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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

    Directory of Open Access Journals (Sweden)

    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

    Directory of Open Access Journals (Sweden)

    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. Identification of the Allosteric Regulatory Site of Insulysin

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. Prediction of allosteric sites and mediating interactions through bond-to-bond propensities

    CERN Document Server

    Amor, Benjamin R C; Yaliraki, Sophia N; Barahona, Mauricio

    2016-01-01

    Allosteric regulation is central to many biochemical processes. Allosteric sites provide a target to fine-tune protein activity, yet we lack computational methods to predict them. Here, we present an efficient graph-theoretical approach for identifying allosteric sites and the mediating interactions that connect them to the active site. Using an atomistic graph with edges weighted by covalent and non-covalent bond energies, we obtain a bond-to-bond propensity that quantifies the effect of instantaneous bond fluctuations propagating through the protein. We use this propensity to detect the sites and communication pathways most strongly linked to the active site, assessing their significance through quantile regression and comparison against a reference set of 100 generic proteins. We exemplify our method in detail with three well-studied allosteric proteins: caspase-1, CheY, and h-Ras, correctly predicting the location of the allosteric site and identifying key allosteric interactions. Consistent prediction of...

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

    DEFF Research Database (Denmark)

    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....

  6. Prediction of allosteric sites and mediating interactions through bond-to-bond propensities

    Science.gov (United States)

    Amor, B. R. C.; Schaub, M. T.; Yaliraki, S. N.; Barahona, M.

    2016-01-01

    Allostery is a fundamental mechanism of biological regulation, in which binding of a molecule at a distant location affects the active site of a protein. Allosteric sites provide targets to fine-tune protein activity, yet we lack computational methodologies to predict them. Here we present an efficient graph-theoretical framework to reveal allosteric interactions (atoms and communication pathways strongly coupled to the active site) without a priori information of their location. Using an atomistic graph with energy-weighted covalent and weak bonds, we define a bond-to-bond propensity quantifying the non-local effect of instantaneous bond fluctuations propagating through the protein. Significant interactions are then identified using quantile regression. We exemplify our method with three biologically important proteins: caspase-1, CheY, and h-Ras, correctly predicting key allosteric interactions, whose significance is additionally confirmed against a reference set of 100 proteins. The almost-linear scaling of our method renders it suitable for high-throughput searches for candidate allosteric sites. PMID:27561351

  7. Common Internal Allosteric Network Links Anesthetic Binding Sites in a Pentameric Ligand-Gated Ion Channel.

    Science.gov (United States)

    Joseph, Thomas T; Mincer, Joshua S

    2016-01-01

    General anesthetics bind reversibly to ion channels, modifying their global conformational distributions, but the underlying atomic mechanisms are not completely known. We examine this issue by way of the model protein Gloeobacter violaceous ligand-gated ion channel (GLIC) using computational molecular dynamics, with a coarse-grained model to enhance sampling. We find that in flooding simulations, both propofol and a generic particle localize to the crystallographic transmembrane anesthetic binding region, and that propofol also localizes to an extracellular region shared with the crystallographic ketamine binding site. Subsequent simulations to probe these binding modes in greater detail demonstrate that ligand binding induces structural asymmetry in GLIC. Consequently, we employ residue interaction correlation analysis to describe the internal allosteric network underlying the coupling of ligand and distant effector sites necessary for conformational change. Overall, the results suggest that the same allosteric network may underlie the actions of various anesthetics, regardless of binding site. PMID:27403526

  8. Allosteric vs. spontaneous exit-site (E-site) tRNA dissociation early in protein synthesis

    OpenAIRE

    Chen, Chunlai; Stevens, Benjamin; Kaur, Jaskiran; Smilansky, Zeev; Cooperman, Barry S.; Goldman, Yale E.

