover promising hits. Interestingly, although some GAGs bound to FXIIIa better than NSGMs, no GAG displayed any inhibition. An undecasulfated quercetin analog was found to inhibit FXIIIa with reasonable potency (efficacy of 98%. Michaelis-Menten kinetic studies revealed an allosteric mechanism of inhibition. Fluorescence studies confirmed close correspondence between binding affinity and inhibition potency, as expected for an allosteric process. The inhibitor was reversible and at least 9-fold- and 26-fold selective over two GAG-binding proteins factor Xa (efficacy of 71% and thrombin, respectively, and at least 27-fold selective over a cysteine protease papain. The inhibitor also inhibited the FXIIIa-mediated polymerization of fibrin in vitro. Overall, our work presents the proof-of-principle that FXIIIa can be allosterically modulated by sulfated non-saccharide agents much smaller than GAGs, which should enable the design of selective and safe anticoagulants.

  15. Allosteric control of internal electron transfer in cytochrome cd1 nitrite reductase

    DEFF Research Database (Denmark)

    Farver, Ole; Kroneck, Peter M H; Zumft, Walter G

    2003-01-01

    Cytochrome cd1 nitrite reductase is a bifunctional multiheme enzyme catalyzing the one-electron reduction of nitrite to nitric oxide and the four-electron reduction of dioxygen to water. Kinetics and thermodynamics of the internal electron transfer process in the Pseudomonas stutzeri enzyme have...... been studied and found to be dominated by pronounced interactions between the c and the d1 hemes. The interactions are expressed both in dramatic changes in the internal electron-transfer rates between these sites and in marked cooperativity in their electron affinity. The results constitute a prime...... example of intraprotein control of the electron-transfer rates by allosteric interactions....

  16. Screening and identification of potential PTP1B allosteric inhibitors using in silico and in vitro approaches.

    Science.gov (United States)

    Shinde, Ranajit Nivrutti; Kumar, G Siva; Eqbal, Shahbaz; Sobhia, M Elizabeth

    2018-01-01

    Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for Type 2 diabetes due to its specific role as a negative regulator of insulin signaling pathways. Discovery of active site directed PTP1B inhibitors is very challenging due to highly conserved nature of the active site and multiple charge requirements of the ligands, which makes them non-selective and non-permeable. Identification of the PTP1B allosteric site has opened up new avenues for discovering potent and selective ligands for therapeutic intervention. Interactions made by potent allosteric inhibitor in the presence of PTP1B were studied using Molecular Dynamics (MD). Computationally optimized models were used to build separate pharmacophore models of PTP1B and TCPTP, respectively. Based on the nature of interactions the target residues offered, a receptor based pharmacophore was developed. The pharmacophore considering conformational flexibility of the residues was used for the development of pharmacophore hypothesis to identify potentially active inhibitors by screening large compound databases. Two pharmacophore were successively used in the virtual screening protocol to identify potential selective and permeable inhibitors of PTP1B. Allosteric inhibition mechanism of these molecules was established using molecular docking and MD methods. The geometrical criteria values confirmed their ability to stabilize PTP1B in an open conformation. 23 molecules that were identified as potential inhibitors were screened for PTP1B inhibitory activity. After screening, 10 molecules which have good permeability values were identified as potential inhibitors of PTP1B. This study confirms that selective and permeable inhibitors can be identified by targeting allosteric site of PTP1B.

  17. Data Mining and Privacy of Social Network Sites' Users: Implications of the Data Mining Problem.

    Science.gov (United States)

    Al-Saggaf, Yeslam; Islam, Md Zahidul

    2015-08-01

    This paper explores the potential of data mining as a technique that could be used by malicious data miners to threaten the privacy of social network sites (SNS) users. It applies a data mining algorithm to a real dataset to provide empirically-based evidence of the ease with which characteristics about the SNS users can be discovered and used in a way that could invade their privacy. One major contribution of this article is the use of the decision forest data mining algorithm (SysFor) to the context of SNS, which does not only build a decision tree but rather a forest allowing the exploration of more logic rules from a dataset. One logic rule that SysFor built in this study, for example, revealed that anyone having a profile picture showing just the face or a picture showing a family is less likely to be lonely. Another contribution of this article is the discussion of the implications of the data mining problem for governments, businesses, developers and the SNS users themselves.

  18. Defining the Structural Basis for Allosteric Product Release from E. coli Dihydrofolate Reductase Using NMR Relaxation Dispersion.

    Science.gov (United States)

    Oyen, David; Fenwick, R Bryn; Aoto, Phillip C; Stanfield, Robyn L; Wilson, Ian A; Dyson, H Jane; Wright, Peter E

    2017-08-16

    The rate-determining step in the catalytic cycle of E. coli dihydrofolate reductase is tetrahydrofolate (THF) product release, which can occur via an allosteric or an intrinsic pathway. The allosteric pathway, which becomes accessible when the reduced cofactor NADPH is bound, involves transient sampling of a higher energy conformational state, greatly increasing the product dissociation rate as compared to the intrinsic pathway that obtains when NADPH is absent. Although the kinetics of this process are known, the enzyme structure and the THF product conformation in the transiently formed excited state remain elusive. Here, we use side-chain proton NMR relaxation dispersion measurements, X-ray crystallography, and structure-based chemical shift predictions to explore the structural basis of allosteric product release. In the excited state of the E:THF:NADPH product release complex, the reduced nicotinamide ring of the cofactor transiently enters the active site where it displaces the pterin ring of the THF product. The p-aminobenzoyl-l-glutamate tail of THF remains weakly bound in a widened binding cleft. Thus, through transient entry of the nicotinamide ring into the active site, the NADPH cofactor remodels the enzyme structure and the conformation of the THF to form a weakly populated excited state that is poised for rapid product release.

  19. Deriving amplification factors from simple site parameters using generalized regression neural networks: implications for relevant site proxies

    Science.gov (United States)

    Boudghene Stambouli, Ahmed; Zendagui, Djawad; Bard, Pierre-Yves; Derras, Boumédiène

    2017-07-01

    Most modern seismic codes account for site effects using an amplification factor (AF) that modifies the rock acceleration response spectra in relation to a "site condition proxy," i.e., a parameter related to the velocity profile at the site under consideration. Therefore, for practical purposes, it is interesting to identify the site parameters that best control the frequency-dependent shape of the AF. The goal of the present study is to provide a quantitative assessment of the performance of various site condition proxies to predict the main AF features, including the often used short- and mid-period amplification factors, Fa and Fv, proposed by Borcherdt (in Earthq Spectra 10:617-653, 1994). In this context, the linear, viscoelastic responses of a set of 858 actual soil columns from Japan, the USA, and Europe are computed for a set of 14 real accelerograms with varying frequency contents. The correlation between the corresponding site-specific average amplification factors and several site proxies (considered alone or as multiple combinations) is analyzed using the generalized regression neural network (GRNN). The performance of each site proxy combination is assessed through the variance reduction with respect to the initial amplification factor variability of the 858 profiles. Both the whole period range and specific short- and mid-period ranges associated with the Borcherdt factors Fa and Fv are considered. The actual amplification factor of an arbitrary soil profile is found to be satisfactorily approximated with a limited number of site proxies (4-6). As the usual code practice implies a lower number of site proxies (generally one, sometimes two), a sensitivity analysis is conducted to identify the "best performing" site parameters. The best one is the overall velocity contrast between underlying bedrock and minimum velocity in the soil column. Because these are the most difficult and expensive parameters to measure, especially for thick deposits, other

  20. Site-to-site genetic correlations and their implications on breeding zone size and optimum number of progeny test sites for Coastal Douglas-fir.

    Science.gov (United States)

    G.R. Johnson

    1997-01-01

    Type B genetic correlations were used to examine the relation among geographic differences between sites and their site-to-site genetic (Type B) correlations. Examination of six local breeding zones in Oregon indicated that breeding zones were, for the most part, not too large because few environmental variables were correlated with Type B genetic correlations. The...

  1. In vitro pharmacological characterization of RXFP3 allosterism: an example of probe dependency.

    Directory of Open Access Journals (Sweden)

    Lily Alvarez-Jaimes

    Full Text Available Recent findings suggest that the relaxin-3 neural network may represent a new ascending arousal pathway able to modulate a range of neural circuits including those affecting circadian rhythm and sleep/wake states, spatial and emotional memory, motivation and reward, the response to stress, and feeding and metabolism. Therefore, the relaxin-3 receptor (RXFP3 is a potential therapeutic target for the treatment of various CNS diseases. Here we describe a novel selective RXFP3 receptor positive allosteric modulator (PAM, 3-[3,5-Bis(trifluoromethylphenyl]-1-(3,4-dichlorobenzyl-1-[2-(5-methoxy-1H-indol-3-ylethyl]urea (135PAM1. Calcium mobilization and cAMP accumulation assays in cell lines expressing the cloned human RXFP3 receptor show the compound does not directly activate RXFP3 receptor but increases functional responses to amidated relaxin-3 or R3/I5, a chimera of the INSL5 A chain and the Relaxin-3 B chain. 135PAM1 increases calcium mobilization in the presence of relaxin-3(NH2 and R3/I5(NH2 with pEC50 values of 6.54 (6.46 to 6.64 and 6.07 (5.94 to 6.20, respectively. In the cAMP accumulation assay, 135PAM1 inhibits the CRE response to forskolin with a pIC50 of 6.12 (5.98 to 6.27 in the presence of a probe (10 nM concentration of relaxin-3(NH2. 135PAM1 does not compete for binding with the orthosteric radioligand, [(125I] R3I5 (amide, in membranes prepared from cells expressing the cloned human RXFP3 receptor. 135PAM1 is selective for RXFP3 over RXFP4, which also responds to relaxin-3. However, when using the free acid (native form of relaxin-3 or R3/I5, 135PAM1 doesn't activate RXFP3 indicating that the compound's effect is probe dependent. Thus one can exchange the entire A-chain of the probe peptide while retaining PAM activity, but the state of the probe's c-terminus is crucial to allosteric activity of the PAM. These data demonstrate the existence of an allosteric site for modulation of this GPCR as well as the subtlety of changes in probe

  2. Assessment of economic factors affecting the satellite power system. Volume 2: The systems implications of rectenna siting issues

    Science.gov (United States)

    Chapman, P. K.; Bugos, B. J.; Csigi, K. I.; Glaser, P. E.; Schimke, G. R.; Thomas, R. G.

    1979-01-01

    The feasibility was evaluated of finding potential sites for Solar Power Satellite (SPS) receiving antennas (rectennas) in the continental United States, in sufficient numbers to permit the SPS to make a major contribution to U.S. generating facilities, and to give statistical validity to an assessment of the characteristics of such sites and their implications for the design of the SPS system. It is found that the cost-optimum power output of the SPS does not depend on the particular value assigned to the cost per unit area of a rectenna and its site, as long as it is independent of rectenna area. Many characteristics of the sites chosen affect the optimum design of the rectenna itself.

  3. Allosteric mechanism of action of the therapeutic anti-IgE antibody omalizumab.

    Science.gov (United States)

    Davies, Anna M; Allan, Elizabeth G; Keeble, Anthony H; Delgado, Jean; Cossins, Benjamin P; Mitropoulou, Alkistis N; Pang, Marie O Y; Ceska, Tom; Beavil, Andrew J; Craggs, Graham; Westwood, Marta; Henry, Alistair J; McDonnell, James M; Sutton, Brian J

    2017-06-16

    Immunoglobulin E and its interactions with receptors FcϵRI and CD23 play a central role in allergic disease. Omalizumab, a clinically approved therapeutic antibody, inhibits the interaction between IgE and FcϵRI, preventing mast cell and basophil activation, and blocks IgE binding to CD23 on B cells and antigen-presenting cells. We solved the crystal structure of the complex between an omalizumab-derived Fab and IgE-Fc, with one Fab bound to each Cϵ3 domain. Free IgE-Fc adopts an acutely bent structure, but in the complex it is only partially bent, with large-scale conformational changes in the Cϵ3 domains that inhibit the interaction with FcϵRI. CD23 binding is inhibited sterically due to overlapping binding sites on each Cϵ3 domain. Studies of omalizumab Fab binding in solution demonstrate the allosteric basis for FcϵRI inhibition and, together with the structure, reveal how omalizumab may accelerate dissociation of receptor-bound IgE from FcϵRI, exploiting the intrinsic flexibility and allosteric potential of IgE. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Structural Insights into the Allosteric Operation of the Lon AAA+ Protease.

    Science.gov (United States)

    Lin, Chien-Chu; Su, Shih-Chieh; Su, Ming-Yuan; Liang, Pi-Hui; Feng, Chia-Cheng; Wu, Shih-Hsiung; Chang, Chung-I

    2016-05-03

    The Lon AAA+ protease (LonA) is an evolutionarily conserved protease that couples the ATPase cycle into motion to drive substrate translocation and degradation. A hallmark feature shared by AAA+ proteases is the stimulation of ATPase activity by substrates. Here we report the structure of LonA bound to three ADPs, revealing the first AAA+ protease assembly where the six protomers are arranged alternately in nucleotide-free and bound states. Nucleotide binding induces large coordinated movements of conserved pore loops from two pairs of three non-adjacent protomers and shuttling of the proteolytic groove between the ATPase site and a previously unknown Arg paddle. Structural and biochemical evidence supports the roles of the substrate-bound proteolytic groove in allosteric stimulation of ATPase activity and the conserved Arg paddle in driving substrate degradation. Altogether, this work provides a molecular framework for understanding how ATP-dependent chemomechanical movements drive allosteric processes for substrate degradation in a major protein-destruction machine. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis

    Science.gov (United States)

    Kachaner, David; Pinson, Xavier; El Kadhi, Khaled Ben; Normandin, Karine; Talje, Lama; Lavoie, Hugo; Lépine, Guillaume; Carréno, Sébastien; Kwok, Benjamin H.; Hickson, Gilles R.

    2014-01-01

    Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks. PMID:25332165

  6. Mechanism of allosteric regulation of β2-adrenergic receptor by cholesterol

    DEFF Research Database (Denmark)

    Manna, Moutusi; Niemelä, Miia; Tynkkynen, Joona

    2016-01-01

    ) - a prototypical G protein-coupled receptor - is modulated by cholesterol in an allosteric fashion. Extensive atomistic simulations show that cholesterol regulates b2AR by limiting its conformational variability. The mechanism of action is based on the binding of cholesterol at specific high-affinity sites located...... near the transmembrane helices 5-7 of the receptor. The alternative mechanism, where the β2AR conformation would be modulated by membrane-mediated interactions, plays only a minor role. Cholesterol analogues also bind to cholesterol binding sites and impede the structural flexibility of β2AR, however...... cholesterol generates the strongest effect. The results highlight the capacity of lipids to regulate the conformation of membrane receptors through specific interactions....

  7. Gs protein peptidomimetics as allosteric modulators of the β2-adrenergic receptor

    DEFF Research Database (Denmark)

    Boyhus, Lotte Emilie; Danielsen, Mia; Bengtson, Nina Smidt

    2018-01-01

    A series of Gs protein peptidomimetics were designed and synthesised based on the published X-ray crystal structure of the active state β2-Adrenergic receptor (β2AR) in complex with the Gs protein (PDB 3SN6). We hypothesised that such peptidomimetics may function as allosteric modulators...... that target the intracellular Gs protein binding site of the β2AR. Peptidomimetics were designed to mimic the 15 residue C-Terminal α-helix of the Gs protein and were pre-organised in a helical conformation by (i, i + 4)-stapling using copper catalysed azide alkyne cycloaddition. Linear and stapled...... be able to compete with the native Gs protein for the intracellular binding site to block ISO-induced cAMP formation, but are unable to stabilise an active-like receptor conformation....

  8. Extracellular loop 2 of the free Fatty Acid receptor 2 mediates allosterism of a phenylacetamide ago-allosteric modulator

    DEFF Research Database (Denmark)

    Smith, Nicola J; Ward, Richard J; Stoddart, Leigh A

    2011-01-01

    Allosteric agonists are powerful tools for exploring the pharmacology of closely related G protein-coupled receptors that have nonselective endogenous ligands, such as the short chain fatty acids at free fatty acid receptors 2 and 3 (FFA2/GPR43 and FFA3/GPR41, respectively). We explored the molec...

  9. A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

    Science.gov (United States)

    Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

    2011-05-01

    A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

  10. In search of allosteric modulators of a7-nAChR by solvent density guided virtual screening.

    Science.gov (United States)

    Dey, Raja; Chen, Lin

    2011-04-01

    Nicotinic acetylcholine receptors (nAChR) are pentameric ligand gated ion channels whose activity can be modulated by endogenous neurotransmitters as well as by synthetic ligands that bind the same or distinct sites from the natural ligand. The subtype of α7 nAChR has been considered as a potenial therapeutic target for Alzheimer's disease, schizophrenia and other neurological and psychiatric disorders. Here we have developed a homology model of α7 nAChR based on two high resolution crystal structures with Brookhaven Protein Data Bank (PDB) codes 2QC1 and 2WN9 for threading on one monomer and then for building a pentamer, respectively. A number of small molecule binding sites are identified using Pocket Finder (J. An, M. Tortov, and R. Abagyan, Molecular & Cellular Proteomics, 4.6, 752-761 (2005)) of Internal Coordinate Mechanics (ICM). Remarkably, these computer-identified sites match perfectly with ordered solvent densities found in the high-resolution crystal structure of α1 nAChR, suggesting that the surface cavities in the α7 nAChR model are likely binding sites of small molecules. A high throughput virtual screening by flexible ligand docking of 5008 small molecule compounds was performed at three potential allosteric modulator (AM) binding sites of α7 nAChR using Molsoft ICM software (R. Abagyan, M. Tortov and D. Kuznetsov, J Comput Chem 15, 488-506, (1994)). Some experimentally verified allosteric modulators of α7 like CCMI comp-6, LY 7082101, 5-HI, TQS, PNU-120596, genistein, and NS-1738 ranked among top 100 compounds, while the rest of the compounds in the list could guide further search for new allosteric modulators.

  11. Association of actinides with microorganisms and clay: Implications for radionuclide migration from waste-repository sites

    International Nuclear Information System (INIS)

    Ohnuki, T.; Francis, A.; Kozai, N.; Sakamoto, F.; Ozaki, T.; Nankawa, T.; Suzuki, Y.

    2010-01-01

    We conducted a series of basic studies on the microbial accumulation of actinides to elucidate their migration behavior around backfill materials used in the geological disposal of radioactive wastes. We explored the interactions of U(VI) and Pu(VI) with Bacillus subtilis, kaolinite clay, and within a mixture of the two, directly analyzing their association with the bacterium in the mixture by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The accumulation of U by the mixture rose as the numbers of B. subtilis cells increased. Treating the kaolinite with potassium acetate (CH 3 COOK) removed approximately 80% of the associated uranium while only 65% was removed in the presence of B. subtilis. TEM-EDS analysis confirmed that most of the U taken from solution was associated with B. subtilis. XANES analyses revealed that the oxidation state of uranium associated with B. subtilis, kaolinite, and with the mixture containing both was U(VI). The amount of Pu sorbed by B. subtilis increased with time, but did not reach equilibrium in 48 h; in kaolinite alone, equilibrium was attained within 8 h. After 48 h, the oxidation state of Pu in the solutions exposed to B. subtilis and to the mixture had changed to Pu(V), whereas the oxidation state of the Pu associated with both was Pu(IV). In contrast, there was no change in the oxidation state of Pu in the solution nor on kaolinite after exposure to Pu(VI). SEM-EDS analysis indicated that most of the Pu in the mixture was associated with the bacteria. These results suggest that U(VI) and Pu(VI) preferentially are sorbed to bacterial cells in the presence of kaolinite clay, and that the mechanism of accumulation of U and Pu differs. U(VI) is sorbed directly to the bacterial cells, whereas Pu(VI) first is reduced to Pu(V) and then to Pu(IV), and the latter is associated with the cells. These results have important implications on the migrations of radionuclides around the repository sites of

  12. Privacy Practices of Health Social Networking Sites: Implications for Privacy and Data Security in Online Cancer Communities.

    Science.gov (United States)

    Charbonneau, Deborah H

    2016-08-01

    While online communities for social support continue to grow, little is known about the state of privacy practices of health social networking sites. This article reports on a structured content analysis of privacy policies and disclosure practices for 25 online ovarian cancer communities. All of the health social networking sites in the study sample provided privacy statements to users, yet privacy practices varied considerably across the sites. The majority of sites informed users that personal information was collected about participants and shared with third parties (96%, n = 24). Furthermore, more than half of the sites (56%, n = 14) stated that cookies technology was used to track user behaviors. Despite these disclosures, only 36% (n = 9) offered opt-out choices for sharing data with third parties. In addition, very few of the sites (28%, n = 7) allowed individuals to delete their personal information. Discussions about specific security measures used to protect personal information were largely missing. Implications for privacy, confidentiality, consumer choice, and data safety in online environments are discussed. Overall, nurses and other health professionals can utilize these findings to encourage individuals seeking online support and participating in social networking sites to build awareness of privacy risks to better protect their personal health information in the digital age.

  13. Escitalopram, an antidepressant with an allosteric effect at the serotonin transporter--a review of current understanding of its mechanism of action.

    Science.gov (United States)

    Zhong, Huailing; Haddjeri, Nasser; Sánchez, Connie

    2012-01-01

    Escitalopram is a widely used antidepressant for the treatment of patients with major depression. It is the pure S-enantiomer of racemic citalopram. Several clinical trials and meta-analyses indicate that escitalopram is quantitatively more efficacious than many other antidepressants with a faster onset of action. This paper reviews current knowledge about the mechanism of action of escitalopram. The primary target for escitalopram is the serotonin transporter (SERT), which is responsible for serotonin (or 5-hydroxytryptamine [5-HT]) reuptake at the terminals and cell bodies of serotonergic neurons. Escitalopram and selective serotonin reuptake inhibitors bind with high affinity to the 5-HT binding site (orthosteric site) on the transporter. This leads to antidepressant effects by increasing extracellular 5-HT levels which enhance 5-HT neurotransmission. SERT also has one or more allosteric sites, binding to which modulates activity at the orthosteric binding site but does not directly affect 5-HT reuptake by the transporter. In vitro studies have shown that through allosteric binding, escitalopram decreases its own dissociation rate from the orthosteric site on the SERT. R-citalopram, the nontherapeutic enantiomer in citalopram, is also an allosteric modulator of SERT but can inhibit the actions of escitalopram by interfering negatively with its binding. Both nonclinical studies and some clinical investigations have demonstrated the cellular, neurochemical, neuroadaptive, and neuroplastic changes induced by escitalopram with acute and chronic administration. The findings from binding, neurochemical, and neurophysiological studies may provide a mechanistic rationale for the clinical difference observed with escitalopram compared to other antidepressant therapies.

  14. Computational redesign reveals allosteric mutation hotspots of organophosphate hydrolase that enhance organophosphate hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Jacob, Reed B. [Univ. of North Carolina, Chapel Hill, NC (United States); Ding, Feng [Clemson Univ., SC (United States); Ye, Dongmei [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Ackerman, Eric [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Dokholyan, Nikolay V. [Univ. of North Carolina, Chapel Hill, NC (United States)

    2015-04-01

    Organophosphates are widely used for peaceful (agriculture) and military purposes (chemical warfare agents). The extraordinary toxicity of organophosphates and the risk of deployment, make it critical to develop means for their rapid and efficient deactivation. Organophosphate hydrolase (OPH) already plays an important role in organophosphate remediation, but is insufficient for therapeutic or prophylactic purposes primarily due to low substrate affinity. Current efforts focus on directly modifying the active site to differentiate substrate specificity and increase catalytic activity. Here, we present a novel strategy for enhancing the general catalytic efficiency of OPH through computational redesign of the residues that are allosterically coupled to the active site and validated our design by mutagenesis. Specifically, we identify five such hot-spot residues for allosteric regulation and assay these mutants for hydrolysis activity against paraoxon, a chemical-weapons simulant. A high percentage of the predicted mutants exhibit enhanced activity over wild-type (kcat =16.63 s-1), such as T199I/T54I (899.5 s-1) and C227V/T199I/T54I (848 s-1), while the Km remains relatively unchanged in our high-throughput cell-free expression system. Further computational studies of protein dynamics reveal four distinct distal regions coupled to the active site that display significant changes in conformation dynamics upon these identified mutations. These results validate a computational design method that is both efficient and easily adapted as a general procedure for enzymatic enhancement.

  15. Sleeping sites and latrines of spider monkeys in continuous and fragmented rainforests: implications for seed dispersal and forest regeneration.

    Directory of Open Access Journals (Sweden)

    Arturo González-Zamora

    Full Text Available Spider monkeys (Ateles geoffroyi use sites composed of one or more trees for sleeping (sleeping sites and sleeping trees, respectively. Beneath these sites/trees they deposit copious amounts of dung in latrines. This behavior results in a clumped deposition pattern of seeds and nutrients that directly impacts the regeneration of tropical forests. Therefore, information on the density and spatial distribution of sleeping sites and latrines, and the characteristics (i.e., composition and structure of sleeping trees are needed to improve our understanding of the ecological significance of spider monkeys in influencing forest composition. Moreover, since primate populations are increasingly forced to inhabit fragmented landscapes, it is important to assess if these characteristics differ between continuous and fragmented forests. We assessed this novel information from eight independent spider monkey communities in the Lacandona rainforest, Mexico: four continuous forest sites and four forest fragments. Both the density of sleeping sites and latrines did not differ between forest conditions. Latrines were uniformly distributed across sleeping sites, but the spatial distribution of sleeping sites within the areas was highly variable, being particularly clumped in forest fragments. In fact, the average inter-latrine distances were almost double in continuous forest than in fragments. Latrines were located beneath only a few tree species, and these trees were larger in diameter in continuous than fragmented forests. Because latrines may represent hotspots of seedling recruitment, our results have important ecological and conservation implications. The variation in the spatial distribution of sleeping sites across the forest indicates that spider monkeys likely create a complex seed deposition pattern in space and time. However, the use of a very few tree species for sleeping could contribute to the establishment of specific vegetation associations

  16. Sleeping sites and latrines of spider monkeys in continuous and fragmented rainforests: implications for seed dispersal and forest regeneration.

    Science.gov (United States)

    González-Zamora, Arturo; Arroyo-Rodríguez, Víctor; Oyama, Ken; Sork, Victoria; Chapman, Colin A; Stoner, Kathryn E

    2012-01-01

    Spider monkeys (Ateles geoffroyi) use sites composed of one or more trees for sleeping (sleeping sites and sleeping trees, respectively). Beneath these sites/trees they deposit copious amounts of dung in latrines. This behavior results in a clumped deposition pattern of seeds and nutrients that directly impacts the regeneration of tropical forests. Therefore, information on the density and spatial distribution of sleeping sites and latrines, and the characteristics (i.e., composition and structure) of sleeping trees are needed to improve our understanding of the ecological significance of spider monkeys in influencing forest composition. Moreover, since primate populations are increasingly forced to inhabit fragmented landscapes, it is important to assess if these characteristics differ between continuous and fragmented forests. We assessed this novel information from eight independent spider monkey communities in the Lacandona rainforest, Mexico: four continuous forest sites and four forest fragments. Both the density of sleeping sites and latrines did not differ between forest conditions. Latrines were uniformly distributed across sleeping sites, but the spatial distribution of sleeping sites within the areas was highly variable, being particularly clumped in forest fragments. In fact, the average inter-latrine distances were almost double in continuous forest than in fragments. Latrines were located beneath only a few tree species, and these trees were larger in diameter in continuous than fragmented forests. Because latrines may represent hotspots of seedling recruitment, our results have important ecological and conservation implications. The variation in the spatial distribution of sleeping sites across the forest indicates that spider monkeys likely create a complex seed deposition pattern in space and time. However, the use of a very few tree species for sleeping could contribute to the establishment of specific vegetation associations typical of the

  17. Defying c-Abl signaling circuits through small allosteric compounds

    Directory of Open Access Journals (Sweden)

    Stefania eGonfloni

    2014-11-01

    Full Text Available Many extracellular and intracellular signals promote the c-Abl tyrosine kinase activity. c-Abl in turn triggers a multitude of changes either in protein phosphorylation or in gene expression in the cell. Yet, c-Abl takes part in diverse signaling routes because of several domains linked to its catalytic core. Complex conformational changes turn on and off its kinase activity. These changes affect surface features of the c-Abl kinase and likely its capability to bind actin and/or DNA. Two specific inhibitors (ATP-competitive or allosteric compounds regulate the c-Abl kinase through different mechanisms. NMR studies show that a c-Abl fragment (SH3-SH2-linker-SH1 adopts different conformational states upon binding to each inhibitor. This supports an unconventional use for allosteric compounds to unraveling physiological c-Abl signaling circuits.

  18. Correction for Inhibition Leads to an Allosteric Co-Agonist Model for Pentobarbital Modulation and Activation of α1β3γ2L GABAA Receptors.

    Directory of Open Access Journals (Sweden)

    Alexis M Ziemba

    Full Text Available Pentobarbital, like propofol and etomidate, produces important general anesthetic effects through GABAA receptors. Photolabeling also indicates that pentobarbital binds to some of the same sites where propofol and etomidate act. Quantitative allosteric co-agonist models for propofol and etomidate account for modulatory and agonist effects in GABAA receptors and have proven valuable in establishing drug site characteristics and for functional analysis of mutants. We therefore sought to establish an allosteric co-agonist model for pentobarbital activation and modulation of α1β3γ2L receptors, using a novel approach to first correct pentobarbital activation data for inhibitory effects in the same concentration range.Using oocyte-expressed α1β3γ2L GABAA receptors and two-microelectrode voltage-clamp, we quantified modulation of GABA responses by a low pentobarbital concentration and direct effects of high pentobarbital concentrations, the latter displaying mixed agonist and inhibitory effects. We then isolated and quantified pentobarbital inhibition in activated receptors using a novel single-sweep "notch" approach, and used these results to correct steady-state direct activation for inhibition.Combining results for GABA modulation and corrected direct activation, we estimated receptor open probability and optimized parameters for a Monod-Wyman-Changeux allosteric co-agonist model. Inhibition by pentobarbital was consistent with two sites with IC50s near 1 mM, while co-agonist model parameters suggest two allosteric pentobarbital agonist sites characterized by KPB ≈ 5 mM and high efficacy. The results also indicate that pentobarbital may be a more efficacious agonist than GABA.Our novel approach to quantifying both inhibitory and co-agonist effects of pentobarbital provides a basis for future structure-function analyses of GABAA receptor mutations in putative pentobarbital binding sites.

  19. Covalent Allosteric Inactivation of Protein Tyrosine Phosphatase 1B (PTP1B) by an Inhibitor-Electrophile Conjugate.

    Science.gov (United States)

    Punthasee, Puminan; Laciak, Adrian R; Cummings, Andrea H; Ruddraraju, Kasi Viswanatharaju; Lewis, Sarah M; Hillebrand, Roman; Singh, Harkewal; Tanner, John J; Gates, Kent S

    2017-04-11

    Protein tyrosine phosphatase 1B (PTP1B) is a validated drug target, but it has proven difficult to develop medicinally useful, reversible inhibitors of this enzyme. Here we explored covalent strategies for the inactivation of PTP1B using a conjugate composed of an active site-directed 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide inhibitor connected via a short linker to an electrophilic α-bromoacetamide moiety. Inhibitor-electrophile conjugate 5a caused time-dependent loss of PTP1B activity consistent with a covalent inactivation mechanism. The inactivation occurred with a second-order rate constant of (1.7 ± 0.3) × 10 2 M -1 min -1 . Mass spectrometric analysis of the inactivated enzyme indicated that the primary site of modification was C121, a residue distant from the active site. Previous work provided evidence that covalent modification of the allosteric residue C121 can cause inactivation of PTP1B [Hansen, S. K., Cancilla, M. T., Shiau, T. P., Kung, J., Chen, T., and Erlanson, D. A. (2005) Biochemistry 44, 7704-7712]. Overall, our results are consistent with an unusual enzyme inactivation process in which noncovalent binding of the inhibitor-electrophile conjugate to the active site of PTP1B protects the nucleophilic catalytic C215 residue from covalent modification, thus allowing inactivation of the enzyme via selective modification of allosteric residue C121.

  20. Methods for assessing the socioeconomic impacts of large-scale resource developments: implications for nuclear repository siting

    International Nuclear Information System (INIS)

    Murdock, S.H.; Leistritz, F.L.

    1983-03-01

    An overview of the major methods presently available for assessing the socioeconomic impacts of large-scale resource developments and includes discussion of the implications and applications of such methods for nuclear-waste-repository siting are provided. The report: (1) summarizes conceptual approaches underlying, and methodological alternatives for, the conduct of impact assessments in each substantive area, and then enumerates advantages and disadvantages of each alternative; (2) describes factors related to the impact-assessment process, impact events, and the characteristics of rural areas that affect the magnitude and distribution of impacts and the assessment of impacts in each area; (3) provides a detailed review of those methodologies actually used in impact assessment for each area, describes advantages and problems encountered in the use of each method, and identifies the frequency of use and the general level of acceptance of each technique; and (4) summarizes the implications of each area of projection for the repository-siting process, the applicability of the methods for each area to the special and standard features of repositories, and makes general recommendations concerning specific methods and procedures that should be incorporated in assessments for siting areas

  1. Substrate-Induced Allosteric Change in the Quaternary Structure of the Spermidine N-Acetyltransferase SpeG

    OpenAIRE

    Filippova, Ekaterina V.; Weigand, Steven; Osipiuk, Jerzy; Kiryukhina, Olga; Joachimiak, Andrzej; Anderson, Wayne F.

    2015-01-01

    The spermidine N-acetyltransferase SpeG is a dodecameric enzyme that catalyzes the transfer of an acetyl group from acetyl-coenzyme A to polyamines such as spermidine and spermine. SpeG has an allosteric polyamine-binding site and acetylating polyamines regulates their intracellular concentrations. The structures of SpeG from Vibrio cholerae in complexes with polyamines and cofactor have been characterized earlier. Here, we present the dodecameric structure of SpeG from V. cholerae in a ligan...

  2. Quality assurance of nursing web sites: development and implications of the ALEU method.

    Science.gov (United States)

    Cambil-Martín, Jacobo; Flynn, Maria; Villaverde-Gutiérrez, Carmen

    2011-09-01

    This article presents a study that evaluated the physical accessibility, readability, and usability of Spanish nursing Web sites and discusses the quality assurance issues raised, which are relevant to the wider nursing community. The Internet is recognized as an important source of health information for both nurses and the general public. Although it makes health-related information universally available, the wide variation in the overall quality of health Web sites is problematic. This raises many questions for the nursing profession: about what constitutes a good-quality Web site, about the nature of the information that nurses are finding and using to support their professional education, research, and clinical practice, and about the impact that Internet information ultimately has on health interactions and nursing care. The process of completing this small study showed that it is possible to usefully assess dimensions of Web site quality and suggested that it may be feasible to develop tools to help nurses evaluate national and international nursing Web sites. More research is needed to understand how nurses use the Internet to support their everyday professional practices, but the development and application of international Web site quality assurance tools may be important for maintaining professional nursing standards in the Internet age.

  3. Analysis of orientation patterns in Olduvai Bed I assemblages using GIS techniques: implications for site formation processes.

    Science.gov (United States)

    Benito-Calvo, Alfonso; de la Torre, Ignacio

    2011-07-01

    Mary Leakey's excavations at Olduvai Beds I and II provided an unparalleled wealth of data on the archaeology of the early Pleistocene. We have been able to obtain axial orientations of the Bed I bone and stone tools by applying GIS methods to the site plans contained in the Olduvai Volume 3 monograph (Leakey, 1971). Our analysis indicates that the Bed I assemblages show preferred orientations, probably caused by natural agents such as water disturbance. These results, based on new GIS techniques applied to paleoanthropological studies, have important implications for the understanding of the formative agents of Olduvai sites and the behavioral meaning of the bone and lithic accumulations in Bed I. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Structural and functional implications in the eubacterial ribosome as revealed by protein-rRNA and antibiotic contact sites.

    Science.gov (United States)

    Wittmann-Liebold, B; Uhlein, M; Urlaub, H; Müller, E C; Otto, A; Bischof, O

    1995-01-01

    Contact sites between protein and rRNA in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus were investigated at the molecular level using UV and 2-iminothiolane as cross-linkers. Thirteen ribosomal proteins (S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29, and L36) from these organisms were cross-linked in direct contact with the RNAs, and the peptide stretches as well as amino acids involved were identified. Further, the binding sites of puromycin and spiramycin were established at the peptide level in several proteins that were found to constitute the antibiotic-binding sites. Peptide stretches of puromycin binding were identified from proteins S7, S14, S18, L18, AND L29; those of spiramycin attachment were derived from proteins S12, S14, L17, L18, L27, and L35. Comparison of the RNA-peptide contact sites with the peptides identified for antibiotic binding and with those altered in antibiotic-resistant mutants clearly showed identical peptide areas to be involved and, hence, demonstrated the functional importance of these peptides. Further evidence for a functional implication of ribosomal proteins in the translational process came from complementation experiments in which protein L2 from Halobacterium marismortui was incorporated into the E. coli ribosomes that were active. The incorporated protein was present in 50S subunits and 70S particles, in disomes, and in higher polysomes. These results clearly demonstrate the functional implication of protein L2 in protein biosynthesis. Incorporation studies with a mutant of HmaL2 with a replacement of histidine-229 by glycine completely abolished the functional activity of the ribosome. Accordingly, protein L2 with histidine-229 is a crucial element of the translational machinery.

  5. Management Implications for the Most Attractive Scenic Sites along the Andalusia Coast (SW Spain

    Directory of Open Access Journals (Sweden)

    Alexis Mooser

    2018-04-01

    Full Text Available A coastal scenery assessment was carried out at 50 sites along the 910 km long Andalusia coast (SW Spain using a checklist of 26 natural and human parameters, parameter weighting matrices, and fuzzy logic. A scenic classification was utilised that can rate sites as Class I (natural areas of great scenic beauty to Class V (urbanised areas of poor scenic interest, but, for this study, only natural sites of great scenic value were investigated; 41 sites were included in Class I, 9 in Class II and, apart from four, all of the sites were under some feature of protection—managed by the Andalusia Environmental Agency (RENPA, in Spanish. Sites belong to the Natural Park Cabo de Gata-Nijar (24% of sites, the Natural Park of Gibraltar Strait (18%, the Natural Place Acantilado de Maro-Cerro Gordo (12%, and the Natural and National parks of Doñana (8%. Results obtained by means of scenic evaluation constitute a sound scientific basis for any envisaged management plan for investigated coastal areas preservation/conservation and responsible future developments, especially for natural protected areas, which represent the most attractive coastal tourist destinations. With respect to natural parameters, excellent scenic values appeared to be linked to the geological setting and the presence of mountainous landscapes related to the Betic Chain. Human parameters usually show good scores because null or extremely reduced human impacts are recorded, but, at places, conflicts arose between conservation and recreational activities because visitors are often interested in beach activities more so than ecotourism. Low scores of human parameters were often related to litter presence or the unsuitable emplacement of utilities, such as informative panels, litter bins, etc.

  6. Heat flow at the proposed Appalachian Ultradeep Core Hole (ADCOH) Site: Tectonic implications

    Science.gov (United States)

    Costain, John K.; Decker, Edward R.

    The heat flow in northwestern South Carolina at the Appalachian Ultradeep Core Hole (ADCOH) site area is approximately 55 mW/m². This data supplements other data to the east in the Piedmont and Atlantic Coastal Plain provinces where heat flows > 55 mW/m² are characteristic of post- and late-synmetamorphic granitoids. Piedmont heat flow and heat generation data for granites, metagranites, and one Slate Belt site, in a zone approximately parallel to major structural Appalachian trends, define a linear relation. Tectonic truncation of heat-producing crust at a depth of about 8 km (a depth equal to the slope of the heat flow-heat production line) is proposed to explain the linear relation. Using the value of reduced heat flow estimated from this empirical relation, and assuming thicknesses of heat-producing crust defined by new ADCOH seismic data, the heat flow and heat production at the ADCOH site are consistent with a depth to the base of the Inner Piedmont crystalline allochthon of about 5.5 km. Seismic data at the ADCOH site confirm that the Inner Piedmont is tectonically truncated at about 5.5 km by the Blue Ridge master decollement. Temperatures at 10 km at the ADCOH site are predicted to be less than 200 °C.

  7. Stone tool function at the paleolithic sites of Starosele and Buran Kaya III, Crimea: behavioral implications.

    Science.gov (United States)

    Hardy, B L; Kay, M; Marks, A E; Monigal, K

    2001-09-11

    Stone tools are often the most abundant type of cultural remains at Paleolithic sites, yet their function is often poorly understood. Investigations of stone tool function, including microscopic use-wear and residue analyses, were performed on a sample of artifacts from the Paleolithic sites of Starosele (40,000-80,000 years BP) and Buran Kaya III (32,000-37,000 years BP). The Middle Paleolithic levels at Starosele exhibit a typical variant of the local Micoquian Industry. The artifacts from Buran Kaya III most closely resemble an Early Streletskayan Industry associated with the early Upper Paleolithic. The results of the functional analyses suggest that hominids at both sites were exploiting woody and starchy plant material as well as birds and mammals. Both sites show evidence of hafting of a wide variety of tools and the possible use of projectile or thrusting spears. These analyses were performed by using two different techniques conducted by independent researchers. Combined residue and use-wear analyses suggest that both the Upper Paleolithic and Middle Paleolithic hominids at these sites were broad-based foragers capable of exploiting a wide range of resources.

  8. Positive allosteric modulation of mGluR5 accelerates extinction learning but not relearning following methamphetamine self-administration

    Directory of Open Access Journals (Sweden)

    Peter R Kufahl

    2012-11-01

    Full Text Available Recent studies have implicated glutamate neurotransmission as an important substrate for the extinction of conditioned behaviors, including responding for drug reinforcement. Positive allosteric modulation of the type-5 metabotropic glutamate receptor (mGluR5 in particular has emerged as a treatment strategy for the enhancement of extinction of drug-motivated behaviors. Here, we investigated the effects of the mGluR5 positive allosteric modulator CDPPB, a compound known for its cognitive enhancing effects in rodents, on extinction learning in rats with different histories of methamphetamine (METH training. Rats were trained to self-administer METH under two conditions: 16 daily sessions of short access (90 min/day, ShA, or 8 daily sessions of short access followed by 8 sessions of long access (6 hr/day, LgA. Control rats self-administered sucrose pellets in daily 30 min sessions. Next, rats were administered vehicle or 30 mg/kg CDPPB prior to 7 consecutive daily extinction sessions, subjected to additional extinction sessions to re-establish a post-treatment baseline, and then tested for reinstatement of behavior in the presence of METH- or sucrose-paired cues. Rats were then subjected to a second series of extinction sessions, preceded by vehicle or 30 mg/kg CDPPB, and an additional test for cue-triggered reinstatement. CDPPB treatment resulted in a more rapid extinction of responding on the active lever, especially in the early sessions of the first extinction sequence. However, treatment effects were minimal during subsequent cue reinstatement tests and nonexistent during the second series of extinction sessions. Rats with histories of ShA, LgA and sucrose training expressed similar behavioral sensitivities to CDPPB, with LgA rats demonstrating a modestly higher treatment effect. Positive allosteric modulation of mGluR5 may therefore have some beneficial effects on efforts to facilitate extinction learning and reduce methamphetamine seeking.

  9. In silico determination of intracellular glycosylation and phosphorylation sites in human selectins: Implications for biological function

    DEFF Research Database (Denmark)

    Ahmad, I.; Hoessli, D.C.; Gupta, Ramneek

    2007-01-01

    Post-translational modifications provide the proteins with the possibility to perform functions in addition to those determined by their primary sequence. However, analysis of multifunctional protein structures in the environment of cells and body fluids is made especially difficult by the presence...... both modifications are likely to occur can also be predicted (YinYang sites), to suggest further functional versatility. Structural modifications of hydroxyl groups of P-, E-, and L-selectins have been predicted and possible functions resulting from such modifications are proposed. Functional changes...... of the three selectins are based on the assumption that transitory and reversible protein modifications by phosphate and O-GlcNAc cause specific conformational changes and generate binding sites for other proteins. The computer-assisted prediction of glycosylation and phosphorylation sites in selectins should...

  10. Environmental justice: Implications for siting of Federal Radioactive Waste Management Facilities

    International Nuclear Information System (INIS)

    Easterling, J.B.; Poles, J.S.

    1994-01-01

    Environmental justice is a term that has developed as a result of our need to address whether some of the environmental decisions we have made -- and others we will make -- are fair. The idea of environmental justice has been actively pursued by the Clinton Administration, and this consideration has resulted in Executive Order 12898, which was signed by President Clinton on February 11, 1994. The Executive Order calls for adverse impacts of Federal actions on minority or low-income populations to be identified before decisions implementing those actions are made. Numerous studies show that noxious facilities, such as incinerators and landfills, have been constructed in minority or low-income communities. And since the Department has not yet decided on sites for high-level waste storage or disposal facilities, it will have to take the new Executive Order into consideration as another piece in the complicated quilt of requirements that cover facility siting. An interesting twist to this is the fact that twenty Native American Indian Tribes expressed interest in voluntarily hosting a high-level radioactive waste management facility for temporary storage. They made these expressions on their own initiative, and several Tribes continue to pursue the idea of negotiations with either the Federal Government or private entities to locate a temporary storage site on Tribal land. The Executive Order goes beyond simply studying the effect of siting a facility and addresses in spirit a criticism that the Federal Government has been guilty of open-quotes environmental racismclose quotes in its siting policies -- that it has intentionally picked minority or low-income communities for waste management facilities. What Department of Energy staff and others may have considered foregone conclusions in terms of interim storage facility siting and transportation options will have to be reevaluated for compatibility with provisions of the new Executive Order

  11. Mine tailings composition in a historic site: implications for ecological restoration.

    Science.gov (United States)

    Courtney, R

    2013-02-01

    Ecological restoration, using tolerant plant species and nutrient additions, is a low-cost option to decrease environmental risks associated with mine tailings. An attempt was previously made to establish such a vegetation cover on an abandoned tailings facility in Southern Ireland. Historically, the tailings site has been prone to dusting and is a potential source of contamination to the surrounding environment. The site was examined to determine the success of the previous restoration plan used to revegetate the site and to determine its suitability for further restoration. Three distinct floristic areas were identified (grassland, poor grassland and bare area) based on herbage compositions and elemental analysis. Surface and subsurface samples were taken to characterise tailings from within these areas of the tailings site. The pH of bare surface tailings (pH, 2.7) was significantly more acidic (p tailings being hostile to plant growth. Total metal concentrations in tailings were high (c. 10,000 mg kg(-1) for Pb and up to 20,000 mg kg(-1) for Zn). DTPA-extractable Zn and Pb were 16 and 11 % of the total amount, respectively. Metal content in grasses growing on some areas of the tailings were elevated and demonstrated the inability of the tailings to support sustainable plant growth. Due to the inherently hostile characteristics of these areas, future restoration work will employ capping with a barrier layer.

  12. On the Rapid Rise of Social Networking Sites: New Findings and Policy Implications

    Science.gov (United States)

    Livingstone, Sonia; Brake, David R

    2010-01-01

    Social networking sites have been rapidly adopted by children and, especially, teenagers and young people worldwide, enabling new opportunities for the presentation of the self, learning, construction of a wide circle of relationships, and the management of privacy and intimacy. On the other hand, there are also concerns that social networking…

  13. Consumer adoption of social networking sites: implications for theory and practice

    NARCIS (Netherlands)

    Lorenzo Romero, Carlota; Constantinides, Efthymios; Alarcon-del-Amo, Maria-del-Carmen

    2011-01-01

    Purpose – The purpose of this paper is to study factors affecting the acceptance of social networking sites (SNS), analyze users' practices and behavior in these environments and assess the degree of acceptance of SNS in The Netherlands. Design/methodology/approach – An extended technology

  14. Marketing of Academic Library Services through Social Networking Sites: Implications of Electronic Word-of-Mouth

    Science.gov (United States)

    Siddike, Md. Abul Kalam; Kiran, K.

    2015-01-01

    The main objective of this study is to investigate the perceptions of academic librarians towards the marketing of library services through social networking sites (SNSs) and their understanding of using electronic word-of-mouth (eWOM) as a marketing tool in academic libraries. This study follows a qualitative data-gathering approach of structured…

  15. Life cycle of petroleum biodegradation metabolite plumes, and implications for risk management at fuel release sites.

    Science.gov (United States)

    Zemo, Dawn A; O'Reilly, Kirk T; Mohler, Rachel E; Magaw, Renae I; Espino Devine, Catalina; Ahn, Sungwoo; Tiwary, Asheesh K

    2017-07-01

    This paper summarizes the results of a 5-y research study of the nature and toxicity of petroleum biodegradation metabolites in groundwater at fuel release sites that are quantified as diesel-range "Total Petroleum Hydrocarbons" (TPH; also known as TPHd, diesel-range organics (DRO), etc.), unless a silica gel cleanup (SGC) step is used on the sample extract prior to the TPH analysis. This issue is important for site risk management in regulatory jurisdictions that use TPH as a metric; the presence of these metabolites may preclude site closure even if all other factors can be considered "low-risk." Previous work has shown that up to 100% of the extractable organics in groundwater at petroleum release sites can be biodegradation metabolites. The metabolites can be separated from the hydrocarbons by incorporating an SGC step; however, regulatory agency acceptance of SGC has been inconsistent because of questions about the nature and toxicity of the metabolites. The present study was conducted to answer these specific questions. Groundwater samples collected from source and downgradient wells at fuel release sites were extracted and subjected to targeted gas chromatography-mass spectrometry (GC-MS) and nontargeted two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC-MS) analyses, and the metabolites identified in each sample were classified according to molecular structural classes and assigned an oral reference dose (RfD)-based toxicity ranking. Our work demonstrates that the metabolites identified in groundwater at biodegrading fuel release sites are in classes ranked as low toxicity to humans and are not expected to pose significant risk to human health. The identified metabolites naturally attenuate in a predictable manner, with an overall trend to an increasingly higher proportion of organic acids and esters, and a lower human toxicity profile, and a life cycle that is consistent with the low-risk natural attenuation paradigm adopted

  16. Oviposition Site Selection by the Dengue Vector Aedes aegypti and Its Implications for Dengue Control

    Science.gov (United States)

    Wong, Jacklyn; Stoddard, Steven T.; Astete, Helvio; Morrison, Amy C.; Scott, Thomas W.