    2011-01-01

    During protein synthesis, deacylated transfer RNAs leave the ribosome via an exit (E) site after mRNA translocation. How the ribosome regulates tRNA dissociation and whether functional linkages between the aminoacyl (A) and E sites modulate the dynamics of protein synthesis have long been debated. Using single molecule fluorescence resonance energy transfer experiments, we find that, during early cycles of protein elongation, tRNAs are often held in the E site until being allosterically relea...

  9. FR258900, a potential anti-hyperglycemic drug, binds at the allosteric site of glycogen phosphorylase

    OpenAIRE

    Tiraidis, C.; Alexacou, K. M.; Zographos, Spyros E.; Leonidas, Demetres D.; Gimisis, T.; Oikonomakos, Nikos G.

    2007-01-01

    FR258900 has been discovered as a novel inhibitor of human liver glycogen phosphorylase a and proved to suppress hepatic glycogen breakdown and reduce plasma glucose concentrations in diabetic mice models. To elucidate the mechanism of inhibition, we have determined the crystal structure of the cocrystallized rabbit muscle glycogen phosphorylase b–FR258900 complex and refined it to 2.2 Å resolution. The structure demonstrates that the inhibitor binds at the allosteric activator site, where th...

  10. Predicting Allosteric Effects from Orthosteric Binding in Hsp90-Ligand Interactions: Implications for Fragment-Based Drug Design

    Science.gov (United States)

    Larsson, Andreas; Nordlund, Paer; Jansson, Anna; Anand, Ganesh S.

    2016-01-01

    A key question in mapping dynamics of protein-ligand interactions is to distinguish changes at binding sites from those associated with long range conformational changes upon binding at distal sites. This assumes a greater challenge when considering the interactions of low affinity ligands (dissociation constants, KD, in the μM range or lower). Amide hydrogen deuterium Exchange mass spectrometry (HDXMS) is a robust method that can provide both structural insights and dynamics information on both high affinity and transient protein-ligand interactions. In this study, an application of HDXMS for probing the dynamics of low affinity ligands to proteins is described using the N-terminal ATPase domain of Hsp90. Comparison of Hsp90 dynamics between high affinity natural inhibitors (KD ~ nM) and fragment compounds reveal that HDXMS is highly sensitive in mapping the interactions of both high and low affinity ligands. HDXMS reports on changes that reflect both orthosteric effects and allosteric changes accompanying binding. Orthosteric sites can be identified by overlaying HDXMS onto structural information of protein-ligand complexes. Regions distal to orthosteric sites indicate long range conformational changes with implications for allostery. HDXMS, thus finds powerful utility as a high throughput method for compound library screening to identify binding sites and describe allostery with important implications for fragment-based ligand discovery (FBLD). PMID:27253209

  11. Predicting Allosteric Effects from Orthosteric Binding in Hsp90-Ligand Interactions: Implications for Fragment-Based Drug Design.

    Directory of Open Access Journals (Sweden)

    Arun Chandramohan

    2016-06-01

    Full Text Available A key question in mapping dynamics of protein-ligand interactions is to distinguish changes at binding sites from those associated with long range conformational changes upon binding at distal sites. This assumes a greater challenge when considering the interactions of low affinity ligands (dissociation constants, KD, in the μM range or lower. Amide hydrogen deuterium Exchange mass spectrometry (HDXMS is a robust method that can provide both structural insights and dynamics information on both high affinity and transient protein-ligand interactions. In this study, an application of HDXMS for probing the dynamics of low affinity ligands to proteins is described using the N-terminal ATPase domain of Hsp90. Comparison of Hsp90 dynamics between high affinity natural inhibitors (KD ~ nM and fragment compounds reveal that HDXMS is highly sensitive in mapping the interactions of both high and low affinity ligands. HDXMS reports on changes that reflect both orthosteric effects and allosteric changes accompanying binding. Orthosteric sites can be identified by overlaying HDXMS onto structural information of protein-ligand complexes. Regions distal to orthosteric sites indicate long range conformational changes with implications for allostery. HDXMS, thus finds powerful utility as a high throughput method for compound library screening to identify binding sites and describe allostery with important implications for fragment-based ligand discovery (FBLD.