    2011-01-01

    Background Because no dengue vaccine or antiviral therapy is commercially available, controlling the primary mosquito vector, Aedes aegypti, is currently the only means to prevent dengue outbreaks. Traditional models of Ae. aegypti assume that population dynamics are regulated by density-dependent larval competition for food and little affected by oviposition behavior. Due to direct impacts on offspring survival and development, however, mosquito choice in oviposition site can have important consequences for population regulation that should be taken into account when designing vector control programs. Methodology/Principal Findings We examined oviposition patterns by Ae. aegypti among 591 naturally occurring containers and a set of experimental containers in Iquitos, Peru. Using larval starvation bioassays as an indirect measure of container food content, we assessed whether females select containers with the most food for their offspring. Our data indicate that choice of egg-laying site is influenced by conspecific larvae and pupae, container fill method, container size, lid, and sun exposure. Although larval food positively influenced oviposition, our results did not support the hypothesis that females act primarily to maximize food for larvae. Females were most strongly attracted to sites containing immature conspecifics, even when potential competitors for their progeny were present in abundance. Conclusion/Significance Due to strong conspecific attraction, egg-laying behavior may contribute more to regulating Ae. aegypti populations than previously thought. If highly infested containers are targeted for removal or larvicide application, females that would have preferentially oviposited in those sites may instead distribute their eggs among other suitable, previously unoccupied containers. Strategies that kill mosquitoes late in their development (i.e., insect growth regulators that kill pupae rather than larvae) will enhance vector control by creating

  17. Theoretical Analysis of Allosteric and Operator Binding for Cyclic-AMP Receptor Protein Mutants

    Science.gov (United States)

    Einav, Tal; Duque, Julia; Phillips, Rob

    2018-02-01

    Allosteric transcription factors undergo binding events both at their inducer binding sites as well as at distinct DNA binding domains, and it is often difficult to disentangle the structural and functional consequences of these two classes of interactions. In this work, we compare the ability of two statistical mechanical models - the Monod-Wyman-Changeux (MWC) and the Koshland-N\\'emethy-Filmer (KNF) models of protein conformational change - to characterize the multi-step activation mechanism of the broadly acting cyclic-AMP receptor protein (CRP). We first consider the allosteric transition resulting from cyclic-AMP binding to CRP, then analyze how CRP binds to its operator, and finally investigate the ability of CRP to activate gene expression. In light of these models, we examine data from a beautiful recent experiment that created a single-chain version of the CRP homodimer, thereby enabling each subunit to be mutated separately. Using this construct, six mutants were created using all possible combinations of the wild type subunit, a D53H mutant subunit, and an S62F mutant subunit. We demonstrate that both the MWC and KNF models can explain the behavior of all six mutants using a small, self-consistent set of parameters. In comparing the results, we find that the MWC model slightly outperforms the KNF model in the quality of its fits, but more importantly the parameters inferred by the MWC model are more in line with structural knowledge of CRP. In addition, we discuss how the conceptual framework developed here for CRP enables us to not merely analyze data retrospectively, but has the predictive power to determine how combinations of mutations will interact, how double mutants will behave, and how each construct would regulate gene expression.

  18. Biochemistry and structural studies of kynurenine 3-monooxygenase reveal allosteric inhibition by Ro 61-8048.

    Science.gov (United States)

    Gao, Jingjing; Yao, Licheng; Xia, Tingting; Liao, Xuebin; Zhu, Deyu; Xiang, Ye

    2018-04-01

    The human kynurenine 3-monooxygenase (hKMO) is a potential therapeutic target for neurodegenerative and neurologic disorders. Inhibition of KMO by Ro 61-8048, a potent, selective, and the most widely used inhibitor of KMO, was shown effective in various models of neurodegenerative or neurologic disorders. However, the molecular basis of hKMO inhibition by Ro 61-8048 is not clearly understood. Here, we report biochemistry studies on hKMO and crystal structures of an hKMO homolog, pfKMO from Pseudomonas fluorescens, in complex with the substrate l-kynurenine and Ro 61-8048. We found that the C-terminal ∼110 aa are essential for the enzymatic activity of hKMO and the homologous C-terminal region of pfKMO folds into a distinct, all-α-helical domain, which associates with the N-terminal catalytic domain to form a unique tunnel in proximity to the substrate-binding pocket. The tunnel binds the Ro 61-8048 molecule, which fills most of the tunnel, and Ro 61-8048 is hydrogen bonded with several completely conserved residues, including an essential catalytic residue. Modification of Ro 61-8048 and biochemical studies of the modified Ro 61-8048 derivatives suggested that Ro 61-8048 inhibits the enzyme in an allosteric manner by affecting the conformation of the essential catalytic residue and by blocking entry of the substrate or product release. The unique binding sites distinguish Ro 61-8048 as a noncompetitive and highly selective inhibitor from other competitive inhibitors, which should facilitate further optimization of Ro 61-8048 and the development of new inhibitory drugs to hKMO.-Gao, J., Yao, L., Xia, T., Liao, X., Zhu, D., Xiang, Y. Biochemistry and structural studies of kynurenine 3-monooxygenase reveal allosteric inhibition by Ro 61-8048.

  19. Structural Dynamics Control Allosteric Activation of Cytohesin Family Arf GTPase Exchange Factors

    Energy Technology Data Exchange (ETDEWEB)

    Malaby, Andrew W.; Das, Sanchaita; Chakravarthy, Srinivas; Irving, Thomas C.; Bilsel, Osman; Lambright, David G.

    2018-01-01

    Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (GTPase) activation by guanine nucleotide exchange factors (GEFs) containing a catalytic Sec7 domain and a membrane-targeting module such as a pleckstrin homology (PH) domain. The catalytic output of cytohesin family Arf GEFs is controlled by autoinhibitory interactions that impede accessibility of the exchange site in the Sec7 domain. These restraints can be relieved through activator Arf-GTP binding to an allosteric site comprising the PH domain and proximal autoinhibitory elements (Sec7-PH linker and C-terminal helix). Small-angle X-ray scattering and negative-stain electron microscopy were used to investigate the structural organization and conformational dynamics of cytohesin-3 (Grp1) in autoinhibited and active states. The results support a model in which hinge dynamics in the autoinhibited state expose the activator site for Arf-GTP binding, while subsequent C-terminal helix unlatching and repositioning unleash conformational entropy in the Sec7-PH linker to drive exposure of the exchange site.

  20. Comparison of crystal and solution hemoglobin binding of selected antigelling agents and allosteric modifiers

    International Nuclear Information System (INIS)

    Mehanna, A.S.; Abraham, D.J.

    1990-01-01

    This paper details comprehensive binding studies (solution and X-ray) of human hemoglobin A with a group of halogenated carboxylic acids that were investigated as potential antisickling agents. It is, to our knowledge, the first study to compare solution and crystal binding for a series of compounds under similar high-salt conditions used for cocrystallization. The compounds include [(3,4-dichlorobenzyl)oxy]acetic acid, [(p-bromobenzyl)oxy]acetic acid, clofibric acid, and bezafibrate. The location and stereochemistry of binding sites have been established by X-ray crystallography, while the number of binding sites and affinity constants were measured by using equilibrium dialysis. The observed crystal structures are consistent with the binding observed in solution and that the number of binding sites is independent of salt concentration, while the binding constant increases with increasing salt concentration. The studies also reveal that relatively small changes in the chemical structure of a drug molecule can result in entirely different binding sites on the protein. Moreover, the X-ray studies provide a possible explanation for the multiplicity in function exhibited by these compounds as allosteric modulators and/or antisickling agents. Finally, the studies indicate that these compounds bind differently to the R and T states of hemoglobin, and observation of special significance to the original design of these agents

  1. Differentially expressed genes distributed over chromosomes and implicated in certain biological processes for site insertion genetically modified rice Kemingdao.

    Science.gov (United States)

    Liu, Zhi; Li, Yunhe; Zhao, Jie; Chen, Xiuping; Jian, Guiliang; Peng, Yufa; Qi, Fangjun

    2012-01-01

    Release of genetically modified (GM) plants has sparked off intensive debates worldwide partly because of concerns about potential adverse unintended effects of GM plants to the agro system and the safety of foods. In this study, with the aim of revealing the molecular basis for unintended effects of a single site insertion GM Kemingdao (KMD) rice transformed with a synthetic cry1Ab gene, and bridging unintended effects of KMD rice through clues of differentially expressed genes, comparative transcriptome analyses were performed for GM KMD rice and its parent rice of Xiushui11 (XS11). The results showed that 680 differentially expressed transcripts were identified from 30-day old seedlings of GM KMD rice. The absolute majority of these changed expression transcripts dispersed and located over all rice chromosomes, and existed physical distance on chromosome from the insertion site, while only two transcripts were found to be differentially expressed within the 21 genes located within 100 kb up and down-stream of the insertion site. Pathway and biology function analyses further revealed that differentially expressed transcripts of KMD rice were involved in certain biological processes, and mainly implicated in two types of pathways. One type was pathways implicated in plant stress/defense responses, which were considerably in coordination with the reported unintended effects of KMD rice, which were more susceptible to rice diseases compared to its parent rice XS11; the other type was pathways associated with amino acids metabolism. With this clue, new unintended effects for changes in amino acids synthesis of KMD rice leaves were successfully revealed. Such that an actual case was firstly provided for identification of unintended effects in GM plants by comparative transciptome analysis.

  2. Non equivalence of the chains in the allosteric interaction of the hemoglobin

    International Nuclear Information System (INIS)

    Jacchieri, S.G.

    1983-01-01

    The importance, for the temperature dependence of the cooperative behaviour of hemoglobin, of the functional non equivalence of the polypeptide chains from which the hemoglobin molecule is built is studied. With such purpose thermodynamic allosteric parameters are introduced called 'mean allosteric parameters' which relate the last two oxygen bindings to the firsttwo ones. It is shown that the mean allosteric free energy is strongly correlated to the Hill parameter which is a classic measure of cooperativity; hence, the mean allosteric free energy measures the hemoglobin cooperativity. Recent experimental data show that the mean allosteric free energy decreasses with temperature; this is due to the mean allosteric enthalphy and entropy being positive quantities. To analise such behaviour in terms of thermodynamic's arguments equations are derived for the thermodynamic parameters of oxygen binding to hemoglobin in terms of those of its chains. Since the obtained equations have a great number of terms the same treatment is applied to a hypothetic dimer from which simpler relations are derived. From both cases it is concluded that the positive character of the mean allosteric enthalpy and entropy is due to the presence of cooperative and anticooperative terms. Since the last terms are absent in the equations of allosteric homoproteins, the characteristic temperature-dependence of hemoglobin's cooperativity depends on the presence of non-equivalent chains. (Author) [pt

  3. Forward-looking farmers owning multiple potential wetland restoration sites: implications for efficient restoration

    Science.gov (United States)

    Schroder (Kushch), Svetlana; Lang, Zhengxin; Rabotyagov, Sergey

    2018-04-01

    Wetland restoration can increase the provision of multiple non-market ecosystem services. Environmental and socio-economic factors need to be accounted for when land is withdrawn from agriculture and wetlands are restored. We build multi-objective optimization models to provide decision support for wetland restoration in the Le Sueur river watershed in Southern Minnesota. We integrate environmental objectives of sediment reduction and habitat protection with socio-economic factors associated with the overlap of private land with potential wetland restoration sites in the watershed and the costs representing forward-looking farmers voluntarily taking land out of agricultural production in favor of wetland restoration. Our results demonstrate that the inclusion of these factors early on in the restoration planning process affects both the total costs of the restoration project and the spatial distribution of optimally selected wetland restoration sites.

  4. Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-chemokine Receptor 5 (CCR5).

    Science.gov (United States)

    Karlshøj, Stefanie; Amarandi, Roxana Maria; Larsen, Olav; Daugvilaite, Viktorija; Steen, Anne; Brvar, Matjaž; Pui, Aurel; Frimurer, Thomas Michael; Ulven, Trond; Rosenkilde, Mette Marie

    2016-12-23

    The small molecule metal ion chelators bipyridine and terpyridine complexed with Zn 2+ (ZnBip and ZnTerp) act as CCR5 agonists and strong positive allosteric modulators of CCL3 binding to CCR5, weak modulators of CCL4 binding, and competitors for CCL5 binding. Here we describe their binding site using computational modeling, binding, and functional studies on WT and mutated CCR5. The metal ion Zn 2+ is anchored to the chemokine receptor-conserved Glu-283 VII:06/7.39 Both chelators interact with aromatic residues in the transmembrane receptor domain. The additional pyridine ring of ZnTerp binds deeply in the major binding pocket and, in contrast to ZnBip, interacts directly with the Trp-248 VI:13/6.48 microswitch, contributing to its 8-fold higher potency. The impact of Trp-248 was further confirmed by ZnClTerp, a chloro-substituted version of ZnTerp that showed no inherent agonism but maintained positive allosteric modulation of CCL3 binding. Despite a similar overall binding mode of all three metal ion chelator complexes, the pyridine ring of ZnClTerp blocks the conformational switch of Trp-248 required for receptor activation, thereby explaining its lack of activity. Importantly, ZnClTerp becomes agonist to the same extent as ZnTerp upon Ala mutation of Ile-116 III:16/3.40 , a residue that constrains the Trp-248 microswitch in its inactive conformation. Binding studies with 125 I-CCL3 revealed an allosteric interface between the chemokine and the small molecule binding site, including residues Tyr-37 I:07/1.39 , Trp-86 II:20/2.60 , and Phe-109 III:09/3.33 The small molecules and CCL3 approach this interface from opposite directions, with some residues being mutually exploited. This study provides new insight into the molecular mechanism of CCR5 activation and paves the way for future allosteric drugs for chemokine receptors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein*

    Science.gov (United States)

    Townsend, Philip D.; Rodgers, Thomas L.; Glover, Laura C.; Korhonen, Heidi J.; Richards, Shane A.; Colwell, Lucy J.; Pohl, Ehmke; Wilson, Mark R.; Hodgson, David R. W.; McLeish, Tom C. B.; Cann, Martin J.

    2015-01-01

    Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring. PMID:26187469

  6. The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein.

    Science.gov (United States)

    Townsend, Philip D; Rodgers, Thomas L; Glover, Laura C; Korhonen, Heidi J; Richards, Shane A; Colwell, Lucy J; Pohl, Ehmke; Wilson, Mark R; Hodgson, David R W; McLeish, Tom C B; Cann, Martin J

    2015-09-04

    Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Correlations Between Life-Detection Techniques and Implications for Sampling Site Selection in Planetary Analog Missions

    Science.gov (United States)

    Gentry, Diana M.; Amador, Elena S.; Cable, Morgan L.; Chaudry, Nosheen; Cullen, Thomas; Jacobsen, Malene B.; Murukesan, Gayathri; Schwieterman, Edward W.; Stevens, Adam H.; Stockton, Amanda; Tan, George; Yin, Chang; Cullen, David C.; Geppert, Wolf

    2017-10-01

    We conducted an analog sampling expedition under simulated mission constraints to areas dominated by basaltic tephra of the Eldfell and Fimmvörðuháls lava fields (Iceland). Sites were selected to be "homogeneous" at a coarse remote sensing resolution (10-100 m) in apparent color, morphology, moisture, and grain size, with best-effort realism in numbers of locations and replicates. Three different biomarker assays (counting of nucleic-acid-stained cells via fluorescent microscopy, a luciferin/luciferase assay for adenosine triphosphate, and quantitative polymerase chain reaction (qPCR) to detect DNA associated with bacteria, archaea, and fungi) were characterized at four nested spatial scales (1 m, 10 m, 100 m, and >1 km) by using five common metrics for sample site representativeness (sample mean variance, group F tests, pairwise t tests, and the distribution-free rank sum H and u tests). Correlations between all assays were characterized with Spearman's rank test. The bioluminescence assay showed the most variance across the sites, followed by qPCR for bacterial and archaeal DNA; these results could not be considered representative at the finest resolution tested (1 m). Cell concentration and fungal DNA also had significant local variation, but they were homogeneous over scales of >1 km. These results show that the selection of life detection assays and the number, distribution, and location of sampling sites in a low biomass environment with limited a priori characterization can yield both contrasting and complementary results, and that their interdependence must be given due consideration to maximize science return in future biomarker sampling expeditions.

  8. Positive selection neighboring functionally essential sites and disease-implicated regions of mammalian reproductive proteins.

    LENUS (Irish Health Repository)

    Morgan, Claire C

    2010-01-01

    ABSTRACT: BACKGROUND: Reproductive proteins are central to the continuation of all mammalian species. The evolution of these proteins has been greatly influenced by environmental pressures induced by pathogens, rival sperm, sexual selection and sexual conflict. Positive selection has been demonstrated in many of these proteins with particular focus on primate lineages. However, the mammalia are a diverse group in terms of mating habits, population sizes and germ line generation times. We have examined the selective pressures at work on a number of novel reproductive proteins across a wide variety of mammalia. RESULTS: We show that selective pressures on reproductive proteins are highly varied. Of the 10 genes analyzed in detail, all contain signatures of positive selection either across specific sites or in specific lineages or a combination of both. Our analysis of SP56 and Col1a1 are entirely novel and the results show positively selected sites present in each gene. Our findings for the Col1a1 gene are suggestive of a link between positive selection and severe disease type. We find evidence in our dataset to suggest that interacting proteins are evolving in symphony: most likely to maintain interacting functionality. CONCLUSION: Our in silico analyses show positively selected sites are occurring near catalytically important regions suggesting selective pressure to maximize efficient fertilization. In those cases where a mechanism of protein function is not fully understood, the sites presented here represent ideal candidates for mutational study. This work has highlighted the widespread rate heterogeneity in mutational rates across the mammalia and specifically has shown that the evolution of reproductive proteins is highly varied depending on the species and interacting partners. We have shown that positive selection and disease are closely linked in the Col1a1 gene.

  9. Progress in centralised ethics review processes: Implications for multi-site health evaluations.

    Science.gov (United States)

    Prosser, Brenton; Davey, Rachel; Gibson, Diane

    2015-04-01

    Increasingly, public sector programmes respond to complex social problems that intersect specific fields and individual disciplines. Such responses result in multi-site initiatives that can span nations, jurisdictions, sectors and organisations. The rigorous evaluation of public sector programmes is now a baseline expectation. For evaluations of large and complex multi-site programme initiatives, the processes of ethics review can present a significant challenge. However in recent years, there have been new developments in centralised ethics review processes in many nations. This paper provides the case study of an evaluation of a national, inter-jurisdictional, cross-sector, aged care health initiative and its encounters with Australian centralised ethics review processes. Specifically, the paper considers progress against the key themes of a previous five-year, five nation study (Fitzgerald and Phillips, 2006), which found that centralised ethics review processes would save time, money and effort, as well as contribute to more equitable workloads for researchers and evaluators. The paper concludes with insights for those charged with refining centralised ethics review processes, as well as recommendations for future evaluators of complex multi-site programme initiatives. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Spatio-temporal evolution of fault networks: implications for deep radioactive waste disposal sites

    International Nuclear Information System (INIS)

    Hardacre, K.; Scotti, O.

    2001-01-01

    The objective of this work is to provide estimates of both vertical and lateral propagation rates, on time scales of 100 000 years, for the faults systems known to be present today in the region of Bure, the site of an underground rock laboratory. The project is divided into three parts: 1) literature review (fault growth processes and data), 2) benchmarking against data a numerical code that allows for spontaneous development and growth of faults and 3) application to the Bure site. A brief overview of fault growth processes and observed fault propagation rates shows that non-negligible values (20-50 mm/yrs or roughly 5 km in 100 000 years) can be reached. Preliminary results obtained from two numerical simulations 1) fault growth of a pre-existing weaknesses and 2) fault growth of a spontaneously generated fault system, provide encouraging results with values that are comparable with those observed in nature for the growth of normal fault systems. The application to strike-slip system that characterizes the Bure site is still underway. (authors)

  11. Implications of matrix diffusion on 1,4-dioxane persistence at contaminated groundwater sites.

    Science.gov (United States)

    Adamson, David T; de Blanc, Phillip C; Farhat, Shahla K; Newell, Charles J

    2016-08-15

    Management of groundwater sites impacted by 1,4-dioxane can be challenging due to its migration potential and perceived recalcitrance. This study examined the extent to which 1,4-dioxane's persistence was subject to diffusion of mass into and out of lower-permeability zones relative to co-released chlorinated solvents. Two different release scenarios were evaluated within a two-layer aquifer system using an analytical modeling approach. The first scenario simulated a 1,4-dioxane and 1,1,1-TCA source zone where spent solvent was released. The period when 1,4-dioxane was actively loading the low-permeability layer within the source zone was estimated to be high effective solubility. While this was approximately an order-of-magnitude shorter than the loading period for 1,1,1-TCA, the mass of 1,4-dioxane stored within the low-permeability zone at the end of the simulation period (26kg) was larger than that predicted for 1,1,1-TCA (17kg). Even 80years after release, the aqueous 1,4-dioxane concentration was still several orders-of-magnitude higher than potentially-applicable criteria. Within the downgradient plume, diffusion contributed to higher concentrations and enhanced penetration of 1,4-dioxane into the low-permeability zones relative to 1,1,1-TCA. In the second scenario, elevated 1,4-dioxane concentrations were predicted at a site impacted by migration of a weak source from an upgradient site. Plume cutoff was beneficial because it could be implemented in time to prevent further loading of the low-permeability zone at the downgradient site. Overall, this study documented that 1,4-dioxane within transmissive portions of the source zone is quickly depleted due to characteristics that favor both diffusion-based storage and groundwater transport, leaving little mass to treat using conventional means. Furthermore, the results highlight the differences between 1,4-dioxane and chlorinated solvent source zones, suggesting that back diffusion of 1,4-dioxane mass may be

  12. Assessing Climate Vulnerabilities of Food Distribution Center Sites in Greater Boston and Their Regional Implications: Climate Adaptation Planning in Practice

    Science.gov (United States)

    Teferra, A.; Watson, C.; Douglas, E. M.

    2016-12-01

    The Metro Boston region, an area whose civic leaders have been at the forefront of climate resilience initiatives in recent years, is finalizing a flood vulnerability assessment of food distribution center sites located north of Boston, with the support of the University of Massachusetts Boston and the American Geophysical Union's Thriving Earth Exchange program. The community-scientist collaboration emerged because of the need for more local analyses of the area to inform climate resiliency policy and planning actions for the region. A significant amount of the metro region's food supply passes through two major distribution centers in the cities of Everett and Chelsea, just north of the Mystic River. The Metropolitan Area Planning Council (MAPC), on behalf of the Metro Boston Climate Preparedness Taskforce, is working with Chris Watson and Ellen Douglas of UMass Boston to build on existing analyses of the region's food system and climate vulnerabilities and to develop a report identifying flood risk exposure to the sites. The analysis brings in dynamic modeling techniques that incorporate storm surge and sea level rise projections under different climate scenarios, and aims to align methodologies with those of other regional analyses, such as Climate Ready Boston and the City of Cambridge's Vulnerability Assessment. The study is helping to inform MAPC's and the Metro Boston Climate Preparedness Taskforce's understanding of this critical food distribution infrastructure, illustrate the larger regional implications of climate impacts on food distribution in the Greater Boston area, and guide the development of site-specific strategies for addressing identified vulnerabilities.

  13. Substrate-Induced Allosteric Change in the Quaternary Structure of the Spermidine N-Acetyltransferase SpeG.

    Science.gov (United States)

    Filippova, Ekaterina V; Weigand, Steven; Osipiuk, Jerzy; Kiryukhina, Olga; Joachimiak, Andrzej; Anderson, Wayne F

    2015-11-06

    The spermidine N-acetyltransferase SpeG is a dodecameric enzyme that catalyzes the transfer of an acetyl group from acetyl coenzyme A to polyamines such as spermidine and spermine. SpeG has an allosteric polyamine-binding site and acetylating polyamines regulate their intracellular concentrations. The structures of SpeG from Vibrio cholerae in complexes with polyamines and cofactor have been characterized earlier. Here, we present the dodecameric structure of SpeG from V. cholerae in a ligand-free form in three different conformational states: open, intermediate and closed. All structures were crystallized in C2 space group symmetry and contain six monomers in the asymmetric unit cell. Two hexamers related by crystallographic 2-fold symmetry form the SpeG dodecamer. The open and intermediate states have a unique open dodecameric ring. This SpeG dodecamer is asymmetric except for the one 2-fold axis and is unlike any known dodecameric structure. Using a fluorescence thermal shift assay, size-exclusion chromatography with multi-angle light scattering, small-angle X-ray scattering analysis, negative-stain electron microscopy and structural analysis, we demonstrate that this unique open dodecameric state exists in solution. Our combined results indicate that polyamines trigger conformational changes and induce the symmetric closed dodecameric state of the protein when they bind to their allosteric sites. Copyright © 2015. Published by Elsevier Ltd.

  14. A key agonist-induced conformational change in the cannabinoid receptor CB1 is blocked by the allosteric ligand Org 27569.

    Science.gov (United States)

    Fay, Jonathan F; Farrens, David L

    2012-09-28

    Allosteric ligands that modulate how G protein-coupled receptors respond to traditional orthosteric drugs are an exciting and rapidly expanding field of pharmacology. An allosteric ligand for the cannabinoid receptor CB1, Org 27569, exhibits an intriguing effect; it increases agonist binding, yet blocks agonist-induced CB1 signaling. Here we explored the mechanism behind this behavior, using a site-directed fluorescence labeling approach. Our results show that Org 27569 blocks conformational changes in CB1 that accompany G protein binding and/or activation, and thus inhibit formation of a fully active CB1 structure. The underlying mechanism behind this behavior is that simultaneous binding of Org 27569 produces a unique agonist-bound conformation, one that may resemble an intermediate structure formed on the pathway to full receptor activation.

  15. Positive versus negative modulation of different endogenous chemokines for CC-chemokine receptor 1 by small molecule agonists through allosteric versus orthosteric binding

    DEFF Research Database (Denmark)

    Jensen, Pia C; Thiele, Stefanie; Ulven, Trond

    2008-01-01

    7 transmembrane-spanning (7TM) chemokine receptors having multiple endogenous ligands offer special opportunities to understand the molecular basis for allosteric mechanisms. Thus, CC-chemokine receptor 1 (CCR1) binds CC-chemokine 3 and 5 (CCL3 and CCL5) with K(d) values of 7.3 and 0.16 nm......5 and not CCL3 activation is affected by substitutions in the main ligand binding pocket including the conserved GluVII:06 anchor point. A series of metal ion chelator complexes were found to act as full agonists on CCR1 and to be critically affected by the same substitutions in the main ligand...... binding pocket as CCL5 but not by mutations in the extracellular domain. In agreement with the overlapping binding sites, the small non-peptide agonists displaced radiolabeled CCL5 with high affinity. Interestingly, the same compounds acted as allosteric enhancers of the binding of CCL3, with which...

  16. A New Negative Allosteric Modulator AP14145 for the Study of Small Conductance Calcium-Activated Potassium Channels

    DEFF Research Database (Denmark)

    Simo Vicens, Rafel; Kirchhoff, Jeppe Egedal; Dolce, Bernardo

    2017-01-01

    ) prolongation in anaesthetised rats and a beam walk test was performed in mice to determine acute CNS related effects of the drug. Key results: AP14145 was found to be an equipotent negative allosteric modulator of KCa2.2 and KCa2.3 channels (IC50 = 1.1 ± 0.3 μM L-1). The presence of AP14145 (10 μM L-1......) increased the EC50 of Ca2+ on KCa2.3 from 0.36 ± 0.02 μM L-1 to 1.2 ± 0.1 μM L-1. The inhibitory effect strongly depended on two amino acids, S508 and A533. AP14145 concentration-dependently prolonged AERP in rats. Moreover, AP14145 (10 mg kg-1) did not trigger any apparent CNS effects in mice. Conclusion...... and implications: AP14145 is a negative allosteric modulator of KCa2.2 and KCa2.3 that shifts the calcium dependence of channel activation, an effect strongly dependent on two identified amino acids. AP14145 prolongs AERP in rats and does not trigger any acute CNS effects in mice. The understanding of how KCa2...

  17. Parallel sites implicate functional convergence of the hearing gene prestin among echolocating mammals.

    Science.gov (United States)

    Liu, Zhen; Qi, Fei-Yan; Zhou, Xin; Ren, Hai-Qing; Shi, Peng

    2014-09-01

    Echolocation is a sensory system whereby certain mammals navigate and forage using sound waves, usually in environments where visibility is limited. Curiously, echolocation has evolved independently in bats and whales, which occupy entirely different environments. Based on this phenotypic convergence, recent studies identified several echolocation-related genes with parallel sites at the protein sequence level among different echolocating mammals, and among these, prestin seems the most promising. Although previous studies analyzed the evolutionary mechanism of prestin, the functional roles of the parallel sites in the evolution of mammalian echolocation are not clear. By functional assays, we show that a key parameter of prestin function, 1/α, is increased in all echolocating mammals and that the N7T parallel substitution accounted for this functional convergence. Moreover, another parameter, V1/2, was shifted toward the depolarization direction in a toothed whale, the bottlenose dolphin (Tursiops truncatus) and a constant-frequency (CF) bat, the Stoliczka's trident bat (Aselliscus stoliczkanus). The parallel site of I384T between toothed whales and CF bats was responsible for this functional convergence. Furthermore, the two parameters (1/α and V1/2) were correlated with mammalian high-frequency hearing, suggesting that the convergent changes of the prestin function in echolocating mammals may play important roles in mammalian echolocation. To our knowledge, these findings present the functional patterns of echolocation-related genes in echolocating mammals for the first time and rigorously demonstrate adaptive parallel evolution at the protein sequence level, paving the way to insights into the molecular mechanism underlying mammalian echolocation. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Groundwater geochemistry near the storage sites of low-level radioactive waste: Implications for uranium migration

    Energy Technology Data Exchange (ETDEWEB)

    Gaskova, Olga L.; Boguslavsky, Anatoly E. [Institute of Geology and Mineralogy SB RAS, Ac. Koptyug prosp. 3, Novosibirsk 630090 (Russian Federation)

    2013-07-01

    This paper presents results of detailed sampling of groundwater and surface water near the storage sites of radioactive waste from the Electrochemical Plant ECP (Zelenogorsk, Krasnoyarsk region, Russia) and the Angarsk Electrolysis Chemical Complex AEC (Angarsk, Irkutsk region, Russia), both of which have produced enriched uranium since 1960's. The liquid (LRW) and solid (SRW) radioactive wastes belong to the category of low-level activity waste. The main result is that the uranium is below the recommended MPC for drinking waters in all types of groundwater around the sludge of ECP and AEC. But alkaline nitrate solutions have been penetrating and spreading into the aquifers under the LRW sludge pits. According to our calculations, redox conditions in the groundwater influenced by discharge are controlled by the couple NO{sub 3}{sup -}/NO{sub 2}{sup -} that facilitates U(VI) migration. The groundwater under SRW repositories is distinguished by its low mineralization and neutral pH. Co-contaminants, such as Mo, V, and Zr may serve as markers of techno-genous contamination in storage sites of the LRW sludge. (authors)

  19. Dynamic temperature fields under Mars landing sites and implications for supporting microbial life.

    Science.gov (United States)

    Ulrich, Richard; Kral, Tim; Chevrier, Vincent; Pilgrim, Robert; Roe, Larry

    2010-01-01

    While average temperatures on Mars may be too low to support terrestrial life-forms or aqueous liquids, diurnal peak temperatures over most of the planet can be high enough to provide for both, down to a few centimeters beneath the surface for some fraction of the time. A thermal model was applied to the Viking 1, Viking 2, Pathfinder, Spirit, and Opportunity landing sites to demonstrate the dynamic temperature fields under the surface at these well-characterized locations. A benchmark temperature of 253 K was used as a lower limit for possible metabolic activity, which corresponds to the minimum found for specific terrestrial microorganisms. Aqueous solutions of salts known to exist on Mars can provide liquid solutions well below this temperature. Thermal modeling has shown that 253 K is reached beneath the surface at diurnal peak heating for at least some parts of the year at each of these landing sites. Within 40 degrees of the equator, 253 K beneath the surface should occur for at least some fraction of the year; and, within 20 degrees , it will be seen for most of the year. However, any life-form that requires this temperature to thrive must also endure daily excursions to far colder temperatures as well as periods of the year where 253 K is never reached at all.

  20. The condensed chromatin fiber: an allosteric chemo-mechanical machine for signal transduction and genome processing

    International Nuclear Information System (INIS)

    Lesne, Annick; Victor, Jean–Marc; Bécavin, Christophe

    2012-01-01

    Allostery is a key concept of molecular biology which refers to the control of an enzyme activity by an effector molecule binding the enzyme at another site rather than the active site (allos = other in Greek). We revisit here allostery in the context of chromatin and argue that allosteric principles underlie and explain the functional architecture required for spacetime coordination of gene expression at all scales from DNA to the whole chromosome. We further suggest that this functional architecture is provided by the chromatin fiber itself. The structural, mechanical and topological features of the chromatin fiber endow chromosomes with a tunable signal transduction from specific (or nonspecific) effectors to specific (or nonspecific) active sites. Mechanical constraints can travel along the fiber all the better since the fiber is more compact and regular, which speaks in favor of the actual existence of the (so-called 30 nm) chromatin fiber. Chromatin fiber allostery reconciles both the physical and biochemical approaches of chromatin. We illustrate this view with two supporting specific examples. Moreover, from a methodological point of view, we suggest that the notion of chromatin fiber allostery is particularly relevant for systemic approaches. Finally we discuss the evolutionary power of allostery in the context of chromatin and its relation to modularity. (perspective)

  1. The condensed chromatin fiber: an allosteric chemo-mechanical machine for signal transduction and genome processing

    Science.gov (United States)

    Lesne, Annick; Bécavin, Christophe; Victor, Jean–Marc

    2012-02-01

    Allostery is a key concept of molecular biology which refers to the control of an enzyme activity by an effector molecule binding the enzyme at another site rather than the active site (allos = other in Greek). We revisit here allostery in the context of chromatin and argue that allosteric principles underlie and explain the functional architecture required for spacetime coordination of gene expression at all scales from DNA to the whole chromosome. We further suggest that this functional architecture is provided by the chromatin fiber itself. The structural, mechanical and topological features of the chromatin fiber endow chromosomes with a tunable signal transduction from specific (or nonspecific) effectors to specific (or nonspecific) active sites. Mechanical constraints can travel along the fiber all the better since the fiber is more compact and regular, which speaks in favor of the actual existence of the (so-called 30 nm) chromatin fiber. Chromatin fiber allostery reconciles both the physical and biochemical approaches of chromatin. We illustrate this view with two supporting specific examples. Moreover, from a methodological point of view, we suggest that the notion of chromatin fiber allostery is particularly relevant for systemic approaches. Finally we discuss the evolutionary power of allostery in the context of chromatin and its relation to modularity.

  2. Allosteric behavior in the activation of transducin mediated by rhodopsin

    International Nuclear Information System (INIS)

    Wessling-Resnick, M.; Johnson, G.I.

    1986-01-01

    Transducin is a member of the family of regulatory GTP-binding proteins which provide a signal transduction mechanism for many cell surface receptors. These receptors act in a catalytic manner to displace GDP bound to the G protein in exchange for GTP during a process referred to as activation. The authors have studied the steady-state kinetics of the activation of transducin mediated by rhodopsin by employing the non-hydrolyzable GTP analog, [ 35 S]-GTPγS. The substrate-velocity curves display remarkable allosteric behavior with a Hill coefficient, n/sub H/ = 2. Lineweaver-Burke plots with respect to reciprocal [transducin] show curvilinearity indicative of positive cooperativity. However, a series of parallel lines are generated by plotting the linear transformation as [transducin] -2 . The double reciprocal plots with respect to [GTPγS] are a series of parallel lines. The initial rate analysis supports a double displacement catalytic mechanism for the molecular interactions between the photon receptor, G protein, and guanine nucleotides. It remains to be determined whether the positive cooperative behavior the authors observe can be assigned to the interaction of multiple transducins with rhodopsin, the presence of an allosteric effector, or hysteresis in the receptor's activity. These unique observations also provide insight into the molecular interactions of members of the family of G protein-coupled receptors

  3. Implication of Crystal Water Molecules in Inhibitor Binding at ALR2 Active Site

    Directory of Open Access Journals (Sweden)

    Hymavati

    2012-01-01

    Full Text Available Water molecules play a crucial role in mediating the interaction between a ligand and a macromolecule. The solvent environment around such biomolecule controls their structure and plays important role in protein-ligand interactions. An understanding of the nature and role of these water molecules in the active site of a protein could greatly increase the efficiency of rational drug design approaches. We have performed the comparative crystal structure analysis of aldose reductase to understand the role of crystal water in protein-ligand interaction. Molecular dynamics simulation has shown the versatile nature of water molecules in bridge H bonding during interaction. Occupancy and life time of water molecules depend on the type of cocrystallized ligand present in the structure. The information may be useful in rational approach to customize the ligand, and thereby longer occupancy and life time for bridge H-bonding.

  4. Radionuclide dynamics and health implications for the New York nuclear service center's radioactive waste burial site

    International Nuclear Information System (INIS)

    Matuszek, J.M.; Strnisa, F.V.; Baxter, C.F.

    1976-01-01

    A commercial radioactive waste burial site has operated since 1963 at the Western New York Nuclear Service Center. Solid low-level radioactive wastes are buried in trenches excavated from a very fine-grained heterogeneous mixture of silt and clay (silty till) and are then covered with the excavated material. Despite many operational precautions, water levels in three burial trenches rose to within a few centimeters of the covering material by late 1973. Activity levels of HTO, 90 Sr, and 137 Cs in trench water and core samples were measured to obtain preliminary information on the degree of subsurface radionuclide migration from the burial trenches into the surrounding soil. Tritium concentrations measured in void-space water from vertical cores appeared to peak in the cover material 1.5 to 2m below the ground surface. Concentrations of 90 Sr and 137 Cs in the silty till were greatest near the surface of the cover material. Concentrations of HTO and 90 Sr, measured in a series of slant-hole core samples collected until the trench was intercepted, showed tritium migration to have progressed less than 0.3m, while 90 Sr migration appeared to be somewhat less. The preliminary data suggest that: (a) radionuclide migration from the burial trenches into the undisturbed silty till is slight; (b) radioactivity in the surface soil is not necessarily caused by migration of trench water; (c) groundwater movement is not massive; (d) rainwater infiltration, with settlement and compaction of buried wastes, is the most likely cause of rising trench water levels; and (e) surface contamination may occur from spills during burial operations, from trench digging, and from deposition of stack effluents from a nearby nuclear fuel reprocessing plant. By January 1975 the steadily rising water levels in three trenches were approximately 1m above the undisturbed soil from which the trenches were excavated, resulting in increased radioactivity levels in local streams draining the site. To

  5. Behind the curtain: cellular mechanisms for allosteric modulation of calcium-sensing receptors

    Science.gov (United States)

    Cavanaugh, Alice; Huang, Ying; Breitwieser, Gerda E

    2012-01-01

    Calcium-sensing receptors (CaSR) are integral to regulation of systemic Ca2+ homeostasis. Altered expression levels or mutations in CaSR cause Ca2+ handling diseases. CaSR is regulated by both endogenous allosteric modulators and allosteric drugs, including the first Food and Drug Administration-approved allosteric agonist, Cinacalcet HCl (Sensipar®). Recent studies suggest that allosteric modulators not only alter function of plasma membrane-localized CaSR, but regulate CaSR stability at the endoplasmic reticulum. This brief review summarizes our current understanding of the role of membrane-permeant allosteric agonists in cotranslational stabilization of CaSR, and highlights additional, indirect, signalling-dependent role(s) for membrane-impermeant allosteric drugs. Overall, these studies suggest that allosteric drugs act at multiple cellular organelles to control receptor abundance and hence function, and that drug hydrophobicity can bias the relative contributions of plasma membrane and intracellular organelles to CaSR abundance and signalling. LINKED ARTICLES This article is part of a themed section on the Molecular Pharmacology of G Protein-Coupled Receptors (GPCRs). To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-6. To view the 2010 themed section on the same topic visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2010.159.issue-5/issuetoc PMID:21470201

  6. Ichthyoplankton entrainment at Wylfa power station, Anglesey and implications for a further siting proposal

    International Nuclear Information System (INIS)

    Dempsey, C.H.; Rogers, S.I.

    1989-07-01

    A 12 month survey of ichthyoplankton in the cooling water system of Wylfa Power Station and the surrounding 40 km 2 of sea, was carried out between October 1986 and September 1987. The larvae of 31 species and the eggs of 8 species were identified in the survey. Samples taken from the cooling water system and by boat from offshore were largely similar in respect of species diversity and density. Estimates of annual losses due to entrainment are given both in terms of immediate losses and consequential losses of adults to the population. Estimates of losses of six commercially exploited species are considered in terms of loss to the commercial fishery. Assuming the 'worst case' of a 100% mortality of eggs and larvae passing through the cooling system, losses of ichthyoplankton due to entrainment at the existing 'magnox' nuclear power station at Wylfa Point are small and could have no significant adverse effect on fish populations of those species entrained. The operation of the proposed 'pressurised water reactor' nuclear power station on the same site would increase losses by up to 100%. Such an increase would still not alter the existing situation. No significant adverse effect is likely. (author)

  7. Rodent burrows in late Pleistocene paleosols at Korean Palaeolithic sites and their implications for paleoclimate changes

    Science.gov (United States)

    Lim, H.; Park, S.; Lee, J.; Lee, Y.

    2013-12-01

    Rodent burrows are commonly found at many Paleolithic archaeological sites in Korea. They are nearly straight in horizontal view and gently inclined in lateral view. Burrow diameters are mostly 7 - 10cm, and burrow length may reach a few meters. Vertical penetration depths are generally about 1 m from the surface, and the thickness of the burrow-bearing layer is about 1-2 m. Although no remains (bones, teeth, claws, and coprolites) were found within burrows, they are interpreted to have been produced by rodent-like mammals (probably ground squirrels) based on the size and architecture. According to the previous study, the age of these burrows was constrained to be between ca. 40,000 and 25,000 yr BP by tephrochronology, radiocarbon and optically stimulated luminescence dating results (Lim et al., 2007). However, little is known about the reason why these burrows have disappeared after late Pleistocene time. For this question, two explanations can be considered: extinction or migration. Since same kinds of burrows are still found in the high-latitude regions, such as Mongolia and North America, the possibility of extinction can be ruled out. Therefore, migration seems to be the most likely explanation. Our results show that the destruction of habitat caused by climate change during this period is the main reason for the northward migration of burrowing animals. This study suggests that rodent burrows found in the late Pleistocene paleosols can provide useful information on paleoclimate and paleoenvironmental changes.

  8. A terrain-based site characterization map of California with implications for the contiguous United States

    Science.gov (United States)

    Yong, Alan K.; Hough, Susan E.; Iwahashi, Junko; Braverman, Amy

    2012-01-01

    We present an approach based on geomorphometry to predict material properties and characterize site conditions using the VS30 parameter (time‐averaged shear‐wave velocity to a depth of 30 m). Our framework consists of an automated terrain classification scheme based on taxonomic criteria (slope gradient, local convexity, and surface texture) that systematically identifies 16 terrain types from 1‐km spatial resolution (30 arcsec) Shuttle Radar Topography Mission digital elevation models (SRTM DEMs). Using 853 VS30 values from California, we apply a simulation‐based statistical method to determine the mean VS30 for each terrain type in California. We then compare the VS30 values with models based on individual proxies, such as mapped surface geology and topographic slope, and show that our systematic terrain‐based approach consistently performs better than semiempirical estimates based on individual proxies. To further evaluate our model, we apply our California‐based estimates to terrains of the contiguous United States. Comparisons of our estimates with 325 VS30 measurements outside of California, as well as estimates based on the topographic slope model, indicate our method to be statistically robust and more accurate. Our approach thus provides an objective and robust method for extending estimates of VS30 for regions where in situ measurements are sparse or not readily available.

  9. Paleoclimatic implications from fluid inclusion data in Messinian halite of Italian sites

    Science.gov (United States)

    Speranza, G.; Tecce, F.; Cosentino, D.; Faccenna, C.

    2012-12-01

    The Neogene sedimentary succession of the Mediterranean Basin includes a thick evaporitic succession (gypsum and halite) deposited during the Messinian Salinity Crisis (MSC), which occurred between 5.96 and 5,33 Ma. While several studies have been carried out to define the water budget of the MSC, the temperature of the Mediterranean water system is poorly constrained. The purpose of this work is to collect the first dataset of homogenization temperatures (Th) from primary fluid inclusions in Messinian halite from different Italian sites. Such data yield very useful information on water temperature at salt deposition time and thus on the climatic conditions in the peak desiccation stage of the Mediterranean sea. We focused our attention on three areas: the Volterra Basin (Tuscany), the Crotone Basin (Calabria) and the Caltanissetta Basin (Sicily). These basins are filled by Neogene sedimentary sequences, including Messinian gypsum deposits and halite. Halite samples were taken directly from salt diapirs outcrops (Crotone Basin), from borehole S1113 cores drilled by the Italian Solvay company (Volterra Basin) and inside salt mines of Petralia Sottana, Racalmuto and Realmonte (Caltanissetta Basin). Halite chips were manually prepared carefully avoiding water and controlling the temperature. Halite minerals contain abundant fluid inclusions. The majority of them are monophase liquid inclusions, showing a very regular cubic or rectangular shape. They occur along chevron and growth planes and thus were considered to have a primary origin. Some others contain solids and/or organic matter. During microthermometry, vapor bubbles nucleation has been produced directly into the stage chamber, slightly modifying the traditional "cooling" method; we could then nucleate the bubbles and at the same time constantly control the sample temperature, avoiding any sudden change that can lead to useless altered data. Microthermometric data were measured from 218 primary all liquid fluid

  10. Dust devil track survey at Elysium Planitia, Mars: Implications for the InSight landing sites

    Science.gov (United States)

    Reiss, Dennis; Lorenz, Ralph D.

    2016-03-01

    The InSight (Interior Exploration using Seismic Investigations, Geodesy and Heat Transport) robotic lander is scheduled to land in Elysium Planitia on Mars in September 2016. InSight will perform the first comprehensive surface-based geophysical investigation including seismic measurements. Knowledge about encounter rates of dust devils with the InSight lander are important for two main reasons: (1) dust devils will affect the scientific measurements, i.e., wind-induced seismic noise, and (2) the power-supply of the InSight lander and instruments is provided by solar arrays and previous landers and rovers on Mars were affected by a steady decline in electrical power output due to atmospheric dust deposition on the solar panels. Long term science operations were only made possible by dust clearing events of the solar arrays caused by wind gusts and dust devils. In this study we analyzed dust devil tracks (DDTs) at the final InSight landing site region in Elysium Planitia. Formation of DDTs is caused by the removal of a layer of dust by passing dust devils, hence in principle the same process as clearing of dust from solar panels. We mapped the number, size (width and length), and orientation of DDTs in repeat observations using High Resolution Imaging Science Experiment (HiRISE) images covering the exact same surface area acquired within a relatively short time span (solar panel clearing recurrence interval estimate of ∼11 Mars years using the mean annual DDT formation rate, and the mean DDT width and length from all measured DDTs. Due to several uncertainties this solar panel clearing recurrence interval for the InSight landing should be seen as an upper limit estimate.

  11. Lacustrine sedimentation in an altitude forest site, central Andes, Bolivia. Palaeo-climatic implications

    International Nuclear Information System (INIS)

    Sifeddine, A.; Bertaux, J.; Mourguiart, Ph.; Disnar, J.R.; Laggoun-Defarge, F.; Argollo, J.

    1998-01-01

    A sedimentological study of a 755 cm length core sampled in the middle of a marshy depression surrounded by a cloud forest in the central Andes reveals that this site has recorded important environmental variations during the last 50 000 years. For the most part (625 cm) the core is composed of detrital rich sediments deposited during the Upper Pleistocene. The highest amount of detrital influx underlines the Last Glacial Maximum which ranges from ca 29,000 14 C yr B.P. to ca 16,000 14 C yr B.P. (ca 18,500 cal yr B.P.), between two relatively humid phases. The sedimentation of the present Interglacial, starting at ca 12,500 14 C yr B.P. (14,500 cal yr B.P.), is mainly organic, as a consequence of the great development of soils and the forest vegetal cover the catchment area. The maximum extension of this vegetal cover ranging from 12,500 to ca 10,500 14 C yr B.P. (14,500 and 12,400 cal yr B.P.) is followed from 10,500 to 8,000 14 C yr B.P. (12,400 and 8,800 cal yr B.P.) by a drier period is revealed by the occurrence of micro-charcoals in the sediment. Between ca 8,000 and 4,000 14 C yr B.P. (8,800 and 4,500 cal yr B.P.), the sharp increase of micro-charcoals content, likely related to palaeo-fires, underlines an intensification of this dry trend. (authors)

  12. Nest-site competition between invasive and native cavity nesting birds and its implication for conservation.

    Science.gov (United States)

    Charter, Motti; Izhaki, Ido; Ben Mocha, Yitzchak; Kark, Salit

    2016-10-01

    Nesting cavities are often a limited resource that multiple species use. There is an ongoing discussion on whether invasive cavity nesting birds restrict the availability of this key limited resource. While the answer to this question has important conservation implications, little experimental work has been done to examine it. Here, we aimed to experimentally test whether alien cavity nesting birds affect the occupancy of cavities and the resulting breeding success of native cavity breeders in a large urban park located in Tel Aviv, Israel. Over three breeding seasons, we manipulated the entry size of nest boxes and compared the occupancy and breeding success of birds in nest boxes of two treatments. These included nest boxes with large-entrance and small-entrance holes. The large-entrance holes allowed access for both the native and invasive birds (the two main aliens in the park are the common mynas and rose-ringed parakeets). The smaller-entrance boxes, on the other hand, allowed only the smaller sized native cavity breeders (great tits and house sparrows) to enter the boxes but prevented the alien species from entering. We found that the large-entrance nest boxes were occupied by five different bird species, comprising three natives (great tit, house sparrow, Scops owl) and two invasive species (common myna, rose-ringed parakeet) while the small-entrance boxes were only occupied by the two native species. The alien common mynas and rose-ringed parakeets occupied 77.5% of the large-entrance nest boxes whereas native species, mainly great tits, occupied less than 9% of the large-entrance boxes and 36.5% of the small-entrance boxes. When examining the occupancy of those cavities that were not occupied by the aliens, natives occupied both the small and large-entrance nest boxes equally. Three quarters (78%) of the great tits breeding in the large-entrance boxes were usurped by common mynas during the breeding season and as a result breeding success was

  13. Structural basis for the cooperative allosteric activation of the free fatty acid receptor GPR40

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Jun; Byrne, Noel; Wang, John; Bricogne, Gerard; Brown, Frank K.; Chobanian, Harry R.; Colletti, Steven L.; Di Salvo, Jerry; Thomas-Fowlkes, Brande; Guo, Yan; Hall, Dawn L.; Hadix, Jennifer; Hastings, Nicholas B.; Hermes, Jeffrey D.; Ho, Thu; Howard, Andrew D.; Josien, Hubert; Kornienko, Maria; Lumb, Kevin J.; Miller, Michael W.; Patel, Sangita B.; Pio, Barbara; Plummer, Christopher W.; Sherborne, Bradley S.; Sheth, Payal; Souza, Sarah; Tummala, Srivanya; Vonrhein, Clemens; Webb, Maria; Allen, Samantha J.; Johnston, Jennifer M.; Weinglass, Adam B.; Sharma, Sujata; Soisson, Stephen M. (Merck); (Globel Phasing)

    2017-06-05

    Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40–MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.

  14. Allosteric inhibition enhances the efficacy of ABL kinase inhibitors to target unmutated BCR-ABL and BCR-ABL-T315I

    Directory of Open Access Journals (Sweden)

    Mian Afsar

    2012-09-01

    Full Text Available Abstract Background Chronic myelogenous leukemia (CML and Philadelphia chromosome-positive (Ph+ acute lymphatic leukemia (Ph + ALL are caused by the t(9;22, which fuses BCR to ABL resulting in deregulated ABL-tyrosine kinase activity. The constitutively activated BCR/ABL-kinase “escapes” the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. The ABL-kinase inhibitors (AKIs Imatinib, Nilotinib or Dasatinib, which target the ATP-binding site, are effective in Ph + leukemia. Another molecular therapy approach targeting BCR/ABL restores allosteric inhibition. Given the fact that all AKIs fail to inhibit BCR/ABL harboring the ‘gatekeeper’ mutation T315I, we investigated the effects of AKIs in combination with the allosteric inhibitor GNF2 in Ph + leukemia. Methods The efficacy of this approach on the leukemogenic potential of BCR/ABL was studied in Ba/F3 cells, primary murine bone marrow cells, and untransformed Rat-1 fibroblasts expressing BCR/ABL or BCR/ABL-T315I as well as in patient-derived long-term cultures (PDLTC from Ph + ALL-patients. Results Here, we show that GNF-2 increased the effects of AKIs on unmutated BCR/ABL. Interestingly, the combination of Dasatinib and GNF-2 overcame resistance of BCR/ABL-T315I in all models used in a synergistic manner. Conclusions Our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants using a combination of AKIs and allosteric inhibitors.

  15. Evidence for allosterism in ribulose-1,5-bisphosphate carboxylase/oxygenase from comfrey

    International Nuclear Information System (INIS)

    Mueller, D.D.; Bolden, T.D.

    1986-01-01

    Evidence has been obtained suggesting that ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is an allosteric enzyme in the sense that it shows cooperative active site binding, cooperative interactions between the activation and active sites and significant binding of some metabolites at a second site. Investigation of the binding of a potent competitive inhibitor. 2-carboxymannitol-1,6-bisphosphate (CMBP) by 31 P-NMR indicated essentially 1:1 binding with the active sites of comfrey RuBisCo. Among the interactions of competitive inhibitors, as measured by difference UV spectroscopy, the binding curves for ortho-phosphate and ribose-5-phosphate were better fitted by a Monod-Wyman-Changeux model than by an independent site model, whereas the binding of CMBP and 2-phosphoglycolate were not. Difference UV methods also were used to study activation by CO 2 which at pH 7.9 in 10 mM MgCl 2 showed positive cooperativity with k = 100 +/- 3 μM (based on pK/sub a/ = 6.4 for the CO 2 -HCO 3 - equilibrium) and L = 3.5 +/- 0.7. Addition of saturating amounts of CMBP and lowering the MgCl 2 to 2 mM still gave a sigmoidal curve but it was shifted to higher CO 2 concentrations (k = 124 +/- 2 μM and L = 31 +/- 3). In the absence of CMBP the same conditions gave k = 26 +/- 2 μM for L = 3.5. Conversely, k was 0.96 +/- 0.08 μM for CMBP in 0.5 mM MgCl 2 without added NaHCO 3 but was 21 +/- 0.06 μM in 10 MgCl 2 and 2 mM NaHCO 3 , pH 7.3

  16. Ras activation by SOS: Allosteric regulation by altered fluctuation dynamics

    Science.gov (United States)

    Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen; Christensen, Sune M.; Abel, Steven M.; Iwig, Jeff; Wu, Hung-Jen; Gureasko, Jodi; Rhodes, Christopher; Petit, Rebecca S.; Hansen, Scott D.; Thill, Peter; Yu, Cheng-Han; Stamou, Dimitrios; Chakraborty, Arup K.; Kuriyan, John; Groves, Jay T.

    2014-01-01

    Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras–guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average. PMID:24994643

  17. Enhancing NMDA Receptor Function: Recent Progress on Allosteric Modulators

    Directory of Open Access Journals (Sweden)

    Lulu Yao

    2017-01-01

    Full Text Available The N-methyl-D-aspartate receptors (NMDARs are subtype glutamate receptors that play important roles in excitatory neurotransmission and synaptic plasticity. Their hypo- or hyperactivation are proposed to contribute to the genesis or progression of various brain diseases, including stroke, schizophrenia, depression, and Alzheimer’s disease. Past efforts in targeting NMDARs for therapeutic intervention have largely been on inhibitors of NMDARs. In light of the discovery of NMDAR hypofunction in psychiatric disorders and perhaps Alzheimer’s disease, efforts in boosting NMDAR activity/functions have surged in recent years. In this review, we will focus on enhancing NMDAR functions, especially on the recent progress in the generation of subunit-selective, allosteric positive modulators (PAMs of NMDARs. We shall also discuss the usefulness of these newly developed NMDAR-PAMs.

  18. Chemical conditions in present and future ecosystems in Forsmark - implications for selected radionuclides in the safety assessment SR-Site

    International Nuclear Information System (INIS)

    Troejbom, Mats; Grolander, Sara

    2010-12-01

    This report is a background report for the biosphere analysis of the SR-Site Safety Assessment. This work aims to describe the future development of the chemical conditions at Forsmark, based on the present chemical conditions at landscape level taking landscape development and climate cases into consideration. The results presented contribute to the overall understanding of the present and future chemistry in the Forsmark area, and specifically, to the understanding of the behaviour of some selected radionuclides in the surface system. The future development of the chemistry at the site is qualitatively discussed with focus on the interglacial within the next 10,000 years. The effects on the chemical environment of future climate cases as Global Warming and cold permafrost climates are also briefly discussed. The work is presented in two independent parts describing background radionuclide activities in the Forsmark area and the distribution and behaviour of a large number of stable elements in the landscape. In a concluding section, implications of the future chemical environment of a selection of radionuclides important in the Safety Assessment are discussed based on the knowledge of stable elements. The broad range of elements studied show that there are general and expected patterns for the distribution and behaviour in the landscape of different groups of elements. Mass balances reveal major sources and sinks, pool estimations show where elements are accumulated in the landscape and estimations of time-scales give indications of the potential future development. This general knowledge is transferred to radionuclides not measured in order to estimate their behaviour and distribution in the landscape. It could be concluded that the future development of the chemical environment in the Forsmark area might affect element specific parameters used in de radionuclide model in different directions depending on element. The alternative climate cases, Global Warming

  19. Chemical conditions in present and future ecosystems in Forsmark - implications for selected radionuclides in the safety assessment SR-Site

    Energy Technology Data Exchange (ETDEWEB)

    Troejbom, Mats (Mats Troejbom Konsult AB (Sweden)); Grolander, Sara (Facilia AB (Sweden))

    2010-12-15

    This report is a background report for the biosphere analysis of the SR-Site Safety Assessment. This work aims to describe the future development of the chemical conditions at Forsmark, based on the present chemical conditions at landscape level taking landscape development and climate cases into consideration. The results presented contribute to the overall understanding of the present and future chemistry in the Forsmark area, and specifically, to the understanding of the behaviour of some selected radionuclides in the surface system. The future development of the chemistry at the site is qualitatively discussed with focus on the interglacial within the next 10,000 years. The effects on the chemical environment of future climate cases as Global Warming and cold permafrost climates are also briefly discussed. The work is presented in two independent parts describing background radionuclide activities in the Forsmark area and the distribution and behaviour of a large number of stable elements in the landscape. In a concluding section, implications of the future chemical environment of a selection of radionuclides important in the Safety Assessment are discussed based on the knowledge of stable elements. The broad range of elements studied show that there are general and expected patterns for the distribution and behaviour in the landscape of different groups of elements. Mass balances reveal major sources and sinks, pool estimations show where elements are accumulated in the landscape and estimations of time-scales give indications of the potential future development. This general knowledge is transferred to radionuclides not measured in order to estimate their behaviour and distribution in the landscape. It could be concluded that the future development of the chemical environment in the Forsmark area might affect element specific parameters used in de radionuclide model in different directions depending on element. The alternative climate cases, Global Warming

  20. Zinc-mediated Allosteric Inhibition of Caspase-6*

    Science.gov (United States)

    Velázquez-Delgado, Elih M.; Hardy, Jeanne A.

    2012-01-01

    Zinc and caspase-6 have independently been implicated in several neurodegenerative disorders. Depletion of zinc intracellularly leads to apoptosis by an unknown mechanism. Zinc inhibits cysteine proteases, including the apoptotic caspases, leading to the hypothesis that zinc-mediated inhibition of caspase-6 might contribute to its regulation in a neurodegenerative context. Using inductively coupled plasma optical emission spectroscopy, we observed that caspase-6 binds one zinc per monomer, under the same conditions where the zinc leads to complete loss of enzymatic activity. To understand the molecular details of zinc binding and inhibition, we performed an anomalous diffraction experiment above the zinc edge. The anomalous difference maps showed strong 5σ peaks, indicating the presence of one zinc/monomer bound at an exosite distal from the active site. Zinc was not observed bound to the active site. The zinc in the exosite was liganded by Lys-36, Glu-244, and His-287 with a water molecule serving as the fourth ligand, forming a distorted tetrahedral ligation sphere. This exosite appears to be unique to caspase-6, as the residues involved in zinc binding were not conserved across the caspase family. Our data suggest that binding of zinc at the exosite is the primary route of inhibition, potentially locking caspase-6 into the inactive helical conformation. PMID:22891250

  1. Endogenous vs Exogenous Allosteric Modulators in GPCRs: A dispute for shuttling CB1 among different membrane microenvironments

    Science.gov (United States)

    Stornaiuolo, Mariano; Bruno, Agostino; Botta, Lorenzo; Regina, Giuseppe La; Cosconati, Sandro; Silvestri, Romano; Marinelli, Luciana; Novellino, Ettore

    2015-10-01

    A Cannabinoid Receptor 1 (CB1) binding site for the selective allosteric modulator ORG27569 is here identified through an integrate approach of consensus pocket prediction, mutagenesis studies and Mass Spectrometry. This unprecedented ORG27569 pocket presents the structural features of a Cholesterol Consensus Motif, a cholesterol interacting region already found in other GPCRs. ORG27569 and cholesterol affects oppositely CB1 affinity for orthosteric ligands. Moreover, the rise in cholesterol intracellular level results in CB1 trafficking to the axonal region of neuronal cells, while, on the contrary, ORG27568 binding induces CB1 enrichment at the soma. This control of receptor migration among functionally different membrane regions of the cell further contributes to downstream signalling and adds a previously unknown mechanism underpinning CB1 modulation by ORG27569 , that goes beyond a mere control of receptor affinity for orthosteric ligands.

  2. Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-chemokine Receptor 5 (CCR5)

    DEFF Research Database (Denmark)

    Karlshøj, Stefanie; Amarandi, Roxana Maria; Larsen, Olav

    2016-01-01

    The small molecule metal ion chelators bipyridine and terpyridine complexed with Zn(2+) (ZnBip and ZnTerp) act as CCR5 agonists and strong positive allosteric modulators of CCL3 binding to CCR5, weak modulators of CCL4 binding, and competitors for CCL5 binding. Here we describe their binding site......Terp binds deeply in the major binding pocket and, in contrast to ZnBip, interacts directly with the Trp-248(VI:13/6.48) microswitch, contributing to its 8-fold higher potency. The impact of Trp-248 was further confirmed by ZnClTerp, a chloro-substituted version of ZnTerp that showed no inherent agonism...

  3. Shift in the Equilibrium between On and Off States of the Allosteric Switch in Ras-GppNHp Affected by Small Molecules and Bulk Solvent Composition

    Energy Technology Data Exchange (ETDEWEB)

    Holzapfel, Genevieve; Buhrman, Greg; Mattos, Carla (NCSU)

    2012-08-31

    Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly(ethylene glycol) (PEG) can selectively shift the equilibrium to the 'on' state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the 'ordered off' state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-{beta}. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE.

  4. Mesozoic and Cenozoic structural geology of the CP Hills, Nevada Test Site, Nye County, Nevada; and regional implications

    Energy Technology Data Exchange (ETDEWEB)

    Caskey, S. John [Univ. of Nevada, Reno, NV (United States)

    1991-08-01

    Detailed mapping and structural analysis of upper Proterozoic and Paleozoic rocks in the CP Hills of the Nevada Test Site, together with analysis of published maps and cross sections and a reconnaissance of regional structural relations indicate that the CP thrust of Barnes and Poole (1968) actually comprises two separate, oppositely verging Mesozoic thrust systems: (1) the west-vergent CP thrust which is well exposed in the CP Hills and at Mine Mountain, and (2) the east-vergent Belted Range thrust located northwest of Yucca Flat. West-vergence of the CP thrust is indicated by large scale west-vergent recumbent folds in both its hangingwall and footwall and by the fact that the CP thrust ramps up section through hangingwall strata toward the northwest. Regional structural relations indicate that the CP thrust forms part of a narrow sigmoidal belt of west-vergent folding and thrusting traceable for over 180 km along strike. The Belted Range thrust represents earlier Mesozoic deformation that was probably related to the Last Chance thrust system in southeastern California, as suggested by earlier workers. A pre-Tertiary reconstruction of the Cordilleran fold and thrust belt in the region between the NTS and the Las Vegas Range bears a close resemblance to other regions of the Cordillera and has important implications for the development of hinterland-vergent deformation as well as for the probable magnitude of Tertiary extension north of Las Vegas Valley. Subsequent to Mesozoic deformation, the CP Hills were disrupted by at least two episodes of Tertiary extensional deformation: (1) an earlier episode represented by pre-middle Miocene low-angle normal faults, and (2) a later, post-11 Ma episode of high-angle normal faulting. Both episodes of extension were related to regional deformation, the latter of which has resulted in the present basin and range topography of the NTS region.

  5. Adolescent pornographic internet site use: a multivariate regression analysis of the predictive factors of use and psychosocial implications.

    Science.gov (United States)

    Tsitsika, Artemis; Critselis, Elena; Kormas, Georgios; Konstantoulaki, Eleftheria; Constantopoulos, Andreas; Kafetzis, Dimitrios

    2009-10-01

    The study objectives were to evaluate the prevalence, predictors, and implications of pornographic Internet site (PIS) use among Greek adolescents. A cross-sectional study was conducted among 529 randomly selected Greek high school students. The prevalence of overall PIS use was 19.47% (n = 96). Among PIS users, 55 (57.29%) reported infrequent and 41 (42.71%) reported frequent PIS use. The predictors of infrequent PIS use included male gender (adjusted odds ratio [AOR] = 8.33; 95% confidence interval [CI] = 3.52-19.61), Internet use for sexual education (AOR = 5.26; 95% CI = 1.78-15.55), chat rooms (AOR = 2.95; 95% CI = 1.48-5.91), and purchases (AOR = 3.06; 95% CI = 1.22-7.67). The predictors of frequent PIS use were male gender (AOR = 19.61; 95% CI = 4.46-83.33), Internet use for sexual education (AOR = 7.39; 95% CI = 2.37-23.00), and less than 10 hours per week Internet use (AOR = 1.32; 95% CI = 1.10-1.59). Compared to non-PIS users, infrequent PIS users were twice as likely to have abnormal conduct problems (odds ratio [OR] = 2.74; 95% CI = 1.19-6.28); frequent PIS users were significantly more likely to have abnormal conduct problems (OR = 4.05; 95% CI = 1.57-10.46) and borderline prosocial score (OR = 4.22; 95% CI = 1.64-10.85). Thus, both infrequent and frequent PIS use are prevalent and significantly associated with social maladjustment among Greek adolescents.

  6. Mesozoic and Cenozoic structural geology of the CP Hills, Nevada Test Site, Nye County, Nevada; and regional implications

    International Nuclear Information System (INIS)

    Caskey, S.J.

    1991-08-01

    Detailed mapping and structural analysis of upper Proterozoic and Paleozoic rocks in the CP Hills of the Nevada Test Site, together with analysis of published maps and cross sections and a reconnaissance of regional structural relations indicate that the CP thrust of Barnes and Poole (1968) actually comprises two separate, oppositely verging Mesozoic thrust systems: (1) the west-vergent CP thrust which is well exposed in the CP Hills and at Mine Mountain, and (2) the east-vergent Belted Range thrust located northwest of Yucca Flat. West-vergence of the CP thrust is indicated by large scale west-vergent recumbent folds in both its hangingwall and footwall and by the fact that the CP thrust ramps up section through hangingwall strata toward the northwest. Regional structural relations indicate that the CP thrust forms part of a narrow sigmoidal belt of west-vergent folding and thrusting traceable for over 180 km along strike. The Belted Range thrust represents earlier Mesozoic deformation that was probably related to the Last Chance thrust system in southeastern California, as suggested by earlier workers. A pre-Tertiary reconstruction of the Cordilleran fold and thrust belt in the region between the NTS and the Las Vegas Range bears a close resemblance to other regions of the Cordillera and has important implications for the development of hinterland-vergent deformation as well as for the probable magnitude of Tertiary extension north of Las Vegas Valley. Subsequent to Mesozoic deformation, the CP Hills were disrupted by at least two episodes of Tertiary extensional deformation: (1) an earlier episode represented by pre-middle Miocene low-angle normal faults, and (2) a later, post-11 Ma episode of high-angle normal faulting. Both episodes of extension were related to regional deformation, the latter of which has resulted in the present basin and range topography of the NTS region

  7. Structure-based network analysis of activation mechanisms in the ErbB family of receptor tyrosine kinases: the regulatory spine residues are global mediators of structural stability and allosteric interactions.

    Directory of Open Access Journals (Sweden)

    Kevin A James

    Full Text Available The ErbB protein tyrosine kinases are among the most important cell signaling families and mutation-induced modulation of their activity is associated with diverse functions in biological networks and human disease. We have combined molecular dynamics simulations of the ErbB kinases with the protein structure network modeling to characterize the reorganization of the residue interaction networks during conformational equilibrium changes in the normal and oncogenic forms. Structural stability and network analyses have identified local communities integrated around high centrality sites that correspond to the regulatory spine residues. This analysis has provided a quantitative insight to the mechanism of mutation-induced "superacceptor" activity in oncogenic EGFR dimers. We have found that kinase activation may be determined by allosteric interactions between modules of structurally stable residues that synchronize the dynamics in the nucleotide binding site and the αC-helix with the collective motions of the integrating αF-helix and the substrate binding site. The results of this study have pointed to a central role of the conserved His-Arg-Asp (HRD motif in the catalytic loop and the Asp-Phe-Gly (DFG motif as key mediators of structural stability and allosteric communications in the ErbB kinases. We have determined that residues that are indispensable for kinase regulation and catalysis often corresponded to the high centrality nodes within the protein structure network and could be distinguished by their unique network signatures. The optimal communication pathways are also controlled by these nodes and may ensure efficient allosteric signaling in the functional kinase state. Structure-based network analysis has quantified subtle effects of ATP binding on conformational dynamics and stability of the EGFR structures. Consistent with the NMR studies, we have found that nucleotide-induced modulation of the residue interaction networks is not

  8. Allosteric mechanism controls traffic in the chaperone/usher pathway.

    Science.gov (United States)

    Di Yu, Xiao; Dubnovitsky, Anatoly; Pudney, Alex F; Macintyre, Sheila; Knight, Stefan D; Zavialov, Anton V

    2012-11-07

    Many virulence organelles of Gram-negative bacterial pathogens are assembled via the chaperone/usher pathway. The chaperone transports organelle subunits across the periplasm to the outer membrane usher, where they are released and incorporated into growing fibers. Here, we elucidate the mechanism of the usher-targeting step in assembly of the Yersinia pestis F1 capsule at the atomic level. The usher interacts almost exclusively with the chaperone in the chaperone:subunit complex. In free chaperone, a pair of conserved proline residues at the beginning of the subunit-binding loop form a "proline lock" that occludes the usher-binding surface and blocks usher binding. Binding of the subunit to the chaperone rotates the proline lock away from the usher-binding surface, allowing the chaperone-subunit complex to bind to the usher. We show that the proline lock exists in other chaperone/usher systems and represents a general allosteric mechanism for selective targeting of chaperone:subunit complexes to the usher and for release and recycling of the free chaperone. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Are AMPA Receptor Positive Allosteric Modulators Potential Pharmacotherapeutics for Addiction?

    Directory of Open Access Journals (Sweden)

    Lucas R. Watterson

    2013-12-01

    Full Text Available Positive allosteric modulators (PAMs of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA receptors are a diverse class of compounds that increase fast excitatory transmission in the brain. AMPA PAMs have been shown to facilitate long-term potentiation, strengthen communication between various cortical and subcortical regions, and some of these compounds increase the production and release of brain-derived neurotrophic factor (BDNF in an activity-dependent manner. Through these mechanisms, AMPA PAMs have shown promise as broad spectrum pharmacotherapeutics in preclinical and clinical studies for various neurodegenerative and psychiatric disorders. In recent years, a small collection of preclinical animal studies has also shown that AMPA PAMs may have potential as pharmacotherapeutic adjuncts to extinction-based or cue-exposure therapies for the treatment of drug addiction. The present paper will review this preclinical literature, discuss novel data collected in our laboratory, and recommend future research directions for the possible development of AMPA PAMs as anti-addiction medications.

  10. Development of an experimental activity for teaching cooperativity and allosterism

    Directory of Open Access Journals (Sweden)

    B. Manta

    2006-07-01

    Full Text Available Although  enzyme  control  and  regulation  is  an  important  topic  in  most  Biochemistry  and  Enzymology  courses, laboratory  activities  that  allow  an  experimental  approach  to  cooperativity  and  allosterism  are  difficult  to  implement. The objective of this work was to develop a simple and inexpensive experimental activity to teach this topic in basic courses.  We  decided  to  use  the  enzyme  glucosamine-6-phosphate  deaminase  (GNPD,  E.C.  3.5.99.6  from Escherichia coli,  that  is  both  kinetically  and  structurally  well-known.  GNPD  is  an  allosteric  enzyme,  activated  by  N-acetylglucosamine 6-phosphate, that catalyzes the conversion of glucosamine 6-phosphate into fructose 6-phosphate and  ammonia.  The  enzyme  is  a  typical  allosteric  K-system  and  can  be  well  described  by  the  Monod-Wyman-Changeux  (MWC  model.  GNPD  was  partially  purified  through  anionic-exchange  chromatography  from  a  mutant E.coli strain  which  expresses  constitutively  high  levels  of the  enzyme.  In  order  to  measure  activity  we  used  an end point  method  which  consists  in  stopping  the  reaction  at  a  certain  time  point  with  HCl  10  N,  and  quantifying  the fructose-6-phosphate  formed  with  resorcinol  (Selliwanoff  reaction  through  the  formation  of  a  red  color  that  is measured  spectrophotometrically.  We  developed  a  protocol  that  consisted  in  a  4-hour  experiment  in  which  the students  measured  the  activity  of  the  GNPD  with  increasing  concentrations  of  the  substrate,  in  the  presence  or absence  of  allosteric  modulator.  The  students  obtained  a  good  quality  data  set  that  they  analyzed  based  on  the equations  of  Hill,  MWC  and  Acerenza-Mirzaji

  11. Allosteric activation of the follicle-stimulating hormone (FSH) receptor by selective, nonpeptide agonists.

    Science.gov (United States)

    Yanofsky, Stephen D; Shen, Emily S; Holden, Frank; Whitehorn, Erik; Aguilar, Barbara; Tate, Emily; Holmes, Christopher P; Scheuerman, Randall; MacLean, Derek; Wu, May M; Frail, Donald E; López, Francisco J; Winneker, Richard; Arey, Brian J; Barrett, Ronald W

    2006-05-12

    The pituitary glycoprotein hormones, luteinizing hormone and follicle-stimulating hormone (FSH), act through their cognate receptors to initiate a series of coordinated physiological events that results in germ cell maturation. Given the importance of FSH in regulating folliculogenesis and fertility, the development of FSH mimetics has been sought to treat infertility. Currently, purified and recombinant human FSH are the only FSH receptor (FSH-R) agonists available for infertility treatment. By screening unbiased combinatorial chemistry libraries, using a cAMP-responsive luciferase reporter assay, we discovered thiazolidinone agonists (EC50's = 20 microm) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC50 = 2 nm) with full efficacy compared with FSH that was FSH-R-selective and -dependent. The compound mediated progesterone production in Y1 cells transfected with the human FSH-R (EC50 = 980 nm) and estradiol production from primary rat ovarian granulosa cells (EC50 = 10.5 nm). This and related compounds did not compete with FSH for binding to the FSH-R. Use of human FSH/thyroid-stimulating hormone (TSH) receptor chimeras suggested a novel mechanism for receptor activation through a binding site independent of the natural hormone binding site. This study is the first report of a high affinity small molecule agonist that activates a glycoprotein hormone receptor through an allosteric mechanism. The small molecule FSH receptor agonists described here could lead to an oral alternative to the current parenteral FSH treatments used clinically to induce ovarian stimulation for both in vivo and in vitro fertilization therapy.

  12. Cyclophilin40 isomerase activity is regulated by a temperature-dependent allosteric interaction with Hsp90.

    Science.gov (United States)

    Blackburn, Elizabeth A; Wear, Martin A; Landré, Vivian; Narayan, Vikram; Ning, Jia; Erman, Burak; Ball, Kathryn L; Walkinshaw, Malcolm D

    2015-09-01

    Cyclophilin 40 (Cyp40) comprises an N-terminal cyclophilin domain with peptidyl-prolyl isomerase (PPIase) activity and a C-terminal tetratricopeptide repeat (TPR) domain that binds to the C-terminal-EEVD sequence common to both heat shock protein 70 (Hsp70) and Hsp90. We show in the present study that binding of peptides containing the MEEVD motif reduces the PPIase activity by ∼30%. CD and fluorescence assays show that the TPR domain is less stable than the cyclophilin domain and is stabilized by peptide binding. Isothermal titration calorimetry (ITC) shows that the affinity for the-MEEVD peptide is temperature sensitive in the physiological temperature range. Results from these biophysical studies fit with the MD simulations of the apo and holo (peptide-bound) structures which show a significant reduction in root mean square (RMS) fluctuation in both TPR and cyclophilin domains when-MEEVD is bound. The MD simulations of the apo-protein also highlight strong anti-correlated motions between residues around the PPIase-active site and a band of residues running across four of the seven helices in the TPR domain. Peptide binding leads to a distortion in the shape of the active site and a significant reduction in these strongly anti-correlated motions, providing an explanation for the allosteric effect of ligand binding and loss of PPIase activity. Together the experimental and MD results suggest that on heat shock, dissociation of Cyp40 from complexes mediated by the TPR domain leads to an increased pool of free Cyp40 capable of acting as an isomerase/chaperone in conditions of cellular stress. © 2015 Authors.

  13. A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease.

    Directory of Open Access Journals (Sweden)

    Matthew Brecher

    2017-05-01

    Full Text Available The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2 in vitro, with IC50 values of 1.8 μM, 11.4 μM, and 4.8 μM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV, West Nile virus (WNV, and Yellow fever virus (YFV on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 μM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and

  14. The allosteric behavior of Fur mediates oxidative stress signal transduction in Helicobacter pylori

    Directory of Open Access Journals (Sweden)

    Simone ePelliciari

    2015-08-01

    Full Text Available The microaerophilic gastric pathogen Helicobacter pylori is exposed to oxidative stress originating from the aerobic environment, the oxidative burst of phagocytes and the formation of reactive oxygen species, catalyzed by iron excess. Accordingly, the expression of genes involved in oxidative stress defense have been repeatedly linked to the ferric uptake regulator Fur. Moreover, mutations in the Fur protein affect the resistance to metronidazole, likely due to loss-of-function in the regulation of genes involved in redox control. Although many advances in the molecular understanding of HpFur function were made, little is known about the mechanisms that enable Fur to mediate the responses to oxidative stress.Here we show that iron-inducible, apo-Fur repressed genes, such as pfr and hydA, are induced shortly after oxidative stress, while their oxidative induction is lost in a fur knockout strain. On the contrary, holo-Fur repressed genes, such as frpB1 and fecA1, vary modestly in response to oxidative stress. This indicates that the oxidative stress signal specifically targets apo-Fur repressed genes, rather than impairing indiscriminately the regulatory function of Fur. Footprinting analyses showed that the oxidative signal strongly impairs the binding affinity of Fur towards apo-operators, while the binding towards holo-operators is less affected. Further evidence is presented that a reduced state of Fur is needed to maintain apo-repression, while oxidative conditions shift the preferred binding architecture of Fur towards the holo-operator binding conformation, even in the absence of iron. Together the results demonstrate that the allosteric regulation of Fur enables transduction of oxidative stress signals in H. pylori, supporting the concept that apo-Fur repressed genes can be considered oxidation inducible Fur regulatory targets. These findings may have important implications in the study of H. pylori treatment and resistance to

  15. Negative Allosteric Modulators of Metabotropic Glutamate Receptors Subtype 5 in Addiction: a Therapeutic Window

    Science.gov (United States)

    2016-01-01

    Background: Abundant evidence at the anatomical, electrophysiological, and molecular levels implicates metabotropic glutamate receptor subtype 5 (mGluR5) in addiction. Consistently, the effects of a wide range of doses of different mGluR5 negative allosteric modulators (NAMs) have been tested in various animal models of addiction. Here, these studies were subjected to a systematic review to find out if mGluR5 NAMs have a therapeutic potential that can be translated to the clinic. Methods: Literature on consumption/self-administration and reinstatement of drug seeking as outcomes of interest published up to April 2015 was retrieved via PubMed. The review focused on the effects of systemic (i.p., i.v., s.c.) administration of the mGluR5 NAMs 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP) and 2-Methyl-6-(phenylethynyl)pyridine (MPEP) on paradigms with cocaine, ethanol, nicotine, and food in rats. Results: MTEP and MPEP were found to reduce self-administration of cocaine, ethanol, and nicotine at doses ≥1mg/kg and 2.5mg/kg, respectively. Dose-response relationship resembled a sigmoidal curve, with low doses not reaching statistical significance and high doses reliably inhibiting self-administration of drugs of abuse. Importantly, self-administration of cocaine, ethanol, and nicotine, but not food, was reduced by MTEP and MPEP in the dose range of 1 to 2mg/kg and 2.5 to 3.2mg/kg, respectively. This dose range corresponds to approximately 50% to 80% mGluR5 occupancy. Interestingly, the limited data found in mice and monkeys showed a similar therapeutic window. Conclusion: Altogether, this review suggests a therapeutic window for mGluR5 NAMs that can be translated to the treatment of substance-related and addictive disorders. PMID:26802568

  16. Causality, transfer entropy, and allosteric communication landscapes in proteins with harmonic interactions.

    Science.gov (United States)

    Hacisuleyman, Aysima; Erman, Burak

    2017-06-01

    A fast and approximate method of generating allosteric communication landscapes in proteins is presented by using Schreiber's entropy transfer concept in combination with the Gaussian Network Model of proteins. Predictions of the model and the allosteric communication landscapes generated show that information transfer in proteins does not necessarily take place along a single path, but an ensemble of pathways is possible. The model emphasizes that knowledge of entropy only is not sufficient for determining allosteric communication and additional information based on time delayed correlations should be introduced, which leads to the presence of causality in proteins. The model provides a simple tool for mapping entropy sink-source relations into pairs of residues. By this approach, residues that should be manipulated to control protein activity may be determined. This should be of great importance for allosteric drug design and for understanding the effects of mutations on function. The model is applied to determine allosteric communication in three proteins, Ubiquitin, Pyruvate Kinase, and the PDZ domain. Predictions are in agreement with molecular dynamics simulations and experimental evidence. Proteins 2017; 85:1056-1064. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  17. New sites of Australasian microtektites in the central Indian Ocean: Implications for the location and size of source crater

    Digital Repository Service at National Institute of Oceanography (India)

    ShyamPrasad, M.; Mahale, V.P.; Kodagali, V.N.

    Fifteen new Australasian microtektite sites have been identified along a transect roughly N-S in the central Indian Ocean. These locations, in addition to the existing 46 sites, total to 61 microtektite sites in the oceans. We carried out regression...

  18. Evidence for allosterism in ribulose-1,5-bisphosphate carboxylase/oxygenase from comfrey

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, D.D.; Bolden, T.D.

    1986-05-01

    Evidence has been obtained suggesting that ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is an allosteric enzyme in the sense that it shows cooperative active site binding, cooperative interactions between the activation and active sites and significant binding of some metabolites at a second site. Investigation of the binding of a potent competitive inhibitor. 2-carboxymannitol-1,6-bisphosphate (CMBP) by /sup 31/P-NMR indicated essentially 1:1 binding with the active sites of comfrey RuBisCo. Among the interactions of competitive inhibitors, as measured by difference UV spectroscopy, the binding curves for ortho-phosphate and ribose-5-phosphate were better fitted by a Monod-Wyman-Changeux model than by an independent site model, whereas the binding of CMBP and 2-phosphoglycolate were not. Difference UV methods also were used to study activation by CO/sub 2/ which at pH 7.9 in 10 mM MgCl/sub 2/ showed positive cooperativity with k = 100 +/- 3 ..mu..M (based on pK/sub a/ = 6.4 for the CO/sub 2/-HCO/sub 3//sup -/ equilibrium) and L = 3.5 +/- 0.7. Addition of saturating amounts of CMBP and lowering the MgCl/sub 2/ to 2 mM still gave a sigmoidal curve but it was shifted to higher CO/sub 2/ concentrations (k = 124 +/- 2 ..mu..M and L = 31 +/- 3). In the absence of CMBP the same conditions gave k = 26 +/- 2 ..mu..M for L = 3.5. Conversely, k was 0.96 +/- 0.08 ..mu..M for CMBP in 0.5 mM MgCl/sub 2/ without added NaHCO/sub 3/ but was 21 +/- 0.06 ..mu..M in 10 MgCl/sub 2/ and 2 mM NaHCO/sub 3/, pH 7.3.

  19. A novel antidiabetic drug, fasiglifam/TAK-875, acts as an ago-allosteric modulator of FFAR1.

    Directory of Open Access Journals (Sweden)

    Chiori Yabuki

    Full Text Available Selective free fatty acid receptor 1 (FFAR1/GPR40 agonist fasiglifam (TAK-875, an antidiabetic drug under phase 3 development, potentiates insulin secretion in a glucose-dependent manner by activating FFAR1 expressed in pancreatic β cells. Although fasiglifam significantly improved glycemic control in type 2 diabetes patients with a minimum risk of hypoglycemia in a phase 2 study, the precise mechanisms of its potent pharmacological effects are not fully understood. Here we demonstrate that fasiglifam acts as an ago-allosteric modulator with a partial agonistic activity for FFAR1. In both Ca(2+ influx and insulin secretion assays using cell lines and mouse islets, fasiglifam showed positive cooperativity with the FFAR1 ligand γ-linolenic acid (γ-LA. Augmentation of glucose-induced insulin secretion by fasiglifam, γ-LA, or their combination was completely abolished in pancreatic islets of FFAR1-knockout mice. In diabetic rats, the insulinotropic effect of fasiglifam was suppressed by pharmacological reduction of plasma free fatty acid (FFA levels using a lipolysis inhibitor, suggesting that fasiglifam potentiates insulin release in conjunction with plasma FFAs in vivo. Point mutations of FFAR1 differentially affected Ca(2+ influx activities of fasiglifam and γ-LA, further indicating that these agonists may bind to distinct binding sites. Our results strongly suggest that fasiglifam is an ago-allosteric modulator of FFAR1 that exerts its effects by acting cooperatively with endogenous plasma FFAs in human patients as well as diabetic animals. These findings contribute to our understanding of fasiglifam as an attractive antidiabetic drug with a novel mechanism of action.

  20. Mutation of I696 and W697 in the TRP box of vanilloid receptor subtype I modulates allosteric channel activation.

    Science.gov (United States)

    Gregorio-Teruel, Lucia; Valente, Pierluigi; González-Ros, José Manuel; Fernández-Ballester, Gregorio; Ferrer-Montiel, Antonio

    2014-03-01

    The transient receptor potential vanilloid receptor subtype I (TRPV1) channel acts as a polymodal sensory receptor gated by chemical and physical stimuli. Like other TRP channels, TRPV1 contains in its C terminus a short, conserved domain called the TRP box, which is necessary for channel gating. Substitution of two TRP box residues-I696 and W697-with Ala markedly affects TRPV1's response to all activating stimuli, which indicates that these two residues play a crucial role in channel gating. We systematically replaced I696 and W697 with 18 native l-amino acids (excluding cysteine) and evaluated the effect on voltage- and capsaicin-dependent gating. Mutation of I696 decreased channel activation by either voltage or capsaicin; furthermore, gating was only observed with substitution of hydrophobic amino acids. Substitution of W697 with any of the 18 amino acids abolished gating in response to depolarization alone, shifting the threshold to unreachable voltages, but not capsaicin-mediated gating. Moreover, vanilloid-activated responses of W697X mutants showed voltage-dependent gating along with a strong voltage-independent component. Analysis of the data using an allosteric model of activation indicates that mutation of I696 and W697 primarily affects the allosteric coupling constants of the ligand and voltage sensors to the channel pore. Together, our findings substantiate the notion that inter- and/or intrasubunit interactions at the level of the TRP box are critical for efficient coupling of stimulus sensing and gate opening. Perturbation of these interactions markedly reduces the efficacy and potency of the activating stimuli. Furthermore, our results identify these interactions as potential sites for pharmacological intervention.

  1. PATTERN BASED DETECTION OF POTENTIALLY DRUGGABLE BINDING SITES BY LIGAND SCREENING

    Directory of Open Access Journals (Sweden)

    Uttam Pal

    2018-03-01

    Full Text Available This article describes an innovative way of finding the potentially druggable sites on a target protein, which can be used for orthosteric and allosteric lead detection in a single virtual screening setup. Druggability estimation for an alternate binding site other than the canonical ligand-binding pocket of an enzyme is rewarding for several inherent benefits. Allostery is a direct and efficient way of regulating biomacromolecule function. The allosteric modulators can fine-tune protein mechanics. Besides, allosteric sites are evolutionarily less conserved/more diverse even in very similarly related proteins, thus, provides high degree of specificity in targeting a particular protein. Therefore, targeting of allosteric sites is gaining attention as an emerging strategy in rational drug design. However, the experimental approaches provide a limited degree of characterization of new allosteric sites. Computational approaches are useful to analyze and select potential allosteric sites for drug discovery. Here, the use of molecular docking, which has become an integral part of the drug discovery process, has been discussed to predict the druggability of novel allosteric sites as well as the active site on target proteins by ligand screening. Genetic algorithm was used for docking and the whole protein was placed in the search space. For each ligand in the library of small molecules, the genetic algorithm was run for multiple times to populate all the druggable sites in the target protein, which was then translated into two dimensional density maps or “patterns”. High density clusters were observed for lead like molecules in these pattern diagrams. Each cluster in such a pattern diagram indicated a plausible binding site and the density gave its druggability score in terms of weighted probabilities. The patterns were filtered to find the leads for each of the druggable sites on the target protein. Such a novel pattern based analysis of the

  2. Implications for reconstruction of the relationship between nuclear industry and siting areas in Japan. Lessons learnt from the cases of Site Stakeholder Groups in United Kingdom

    International Nuclear Information System (INIS)

    Sugawara, Shin-etsu

    2014-01-01

    After the Fukushima nuclear accident, the Japanese nuclear community began to recognize the need for establishing local stakeholder meetings in nuclear siting areas for information sharing and communication in reference to the experiences in European countries. This report shows the patterns of institutional design and the management style of stakeholder meetings in UK based on the interview survey, including SSGs (Site Stakeholder Groups) around the NDA (Nuclear Decommissioning Authority) sites and LCLCs (Local Community Liaison Councils) around the nuclear power stations in operation. SSGs have developed a local-oriented management style under the leadership of independent chairs elected from local stakeholders, in contrast to LCLCs where the operator of nuclear energy facilities has the initiative of management. SSGs play critical roles in improving quality of decision-making and enhancing its legitimacy through providing forums for local stakeholder engagement in the process of NDA's consultations and BPEO (Best Practicable Environmental Option) implementation. Based on these insights from UK's cases, the author suggests the following remedies for the relationship between the nuclear industry and siting areas in Japan: (1) introducing evaluation systems of stakeholder engagement linked to the insurance rates of nuclear energy liability, and (2) modifying the nuclear safety agreements into risk management principles. (author)

  3. Assessments of wind-energy potential in selected sites from three geopolitical zones in Nigeria: implications for renewable/sustainable rural electrification.

    Science.gov (United States)

    Okeniyi, Joshua Olusegun; Ohunakin, Olayinka Soledayo; Okeniyi, Elizabeth Toyin

    2015-01-01

    Electricity generation in rural communities is an acute problem militating against socioeconomic well-being of the populace in these communities in developing countries, including Nigeria. In this paper, assessments of wind-energy potential in selected sites from three major geopolitical zones of Nigeria were investigated. For this, daily wind-speed data from Katsina in northern, Warri in southwestern and Calabar in southeastern Nigeria were analysed using the Gumbel and the Weibull probability distributions for assessing wind-energy potential as a renewable/sustainable solution for the country's rural-electrification problems. Results showed that the wind-speed models identified Katsina with higher wind-speed class than both Warri and Calabar that were otherwise identified as low wind-speed sites. However, econometrics of electricity power simulation at different hub heights of low wind-speed turbine systems showed that the cost of electric-power generation in the three study sites was converging to affordable cost per kWh of electric energy from the wind resource at each site. These power simulations identified cost/kWh of electricity generation at Kaduna as €0.0507, at Warri as €0.0774, and at Calabar as €0.0819. These bare positive implications on renewable/sustainable rural electrification in the study sites even as requisite options for promoting utilization of this viable wind-resource energy in the remote communities in the environs of the study sites were suggested.

  4. Assessments of Wind-Energy Potential in Selected Sites from Three Geopolitical Zones in Nigeria: Implications for Renewable/Sustainable Rural Electrification

    Science.gov (United States)

    Okeniyi, Joshua Olusegun; Ohunakin, Olayinka Soledayo; Okeniyi, Elizabeth Toyin

    2015-01-01

    Electricity generation in rural communities is an acute problem militating against socioeconomic well-being of the populace in these communities in developing countries, including Nigeria. In this paper, assessments of wind-energy potential in selected sites from three major geopolitical zones of Nigeria were investigated. For this, daily wind-speed data from Katsina in northern, Warri in southwestern and Calabar in southeastern Nigeria were analysed using the Gumbel and the Weibull probability distributions for assessing wind-energy potential as a renewable/sustainable solution for the country's rural-electrification problems. Results showed that the wind-speed models identified Katsina with higher wind-speed class than both Warri and Calabar that were otherwise identified as low wind-speed sites. However, econometrics of electricity power simulation at different hub heights of low wind-speed turbine systems showed that the cost of electric-power generation in the three study sites was converging to affordable cost per kWh of electric energy from the wind resource at each site. These power simulations identified cost/kWh of electricity generation at Kaduna as €0.0507, at Warri as €0.0774, and at Calabar as €0.0819. These bare positive implications on renewable/sustainable rural electrification in the study sites even as requisite options for promoting utilization of this viable wind-resource energy in the remote communities in the environs of the study sites were suggested. PMID:25879063

  5. Biased signaling of lipids and allosteric actions of synthetic molecules for GPR119

    DEFF Research Database (Denmark)

    Hassing, Helle A; Fares, Suzan; Larsen, Olav

    2016-01-01

    for 2h with the 2-MAG-lipase inhibitor JZL84 doubled the constitutive activity, indicating that endogenous lipids contribute to the apparent constitutive activity. Finally, besides being an agonist, AR231453 acted as a positive allosteric modulator of OEA and increased its potency by 54-fold at 100nM AR......231453. Our studies uncovering broad and biased signaling, masked constitutive activity by endogenous MAGs, and ago-allosteric properties of synthetic ligands may explain why many GPR119 drug-discovery programs have failed so far....

  6. First steps in the direction of synthetic, allosteric, direct inhibitors of thrombin and factor Xa.

    Science.gov (United States)

    Verghese, Jenson; Liang, Aiye; Sidhu, Preet Pal Singh; Hindle, Michael; Zhou, Qibing; Desai, Umesh R

    2009-08-01

    Designing non-saccharide functional mimics of heparin is a major challenge. In this work, a library of small, aromatic molecules based on the sulfated DHP scaffold was synthesized and screened against thrombin and factor Xa. The results reveal that (i) selected monomeric benzofuran derivatives inhibit the two enzymes, albeit weakly; (ii) the two enzymes recognize different structural features in the benzofurans studied suggesting significant selectivity of recognition; and (iii) the mechanism of inhibition is allosteric. The molecules represent the first allosteric small molecule inhibitors of the two enzymes.

  7. First Steps in the Direction of Synthetic, Allosteric, Direct Inhibitors of Thrombin and Factor Xa

    Science.gov (United States)

    Verghese, Jenson; Liang, Aiye; Sidhu, Preet Pal Singh; Hindle, Michael; Zhou, Qibing; Desai, Umesh R.

    2009-01-01

    Designing non-saccharide functional mimics of heparin is a major challenge. In this work, a library of small, aromatic molecules based on the sulfated DHP scaffold was synthesized and screened against thrombin and factor Xa. The results reveal that i) selected monomeric benzofuran derivatives inhibit the two enzymes, albeit weakly; ii) the two enzymes recognize different structural features in the benzofurans studied suggesting significant selectivity of recognition; and iii) the mechanism of inhibition is allosteric. The molecules represent the first allosteric small molecule inhibitors of the two enzymes. PMID:19540113

  8. Allosteric communication in myosin V: from small conformational changes to large directed movements.

    Directory of Open Access Journals (Sweden)

    M Cecchini

    Full Text Available The rigor to post-rigor transition in myosin, a consequence of ATP binding, plays an essential role in the Lymn-Taylor functional cycle because it results in the dissociation of the actomyosin complex after the powerstroke. On the basis of the X-ray structures of myosin V, we have developed a new normal mode superposition model for the transition path between the two states. Rigid-body motions of the various subdomains and specific residues at the subdomain interfaces are key elements in the transition. The allosteric communication between the nucleotide binding site and the U50/L50 cleft is shown to result from local changes due to ATP binding, which induce large amplitude motions that are encoded in the structure of the protein. The triggering event is the change in the interaction of switch I and the P-loop, which is stabilized by ATP binding. The motion of switch I, which is a relatively rigid element of the U50 subdomain, leads directly to a partial opening of the U50/L50 cleft; the latter is expected to weaken the binding of myosin to actin. The calculated transition path demonstrates the nature of the subdomain coupling and offers an explanation for the mutual exclusion of ATP and actin binding. The mechanism of the uncoupling of the converter from the motor head, an essential part of the transition, is elucidated. The origin of the partial untwisting of the central beta-sheet in the rigor to post-rigor transition is described.

  9. A mechanistic understanding of allosteric immune escape pathways in the HIV-1 envelope glycoprotein.

    Directory of Open Access Journals (Sweden)

    Anurag Sethi

    Full Text Available The HIV-1 envelope (Env spike, which consists of a compact, heterodimeric trimer of the glycoproteins gp120 and gp41, is the target of neutralizing antibodies. However, the high mutation rate of HIV-1 and plasticity of Env facilitates viral evasion from neutralizing antibodies through various mechanisms. Mutations that are distant from the antibody binding site can lead to escape, probably by changing the conformation or dynamics of Env; however, these changes are difficult to identify and define mechanistically. Here we describe a network analysis-based approach to identify potential allosteric immune evasion mechanisms using three known HIV-1 Env gp120 protein structures from two different clades, B and C. First, correlation and principal component analyses of molecular dynamics (MD simulations identified a high degree of long-distance coupled motions that exist between functionally distant regions within the intrinsic dynamics of the gp120 core, supporting the presence of long-distance communication in the protein. Then, by integrating MD simulations with network theory, we identified the optimal and suboptimal communication pathways and modules within the gp120 core. The results unveil both strain-dependent and -independent characteristics of the communication pathways in gp120. We show that within the context of three structurally homologous gp120 cores, the optimal pathway for communication is sequence sensitive, i.e. a suboptimal pathway in one strain becomes the optimal pathway in another strain. Yet the identification of conserved elements within these communication pathways, termed inter-modular hotspots, could present a new opportunity for immunogen design, as this could be an additional mechanism that HIV-1 uses to shield vulnerable antibody targets in Env that induce neutralizing antibody breadth.

  10. An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behaviour.

    Science.gov (United States)

    Motamedi-Shad, Neda; Jagger, Alistair M; Liedtke, Maximilian; Faull, Sarah V; Nanda, Arjun Scott; Salvadori, Enrico; Wort, Joshua L; Kay, Christopher W M; Heyer-Chauhan, Narinder; Miranda, Elena; Perez, Juan; Ordóñez, Adriana; Haq, Imran; Irving, James A; Lomas, David A

    2016-10-01

    Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A - on the opposite face of the molecule - more liable to adopt an 'open' state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin-enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the 'open' state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation. © 2016 The Author(s).

  11. Hemoglobin function and allosteric regulation in semi-fossorial rodents (family Sciuridae) with different altitudinal ranges

    Science.gov (United States)

    Revsbech, Inge G.; Tufts, Danielle M.; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E.; Storz, Jay F.; Fago, Angela

    2013-01-01

    SUMMARY Semi-fossorial ground squirrels face challenges to respiratory gas transport associated with the chronic hypoxia and hypercapnia of underground burrows, and such challenges are compounded in species that are native to high altitude. During hibernation, such species must also contend with vicissitudes of blood gas concentrations and plasma pH caused by episodic breathing. Here, we report an analysis of hemoglobin (Hb) function in six species of marmotine ground squirrels with different altitudinal distributions. Regardless of their native altitude, all species have high Hb–O2 affinities, mainly due to suppressed sensitivities to allosteric effectors [2,3-diphosphoglycerate (DPG) and chloride ions]. This suppressed anion sensitivity is surprising given that all canonical anion-binding sites are conserved. Two sciurid species, the golden-mantled and thirteen-lined ground squirrel, have Hb–O2 affinities that are characterized by high pH sensitivity and low thermal sensitivity relative to the Hbs of humans and other mammals. The pronounced Bohr effect is surprising in light of highly unusual amino acid substitutions at the C-termini that are known to abolish the Bohr effect in human HbA. Taken together, the high O2 affinity of sciurid Hbs suggests an enhanced capacity for pulmonary O2 loading under hypoxic and hypercapnic conditions, while the large Bohr effect should help to ensure efficient O2 unloading in tissue capillaries. In spite of the relatively low thermal sensitivities of the sciurid Hbs, our results indicate that the effect of hypothermia on Hb oxygenation is the main factor contributing to the increased blood–O2 affinity in hibernating ground squirrels. PMID:24172889

  12. Structural Bioinformatics and Protein Docking Analysis of the Molecular Chaperone-Kinase Interactions: Towards Allosteric Inhibition of Protein Kinases by Targeting the Hsp90-Cdc37 Chaperone Machinery

    Directory of Open Access Journals (Sweden)

    Gennady Verkhivker

    2013-11-01

    Full Text Available A fundamental role of the Hsp90-Cdc37 chaperone system in mediating maturation of protein kinase clients and supporting kinase functional activity is essential for the integrity and viability of signaling pathways involved in cell cycle control and organism development. Despite significant advances in understanding structure and function of molecular chaperones, the molecular mechanisms and guiding principles of kinase recruitment to the chaperone system are lacking quantitative characterization. Structural and thermodynamic characterization of Hsp90-Cdc37 binding with protein kinase clients by modern experimental techniques is highly challenging, owing to a transient nature of chaperone-mediated interactions. In this work, we used experimentally-guided protein docking to probe the allosteric nature of the Hsp90-Cdc37 binding with the cyclin-dependent kinase 4 (Cdk4 kinase clients. The results of docking simulations suggest that the kinase recognition and recruitment to the chaperone system may be primarily determined by Cdc37 targeting of the N-terminal kinase lobe. The interactions of Hsp90 with the C-terminal kinase lobe may provide additional “molecular brakes” that can lock (or unlock kinase from the system during client loading (release stages. The results of this study support a central role of the Cdc37 chaperone in recognition and recruitment of the kinase clients. Structural analysis may have useful implications in developing strategies for allosteric inhibition of protein kinases by targeting the Hsp90-Cdc37 chaperone machinery.