  12. FR258900, a potential anti-hyperglycemic drug, binds at the allosteric site of glycogen phosphorylase.

    Science.gov (United States)

    Tiraidis, Costas; Alexacou, Kyra-Melinda; Zographos, Spyros E; Leonidas, Demetres D; Gimisis, Thanasis; Oikonomakos, Nikos G

    2007-08-01

    FR258900 has been discovered as a novel inhibitor of human liver glycogen phosphorylase a and proved to suppress hepatic glycogen breakdown and reduce plasma glucose concentrations in diabetic mice models. To elucidate the mechanism of inhibition, we have determined the crystal structure of the cocrystallized rabbit muscle glycogen phosphorylase b-FR258900 complex and refined it to 2.2 A resolution. The structure demonstrates that the inhibitor binds at the allosteric activator site, where the physiological activator AMP binds. The contacts from FR258900 to glycogen phosphorylase are dominated by nonpolar van der Waals interactions with Gln71, Gln72, Phe196, and Val45' (from the symmetry-related subunit), and also by ionic interactions from the carboxylate groups to the three arginine residues (Arg242, Arg309, and Arg310) that form the allosteric phosphate-recognition subsite. The binding of FR258900 to the protein promotes conformational changes that stabilize an inactive T-state quaternary conformation of the enzyme. The ligand-binding mode is different from those of the potent phenoxy-phthalate and acyl urea inhibitors, previously described, illustrating the broad specificity of the allosteric site. PMID:17600143

  13. Targeting the Akt1 allosteric site to identify novel scaffolds through virtual screening.

    Science.gov (United States)

    Yilmaz, Oya Gursoy; Olmez, Elif Ozkirimli; Ulgen, Kutlu O

    2014-02-01

    Preclinical data and tumor specimen studies report that AKT kinases are related to many human cancers. Therefore, identification and development of small molecule inhibitors targeting AKT and its signaling pathway can be therapeutic in treatment of cancer. Numerous studies report inhibitors that target the ATP-binding pocket in the kinase domains, but the similarity of this site, within the kinase family makes selectivity a major problem. The sequence identity amongst PH domains is significantly lower than that in kinase domains and developing more selective inhibitors is possible if PH domain is targeted. This in silico screening study is the first time report toward the identification of potential allosteric inhibitors expected to bind the cavity between kinase and PH domains of Akt1. Structural information of Akt1 was used to develop structure-based pharmacophore models comprising hydrophobic, acceptor, donor and ring features. The 3D structural information of previously identified allosteric Akt inhibitors obtained from literature was employed to develop a ligand-based pharmacophore model. Database was generated with drug like subset of ZINC and screening was performed based on 3D similarity to the selected pharmacophore hypotheses. Binding modes and affinities of the ligands were predicted by Glide software. Top scoring hits were further analyzed considering 2D similarity between the compounds, interactions with Akt1, fitness to pharmacophore models, ADME, druglikeness criteria and Induced-Fit docking. Using virtual screening methodologies, derivatives of 3-methyl-xanthine, quinoline-4-carboxamide and 2-[4-(cyclohexa-1,3-dien-1-yl)-1H-pyrazol-3-yl]phenol were proposed as potential leads for allosteric inhibition of Akt1. PMID:24291487

  14. Mapping of the Allosteric Site in Cholesterol Hydroxylase CYP46A1 for Efavirenz, a Drug That Stimulates Enzyme Activity.

    Science.gov (United States)

    Anderson, Kyle W; Mast, Natalia; Hudgens, Jeffrey W; Lin, Joseph B; Turko, Illarion V; Pikuleva, Irina A

    2016-05-27

    Cytochrome P450 46A1 (CYP46A1) is a microsomal enzyme and cholesterol 24-hydroxylase that controls cholesterol elimination from the brain. This P450 is also a potential target for Alzheimer disease because it can be activated pharmacologically by some marketed drugs, as exemplified by efavirenz, the anti-HIV medication. Previously, we suggested that pharmaceuticals activate CYP46A1 allosterically through binding to a site on the cytosolic protein surface, which is different from the enzyme active site facing the membrane. Here we identified this allosteric site for efavirenz on CYP46A1 by using a combination of hydrogen-deuterium exchange coupled to MS, computational modeling, site-directed mutagenesis, and analysis of the CYP46A1 crystal structure. We also mapped the binding region for the CYP46A1 redox partner oxidoreductase and found that the allosteric and redox partner binding sites share a common border. On the basis of the data obtained, we propose the mechanism of CYP46A1 allostery and the pathway for the signal transmission from the P450 allosteric site to the active site. PMID:27056331

  15. An external sodium ion binding site controls allosteric gating in TRPV1 channels.

    Science.gov (United States)

    Jara-Oseguera, Andres; Bae, Chanhyung; Swartz, Kenton J

    2016-01-01

    TRPV1 channels in sensory neurons are integrators of painful stimuli and heat, yet how they integrate diverse stimuli and sense temperature remains elusive. Here, we show that external sodium ions stabilize the TRPV1 channel in a closed state, such that removing the external ion leads to channel activation. In studying the underlying mechanism, we find that the temperature sensors in TRPV1 activate in two steps to favor opening, and that the binding of sodium to an extracellular site exerts allosteric control over temperature-sensor activation and opening of the pore. The binding of a tarantula toxin to the external pore also exerts control over temperature-sensor activation, whereas binding of vanilloids influences temperature-sensitivity by largely affecting the open/closed equilibrium. Our results reveal a fundamental role of the external pore in the allosteric control of TRPV1 channel gating and provide essential constraints for understanding how these channels can be tuned by diverse stimuli. PMID:26882503

  16. Allosteric vs. spontaneous exit-site (E-site) tRNA dissociation early in protein synthesis.

    Science.gov (United States)

    Chen, Chunlai; Stevens, Benjamin; Kaur, Jaskiran; Smilansky, Zeev; Cooperman, Barry S; Goldman, Yale E

    2011-10-11

    During protein synthesis, deacylated transfer RNAs leave the ribosome via an exit (E) site after mRNA translocation. How the ribosome regulates tRNA dissociation and whether functional linkages between the aminoacyl (A) and E sites modulate the dynamics of protein synthesis have long been debated. Using single molecule fluorescence resonance energy transfer experiments, we find that, during early cycles of protein elongation, tRNAs are often held in the E site until being allosterically released when the next aminoacyl tRNA binds to the A site. This process is regulated by the length and sequence of the nascent peptide and by the conformational state, detected by tRNA proximity, prior to translocation. In later cycles, E-site tRNA dissociates spontaneously. Our results suggest that the distribution of pretranslocation tRNA states and posttranslocation pathways are correlated within each elongation cycle via communication between distant subdomains in the ribosome, but that this correlation between elongation cycle intermediates does not persist into succeeding cycles. PMID:21969541

  17. Exploring the allosteric mechanism of dihydrodipicolinate synthase by reverse engineering of the allosteric inhibitor binding sites and its application for lysine production.

    Science.gov (United States)