  13. Scalp flora in Indian patients undergoing craniotomy for brain tumors - Implications for pre-surgical site preparation and surgical site infection

    Directory of Open Access Journals (Sweden)

    Aliasgar V Moiyadi

    2012-01-01

    Full Text Available Introduction: Causation of surgical site infection (SSI following craniotomy is multifactorial. Most preventive strategies (including site preparation and antibiotic prophylaxis revolve around reducing preoperative contamination of the local site. There is little evidence, however, linking site contamination with postoperative infections. This is important given the preference for performing non-shaved cranial surgery. We undertook a prospective study to document the scalp flora in neurosurgical patients in an Indian setting and to assess possible association with SSI. Materials and Methods: A prospective study recruited 45 patients undergoing non-shaved clean craniotomies for various brain tumors. Standard perioperative procedures and antibiotic policy were employed. Prior to and immediately following the pre-surgical scrubbing, we collected swabs and evaluated their growth qualitatively. SSI was documented adhering to CDC guidelines. The association of swab-positivity with various parameters (including SSI was evaluated. Results: Pre-scrub positivity was seen in 18 of 44 patients, three of them developed subsequent SSI. Most were known skin contaminants. Five patients had swab positivity after scrubbing, though none of these developed any SSI. Four of these five had pre-scrub positivity. In three the same organisms persisted (two being Staphylococcus aureus, and one had different growth post-scrub, whereas one patient developed new growth (contaminant mycelial fungus in the post-scrub swab. We did not find any association between swab positivity and SSI. Swab positivity was also not related to hair-length or hygiene. Conclusion: Scalp flora in Indian patients is similar to that described. Pre-surgical preparation does not always eliminate this contamination (especially staphylococcus. However, this does not necessarily translate into increased SSI. Moreover, the results also provide objective evidence to support the performance of non

  14. The understanding of the formation of valleys and its implication on site characterization: Moredalen and Pukedalen, south-eastern Sweden

    International Nuclear Information System (INIS)

    Tiren, Sven A.; Waenstedt, Stefan; Straeng, Thomas

    2010-11-01

    In south-eastern Sweden, there are a number of over-deepened narrow valleys, more than 20 m deep, formed in Precambrian bedrock located above the highest post-glacial shoreline. Canyon-like valleys, called 'kursu' or kursu valleys, are generally interpreted to be formed by glaciofluvial erosion. An example of such a valley is Moredalen, a canyon in the Fennoscandian Shield, which has an implication on site selection for radioactive waste disposal. There are also more open over-deepened valleys along which sub-glacial flow has occurred, e.g. Pukedalen. The main part of this paper discusses a combined geological and geophysical investigation of Moredalen, with the aim to investigate possible reasons for the formation of such an unusual feature formed in acid vulcanite and foliated tonalitic to granodioritic rocks. Moredalen is a marked, approximately 7 km long, E-W striking valley that cuts through a plateau (c. 140 m a.s.l.), and an elevated block of the sub-Cambrian peneplain. Glaciofluvial sediments can be found up-streams where the canyon widens to the west. Just east of the valley is a larger delta deposited at the highest post-glacial shoreline (c. 105 m a.s.l). Further east of, and in line with the Moredalen valley there is an esker. Rock debris in the valley is angular. Pukedalen is a northwest-southeast trending valley incised in massive granite. The valley is in its northern parts relatively open and becomes narrow in its south-eastern part having partly a vertical south-western wall. Rock surfaces are smooth along the valley and rock debris in the valley consists generally of rounded blocks. In line with Pukedalen, on both sides at great distances though, there are eskers. Geomorphological features of this kind indicate certain characteristics of the bedrock that need to be considered during safety analysis of repositories for nuclear waste. The distinct weakness zones along which the kursu-valleys are formed create prominent transport paths for

  15. The understanding of the formation of valleys and its implication on site characterization: Moredalen and Pukedalen, south-eastern Sweden

    Energy Technology Data Exchange (ETDEWEB)

    Tiren, Sven A.; Waenstedt, Stefan; Straeng, Thomas (GEOSIGMA AB (Sweden))

    2010-11-15

    In south-eastern Sweden, there are a number of over-deepened narrow valleys, more than 20 m deep, formed in Precambrian bedrock located above the highest post-glacial shoreline. Canyon-like valleys, called 'kursu' or kursu valleys, are generally interpreted to be formed by glaciofluvial erosion. An example of such a valley is Moredalen, a canyon in the Fennoscandian Shield, which has an implication on site selection for radioactive waste disposal. There are also more open over-deepened valleys along which sub-glacial flow has occurred, e.g. Pukedalen. The main part of this paper discusses a combined geological and geophysical investigation of Moredalen, with the aim to investigate possible reasons for the formation of such an unusual feature formed in acid vulcanite and foliated tonalitic to granodioritic rocks. Moredalen is a marked, approximately 7 km long, E-W striking valley that cuts through a plateau (c. 140 m a.s.l.), and an elevated block of the sub-Cambrian peneplain. Glaciofluvial sediments can be found up-streams where the canyon widens to the west. Just east of the valley is a larger delta deposited at the highest post-glacial shoreline (c. 105 m a.s.l). Further east of, and in line with the Moredalen valley there is an esker. Rock debris in the valley is angular. Pukedalen is a northwest-southeast trending valley incised in massive granite. The valley is in its northern parts relatively open and becomes narrow in its south-eastern part having partly a vertical south-western wall. Rock surfaces are smooth along the valley and rock debris in the valley consists generally of rounded blocks. In line with Pukedalen, on both sides at great distances though, there are eskers. Geomorphological features of this kind indicate certain characteristics of the bedrock that need to be considered during safety analysis of repositories for nuclear waste. The distinct weakness zones along which the kursu-valleys are formed create prominent transport paths for

  16. Family-site interaction in Pinus radiata: implications for progeny testing strategy and regionalised breeding in New Zealand.

    Science.gov (United States)

    G.R. Johnson; R.D. Brudon

    1990-01-01

    A progeny test of 170 open-pollinated families from second-generation plus trees of Pinus radiata was established on four sites in New Zealand in 1981. Two test sites were on volcanic purnice soils in the Central North Island region and two were on phosphate-retentive clay soils in the Northland region.Assessments of volume growth, stem straightness, mal-...

  17. Implications of next generation attenuation ground motion prediction equations for site coefficients used in earthquake resistant design

    Science.gov (United States)

    Borcherdt, Roger D.

    2014-01-01

    Proposals are developed to update Tables 11.4-1 and 11.4-2 of Minimum Design Loads for Buildings and Other Structures published as American Society of Civil Engineers Structural Engineering Institute standard 7-10 (ASCE/SEI 7–10). The updates are mean next generation attenuation (NGA) site coefficients inferred directly from the four NGA ground motion prediction equations used to derive the maximum considered earthquake response maps adopted in ASCE/SEI 7–10. Proposals include the recommendation to use straight-line interpolation to infer site coefficients at intermediate values of (average shear velocity to 30-m depth). The NGA coefficients are shown to agree well with adopted site coefficients at low levels of input motion (0.1 g) and those observed from the Loma Prieta earthquake. For higher levels of input motion, the majority of the adopted values are within the 95% epistemic-uncertainty limits implied by the NGA estimates with the exceptions being the mid-period site coefficient, Fv, for site class D and the short-period coefficient, Fa, for site class C, both of which are slightly less than the corresponding 95% limit. The NGA data base shows that the median value  of 913 m/s for site class B is more typical than 760 m/s as a value to characterize firm to hard rock sites as the uniform ground condition for future maximum considered earthquake response ground motion estimates. Future updates of NGA ground motion prediction equations can be incorporated easily into future adjustments of adopted site coefficients using procedures presented herein. 

  18. Analyses of Rock Size-Frequency Distributions and Morphometry of Modified Hawaiian Lava Flows: Implications for Future Martian Landing Sites

    Science.gov (United States)

    Craddock, Robert A.; Golombek, Matthew; Howard, Alan D.

    2000-01-01

    Both the size-frequency distribution and morphometry of rock populations emplaced by a variety of geologic processes in Hawaii indicate that such information may be useful in planning future landing sites on Mars and interpreting the surface geology.

  19. Conservation implications for the Himalayan wolf Canis (lupus) himalayensis based on observations of packs and home sites in Nepal

    OpenAIRE

    Werhahn, G; Kusi, N; Sillero-Zubiri, C; Macdonald, DW

    2017-01-01

    We provide insights into pack composition and den site parameters of the Himalayan wolf Canis (lupus) himalayensis based on observations of free-ranging wolves in three study areas in Nepal. We combine this with a social survey of the local Buddhist communities regarding human–carnivore conflict, to draw inferences for conservation practice in the Nepalese Himalayas. We recorded eight wolf packs (with an average composition of two adults and three pups), and found five home sites in high-alti...

  20. Structural changes at the myrtenol backbone reverse its positive allosteric potential into inhibitory GABAA receptor modulation

    DEFF Research Database (Denmark)

    Milanos, Sinem; Kuenzel, Katharina; Gilbert, Daniel F

    2017-01-01

    monoterpenes, e.g. myrtenol as positive allosteric modulator at α1β2 GABAA receptors. Here, along with pharmacophore-based virtual screening studies, we demonstrate that scaffold modifications of myrtenol resulted in loss of modulatory activity. Two independent approaches, fluorescence-based compound analysis...

  1. Entropy Transfer between Residue Pairs and Allostery in Proteins: Quantifying Allosteric Communication in Ubiquitin.

    Directory of Open Access Journals (Sweden)

    Aysima Hacisuleyman

    2017-01-01

    Full Text Available It has recently been proposed by Gunasakaran et al. that allostery may be an intrinsic property of all proteins. Here, we develop a computational method that can determine and quantify allosteric activity in any given protein. Based on Schreiber's transfer entropy formulation, our approach leads to an information transfer landscape for the protein that shows the presence of entropy sinks and sources and explains how pairs of residues communicate with each other using entropy transfer. The model can identify the residues that drive the fluctuations of others. We apply the model to Ubiquitin, whose allosteric activity has not been emphasized until recently, and show that there are indeed systematic pathways of entropy and information transfer between residues that correlate well with the activities of the protein. We use 600 nanosecond molecular dynamics trajectories for Ubiquitin and its complex with human polymerase iota and evaluate entropy transfer between all pairs of residues of Ubiquitin and quantify the binding susceptibility changes upon complex formation. We explain the complex formation propensities of Ubiquitin in terms of entropy transfer. Important residues taking part in allosteric communication in Ubiquitin predicted by our approach are in agreement with results of NMR relaxation dispersion experiments. Finally, we show that time delayed correlation of fluctuations of two interacting residues possesses an intrinsic causality that tells which residue controls the interaction and which one is controlled. Our work shows that time delayed correlations, entropy transfer and causality are the required new concepts for explaining allosteric communication in proteins.

  2. Entropy Transfer between Residue Pairs and Allostery in Proteins: Quantifying Allosteric Communication in Ubiquitin.

    Science.gov (United States)

    Hacisuleyman, Aysima; Erman, Burak

    2017-01-01

    It has recently been proposed by Gunasakaran et al. that allostery may be an intrinsic property of all proteins. Here, we develop a computational method that can determine and quantify allosteric activity in any given protein. Based on Schreiber's transfer entropy formulation, our approach leads to an information transfer landscape for the protein that shows the presence of entropy sinks and sources and explains how pairs of residues communicate with each other using entropy transfer. The model can identify the residues that drive the fluctuations of others. We apply the model to Ubiquitin, whose allosteric activity has not been emphasized until recently, and show that there are indeed systematic pathways of entropy and information transfer between residues that correlate well with the activities of the protein. We use 600 nanosecond molecular dynamics trajectories for Ubiquitin and its complex with human polymerase iota and evaluate entropy transfer between all pairs of residues of Ubiquitin and quantify the binding susceptibility changes upon complex formation. We explain the complex formation propensities of Ubiquitin in terms of entropy transfer. Important residues taking part in allosteric communication in Ubiquitin predicted by our approach are in agreement with results of NMR relaxation dispersion experiments. Finally, we show that time delayed correlation of fluctuations of two interacting residues possesses an intrinsic causality that tells which residue controls the interaction and which one is controlled. Our work shows that time delayed correlations, entropy transfer and causality are the required new concepts for explaining allosteric communication in proteins.

  3. Nootropic α7 nicotinic receptor allosteric modulator derived from GABAA receptor modulators

    Science.gov (United States)

    Ng, Herman J.; Whittemore, Edward R.; Tran, Minhtam B.; Hogenkamp, Derk J.; Broide, Ron S.; Johnstone, Timothy B.; Zheng, Lijun; Stevens, Karen E.; Gee, Kelvin W.

    2007-01-01

    Activation of brain α7 nicotinic acetylcholine receptors (α7 nAChRs) has broad therapeutic potential in CNS diseases related to cognitive dysfunction, including Alzheimer's disease and schizophrenia. In contrast to direct agonist activation, positive allosteric modulation of α7 nAChRs would deliver the clinically validated benefits of allosterism to these indications. We have generated a selective α7 nAChR-positive allosteric modulator (PAM) from a library of GABAA receptor PAMs. Compound 6 (N-(4-chlorophenyl)-α-[[(4-chloro-phenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide) evokes robust positive modulation of agonist-induced currents at α7 nAChRs, while preserving the rapid native characteristics of desensitization, and has little to no efficacy at other ligand-gated ion channels. In rodent models, it corrects sensory-gating deficits and improves working memory, effects consistent with cognitive enhancement. Compound 6 represents a chemotype for allosteric activation of α7 nAChRs, with therapeutic potential in CNS diseases with cognitive dysfunction. PMID:17470817

  4. A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Wellington, Samantha; Nag, Partha P.; Michalska, Karolina; Johnston, Stephen E.; Jedrzejczak, Robert P.; Kaushik, Virendar K.; Clatworthy, Anne E.; Siddiqi, Noman; McCarren, Patrick; Bajrami, Besnik; Maltseva, Natalia I.; Combs, Senya; Fisher, Stewart L.; Joachimiak, Andrzej; Schreiber, Stuart L.; Hung, Deborah T.

    2017-07-03

    New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes—primarily those involved in macromolecular synthesis—are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB a–b-subunit interface and affects multiple steps in the enzyme’s overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

  5. A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Wellington, Samantha; Nag, Partha P.; Michalska, Karolina; Johnston, Stephen E.; Jedrzejczak, Robert P.; Kaushik, Virendar K.; Clatworthy, Anne E.; Siddiqi, Noman; McCarren, Patrick; Bajrami, Besnik; Maltseva, Natalia I.; Combs, Senya; Fisher, Stewart L.; Joachimiak, Andrzej; Schreiber, Stuart L.; Hung, Deborah T.

    2017-07-03

    New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes—primarily those involved in macromolecular synthesis—are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB α–β-subunit interface and affects multiple steps in the enzyme's overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

  6. Divergence of allosteric effects of rapacuronium on binding and function of muscarinic receptors

    Czech Academy of Sciences Publication Activity Database

    Jakubík, Jan; Randáková, Alena; El-Fakahany, E. E.; Doležal, Vladimír

    2009-01-01

    Roč. 9, č. 15 (2009), s. 1-20 ISSN 1471-2210 R&D Projects: GA ČR GA305/09/0681; GA MŠk(CZ) LC554; GA AV ČR(CZ) IAA500110703 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic receptors * allosteric modulation * rapacuronium Subject RIV: ED - Physiology

  7. A generalized allosteric mechanism for cis-regulated cyclic nucleotide binding domains.

    Directory of Open Access Journals (Sweden)

    Alexandr P Kornev

    2008-04-01

    Full Text Available Cyclic nucleotides (cAMP and cGMP regulate multiple intracellular processes and are thus of a great general interest for molecular and structural biologists. To study the allosteric mechanism of different cyclic nucleotide binding (CNB domains, we compared cAMP-bound and cAMP-free structures (PKA, Epac, and two ionic channels using a new bioinformatics method: local spatial pattern alignment. Our analysis highlights four major conserved structural motifs: 1 the phosphate binding cassette (PBC, which binds the cAMP ribose-phosphate, 2 the "hinge," a flexible helix, which contacts the PBC, 3 the beta(2,3 loop, which provides precise positioning of an invariant arginine from the PBC, and 4 a conserved structural element consisting of an N-terminal helix, an eight residue loop and the A-helix (N3A-motif. The PBC and the hinge were included in the previously reported allosteric model, whereas the definition of the beta(2,3 loop and the N3A-motif as conserved elements is novel. The N3A-motif is found in all cis-regulated CNB domains, and we present a model for an allosteric mechanism in these domains. Catabolite gene activator protein (CAP represents a trans-regulated CNB domain family: it does not contain the N3A-motif, and its long range allosteric interactions are substantially different from the cis-regulated CNB domains.

  8. Molecular mechanism of allosteric communication in Hsp70 revealed by molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Federica Chiappori

    Full Text Available Investigating ligand-regulated allosteric coupling between protein domains is fundamental to understand cell-life regulation. The Hsp70 family of chaperones represents an example of proteins in which ATP binding and hydrolysis at the Nucleotide Binding Domain (NBD modulate substrate recognition at the Substrate Binding Domain (SBD. Herein, a comparative analysis of an allosteric (Hsp70-DnaK and a non-allosteric structural homolog (Hsp110-Sse1 of the Hsp70 family is carried out through molecular dynamics simulations, starting from different conformations and ligand-states. Analysis of ligand-dependent modulation of internal fluctuations and local deformation patterns highlights the structural and dynamical changes occurring at residue level upon ATP-ADP exchange, which are connected to the conformational transition between closed and open structures. By identifying the dynamically responsive protein regions and specific cross-domain hydrogen-bonding patterns that differentiate Hsp70 from Hsp110 as a function of the nucleotide, we propose a molecular mechanism for the allosteric signal propagation of the ATP-encoded conformational signal.

  9. Uptake of heavy metals by Typha capensis from wetland sites polluted by effluent from mineral processing plants: implications of metal-metal interactions.

    Science.gov (United States)

    Zaranyika, M F; Nyati, W

    2017-10-01

    The aim of the present work was to demonstrate the existence of metal-metal interactions in plants and their implications for the absorption of toxic elements like Cr. Typha capensis , a good accumulator of heavy metals, was chosen for the study. Levels of Fe, Cr, Ni, Cd, Pb, Cu and Zn were determined in the soil and roots, rhizomes, stems and leaves of T. capensis from three Sites A, B and C polluted by effluent from a chrome ore processing plant, a gold ore processing plant, and a nickel ore processing plant, respectively. The levels of Cr were extremely high at Site A at 5415 and 786-16,047 μg g -1 dry weight in the soil and the plant, respectively, while the levels of Ni were high at Site C at 176 and 24-891 μg g -1 in the soil and the plant, respectively. The levels of Fe were high at all three sites at 2502-7500 and 906-13,833 μg g -1 in the soil and plant, respectively. For the rest of the metals, levels were modest at 8.5-148 and 2-264 μg g -1 in the soil and plant, respectively. Pearson's correlation analysis confirmed mutual synergistic metal-metal interactions in the uptake of Zn, Cu, Co, Ni, Fe, and Cr, which are attributed to the similarity in the radii and coordination geometry of the cations of these elements. The implications of such metal-metal interactions (or effects of one metal on the behaviour of another) on the uptake of Cr, a toxic element, and possible Cr detoxification mechanism within the plant, are discussed.

  10. Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2

    DEFF Research Database (Denmark)

    Zweemer, Annelien J M; Bunnik, Julia; Veenhuizen, Margo

    2014-01-01

    be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor...

  11. Scalable rule-based modelling of allosteric proteins and biochemical networks.

    Directory of Open Access Journals (Sweden)

    Julien F Ollivier

    2010-11-01

    Full Text Available Much of the complexity of biochemical networks comes from the information-processing abilities of allosteric proteins, be they receptors, ion-channels, signalling molecules or transcription factors. An allosteric protein can be uniquely regulated by each combination of input molecules that it binds. This "regulatory complexity" causes a combinatorial increase in the number of parameters required to fit experimental data as the number of protein interactions increases. It therefore challenges the creation, updating, and re-use of biochemical models. Here, we propose a rule-based modelling framework that exploits the intrinsic modularity of protein structure to address regulatory complexity. Rather than treating proteins as "black boxes", we model their hierarchical structure and, as conformational changes, internal dynamics. By modelling the regulation of allosteric proteins through these conformational changes, we often decrease the number of parameters required to fit data, and so reduce over-fitting and improve the predictive power of a model. Our method is thermodynamically grounded, imposes detailed balance, and also includes molecular cross-talk and the background activity of enzymes. We use our Allosteric Network Compiler to examine how allostery can facilitate macromolecular assembly and how competitive ligands can change the observed cooperativity of an allosteric protein. We also develop a parsimonious model of G protein-coupled receptors that explains functional selectivity and can predict the rank order of potency of agonists acting through a receptor. Our methodology should provide a basis for scalable, modular and executable modelling of biochemical networks in systems and synthetic biology.

  12. Phytophthora cinnamomi Colonized Reclaimed Surface Mined Sites in Eastern Kentucky: Implications for the Restoration of Susceptible Species

    Directory of Open Access Journals (Sweden)

    Kenton L. Sena

    2018-04-01

    Full Text Available Appalachian forests are threatened by a number of factors, especially introduced pests and pathogens. Among these is Phytophthora cinnamomi, a soil-borne oomycete pathogen known to cause root rot in American chestnut, shortleaf pine, and other native tree species. This study was initiated to characterize the incidence of P. cinnamomi on surface mined lands in eastern Kentucky, USA, representing a range of time since reclamation (10, 12, 15, and 20 years since reclamation. Incidence of P. cinnamomi was correlated to soil properties including overall soil development, as indicated by a variety of measured soil physical and chemical parameters, especially the accumulation of soil organic carbon. P. cinnamomi was detected in only two of the four sites studied, aged 15 and 20 years since reclamation. These sites were generally characterized by higher organic matter accumulation than the younger sites in which P. cinnamomi was not detected. These results demonstrate that P. cinnamomi is capable of colonizing reclaimed mine sites in Appalachia; additional research is necessary to determine the impact of P. cinnamomi on susceptible tree species at these sites.

  13. Effects of intraperitoneal administration of the GABAB receptor positive allosteric modulator 2,6-di tert-butyl-4-(2-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930) on food intake in non-deprived rats.

    Science.gov (United States)

    Ebenezer, Ivor S

    2012-09-05

    γ-Aminobutyric acid-(B) (GABA(B)) receptor positive allosteric modulators (PAMs) act on an allosteric site on the GABA(B) receptor to potentiate the effects of GABA and GABA(B) receptor agonists. It has previously been demonstrated that the GABA(B) receptor agonist baclofen increases food intake in non-deprived rats. The aim of this study was to investigate whether the GABA(B) receptor PAM 2,6-di tert-butyl-4-(2-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930) would (i) increase food intake, and (ii) potentiate the hyperphagic effects of baclofen in rats. In Experiment 1, the effects of intraperitoneal (i.p.) administration of CGP7930 (1, 6 and 12 mg/kg) was investigated on food intake in non-deprived male Wistar rats. The 12 mg/kg dose of CGP7930 significantly increased cumulative food intake 30, 60 and 120 min (PGABA and GABA(B) receptor agonists by allosteric modulation of the GABA(B) receptor. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Prediction of consensus binding mode geometries for related chemical series of positive allosteric modulators of adenosine and muscarinic acetylcholine receptors.

    Science.gov (United States)

    Sakkal, Leon A; Rajkowski, Kyle Z; Armen, Roger S

    2017-06-05

    Following insights from recent crystal structures of the muscarinic acetylcholine receptor, binding modes of Positive Allosteric Modulators (PAMs) were predicted under the assumption that PAMs should bind to the extracellular surface of the active state. A series of well-characterized PAMs for adenosine (A 1 R, A 2A R, A 3 R) and muscarinic acetylcholine (M 1 R, M 5 R) receptors were modeled using both rigid and flexible receptor CHARMM-based molecular docking. Studies of adenosine receptors investigated the molecular basis of the probe-dependence of PAM activity by modeling in complex with specific agonist radioligands. Consensus binding modes map common pharmacophore features of several chemical series to specific binding interactions. These models provide a rationalization of how PAM binding slows agonist radioligand dissociation kinetics. M 1 R PAMs were predicted to bind in the analogous M 2 R PAM LY2119620 binding site. The M 5 R NAM (ML-375) was predicted to bind in the PAM (ML-380) binding site with a unique induced-fit receptor conformation. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling.

    Directory of Open Access Journals (Sweden)

    Siew Pheng Lim

    2016-08-01

    Full Text Available Flaviviruses comprise major emerging pathogens such as dengue virus (DENV or Zika virus (ZIKV. The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp domain of non-structural protein 5 (NS5. This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a "de novo" initiation mechanism. Crystal structures of the flavivirus RdRp revealed a "closed" conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the "GDD" active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed "N pocket". Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1-2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses.

  16. Exploring Covalent Allosteric Inhibition of Antigen 85C from Mycobacterium tuberculosis by Ebselen Derivatives.

    Science.gov (United States)

    Goins, Christopher M; Dajnowicz, Steven; Thanna, Sandeep; Sucheck, Steven J; Parks, Jerry M; Ronning, Donald R

    2017-05-12

    rearrangement due to covalent allosteric modification creates a sizable solvent network that encompasses the active site and extends to the modified Cys209 residue. In all, this study outlines factors that influence enzyme inhibition by ebselen and its derivatives while further highlighting the effects of the covalent modification of Cys209 by said inhibitors on the structure and stability of Ag85C. Furthermore, the results suggest a strategy for developing new classes of Ag85 inhibitors with increased specificity and potency.

  17. Mining-related sediment and soil contamination in a large Superfund site: Characterization, habitat implications, and remediation

    Science.gov (United States)

    Juracek, Kyle E.; Drake, K. D.

    2016-01-01

    Historical mining activity (1850–1970) in the now inactive Tri-State Mining District provided an ongoing source of lead and zinc to the environment including the US Environmental Protection Agency Superfund site located in Cherokee County, southeast Kansas, USA. The resultant contamination adversely affected biota and caused human health problems and risks. Remediation in the Superfund site requires an understanding of the magnitude and extent of contamination. To provide some of the required information, a series of sediment and soil investigations were conducted in and near the Superfund site to characterize lead and zinc contamination in the aquatic and floodplain environments along the main-stem Spring River and its major tributaries. In the Superfund site, the most pronounced lead and zinc contamination, with concentrations that far exceed sediment quality guidelines associated with potential adverse biological effects, was measured for streambed sediments and floodplain soils located within or downstream from the most intensive mining-affected areas. Tributary streambeds and floodplains in affected areas are heavily contaminated with some sites having lead and zinc concentrations that are an order of magnitude (or more) greater than the sediment quality guidelines. For the main-stem Spring River, the streambed is contaminated but the floodplain is mostly uncontaminated. Measured lead and zinc concentrations in streambed sediments, lakebed sediments, and floodplain soils documented a persistence of the post-mining contamination on a decadal timescale. These results provide a basis for the prioritization, development, and implementation of plans to remediate contamination in the affected aquatic and floodplain environments within the Superfund site.

  18. Mining-Related Sediment and Soil Contamination in a Large Superfund Site: Characterization, Habitat Implications, and Remediation.

    Science.gov (United States)

    Juracek, K E; Drake, K D

    2016-10-01

    Historical mining activity (1850-1970) in the now inactive Tri-State Mining District provided an ongoing source of lead and zinc to the environment including the US Environmental Protection Agency Superfund site located in Cherokee County, southeast Kansas, USA. The resultant contamination adversely affected biota and caused human health problems and risks. Remediation in the Superfund site requires an understanding of the magnitude and extent of contamination. To provide some of the required information, a series of sediment and soil investigations were conducted in and near the Superfund site to characterize lead and zinc contamination in the aquatic and floodplain environments along the main-stem Spring River and its major tributaries. In the Superfund site, the most pronounced lead and zinc contamination, with concentrations that far exceed sediment quality guidelines associated with potential adverse biological effects, was measured for streambed sediments and floodplain soils located within or downstream from the most intensive mining-affected areas. Tributary streambeds and floodplains in affected areas are heavily contaminated with some sites having lead and zinc concentrations that are an order of magnitude (or more) greater than the sediment quality guidelines. For the main-stem Spring River, the streambed is contaminated but the floodplain is mostly uncontaminated. Measured lead and zinc concentrations in streambed sediments, lakebed sediments, and floodplain soils documented a persistence of the post-mining contamination on a decadal timescale. These results provide a basis for the prioritization, development, and implementation of plans to remediate contamination in the affected aquatic and floodplain environments within the Superfund site.

  19. Characterization of an allosteric citalopram-binding site at the serotonin transporter

    DEFF Research Database (Denmark)

    Chen, Fenghua; Breum Larsen, Mads; Neubauer, Henrik Amtoft

    2005-01-01

    The serotonin transporter (SERT), which belongs to a family of       sodium/chloride-dependent transporters, is the major pharmacological       target in the treatment of several clinical disorders, including       depression and anxiety. In the present study we show that the dissociation       r...

  20. VU0477573: Partial Negative Allosteric Modulator of the Subtype 5 Metabotropic Glutamate Receptor with In Vivo Efficacy.

    Science.gov (United States)

    Nickols, Hilary Highfield; Yuh, Joannes P; Gregory, Karen J; Morrison, Ryan D; Bates, Brittney S; Stauffer, Shaun R; Emmitte, Kyle A; Bubser, Michael; Peng, Weimin; Nedelcovych, Michael T; Thompson, Analisa; Lv, Xiaohui; Xiang, Zixiu; Daniels, J Scott; Niswender, Colleen M; Lindsley, Craig W; Jones, Carrie K; Conn, P Jeffrey

    2016-01-01

    Negative allosteric modulators (NAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) have potential applications in the treatment of fragile X syndrome, levodopa-induced dyskinesia in Parkinson disease, Alzheimer disease, addiction, and anxiety; however, clinical and preclinical studies raise concerns that complete blockade of mGlu5 and inverse agonist activity of current mGlu5 NAMs contribute to adverse effects that limit the therapeutic use of these compounds. We report the discovery and characterization of a novel mGlu5 NAM, N,N-diethyl-5-((3-fluorophenyl)ethynyl)picolinamide (VU0477573) that binds to the same allosteric site as the prototypical mGlu5 NAM MPEP but displays weak negative cooperativity. Because of this weak cooperativity, VU0477573 acts as a "partial NAM" so that full occupancy of the MPEP site does not completely inhibit maximal effects of mGlu5 agonists on intracellular calcium mobilization, inositol phosphate (IP) accumulation, or inhibition of synaptic transmission at the hippocampal Schaffer collateral-CA1 synapse. Unlike previous mGlu5 NAMs, VU0477573 displays no inverse agonist activity assessed using measures of effects on basal [(3)H]inositol phosphate (IP) accumulation. VU0477573 acts as a full NAM when measuring effects on mGlu5-mediated extracellular signal-related kinases 1/2 phosphorylation, which may indicate functional bias. VU0477573 exhibits an excellent pharmacokinetic profile and good brain penetration in rodents and provides dose-dependent full mGlu5 occupancy in the central nervous system (CNS) with systemic administration. Interestingly, VU0477573 shows robust efficacy, comparable to the mGlu5 NAM MTEP, in models of anxiolytic activity at doses that provide full CNS occupancy of mGlu5 and demonstrate an excellent CNS occupancy-efficacy relationship. VU0477573 provides an exciting new tool to investigate the efficacy of partial NAMs in animal models. Copyright © 2015 by The American Society for Pharmacology and

  1. Tritium in a deciduous forest adjacent to a commercial shallow land burial site: implications for monitoring to detect radionuclide migration

    International Nuclear Information System (INIS)

    Rickard, W.H.; Kirby, L.J.

    1983-09-01

    Tritium as tritiated water was measured in the sap taken from the trunks of 26 maple trees growing in the vicinity of the shallow-land, low-level radioactive waste disposal site at Maxey Flats, Kentucky. Tritium values ranged between 10,000 and 290,000 pCi/l with the highest levels measured in sap from trees growing downslope from the burial site's western boundary. Levels of tritium of less than 1000 pCi/l were measured at a distance of 20 kilometers from the site: The source of elevated tritium levels in the vicinity of the disposal site is the evaporator facility which has released tritiated water vapor into the air more or less continuously for 10 years. Another possible source of at least some of the tritium is subterranean leakage from the trenches located near the western boundary. The evaporator facility has been shut down since December 1982. With the shutdown of the evaporator the levels of tritium in tree sap in future years is expected to show a marked decline as the tritiated soil water in the root zone becomes increasingly diluted with fresh rainwater and the residual tritium is dissipated to the air by evaporation and plant transpiration processes. 11 references

  2. Site productivity and forest carbon stocks in the United States: Analysis and implications for forest offset project planning

    Science.gov (United States)

    Coeli M. Hoover; James E. Smith

    2012-01-01

    The documented role of United States forests in sequestering carbon, the relatively low cost of forest-based mitigation, and the many co-benefits of increasing forest carbon stocks all contribute to the ongoing trend in the establishment of forest-based carbon offset projects. We present a broad analysis of forest inventory data using site quality indicators to provide...

  3. Chemostratigraphy at DSDP Sites 386 (Bermuda Rise) and 144 (Demerara Rise), Implications for Euxinic Conditions During OAE-2

    Science.gov (United States)

    Horst, P. A.; Maurrasse, F. J.; Sinninghe-Damsté, J. S.; Sandler, A.

    2008-05-01

    Chemostratigraphic studies of DSDP Site 386 on the Bermuda Rise and Site 144 on the Demerara Rise indicate that euxinic conditions developed at these deep-water sites during the time interval that corresponds to Oceanic Anoxic Event 2 (OAE 2). The data show a large increase in Fe/Al ratios, and dispersed pyrite aggregates (Site 386 Core 43, Section 3). Such findings at these deep oceanic sites are compatible with earlier studies showing that sediments in euxinic settings display increases in Fe/Al ratios due to the scavenging of dissolved Fe, and is also in agreement with previous Pr/Ph ratio of cyanobacteria showing low thermal stress, supporting in situ derivation. Elemental analyses at Site 386 also show that relatively high Sr/CaO ratios are present before and after OAE 2, indicating an increased contribution of biogenic carbonates, but not during the C/T boundary event. When Cr is plotted against Al2O3 in conjunction with a solid line representing the Cr/Al2O3 ratio in average shale, half of the samples fall above and half fall below this line. The values that plot above this line are all from Cores 47, 44, 43, and 42, which contain higher TOC. Their strong Cr enrichment with respect to the average shale can be indicative of an algal source of the OM, as this biota preferentially concentrates Cr. Competitive exclusion due to dominance of opportunistic prokaryotic blooms in combination with oxygen depletion can be invoked to explain the conditions that developed and were unfavorable to most other organisms throughout the water column during OAE 2. Sediments from DSDP Site 144 also reveal increased molecular fossils indicative of green sulfur bacteria, which are further characteristic of euxinic conditions (Kuypers et al., 2002; Forster et al., 2004). These results are in agreement with earlier works that showed lipids at DSDP Site 144 are predominantly of an autochthonous origin with primary production as the dominant source (Simoneit and Stuermer, 1982

  4. Alanine scanning of the rabies virus glycoprotein antigenic site III using recombinant rabies virus: implication for post-exposure treatment.

    Science.gov (United States)

    Papaneri, Amy B; Wirblich, Christoph; Marissen, Wilfred E; Schnell, Matthias J

    2013-12-02

    The safety and availability of the human polyclonal sera that is currently utilized for post-exposure treatment (PET) of rabies virus (RABV) infection remain a concern. Recombinant monoclonal antibodies have been postulated as suitable alternatives by WHO. To this extent, CL184, the RABV human antibody combination comprising monoclonal antibodies (mAbs) CR57 and CR4098, has been developed and has delivered promising clinical data to support its use for RABV PET. For this fully human IgG1 cocktail, mAbs CR57 and CR4098 are produced in the PER.C6 human cell line and combined in equal amounts in the final product. During preclinical evaluation, CR57 was shown to bind to antigenic site I whereas CR4098 neutralization was influenced by a mutation of position 336 (N336) located within antigenic site III. Here, alanine scanning was used to analyze the influence of mutations within the potential binding site for CR4098, antigenic site III, in order to evaluate the possibility of mutated rabies viruses escaping neutralization. For this approach, twenty flanking amino acids (10 upstream and 10 downstream) of the RABV glycoprotein (G) asparagine (N336) were exchanged to alanine (or serine, if already alanine) by site-directed mutagenesis. Analysis of G expression revealed four of the twenty mutant Gs to be non-functional, as shown by their lack of cell surface expression, which is a requirement for the production of infectious RABV. Therefore, these mutants were excluded from further study. The remaining sixteen mutants were introduced in an infectious clone of RABV, and recombinant RABVs (rRABVs) were recovered and utilized for in vitro neutralization assays. All of the viruses were effectively neutralized by CR4098 as well as by CR57, indicating that single amino acid exchanges in this region does not affect the broad neutralizing capability of the CL184 mAb combination. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. KatB, a cyanobacterial Mn-catalase with unique active site configuration: Implications for enzyme function.

    Science.gov (United States)

    Bihani, Subhash C; Chakravarty, Dhiman; Ballal, Anand

    2016-04-01

    Manganese catalases (Mn-catalases), a class of H2O2 detoxifying proteins, are structurally and mechanistically distinct from the commonly occurring catalases, which contain heme. Active site of Mn-catalases can serve as template for the synthesis of catalase mimetics for therapeutic intervention in oxidative stress related disorders. However, unlike the heme catalases, structural aspects of Mn-catalases remain inadequately explored. The genome of the ancient cyanobacterium Anabaena PCC7120, shows the presence of two Mn-catalases, KatA and KatB. Here, we report the biochemical and structural characterization of KatB. The KatB protein (with a C-terminal his-tag) was over-expressed in Escherichia coli and purified by affinity chromatography. On the addition of Mn(2+) to the E. coli growth medium, a substantial increase in production of the soluble KatB protein was observed. The purified KatB protein was an efficient catalase, which was relatively insensitive to inhibition by azide. Crystal structure of KatB showed a hexameric assembly with four-helix bundle fold, characteristic of the Ferritin-like superfamily. With canonical Glu4His2 coordination geometry and two terminal water ligands, the KatB active site was distinctly different from that of other Mn-catalases. Interestingly, the KatB active site closely resembled the active sites of ruberythrin/bacterioferritin, bi-iron members of the Ferritin-like superfamily. The KatB crystal structure provided fundamental insights into the evolutionary relationship within the Ferritin-like superfamily and further showed that Mn-catalases can be sub-divided into two groups, each with a distinct active site configuration. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Radiological and Environmental Monitoring at the Clean Slate I and III Sites, Tonopah Test Range, Nevada, With Emphasis on the Implications for Off-site Transport

    Energy Technology Data Exchange (ETDEWEB)

    Mizell, Steve A [Desert Research Inst. (DRI), Las Vegas, NV (United States); Etyemezian, Vic [Desert Research Inst. (DRI), Las Vegas, NV (United States); McCurdy, Greg [Desert Research Inst. (DRI), Las Vegas, NV (United States); Nikolich, George [Desert Research Inst. (DRI), Las Vegas, NV (United States); Shadel, Craig [Desert Research Inst. (DRI), Las Vegas, NV (United States); Miller, Julianne J [Desert Research Inst. (DRI), Las Vegas, NV (United States)

    2014-09-01

    In 1963, the U.S. Department of Energy (DOE) (formerly the Atomic Energy Commission [AEC]) implemented Operation Roller Coaster on the Tonopah Test Range (TTR) and an adjacent area of the Nevada Test and Training Range (NTTR) (formerly the Nellis Air Force Range [NAFR]). Operation Roller Coaster consisted of four tests in which chemical explosions were detonated in the presence of nuclear devices to assess the dispersal of radionuclides and evaluate the effectiveness of storage structures to contain the ejected radionuclides. These tests resulted in the dispersal of plutonium over the ground surface downwind of the test ground zero (GZ). Three tests—Clean Slate I, II, and III—were conducted on the TTR in Cactus Flat. The fourth, Double Tracks, was conducted in Stonewall Flat on the NTTR. The Desert Research Institute (DRI) installed two monitoring stations in 2008, Station 400 at the Sandia National Laboratories (SNL) Range Operations Center (ROC) and Station 401 at Clean Slate III. Station 402 was installed at Clean Slate I in 2011 to measure radiological, meteorological, and dust conditions. The monitoring activity was implemented to determine if radionuclide contamination in the soil at the Clean Slate sites was being transported beyond the contamination area boundaries. Some of the data collected also permits comparison of radiological exposure at the TTR monitoring stations to conditions observed at Community Environmental Monitoring Program (CEMP) stations around the NTTR. Annual average gross alpha values from the TTR monitoring stations are higher than values from the surrounding CEMP stations. Annual average gross beta values from the TTR monitoring stations are generally lower than values observed for the surrounding CEMP stations. This may be due to use of sample filters with larger pore space because when glass-fiber filters began to be used at TTR Station 400, gross beta values increased. Gamma spectroscopy typically identified only naturally

  7. Vegetative changes in boreal peatlands along salinity gradients resulting from produced water spills : implications for the environmental assessment and remediation of upstream oil and gas sites

    Energy Technology Data Exchange (ETDEWEB)

    Bright, D.; Harris, C.; Meier, M. [AECOM Canada Ltd., Ottawa, ON (Canada)

    2010-07-01

    In the province of Alberta, there are approximately 8,000 registered oil effluent and produced pipelines that have significant potential for ruptures and spills due to the highly corrosive nature of the emulsions and produced water they transport. Most releases occur in or adjacent to northern boreal wetlands. The first objective in spill response involves assessment and remediation for residuals, which involves handling and disposal of large volumes of salinized water. This presentation reported on a study that addressed the issues regarding the ecological features of semi-terrestrial and semi-aquatic components of boreal wetland environments as a basis for environmental protection at salt release sites. The fate of salt ions in such environments was examined along with the implications for secondary succession and ecological restoration. The study also examined the reasonable threshold concentration of salt ions in soils or water beyond which there is an inhibition to wetlands plants and bryophyte secondary succession; the reasonable threshold concentration of salt ions in water beyond which there may be adverse effects on invertebrates, vertebrates and other non-plant taxa; and species sensitivity distributions for floral and faunal assemblages found in boreal wetland habitats. The presentation summarized the 3 phases of a project that examined pipeline ruptures at 9 sites. Field methods and site sampling summaries were presented. It was difficult to locate study sites with residual salt contamination in surface media at concentrations above effects threshold for many species. It was concluded that the departures between surface and subsurface salinity indicates a smaller potential for effects on site vegetation. tabs., figs.

  8. Vegetative changes in boreal peatlands along salinity gradients resulting from produced water spills : implications for the environmental assessment and remediation of upstream oil and gas sites

    International Nuclear Information System (INIS)

    Bright, D.; Harris, C.; Meier, M.

    2010-01-01

    In the province of Alberta, there are approximately 8,000 registered oil effluent and produced pipelines that have significant potential for ruptures and spills due to the highly corrosive nature of the emulsions and produced water they transport. Most releases occur in or adjacent to northern boreal wetlands. The first objective in spill response involves assessment and remediation for residuals, which involves handling and disposal of large volumes of salinized water. This presentation reported on a study that addressed the issues regarding the ecological features of semi-terrestrial and semi-aquatic components of boreal wetland environments as a basis for environmental protection at salt release sites. The fate of salt ions in such environments was examined along with the implications for secondary succession and ecological restoration. The study also examined the reasonable threshold concentration of salt ions in soils or water beyond which there is an inhibition to wetlands plants and bryophyte secondary succession; the reasonable threshold concentration of salt ions in water beyond which there may be adverse effects on invertebrates, vertebrates and other non-plant taxa; and species sensitivity distributions for floral and faunal assemblages found in boreal wetland habitats. The presentation summarized the 3 phases of a project that examined pipeline ruptures at 9 sites. Field methods and site sampling summaries were presented. It was difficult to locate study sites with residual salt contamination in surface media at concentrations above effects threshold for many species. It was concluded that the departures between surface and subsurface salinity indicates a smaller potential for effects on site vegetation. tabs., figs.

  9. Size-Frequency Distributions of Rocks on Mars and Earth Analog Sites: Implications for Future Landed Missions

    Science.gov (United States)

    Golombeck, M.; Rapp, D.

    1996-01-01

    The size-frequency distribution of rocks and the Vicking landing sites and a variety of rocky locations on the Earth that formed from a number of geologic processes all have the general shape of simple exponential curves, which have been combined with remote sensing data and models on rock abundance to predict the frequency of boulders potentially hazardous to future Mars landers and rovers.

  10. ‘All Museums Will Become Department Stores’: The Development and Implications of Retailing at Museums and Heritage Sites

    OpenAIRE

    Larkin, Jamie

    2016-01-01

    Museums and heritage sites have provided merchandise for visitors to purchase since their earliest incarnations as public attractions in the 18th century. Despite this longevity scant academic research has been directed towards such activities. However, retailing – formalised in the emergence of the museum shop – offers insights into a range of issues, from cultural representation and education, to eco- nomic sustainability. This paper outlines the historical development of retailing at museu...

  11. Sites of local recurrence after surgery, with or without chemotherapy, for rectal cancer: implications for radiotherapy field design

    International Nuclear Information System (INIS)

    Hruby, George; Barton, Michael; Miles, Sharon; Carroll, Susan; Nasser, Elias; Stevens, Graham

    2003-01-01

    Purpose: To examine the sites of pelvic recurrence in patients with rectal cancer previously untreated with radiotherapy to determine the relative frequency and location of recurrence within the pelvis. Methods and Materials: The records of patients with locally recurrent rectal cancer referred to three radiation oncology departments between 1984 and 1997 were reviewed. The data collected included the date and type of the initial resection and the pathologic findings. The site of recurrence within the pelvis, presence of metastasis, and date of recurrence were documented. Results: A total of 269 patients were included. Tumor had invaded through the muscularis in 74% and involved other organs in 9%. Fifty-two percent of patients were node positive at initial surgery. The median time to local recurrence from surgery was 18 months (range 15-20) and from local recurrence to death was 14 months (range 12-17). Both the initial tumor stage and the resection type influenced the recurrence location within the pelvis (p<0.01). T4 tumors comprised only 9% of initial T stage tumors but accounted for 38% of anterior central pelvic recurrences (p<0.01). All perineal recurrences occurred after abdominoperineal resection. The sites of recurrence within the pelvis were the posterior central pelvis (47%) and anastomotic (21%). Conclusion: If those patients with T4 tumors at presentation were excluded, 89% had local recurrence at, or posterior to, the anastomosis. Furthermore, if we exclude both patients who underwent abdominoperineal resection and those with T4 tumors at presentation, the rate increases to 93%. The rate of recurrence anteriorly (7%) does not justify routine radiation of the anterior pelvis beyond that required to adequately cover the anastomotic site

  12. A survey of the practice of nurses' skills in Wenchuan earthquake disaster sites: implications for disaster training.

    Science.gov (United States)

    Yin, Huahua; He, Haiyan; Arbon, Paul; Zhu, Jingci

    2011-10-01

    To determine nursing skills most relevant for nurses participating in disaster response medical teams; make recommendations to enhance training of nurses who will be first responders to a disaster site; to improve the capacity of nurses to prepare and respond to severe natural disasters. Worldwide, nurses play a key role in disaster response teams at disaster sites. They are often not prepared for the challenges of dealing with mass casualties; little research exists into what basic nursing skills are required by nurses who are first responders to a disaster situation. This study assessed the most relevant disaster nursing skills of first responder nurses at the 2008 Wenchuan earthquake disaster site. Data were collected in China in 2008 using a self-designed questionnaire, with 24 participants who had been part of the medical teams that were dispatched to the disaster sites. The top three skills essential for nurses were: intravenous insertion; observation and monitoring; mass casualty triage. The three most frequently used skills were: debridement and dressing; observation and monitoring; intravenous insertion. The three skills performed most proficiently were: intravenous insertion; observation and monitoring; urethral catheterization. The top three ranking skills most important for training were: mass casualty transportation; emergency management; haemostasis, bandaging, fixation, manual handling. The core nursing skills for disaster response training are: mass casualty transportation; emergency management; haemostasis, bandaging, fixation, manual handling; observation and monitoring; mass casualty triage; controlling specific infection; psychological crisis intervention; cardiopulmonary resuscitation; debridement and dressing; central venous catheter insertion; patient care recording. © 2011 The Authors. Journal of Advanced Nursing © 2011 Blackwell Publishing Ltd.

  13. Formation of 3D cholesterol crystals from 2D nucleation sites in lipid bilayer membranes: implications for atherosclerosis.

    Science.gov (United States)

    Varsano, Neta; Fargion, Iael; Wolf, Sharon G; Leiserowitz, Leslie; Addadi, Lia

    2015-02-04

    Atherosclerosis is the major precursor of cardiovascular disease. The formation of cholesterol crystals in atherosclerotic plaques is associated with the onset of acute pathology. The cholesterol crystals induce physical injury in the plaque core, promoting cell apoptosis and triggering an increased inflammatory response. Herein we address the question of how cholesterol crystal formation occurs in atherosclerosis. We demonstrate that three-dimensional (3D) cholesterol crystals can undergo directed nucleation from bilayer membranes containing two-dimensional (2D) cholesterol crystalline domains. We studied crystal formation on supported lipid bilayers loaded with exogenous cholesterol and labeled using a monoclonal antibody that specifically recognizes ordered cholesterol arrays. Our findings show that 3D crystals are formed exclusively on the bilayer regions where there are segregated 2D cholesterol crystalline domains and that they form on the domains. This study has potentially significant implications for our understanding of the crucial step in the mechanism by which atherosclerotic lesions form.

  14. Sniffer patch laser uncaging response (SPLURgE): an assay of regional differences in allosteric receptor modulation and neurotransmitter clearance.

    Science.gov (United States)

    Christian, Catherine A; Huguenard, John R

    2013-10-01

    Allosteric modulators exert actions on neurotransmitter receptors by positively or negatively altering the effective response of these receptors to their respective neurotransmitter. γ-Aminobutyric acid (GABA) type A ionotropic receptors (GABAARs) are major targets for allosteric modulators such as benzodiazepines, neurosteroids, and barbiturates. Analysis of substances that produce similar effects has been hampered by the lack of techniques to assess the localization and function of such agents in brain slices. Here we describe measurement of the sniffer patch laser uncaging response (SPLURgE), which combines the sniffer patch recording configuration with laser photolysis of caged GABA. This methodology enables the detection of allosteric GABAAR modulators endogenously present in discrete areas of the brain slice and allows for the application of exogenous GABA with spatiotemporal control without altering the release and localization of endogenous modulators within the slice. Here we demonstrate the development and use of this technique for the measurement of allosteric modulation in different areas of the thalamus. Application of this technique will be useful in determining whether a lack of modulatory effect on a particular category of neurons or receptors is due to insensitivity to allosteric modulation or a lack of local release of endogenous ligand. We also demonstrate that this technique can be used to investigate GABA diffusion and uptake. This method thus provides a biosensor assay for rapid detection of endogenous GABAAR modulators and has the potential to aid studies of allosteric modulators that exert effects on other classes of neurotransmitter receptors, such as glutamate, acetylcholine, or glycine receptors.

  15. Bay breeze climatology at two sites along the Chesapeake bay from 1986-2010: Implications for surface ozone.

    Science.gov (United States)

    Stauffer, Ryan M; Thompson, Anne M

    Hourly surface meteorological measurements were coupled with surface ozone (O 3 ) mixing ratio measurements at Hampton, Virginia and Baltimore, Maryland, two sites along the Chesapeake Bay in the Mid-Atlantic United States, to examine the behavior of surface O 3 during bay breeze events and quantify the impact of the bay breeze on local O 3 pollution. Analyses were performed for the months of May through September for the years 1986 to 2010. The years were split into three groups to account for increasingly stringent environmental regulations that reduced regional emissions of nitrogen oxides (NO x ): 1986-1994, 1995-2002, and 2003-2010. Each day in the 25-year record was marked either as a bay breeze day, a non-bay breeze day, or a rainy/cloudy day based on the meteorological data. Mean eight hour (8-h) averaged surface O 3 values during bay breeze events were 3 to 5 parts per billion by volume (ppbv) higher at Hampton and Baltimore than on non-bay breeze days in all year periods. Anomalies from mean surface O 3 were highest in the afternoon at both sites during bay breeze days in the 2003-2010 study period. In conjunction with an overall lowering of baseline O 3 after the 1995-2002 period, the percentage of total exceedances of the Environmental Protection Agency (EPA) 75 ppbv 8-h O 3 standard that occurred on bay breeze days increased at Hampton for 2003-2010, while remaining steady at Baltimore. These results suggest that bay breeze circulations are becoming more important to causing exceedance events at particular sites in the region, and support the hypothesis of Martins et al. (2012) that highly localized meteorology increasingly drives air quality events at Hampton.

  16. Environmental hazard assessment of a marine mine tailings deposit site and potential implications for deep-sea mining.