    Geng, Feng; Chen, Zhen; Zheng, Ping; Sun, Jibin; Zeng, An-Ping

    2013-03-01

    Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyzes the first committed reaction of L-lysine biosynthesis in bacteria and plants and is allosterically regulated by L-lysine. In previous studies, DHDPSs from different species were proved to have different sensitivity to L-lysine inhibition. In this study, we investigated the key determinants of feedback regulation between two industrially important DHDPSs, the L-lysine-sensitive DHDPS from Escherichia coli and L-lysine-insensitive DHDPS from Corynebacterium glutamicum, by sequence and structure comparisons and site-directed mutation. Feedback inhibition of E. coli DHDPS was successfully alleviated after substitution of the residues around the inhibitor's binding sites with those of C. glutamicum DHDPS. Interestingly, mutagenesis of the lysine binding sites of C. glutamicum DHDPS according to E. coli DHDPS did not recover the expected feedback inhibition but an activation of DHDPS by L-lysine, probably due to differences in the allosteic signal transduction in the DHDPS of these two organisms. Overexpression of L-lysine-insensitive E. coli DHDPS mutants in E. coli MG1655 resulted in an improvement of L-lysine production yield by 46 %. PMID:22644522

  18. Mapping Cannabinoid 1 Receptor Allosteric Site(s): Critical Molecular Determinant and Signaling Profile of GAT100, a Novel, Potent, and Irreversibly Binding Probe.

    Science.gov (United States)

    Laprairie, Robert B; Kulkarni, Abhijit R; Kulkarni, Pushkar M; Hurst, Dow P; Lynch, Diane; Reggio, Patricia H; Janero, David R; Pertwee, Roger G; Stevenson, Lesley A; Kelly, Melanie E M; Denovan-Wright, Eileen M; Thakur, Ganesh A

    2016-06-15

    agonism associated with Org27569 and PSNCBAM-1. Computational docking studies implicate C7.38(382) as a key feature of GAT100 ligand-binding motif. These data help inform the engineering of newer-generation, druggable CB1R allosteric modulators and demonstrate the utility of GAT100 as a covalent probe for mapping structure-function correlates characteristic of the druggable CB1R allosteric space. PMID:27046127

  19. Multiple Transmembrane Binding Sites for p-Trifluoromethyldiazirinyl-etomidate, a Photoreactive Torpedo Nicotinic Acetylcholine Receptor Allosteric Inhibitor*

    OpenAIRE

    Hamouda, Ayman K.; Stewart, Deirdre S.; Husain, S. Shaukat; Cohen, Jonathan B.

    2011-01-01

    Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [3H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[3H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our unders...

  20. Presence of a putative steroidal allosteric site on glycoprotein hormone receptors.

    Science.gov (United States)

    Rossi, Mario; Dimida, Antonio; Ferrarini, Eleonora; Silvano, Elena; De Marco, Giuseppina; Agretti, Patrizia; Aloisi, Gabriella; Simoncini, Tommaso; Di Bari, Lorenzo; Tonacchera, Massimo; Giorgi, Franco; Maggio, Roberto

    2009-11-25

    In a previous work we found that the insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), inhibits the accumulation of cAMP as induced by the bovine thyroid stimulating hormone (bTSH) in cells transfected with the TSH receptor. In this work, we demonstrate that the DDT molecular analogues, diethylstilbestrol and quercetine, are more potent inhibitors of the TSH receptor activity than DDT itself. The notion that all these compounds interfere with nuclear estrogen receptors, as either agonists (DDT and diethylstilbestrol) or antagonists (quercetin), prompted us to test the ability of the steroid hormone 17-beta-estradiol to inhibit the TSH receptor activity. We found that estrogen exposure causes a modest but significant inhibition of the bTSH induced cAMP accumulation both in transfected CHO-TSH receptor and Fischer Rat Thyroid Low Serum 5% (FRTL-5) cells. When applied to CHO cells transfected with the luteinizing hormone receptor, 17-beta-estradiol proved capable of inhibiting the hCG induced cAMP accumulation at a concentration as low as 10nM, though the effect was not greater than 35%. The effect of 17-beta-estradiol was not estrogen receptors mediated, as co-transfection of the estrogen receptor alpha and beta subunits with LH receptor caused cAMP to increase above the level attained by the sole hCG stimulation, and not to decrease it as expected. These data suggest the presence of a steroidal-like allosteric binding site on glycoprotein hormone receptors. PMID:19766106

  1. 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.

  2. 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

    -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 is...