    Science.gov (United States)

    Mestre, Nélia C; Rocha, Thiago L; Canals, Miquel; Cardoso, Cátia; Danovaro, Roberto; Dell'Anno, Antonio; Gambi, Cristina; Regoli, Francesco; Sanchez-Vidal, Anna; Bebianno, Maria João

    2017-09-01

    Portmán Bay is a heavily contaminated area resulting from decades of metal mine tailings disposal, and is considered a suitable shallow-water analogue to investigate the potential ecotoxicological impact of deep-sea mining. Resuspension plumes were artificially created by removing the top layer of the mine tailings deposit by bottom trawling. Mussels were deployed at three sites: i) off the mine tailings deposit area; ii) on the mine tailings deposit beyond the influence from the resuspension plumes; iii) under the influence of the artificially generated resuspension plumes. Surface sediment samples were collected at the same sites for metal analysis and ecotoxicity assessment. Metal concentrations and a battery of biomarkers (oxidative stress, metal exposure, biotransformation and oxidative damage) were measured in different mussel tissues. The environmental hazard posed by the resuspension plumes was investigated by a quantitative weight of evidence (WOE) model that integrated all the data. The resuspension of sediments loaded with metal mine tails demonstrated that chemical contaminants were released by trawling subsequently inducing ecotoxicological impact in mussels' health. Considering as sediment quality guidelines (SQGs) those indicated in Spanish action level B for the disposal of dredged material at sea, the WOE model indicates that the hazard is slight off the mine tailings deposit, moderate on the mine tailings deposit without the influence from the resuspension plumes, and major under the influence of the resuspension plumes. Portmán Bay mine tailings deposit is a by-product of sulphide mining, and despite differences in environmental setting, it can reflect the potential ecotoxic effects to marine fauna from the impact of resuspension of plumes created by deep-sea mining of polymetallic sulphides. A similar approach as in this study could be applied in other areas affected by sediment resuspension and for testing future deep-sea mining sites in

  17. Networks of high mutual information define the structural proximity of catalytic sites: implications for catalytic residue identification.

    Directory of Open Access Journals (Sweden)

    Cristina Marino Buslje

    Full Text Available Identification of catalytic residues (CR is essential for the characterization of enzyme function. CR are, in general, conserved and located in the functional site of a protein in order to attain their function. However, many non-catalytic residues are highly conserved and not all CR are conserved throughout a given protein family making identification of CR a challenging task. Here, we put forward the hypothesis that CR carry a particular signature defined by networks of close proximity residues with high mutual information (MI, and that this signature can be applied to distinguish functional from other non-functional conserved residues. Using a data set of 434 Pfam families included in the catalytic site atlas (CSA database, we tested this hypothesis and demonstrated that MI can complement amino acid conservation scores to detect CR. The Kullback-Leibler (KL conservation measurement was shown to significantly outperform both the Shannon entropy and maximal frequency measurements. Residues in the proximity of catalytic sites were shown to be rich in shared MI. A structural proximity MI average score (termed pMI was demonstrated to be a strong predictor for CR, thus confirming the proposed hypothesis. A structural proximity conservation average score (termed pC was also calculated and demonstrated to carry distinct information from pMI. A catalytic likeliness score (Cls, combining the KL, pC and pMI measures, was shown to lead to significantly improved prediction accuracy. At a specificity of 0.90, the Cls method was found to have a sensitivity of 0.816. In summary, we demonstrate that networks of residues with high MI provide a distinct signature on CR and propose that such a signature should be present in other classes of functional residues where the requirement to maintain a particular function places limitations on the diversification of the structural environment along the course of evolution.

  18. Networks of high mutual information define the structural proximity of catalytic sites: implications for catalytic residue identification.

    Science.gov (United States)

    Marino Buslje, Cristina; Teppa, Elin; Di Doménico, Tomas; Delfino, José María; Nielsen, Morten

    2010-11-04

    Identification of catalytic residues (CR) is essential for the characterization of enzyme function. CR are, in general, conserved and located in the functional site of a protein in order to attain their function. However, many non-catalytic residues are highly conserved and not all CR are conserved throughout a given protein family making identification of CR a challenging task. Here, we put forward the hypothesis that CR carry a particular signature defined by networks of close proximity residues with high mutual information (MI), and that this signature can be applied to distinguish functional from other non-functional conserved residues. Using a data set of 434 Pfam families included in the catalytic site atlas (CSA) database, we tested this hypothesis and demonstrated that MI can complement amino acid conservation scores to detect CR. The Kullback-Leibler (KL) conservation measurement was shown to significantly outperform both the Shannon entropy and maximal frequency measurements. Residues in the proximity of catalytic sites were shown to be rich in shared MI. A structural proximity MI average score (termed pMI) was demonstrated to be a strong predictor for CR, thus confirming the proposed hypothesis. A structural proximity conservation average score (termed pC) was also calculated and demonstrated to carry distinct information from pMI. A catalytic likeliness score (Cls), combining the KL, pC and pMI measures, was shown to lead to significantly improved prediction accuracy. At a specificity of 0.90, the Cls method was found to have a sensitivity of 0.816. In summary, we demonstrate that networks of residues with high MI provide a distinct signature on CR and propose that such a signature should be present in other classes of functional residues where the requirement to maintain a particular function places limitations on the diversification of the structural environment along the course of evolution.

  19. Directional topographic site response at Tarzana observed in aftershocks of the 1994 Northridge, California, earthquake: Implications for mainshock motions

    Science.gov (United States)

    Spudich, P.; Hellweg, M.; Lee, W.H.K.

    1996-01-01

    The Northridge earthquake caused 1.78 g acceleration in the east-west direction at a site in Tarzana, California, located about 6 km south of the mainshock epicenter. The accelerograph was located atop a hill about 15-m high, 500-m long, and 130-m wide, striking about N78??E. During the aftershock sequence, a temporary array of 21 three-component geophones was deployed in six radial lines centered on the accelerograph, with an average sensor spacing of 35 m. Station COO was located about 2 m from the accelerograph. We inverted aftershock spectra to obtain average relative site response at each station as a function of direction of ground motion. We identified a 3.2-Hz resonance that is a transverse oscillation of the hill (a directional topographic effect). The top/base amplification ratio at 3.2 Hz is about 4.5 for horizontal ground motions oriented approximately perpendicular to the long axis of the hill and about 2 for motions parallel to the hill. This resonance is seen most strongly within 50 m of COO. Other resonant frequencies were also observed. A strong lateral variation in attenuation, probably associated with a fault, caused substantially lower motion at frequencies above 6 Hz at the east end of the hill. There may be some additional scattered waves associated with the fault zone and seen at both the base and top of the hill, causing particle motions (not spectral ratios) at the top of the hill to be rotated about 20?? away from the direction transverse to the hill. The resonant frequency, but not the amplitude, of our observed topographic resonance agrees well with theory, even for such a low hill. Comparisons of our observations with theoretical results indicate that the 3D shape of the hill and its internal structure are important factors affecting its response. The strong transverse resonance of the hill does not account for the large east-west mainshock motions. Assuming linear soil response, mainshock east-west motions at the Tarzana accelerograph

  20. Networks of High Mutual Information Define the Structural Proximity of Catalytic Sites: Implications for Catalytic Residue Identification

    DEFF Research Database (Denmark)

    Buslje, Cristina Marino; Teppa, Elin; Di Doménico, Tomas

    2010-01-01

    other non-functional conserved residues. Using a data set of 434 Pfam families included in the catalytic site atlas (CSA) database, we tested this hypothesis and demonstrated that MI can complement amino acid conservation scores to detect CR. The Kullback-Leibler (KL) conservation measurement was shown.......90, the Cls method was found to have a sensitivity of 0.816. In summary, we demonstrate that networks of residues with high MI provide a distinct signature on CR and propose that such a signature should be present in other classes of functional residues where the requirement to maintain a particular function...

  1. Thyroid nodules in the population living around semipalatinsk nuclear test site. Possible implications for dose-response relationships study

    International Nuclear Information System (INIS)

    Zhumadilov, Z.

    2006-01-01

    The risk of radiation-induced nodules is higher than the risk for radiation-induced cancer. Risk factors and specific modifiers of the dose-response relationship may vary among different populations and not be well recognized. Many thyroid studies have considered thyroid nodularity itself, but not specific morphological types of thyroid nodules. There are many specific types of thyroid nodules which follow a morphological classification of thyroid lesions, including some congenital and tumor-like conditions. Modern equipment and technique can help us to identify particular specific types of thyroid nodules. In this study we report some results of a clinically applicable approach to materials derived from three studies. From 1999 through 2002, we have screened 571 current residents from 4 exposed and 1 control village near the Semipalatinsk Nuclear Test Site area, who were of similar ages (<20) at the time of major radiation fallout events at the Semipalatinsk Nuclear Test Site (SNTS). Prevalent nodules were identified by ultrasound and fine-needle aspiration biopsy, cytopathology results. Analysis of ultrasound images and cytopathology of thyroid lesions among exposed and non-exposed population allowed us to distinguish some interesting ultrasound features for specific types of thyroid nodules. We believe that it would be interesting and possibly more informative for thyroid dosimetry studies to consider specific morphological types of thyroid nodules. We need more detailed research to clarify the feasibility of applying these findings for study of the dose-response relationship. (author)

  2. Physical and numerical modelling of permafrost dynamic during a climatic cycle: implications for Meuse - Haute-Marne site

    International Nuclear Information System (INIS)

    Regnier, D.

    2012-01-01

    This manuscript deals about works realized on the permafrost modelling in porous media and its impact on the hydrogeological circulations. These are parts of the Andra's studies on the nuclear waste storage and, on the environmental studies of the Meuse/Haute-Marne (MHM) site. During a climatic cycle, cold periods can generate permafrost (ground with temperature lower than 0 C for 2 consecutive years). This peri-glacial structure propagates towards deep geological layers, and, due to its very low permeability, can stop the flow of water bodies like aquifers. This work presents the elaboration of two numerical models (with Cast3M code (CEA)): (i) a model with thermal conduction, used for the study of a cold wave propagation in porous media with phase transition (water-ice); (ii) a more complex model, managing the thermo-hydraulic coupling of ground phenomenon (conduction, convection and transition of phase). After validation, these two models offer three axes of development: (i) benchmark proposition by the study of two generic test-cases; (ii) study of the local air temperature signal on MHM site: importance of high frequency temperature variations (centennial scale) for permafrost depth and stability; (iii) study of the dynamics of a thermal discontinuity in a typical hydrological system river-plain: closure time of the system by the permafrost according to various parameters (temperatures, geothermal flow, hydrological flow directions). (author) [fr

  3. Spatial evaluation of larvae of Culicidae (Diptera from different breeding sites: application of a geospatial method and implications for vector control

    Directory of Open Access Journals (Sweden)

    Rafael Piovezan

    2012-09-01

    Full Text Available Spatial evaluation of Culicidae (Diptera larvae from different breeding sites: application of a geospatial method and implications for vector control. This study investigates the spatial distribution of urban Culicidae and informs entomological monitoring of species that use artificial containers as larval habitats. Collections of mosquito larvae were conducted in the São Paulo State municipality of Santa Bárbara d' Oeste between 2004 and 2006 during house-to-house visits. A total of 1,891 samples and nine different species were sampled. Species distribution was assessed using the kriging statistical method by extrapolating municipal administrative divisions. The sampling method followed the norms of the municipal health services of the Ministry of Health and can thus be adopted by public health authorities in disease control and delimitation of risk areas. Moreover, this type of survey and analysis can be employed for entomological surveillance of urban vectors that use artificial containers as larval habitat.

  4. A Coincidence Detection Mechanism Controls PX-BAR Domain-Mediated Endocytic Membrane Remodeling via an Allosteric Structural Switch.

    Science.gov (United States)

    Lo, Wen-Ting; Vujičić Žagar, Andreja; Gerth, Fabian; Lehmann, Martin; Puchkov, Dymtro; Krylova, Oxana; Freund, Christian; Scapozza, Leonardo; Vadas, Oscar; Haucke, Volker

    2017-11-20

    Clathrin-mediated endocytosis occurs by bending and remodeling of the membrane underneath the coat. Bin-amphiphysin-rvs (BAR) domain proteins are crucial for endocytic membrane remodeling, but how their activity is spatiotemporally controlled is largely unknown. We demonstrate that the membrane remodeling activity of sorting nexin 9 (SNX9), a late-acting endocytic PX-BAR domain protein required for constriction of U-shaped endocytic intermediates, is controlled by an allosteric structural switch involving coincident detection of the clathrin adaptor AP2 and phosphatidylinositol-3,4-bisphosphate (PI(3,4)P 2 ) at endocytic sites. Structural, biochemical, and cell biological data show that SNX9 is autoinhibited in solution. Binding to PI(3,4)P 2 via its PX-BAR domain, and concomitant association with AP2 via sequences in the linker region, releases SNX9 autoinhibitory contacts to enable membrane constriction. Our results reveal a mechanism for restricting the latent membrane remodeling activity of BAR domain proteins to allow spatiotemporal coupling of membrane constriction to the progression of the endocytic pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Abacavir and warfarin modulate allosterically kinetics of NO dissociation from ferrous nitrosylated human serum heme-albumin

    International Nuclear Information System (INIS)

    Ascenzi, Paolo; Imperi, Francesco; Coletta, Massimo; Fasano, Mauro

    2008-01-01

    Human serum albumin (HSA) participates to heme scavenging, in turn HSA-heme binds gaseous diatomic ligands at the heme-Fe-atom. Here, the effect of abacavir and warfarin on denitrosylation kinetics of HSA-heme-Fe(II)-NO (i.e., k off ) is reported. In the absence of drugs, the value of k off is (1.3 ± 0.2) x 10 -4 s -1 . Abacavir and warfarin facilitate NO dissociation from HSA-heme-Fe(II)-NO, the k off value increases to (8.6 ± 0.9) x 10 -4 s -1 . From the dependence of k off on the drug concentration, values of the dissociation equilibrium constant for the abacavir and warfarin binding to HSA-heme-Fe(II)-NO (i.e., K = (1.2 ± 0.2) x 10 -3 M and (6.2 ± 0.7) x 10 -5 M, respectively) were determined. The increase of k off values reflects the stabilization of the basic form of HSA-heme-Fe by ligands (e.g., abacavir and warfarin) that bind to Sudlow's site I. This event parallels the stabilization of the six-coordinate derivative of the HSA-heme-Fe(II)-NO atom. Present data highlight the allosteric modulation of HSA-heme-Fe(II) reactivity by heterotropic effectors

  6. Molecular sampling of the allosteric binding pocket of the TSH receptor provides discriminative pharmacophores for antagonist and agonists.

    Science.gov (United States)

    Hoyer, Inna; Haas, Ann-Karin; Kreuchwig, Annika; Schülein, Ralf; Krause, Gerd

    2013-02-01

    The TSHR (thyrotropin receptor) is activated endogenously by the large hormone thyrotropin and activated pathologically by auto-antibodies. Both activate and bind at the extracellular domain. Recently, SMLs (small-molecule ligands) have been identified, which bind in an allosteric binding pocket within the transmembrane domain. Modelling driven site-directed mutagenesis of amino acids lining this pocket led to the delineation of activation and inactivation sensitive residues. Modified residues showing CAMs (constitutively activating mutations) indicate signalling-sensitive positions and mark potential trigger points for agonists. Silencing mutations lead to an impairment of basal activity and mark contact points for antagonists. Mapping these residues on to a structural model of TSHR indicates locations where an SML may switch the receptor to an inactive or active conformation. In the present article, we report the effects of SMLs on these signalling-sensitive amino acids at the TSHR. Surprisingly, the antagonistic effect of SML compound 52 was reversed to an agonistic effect, when tested at the CAM Y667A. Switching agonism to antagonism and the reverse by changing either SMLs or residues covering the binding pocket provides detailed knowledge about discriminative pharmacophores. It prepares the basis for rational optimization of new high-affinity antagonists to interfere with the pathogenic activation of the TSHR.

  7. Tissue factor activates allosteric networks in factor VIIa through structural and dynamic changes

    DEFF Research Database (Denmark)

    Madsen, Jesper Jonasson; Persson, E.; Olsen, O. H.

    2015-01-01

    that are not likely to be inferred from mutagenesis studies. Furthermore, paths from Met306 to Ile153 (N-terminus) and Trp364, both representing hallmark residues of allostery, are 7% and 37% longer, respectively, in free FVIIa. Thus, there is significantly weaker coupling between the TF contact point and key......Background: Tissue factor (TF) promotes colocalization of enzyme (factorVIIa) and substrate (FX or FIX), and stabilizes the active conformation of FVIIa. Details on how TF induces structural and dynamic changes in the catalytic domain of FVIIa to enhance its efficiency remain elusive. Objective......: To elucidate the activation of allosteric networks in the catalytic domain of the FVIIa protease it is when bound to TF.MethodsLong-timescale molecular dynamics simulations of FVIIa, free and in complex with TF, were executed and analyzed by dynamic network analysis. Results: Allosteric paths of correlated...

  8. Atmospheric mercury concentration and chemical speciation at a rural site in Beijing, China: implications of mercury emission sources

    Directory of Open Access Journals (Sweden)

    L. Zhang

    2013-10-01

    Full Text Available Continuous measurements of atmospheric mercury concentration and speciation play a key role in identifying mercury sources and its behavior in the atmosphere. In this study, speciated atmospheric mercury including gaseous elemental mercury (GEM, reactive gaseous mercury (RGM and particle-bound mercury (PBM were continuously measured at Miyun, a rural site in Beijing, China, from December 2008 to November 2009. The average GEM, RGM and PBM concentrations were found to be 3.22 ± 1.74, 10.1 ± 18.8 and 98.2 ± 112.7 pg m−3, respectively, about 2–20 times higher than the background concentration of the Northern Hemisphere. The results indicated that atmospheric mercury concentrations in northern China were highly affected by anthropogenic emissions. The atmospheric mercury showed obvious seasonal variations, with the highest seasonal average GEM concentration in summer (3.48 ng m−3 and the lowest value in winter (2.66 ng m−3. In autumn and winter a diurnal variation of GEM was observed, with peak levels in the late afternoon till midnight. Most of the high RGM concentration values occurred in the afternoon of all seasons due to the higher oxidation. The PBM concentration was higher in early morning of all seasons because of the the temperature inversion that increases in depth as the night proceeds. The ratio of GEM to CO indicates that residential boilers play an important role in the elevation of GEM in winter. The ratio of RGM to O3 could be an indicator of the contribution of local primary sources. The ratio of PBM to PM2.5 reveals that the air mass from the east and southwest of the site in spring and summer carries more atmospheric mercury. The HYSPLIT back-trajectory analysis indicated that the monitoring site is affected by local, regional and interregional sources simultaneously during heavy pollution episodes. The results from the potential source contribution function (PSCF model indicate that the atmospheric transport

  9. mGluR5 Positive Allosteric Modulation Enhances Extinction Learning Following Cocaine Self-Administration

    OpenAIRE

    Cleva, Richard M.; Hicks, Megan P.; Gass, Justin T.; Wischerath, Kelly C.; Plasters, Elizabeth T.; Widholm, John J.; Olive, M. Foster

    2011-01-01

    Extinction of classically and instrumentally conditioned behaviors, such as conditioned fear and drug-seeking behavior, is a process of active learning, and recent studies indicate that potentiation of glutamatergic transmission facilitates extinction learning. In this study we investigated the effects of the type 5 metabotropic glutamate receptors (mGluR5) positive allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) on the extinction of cocaine-seeking behavior in ...

  10. The therapeutic potential of allosteric ligands for free fatty acid sensitive GPCRs

    DEFF Research Database (Denmark)

    Hudson, Brian D; Ulven, Trond; Milligan, Graeme

    2013-01-01

    G protein coupled receptors (GPCRs) are the most historically successful therapeutic targets. Despite this success there are many important aspects of GPCR pharmacology and function that have yet to be exploited to their full therapeutic potential. One in particular that has been gaining attention...... safety, more physiologically appropriate responses, better target selectivity, and reduced likelihood of desensitisation and tachyphylaxis. Despite these advantages, the development of allosteric ligands is often difficult from a medicinal chemistry standpoint due to the more complex challenge...

  11. Modulation in selectivity and allosteric properties of small-molecule ligands for CC-chemokine receptors

    DEFF Research Database (Denmark)

    Thiele, Stefanie; Malmgaard-Clausen, Mikkel; Engel-Andreasen, Jens

    2012-01-01

    Among 18 human chemokine receptors, CCR1, CCR4, CCR5, and CCR8 were activated by metal ion Zn(II) or Cu(II) in complex with 2,2'-bipyridine or 1,10-phenanthroline with similar potencies (EC(50) from 3.9 to 172 μM). Besides being agonists, they acted as selective allosteric enhancers of CCL3. Thes...

  12. Structure and allosteric effects of low-molecular-weight activators on the protein kinase PDK1

    DEFF Research Database (Denmark)

    Hindie, Valerie; Stroba, Adriana; Zhang, Hua

    2009-01-01

    -dependent activation of AGC kinases. The AGC kinase PDK1 is activated by the docking of a phosphorylated motif from substrates. Here we present the crystallography of PDK1 bound to a rationally developed low-molecular-weight activator and describe the conformational changes induced by small compounds in the crystal...... molecular details of the allosteric changes induced by small compounds that trigger the activation of PDK1 through mimicry of phosphorylation-dependent conformational changes....

  13. The effects of site, supplemental food, and age on survivorship of Carolina Chickadees and implications for dispersal through- riparian corridors

    Science.gov (United States)

    Doherty, P.F.; Grubb, T.G.

    2000-01-01

    Few studies have examined survivorship of animals in forest fragments differing in size, and none has used appropriate mark-recapture analysis techniques taking into account probability of recapture. Using Program MARK, a flexible mark-recapture software package, we estimated annual survival rates of Carolina Chickadees over a 5-yr period in a fragmented landscape in Ohio. The probability of survival was related to site (riparian woodland or woodlot area) and increased with the presence of supplemental food. While there was little evidence for an age difference in apparent survival in woodlots, young birds appeared to survive less well in forested river corridors. This last result was quite likely due, at least in part, to age-specific dispersal, suggesting that river corridors function as important dispersal routes for young birds.

  14. A Review of HIV Prevention Studies that Use Social Networking Sites: Implications for Recruitment, Health Promotion Campaigns, and Efficacy Trials.

    Science.gov (United States)

    Jones, Jamal; Salazar, Laura F

    2016-11-01

    This review describes the use of social networking sites (SNS) in the context of primary prevention of HIV. A review was conducted to assess the published literature for HIV interventions using SNS. Sixteen articles describing twelve interventions were included. SNS were instrumental in recruiting hard-to-reach populations within a short amount of time; were able to reach wide audiences beyond the targeted population for HIV prevention campaigns; and helped to significantly reduce sexual risk behaviors and increase HIV testing. SNS are a viable option to recruit hidden populations, engage the target audience, and disseminate HIV prevention messages. Researchers should use SNS to generate sampling frames that can be used to select participants. Practitioners should use SNS to post images of preventive behavior within health promotion campaigns. Researchers should use multiple SNS platforms to engage participants. As more studies are published using SNS for HIV prevention, meta-analyses will be needed.

  15. Prevalence of conduction delay of the right atrium in patients with SSS: implications for pacing site selection.

    Science.gov (United States)

    Verlato, Roberto; Zanon, Francesco; Bertaglia, Emanuele; Turrini, Pietro; Baccillieri, Maria Stella; Baracca, Enrico; Bongiorni, Maria Grazia; Zampiero, Aldo; Zonzin, Pietro; Pascotto, Pietro; Venturini, Diego; Corbucci, Giorgio

    2007-09-01

    To evaluate the prevalence of severe right atrial conduction delay in patients with sinus node dysfunction (SND) and atrial fibrillation (AF) and the effects of pacing in the right atrial appendage (RAA) and in the inter-atrial septum (IAS). Forty-two patients (15 male, 72 +/- 7 years) underwent electrophysiologic study to measure the difference between the conduction time from RAA to coronary sinus ostium during stimulation at 600 ms and after extrastimulus (DeltaCTos). Patients were classified as group A if DeltaCTos > 60 ms and group B if IAS pacing and algorithms ON/OFF. Fifteen patients (36%, group A) had DeltaCTos = 76 +/- 11 ms and 27 patients (64%, group B) had DeltaCTos = 36 +/- 20 ms. Twenty-two patients were paced at the RAA and 20 at the IAS. During the study, no AF recurrences were reported in 11 of 42 (26%) patients, independently of RAA or IAS pacing. Patients from group A and RAA pacing had 0.79 +/- 0.81 episodes of AF/day during DDD, which increased to 1.52 +/- 1.41 episodes of AF/day during DDDR + Alg (P = 0.046). Those with IAS pacing had 0.5 +/- 0.24 episodes of AF/day during DDD, which decreased to 0.06 +/- 0.08 episodes of AF/day during DDDR + Alg (P = 0.06). In group B, no differences were reported between pacing sites and pacing modes. Severe right atrial conduction delay is present in one-third of patients with SND and AF: continuous pacing at the IAS is superior to RAA for AF recurrences. In patients without severe conduction delay, no differences between pacing site or mode were observed.

  16. Tuning Transcriptional Regulation through Signaling: A Predictive Theory of Allosteric Induction.

    Science.gov (United States)

    Razo-Mejia, Manuel; Barnes, Stephanie L; Belliveau, Nathan M; Chure, Griffin; Einav, Tal; Lewis, Mitchell; Phillips, Rob

    2018-04-25

    Allosteric regulation is found across all domains of life, yet we still lack simple, predictive theories that directly link the experimentally tunable parameters of a system to its input-output response. To that end, we present a general theory of allosteric transcriptional regulation using the Monod-Wyman-Changeux model. We rigorously test this model using the ubiquitous simple repression motif in bacteria by first predicting the behavior of strains that span a large range of repressor copy numbers and DNA binding strengths and then constructing and measuring their response. Our model not only accurately captures the induction profiles of these strains, but also enables us to derive analytic expressions for key properties such as the dynamic range and [EC 50 ]. Finally, we derive an expression for the free energy of allosteric repressors that enables us to collapse our experimental data onto a single master curve that captures the diverse phenomenology of the induction profiles. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  17. The structure of brain glycogen phosphorylase-from allosteric regulation mechanisms to clinical perspectives.

    Science.gov (United States)

    Mathieu, Cécile; Dupret, Jean-Marie; Rodrigues Lima, Fernando

    2017-02-01

    Glycogen phosphorylase (GP) is the key enzyme that regulates glycogen mobilization in cells. GP is a complex allosteric enzyme that comprises a family of three isozymes: muscle GP (mGP), liver GP (lGP), and brain GP (bGP). Although the three isozymes display high similarity and catalyze the same reaction, they differ in their sensitivity to the allosteric activator adenosine monophosphate (AMP). Moreover, inactivating mutations in mGP and lGP have been known to be associated with glycogen storage diseases (McArdle and Hers disease, respectively). The determination, decades ago, of the structure of mGP and lGP have allowed to better understand the allosteric regulation of these two isoforms and the development of specific inhibitors. Despite its important role in brain glycogen metabolism, the structure of the brain GP had remained elusive. Here, we provide an overview of the human brain GP structure and its relationship with the two other members of this key family of the metabolic enzymes. We also summarize how this structure provides valuable information to understand the regulation of bGP and to design specific ligands of potential pharmacological interest. © 2016 Federation of European Biochemical Societies.

  18. Astronomical tunings of the Oligocene-Miocene transition from Pacific Ocean Site U1334 and implications for the carbon cycle

    Science.gov (United States)

    Beddow, Helen M.; Liebrand, Diederik; Wilson, Douglas S.; Hilgen, Frits J.; Sluijs, Appy; Wade, Bridget S.; Lourens, Lucas J.

    2018-03-01

    Astronomical tuning of sediment sequences requires both unambiguous cycle pattern recognition in climate proxy records and astronomical solutions, as well as independent information about the phase relationship between these two. Here we present two different astronomically tuned age models for the Oligocene-Miocene transition (OMT) from Integrated Ocean Drilling Program Site U1334 (equatorial Pacific Ocean) to assess the effect tuning has on astronomically calibrated ages and the geologic timescale. These alternative age models (roughly from ˜ 22 to ˜ 24 Ma) are based on different tunings between proxy records and eccentricity: the first age model is based on an aligning CaCO3 weight (wt%) to Earth's orbital eccentricity, and the second age model is based on a direct age calibration of benthic foraminiferal stable carbon isotope ratios (δ13C) to eccentricity. To independently test which tuned age model and associated tuning assumptions are in best agreement with independent ages based on tectonic plate-pair spreading rates, we assign the tuned ages to magnetostratigraphic reversals identified in deep-marine magnetic anomaly profiles. Subsequently, we compute tectonic plate-pair spreading rates based on the tuned ages. The resultant alternative spreading-rate histories indicate that the CaCO3 tuned age model is most consistent with a conservative assumption of constant, or linearly changing, spreading rates. The CaCO3 tuned age model thus provides robust ages and durations for polarity chrons C6Bn.1n-C7n.1r, which are not based on astronomical tuning in the latest iteration of the geologic timescale. Furthermore, it provides independent evidence that the relatively large (several 10 000 years) time lags documented in the benthic foraminiferal isotope records relative to orbital eccentricity constitute a real feature of the Oligocene-Miocene climate system and carbon cycle. The age constraints from Site U1334 thus indicate that the delayed responses of the

  19. One Dimensional Backstripping Results from IODP Expedition 318, Site U1356: Tectonic Implications for the Wilkes Land Margin of Antarctica

    Science.gov (United States)

    Hayden, T. G.; Kominz, M. A.; González, J. J.; Escutia, C.; Brinkhuis, H.; Scientific Party of IODP Expedition 318

    2011-12-01

    The Wilkes Land margin of Antarctica is the conjugate margin of the Great Australian Bight, which underwent extension, thinning and rifting from ~160 Ma until breakup at ~83 Ma. Both Wilkes Land and the Great Australian Bight are considered passive margins, and were thought to be tectonically inactive since breakup at 83 Ma. We have backstripped the U1356 Core recovered from the continental rise off Wilkes Land, Antarctica by IODP Expedition 318. Backstripping input included lithological and sedimentary analysis, paleo-environmental indicators, combined paleomagnetic and biostratigraphic chronologies, and physical properties measurements. Tectonic subsidence shows a major event between 50 and 33.6 Ma, a time represented by a hiatus in the U1356 core. The magnitude of subsidence requires it to be tectonic in origin, and the timing matches with a reorganization of plate motions that represents the transition from slow spreading to fast spreading between Antarctica and Australia, which occurred at approximately 43 Ma. Coupled with a regional seismic framework, and using other Expedition 318 site analyses, the Wilkes Land margin is shown to be far more complex then the simple passive margin currently assumed. We explore several possible mechanisms for the subsidence and erosion observed; including thermal uplift due to continental insulation of the asthenosphere and it's interaction with a recently rifted margin, asthenospheric convection, transtensional or transpressional basin development and loading, and edge-driven asthenospheric convection.

  20. Tiechanshan-Tunghsiao anticline earthquake analysis: Implications for northwestern Taiwan potential carbon dioxide storage site seismic hazard

    Directory of Open Access Journals (Sweden)

    Ruey-Juin Rau

    2017-01-01

    Full Text Available We analyze the seismicity and earthquake focal mechanisms beneath the Tiechanshan-Tunghsiao (TCS-TH anticline over the last two decades for seismic hazard evaluation of a potential carbon dioxide storage site in northwestern Taiwan. Seismicity in the TCS-TH anticline indicates both spatial and temporal clustering at a depth range of 7 - 12 km. Thirteen 3.0 ≤ ML ≤ 5.2 earthquake focal mechanisms show a combination of thrust, strike-slip, and normal faulting mechanisms under the TCS-TH anticline. A 1992 ML 5.2 earthquake with a focal depth of ~10 km, the largest event ever recorded beneath the TCS-TH anticline during the last two decades, has a normal fault mechanism with the T-axis trending NNE-SSW and nodal planes oriented NNW-SSE, dipping either gently to the NNE or steeply to the SSW. Thrust fault mechanisms that occurred with mostly E-W or NWW-SEE striking P-axes and strike-slip faulting events indicate NWW-SEE striking P-axes and NNE-SSW trending T-axes, which are consistent with the regional plate convergence direction. For the strike-slip faulting events, if we take the N-S or NNW-SSE striking nodal planes as the fault planes, the strike-slip faults are sinistral motions and correspond to the Tapingting fault, which is a strike-slip fault reactivated from the inherited normal fault and intersects the Tiechanshan and Tunghsiao anticlines.

  1. CO2 and its correlation with CO at a rural site near Beijing: implications for combustion efficiency in China

    Directory of Open Access Journals (Sweden)

    H. Ma

    2010-09-01

    Full Text Available Although China has surpassed the United States as the world's largest carbon dioxide emitter, in situ measurements of atmospheric CO2 have been sparse in China. This paper analyzes hourly CO2 and its correlation with CO at Miyun, a rural site near Beijing, over a period of 51 months (Dec 2004 through Feb 2009. The CO2-CO correlation analysis evaluated separately for each hour of the day provides useful information with statistical significance even in the growing season. We found that the intercept, representing the initial condition imposed by global distribution of CO2 with influence of photosynthesis and respiration, exhibits diurnal cycles differing by season. The background CO2 (CO2,b derived from Miyun observations is comparable to CO2 observed at a Mongolian background station to the northwest. Annual growth of overall mean CO2 at Miyun is estimated at 2.7 ppm yr−1 while that of CO2,b is only 1.7 ppm yr−1 similar to the mean growth rate at northern mid-latitude background stations. This suggests a relatively faster increase in the regional CO2 sources in China than the global average, consistent with bottom-up studies of CO2 emissions. For air masses with trajectories through the northern China boundary layer, mean winter CO2/CO correlation slopes (dCO2/dCO increased by 2.8 ± 0.9 ppmv/ppmv or 11% from 2005–2006 to 2007–2008, with CO2 increasing by 1.8 ppmv. The increase in dCO2/dCO indicates improvement in overall combustion efficiency over northern China after winter 2007, attributed to pollution reduction measures associated with the 2008 Beijing Olympics. The observed CO2/CO ratio at Miyun is 25% higher than the bottom-up CO2/CO emission ratio, suggesting a contribution of respired CO2 from urban residents as well as agricultural soils and livestock in the observations and uncertainty in the emission estimates.

  2. Late Pleistocene Age Model for Site U1460, Perth Basin, SW Australian Shelf: Implications for Leeuwin Current History

    Science.gov (United States)

    Christensen, B. A.; Takayanagi, H.; Petrick, B.; Ishiwa, T.; Henderiks, J.; Groeneveld, J.; Mamo, B. L.; De Vleeschouwer, D.; Auer, G.; Deik, H.; Fulthorpe, C.; Gallagher, S. J.; McHugh, C.; Reuning, L.; Yokoyama, Y.

    2017-12-01

    The Leeuwin Current (LC) exerts an important control on modern Australian climate, but its onset is not well defined. The LC is the only southward flowing eastern boundary current. Driven by a pressure gradient set up in the Indonesian Throughflow, its warm waters support reefs to 29°S. It is seasonally controlled south of the Western Cape. Determination of the onset of the LC was a major objective of IODP Expedition 356. Expedition 356 drilling on the western Australian margin provides an opportunity to explore depositional patterns and timing in the region influenced by the current. Site U1460 was drilled in 214.5 m w.d. (Gallagher et al., 2017). Integrated calcareous nannofossil and planktonic foraminiferal biostratigraphy places the upper 86.5 m firmly within the late Pleistocene. However, the glacial- interglacial stratigraphy is uncertain because of the complexity of this shelfal depositional environment. Here we present a likely late Pleistocene stratigraphy based on integrated geochemical and paleontological datasets. A benthic foraminifer (Uvigerina peregrina) stable isotope record provides the foundation for the age model and the data are supported by SST estimates based on Tex86 and alkenones. Our age model places MIS 16 between 104 and 99 m-CSF-A, followed by an expanded MIS 15 section (99 - 50 m-CSF-A). We correlate the interval from 50 - 5 m-CSF-A with MIS 14 to MIS 8, with the largest magnitude glacial events (MIS 12, MIS 6) either condensed or represented as depositional hiatuses. A Recent 14C date at 0.34 mbsf constrains the interval from 2 - 0.5 m-CSF-A to MIS 4-3, which is in good agreement with the base of common Emiliania huxleyi (0.09 Ma) at 2.13 m-CSF-A. Thus MIS 5 is equivalent to the interval from 5 to 2 m-CSF-A. The expanded MIS 15 section follows a geometric change from slope to prograding shelf. It is associated with a shift to infaunal benthic foraminiferal assemblages, abundant sponge spicules, and a reduction in CaCO3%, suggesting

  3. Chemical Kinetics of Hydrogen Atom Abstraction from Allylic Sites by 3O2; Implications for Combustion Modeling and Simulation.

    Science.gov (United States)

    Zhou, Chong-Wen; Simmie, John M; Somers, Kieran P; Goldsmith, C Franklin; Curran, Henry J

    2017-03-09

    Hydrogen atom abstraction from allylic C-H bonds by molecular oxygen plays a very important role in determining the reactivity of fuel molecules having allylic hydrogen atoms. Rate constants for hydrogen atom abstraction by molecular oxygen from molecules with allylic sites have been calculated. A series of molecules with primary, secondary, tertiary, and super secondary allylic hydrogen atoms of alkene, furan, and alkylbenzene families are taken into consideration. Those molecules include propene, 2-butene, isobutene, 2-methylfuran, and toluene containing the primary allylic hydrogen atom; 1-butene, 1-pentene, 2-ethylfuran, ethylbenzene, and n-propylbenzene containing the secondary allylic hydrogen atom; 3-methyl-1-butene, 2-isopropylfuran, and isopropylbenzene containing tertiary allylic hydrogen atom; and 1-4-pentadiene containing super allylic secondary hydrogen atoms. The M06-2X/6-311++G(d,p) level of theory was used to optimize the geometries of all of the reactants, transition states, products and also the hinder rotation treatments for lower frequency modes. The G4 level of theory was used to calculate the electronic single point energies for those species to determine the 0 K barriers to reaction. Conventional transition state theory with Eckart tunnelling corrections was used to calculate the rate constants. The comparison between our calculated rate constants with the available experimental results from the literature shows good agreement for the reactions of propene and isobutene with molecular oxygen. The rate constant for toluene with O 2 is about an order magnitude slower than that experimentally derived from a comprehensive model proposed by Oehlschlaeger and coauthors. The results clearly indicate the need for a more detailed investigation of the combustion kinetics of toluene oxidation and its key pyrolysis and oxidation intermediates. Despite this, our computed barriers and rate constants retain an important internal consistency. Rate constants

  4. Past climate changes and permafrost depth at the Lake El'gygytgyn site: implications from data and thermal modeling

    Directory of Open Access Journals (Sweden)

    D. Mottaghy

    2013-01-01

    Full Text Available This study focuses on the temperature field observed in boreholes drilled as part of interdisciplinary scientific campaign targeting the El'gygytgyn Crater Lake in NE Russia. Temperature data are available from two sites: the lake borehole 5011-1 located near the center of the lake reaching 400 m depth, and the land borehole 5011-3 at the rim of the lake, with a depth of 140 m. Constraints on permafrost depth and past climate changes are derived from numerical simulation of the thermal regime associated with the lake-related talik structure. The thermal properties of the subsurface needed for these simulations are based on laboratory measurements of representative cores from the quaternary sediments and the underlying impact-affected rock, complemented by further information from geophysical logs and data from published literature. The temperature observations in the lake borehole 5011-1 are dominated by thermal perturbations related to the drilling process, and thus only give reliable values for the lowermost value in the borehole. Undisturbed temperature data recorded over more than two years are available in the 140 m deep land-based borehole 5011-3. The analysis of these observations allows determination of not only the recent mean annual ground surface temperature, but also the ground surface temperature history, though with large uncertainties. Although the depth of this borehole is by far too insufficient for a complete reconstruction of past temperatures back to the Last Glacial Maximum, it still affects the thermal regime, and thus permafrost depth. This effect is constrained by numerical modeling: assuming that the lake borehole observations are hardly influenced by the past changes in surface air temperature, an estimate of steady-state conditions is possible, leading to a meaningful value of 14 ± 5 K for the post-glacial warming. The strong curvature of the temperature data in shallower depths around 60 m can be explained by a

  5. Allosteric analysis of glucocorticoid receptor-DNA interface induced by cyclic Py-Im polyamide: a molecular dynamics simulation study.

    Directory of Open Access Journals (Sweden)

    Yaru Wang

    Full Text Available BACKGROUND: It has been extensively developed in recent years that cell-permeable small molecules, such as polyamide, can be programmed to disrupt transcription factor-DNA interfaces and can silence aberrant gene expression. For example, cyclic pyrrole-imidazole polyamide that competes with glucocorticoid receptor (GR for binding to glucocorticoid response elements could be expected to affect the DNA dependent binding by interfering with the protein-DNA interface. However, how such small molecules affect the transcription factor-DNA interfaces and gene regulatory pathways through DNA structure distortion is not fully understood so far. METHODOLOGY/PRINCIPAL FINDINGS: In the present work, we have constructed some models, especially the ternary model of polyamides+DNA+GR DNA-binding domain (GRDBD dimer, and carried out molecular dynamics simulations and free energy calculations for them to address how polyamide molecules disrupt the GRDBD and DNA interface when polyamide and protein bind at the same sites on opposite grooves of DNA. CONCLUSIONS/SIGNIFICANCE: We found that the cyclic polyamide binding in minor groove of DNA can induce a large structural perturbation of DNA, i.e. a >4 Å widening of the DNA minor groove and a compression of the major groove by more than 4 Å as compared with the DNA molecule in the GRDBD dimer+DNA complex. Further investigations for the ternary system of polyamides+DNA+GRDBD dimer and the binary system of allosteric DNA+GRDBD dimer revealed that the compression of DNA major groove surface causes GRDBD to move away from the DNA major groove with the initial average distance of ∼4 Å to the final average distance of ∼10 Å during 40 ns simulation course. Therefore, this study straightforward explores how small molecule targeting specific sites in the DNA minor groove disrupts the transcription factor-DNA interface in DNA major groove, and consequently modulates gene expression.

  6. Oncogenic exon 2 mutations in Mediator subunit MED12 disrupt allosteric activation of cyclin C-CDK8/19.

    Science.gov (United States)

    Park, Min Ju; Shen, Hailian; Spaeth, Jason M; Tolvanen, Jaana H; Failor, Courtney; Knudtson, Jennifer F; McLaughlin, Jessica; Halder, Sunil K; Yang, Qiwei; Bulun, Serdar E; Al-Hendy, Ayman; Schenken, Robert S; Aaltonen, Lauri A; Boyer, Thomas G

    2018-03-30

    Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at high frequency in uterine fibroids (UFs) and breast fibroepithelial tumors as well as recurrently, albeit less frequently, in malignant uterine leimyosarcomas, chronic lymphocytic leukemias, and colorectal cancers. Previously, we reported that UF-linked mutations in MED12 disrupt its ability to activate cyclin C (CycC)-dependent kinase 8 (CDK8) in Mediator, implicating impaired Mediator-associated CDK8 activity in the molecular pathogenesis of these clinically significant lesions. Notably, the CDK8 paralog CDK19 is also expressed in myometrium, and both CDK8 and CDK19 assemble into Mediator in a mutually exclusive manner, suggesting that CDK19 activity may also be germane to the pathogenesis of MED12 mutation-induced UFs. However, whether and how UF-linked mutations in MED12 affect CDK19 activation is unknown. Herein, we show that MED12 allosterically activates CDK19 and that UF-linked exon 2 mutations in MED12 disrupt its CDK19 stimulatory activity. Furthermore, we find that within the Mediator kinase module, MED13 directly binds to the MED12 C terminus, thereby suppressing an apparent UF mutation-induced conformational change in MED12 that otherwise disrupts its association with CycC-CDK8/19. Thus, in the presence of MED13, mutant MED12 can bind, but cannot activate, CycC-CDK8/19. These findings indicate that MED12 binding is necessary but not sufficient for CycC-CDK8/19 activation and reveal an additional step in the MED12-dependent activation process, one critically dependent on MED12 residues altered by UF-linked exon 2 mutations. These findings confirm that UF-linked mutations in MED12 disrupt composite Mediator-associated kinase activity and identify CDK8/19 as prospective therapeutic targets in UFs. © 2018 Park et al.

  7. The cognition-enhancing activity of E1R, a novel positive allosteric modulator of sigma-1 receptors

    Science.gov (United States)

    Zvejniece, L; Vavers, E; Svalbe, B; Vilskersts, R; Domracheva, I; Vorona, M; Veinberg, G; Misane, I; Stonans, I; Kalvinsh, I; Dambrova, M

    2014-01-01

    Background and Purpose Here, we describe the in vitro and in vivo effects of (4R,5S)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide (E1R), a novel positive allosteric modulator of sigma-1 receptors. Experimental Approach E1R was tested for sigma receptor binding activity in a [3H](+)-pentazocine assay, in bradykinin (BK)-induced intracellular Ca2+ concentration ([Ca2+]i) assays and in an electrically stimulated rat vas deferens model. E1R's effects on cognitive function were tested using passive avoidance (PA) and Y-maze tests in mice. A selective sigma-1 receptor antagonist (NE-100), was used to study the involvement of the sigma-1 receptor in the effects of E1R. The open-field test was used to detect the effects of E1R on locomotion. Key Results Pretreatment with E1R enhanced the selective sigma-1 receptor agonist PRE-084's stimulating effect during a model study employing electrically stimulated rat vasa deferentia and an assay measuring the BK-induced [Ca2+]i increase. Pretreatment with E1R facilitated PA retention in a dose-related manner. Furthermore, E1R alleviated the scopolamine-induced cognitive impairment during the PA and Y-maze tests in mice. The in vivo and in vitro effects of E1R were blocked by treatment with the selective sigma-1 receptor antagonist NE-100. E1R did not affect locomotor activity. Conclusion and Implications E1R is a novel 4,5-disubstituted derivative of piracetam that enhances cognition and demonstrates efficacy against scopolamine-induced cholinergic dysfunction in mice. These effects are attributed to its positive modulatory action on the sigma-1 receptor and this activity may be relevant when developing new drugs for treating cognitive symptoms related to neurodegenerative diseases. PMID:24490863

  8. Sex-dependent anti-stress effect of an α5 subunit containing GABAA receptor positive allosteric modulator

    Directory of Open Access Journals (Sweden)

    Sean C. Piantadosi

    2016-11-01

    Full Text Available Rationale: Current first-line treatments for stress-related disorders such as Major Depressive Disorder (MDD act on monoaminergic systems and take weeks to achieve a therapeutic effect with poor response and low remission rates. Recent research has implicated the GABAergic system in the pathophysiology of depression, including deficits in interneurons targeting the dendritic compartment of cortical pyramidal cells. Objectives: The present study evaluates whether SH-053-2'F-R-CH3 (denoted α5-PAM, a positive allosteric modulator selective for α5-subunit containing GABAA receptors found predominantly on cortical pyramidal cell dendrites has anti-stress effects. Methods: Female and male C57BL6/J mice were exposed to unpredictable chronic mild stress (UCMS and treated with α5-PAM acutely (30 minutes prior to assessing behavior or chronically before being assessed behaviorally. Results: Acute and chronic α5-PAM treatments produce a pattern of decreased stress-induced behaviors (denoted as behavioral emotionality across various tests in female, but not in male mice. Behavioral Z-scores calculated across a panel of tests designed to best model the range and heterogeneity of human symptomatology confirmed that acute and chronic α5-PAM treatments consistently produce significant decreases in behavioral emotionality in several independent cohorts of females. The behavioral responses to α5-PAM could not be completely accounted for by differences in drug brain disposition between female and male mice. In mice exposed to UCMS, expression of the Gabra5 gene was increased in the frontal cortex after acute treatment and in hippocampus after chronic treatment with α5-PAM in females only, and these expression changes correlated with behavioral emotionality. Conclusions: We showed that acute and chronic positive modulation of α5 subunit-containing GABAA receptors elicit anti-stress effects in a sex-dependent manner, suggesting novel therapeutic modalities.

  9. Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

    Energy Technology Data Exchange (ETDEWEB)

    Singh,S.; Yamashita, A.; Gouaux, E.

    2007-01-01

    Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 {angstrom} above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the

  10. Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

    International Nuclear Information System (INIS)

    Singh, S.; Yamashita, A.; Gouaux, E.

    2007-01-01

    Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 (angstrom) above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational

  11. On Allosteric Modulation of P-Type Cu+-ATPases

    DEFF Research Database (Denmark)

    Mattle, Daniel; Sitsel, Oleg; Autzen, Henriette Elisabeth

    2013-01-01

    P-type ATPases perform active transport of various compounds across biological membranes and are crucial for ion homeostasis and the asymmetric composition of lipid bilayers. Although their functional cycle share principles of phosphoenzyme intermediates, P-type ATPases also show subclass...... of intramembranous Cu+ binding, and we suggest an alternative role for the proposed second site in copper translocation and proton exchange. The class-specific features demonstrate that topological diversity in P-type ATPases may tune a general energy coupling scheme to the translocation of compounds with remarkably...

  12. AIM for Allostery: Using the Ising Model to Understand Information Processing and Transmission in Allosteric Biomolecular Systems.

    Science.gov (United States)

    LeVine, Michael V; Weinstein, Harel

    2015-05-01

    In performing their biological functions, molecular machines must process and transmit information with high fidelity. Information transmission requires dynamic coupling between the conformations of discrete structural components within the protein positioned far from one another on the molecular scale. This type of biomolecular "action at a distance" is termed allostery . Although allostery is ubiquitous in biological regulation and signal transduction, its treatment in theoretical models has mostly eschewed quantitative descriptions involving the system's underlying structural components and their interactions. Here, we show how Ising models can be used to formulate an approach to allostery in a structural context of interactions between the constitutive components by building simple allosteric constructs we termed Allosteric Ising Models (AIMs). We introduce the use of AIMs in analytical and numerical calculations that relate thermodynamic descriptions of allostery to the structural context, and then show that many fundamental properties of allostery, such as the multiplicative property of parallel allosteric channels, are revealed from the analysis of such models. The power of exploring mechanistic structural models of allosteric function in more complex systems by using AIMs is demonstrated by building a model of allosteric signaling for an experimentally well-characterized asymmetric homodimer of the dopamine D2 receptor.

  13. Two-state dynamics of the SH3-SH2 tandem of Abl kinase and the allosteric role of the N-cap.

    Science.gov (United States)

    Corbi-Verge, Carles; Marinelli, Fabrizio; Zafra-Ruano, Ana; Ruiz-Sanz, Javier; Luque, Irene; Faraldo-Gómez, José D

    2013-09-03

    The regulation and localization of signaling enzymes is often mediated by accessory modular domains, which frequently function in tandems. The ability of these tandems to adopt multiple conformations is as important for proper regulation as the individual domain specificity. A paradigmatic example is Abl, a ubiquitous tyrosine kinase of significant pharmacological interest. SH3 and SH2 domains inhibit Abl by assembling onto the catalytic domain, allosterically clamping it in an inactive state. We investigate the dynamics of this SH3-SH2 tandem, using microsecond all-atom simulations and differential scanning calorimetry. Our results indicate that the Abl tandem is a two-state switch, alternating between the conformation observed in the structure of the autoinhibited enzyme and another configuration that is consistent with existing scattering data for an activated form. Intriguingly, we find that the latter is the most probable when the tandem is disengaged from the catalytic domain. Nevertheless, an amino acid stretch preceding the SH3 domain, the so-called N-cap, reshapes the free-energy landscape of the tandem and favors the interaction of this domain with the SH2-kinase linker, an intermediate step necessary for assembly of the autoinhibited complex. This allosteric effect arises from interactions between N-cap and the SH2 domain and SH3-SH2 connector, which involve a phosphorylation site. We also show that the SH3-SH2 connector plays a determinant role in the assembly equilibrium of Abl, because mutations thereof hinder the engagement of the SH2-kinase linker. These results provide a thermodynamic rationale for the involvement of N-cap and SH3-SH2 connector in Abl regulation and expand our understanding of the principles of modular domain organization.