  3. Multiple transmembrane binding sites for p-trifluoromethyldiazirinyl-etomidate, a photoreactive Torpedo nicotinic acetylcholine receptor allosteric inhibitor.

    Science.gov (United States)

    Hamouda, Ayman K; Stewart, Deirdre S; Husain, S Shaukat; Cohen, Jonathan B

    2011-06-10

    Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [(3)H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[(3)H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate). TFD-etomidate inhibited acetylcholine-induced currents with an IC(50) = 4 μM, whereas it inhibited the binding of [(3)H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC(50) values of 2.5 and 0.7 mm, respectively. Similar to [(3)H]TDBzl-etomidate, [(3)H]TFD-etomidate bound to a site at the γ-α subunit interface, photolabeling αM2-10 (αSer-252) and γMet-295 and γMet-299 within γM3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [(3)H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the δ subunit M2-M3 loop (δIle-288) and the δ subunit transmembrane helix bundle (δPhe-232 and δCys-236 within δM1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the γ-α subunit interface and/or within δ subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the γ-α subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive

  4. Docking of noncompetitive inhibitors into dengue virus type 2 protease: understanding the interactions with allosteric binding sites.

    Science.gov (United States)

    Othman, Rozana; Kiat, Tan Siew; Khalid, Norzulaani; Yusof, Rohana; Newhouse, E Irene; Newhouse, James S; Alam, Masqudul; Rahman, Noorsaadah Abdul

    2008-08-01

    A group of flavanones and their chalcones, isolated from Boesenbergia rotunda L., were previously reported to show varying degrees of noncompetitive inhibitory activities toward Dengue virus type 2 (Den2) protease. Results obtained from automated docking studies are in agreement with experimental data in which the ligands were shown to bind to sites other than the active site of the protease. The calculated K(i) values are very small, indicating that the ligands bind quite well to the allosteric binding site. Greater inhibition by pinostrobin, compared to the other compounds, can be explained by H-bonding interaction with the backbone carbonyl of Lys74, which is bonded to Asp75 (one of the catalytic triad residues). In addition, structure-activity relationship analysis yields structural information that may be useful for designing more effective therapeutic drugs against dengue virus infections. PMID:18656912

  5. Allosteric-Site and Catalytic-Site Ligand Effects on PDE5 Functions are Associated with Distinct Changes in Physical Form of the Enzyme

    OpenAIRE

    Corbin, Jackie D.; Zoraghi, Roya; Francis, Sharron H.

    2009-01-01

    Native phosphodiesterase-5 (PDE5) homodimer contains distinct non-catalytic cGMP allosteric sites and catalytic sites for cGMP hydrolysis. Purified recombinant PDE5 was activated by pre-incubation with cGMP. Relatively low concentrations of cGMP produced a Native PAGE gel-shift of PDE5 from a single band position (lower band) to a band with decreased mobility (upper band); higher concentrations of cGMP produced a band of intermediate mobility (middle band) in addition to the upper band. Two p...

  6. 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

    Metabotropic glutamate receptor subtype 5 (mGluR5) is a potential drug target in neurological and psychiatric disorders, and subtype-selective allosteric modulators have attracted much attention as potential drug candidates. In this study, the binding sites of three novel 2-methyl-6-(phenylethynyl......)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...

  7. Elucidation of the ATP7B N-domain Mg2+-ATP coordination site and its allosteric regulation.

    Directory of Open Access Journals (Sweden)

    Claude Hercend

    Full Text Available The diagnostic of orphan genetic disease is often a puzzling task as less attention is paid to the elucidation of the pathophysiology of these rare disorders at the molecular level. We present here a multidisciplinary approach using molecular modeling tools and surface plasmonic resonance to study the function of the ATP7B protein, which is impaired in the Wilson disease. Experimentally validated in silico models allow the elucidation in the Nucleotide binding domain (N-domain of the Mg(2+-ATP coordination site and answer to the controversial role of the Mg(2+ ion in the nucleotide binding process. The analysis of protein motions revealed a substantial effect on a long flexible loop branched to the N-domain protein core. We demonstrated the capacity of the loop to disrupt the interaction between Mg(2+-ATP complex and the N-domain and propose a role for this loop in the allosteric regulation of the nucleotide binding process.