  14. Two-state dynamics of the SH3–SH2 tandem of Abl kinase and the allosteric role of the N-cap

    Science.gov (United States)

    Corbi-Verge, Carles; Marinelli, Fabrizio; Zafra-Ruano, Ana; Ruiz-Sanz, Javier; Luque, Irene; Faraldo-Gómez, José D.

    2013-01-01

    The regulation and localization of signaling enzymes is often mediated by accessory modular domains, which frequently function in tandems. The ability of these tandems to adopt multiple conformations is as important for proper regulation as the individual domain specificity. A paradigmatic example is Abl, a ubiquitous tyrosine kinase of significant pharmacological interest. SH3 and SH2 domains inhibit Abl by assembling onto the catalytic domain, allosterically clamping it in an inactive state. We investigate the dynamics of this SH3–SH2 tandem, using microsecond all-atom simulations and differential scanning calorimetry. Our results indicate that the Abl tandem is a two-state switch, alternating between the conformation observed in the structure of the autoinhibited enzyme and another configuration that is consistent with existing scattering data for an activated form. Intriguingly, we find that the latter is the most probable when the tandem is disengaged from the catalytic domain. Nevertheless, an amino acid stretch preceding the SH3 domain, the so-called N-cap, reshapes the free-energy landscape of the tandem and favors the interaction of this domain with the SH2-kinase linker, an intermediate step necessary for assembly of the autoinhibited complex. This allosteric effect arises from interactions between N-cap and the SH2 domain and SH3–SH2 connector, which involve a phosphorylation site. We also show that the SH3–SH2 connector plays a determinant role in the assembly equilibrium of Abl, because mutations thereof hinder the engagement of the SH2-kinase linker. These results provide a thermodynamic rationale for the involvement of N-cap and SH3–SH2 connector in Abl regulation and expand our understanding of the principles of modular domain organization. PMID:23959873

  15. The location and nature of general anesthetic binding sites on the active conformation of firefly luciferase; a time resolved photolabeling study.

    Directory of Open Access Journals (Sweden)

    Sivananthaperumal Shanmugasundararaj

    Full Text Available Firefly luciferase is one of the few soluble proteins that is acted upon by a wide variety of general anesthetics and alcohols; they inhibit the ATP-driven production of light. We have used time-resolved photolabeling to locate the binding sites of alcohols during the initial light output, some 200 ms after adding ATP. The photolabel 3-azioctanol inhibited the initial light output with an IC50 of 200 µM, close to its general anesthetic potency. Photoincorporation of [(3H]3-azioctanol into luciferase was saturable but weak. It was enhanced 200 ms after adding ATP but was negligible minutes later. Sequencing of tryptic digests by HPLC-MSMS revealed a similar conformation-dependence for photoincorporation of 3-azioctanol into Glu-313, a residue that lines the bottom of a deep cleft (vestibule whose outer end binds luciferin. An aromatic diazirine analog of benzyl alcohol with broader side chain reactivity reported two sites. First, it photolabeled two residues in the vestibule, Ser-286 and Ile-288, both of which are implicated with Glu-313 in the conformation change accompanying activation. Second, it photolabeled two residues that contact luciferin, Ser-316 and Ser-349. Thus, time resolved photolabeling supports two mechanisms of action. First, an allosteric one, in which anesthetics bind in the vestibule displacing water molecules that are thought to be involved in light output. Second, a competitive one, in which anesthetics bind isosterically with luciferin. This work provides structural evidence that supports the competitive and allosteric actions previously characterized by kinetic studies.

  16. An engineered allosteric switch in leucine-zipper oligomerization.

    Science.gov (United States)

    Gonzalez, L; Plecs, J J; Alber, T

    1996-06-01

    Controversy remains about the role of core side-chain packing in specifying protein structure. To investigate the influence of core packing on the oligomeric structure of a coiled coil, we engineered a GCN4 leucine zipper mutant that switches from two to three strands upon binding the hydrophobic ligands cyclohexane and benzene. In solution these ligands increased the apparent thermal stability and the oligomerization order of the mutant leucine zipper. The crystal structure of the peptide-benzene complex shows a single benzene molecule bound at the engineered site in the core of the trimer. These results indicate that coiled coils are well-suited to function as molecular switches and emphasize that core packing is an important determinant of oligomerization specificity.

  17. The odyssey of a young gene: structure-function studies in human glutamate dehydrogenases reveal evolutionary-acquired complex allosteric regulation mechanisms.

    Science.gov (United States)

    Zaganas, Ioannis V; Kanavouras, Konstantinos; Borompokas, Nikolas; Arianoglou, Giovanna; Dimovasili, Christina; Latsoudis, Helen; Vlassi, Metaxia; Mastorodemos, Vasileios

    2014-01-01

    performed mutagenesis at sites of difference in their amino acid sequence. Results showed that the low basal activity, heat-lability and estrogen sensitivity of hGDH2 could be, at least partially, ascribed to the Arg443Ser evolutionary change, whereas resistance to GTP inhibition has been attributed to the Gly456Ala change. Other amino acid substitutions studied thus far cannot explain all the remaining functional differences between the two iso-enzymes. Also, the Arg443Ser/Gly456Ala double mutation in hGDH1 approached the properties of wild-type hGDH2, without being identical to it. The insights into the structural mechanism of enzymatic regulation and the implications in cell biology provided by these findings are discussed.

  18. Chemogenomic discovery of allosteric antagonists at the GPRC6A receptor

    DEFF Research Database (Denmark)

    Gloriam, David E.; Wellendorph, Petrine; Johansen, Lars Dan

    2011-01-01

    and pharmacological character: (1) chemogenomic lead identification through the first, to our knowledge, ligand inference between two different GPCR families, Families A and C; and (2) the discovery of the most selective GPRC6A allosteric antagonists discovered to date. The unprecedented inference of...... pharmacological activity across GPCR families provides proof-of-concept for in silico approaches against Family C targets based on Family A templates, greatly expanding the prospects of successful drug design and discovery. The antagonists were tested against a panel of seven Family A and C G protein-coupled receptors...

  19. Substituted 3-Benzylcoumarins as Allosteric MEK1 Inhibitors: Design, Synthesis and Biological Evaluation as Antiviral Agents

    Directory of Open Access Journals (Sweden)

    Ping Xu

    2013-05-01

    Full Text Available In order to find novel antiviral agents, a series of allosteric MEK1 inhibitors were designed and synthesized. Based on docking results, multiple optimizations were made on the coumarin scaffold. Some of the derivatives showed excellent MEK1 binding affinity in the appropriate enzymatic assays and displayed obvious inhibitory effects on the ERK pathway in a cellular assay. These compounds also significantly inhibited virus (EV71 replication in HEK293 and RD cells. Several compounds showed potential as agents for the treatment of viral infective diseases, with the most potent compound 18 showing an IC50 value of 54.57 nM in the MEK1 binding assay.

  20. Complex pharmacology of novel allosteric free fatty acid 3 receptor ligands

    DEFF Research Database (Denmark)

    Hudson, Brian D; Christiansen, Elisabeth; Murdoch, Hannah

    2014-01-01

    this series resulted in compounds completely lacking activity, acting as FFA3 PAMs, or appearing to act as FFA3-negative allosteric modulators. However, the pharmacology of this series was further complicated in that certain analogs displaying overall antagonism of FFA3 function actually appeared to generate......, considerable care must be taken to define the pharmacological characteristics of specific compounds before useful predictions of their activity and their use in defining specific roles of FFA3 in either in vitro and in vivo settings can be made....

  1. Dual interaction of agmatine with the rat α2D-adrenoceptor: competitive antagonism and allosteric activation

    Science.gov (United States)

    Molderings, G J; Menzel, S; Kathmann, M; Schlicker, E; Göthert, M

    2000-01-01

    In segments of rat vena cava preincubated with [3H]-noradrenaline and superfused with physiological salt solution, the influence of agmatine on the electrically evoked [3H]-noradrenaline release, the EP3 prostaglandin receptor-mediated and the α2D-adrenoceptor-mediated inhibition of evoked [3H]-noradrenaline release was investigated. Agmatine (0.1–10 μM) by itself was without effect on evoked [3H]-noradrenaline release. In the presence of 10 μM agmatine, the prostaglandin E2(PGE2)-induced EP3-receptor-mediated inhibition of [3H]-noradrenaline release was not modified, whereas the α2D-adrenoceptor-mediated inhibition of [3H]-noradrenaline release induced by noradrenaline, moxonidine or clonidine was more pronounced than in the absence of agmatine. However, 1 mM agmatine antagonized the moxonidine-induced inhibition of [3H]-noradrenaline release. Agmatine concentration-dependently inhibited the binding of [3H]-clonidine and [3H]-rauwolscine to rat brain cortex membranes (Ki values 6 μM and 12 μM, respectively). In addition, 30 and 100 μM agmatine increased the rate of association and decreased the rate of dissociation of [3H]-clonidine resulting in an increased affinity of the radioligand for the α2D-adrenoceptors. [14C]-agmatine labelled specific binding sites on rat brain cortex membranes. In competition experiments. [14C]-agmatine was inhibited from binding to its specific recognition sites by unlabelled agmatine, but not by rauwolscine and moxonidine. In conclusion, the present data indicate that agmatine both acts as an antagonist at the ligand recognition site of the α2D-adrenoceptor and enhances the effects of α2-adrenoceptor agonists probably by binding to an allosteric binding site of the α2D-adrenoceptor which seems to be labelled by [14C]-agmatine. PMID:10928978

  2. Analgesic effect of ADX71441, a positive allosteric modulator (PAM) of GABAB receptor in a rat model of bladder pain.

    Science.gov (United States)

    Kannampalli, Pradeep; Poli, Sonia-Maria; Boléa, Christelle; Sengupta, Jyoti N

    2017-11-01

    Therapeutic use of GABA B receptor agonists for conditions like chronic abdominal pain, overactive bladder (OAB) and gastroesophageal reflux disease (GERD) is severely affected by poor blood-brain barrier permeability and potential side effects. ADX71441 is a novel positive allosteric modulator (PAM) of the GABA B receptor that has shown encouraging results in pre-clinical models of anxiety, pain, OAB and alcohol addiction. The present study investigates the analgesic effect of ADX71441 to noxious stimulation of the urinary bladder and colon in rats. In female Sprague-Dawley rats, systemic (i.p), but not intrathecal (i.t), administration of ADX71441 produced a dose-dependent decrease in viscero-motor response (VMR) to graded urinary bladder distension (UBD) and colorectal distension (CRD). Additionally, intra-cerebroventricular (i.c.v.) administration of ADX71441 significantly decreased the VMRs to noxious UBD. In electrophysiology experiments, the drug did not attenuate the responses of UBD-sensitive pelvic nerve afferent (PNA) fibers to UBD. In contrast, ADX71441 significantly decreased the responses of UBD-responsive lumbosacral (LS) spinal neurons in spinal intact rats. However, ADX71441 did not attenuate these LS neurons in cervical (C1-C2) spinal transected rats. During cystometrogram (CMG) recordings, ADX71441 (i.p.) significantly decreased the VMR to slow infusion without affecting the number of voiding contraction. These results indicate that ADX71441 modulate bladder nociception via its effect at the supra-spinal sites without affecting the normal bladder motility and micturition reflex in naïve adult rats. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Aryloxyalkanoic Acids as Non-Covalent Modifiers of the Allosteric Properties of Hemoglobin

    Directory of Open Access Journals (Sweden)

    Abdelsattar M. Omar

    2016-08-01

    Full Text Available Hemoglobin (Hb modifiers that stereospecifically inhibit sickle hemoglobin polymer formation and/or allosterically increase Hb affinity for oxygen have been shown to prevent the primary pathophysiology of sickle cell disease (SCD, specifically, Hb polymerization and red blood cell sickling. Several such compounds are currently being clinically studied for the treatment of SCD. Based on the previously reported non-covalent Hb binding characteristics of substituted aryloxyalkanoic acids that exhibited antisickling properties, we designed, synthesized and evaluated 18 new compounds (KAUS II series for enhanced antisickling activities. Surprisingly, select test compounds showed no antisickling effects or promoted erythrocyte sickling. Additionally, the compounds showed no significant effect on Hb oxygen affinity (or in some cases, even decreased the affinity for oxygen. The X-ray structure of deoxygenated Hb in complex with a prototype compound, KAUS-23, revealed that the effector bound in the central water cavity of the protein, providing atomic level explanations for the observed functional and biological activities. Although the structural modification did not lead to the anticipated biological effects, the findings provide important direction for designing candidate antisickling agents, as well as a framework for novel Hb allosteric effectors that conversely, decrease the protein affinity for oxygen for potential therapeutic use for hypoxic- and/or ischemic-related diseases.

  4. Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and Orally Efficacious Phosphatase Inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Fortanet, Jorge Garcia; Chen, Christine Hiu-Tung; Chen, Ying-Nan P.; Chen, Zhouliang; Deng, Zhan; Firestone, Brant; Fekkes, Peter; Fodor, Michelle; Fortin, Pascal D.; Fridrich, Cary; Grunenfelder, Denise; Ho, Samuel; Kang, Zhao B.; Karki, Rajesh; Kato, Mitsunori; Keen, Nick; LaBonte, Laura R.; Larrow, Jay; Lenoir, Francois; Liu, Gang; Liu, Shumei; Lombardo, Franco; Majumdar, Dyuti; Meyer, Matthew J.; Palermo, Mark; Perez, Lawrence; Pu, Minying; Ramsey, Timothy; Sellers, William R.; Shultz, Michael D.; Stams, Travis; Towler, Christopher; Wang, Ping; Williams, Sarah L.; Zhang, Ji-Hu; LaMarche, Matthew J. (Novartis)

    2016-09-08

    SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein–ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor.

  5. Notes on stochastic (bio)-logic gates: computing with allosteric cooperativity.

    Science.gov (United States)

    Agliari, Elena; Altavilla, Matteo; Barra, Adriano; Dello Schiavo, Lorenzo; Katz, Evgeny

    2015-05-15

    Recent experimental breakthroughs have finally allowed to implement in-vitro reaction kinetics (the so called enzyme based logic) which code for two-inputs logic gates and mimic the stochastic AND (and NAND) as well as the stochastic OR (and NOR). This accomplishment, together with the already-known single-input gates (performing as YES and NOT), provides a logic base and paves the way to the development of powerful biotechnological devices. However, as biochemical systems are always affected by the presence of noise (e.g. thermal), standard logic is not the correct theoretical reference framework, rather we show that statistical mechanics can work for this scope: here we formulate a complete statistical mechanical description of the Monod-Wyman-Changeaux allosteric model for both single and double ligand systems, with the purpose of exploring their practical capabilities to express noisy logical operators and/or perform stochastic logical operations. Mixing statistical mechanics with logics, and testing quantitatively the resulting findings on the available biochemical data, we successfully revise the concept of cooperativity (and anti-cooperativity) for allosteric systems, with particular emphasis on its computational capabilities, the related ranges and scaling of the involved parameters and its differences with classical cooperativity (and anti-cooperativity).

  6. Enzyme activity and allosteric characteristics of gamma-irradiated solid aspartate transcarbamylase

    International Nuclear Information System (INIS)

    Bigler, W.N.; Tolbert, B.M.

    1977-01-01

    Aspartate transcarbamylase purified from E. coli was lyophilized, irradiated in vacuo with γ radiation from a cesium-137 source, redissolved in buffer under a nitrogen atmosphere, and assayed for enzyme activity. Lyophilized and redissolved enzyme had normal catalytic and allosteric kinetic characteristics. The average D 37 observed with saturating substrate, 25 mM aspartate, was 4.1 Mrad. With less than saturating substrate, 5 mM aspartate, the activity increases from zero to 1.6 Mrad and then decreases with a D 37 of 7.2 Mrad. Inclusion of 1 mM CTP, an allosteric inhibitor, in the 5 mM aspartate assays results in a more pronounced maximum in the activity curve occurring at slightly higher dose, 2.2 Mrad. Inhibitability by CTP has a D 37 of 2.3 Mrad with doses below the activity maximum. Enzyme lyophilized in the presence of 1 mM CTP has a D 37 of 2.9 Mrad. ATCase activity changes caused by irradiation of lyophylized bacteria were qualitatively like the changes observed in the detailed studies with the purified enzyme. Apparent radiation sensitivities of ATCase in lyophilized bacteria were observed to vary with the technique used to disrupt the resuspended bacteria

  7. Engineering integrated digital circuits with allosteric ribozymes for scaling up molecular computation and diagnostics.

    Science.gov (United States)

    Penchovsky, Robert

    2012-10-19

    Here we describe molecular implementations of integrated digital circuits, including a three-input AND logic gate, a two-input multiplexer, and 1-to-2 decoder using allosteric ribozymes. Furthermore, we demonstrate a multiplexer-decoder circuit. The ribozymes are designed to seek-and-destroy specific RNAs with a certain length by a fully computerized procedure. The algorithm can accurately predict one base substitution that alters the ribozyme's logic function. The ability to sense the length of RNA molecules enables single ribozymes to be used as platforms for multiple interactions. These ribozymes can work as integrated circuits with the functionality of up to five logic gates. The ribozyme design is universal since the allosteric and substrate domains can be altered to sense different RNAs. In addition, the ribozymes can specifically cleave RNA molecules with triplet-repeat expansions observed in genetic disorders such as oculopharyngeal muscular dystrophy. Therefore, the designer ribozymes can be employed for scaling up computing and diagnostic networks in the fields of molecular computing and diagnostics and RNA synthetic biology.

  8. Modeling Np and Pu transport with a surface complexation model and spatially variant sorption capacities: Implications for reactive transport modeling and performance assessments of nuclear waste disposal sites

    Science.gov (United States)

    Glynn, P.D.

    2003-01-01

    simulation conditions. Functional behaviors that cannot be fit include concentration trend reversals and radionuclide desorption spikes. Other simulation results are fit successfully but the fitted parameters (Kd and dispersivity) vary significantly depending on simulation conditions (e.g. "infiltration" vs. "cleanup" conditions). Notably, an increase in the variance of the specified sorption capacities results in a marked increase in the dispersion of the radionuclides. The results presented have implications for the simulation of radionuclide migration in performance assessments of nuclear waste-disposal sites, for the future monitoring of those sites, and more generally for modeling contaminant transport in ground-water environments. ?? 2003 Published by Elsevier Science Ltd.

  9. Phosphorylation of human aquaporin 2 (AQP2) allosterically controls its interaction with the lysosomal trafficking protein LIP5.

    Science.gov (United States)

    Roche, Jennifer Virginia; Survery, Sabeen; Kreida, Stefan; Nesverova, Veronika; Ampah-Korsah, Henry; Gourdon, Maria; Deen, Peter M T; Törnroth-Horsefield, Susanna

    2017-09-01

    The interaction between the renal water channel aquaporin-2 (AQP2) and the lysosomal trafficking regulator-interacting protein LIP5 targets AQP2 to multivesicular bodies and facilitates lysosomal degradation. This interaction is part of a process that controls AQP2 apical membrane abundance in a vasopressin-dependent manner, allowing for urine volume adjustment. Vasopressin regulates phosphorylation at four sites within the AQP2 C terminus (Ser 256 , Ser 261 , Ser 264 , and Thr 269 ), of which Ser 256 is crucial and sufficient for AQP2 translocation from storage vesicles to the apical membrane. However, whether AQP2 phosphorylation modulates AQP2-LIP5 complex affinity is unknown. Here we used far-Western blot analysis and microscale thermophoresis to show that the AQP2 binds LIP5 in a phosphorylation-dependent manner. We constructed five phospho-mimicking mutants (S256E, S261E, S264E, T269E, and S256E/T269E) and a C-terminal truncation mutant (ΔP242) that lacked all phosphorylation sites but retained a previously suggested LIP5-binding site. CD spectroscopy indicated that wild-type AQP2 and the phospho-mimicking mutants had similar overall structure but displayed differences in melting temperatures possibly arising from C-terminal conformational changes. Non-phosphorylated AQP2 bound LIP5 with the highest affinity, whereas AQP2-ΔP242 had 20-fold lower affinity as determined by microscale thermophoresis. AQP2-S256E, S261E, T269E, and S256E/T269E all had reduced affinity. This effect was most prominent for AQP2-S256E, which fits well with its role in apical membrane targeting. AQP2-S264E had affinity similar to non-phosphorylated AQP2, possibly indicating a role in exosome excretion. Our data suggest that AQP2 phosphorylation allosterically controls its interaction with LIP5, illustrating how altered affinities to interacting proteins form the basis for regulation of AQP2 trafficking by post-translational modifications. © 2017 by The American Society for

  10. Positive allosteric modulation of the human metabotropic glutamate receptor 4 (hmGluR4) by SIB-1893 and MPEP

    DEFF Research Database (Denmark)

    Mathiesen, Jesper Mosolff; Svendsen, Nannette; Bräuner-Osborne, Hans

    2003-01-01

    We have identified 2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893) and 2-methyl-6-phenylethynyl pyridine hydrochloride (MPEP) as positive allosteric modulators for the hmGluR4. SIB-1893 and MPEP enhanced the potency and efficacy of L-2-amino-4-phophonobutyrate (L-AP4) in guanosine 5'-O-(3-[(35)S...

  11. A large-scale allosteric transition in cytochrome P450 3A4 revealed by luminescence resonance energy transfer (LRET.

    Directory of Open Access Journals (Sweden)

    Elena V Sineva

    Full Text Available Effector-induced allosteric transitions in cytochrome P450 3A4 (CYP3A4 were investigated by luminescence resonance energy transfer (LRET between two SH-reactive probes attached to various pairs of distantly located cysteine residues, namely the double-cysteine mutants CYP3A4(C64/C468, CYP3A4(C377/C468 and CYP3A4(C64/C121. Successive equimolar labeling of these proteins with the phosphorescent probe erythrosine iodoacetamide (donor and the near-infrared fluorophore DY-731 maleimide (acceptor allowed us to establish donor/acceptor pairs sensitive to conformational motions. The interactions of all three double-labeled mutants with the allosteric activators α-naphthoflavone and testosterone resulted in an increase in the distance between the probes. A similar effect was elicited by cholesterol. These changes in distance vary from 1.3 to 8.5 Å, depending on the position of the donor/acceptor pair and the nature of the effector. In contrast, the changes in the interprobe distance caused by such substrates as bromocriptine or 1-pyrenebutanol were only marginal. Our results provide a decisive support to the paradigm of allosteric modulation of CYP3A4 and indicate that the conformational transition caused by allosteric effectors increases the spatial separation between the beta-domain of the enzyme (bearing residues Cys64 and Cys377 and the alpha-domain, where Cys121 and Cys468 are located.

  12. Thermodynamics and structural analysis of positive allosteric modulation of the ionotropic glutamate receptor GluA2

    DEFF Research Database (Denmark)

    Krintel, Christian; Frydenvang, Karla; Olsen, Lars

    2012-01-01

    Positive allosteric modulators of the ionotropic glutamate receptor-2 (GluA2) are promising compounds for the treatment of cognitive disorders, e.g. Alzheimer's disease. These modulators bind within the dimer interface of the ligand-binding domain and stabilize the agonist-bound conformation slow...

  13. Positive allosteric modulation of GABA-A receptors reduces capsaicin-induced primary and secondary hypersensitivity in rats

    DEFF Research Database (Denmark)

    Hansen, Rikke Rie; Erichsen, Helle K; Brown, David T

    2012-01-01

    GABA-A receptor positive allosteric modulators (PAMs) mediate robust analgesia in animal models of pathological pain, in part via enhancing injury-induced loss of GABA-A-α2 and -α3 receptor function within the spinal cord. As yet, a lack of clinically suitable tool compounds has prevented this co...

  14. Allosteric inactivation of a trypsin-like serine protease by an antibody binding to the 37- and 70-loops

    DEFF Research Database (Denmark)

    Kromann-Hansen, Tobias; Lund, Ida K; Liu, Zhuo

    2013-01-01

    for elucidating fundamental allosteric mechanisms. The monoclonal antibody mU1 has previously been shown to be able to inhibit the function of murine urokinase-type plasminogen activator in vivo. We have now mapped the epitope of mU1 to the catalytic domain's 37- and 70-loops, situated about 20 Å from the S1...

  15. Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface*

    Science.gov (United States)

    Wojcik, John; Lamontanara, Allan Joaquim; Grabe, Grzegorz; Koide, Akiko; Akin, Louesa; Gerig, Barbara; Hantschel, Oliver; Koide, Shohei

    2016-01-01

    Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl. PMID:26912659

  16. Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface.

    Science.gov (United States)

    Wojcik, John; Lamontanara, Allan Joaquim; Grabe, Grzegorz; Koide, Akiko; Akin, Louesa; Gerig, Barbara; Hantschel, Oliver; Koide, Shohei

    2016-04-15

    Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. The Structural Basis for Endotoxin-induced Allosteric Regulation of the Toll-like Receptor 4 (TLR4) Innate Immune Receptor*

    Science.gov (United States)

    Paramo, Teresa; Piggot, Thomas J.; Bryant, Clare E.; Bond, Peter J.

    2013-01-01

    As part of the innate immune system, Toll-like receptor 4 (TLR4) recognizes bacterial cell surface lipopolysaccharide (LPS) by forming a complex with a lipid-binding co-receptor, MD-2. In the presence of agonist, TLR4·MD-2 dimerizes to form an active receptor complex, leading to initiation of intracellular inflammatory signals. TLR4 is of great biomedical interest, but its pharmacological manipulation is complicated because even subtle variations in the structure of LPS can profoundly impact the resultant immunological response. Here, we use atomically detailed molecular simulations to gain insights into the nature of the molecular signaling mechanism. We first demonstrate that MD-2 is extraordinarily flexible. The “clamshell-like” motions of its β-cup fold enable it to sensitively match the volume of its hydrophobic cavity to the size and shape of the bound lipid moiety. We show that MD-2 allosterically transmits this conformational plasticity, in a ligand-dependent manner, to a phenylalanine residue (Phe-126) at the cavity mouth previously implicated in TLR4 activation. Remarkably, within the receptor complex, we observe spontaneous transitions between active and inactive signaling states of Phe-126, and we confirm that Phe-126 is indeed the “molecular switch” in endotoxic signaling. PMID:24178299

  18. The structural basis for endotoxin-induced allosteric regulation of the Toll-like receptor 4 (TLR4) innate immune receptor.

    Science.gov (United States)

    Paramo, Teresa; Piggot, Thomas J; Bryant, Clare E; Bond, Peter J

    2013-12-20

    As part of the innate immune system, Toll-like receptor 4 (TLR4) recognizes bacterial cell surface lipopolysaccharide (LPS) by forming a complex with a lipid-binding co-receptor, MD-2. In the presence of agonist, TLR4·MD-2 dimerizes to form an active receptor complex, leading to initiation of intracellular inflammatory signals. TLR4 is of great biomedical interest, but its pharmacological manipulation is complicated because even subtle variations in the structure of LPS can profoundly impact the resultant immunological response. Here, we use atomically detailed molecular simulations to gain insights into the nature of the molecular signaling mechanism. We first demonstrate that MD-2 is extraordinarily flexible. The "clamshell-like" motions of its β-cup fold enable it to sensitively match the volume of its hydrophobic cavity to the size and shape of the bound lipid moiety. We show that MD-2 allosterically transmits this conformational plasticity, in a ligand-dependent manner, to a phenylalanine residue (Phe-126) at the cavity mouth previously implicated in TLR4 activation. Remarkably, within the receptor complex, we observe spontaneous transitions between active and inactive signaling states of Phe-126, and we confirm that Phe-126 is indeed the "molecular switch" in endotoxic signaling.

  19. The Allosterically Unregulated Isoform of ADP-Glucose Pyrophosphorylase from Barley Endosperm Is the Most Likely Source of ADP-Glucose Incorporated into Endosperm Starch1

    Science.gov (United States)

    Doan, Danny N.P.; Rudi, Heidi; Olsen, Odd-Arne

    1999-01-01

    We present the results of studies of an unmodified version of the recombinant major barley (Hordeum vulgare) endosperm ADP-glucose pyrophoshorylase (AGPase) expressed in insect cells, which corroborate previous data that this isoform of the enzyme acts independently of the allosteric regulators 3-phosphoglycerate and inorganic phosphate. We also present a characterization of the individual subunits expressed separately in insect cells, showing that the SS AGPase is active in the presence of 3-phosphoglycerate and is inhibited by inorganic phosphate. As a step toward the elucidation of the role of the two AGPase isoforms in barley, the temporal and spatial expression profile of the four barley AGPase transcripts encoding these isoforms were studied. The results show that the steady-state level of beps and bepl, the transcripts encoding the major endosperm isoform, correlated positively with the rate of endosperm starch accumulation. In contrast, blps and blpl, the transcripts encoding the major leaf isoform, were constitutively expressed at a very low steady-state level throughout the barley plant. The implications of these findings for the evolution of plant AGPases are discussed. PMID:10557246

  20. The Allosterically Unregulated Isoform of ADP-Glucose Pyrophosphorylase from Barley Endosperm Is the Most Likely Source of ADP-Glucose Incorporated into Endosperm Starch.

    Science.gov (United States)

    Doan; Rudi; Olsen

    1999-11-01

    We present the results of studies of an unmodified version of the recombinant major barley (Hordeum vulgare) endosperm ADP-glucose pyrophoshorylase (AGPase) expressed in insect cells, which corroborate previous data that this isoform of the enzyme acts independently of the allosteric regulators 3-phosphoglycerate and inorganic phosphate. We also present a characterization of the individual subunits expressed separately in insect cells, showing that the SS AGPase is active in the presence of 3-phosphoglycerate and is inhibited by inorganic phosphate. As a step toward the elucidation of the role of the two AGPase isoforms in barley, the temporal and spatial expression profile of the four barley AGPase transcripts encoding these isoforms were studied. The results show that the steady-state level of beps and bepl, the transcripts encoding the major endosperm isoform, correlated positively with the rate of endosperm starch accumulation. In contrast, blps and blpl, the transcripts encoding the major leaf isoform, were constitutively expressed at a very low steady-state level throughout the barley plant. The implications of these findings for the evolution of plant AGPases are discussed.

  1. Fragment Based Optimization of Metabotropic Glutamate Receptor 2 (mGluR2) Positive Allosteric Modulators in the Absence of Structural Information.

    Science.gov (United States)

    Szabó, György; Túrós, György I; Kolok, Sándor; Vastag, Mónika; Sánta, Zsuzsanna; Dékány, Miklós; Lévay, György I; Greiner, István; Natsumi, Minami; Tatsuya, Watanabe; Keserű, György M

    2018-03-14

    Metabotropic glutamate receptor 2 (mGluR2) positive allosteric modulators (PAMs) have been implicated as potential pharmacotherapy for psychiatric conditions. Screening our corporate compound deck, we identified a benzotriazole fragment (4) that was rapidly optimized to a potent and metabolically stable early lead (16). The highly lipophilic character of 16, together with its limited solubility, permeability, and high protein binding, however, did not allow reaching of the proof of concept in vivo. Since further attempts on the optimization of druglike properties were unsuccessful, the original hit 4 has been revisited and was optimized following the principles of fragment based drug discovery (FBDD). Lacking structural information on the receptor-ligand complex, we implemented a group efficiency (GE) based strategy and identified a new fragment like lead (60) with more balanced profile. Significant improvement achieved on the druglike properties nominated the compound for in vivo proof of concept studies that revealed the chemotype being a promising PAM lead targeting mGluR2 receptors.

  2. Evidence for a Common Mechanism of SIRT1 Regulation by Allosteric Activators

    Science.gov (United States)

    Hubbard, Basil P.; Gomes, Ana P.; Dai, Han; Li, Jun; Case, April W.; Considine, Thomas; Riera, Thomas V.; Lee, Jessica E.; Sook Yen, E; Lamming, Dudley W.; Pentelute, Bradley L.; Schuman, Eli R.; Stevens, Linda A.; Ling, Alvin J. Y.; Armour, Sean M.; Michan, Shaday; Zhao, Huizhen; Jiang, Yong; Sweitzer, Sharon M.; Blum, Charles A.; Disch, Jeremy S.; Ng, Pui Yee; Howitz, Konrad T.; Rolo, Anabela P.; Hamuro, Yoshitomo; Moss, Joel; Perni, Robert B.; Ellis, James L.; Vlasuk, George P.; Sinclair, David A.

    2013-01-01

    A molecule that treats multiple age-related diseases would have a major impact on global health and economics. The SIRT1 deacetylase has drawn attention in this regard as a target for drug design. Yet controversy exists around the mechanism of sirtuin-activating compounds (STACs). We found that specific hydrophobic motifs found in SIRT1 substrates such as PGC-1α and FOXO3a facilitate SIRT1 activation by STACs. A single amino acid in SIRT1, Glu230, located in a structured N-terminal domain, was critical for activation by all previously reported STAC scaffolds and a new class of chemically distinct activators. In primary cells reconstituted with activation-defective SIRT1, the metabolic effects of STACs were blocked. Thus, SIRT1 can be directly activated through an allosteric mechanism common to chemically diverse STACs. PMID:23471411

  3. Molecular kinetics. Ras activation by SOS: allosteric regulation by altered fluctuation dynamics.

    Science.gov (United States)

    Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen; Christensen, Sune M; Abel, Steven M; Iwig, Jeff; Wu, Hung-Jen; Gureasko, Jodi; Rhodes, Christopher; Petit, Rebecca S; Hansen, Scott D; Thill, Peter; Yu, Cheng-Han; Stamou, Dimitrios; Chakraborty, Arup K; Kuriyan, John; Groves, Jay T

    2014-07-04

    Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average. Copyright © 2014, American Association for the Advancement of Science.

  4. Design and optimization of selective azaindole amide M1 positive allosteric modulators.

    Science.gov (United States)

    Davoren, Jennifer E; O'Neil, Steven V; Anderson, Dennis P; Brodney, Michael A; Chenard, Lois; Dlugolenski, Keith; Edgerton, Jeremy R; Green, Michael; Garnsey, Michelle; Grimwood, Sarah; Harris, Anthony R; Kauffman, Gregory W; LaChapelle, Erik; Lazzaro, John T; Lee, Che-Wah; Lotarski, Susan M; Nason, Deane M; Obach, R Scott; Reinhart, Veronica; Salomon-Ferrer, Romelia; Steyn, Stefanus J; Webb, Damien; Yan, Jiangli; Zhang, Lei

    2016-01-15

    Selective activation of the M1 receptor via a positive allosteric modulator (PAM) is a new approach for the treatment of the cognitive impairments associated with schizophrenia and Alzheimer's disease. A novel series of azaindole amides and their key pharmacophore elements are described. The nitrogen of the azaindole core is a key design element as it forms an intramolecular hydrogen bond with the amide N-H thus reinforcing the bioactive conformation predicted by published SAR and our homology model. Representative compound 25 is a potent and selective M1 PAM that has well aligned physicochemical properties, adequate brain penetration and pharmacokinetic (PK) properties, and is active in vivo. These favorable properties indicate that this series possesses suitable qualities for further development and studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Optimization of Allosteric With-No-Lysine (WNK) Kinase Inhibitors and Efficacy in Rodent Hypertension Models

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Ken; Levell, Julian; Yoon, Taeyong; Kohls, Darcy; Yowe, David; Rigel, Dean F.; Imase, Hidetomo; Yuan, Jun; Yasoshima, Kayo; DiPetrillo, Keith; Monovich, Lauren; Xu, Lingfei; Zhu, Meicheng; Kato, Mitsunori; Jain, Monish; Idamakanti, Neeraja; Taslimi, Paul; Kawanami, Toshio; Argikar, Upendra A.; Kunjathoor, Vidya; Xie, Xiaoling; Yagi, Yukiko I.; Iwaki, Yuki; Robinson, Zachary; Park, Hyi-Man (Novartis)

    2017-08-03

    The observed structure–activity relationship of three distinct ATP noncompetitive With-No-Lysine (WNK) kinase inhibitor series, together with a crystal structure of a previously disclosed allosteric inhibitor bound to WNK1, led to an overlay hypothesis defining core and side-chain relationships across the different series. This in turn enabled an efficient optimization through scaffold morphing, resulting in compounds with a good balance of selectivity, cellular potency, and pharmacokinetic profile, which were suitable for in vivo proof-of-concept studies. When dosed orally, the optimized compound reduced blood pressure in mice overexpressing human WNK1, and induced diuresis, natriuresis and kaliuresis in spontaneously hypertensive rats (SHR), confirming that this mechanism of inhibition of WNK kinase activity is effective at regulating cardiovascular homeostasis.

  6. Evidence for a common mechanism of SIRT1 regulation by allosteric activators.

    Science.gov (United States)

    Hubbard, Basil P; Gomes, Ana P; Dai, Han; Li, Jun; Case, April W; Considine, Thomas; Riera, Thomas V; Lee, Jessica E; E, Sook Yen; Lamming, Dudley W; Pentelute, Bradley L; Schuman, Eli R; Stevens, Linda A; Ling, Alvin J Y; Armour, Sean M; Michan, Shaday; Zhao, Huizhen; Jiang, Yong; Sweitzer, Sharon M; Blum, Charles A; Disch, Jeremy S; Ng, Pui Yee; Howitz, Konrad T; Rolo, Anabela P; Hamuro, Yoshitomo; Moss, Joel; Perni, Robert B; Ellis, James L; Vlasuk, George P; Sinclair, David A

    2013-03-08

    A molecule that treats multiple age-related diseases would have a major impact on global health and economics. The SIRT1 deacetylase has drawn attention in this regard as a target for drug design. Yet controversy exists around the mechanism of sirtuin-activating compounds (STACs). We found that specific hydrophobic motifs found in SIRT1 substrates such as PGC-1α and FOXO3a facilitate SIRT1 activation by STACs. A single amino acid in SIRT1, Glu(230), located in a structured N-terminal domain, was critical for activation by all previously reported STAC scaffolds and a new class of chemically distinct activators. In primary cells reconstituted with activation-defective SIRT1, the metabolic effects of STACs were blocked. Thus, SIRT1 can be directly activated through an allosteric mechanism common to chemically diverse STACs.

  7. Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

    Science.gov (United States)

    Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J; Smithgall, Thomas E

    2015-01-01

    The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important

  8. Modulation of calmodulin lobes by different targets: an allosteric model with hemiconcerted conformational transitions.

    Directory of Open Access Journals (Sweden)

    Massimo Lai

    2015-01-01

    Full Text Available Calmodulin is a calcium-binding protein ubiquitous in eukaryotic cells, involved in numerous calcium-regulated biological phenomena, such as synaptic plasticity, muscle contraction, cell cycle, and circadian rhythms. It exibits a characteristic dumbell shape, with two globular domains (N- and C-terminal lobe joined by a linker region. Each lobe can take alternative conformations, affected by the binding of calcium and target proteins. Calmodulin displays considerable functional flexibility due to its capability to bind different targets, often in a tissue-specific fashion. In various specific physiological environments (e.g. skeletal muscle, neuron dendritic spines several targets compete for the same calmodulin pool, regulating its availability and affinity for calcium. In this work, we sought to understand the general principles underlying calmodulin modulation by different target proteins, and to account for simultaneous effects of multiple competing targets, thus enabling a more realistic simulation of calmodulin-dependent pathways. We built a mechanistic allosteric model of calmodulin, based on an hemiconcerted framework: each calmodulin lobe can exist in two conformations in thermodynamic equilibrium, with different affinities for calcium and different affinities for each target. Each lobe was allowed to switch conformation on its own. The model was parameterised and validated against experimental data from the literature. In spite of its simplicity, a two-state allosteric model was able to satisfactorily represent several sets of experiments, in particular the binding of calcium on intact and truncated calmodulin and the effect of different skMLCK peptides on calmodulin's saturation curve. The model can also be readily extended to include multiple targets. We show that some targets stabilise the low calcium affinity T state while others stabilise the high affinity R state. Most of the effects produced by calmodulin targets can be

  9. The geology of the Vaalputs radioactive waste disposal site, with implications for granite-charnockite relationships and crustal evolution in western Namaqualand

    International Nuclear Information System (INIS)

    Andreoli, M.A.G.; Brynard, H.J.; Anderson, N.J.B.; Hart, R.J.; Moore, J.M.; Welke, H.

    1990-01-01

    The Precambrian geology of the Valputs site is characterized by extensive sheets of Spektakel Suite syntectonic granite gneisses. Experimental methods used for the geological evaluation of the Vaalputs site included semi-regional and site-specific mapping. All main rock types were petrologically and geochemically classified. In addition, preliminary Rb-Sr whole-rock ages were calculated for the main lithotypes, together with Sm-Nd dating. The investigations for the licensing of the Vaalputs site have revealed numerous intriguing aspects of the crustal history in southwestern Namaqualand. 10 refs

  10. Site-Mutation of Hydrophobic Core Residues Synchronically Poise Super Interleukin 2 for Signaling: Identifying Distant Structural Effects through Affordable Computations

    Directory of Open Access Journals (Sweden)

    Longcan Mei

    2018-03-01

    Full Text Available A superkine variant of interleukin-2 with six site mutations away from the binding interface developed from the yeast display technique has been previously characterized as undergoing a distal structure alteration which is responsible for its super-potency and provides an elegant case study with which to get insight about how to utilize allosteric effect to achieve desirable protein functions. By examining the dynamic network and the allosteric pathways related to those mutated residues using various computational approaches, we found that nanosecond time scale all-atom molecular dynamics simulations can identify the dynamic network as efficient as an ensemble algorithm. The differentiated pathways for the six core residues form a dynamic network that outlines the area of structure alteration. The results offer potentials of using affordable computing power to predict allosteric structure of mutants in knowledge-based mutagenesis.

  11. LIGAND-BINDING SITES ON THE MYCOBACTERIUM TUBERCULOSIS UREASE

    Directory of Open Access Journals (Sweden)

    Lisnyak Yu. V.

    2017-10-01

    Full Text Available Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis that remains a serious medical and social health problem. Despite intensive efforts have been made in the past decade, there are no new efficient anti-tuberculosis drugs today, and that need is growing due to the spread of drug-resistant strains of M.tuberculosis. M. tuberculosis urease (MTU, being an important factor of the bacterium viability and virulence, is an attractive target for anti-tuberculosis drugs acting by inhibition of urease activity. However, the commercially available urease inhibitors are toxic and unstable, that prevent their clinical use. Therefore, new more potent anti-tuberculosis drugs inhibiting new targets are urgently needed. A useful tool for the search of novel inhibitors is a computational drug design. The inhibitor design is significantly easier if binding sites on the enzyme are identified in advance. This paper aimed to determine the probable ligand binding sites on the surface of M. tuberculosis urease. Methods. To identify ligand binding sites on MTU surface, сomputational solvent mapping method FTSite was applied by the use of MTU homology model we have built earlier. The method places molecular probes (small organic molecules containing various functional groups on a dense grid defined around the enzyme, and for each probe finds favorable positions. The selected poses are refined by free energy minimization, the low energy conformations are clustered, and the clusters are ranked on the basis of the average free energy. FTSite server outputs the protein residues delineating a binding sites and the probe molecules representing each cluster. To predict allosteric pockets on MTU, AlloPred and AlloSite servers were applied. AlloPred uses the normal mode analysis (NMA and models how the dynamics of a protein would be altered in the presence of a modulator at a specific pocket. Pockets on the enzyme are predicted using the Fpocket

  12. Structural Fine-Tuning of MIT-Interacting Motif 2 (MIM2) and Allosteric Regulation of ESCRT-III by Vps4 in Yeast.

    Science.gov (United States)

    Kojima, Rieko; Obita, Takayuki; Onoue, Kousuke; Mizuguchi, Mineyuki

    2016-06-05

    The endosomal sorting complex required for transport (ESCRT) facilitates roles in membrane remodeling, such as multivesicular body biogenesis, enveloped virus budding and cell division. In yeast, Vps4 plays a crucial role in intraluminal vesicle formation by disassembling ESCRT proteins. Vps4 is recruited by ESCRT-III proteins to the endosomal membrane through the interaction between the microtubule interacting and trafficking (MIT) domain of Vps4 and the C-terminal MIT-interacting motif (MIM) of ESCRT-III proteins. Here, we have determined the crystal structure of Vps4-MIT in a complex with Vps20, a member of ESCRT-III, and revealed that Vps20 adopts a unique MIM2 conformation. Based on structural comparisons with other known MIM2s, we have refined the consensus sequence of MIM2. We have shown that another ESCRT-III protein, Ist1, binds to Vps4-MIT via its C-terminal MIM1 with higher affinity than Vps2, but lacks MIM2 by surface plasmon resonance. Surprisingly, the Ist1 MIM1 competed with the MIM2 of Vfa1, a regulator of Vps4, for binding to Vps4-MIT, even though these MIMs bind in non-overlapping sites on the MIT. These findings provide insight into the allosteric recognition of MIMs of ESCRT-III by Vps4 and also the regulation of ESCRT machinery at the last step of membrane remodeling. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Can a Positive Allosteric Modulation of GABAergic Receptors Improve Motor Symptoms in Patients with Parkinson's Disease? The Potential Role of Zolpidem in the Treatment of Parkinson's Disease

    Science.gov (United States)

    Daniele, Antonio; Panza, Francesco; Greco, Antonio; Logroscino, Giancarlo; Seripa, Davide

    2016-01-01

    At present, patients with advanced Parkinson's disease (PD) are unsatisfactorily controlled by currently used anti-Parkinsonian dopaminergic drugs. Various studies suggest that therapeutic strategies based on nondopaminergic drugs might be helpful in PD. Zolpidem, an imidazopyridine widely used as sleep inducer, shows high affinity only for GABAA receptors containing the α-1 subunit and facilitates GABAergic neurotransmission through a positive allosteric modulation of GABAA receptors. Various observations, although preliminary, consistently suggest that in PD patients zolpidem may induce beneficial (and sometimes remarkable) effects on motor symptoms even after single doses and may also improve dyskinesias. Since a high density of zolpidem binding sites is in the two main output structures of the basal ganglia which are abnormally overactive in PD (internal globus pallidus, GPi, and substantia nigra pars reticulata, SNr), it was hypothesized that in PD patients zolpidem may induce through GABAA receptors an inhibition of GPi and SNr (and, possibly, of the subthalamic nucleus also), resulting in an increased activity of motor cortical areas (such as supplementary motor area), which may give rise to improvement of motor symptoms of PD. Randomized clinical trials are needed in order to assess the efficacy, safety, and tolerability of zolpidem in treating motor symptoms of PD. PMID:27293955

  14. Identification of halosalicylamide derivatives as a novel class of allosteric inhibitors of HCV NS5B polymerase.

    Science.gov (United States)

    Liu, Yaya; Donner, Pamela L; Pratt, John K; Jiang, Wen W; Ng, Teresa; Gracias, Vijaya; Baumeister, Steve; Wiedeman, Paul E; Traphagen, Linda; Warrior, Usha; Maring, Clarence; Kati, Warren M; Djuric, Stevan W; Molla, Akhteruzzaman

    2008-06-01

    Halosalicylamide derivatives were identified from high-throughput screening as potent inhibitors of HCV NS5B polymerase. The subsequent structure and activity relationship revealed the absolute requirement of the salicylamide moiety for optimum activity. Methylation of either the hydroxyl group or the amide group of the salicylamide moiety abolished the activity while the substitutions on both phenyl rings are acceptable. The halosalicylamide derivatives were shown to be non-competitive with respect to elongation nucleotide and demonstrated broad genotype activity against genotype 1-3 HCV NS5B polymerases. Inhibitor competition studies indicated an additive binding mode to the initiation pocket that is occupied by the thiadiazine class of compounds and an additive binding mode to the elongation pocket that is occupied by diketoacids, but a mutually exclusive binding mode with respect to the allosteric thumb pocket that is occupied by the benzimidazole class of inhibitors. Therefore, halosalicylamides represent a novel class of allosteric inhibitors of HCV NS5B polymerase.

  15. A novel strategy for selection of allosteric ribozymes yields RiboReporter™ sensors for caffeine and aspartame

    Science.gov (United States)

    Ferguson, Alicia; Boomer, Ryan M.; Kurz, Markus; Keene, Sara C.; Diener, John L.; Keefe, Anthony D.; Wilson, Charles; Cload, Sharon T.

    2004-01-01

    We have utilized in vitro selection technology to develop allosteric ribozyme sensors that are specific for the small molecule analytes caffeine or aspartame. Caffeine- or aspartame-responsive ribozymes were converted into fluorescence-based RiboReporter™ sensor systems that were able to detect caffeine or aspartame in solution over a concentration range from 0.5 to 5 mM. With read-times as short as 5 min, these caffeine- or aspartame-dependent ribozymes function as highly specific and facile molecular sensors. Interestingly, successful isolation of allosteric ribozymes for the analytes described here was enabled by a novel selection strategy that incorporated elements of both modular design and activity-based selection methods typically used for generation of catalytic nucleic acids. PMID:15026535

  16. A New Negative Allosteric Modulator AP14145 for the Study of Small Conductance Calcium-Activated Potassium Channels

    DEFF Research Database (Denmark)

    Simo Vicens, Rafel; Kirchhoff, Jeppe Egedal; Dolce, Bernardo

    2017-01-01

    ) prolongation in anaesthetised rats and a beam walk test was performed in mice to determine acute CNS related effects of the drug. Key results: AP14145 was found to be an equipotent negative allosteric modulator of KCa2.2 and KCa2.3 channels (IC50 = 1.1 ± 0.3 μM L-1). The presence of AP14145 (10 μM L-1...

  17. Determinants of positive cooperativity between strychnine-like allosteric modulators and N-methylscopolamine at muscarinic receptors

    Czech Academy of Sciences Publication Activity Database

    Jakubík, Jan; Doležal, Vladimír

    2006-01-01

    Roč. 30, č. 1-2 (2006), s. 111-112 ISSN 0895-8696 R&D Projects: GA ČR(CZ) GA305/05/0452; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic receptors * strychnine -like allosteric modulators * cooperativity Subject RIV: ED - Physiology Impact factor: 2.965, year: 2006

  18. [Pharmacological characteristics of drugs targeted on calcium-sensing receptor.-properties of cinacalcet hydrochloride as allosteric modulator].

    Science.gov (United States)

    Nagano, Nobuo; Tsutsui, Takaaki

    2016-06-01

    Calcimimetics act as positive allosteric modulators of the calcium-sensing receptor (CaSR), thereby decreasing parathyroid hormone (PTH) secretion from the parathyroid glands. On the other hand, negative allosteric modulators of the CaSR with stimulatory effect on PTH secretion are termed calcilytics. The calcimimetic cinacalcet hydrochloride (cinacalcet) is the world's first allosteric modulator of G protein-coupled receptor to enter the clinical market. Cinacalcet just tunes the physiological effects of Ca(2+), an endogenous ligand, therefore, shows high selectivity and low side effects. Calcimimetics also increase cell surface CaSR expression by acting as pharmacological chaperones (pharmacoperones). It is considered that the cinacalcet-induced upper gastrointestinal problems are resulted from enhanced physiological responses to Ca(2+) and amino acids via increased sensitivity of digestive tract CaSR by cinacalcet. While clinical developments of calcilytics for osteoporosis were unfortunately halted or terminated due to paucity of efficacy, it is expected that calcilytics may be useful for the treatment of patients with activating CaSR mutations, asthma, and idiopathic pulmonary artery hypertension.

  19. Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer.

    Science.gov (United States)

    Bonaventura, Jordi; Navarro, Gemma; Casadó-Anguera, Verònica; Azdad, Karima; Rea, William; Moreno, Estefanía; Brugarolas, Marc; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Cortés, Antoni; Volkow, Nora D; Schiffmann, Serge N; Ferré, Sergi; Casadó, Vicent

    2015-07-07

    Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain.

  20. Positive allosteric modulation of TRPV1 as a novel analgesic mechanism

    Directory of Open Access Journals (Sweden)

    Lebovitz Evan E

    2012-09-01

    Full Text Available Abstract Background The prevalence of long-term opiate use in treating chronic non-cancer pain is increasing, and prescription opioid abuse and dependence are a major public health concern. To explore alternatives to opioid-based analgesia, the present study investigates a novel allosteric pharmacological approach operating through the cation channel TRPV1. This channel is highly expressed in subpopulations of primary afferent unmyelinated C- and lightly-myelinated Aδ-fibers that detect low and high rates of noxious heating, respectively, and it is also activated by vanilloid agonists and low pH. Sufficient doses of exogenous vanilloid agonists, such as capsaicin or resiniferatoxin, can inactivate/deactivate primary afferent endings due to calcium overload, and we hypothesized that positive allosteric modulation of agonist-activated TRPV1 could produce a selective, temporary inactivation of nociceptive nerve terminals in vivo. We previously identified MRS1477, a 1,4-dihydropyridine that potentiates vanilloid and pH activation of TRPV1 in vitro, but displays no detectable intrinsic agonist activity of its own. To study the in vivo effects of MRS1477, we injected the hind paws of rats with a non-deactivating dose of capsaicin, MRS1477, or the combination. An infrared diode laser was used to stimulate TRPV1-expressing nerve terminals and the latency and intensity of paw withdrawal responses were recorded. qRT-PCR and immunohistochemistry were performed on dorsal root ganglia to examine changes in gene expression and the cellular specificity of such changes following treatment. Results Withdrawal responses of the capsaicin-only or MRS1477-only treated paws were not significantly different from the untreated, contralateral paws. However, rats treated with the combination of capsaicin and MRS1477 exhibited increased withdrawal latency and decreased response intensity consistent with agonist potentiation and inactivation or lesion of TRPV1-containing

  1. Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

    Directory of Open Access Journals (Sweden)

    Prerna Grover

    Full Text Available The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery

  2. Programmatic implications of implementing the relational algebraic capacitated location (RACL algorithm outcomes on the allocation of laboratory sites, test volumes, platform distribution and space requirements

    Directory of Open Access Journals (Sweden)

    Naseem Cassim

    2017-02-01

    Full Text Available Introduction: CD4 testing in South Africa is based on an integrated tiered service delivery model that matches testing demand with capacity. The National Health Laboratory Service has predominantly implemented laboratory-based CD4 testing. Coverage gaps, over-/under-capacitation and optimal placement of point-of-care (POC testing sites need investigation. Objectives: We assessed the impact of relational algebraic capacitated location (RACL algorithm outcomes on the allocation of laboratory and POC testing sites. Methods: The RACL algorithm was developed to allocate laboratories and POC sites to ensure coverage using a set coverage approach for a defined travel time (T. The algorithm was repeated for three scenarios (A: T = 4; B: T = 3; C: T = 2 hours. Drive times for a representative sample of health facility clusters were used to approximate T. Outcomes included allocation of testing sites, Euclidian distances and test volumes. Additional analysis included platform distribution and space requirement assessment. Scenarios were reported as fusion table maps. Results: Scenario A would offer a fully-centralised approach with 15 CD4 laboratories without any POC testing. A significant increase in volumes would result in a four-fold increase at busier laboratories. CD4 laboratories would increase to 41 in scenario B and 61 in scenario C. POC testing would be offered at two sites in scenario B and 20 sites in scenario C. Conclusion: The RACL algorithm provides an objective methodology to address coverage gaps through the allocation of CD4 laboratories and POC sites for a given T. The algorithm outcomes need to be assessed in the context of local conditions.

  3. Models, theory structure and mechanisms in biochemistry: The case of allosterism.

    Science.gov (United States)

    Alleva, Karina; Díez, José; Federico, Lucia

    2017-06-01

    From the perspective of the new mechanistic philosophy, it has been argued that explanatory causal mechanisms in some special sciences such as biochemistry and neurobiology cannot be captured by any useful notion of theory, or at least by any standard notion. The goal of this paper is to show that a model-theoretic notion of theory, and in particular the structuralist notion of a theory-net already applied to other unified explanatory theories, adequately suits the MWC allosteric mechanism explanatory set-up. We also argue, contra some mechanistic claims questioning the use of laws in biological explanations, that the theory reconstructed in this way essentially contains non-accidental regularities that qualify as laws, and that taking into account these lawful components, it is possible to explicate the unified character of the theory. Finally, we argue that, contrary to what some mechanists also claim, functional explanations that do not fully specify the mechanistic structure are not defective or incomplete in any relevant sense, and that functional components are perfectly explanatory. The conclusion is that, as some authors have emphasized in other fields (Walmsley 2008), particular elements of traditional approaches do not contradict but rather complement the new mechanist philosophy, and taken together they may offer a more complete understanding of special sciences and the variety of explanations they provide. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Self-phosphorylation of epidermal growth factor receptor: evidence for a model of intermolecular allosteric activation

    International Nuclear Information System (INIS)

    Yarden, Y.; Schlessinger, J.

    1987-01-01

    The membrane receptor for epidermal growth factor (EGF) is a 170,000 dalton glycoprotein composed of an extracellular EGF-binding domain and a cytoplasmic kinase domain connected by a stretch of 23 amino acids traversing the plasma membrane. The binding of EGF to the extracellular domain activates the cytoplasmic kinase function even in highly purified preparations of EGF receptor, suggesting that the activation occurs exclusively within the EGF receptor moiety. Conceivably, kinase activation may require the transfer of a conformational change through the single transmembrane region from the ligand binding domain to the cytoplasmic kinase region. Alternatively, ligand-induced receptor-receptor interactions may activate the kinase and thus bypass this requirement. Both mechanisms were contrasted by employing independent experimental approaches. On the basis of these results, an allosteric aggregation model is formulated for the activation of the cytoplasmic kinase function of the receptor by EGF. This model may be relevant to the mechanism by which the mitogenic signal of EGF is transferred across the membrane

  5. Nonequilibrium dissipation-free transport in F₁-ATPase and the thermodynamic role of asymmetric allosterism.

    Science.gov (United States)

    Kawaguchi, Kyogo; Sasa, Shin-Ichi; Sagawa, Takahiro

    2014-06-03

    F1-ATPase (or F1), the highly efficient and reversible biochemical engine, has motivated physicists as well as biologists to imagine the design principles governing machines in the fluctuating world. Recent experiments have clarified yet another interesting property of F1; the dissipative heat inside the motor is very small, irrespective of the velocity of rotation and energy transport. Conceptual interest is devoted to the fact that the amount of internal dissipation is not simply determined by the sequence of equilibrium pictures, but also relies on the rotational-angular dependence of nucleotide affinity, which is a truly nonequilibrium aspect. We propose that the totally asymmetric allosteric model (TASAM), where adenosine triphosphate (ATP) binding to F1 is assumed to have low dependence on the angle of the rotating shaft, produces results that are most consistent with the experiments. Theoretical analysis proves the crucial role of two time scales in the model, which explains the universal mechanism to produce the internal dissipation-free feature. The model reproduces the characteristic torque dependence of the rotational velocity of F1 and predicts that the internal dissipation upon the ATP synthesis direction rotation becomes large at the low nucleotide condition. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  6. Conformational changes and allosteric communications in human serum albumin due to ligand binding.

    Science.gov (United States)

    Ahalawat, Navjeet; Murarka, Rajesh K

    2015-01-01

    It is well recognized that knowledge of structure alone is not sufficient to understand the fundamental mechanism of biomolecular recognition. Information of dynamics is necessary to describe motions involving relevant conformational states of functional importance. We carried out principal component analysis (PCA) of structural ensemble, derived from 84 crystal structures of human serum albumin (HSA) with different ligands and/or different conditions, to identify the functionally important collective motions, and compared with the motions along the low-frequency modes obtained from normal mode analysis of the elastic network model (ENM) of unliganded HSA. Significant overlap is observed in the collective motions derived from PCA and ENM. PCA and ENM analysis revealed that ligand selects the most favored conformation from accessible equilibrium structures of unliganded HSA. Further, we analyzed dynamic network obtained from molecular dynamics simulations of unliganded HSA and fatty acids- bound HSA. Our results show that fatty acids-bound HSA has more robust community network with several routes to communicate among different parts of the protein. Critical nodes (residues) identified from dynamic network analysis are in good agreement with allosteric residues obtained from sequence-based statistical coupling analysis method. This work underscores the importance of intrinsic structural dynamics of proteins in ligand recognition and can be utilized for the development of novel drugs with optimum activity.

  7. Intracellular calcium levels determine differential modulation of allosteric interactions within G protein-coupled receptor heteromers.

    Science.gov (United States)

    Navarro, Gemma; Aguinaga, David; Moreno, Estefania; Hradsky, Johannes; Reddy, Pasham P; Cortés, Antoni; Mallol, Josefa; Casadó, Vicent; Mikhaylova, Marina; Kreutz, Michael R; Lluís, Carme; Canela, Enric I; McCormick, Peter J; Ferré, Sergi

    2014-11-20

    The pharmacological significance of the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer is well established and it is being considered as an important target for the treatment of Parkinson’s disease and other neuropsychiatric disorders. However, the physiological factors that control its distinctive biochemical properties are still unknown. We demonstrate that different intracellular Ca2+ levels exert a differential modulation of A2AR-D2R heteromer-mediated adenylyl-cyclase and MAPK signaling in striatal cells. This depends on the ability of low and high Ca2+ levels to promote a selective interaction of the heteromer with the neuronal Ca2+-binding proteins NCS-1 and calneuron-1, respectively. These Ca2+-binding proteins differentially modulate allosteric interactions within the A2AR-D2R heteromer, which constitutes a unique cellular device that integrates extracellular (adenosine and dopamine) and intracellular (Ca+2) signals to produce a specific functional response.

  8. Energy transfer by way of an exciplex intermediate in flexible boron dipyrromethene-based allosteric architectures.

    Science.gov (United States)

    Mula, Soumyaditya; Elliott, Kristopher; Harriman, Anthony; Ziessel, Raymond

    2010-10-07

    We have designed and synthesized a series of modular, dual-color dyes comprising a conventional boron dipyrromethene (Bodipy) dye, as a yellow emitter, and a Bodipy dye possessing extended conjugation that functions as a red emitter. A flexible tether of variable length, built from ethylene glycol residues, connects the terminal dyes. A critical design element of this type of dyad relates to a secondary amine linkage interposed between the conventional Bodipy and the tether. Cyclic voltammetry shows both Bodipy dyes to be electroactive and indicates that the secondary amine is quite easily oxidized. The ensuing fluorescence quenching is best explained in terms of the rapid formation of an intermediate charge-transfer state. In fact, exciplex-type emission is observed in weakly polar solvents and over a critical temperature range. In the dual-color dyes, direct excitation of the yellow emitter results in the appearance of red fluorescence, indicating that the exciplex is likely involved in the energy-transfer event, and provides for a virtual Stokes shift of 5000 cm(-1). Replacing the red emitter with a higher energy absorber (namely, pyrene) facilitates the collection of near-UV light and extends the virtual Stokes shift to 8000 cm(-1). Modulation of the efficacy of intramolecular energy transfer is achieved by preorganization of the connector in the presence of certain cations. This latter behavior, which is fully reversible, corresponds to an artificial allosteric effect.

  9. Comparison of Cenozoic Faulting at the Savannah River Site to Fault Characteristics of the Atlantic Coast Fault Province: Implications for Fault Capability

    International Nuclear Information System (INIS)

    Cumbest, R.J.

    2000-01-01

    This study compares the faulting observed on the Savannah River Site and vicinity with the faults of the Atlantic Coastal Fault Province and concludes that both sets of faults exhibit the same general characteristics and are closely associated. Based on the strength of this association it is concluded that the faults observed on the Savannah River Site and vicinity are in fact part of the Atlantic Coastal Fault Province. Inclusion in this group means that the historical precedent established by decades of previous studies on the seismic hazard potential for the Atlantic Coastal Fault Province is relevant to faulting at the Savannah River Site. That is, since these faults are genetically related the conclusion of ''not capable'' reached in past evaluations applies.In addition, this study establishes a set of criteria by which individual faults may be evaluated in order to assess their inclusion in the Atlantic Coast Fault Province and the related association of the ''not capable'' conclusion

  10. Fast growing, healthy and resident green turtles (Chelonia mydas at two neritic sites in the central and northern coast of Peru: implications for conservation.

    Directory of Open Access Journals (Sweden)

    Ximena Velez-Zuazo

    Full Text Available In order to enhance protection and conservation strategies for endangered green turtles (Chelonia mydas, the identification of neritic habitats where this species aggregates is mandatory. Herein, we present new information about the population parameters and residence time of two neritic aggregations from 2010 to 2013; one in an upwelling dominated site (Paracas ∼14°S and the other in an ecotone zone from upwelling to warm equatorial conditions (El Ñuro ∼4°S in the Southeast Pacific. We predicted proportionally more adult individuals would occur in the ecotone site; whereas in the site dominated by an upwelling juvenile individuals would predominate. At El Ñuro, the population was composed by (15.3% of juveniles, (74.9% sub-adults, and (9.8% adults, with an adult sex ratio of 1.16 males per female. Times of residence in the area ranged between a minimum of 121 and a maximum of 1015 days (mean 331.1 days. At Paracas the population was composed by (72% of juveniles and (28% sub-adults, no adults were recorded, thus supporting the development habitat hypothesis stating that throughout the neritic distribution there are sites exclusively occupied by juveniles. Residence time ranged between a minimum of 65 days and a maximum of 680 days (mean 236.1. High growth rates and body condition index values were estimated suggesting healthy individuals at both study sites. The population traits recorded at both sites suggested that conditions found in Peruvian neritic waters may contribute to the recovery of South Pacific green turtles. However, both aggregations are still at jeopardy due to pollution, bycatch and illegal catch and thus require immediate enforcing of conservation measurements.

  11. Fast Growing, Healthy and Resident Green Turtles (Chelonia mydas) at Two Neritic Sites in the Central and Northern Coast of Peru: Implications for Conservation

    Science.gov (United States)

    Velez-Zuazo, Ximena; Quiñones, Javier; Pacheco, Aldo S.; Klinge, Luciana; Paredes, Evelyn; Quispe, Sixto; Kelez, Shaleyla

    2014-01-01

    In order to enhance protection and conservation strategies for endangered green turtles (Chelonia mydas), the identification of neritic habitats where this species aggregates is mandatory. Herein, we present new information about the population parameters and residence time of two neritic aggregations from 2010 to 2013; one in an upwelling dominated site (Paracas ∼14°S) and the other in an ecotone zone from upwelling to warm equatorial conditions (El Ñuro ∼4°S) in the Southeast Pacific. We predicted proportionally more adult individuals would occur in the ecotone site; whereas in the site dominated by an upwelling juvenile individuals would predominate. At El Ñuro, the population was composed by (15.3%) of juveniles, (74.9%) sub-adults, and (9.8%) adults, with an adult sex ratio of 1.16 males per female. Times of residence in the area ranged between a minimum of 121 and a maximum of 1015 days (mean 331.1 days). At Paracas the population was composed by (72%) of juveniles and (28%) sub-adults, no adults were recorded, thus supporting the development habitat hypothesis stating that throughout the neritic distribution there are sites exclusively occupied by juveniles. Residence time ranged between a minimum of 65 days and a maximum of 680 days (mean 236.1). High growth rates and body condition index values were estimated suggesting healthy individuals at both study sites. The population traits recorded at both sites suggested that conditions found in Peruvian neritic waters may contribute to the recovery of South Pacific green turtles. However, both aggregations are still at jeopardy due to pollution, bycatch and illegal catch and thus require immediate enforcing of conservation measurements. PMID:25409240

  12. Beach erosion and nest site selection by the leatherback sea turtle Dermochelys coriacea (Testudines: Dermochelyidae and implications for management practices at Playa Gandoca, Costa Rica

    Directory of Open Access Journals (Sweden)

    Matthew J Spanier

    2010-12-01

    Full Text Available Leatherback sea turtles (Dermochelys coriacea nest on dynamic, erosion-prone beaches. Erosive processes and resulting nest loss have long been presumed to be a hindrance to clutch survival. In order to better understand how leatherbacks cope with unstable nesting beaches, I investigated the role of beach erosion in leatherback nest site selection at Playa Gandoca, Costa Rica. I also examined the potential effect of nest relocation, a conservation strategy in place at Playa Gandoca to prevent nest loss to erosion, on the temperature of incubating clutches. I monitored changes in beach structure as a result of erosion at natural nest sites during the time the nest was laid, as well as in subsequent weeks. To investigate slope as a cue for nest site selection, I measured the slope of the beach where turtles ascended from the sea to nest, as well as the slopes at other random locations on the beach for comparison. I examined temperature differences between natural and relocated nest sites with thermocouples placed in the sand at depths typical of leatherback nests. Nests were distributed non-randomly in a clumped distribution along the length of the beach and laid at locations that were not undergoing erosion. The slope at nest sites was significantly different than at randomly chosen locations on the beach. The sand temperature at nest depths was significantly warmer at natural nest sites than at locations of relocated nests. The findings of this study suggest leatherbacks actively select nest sites that are not undergoing erosive processes, with slope potentially being used as a cue for site selection. The relocation of nests appears to be inadvertently cooling the nest environment. Due to the fact that leatherback clutches undergo temperaturedependent sex determination, the relocation of nests may be producing an unnatural male biasing of hatchlings. The results of this study suggest that the necessity of relocation practices, largely in place to

  13. Deciphering complex dynamics of water counteraction around secondary structural elements of allosteric protein complex: Case study of SAP-SLAM system in signal transduction cascade.

    Science.gov (United States)

    Samanta, Sudipta; Mukherjee, Sanchita

    2018-01-28

    The first hydration shell of a protein exhibits heterogeneous behavior owing to several attributes, majorly local polarity and structural flexibility as revealed by solvation dynamics of secondary structural elements. We attempt to recognize the change in complex water counteraction generated due to substantial alteration in flexibility during protein complex formation. The investigation is carried out with the signaling lymphocytic activation molecule (SLAM) family of receptors, expressed by an array of immune cells, and interacting with SLAM-associated protein (SAP), composed of one SH2 domain. All atom molecular dynamics simulations are employed to the aqueous solutions of free SAP and SLAM-peptide bound SAP. We observed that water dynamics around different secondary structural elements became highly affected as well as nicely correlated with the SLAM-peptide induced change in structural rigidity obtained by thermodynamic quantification. A few instances of contradictory dynamic features of water to the change in structural flexibility are explained by means of occluded polar residues by the peptide. For βD, EFloop, and BGloop, both structural flexibility and solvent accessibility of the residues confirm the obvious contribution. Most importantly, we have quantified enhanced restriction in water dynamics around the second Fyn-binding site of the SAP due to SAP-SLAM complexation, even prior to the presence of Fyn. This observation leads to a novel argument that SLAM induced more restricted water molecules could offer more water entropic contribution during the subsequent Fyn binding and provide enhanced stability to the SAP-Fyn complex in the signaling cascade. Finally, SLAM induced water counteraction around the second binding site of the SAP sheds light on the allosteric property of the SAP, which becomes an integral part of the underlying signal transduction mechanism.

  14. Deciphering complex dynamics of water counteraction around secondary structural elements of allosteric protein complex: Case study of SAP-SLAM system in signal transduction cascade

    Science.gov (United States)

    Samanta, Sudipta; Mukherjee, Sanchita

    2018-01-01

    The first hydration shell of a protein exhibits heterogeneous behavior owing to several attributes, majorly local polarity and structural flexibility as revealed by solvation dynamics of secondary structural elements. We attempt to recognize the change in complex water counteraction generated due to substantial alteration in flexibility during protein complex formation. The investigation is carried out with the signaling lymphocytic activation molecule (SLAM) family of receptors, expressed by an array of immune cells, and interacting with SLAM-associated protein (SAP), composed of one SH2 domain. All atom molecular dynamics simulations are employed to the aqueous solutions of free SAP and SLAM-peptide bound SAP. We observed that water dynamics around different secondary structural elements became highly affected as well as nicely correlated with the SLAM-peptide induced change in structural rigidity obtained by thermodynamic quantification. A few instances of contradictory dynamic features of water to the change in structural flexibility are explained by means of occluded polar residues by the peptide. For βD, EFloop, and BGloop, both structural flexibility and solvent accessibility of the residues confirm the obvious contribution. Most importantly, we have quantified enhanced restriction in water dynamics around the second Fyn-binding site of the SAP due to SAP-SLAM complexation, even prior to the presence of Fyn. This observation leads to a novel argument that SLAM induced more restricted water molecules could offer more water entropic contribution during the subsequent Fyn binding and provide enhanced stability to the SAP-Fyn complex in the signaling cascade. Finally, SLAM induced water counteraction around the second binding site of the SAP sheds light on the allosteric property of the SAP, which becomes an integral part of the underlying signal transduction mechanism.

  15. The Role of Aldehyde Oxidase and Xanthine Oxidase in the Biotransformation of a Novel Negative Allosteric Modulator of Metabotropic Glutamate Receptor Subtype 5

    Science.gov (United States)

    Morrison, Ryan D.; Blobaum, Anna L.; Byers, Frank W.; Santomango, Tammy S.; Bridges, Thomas M.; Stec, Donald; Brewer, Katrina A.; Sanchez-Ponce, Raymundo; Corlew, Melany M.; Rush, Roger; Felts, Andrew S.; Manka, Jason; Bates, Brittney S.; Venable, Daryl F.; Rodriguez, Alice L.; Jones, Carrie K.; Niswender, Colleen M.; Conn, P. Jeffrey; Lindsley, Craig W.; Emmitte, Kyle A.

    2012-01-01

    Negative allosteric modulation (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5) represents a therapeutic strategy for the treatment of childhood developmental disorders, such as fragile X syndrome and autism. VU0409106 emerged as a lead compound within a biaryl ether series, displaying potent and selective inhibition of mGlu5. Despite its high clearance and short half-life, VU0409106 demonstrated efficacy in rodent models of anxiety after extravascular administration. However, lack of a consistent correlation in rat between in vitro hepatic clearance and in vivo plasma clearance for the biaryl ether series prompted an investigation into the biotransformation of VU0409106 using hepatic subcellular fractions. An in vitro appraisal in rat, monkey, and human liver S9 fractions indicated that the principal pathway was NADPH-independent oxidation to metabolite M1 (+16 Da). Both raloxifene (aldehyde oxidase inhibitor) and allopurinol (xanthine oxidase inhibitor) attenuated the formation of M1, thus implicating the contribution of both molybdenum hydroxylases in the biotransformation of VU0409106. The use of 18O-labeled water in the S9 experiments confirmed the hydroxylase mechanism proposed, because 18O was incorporated into M1 (+18 Da) as well as in a secondary metabolite (M2; +36 Da), the formation of which was exclusively xanthine oxidase-mediated. This unusual dual and sequential hydroxylase metabolism was confirmed in liver S9 and hepatocytes of multiple species and correlated with in vivo data because M1 and M2 were the principal metabolites detected in rats administered VU0409106. An in vitro-in vivo correlation of predicted hepatic and plasma clearance was subsequently established for VU0409106 in rats and nonhuman primates. PMID:22711749

  16. The Transcription Bubble of the RNA Polymerase-Promoter Open Complex Exhibits Conformational Heterogeneity and Millisecond-Scale Dynamics : Implications for Transcription Start-Site Selection

    NARCIS (Netherlands)

    Robb, Nicole C.; Cordes, Thorben; Hwang, Ling Chin; Gryte, Kristofer; Duchi, Diego; Craggs, Timothy D.; Santoso, Yusdi; Weiss, Shimon; Ebright, Richard H.; Kapanidis, Achillefs N.

    2013-01-01

    Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts similar to 14 bp around the transcription start site and forms a single-stranded "transcription bubble" within a catalytically active RNAP-DNA open complex (RPo). There is significant flexibility in the

  17. Counselling Implications of Teachers' Digital Competencies in the Use of Social Networking Sites (SNSs) in the Teaching-Learning Process in Calabar, Nigeria

    Science.gov (United States)

    Eyo, Mfon

    2016-01-01

    The study investigated teachers' digital competencies in the use of Social Networking Sites (SNSs) in the teaching-learning process. It had five research questions and two hypotheses. Adopting a survey design, it used a sample of 250 teachers from 10 out of 16 secondary schools in Calabar Municipal Local Government. A researcher-developed…

  18. Heavy metal contamination of soil and water in the vicinity of an abandoned e-waste recycling site: implications for dissemination of heavy metals.

    Science.gov (United States)

    Wu, Qihang; Leung, Jonathan Y S; Geng, Xinhua; Chen, Shejun; Huang, Xuexia; Li, Haiyan; Huang, Zhuying; Zhu, Libin; Chen, Jiahao; Lu, Yayin

    2015-02-15

    Illegal e-waste recycling activity has caused heavy metal pollution in many developing countries, including China. In recent years, the Chinese government has strengthened enforcement to impede such activity; however, the heavy metals remaining in the abandoned e-waste recycling site can still pose ecological risk. The present study aimed to investigate the concentrations of heavy metals in soil and water in the vicinity of an abandoned e-waste recycling site in Longtang, South China. Results showed that the surface soil of the former burning and acid-leaching sites was still heavily contaminated with Cd (>0.39 mg kg(-1)) and Cu (>1981 mg kg(-1)), which exceeded their respective guideline levels. The concentration of heavy metals generally decreased with depth in both burning site and paddy field, which is related to the elevated pH and reduced TOM along the depth gradient. The pond water was seriously acidified and contaminated with heavy metals, while the well water was slightly contaminated since heavy metals were mostly retained in the surface soil. The use of pond water for irrigation resulted in considerable heavy metal contamination in the paddy soil. Compared with previous studies, the reduced heavy metal concentrations in the surface soil imply that heavy metals were transported to the other areas, such as pond. Therefore, immediate remediation of the contaminated soil and water is necessary to prevent dissemination of heavy metals and potential ecological disaster. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. The structural evolution of the coastal area between Danger Point and Struisbaai in the southern Cape Fold Belt, with implications for the siting of a nuclear power station

    International Nuclear Information System (INIS)

    Andersen, N.J.B.; Andreoli, M.A.G.

    1990-01-01

    A structural analysis of the coastal area between Danger Point and Struisbaai in the Southern Cape has been undertaken, using the technique of structural domain analysis coupled with geophysical interpretation and geological mapping. This study forms part of the country-wide geological investigations that are being carried out for the purpose of siting South Africa's future nuclear power stations. 30 refs., 18 figs

  20. The Legal Implications of Student Use of Social Networking Sites in the UK and US: Current Concerns and Lessons for the Future

    Science.gov (United States)

    Davies, Mark R.; Lee, Barbara A.

    2008-01-01

    This paper provides a comparative snapshot of the current state of the law in the US and UK with respect to potential liability of university and college students for use (and misuse) of social networking sites. It reviews the limited case law on this topic, highlights the differences in the two nations' laws of defamation and the various possible…

  1. Basement Basalts from IODP Site 1438, Amami-Sankaku Basin: Implications for Sources and Melting Processes during Subduction Initiation in the Izu-Bonin-Mariana System

    Science.gov (United States)

    McCarthy, A. J.; Hickey-Vargas, R.; Yogodzinski, G. M.; Ishizuka, O.; Hocking, B.; Bizimis, M.; Savov, I. P.; Kusano, Y.; Arculus, R. J.

    2016-12-01

    IODP Expedition 351 Site 1438 is located in the Amami-Sankaku basin, just west of the Kyushu-Palau Ridge (KPR), a remnant of the early Izu-Bonin-Mariana (IBM) volcanic arc. 150 meters of basement basalt were drilled beneath 1460 m of volcaniclastic sediments and sedimentary rock. The age range inferred for these basalts is 51-52 Ma, close to the 48-52 Ma age of basalts associated with subduction initiation in the IBM forearc (forearc basalts or FABs). Site 1438 basement basalts form several distinct subunits, all relatively mafic (MgO = 6-14 %; Mg# = 51-83). Non-fluid-mobile incompatible trace element patterns are profoundly depleted. Sm/Nd (0.34-0.43) and Lu/Hf (0.18-0.37) reach values higher than most normal MORBs while La/Yb (0.31-0.98) and Ti/V (15.8-27.0) are lower. These features are shared with basalts drilled just west of the KPR at ODP Site 1201 and DSDP Site 447, and many FABs. Abundances of fluid-mobile incompatible elements vary together and are correlated with subunits defined by flow margins and rock physical properties, suggesting control by post-eruptive seawater alteration rather than varying inputs of subduction fluids. Hf-Nd isotopes for Site 1438 basement basalts range from (present-day) ɛNd of 7.0 to 9.5 and ɛHf of 14.5 to 19.8 in a well-correlated array. Their more radiogenic Hf-isotope character could indicate an Indian-type MORB source, however, basalts with ɛHf >16.5, are more radiogenic than many Indian MORB. Pb isotope data will help distinguish differing mantle source domains and origins for fluid-mobile elements. Overall, the combined geochemical data indicate that the mantle source of basement basalts in drill sites west of the KPR (1438, 1201, 447) are closely similar to those for FAB, and that as a group, these rocks are more depleted than more than 90% of global MORB. Our interpretation is that both IBM forearc basalts and basalts from drill sites immediately west of the KPR formed by melting of the same uniquely depleted mantle

  2. Using X-ray photoelectron spectroscopy to discriminate among different sorption sites of micas: With implications for heterogeneous reduction of chromate at the mica-water interface

    Energy Technology Data Exchange (ETDEWEB)

    Ilton, E.S.; Moses, C.O.; Veblen, D.R.

    2000-04-01

    This contribution uses Cr2p and 3p binding energies (BEs) determined by X-ray photoelectron spectroscopy (XPS) to distinguish Cr{sup III} sorbed to different mica sorption sites. The results were used to better understand mechanisms for coupled sorption-reduction of Cr{sub (aq)}{sup VI} by ferrous micas. The research is important because: (1) Cr contamination is a serious and wide spread problem associated with a variety of industries; (2) Micas are important sorbents for Cr because they are ubiquitous, sorb cations and anions over a wide range of pH, and participate in heterogeneous redox reactions via structural Fe{sup II} and FE{sup III}; and (3) The mobility of cations sorbed by micas will depend, in part, on the dominant sorption site. Micas (two biotites, end member phologopite and muscovite) were reacted with Cr{sup III} solutions that contained variable concentrations of NaCl and KCl. Samples were extracted at times intervals and mica edge orientations were analyzed by ZPS. Cr{sub (aq)}{sup III} sorption was greater in Cr{sup III}-NaCl solutions relative to Cr{sup III}-KCl solutions. Cr2p and 3p BEs were bracketed by those for Cr{sup III} in the structure of silicates and in Cr{sup III}-oxyhydroxides. The BE of Cr{sup III} sorbed to micas was higher by about 0.3 eV after reaction with Cr{sup III}-NaCl solutions compared to Cr{sup III}-KCl aqueous solutions. At the experimental conditions, Na{sup +} and K{sup +} differentially block permanent charge sites but not variably charged edge sites of micas. Using the constraints inherent in the experimental design, the authors interpret the difference in CR{sup III} BE between the Cr{sup III}-NaCl and Cr{sup III}-KCl experiments to reflect a change in the dominant sorption site, where high and low Cr BEs indicate Cr sorbed by the interlamellar region and variably charged functional groups at mica edges, respectively. They conclude that small BE shifts for cations sorbed to silicates can be interpreted with

  3. Parentage of overlapping offspring of an arboreal-breeding frog with no nest defense: implications for nest site selection and reproductive strategy.

    Science.gov (United States)

    Tung, Wan-Ping; Chen, Yi-Huey; Cheng, Wei-Chun; Chuang, Ming-Feng; Hsu, Wan-Tso; Kam, Yeong-Choy; Lehtinen, Richard M

    2015-01-01

    Overlapping offspring occurs when eggs are laid in a nest containing offspring from earlier reproduction. Earlier studies showed that the parentage is not always obvious due to difficulties in field observation and/or alternative breeding tactics. To unveil the parentage between overlapping offspring and parents is critical in understanding oviposition site selection and the reproductive strategies of parents. Amplectant pairs of an arboreal-breeding frog, Kurixalus eiffingeri, lay eggs in tadpole-occupied nests where offspring of different life stages (embryos and tadpoles) coexist. We used five microsatellite DNA markers to assess the parentage between parents and overlapping offspring. We also tested the hypothesis that the male or female frog would breed in the same breeding site because of the scarcity of nest sites. Results showed varied parentage patterns, which may differ from the phenomenon of overlapping egg clutches reported earlier. Parentage analyses showed that only 58 and 25% of the tadpole-occupied stumps were reused by the same male and female respectively, partially confirming our prediction. Re-nesting by the same individual was more common in males than females, which is most likely related to the cost of tadpole feeding and/or feeding schemes of females. On the other hand, results of parentage analyses showed that about 42 and 75% of male and female respectively bred in tadpole-occupied stumps where tadpoles were genetically unrelated. Results of a nest-choice experiment revealed that 40% of frogs chose tadpole-occupied bamboo cups when we presented identical stumps, without or with tadpoles, suggesting that the habitat saturation hypothesis does not fully explain why frogs used the tadpole-occupied stumps. Several possible benefits of overlapping offspring with different life stages were proposed. Our study highlights the importance of integrating molecular data with field observations to better understand the reproductive biology and nest

  4. Bone taphonomy of the Schöningen "Spear Horizon South" and its implications for site formation and hominin meat provisioning.

    Science.gov (United States)

    Starkovich, Britt M; Conard, Nicholas J

    2015-12-01

    This paper presents the faunal remains from the new excavation area at the Lower Paleolithic site of Schöningen. The focus of the study is on the southern extension of the main find horizon (Spear Horizon South), which includes the layer that yielded the famous Schöningen spears (13 II-4). Taxonomic data corroborate previous studies, that hominins primarily hunted Equus mosbachensis, a large Pleistocene horse. Equid body part representation at the site suggests that the animals were hunted and butchered locally. There is no evidence for density-mediated attrition in the assemblage. Weathering damage is uncommon, though there is ample evidence that carnivores had access to the bone. Carnivore bite sizes were measured and compared to experimental data provided by previous authors. Based on relationships between bite size and carnivore behavior and body size, we conclude that the primary modifying agents were large carnivores (i.e., wolves or saber-toothed cats). Previous studies show that carnivores often had secondary access to the remains, after hominins. Cut marks are commonly arranged haphazardly on the bones. This may indicate that multiple hominins participated in the butchery of horse skeletons, or that they were butchered over the course of hours or days. Cut marks on axial elements are more "orderly," which probably reflects the physical logistics of orienting one's body in relation to a large carcass. These data differ from sites formed by Middle and Upper Paleolithic hominins, which might suggest that in later times, a system of organized meat provisioning was already in place. Taken together, the faunal evidence from the Spear Horizon South indicates that late Lower Paleolithic hominins using the site understood the behaviors of different prey species, hunted socially to take down large game, and successfully competed with large carnivores on the landscape for primary access to ungulate remains. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. The Spirituality of the Leader and its influence on Visitor Experience Management at Sacred Sites in the Island of Ireland: Insights and implications

    OpenAIRE

    Enongene, Vreny; Griffin, Kevin A.

    2017-01-01

    Despite growing interest in understanding the sacred site visitor experience management, scholars have predominantly explored the phenomenon from the visitor’s perspective. There is very little exploration from the managerial perspectives, given that decisions regarding the nature of the experience, the product and service delivery strategies aimed at providing a diversity of visitors with rewarding, satisfying and memorable experiences, solely depends on these individuals, whose personal att...

  6. Heavy metal accumulation and ecosystem engineering by two common mine site-nesting ant species: implications for pollution-level assessment and bioremediation of coal mine soil.

    Science.gov (United States)

    Khan, Shbbir R; Singh, Satish K; Rastogi, Neelkamal

    2017-04-01

    The present study focuses on the abundance, heavy metal content, and the impact of ecosystem engineering activities of two coal mine site-inhabiting ant species, Cataglyphis longipedem and Camponotus compressus. The abundance of Ct. longipedem increased while that of C. compressus decreased, with increasing soil pollution. Correspondence analysis reveals a close association between soil heavy metal concentrations and Ct. longipedem abundance, but this association is lacking in the case of C. compressus. Cataglyphis ants which occupy stress-characterized niches appear to be pre-adapted to tolerate heavy metal pollution. Higher concentrations of Zn and Mn in Ct. longipedem may contribute to the strengthening of the cuticular structures, necessary for nest excavation in the hard, arid soil and for single load carrying. C. compressus ants appear to be pollution sensitive. Their higher Fe content may be related to metal uptake via plant-derived liquids and species-specific regulatory mechanisms. The metal pollution index and biota-to-soil accumulation factors, calculated by using the ant body metal content of the two species, indicate an overall decrease of soil heavy metal concentrations with increase of the site age, which reflects the degree of pollution related to the mine site age. The concentrations of total and available heavy metals (Fe, Zn, Mn, Pb, and Cu) were significantly lower in the ant nest debris soil as compared to the reference soil. The results of the present study highlight the role of ants as bioindicators and in bioremediation of contaminated soil.

  7. Water-Level Reconstruction and its Implications for Late Pleistocene Paleontological Site Formation in Hoyo Negro, a Submerged Subterranean Pit in Quintana Roo, Mexico

    Science.gov (United States)

    Rissolo, D.; Reinhardt, E. G.; Collins, S.; Kovacs, S. E.; Beddows, P. A.; Chatters, J. C.; Nava Blank, A.; Luna Erreguerena, P.

    2014-12-01

    A massive pit deep within the now submerged cave system of Sac Actun, located along the central east coast of the Yucatan Peninsula, contains a diverse fossil assemblage of extinct megafauna as well as a nearly complete human skeleton. The inundated site of Hoyo Negro presents a unique and promising opportunity for interdisciplinary Paleoamerican and paleoenvironmental research in the region. Investigations have thus far revealed a range of associated features and deposits which make possible a multi-proxy approach to identifying and reconstructing the natural and cultural processes that have formed and transformed the site over millennia. Understanding water-level fluctuations (both related to, and independent from, eustatic sea level changes), with respect to cave morphology is central to understanding the movement of humans and animals into and through the cave system. Recent and ongoing studies involve absolute dating of human, faunal, macrobotanical, and geological samples; taphonomic analyses; and a characterization of site hydrogeology and sedimentological facies, including microfossil assemblages and calcite raft deposits.

  8. Organohalogen pollutants in surface particulates from workshop floors of four major e-waste recycling sites in China and implications for emission lists

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Yan-Hong [State Key Laboratory of Organic Geochemistry and Guangdong Key Laboratory of Environmental Protection and Resources Utilization, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640 (China); Tang, Bin [State Key Laboratory of Organic Geochemistry and Guangdong Key Laboratory of Environmental Protection and Resources Utilization, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640 (China); Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Luo, Xiao-Jun [State Key Laboratory of Organic Geochemistry and Guangdong Key Laboratory of Environmental Protection and Resources Utilization, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640 (China); Zheng, Xiao-Bo [College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642 (China); Peng, Ping-An [State Key Laboratory of Organic Geochemistry and Guangdong Key Laboratory of Environmental Protection and Resources Utilization, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640 (China); Mai, Bi-Xian, E-mail: nancymai@gig.ac.cn [State Key Laboratory of Organic Geochemistry and Guangdong Key Laboratory of Environmental Protection and Resources Utilization, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640 (China)

    2016-11-01

    To examine the environmental pollution associated with e-waste recycling activities, the concentrations of organohologenated pollutants (OHPs), i.e., short- and medium-chain chlorinated paraffins (SCCPs and MCCPs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and several other halogenated flame retardants (OHFRs), were investigated in surface particulates from the workshop floors of four major e-waste recycling sites (Taizhou, Guiyu, Dali and Qingyuan) in China. The mean levels of SCCPs, MCCPs, PCBs, PBDEs and OHFRs in surface particulates ranged from 30,000–61,000, 170,000–890,000, 2700–27,000, 52,000–240,000, and 62,000–140,000 ng/g dry weight (dw), respectively. OHFRs, including decabromodiphenyl ethane, dechlorane plus, 1,2-bis(2,4,6-tribromophenoxy)ethane, tetrabromobisphenol A, hexabromocyclododecanes, polybrominated biphenyls, hexabromobenzene, pentabromotoluene, and pentabromoethylbenzene, were frequently (> 50% detection frequency) detected in surface particulates with mean concentration ranges of 39,000–63,000, 310–2700, 98–16,000, 21,000–56,000, 55–5700, 1700–27,000, 42–1600, 3.2–220, and 5.8–12 ng/g dw, respectively. The composition of OHPs varied depend on the e-waste items processing in different regions. Guiyu and Dali were typical sites contaminated by halogenated flame retardants (HFRs) and CPs, respectively, while Qingyuan, and Taizhou were representative PCB-polluted regions. The evidence produced by this preliminary study indicated that electronic devices and plastics may account for the high content of HFRs and the metal products are likely the major source of CPs in these e-waste sites. - Highlights: • Report of characterizing the types and possible sources of OHPs in e-waste sites • Guiyu was a typical site contaminated by HFRs, while Dali was dominated by CPs. • Qingyuan and Taizhou were representative PCB-polluted regions. • Electronic devices and plastics may account for the

  9. Organohalogen pollutants in surface particulates from workshop floors of four major e-waste recycling sites in China and implications for emission lists

    International Nuclear Information System (INIS)

    Zeng, Yan-Hong; Tang, Bin; Luo, Xiao-Jun; Zheng, Xiao-Bo; Peng, Ping-An; Mai, Bi-Xian

    2016-01-01

    To examine the environmental pollution associated with e-waste recycling activities, the concentrations of organohologenated pollutants (OHPs), i.e., short- and medium-chain chlorinated paraffins (SCCPs and MCCPs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and several other halogenated flame retardants (OHFRs), were investigated in surface particulates from the workshop floors of four major e-waste recycling sites (Taizhou, Guiyu, Dali and Qingyuan) in China. The mean levels of SCCPs, MCCPs, PCBs, PBDEs and OHFRs in surface particulates ranged from 30,000–61,000, 170,000–890,000, 2700–27,000, 52,000–240,000, and 62,000–140,000 ng/g dry weight (dw), respectively. OHFRs, including decabromodiphenyl ethane, dechlorane plus, 1,2-bis(2,4,6-tribromophenoxy)ethane, tetrabromobisphenol A, hexabromocyclododecanes, polybrominated biphenyls, hexabromobenzene, pentabromotoluene, and pentabromoethylbenzene, were frequently (> 50% detection frequency) detected in surface particulates with mean concentration ranges of 39,000–63,000, 310–2700, 98–16,000, 21,000–56,000, 55–5700, 1700–27,000, 42–1600, 3.2–220, and 5.8–12 ng/g dw, respectively. The composition of OHPs varied depend on the e-waste items processing in different regions. Guiyu and Dali were typical sites contaminated by halogenated flame retardants (HFRs) and CPs, respectively, while Qingyuan, and Taizhou were representative PCB-polluted regions. The evidence produced by this preliminary study indicated that electronic devices and plastics may account for the high content of HFRs and the metal products are likely the major source of CPs in these e-waste sites. - Highlights: • Report of characterizing the types and possible sources of OHPs in e-waste sites • Guiyu was a typical site contaminated by HFRs, while Dali was dominated by CPs. • Qingyuan and Taizhou were representative PCB-polluted regions. • Electronic devices and plastics may account for the

  10. Two-Dimensional Heat Transfer Modeling of the Formosa Ridge Offshore SW Taiwan: Implication for Fluid Migrating Paths of a Cold Seep Site

    Science.gov (United States)

    Tsai, Y.; Chi, W.; Liu, C.; Shyu, C.

    2011-12-01

    The Formosa Ridge, a small ridge located on the passive China continental slope offshore southwestern Taiwan, is an active cold seep site. Large and dense chemosynthetic communities were found there by the ROV Hyper-Dolphin during the 2007 NT0705 cruise. A vertical blank zone is clearly observed on all the seismic profiles across the cold seep site. This narrow zone is interpreted to be the fluid conduit of the seep site. Previous studies suggest that cold sea water carrying large amount of sulfate could flow into the fluid system from flanks of the ridge, and forms a very effective fluid circulation system that emits both methane and hydrogen sulfide to feed the unusual chemosynthetic communities observed at the Formosa Ridge cold seep site. Here we use thermal signals to study possible fluid flow migration paths. In 2008 and 2010, we have collected vdense thermal probe data at this site. We also study the temperatures at Bottom-Simulating Reflectors (BSRs) based on methane hydrate phase diagram. We perform 2D finite element thermal conductive simulations to study the effects of bathymetry on the temperature field in the ridge, and compare the simulation result with thermal probe and BSR-derived datasets. The boundary conditions include insulated boundaries on both sides, and we assign a fix temperature at the bottom of the model using an average regional geothermal gradient. Sensitivity tests and thermal probe data from a nearby region give a regional background geothermal gradient of 0.04 to 0.05 °C/m. The outputs of the simulation runs include geothermal gradient and temperature at different parts of the model. The model can fit the geothermal gradient at a distance away from the ridge where there is less geophysics evidence of fluid flow. However our model over-predicts the geothermal gradient by 50% at the ridge top. We also compare simulated temperature field and found that under the flanks of the ridge the temperature is cooled by 2 °C compared with the

  11. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying-Nan P.; LaMarche, Matthew J.; Chan, Ho Man; Fekkes, Peter; Garcia-Fortanet, Jorge; Acker, Michael G.; Antonakos, Brandon; Chen, Christine Hiu-Tung; Chen, Zhouliang; Cooke, Vesselina G.; Dobson, Jason R.; Deng, Zhan; Fei, Feng; Firestone, Brant; Fodor, Michelle; Fridrich, Cary; Gao, Hui; Grunenfelder, Denise; Hao, Huai-Xiang; Jacob, Jaison; Ho, Samuel; Hsiao, Kathy; Kang, Zhao B.; Karki, Rajesh; Kato, Mitsunori; Larrow, Jay; La Bonte, Laura R.; Lenoir, Francois; Liu, Gang; Liu, Shumei; Majumdar, Dyuti; Meyer, Matthew J.; Palermo, Mark; Perez, Lawrence; Pu, Minying; Price, Edmund; Quinn, Christopher; Shakya, Subarna; Shultz, Michael D.; Slisz, Joanna; Venkatesan, Kavitha; Wang, Ping; Warmuth, Markus; Williams, Sarah; Yang, Guizhi; Yuan, Jing; Zhang, Ji-Hu; Zhu, Ping; Ramsey, Timothy; Keen, Nicholas J.; Sellers, William R.; Stams, Travis; Fortin , Pascal D. (Novartis)

    2016-06-29

    The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase1. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma1, 2, 3, 4, 5. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway2, 3. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways6, 7. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy8, 9. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

  12. Allosteric Regulation in the Ligand Binding Domain of Retinoic Acid Receptorγ.

    Directory of Open Access Journals (Sweden)

    Yassmine Chebaro

    Full Text Available Retinoic acid (RA plays key roles in cell differentiation and growth arrest through nuclear retinoic acid receptors (RARs, which are ligand-dependent transcription factors. While the main trigger of RAR activation is the binding of RA, phosphorylation of the receptors has also emerged as an important regulatory signal. Phosphorylation of the RARγ N-terminal domain (NTD is known to play a functional role in neuronal differentiation. In this work, we investigated the phosphorylation of RARγ ligand binding domain (LBD, and present evidence that the phosphorylation status of the LBD affects the phosphorylation of the NTD region. We solved the X-ray structure of a phospho-mimetic mutant of the LBD (RARγ S371E, which we used in molecular dynamics simulations to characterize the consequences of the S371E mutation on the RARγ structural dynamics. Combined with simulations of the wild-type LBD, we show that the conformational equilibria of LBD salt bridges (notably R387-D340 are affected by the S371E mutation, which likely affects the recruitment of the kinase complex that phosphorylates the NTD. The molecular dynamics simulations also showed that a conservative mutation in this salt bridge (R387K affects the dynamics of the LBD without inducing large conformational changes. Finally, cellular assays showed that the phosphorylation of the NTD of RARγ is differentially regulated by retinoic acid in RARγWT and in the S371N, S371E and R387K mutants. This multidisciplinary work highlights an allosteric coupling between phosphorylations of the LBD and the NTD of RARγ and supports the importance of structural dynamics involving electrostatic interactions in the regulation of RARs activity.

  13. Adaptive response and genomic instability: allosteric response of genome to negative impact

    International Nuclear Information System (INIS)

    Sasaki, Masao S.

    2010-01-01

    Currently, there is an upsurge concern on the unique response of living cells to low dose ionizing radiation for its inconformity to the existing paradigm of the biological action of radiation and its impact on the current understanding of risk evaluation of health effect of radiation in our workplace and environment. For the allosteric response to have significance, the cells must have an excellent sensing mechanism to discriminate tolerable and intolerable signals. In a series of experiments with mammalian, including human, cells, we demonstrated a novel sensing and signaling mechanism in the low-dose irradiated cells that was mediated by a PKCα-p3BMAPK-PLCδ1 feedback regulatory loop. Upon irradiation, PKCα is immediately activated, which in turn activate p38MAPK. The activation of p38MAPK is feedbacked to the activation of PKCα via PLCδ1, which catalyzes the hydrolysis of PtdInsP2 to generate PKCα-directed second messengers DAG and lnsP3. At low doses, the PKCα and p38MAPK continue to be activated for long time through this feedback loop, but when the cells encounter the high dose (>10 cGy or equivalent), the feedback loop is immediately comes to shutdown by deprivation of PKCα protein, known as down-regulation of PKC signaling. Thus, PKCα plays a key role in the long lasting nature of adaptive response to low doses and a binary switch to the genomic instability by too much signals. Tumor suppressor protein, p53, is a downstream effecter

  14. Rational Design and Tuning of Functional RNA Switch to Control an Allosteric Intermolecular Interaction.

    Science.gov (United States)

    Endoh, Tamaki; Sugimoto, Naoki

    2015-08-04

    Conformational transitions of biomolecules in response to specific stimuli control many biological processes. In natural functional RNA switches, often called riboswitches, a particular RNA structure that has a suppressive or facilitative effect on gene expression transitions to an alternative structure with the opposite effect upon binding of a specific metabolite to the aptamer region. Stability of RNA secondary structure (-ΔG°) can be predicted based on thermodynamic parameters and is easily tuned by changes in nucleobases. We envisioned that tuning of a functional RNA switch that causes an allosteric interaction between an RNA and a peptide would be possible based on a predicted switching energy (ΔΔG°) that corresponds to the energy difference between the RNA secondary structure before (-ΔG°before) and after (-ΔG°after) the RNA conformational transition. We first selected functional RNA switches responsive to neomycin with predicted ΔΔG° values ranging from 5.6 to 12.2 kcal mol(-1). We then demonstrated a simple strategy to rationally convert the functional RNA switch to switches responsive to natural metabolites thiamine pyrophosphate, S-adenosyl methionine, and adenine based on the predicted ΔΔG° values. The ΔΔG° values of the designed RNA switches proportionally correlated with interaction energy (ΔG°interaction) between the RNA and peptide, and we were able to tune the sensitivity of the RNA switches for the trigger molecule. The strategy demonstrated here will be generally applicable for construction of functional RNA switches and biosensors in which mechanisms are based on conformational transition of nucleic acids.

  15. Orthosteric and Allosteric Ligands of Nicotinic Acetylcholine Receptors for Smoking Cessation

    Directory of Open Access Journals (Sweden)

    Tasnim S. Mohamed

    2015-11-01

    Full Text Available Nicotine addiction, the result of tobacco use, leads to over six million premature deaths world-wide, a number that is expected to increase by a third within the next two decades. While more than half of smokers want and attempt to quit, only a small percentage of smokers are able to quit without pharmacological interventions. Therefore, over the past decades, researchers in academia and the pharmaceutical industry have focused their attention on the development of more effective smoking cessation therapies, which is now a growing 1.9 billion dollar market. Because the role of neuronal nicotinic acetylcholine receptors (nAChR in nicotine addiction is well established, nAChR based therapeutics remain the leading strategy for smoking cessation. However, the development of neuronal nAChR drugs that are selective for a nAChR subpopulation is challenging, and only few neuronal nAChR drugs are clinically available. Among the many neuronal nAChR subtypes that have been identified in the brain, the α4β2 subtype is the most abundant and plays a critical role in nicotine addiction. Here, we review the role of neuronal nAChRs, especially the α4β2 subtype, in the development and treatment of nicotine addiction. We also compare available smoking cessation medications and other nAChR orthosteric and allosteric ligands that have been developed with emphasis on the difficulties faced in the development of clinically useful compounds with high nAChR subtype selectivity.

  16. Switch I-dependent allosteric signaling in a G-protein chaperone-B12 enzyme complex.

    Science.gov (United States)

    Campanello, Gregory C; Lofgren, Michael; Yokom, Adam L; Southworth, Daniel R; Banerjee, Ruma

    2017-10-27

    G-proteins regulate various processes ranging from DNA replication and protein synthesis to cytoskeletal dynamics and cofactor assimilation and serve as models for uncovering strategies deployed for allosteric signal transduction. MeaB is a multifunctional G-protein chaperone, which gates loading of the active 5'-deoxyadenosylcobalamin cofactor onto methylmalonyl-CoA mutase (MCM) and precludes loading of inactive cofactor forms. MeaB also safeguards MCM, which uses radical chemistry, against inactivation and rescues MCM inactivated during catalytic turnover by using the GTP-binding energy to offload inactive cofactor. The conserved switch I and II signaling motifs used by G-proteins are predicted to mediate allosteric regulation in response to nucleotide binding and hydrolysis in MeaB. Herein, we targeted conserved residues in the MeaB switch I motif to interrogate the function of this loop. Unexpectedly, the switch I mutations had only modest effects on GTP binding and on GTPase activity and did not perturb stability of the MCM-MeaB complex. However, these mutations disrupted multiple MeaB chaperone functions, including cofactor editing, loading, and offloading. Hence, although residues in the switch I motif are not essential for catalysis, they are important for allosteric regulation. Furthermore, single-particle EM analysis revealed, for the first time, the overall architecture of the MCM-MeaB complex, which exhibits a 2:1 stoichiometry. These EM studies also demonstrate that the complex exhibits considerable conformational flexibility. In conclusion, the switch I element does not significantly stabilize the MCM-MeaB complex or influence the affinity of MeaB for GTP but is required for transducing signals between MeaB and MCM. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Vestigialization of an allosteric switch: genetic and structural mechanisms for the evolution of constitutive activity in a steroid hormone receptor.

    Directory of Open Access Journals (Sweden)

    Jamie T Bridgham

    2014-01-01

    Full Text Available An important goal in molecular evolution is to understand the genetic and physical mechanisms by which protein functions evolve and, in turn, to characterize how a protein's physical architecture influences its evolution. Here we dissect the mechanisms for an evolutionary shift in function in the mollusk ortholog of the steroid hormone receptors (SRs, a family of biologically essential transcription factors. In vertebrates, the activity of SRs allosterically depends on binding a hormonal ligand; in mollusks, however, the SR ortholog (called ER, because of high sequence similarity to vertebrate estrogen receptors activates transcription in the absence of ligand and does not respond to steroid hormones. To understand how this shift in regulation evolved, we combined evolutionary, structural, and functional analyses. We first determined the X-ray crystal structure of the ER of the Pacific oyster Crassostrea gigas (CgER, and found that its ligand pocket is filled with bulky residues that prevent ligand occupancy. To understand the genetic basis for the evolution of mollusk ERs' unique functions, we resurrected an ancient SR progenitor and characterized the effect of historical amino acid replacements on its functions. We found that reintroducing just two ancient replacements from the lineage leading to mollusk ERs recapitulates the evolution of full constitutive activity and the loss of ligand activation. These substitutions stabilize interactions among key helices, causing the allosteric switch to become "stuck" in the active conformation and making activation independent of ligand binding. Subsequent changes filled the ligand pocket without further affecting activity; by degrading the allosteric switch, these substitutions vestigialized elements of the protein's architecture required for ligand regulation and made reversal to the ancestral function more complex. These findings show how the physical architecture of allostery enabled a few large

  18. Vestigialization of an Allosteric Switch: Genetic and Structural Mechanisms for the Evolution of Constitutive Activity in a Steroid Hormone Receptor

    Science.gov (United States)

    Bridgham, Jamie T.; Keay, June; Ortlund, Eric A.; Thornton, Joseph W.

    2014-01-01

    An important goal in molecular evolution is to understand the genetic and physical mechanisms by which protein functions evolve and, in turn, to characterize how a protein's physical architecture influences its evolution. Here we dissect the mechanisms for an evolutionary shift in function in the mollusk ortholog of the steroid hormone receptors (SRs), a family of biologically essential transcription factors. In vertebrates, the activity of SRs allosterically depends on binding a hormonal ligand; in mollusks, however, the SR ortholog (called ER, because of high sequence similarity to vertebrate estrogen receptors) activates transcription in the absence of ligand and does not respond to steroid hormones. To understand how this shift in regulation evolved, we combined evolutionary, structural, and functional analyses. We first determined the X-ray crystal structure of the ER of the Pacific oyster Crassostrea gigas (CgER), and found that its ligand pocket is filled with bulky residues that prevent ligand occupancy. To understand the genetic basis for the evolution of mollusk ERs' unique functions, we resurrected an ancient SR progenitor and characterized the effect of historical amino acid replacements on its functions. We found that reintroducing just two ancient replacements from the lineage leading to mollusk ERs recapitulates the evolution of full constitutive activity and the loss of ligand activation. These substitutions stabilize interactions among key helices, causing the allosteric switch to become “stuck” in the active conformation and making activation independent of ligand binding. Subsequent changes filled the ligand pocket without further affecting activity; by degrading the allosteric switch, these substitutions vestigialized elements of the protein's architecture required for ligand regulation and made reversal to the ancestral function more complex. These findings show how the physical architecture of allostery enabled a few large-effect mutations

  19. Computational study on the inhibitor binding mode and allosteric regulation mechanism in hepatitis C virus NS3/4A protein.

    Directory of Open Access Journals (Sweden)

    Weiwei Xue

    Full Text Available HCV NS3/4A protein is an attractive therapeutic target responsible for harboring serine protease and RNA helicase activities during the viral replication. Small molecules binding at the interface between the protease and helicase domains can stabilize the closed conformation of the protein and thus block the catalytic function of HCV NS3/4A protein via an allosteric regulation mechanism. But the detailed mechanism remains elusive. Here, we aimed to provide some insight into the inhibitor binding mode and allosteric regulation mechanism of HCV NS3/4A protein by using computational methods. Four simulation systems were investigated. They include: apo state of HCV NS3/4A protein, HCV NS3/4A protein in complex with an allosteric inhibitor and the truncated form of the above two systems. The molecular dynamics simulation results indicate HCV NS3/4A protein in complex with the allosteric inhibitor 4VA adopts a closed conformation (inactive state, while the truncated apo protein adopts an open conformation (active state. Further residue interaction network analysis suggests the communication of the domain-domain interface play an important role in the transition from closed to open conformation of HCV NS3/4A protein. However, the inhibitor stabilizes the closed conformation through interaction with several key residues from both the protease and helicase domains, including His57, Asp79, Asp81, Asp168, Met485, Cys525 and Asp527, which blocks the information communication between the functional domains interface. Finally, a dynamic model about the allosteric regulation and conformational changes of HCV NS3/4A protein was proposed and could provide fundamental insights into the allosteric mechanism of HCV NS3/4A protein function regulation and design of new potent inhibitors.

  20. Thermal regime at the Upper Stillwater dam site, Uinta mountains, Utah: Implications for terrain, microclimate and structural corrections in heat flow studies

    Science.gov (United States)

    Bauer, Michael S.; Chapman, David S.

    1986-08-01

    A detailed study of the subsurface thermal regime at the Upper Stillwater dam site, Uinta Mountains, northeast Utah, has been made. Temperature measurements were made in 36 drillholes located within a 1 km 2 area and ranging in depth from 20 to 97 m. Holes less than about 40 m deep were used only to obtain information about spatial variations in mean annual surface temperature. Several holes in or near talus slopes at the sides of the canyons have temperature minima approaching 0°C between 10 and 20 m indicating the presence of year-round ice at the base of the talus. Another set of holes show transient thermal effects of surface warming resulting from clearing of a construction site 3.5 years prior to our measurements. Most of the remaining holes show conductive behavior and have gradients ranging from 13° to 17°C km -1. Measurements made on 44 core samples yield a thermal conductivity of 5.6 (std. dev. 0.35) W m -1 K -1 for the Precambrian quartzite present. Surface heat flow estimates for these holes range from 70 to 100 mW m -2. However, the local disturbance of the thermal field by topography and microclimate is considerable. A finite difference method used to model these effects yielded a locally corrected Upper Stillwater heat flow of about 75 mW m -2. A final correction to account for the effects of refraction of heat from the low conductivity sedimentary rocks in the Uinta Basin into the high conductivity quartzite at the dam site, produced a regionally corrected Upper Stillwater heat flow between 60 and 65 mW m -2. This value is consistent with the observed heat flow of 60 mW m -2 in the Green River Basin to the north and the Uinta Basin to the south.

  1. Atmospheric polybrominated diphenyl ethers (PBDEs) and Pb isotopes at a remote site in Southwestern China: Implications for monsoon-associated transport

    International Nuclear Information System (INIS)

    Xu, Yue; Zhang, Gan; Li, Jun; Liu, Xiang; Li, Xiangdong

    2011-01-01

    A 13-month sampling campaign was conducted at a remote site in southwestern China from October, 2005 to December, 2006. An integrated approach with lead isotopes and air back trajectory analysis was used to investigate the monsoon-associated atmospheric transport of PBDEs in tropical/subtropical Asia regions. The air concentration of PBDEs ranged from 1.6 to 57.5 pg m -3 (15.9 ± 12.0 pg m -3 ), comparable to reported levels at other remote sites in the world. BDE-209, followed by BDE-47 and -99 dominated the PBDE compositions, indicating a mixed deca- and penta-BDE source. Air mass back trajectory analysis revealed that the major potential source regions of BDE-47 and -99 could be southern China and Thailand, while those of BDE-209 are widely distributed in industrialized and urbanized areas in tropical Asia. The different lead isotope compositions of aerosols between trajectory clusters further substantiated the observation that the South Asian monsoon from spring to summer could penetrate deep into southwestern China, and facilitate long-range transport of airborne pollutants from South Asia. - Highlights: →The atmospheric levels of PBDEs and Pb isotopic ratios at a remote site were reported. →Significant high concentrations of BDE-47 and -99 were observed when air masses came from China and Southeast Asia. →High concentrations of BDE-209 and low Pb isotopic ratios were associated with Indian monsoon. →The onset of monsoon could facilitate long-range transport of airborne pollutants from South Asia.

  2. Organohalogen pollutants in surface particulates from workshop floors of four major e-waste recycling sites in China and implications for emission lists.

    Science.gov (United States)

    Zeng, Yan-Hong; Tang, Bin; Luo, Xiao-Jun; Zheng, Xiao-Bo; Peng, Ping-An; Mai, Bi-Xian

    2016-11-01

    To examine the environmental pollution associated with e-waste recycling activities, the concentrations of organohologenated pollutants (OHPs), i.e., short- and medium-chain chlorinated paraffins (SCCPs and MCCPs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and several other halogenated flame retardants (OHFRs), were investigated in surface particulates from the workshop floors of four major e-waste recycling sites (Taizhou, Guiyu, Dali and Qingyuan) in China. The mean levels of SCCPs, MCCPs, PCBs, PBDEs and OHFRs in surface particulates ranged from 30,000-61,000, 170,000-890,000, 2700-27,000, 52,000-240,000, and 62,000-140,000ng/g dry weight (dw), respectively. OHFRs, including decabromodiphenyl ethane, dechlorane plus, 1,2-bis(2,4,6-tribromophenoxy)ethane, tetrabromobisphenol A, hexabromocyclododecanes, polybrominated biphenyls, hexabromobenzene, pentabromotoluene, and pentabromoethylbenzene, were frequently (>50% detection frequency) detected in surface particulates with mean concentration ranges of 39,000-63,000, 310-2700, 98-16,000, 21,000-56,000, 55-5700, 1700-27,000, 42-1600, 3.2-220, and 5.8-12ng/g dw, respectively. The composition of OHPs varied depend on the e-waste items processing in different regions. Guiyu and Dali were typical sites contaminated by halogenated flame retardants (HFRs) and CPs, respectively, while Qingyuan, and Taizhou were representative PCB-polluted regions. The evidence produced by this preliminary study indicated that electronic devices and plastics may account for the high content of HFRs and the metal products are likely the major source of CPs in these e-waste sites. Copyright © 2016. Published by Elsevier B.V.

  3. Binding of 3,4,5,6-Tetrahydroxyazepanes to the Acid-[beta]-glucosidase Active Site: Implications for Pharmacological Chaperone Design for Gaucher Disease

    Energy Technology Data Exchange (ETDEWEB)

    Orwig, Susan D.; Tan, Yun Lei; Grimster, Neil P.; Yu, Zhanqian; Powers, Evan T.; Kelly, Jeffery W.; Lieberman, Raquel L. (Scripps); (GIT)

    2013-03-07

    Pharmacologic chaperoning is a therapeutic strategy being developed to improve the cellular folding and trafficking defects associated with Gaucher disease, a lysosomal storage disorder caused by point mutations in the gene encoding acid-{beta}-glucosidase (GCase). In this approach, small molecules bind to and stabilize mutant folded or nearly folded GCase in the endoplasmic reticulum (ER), increasing the concentration of folded, functional GCase trafficked to the lysosome where the mutant enzyme can hydrolyze the accumulated substrate. To date, the pharmacologic chaperone (PC) candidates that have been investigated largely have been active site-directed inhibitors of GCase, usually containing five- or six-membered rings, such as modified azasugars. Here we show that a seven-membered, nitrogen-containing heterocycle (3,4,5,6-tetrahydroxyazepane) scaffold is also promising for generating PCs for GCase. Crystal structures reveal that the core azepane stabilizes GCase in a variation of its proposed active conformation, whereas binding of an analogue with an N-linked hydroxyethyl tail stabilizes GCase in a conformation in which the active site is covered, also utilizing a loop conformation not seen previously. Although both compounds preferentially stabilize GCase to thermal denaturation at pH 7.4, reflective of the pH in the ER, only the core azepane, which is a mid-micromolar competitive inhibitor, elicits a modest increase in enzyme activity for the neuronopathic G202R and the non-neuronopathic N370S mutant GCase in an intact cell assay. Our results emphasize the importance of the conformational variability of the GCase active site in the design of competitive inhibitors as PCs for Gaucher disease.

  4. Probe-Dependent Negative Allosteric Modulators of the Long-Chain Free Fatty Acid Receptor FFA4

    DEFF Research Database (Denmark)

    Watterson, Kenneth R; Hansen, Steffen V F; Hudson, Brian D

    2017-01-01

    High-affinity and selective antagonists that are able to block the actions of both endogenous and synthetic agonists of G protein-coupled receptors are integral to analysis of receptor function and to support suggestions of therapeutic potential. Although there is great interest in the potential...... of endogenous and synthetic agonists, clear agonist probe dependence in the nature of allosteric modulation was apparent. Although AH-7614 did not antagonize the second long-chain free fatty acid receptor, free fatty acid receptor 1, the simple chemical structure of AH-7614 containing features found in many...

  5. Design, synthesis, and activity of 2,3-diphosphoglycerate analogs as allosteric modulators of hemoglobin O2 affinity.

    Science.gov (United States)

    Kassa, Tigist W; Zhang, Ning; Palmer, Andre F; Matthews, Jason Shastri

    2013-04-01

    Four phosphonate derivates of 2,3-diphosphoglycerate (2,3-DPG), in which the phosphate group is replaced by a methylene or difluoromethylene, were successfully synthesized for use as allosteric modulators of hemoglobin (Hb) O2 affinity. The syntheses were accomplished in four steps and the reagents were converted to their potassium salts to allow for effective binding with Hb in aqueous media. O2 equilibrium measurements of the chemically modified Hbs exhibited P50 values in the range 8.9-12.8 with Hill coefficients in the range of 1.5-2.4.

  6. Subsurface Analysis of the Mesaverde Group on and near the Jicarilla Apache Indian Reservation, New Mexico-its implication on Sites of Oil and Gas Accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Ridgley, Jennie

    2001-08-21

    The purpose of the phase 2 Mesaverde study part of the Department of Energy funded project ''Analysis of oil-bearing Cretaceous Sandstone Hydrocarbon Reservoirs, exclusive of the Dakota Sandstone, on the Jicarilla Apache Indian Reservation, New Mexico'' was to define the facies of the oil-producing units within the subsurface units of the Mesaverde Group and integrate these results with outcrop studies that defined the depositional environments of these facies within a sequence stratigraphic context. The focus of this report will center on (1) integration of subsurface correlations with outcrop correlations of components of the Mesaverde, (2) application of the sequence stratigraphic model determined in the phase one study to these correlations, (3) determination of the facies distribution of the Mesaverde Group and their relationship to sites of oil and gas accumulation, (4) evaluation of the thermal maturity and potential source rocks for oil and gas in the Mesaverde Group, and (5) evaluation of the structural features on the Reservation as they may control sites of oil accumulation.

  7. Binding of the sphingolipid S1P to hTERT stabilizes telomerase at the nuclear periphery by allosterically mimicking protein phosphorylation†

    Science.gov (United States)

    Selvam, Shanmugam P.; De Palma, Ryan M.; Oaks, Joshua J.; Oleinik, Natalia; Peterson, Yuri K.; Stahelin, Robert V.; Skordalakes, Emmanuel; Ponnusamy, Suriyan; Garrett-Mayer, Elizabeth; Smith, Charles D.; Ogretmen, Besim

    2015-01-01

    During DNA replication, the enzyme telomerase maintains the ends of chromosomes, called telomeres. Shortened telomeres trigger cell senescence, and cancer cells often have increased telomerase activity to promote their ability to proliferate indefinitely. The catalytic subunit, human telomerase reverse transcriptase (hTERT), is stabilized by phosphorylation. Here, we found that the lysophospholipid sphingosine 1-phosphate (S1P), generated by sphingosine kinase 2 (SK2), bound hTERT at the nuclear periphery in human and mouse fibroblasts. Docking predictions and mutational analyses revealed that binding occurred between a hydroxyl group (C′3-OH) in S1P and Asp684 in hTERT. Inhibiting or depleting SK2 or mutating the S1P binding site decreased the stability of hTERT in cultured cells and promoted senescence and loss of telomere integrity. S1P binding inhibited the interaction of hTERT with MKRN1, an E3 ubiquitin ligase that tags hTERT for degradation. Murine Lewis lung carcinoma (LLC) cells formed smaller tumors in mice lacking SK2 than in wild-type mice, and knocking down SK2 in LLC cells before implantation into mice suppressed their growth. Pharmacologically inhibiting SK2 decreased the growth of subcutaneous A549 lung cancer cell-derived xenografts in mice, and expression of wild-type hTERT, but not an S1P-binding mutant, restored tumor growth. Thus, our data suggest that S1P binding to hTERT allosterically mimicks phosphorylation, promoting telomerase stability and hence telomere maintenance, cell proliferation, and tumor growth PMID:26082434

  8. Dimerization-Induced Allosteric Changes of the Oxyanion-Hole Loop Activate the Pseudorabies Virus Assemblin pUL26N, a Herpesvirus Serine Protease.

    Directory of Open Access Journals (Sweden)

    Martin Zühlsdorf

    2015-07-01

    Full Text Available Herpesviruses encode a characteristic serine protease with a unique fold and an active site that comprises the unusual triad Ser-His-His. The protease is essential for viral replication and as such constitutes a promising drug target. In solution, a dynamic equilibrium exists between an inactive monomeric and an active dimeric form of the enzyme, which is believed to play a key regulatory role in the orchestration of proteolysis and capsid assembly. Currently available crystal structures of herpesvirus proteases correspond either to the dimeric state or to complexes with peptide mimetics that alter the dimerization interface. In contrast, the structure of the native monomeric state has remained elusive. Here, we present the three-dimensional structures of native monomeric, active dimeric, and diisopropyl fluorophosphate-inhibited dimeric protease derived from pseudorabies virus, an alphaherpesvirus of swine. These structures, solved by X-ray crystallography to respective resolutions of 2.05, 2.10 and 2.03 Å, allow a direct comparison of the main conformational states of the protease. In the dimeric form, a functional oxyanion hole is formed by a loop of 10 amino-acid residues encompassing two consecutive arginine residues (Arg136 and Arg137; both are strictly conserved throughout the herpesviruses. In the monomeric form, the top of the loop is shifted by approximately 11 Å, resulting in a complete disruption of the oxyanion hole and loss of activity. The dimerization-induced allosteric changes described here form the physical basis for the concentration-dependent activation of the protease, which is essential for proper virus replication. Small-angle X-ray scattering experiments confirmed a concentration-dependent equilibrium of monomeric and dimeric protease in solution.

  9. Development of a phosphorylated Momordica charantia protein system for inhibiting susceptible dose-dependent C. albicans to available antimycotics: An allosteric regulation of protein.

    Science.gov (United States)

    Qiao, Yuanbiao; Song, Li; Zhu, Chenchen; Wang, Qian; Guo, Tianyan; Yan, Yanhua; Li, Qingshan

    2017-11-15

    A regulatory Momordica charantia protein system was constructed allosterically by in vitro protein phosphorylation, in an attempt to evaluate antimycological pluripotency against dose-dependent susceptibilities in C. albicans. Fungal strain lineages susceptible to ketoconazole, econazole, miconazole, 5-flucytosine, nystatin and amphotericin B were prepared in laboratory, followed by identification via antifungal susceptibility testing. Protein phosphorylation was carried out in reactions with 5'-adenylic, guanidylic, cytidylic and uridylic acids and cyclic adenosine triphosphate, through catalysis of cyclin-dependent kinase 1, protein kinase A and protein kinase C respectively. Biochemical analysis of enzymatic reactions indicated the apparent Michaelis-Menten constants and maximal velocity values of 16.57-91.97mM and 55.56-208.33μM·min -1 , together with an approximate 1:1 reactant stoichiometric ratio. Three major protein phosphorylation sites were theoretically predicted at Thr255, Thr102 and Thr24 by a KinasePhos tool. Additionally, circular dichroism spectroscopy demonstrated that upon phosphorylation, protein folding structures were decreased in random coil, β6-sheet and α1-helix partial regions. McFarland equivalence standard testing yielded the concentration-dependent inhibition patterns, while fungus was grown in Sabouraud's dextrose agar. The minimal inhibitory concentrations of 0.16-0.51μM (at 50% response) were obtained for free protein and phosphorylated counterparts. With respect to the 3-cycling susceptibility testing regimen, individuals of total protein forms were administrated in-turn at 0.14μM/cycle. Relative inhibition ratios were retained to 66.13-81.04% of initial ones regarding the ketoconazole-susceptible C. albicans growth. An inhibitory protein system, with an advantage of decreasing antifungal susceptibilities to diverse antimycotics, was proposed because of regulatory pluripotency whereas little contribution to susceptibility in

  10. A greenhouse trial to investigate the ameliorative properties of biosolids and plants on physicochemical conditions of iron ore tailings: Implications for an iron ore mine site remediation.

    Science.gov (United States)

    Cele, Emmanuel Nkosinathi; Maboeta, Mark

    2016-01-01

    An iron ore mine site in Swaziland is currently (2015) in a derelict state as a consequence of past (1964-1988) and present (2011 - current) iron ore mining operations. In order to control problems associated with mine wastes, the Swaziland Water Services Corporation (SWSC) recently (2013) proposed the application of biosolids in sites degraded by mining operations. It is thought that this practice could generally improve soil conditions and enhance plant reestablishment. More importantly, the SWSC foresees this as a potential solution to the biosolids disposal problems. In order to investigate the effects of biosolids and plants in soil physicochemical conditions of iron mine soils, we conducted two plant growth trials. Trial 1 consisted of tailings that received biosolids and topsoil (TUSB mix) while in trial 2, tailings received biosolids only (TB mix). In the two trials, the application rates of 0 (control), 10, 25, 50, 75 and 100 t ha(-1) were used. After 30 days of equilibration, 25 seeds of Cynodon dactylon were sown in each pot and thinned to 10 plants after 4 weeks. Plants were watered twice weekly and remained under greenhouse conditions for 12 weeks, subsequent to which soils were subjected to chemical analysis. According to the results obtained, there were significant improvements in soil parameters related to fertility such as organic matter (OM), water holding capacity (WHC), cation exchange capacity (CEC), ammonium [Formula: see text] , magnesium (Mg(2+)), calcium (Ca(2+)) and phosphorus ( [Formula: see text] ). With regard to heavy metals, biosolids led to significant increases in soil total concentrations of Cu, Zn, Cd, Hg and Pb. The higher concentrations of Zn and Cu in treated tailings compared to undisturbed adjacent soils are a cause for concern because in the field, this might work against the broader objectives of mine soil remediation, which include the recolonization of reclaimed sites by soil-dwelling organisms. Therefore, while

  11. Geologic Maps and Cross Sections of the Tuba City Open Dump Site and Vicinity, With Implications for the Occurrence and Flow of Ground Water

    Science.gov (United States)

    Otton, James K.; Johnson, Ray H.; Horton, Robert J.

    2008-01-01

    This report is designed to make available to interested parties geologic and limited hydrologic and geochemical information about the Tuba City Open Dump (TCOD) site. This information has been gathered during studies of the site from January to September 2008. Mapping by the authors and construction of cross sections show that a section of gently northeast-dipping Jurassic sedimentary rocks underlies the TCOD and vicinity. Low mesas in the area are capped by variably cemented gravels and siliceous limestones. Surficial sediments are composed of eolian sand and fluvially reworked eolian sand that overlie bedrock underneath the TCOD. Nearby Pasture Canyon is underlain by fluvial and floodplain sediment consisting of sand and silt. Shallow ground water of the water-table aquifer at the TCOD moves westward through the surficial sediment and the underlying weathered bedrock to Pasture Canyon then southward along the canyon. A fracture zone extends up the wash that passes just to the north of the TCOD and brings deeper ground water of the N-aquifer to the water-table aquifer. Bedrock consists of the Jurassic Navajo Sandstone composed of thick sections of eolian crossbedded sandstone with lesser laterally discontinuous layers of silty sandstone, siltstone, and limestone. Below the Navajo Sandstone is a section informally known as the Kayenta Formation-Navajo Sandstone transition zone. It is composed of calcareous sandstone, silty sandstone, siltstone, and limestone beds that intertongue with crossbedded sandstone. The finer grained rocks in both major bedrock units form aquitards that limit downward movement of ground water. The water-table aquifer is perched on these aquitards, which locally occurs beneath the two open dumps that form the TCOD site. A monocline occupies the position of Pasture Canyon west of the TCOD. Fractures likely related to the monocline are exposed in several localities. Deep ground waters consist of dilute calcium-bicarbonate waters low in all

  12. Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence*

    Science.gov (United States)

    Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L.; Embrey, Kevin J.; Golovanov, Alexander P.

    2016-01-01

    The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly 15N-labeled Ras as well as [13C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions. PMID:26565026

  13. Geochemistry of Volcanic Rocks from International Ocean Discovery Program (IODP) Site 1438, Amami Sankaku Basin: Implications for Izu-Bonin-Mariana (IBM) Arc Initiation

    Science.gov (United States)

    Hickey-Vargas, R.; Ishizuka, O.; Yogodzinski, G. M.; Bizimis, M.; Savov, I. P.; McCarthy, A. J.; Arculus, R. J.; Bogus, K.

    2015-12-01

    IODP Expedition 351 drilled 150 m of volcanic basement overlain by 1461 m of sedimentary material at Site 1438 in the Amami Sankaku basin, just west of the Kyushu Palau Ridge, the locus of IBM arc initiation. Age interpretations based on biostratigraphy (Arculus et al., Nat. Geosci., in-press) determined that the age of the basement section is between 64 and 51 Ma, encompassing the age of the earliest volcanic products of the IBM arc. The Site 1438 volcanic basement consists of multiple flows of aphyric microcrystalline to finely crystalline basalts containing plagioclase and clinopyroxene with rare olivine pseudomorphs. New XRF major and ICPMS trace element data confirm findings of shipboard analysis that the basalts are moderately differentiated (6-14 % MgO; Mg# = 51-83; 73-490 ppm Cr and 58-350 ppm Ni) with downcore variations related to flow units. Ti/V and Ti/Sc ratios are 16-27 and 75-152, respectively, with lowest values at the base of the core. One prominent characteristic of the basalts is their depletion of immobile highly incompatible elements compared with MORB. Basalts have MORB-normalized La/Nd of 0.5 to 0.9, and most have Th/La 3 and primitive mantle normalized La/Yb > 1. Our results suggest that mantle melting at the onset of subduction involved exceptionally depleted sources. Enrichment over time may be related to increasing subduction inputs and/or other processes, such as entrainment of fertile asthenosphere during extension of the overriding plate.

  14. Comparative studies of the endonucleases from two related Xenopus laevis retrotransposons, Tx1L and Tx2L: target site specificity and evolutionary implications.

    Science.gov (United States)

    Christensen, S; Pont-Kingdon, G; Carroll, D

    2000-01-01

    In the genome of the South African frog, Xenopus laevis, there are two complex families of transposable elements, Tx1 and Tx2, that have identical overall structures, but distinct sequences. In each family there are approximately 1500 copies of an apparent DNA-based element (Tx1D and Tx2D). Roughly 10% of these elements in each family are interrupted by a non-LTR retrotransposon (Tx1L and Tx2L). Each retrotransposon is flanked by a 23-bp target duplication of a specific D element sequence. In earlier work, we showed that the endonuclease domain (Tx1L EN) located in the second open reading frame (ORF2) of Tx1L encodes a protein that makes a single-strand cut precisely at the expected site within its target sequence, supporting the idea that Tx1L is a site-specific retrotransposon. In this study, we express the endonuclease domain of Tx2L (Tx2L EN) and compare the target preferences of the two enzymes. Each endonuclease shows some preference for its cognate target, on the order of 5-fold over the non-cognate target. The observed discrimination is not sufficient, however, to explain the observation that no cross-occupancy is observed - that is, L elements of one family have never been found within D elements of the other family. Possible sources of additional specificity are discussed. We also compare two hypotheses regarding the genome duplication event that led to the contemporary pseudotetraploid character of Xenopus laevis in light of the Tx1L and Tx2L data.

  15. HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways

    Science.gov (United States)

    Guo, Fang; Zhao, Qiong; Cheng, Junjun; Qi, Yonghe; Su, Qing; Wei, Lai; Li, Wenhui; Chang, Jinhong

    2017-01-01

    Hepatitis B virus (HBV) core protein assembles viral pre-genomic (pg) RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs) and sulfamoylbenzamides (SBAs), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or “empty” capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc) DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B. PMID:28945802

  16. HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways.

    Science.gov (United States)

    Guo, Fang; Zhao, Qiong; Sheraz, Muhammad; Cheng, Junjun; Qi, Yonghe; Su, Qing; Cuconati, Andrea; Wei, Lai; Du, Yanming; Li, Wenhui; Chang, Jinhong; Guo, Ju-Tao

    2017-09-01

    Hepatitis B virus (HBV) core protein assembles viral pre-genomic (pg) RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs) and sulfamoylbenzamides (SBAs), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or "empty" capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc) DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B.

  17. HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways.

    Directory of Open Access Journals (Sweden)

    Fang Guo

    2017-09-01

    Full Text Available Hepatitis B virus (HBV core protein assembles viral pre-genomic (pg RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs and sulfamoylbenzamides (SBAs, have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or "empty" capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B.

  18. Hotspot mutations in KIT receptor differentially modulate its allosterically coupled conformational dynamics: impact on activation and drug sensitivity.

    Directory of Open Access Journals (Sweden)

    Isaure Chauvot de Beauchêne

    2014-07-01

    Full Text Available Receptor tyrosine kinase KIT controls many signal transduction pathways and represents a typical allosterically regulated protein. The mutation-induced deregulation of KIT activity impairs cellular physiological functions and causes serious human diseases. The impact of hotspots mutations (D816H/Y/N/V and V560G/D localized in crucial regulatory segments, the juxtamembrane region (JMR and the activation (A- loop, on KIT internal dynamics was systematically studied by molecular dynamics simulations. The mutational outcomes predicted in silico were correlated with in vitro and in vivo activation rates and drug sensitivities of KIT mutants. The allosteric regulation of KIT in the native and mutated forms is described in terms of communication between the two remote segments, JMR and A-loop. A strong correlation between the communication profile and the structural and dynamical features of KIT in the native and mutated forms was established. Our results provide new insight on the determinants of receptor KIT constitutive activation by mutations and resistance of KIT mutants to inhibitors. Depiction of an intra-molecular component of the communication network constitutes a first step towards an integrated description of vast communication pathways established by KIT in physiopathological contexts.

  19. Understanding large multiprotein complexes: applying a multiple allosteric networks model to explain the function of the Mediator transcription complex.

    Science.gov (United States)

    Lewis, Brian A

    2010-01-15

    The regulation of transcription and of many other cellular processes involves large multi-subunit protein complexes. In the context of transcription, it is known that these complexes serve as regulatory platforms that connect activator DNA-binding proteins to a target promoter. However, there is still a lack of understanding regarding the function of these complexes. Why do multi-subunit complexes exist? What is the molecular basis of the function of their constituent subunits, and how are these subunits organized within a complex? What is the reason for physical connections between certain subunits and not others? In this article, I address these issues through a model of network allostery and its application to the eukaryotic RNA polymerase II Mediator transcription complex. The multiple allosteric networks model (MANM) suggests that protein complexes such as Mediator exist not only as physical but also as functional networks of interconnected proteins through which information is transferred from subunit to subunit by the propagation of an allosteric state known as conformational spread. Additionally, there are multiple distinct sub-networks within the Mediator complex that can be defined by their connections to different subunits; these sub-networks have discrete functions that are activated when specific subunits interact with other activator proteins.

  20. Late Neogene benthic stable isotope record of ODP Site 999: Implications for Caribbean paleoceanography, organic carbon burial and the Messininian salinity crisis

    Science.gov (United States)

    Bickert, T.; Haug, G.; Tiedemann, R.

    2003-04-01

    The late Neogene closure of the seaway between the North and South American continents is thought to have caused extensive changes in ocean circulation and Northern Hemisphere climate. The timing and consequences of the emergence of the Isthmus of Panama for the ocean circulation have been addressed in several papers which indicate a marked reorganization of surface and deep ocean circulation starting 4.6 million years ago. However, the biogeographic development of marine faunas and floras on both sides of the Panama Isthmus suggests that the paleoceanographic changes related to the closing of the isthmus started much earlier. Furthermore, the closing history of the Panama Seaway overlaps with the tectonic evolution of other ocean gateways in the late Miocene, especially the closure of the Strait of Gibraltar, which led to a transient isolation of the Mediterranean Sea from the Atlantic Ocean, known as the Messinian Salinity Crisis. We report on epibenthic foraminiferal d18O and d13C and percentage sand records of the carbonate fraction from Caribbean ODP Site 999 (12°44´N, 78° 44´W, water depth 2828 m) spanning the interval from 8.6 to 5.3 Ma. Low epibenthic d13C values and low sand contents indicate a poorly ventilated deep Caribbean throughout the late Miocene. At this time the deep Caribbean was dominated by a nutrient-rich Southern Ocean water mass. A mostly constant d13C gradient between the Caribbean and deep Atlantic records suggests that the fluctuations in d13C reflect rather global changes in d13C of the dissolved inorganic carbon due to varying erosion of organic carbon from terrigenous soils and shelf sediments. The observed 100-ky cyclicity of epibenthic d13C is in well accordance with the variability of the terrigenous input to the equatorial Atlantic as recorded by susceptibility records of the Ceara Rise. However, some gradient changes between 6.8 and 5.6 Ma indicate a poorer ventilation of the deep Atlantic related to a reduced production of

  1. Sediment sequence and site formation processes at the Arbreda Cave, NE Iberian Peninsula, and implications on human occupation and climate change during the Last Glacial

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    M. Kehl

    2014-09-01

    Full Text Available The Arbreda Cave provides a detailed archaeological record of the Middle to Upper Palaeolithic and is a key site for studying human occupation and cultural transitions in NE Iberia. Recently, studies of lake archives and archaeological sites presented new evidence on climate changes in NE Iberia correlating with Heinrich events. It, therefore, needs to be determined whether climate signals can be identified in the cave sequence of Arbreda, and if so, whether these signals can be correlated with stratigraphic indicators suggesting the continuity or discontinuity of human occupation. We conducted a high-resolution sedimentological and geochemical study, including micromorphological investigations, to shed light on stratigraphy, processes of sediment accumulation and post-depositional alteration in the cave. Seven major sediment units were distinguished which partly correlate with archaeological levels. The lower part of the sequence including Mousterian levels J and K consists of fluvial deposits truncated by a sharp erosional disconformity between Mousterian levels J and I. Strong enrichment with phosphorus and strontium reflect zoogenic inputs. The transition from Mousterian to Archaic Aurignacian in levels I and H, respectively, is reflected by more gradual changes in colour, grain size and geochemical composition. However, a peak in potentially wind-blown particles (40–125 μm in diameter reflects higher aeolian input, and banded microstructure suggests reworking of sediments at the interface. Both properties correlate with low density of finds suggesting low intensity of human occupation related to a dry spell. More arid conditions than during the Holocene are indicated for the Gravettian to Solutrean levels. These findings are in agreement with previous palaeoclimatic interpretations as based on palaeontological proxies. The detailed multi-proxy analyses of the sequence adds to our understanding on sediment accumulation and alteration in

  2. Cocaine treatment admissions at three sentinel sites in South Africa (1997–2006: findings and implications for policy, practice and research

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    Plüddemann Andreas

    2007-12-01

    Full Text Available Abstract Background Accurate prevalence data on cocaine use, that points to where problems exist and the extent of these problems, is necessary to guide the formulation of effective substance abuse policy and practice. The purpose of this study was to provide surveillance information about the nature and extent of problematic cocaine use in South Africa. Methods Data were collected between January 1997 and December 2006 on admissions for drug abuse treatment through a regular monitoring system involving 56 drug treatment centres and programmes in Cape Town, Gauteng Province (Johannesburg and Pretoria and the Eastern Cape every six months as part of the South African Community Epidemiology Network on Drug Use (SACENDU. A one-page form was completed by treatment centre personnel to obtain demographic data, the patients' primary and secondary substances of abuse, the mode, frequency and age of first use of substance, and information on prior treatment. Results Treatment indicators point to a significant increase in cocaine related admissions over time in all sites, but with substantial inter-site variation, particularly in recent years. The data indicate high levels of crack cocaine use and high levels of daily usage among patients, most of whom were first time admissions. Patients with cocaine related problems continue to be predominantly male, with a mean age of around 30 years. Substantial changes in the racial profile of patients have occurred over time. Poly drug use is high with cocaine often used with alcohol, cannabis and other drugs. Conclusion These trends point to the possibility of cocaine use becoming a serious health and social issue in South Africa and demonstrate the utility of continued monitoring of cocaine treatment admissions in the future. They also highlight the need to address cocaine use in national and provincial policy planning and intervention efforts. In terms of treatment, the findings highlight the need to ensure that

  3. Site suitability analysis for Bay scallop aquaculture and implications for sustainable fisheries management in the Ha Long Bay archipelago, northern Vietnam

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    Pham Thi Khanh

    2013-01-01

    Full Text Available Mollusc culture if properly managed, may help decrease capture fisheries over-exploitation in Vietnam, and possibly become an alternative income for local fishermen. The definition and characterization of zones suitable for aquaculture is pivotal for its success and sustainable development, and this study aims at determining the suitability of Argopecten irradians (Bay scallop culture in the Ha Long Bay Archipelago. Temperature, salinity, chlorophyll-a, total suspended solid and bathymetry, were compiled in an environmental suitability model. Distance of culture sites from landing points and fish markets were instead grouped in an infrastructural suitability model. In both models, developed with Geographic Information Systems, the suitability scores were ranked on a scale from 1 (unsuitable to 6 (very-highly suitable. Results showed that 98 % of the studied area is environmentally suitable for such culture. However, overlaying the infrastructural factors the suitable zone decrease to 38 %. Advantages and disadvantages of two management options were then discussed: (a strengthening fisheries infrastructures or (b developing post harvesting processing plants.

  4. Coupled-processes in the Callovo-Oxfordian shales at the Bure site: implications for the transports of water and solutes

    International Nuclear Information System (INIS)

    Rousseau-gueutin, P.

    2008-09-01

    This research thesis is related to the ANDRA's project of building a deep storage site for long-life, high- and medium-activity radioactive wastes within a Callovo-Oxfordian geological formation which is investigated by the Bure underground laboratory. As previous studies revealed hydrostatic loads greater than expected, the objective of this research is to explain these overpressures by validating or invalidating hypotheses on natural phenomena which could cause them. These hypotheses are: a progressive decrease of rock porosity in relationship with a continuous increase of mechanical stresses in the Callovo-Oxfordian, the existence in the past of hydrostatic pressures greater than now, and an osmotic effect. The author gives an overview of the knowledge about clays and their interactions with surrounding solutes, about coupled flows and overpressures. Based on petro-physical data, she proposes an assessment of the chemical osmotic coupling coefficient. Based on experimental and numerical investigations, she reports the measurement of this coefficient in the Callovo-Oxfordian argillites, and then proposes new interpretations of the measured overpressures

  5. The implications of soil acidification on a future HLW repository. Pt 2. Influence on deep granitic groundwater. The Klipperaas study site as test case

    International Nuclear Information System (INIS)

    Wersin, P.; Bruno, J.; Laaksoharju, M.

    1994-05-01

    The effect of acidification on deep groundwater is assessed with a geochemical box model based on the STEADYQL code. The application of the model to the Klipperaas study site shows a remarkable agreement between observed and predicted groundwater composition and offers an adequate description of the geochemical evolution of the aquifer. Proton fluxes are shown to be controlled mainly by calcite weathering and organic carbon degradation processes. The impact of increased acidification is evaluated on the basis of various test cases and by including the soil compartment in the model framework. The results indicate that calcite weathering will be increased by a factor of two to three as a result of increased acidification. Furthermore, the calculations suggest that, once the powerful carbonate buffer is depleted, the buffer capacity is provided mainly by anaerobic respiration and ion exchange processes. Further ongoing acidic loading would lead to neutralization of alkalinity fluxes leaving the system with a very low buffering capacity towards fluctuations in proton fluxes. Estimation of time scales of aquifer acidification was assessed under the focus of calcite depletion with aid of two acidification scenarios. These predict a time range of 12400 to 370000 years for calcite depletion to take place down to 500 meters depth. It is suggested from inherent model assumptions that these estimated time scales are conservative. 53 refs

  6. Conserved Residues Lys57 and Lys401 of Protein Disulfide Isomerase Maintain an Active Site Conformation for Optimal Activity: Implications for Post-Translational Regulation

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    Cody Caba

    2018-02-01

    Full Text Available Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI, the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys57 and Lys401 of human PDI in vitro. Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys57 and Lys401 was assessed by in vitro treatment using acetylsalicylic acid (aspirin. A total of 28 acetyllysine residues were identified, including acLys57 and acLys401. The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity.

  7. Conserved Residues Lys57 and Lys401 of Protein Disulfide Isomerase Maintain an Active Site Conformation for Optimal Activity: Implications for Post-Translational Regulation.

    Science.gov (United States)

    Caba, Cody; Ali Khan, Hyder; Auld, Janeen; Ushioda, Ryo; Araki, Kazutaka; Nagata, Kazuhiro; Mutus, Bulent

    2018-01-01

    Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI), the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys 57 and Lys 401 of human PDI in vitro . Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys 57 and Lys 401 was assessed by in vitro treatment using acetylsalicylic acid (aspirin). A total of 28 acetyllysine residues were identified, including acLys 57 and acLys 401 . The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity.

  8. Counselling implications of teachers’ digital competencies in the use of Social Networking Sites (SNSs in the teaching-learning process in Calabar, Nigeria

    Directory of Open Access Journals (Sweden)

    Mfon Eyo

    2016-12-01

    Full Text Available The study investigated teachers’ digital competencies in the use of Social Networking Sites (SNSs in the teaching-learning process. It had five research questions and two hypotheses. Adopting a survey design, it used a sample of 250 teachers from 10 out of 16 secondary schools in Calabar Municipal Local Government. A researcher-developed instrument called Digital Competency Questionnaire (DCQ was used in data gathering. The DCQ was validated by three experts, and its reliability was established to be 0.86. Findings of the study indicated that the levels of digital competencies of teachers in the use of SNSs and general usage of SNSs by teachers are moderate. It also revealed that the level of application of SNSs in the teaching-learning process is low. Results of the study also indicated significant differences in the level of digital competencies in the use of SNSs based on gender; and in the level of application of SNSs in the teaching-learning process based on age category of teachers. Recommendations were made to the guidance counsellor to improve these levels and bridge the established gaps based on gender and age category of the teachers.

  9. The implications of soil acidification on a future HLW repository. Pt 2. Influence on deep granitic groundwater. The Klipperaas study site as test case.

    Energy Technology Data Exchange (ETDEWEB)

    Wersin, P; Bruno, J [MBT Tecnologia Ambiental, Cerdanyola (Spain); Laaksoharju, M [Geopoint AB, Spaanga (Sweden)

    1994-05-01

    The effect of acidification on deep groundwater is assessed with a geochemical box model based on the STEADYQL code. The application of the model to the Klipperaas study site shows a remarkable agreement between observed and predicted groundwater composition and offers an adequate description of the geochemical evolution of the aquifer. Proton fluxes are shown to be controlled mainly by calcite weathering and organic carbon degradation processes. The impact of increased acidification is evaluated on the basis of various test cases and by including the soil compartment in the model framework. The results indicate that calcite weathering will be increased by a factor of two to three as a result of increased acidification. Furthermore, the calculations suggest that, once the powerful carbonate buffer is depleted, the buffer capacity is provided mainly by anaerobic respiration and ion exchange processes. Further ongoing acidic loading would lead to neutralization of alkalinity fluxes leaving the system with a very low buffering capacity towards fluctuations in proton fluxes. Estimation of time scales of aquifer acidification was assessed under the focus of calcite depletion with aid of two acidification scenarios. These predict a time range of 12400 to 370000 years for calcite depletion to take place down to 500 meters depth. It is suggested from inherent model assumptions that these estimated time scales are conservative. 53 refs.

  10. Integrated transcriptome and binding sites analysis implicates E2F in the regulation of self-renewal in human pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Hock Chuan Yeo

    Full Text Available Rapid cellular growth and multiplication, limited replicative senescence, calibrated sensitivity to apoptosis, and a capacity to differentiate into almost any cell type are major properties that underline the self-renewal capabilities of human pluripotent stem cells (hPSCs. We developed an integrated bioinformatics pipeline to understand the gene regulation and functions involved in maintaining such self-renewal properties of hPSCs compared to matched fibroblasts. An initial genome-wide screening of transcription factor activity using in silico binding-site and gene expression microarray data newly identified E2F as one of major candidate factors, revealing their significant regulation of the transcriptome. This is underscored by an elevated level of its transcription factor activity and expression in all tested pluripotent stem cell lines. Subsequent analysis of functional gene groups demonstrated the importance of the TFs to self-renewal in the pluripotency-coupled context; E2F directly targets the global signaling (e.g. self-renewal associated WNT and FGF pathways and metabolic network (e.g. energy generation pathways, molecular transports and fatty acid metabolism to promote its canonical functions that are driving the self-renewal of hPSCs. In addition, we proposed a core self-renewal module of regulatory interplay between E2F and, WNT and FGF pathways in these cells. Thus, we conclude that E2F plays a significant role in influencing the self-renewal capabilities of hPSCs.

  11. Implications of the Mw9.0 Tohoku-Oki earthquake for ground motion scaling with source, path, and site parameters

    Science.gov (United States)

    Stewart, Jonathan P.; Midorikawa, Saburoh; Graves, Robert W.; Khodaverdi, Khatareh; Kishida, Tadahiro; Miura, Hiroyuki; Bozorgnia, Yousef; Campbell, Kenneth W.

    2013-01-01

    The Mw9.0 Tohoku-oki Japan earthquake produced approximately 2,000 ground motion recordings. We consider 1,238 three-component accelerograms corrected with component-specific low-cut filters. The recordings have rupture distances between 44 km and 1,000 km, time-averaged shear wave velocities of VS30 = 90 m/s to 1,900 m/s, and usable response spectral periods of 0.01 sec to >10 sec. The data support the notion that the increase of ground motions with magnitude saturates at large magnitudes. High-frequency ground motions demonstrate faster attenuation with distance in backarc than in forearc regions, which is only captured by one of the four considered ground motion prediction equations for subduction earthquakes. Recordings within 100 km of the fault are used to estimate event terms, which are generally positive (indicating model underprediction) at short periods and zero or negative (overprediction) at long periods. We find site amplification to scale minimally with VS30 at high frequencies, in contrast with other active tectonic regions, but to scale strongly with VS30 at low frequencies.

  12. [3H]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors

    International Nuclear Information System (INIS)

    Benke, D.; Honer, M.; Mohler, H.; Heckendorn, R.; Pozza, M.F.; Allgeier, H.; Angst, C.

    1999-01-01

    Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [ 3 H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [ 3 H]CGP 61594 labeled with high affinity (K D =23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [ 3 H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [ 3 H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [ 3 H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  13. Localization of polycyclic aromatic hydrocarbons and heavy metals in surface soil of Asia’s oldest oil and gas drilling site in Assam, north-east India: Implications for the bio-economy

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    Hemen Sarma

    2016-09-01

    Full Text Available The environmental influx of hazardous contaminants viz PAHs and HMs occurs due to oil and gas drilling, and processing of petroleum products in industrial facilities and refineries. This problem plagues crude oil drilling sites as PAHs are an essential component of and HMs coexist with crude oil. We analyzed the spatial distribution of 16 PAHs and 8 HMs in 10 contaminated sites of Assam, a state in India. These included Digboi, where crude oil was drilled in 1867 and the first oil well in Asia that was drilled. The Ʃ16 PAHs in soil were detected with a minimum of 13.48 and a maximum of 86.3 mgkg−1 and Ʃ 8 heavy metal concentrations in the soil ranged between 69.51 and 336.06 mgkg−1. A negative correlation was detected between the relative concentrations of PAHs and HMs. The results confirmed that the non-biodegradable nature of HMs made them stay in the soil for longer periods of time. In our study, we found that the levels of lead, copper, nickel, and chromium (total in soil were 73.62, 11.86, 58.97 and 158.66 mgkg−1. The recovery percentage for PAHs and HMs were in the range of 67–97% and 90–95% respectively. Spatial distribution indices for Phenanthrene/Anthracene, Naphthalene/Acenapthhylene, Chyrsene/Benzo (g, h, i perylene and Fluranthene/Pyrene calculated for soil samples indicated that the spatial distribution of PAHs in soil is uneven which might be due to variations in contaminates disseminated in soil. Such regionalized concentration has serious implications on the bio-economy both in terms of health and economy, especially since the proximity of crude oil sites to paddy fields and/or tea plantations uniquely marks the landscape of upper Assam.

  14. A survey of domestic wells and pit latrines in rura