  8. Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors.

    Science.gov (United States)

    Ozoe, Yoshihisa; Kita, Tomo; Ozoe, Fumiyo; Nakao, Toshifumi; Sato, Kazuyuki; Hirase, Kangetsu

    2013-11-01

    γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [(3)H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [(3)H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [(3)H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [(3)H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [(3)H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B1a, were potent inhibitors of [(3)H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides

  9. Allosteric modulation of caspases.

    Science.gov (United States)

    Häcker, Hans-Georg; Sisay, Mihiret Tekeste; Gütschow, Michael

    2011-11-01

    Caspases are proteolytic enzymes mainly involved in the induction and execution phases of apoptosis. This type of programmed cell death is an essential regulatory process required to maintain the integrity and homeostasis of multicellular organisms. Inappropriate apoptosis is attributed a key role in many human diseases, including neurodegenerative disorders, ischemic damage, autoimmune diseases and cancer. Allosteric modulation of the function of a protein occurs when the regulatory trigger, such as the binding of a small effector or inhibitor molecule, takes place some distance from the protein's active site. In recent years, several caspases have been identified that possess allosteric sites and binding of small molecule to these sites resulted in the modulation of enzyme activities. Regulation of caspase activity by small molecule allosteric modulators is believed to be of great therapeutic importance. In this review we give brief highlights on recent developments in identifying and characterizing natural and synthetic allosteric inhibitors as well as activators of caspases and discuss their potential in drug discovery and protein engineering. PMID:21807025

  10. 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 Christopo......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...

  11. Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter.

    Science.gov (United States)

    Hohl, Michael; Hürlimann, Lea M; Böhm, Simon; Schöppe, Jendrik; Grütter, Markus G; Bordignon, Enrica; Seeger, Markus A

    2014-07-29

    ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5'-(β,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport. PMID:25030449

  12. NMR Mapping of the IFNAR1-EC binding site on IFNα2 reveals allosteric changes in the IFNAR2-EC binding site

    Science.gov (United States)

    Akabayov, Sabine Ruth; Biron, Zohar; Lamken, Peter; Piehler, Jacob; Anglister, Jacob

    2010-01-01

    All type I interferons (IFNs) bind to a common cell-surface receptor consisting of two subunits. IFNs initiate intracellular signal transduction cascades by simultaneous interaction with the extracellular domains of its receptor subunits IFNAR1 and IFNAR2. In this study we mapped the surface of IFNα2 interacting with the extracellular domain of IFNAR1 (IFNAR1-EC) by following changes in or the disappearance of the [1H,15N]-TROSY-HSQC cross peaks of IFNα2 caused by the binding of the extracellular domain of IFNAR1 (IFNAR1-EC) to the binary complex of IFNα2 with IFNAR2-EC. The NMR study on the 89 kDa complex was conducted at pH 8 and 308 K using an 800 MHz spectrometer. IFNAR1 binding affected a total of 47 out of 165 IFNα2 residues contained in two large patches on the face of the protein opposing the binding site for IFNAR2 and in a third patch located on the face containing the IFNAR2 binding site. The first two patches form the IFNAR1 binding site and one of these matches the IFNAR1 binding site previously identified by site-directed mutagenesis. The third patch partially matches the IFNα2 binding site for IFNAR2-EC indicating allosteric communication between the binding sites for the two receptor subunits. PMID:20047337

  13. Hydrogen/Deuterium Exchange Kinetics Demonstrate Long Range Allosteric Effects of Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase.

    Science.gov (United States)

    Deredge, Daniel; Li, Jiawen; Johnson, Kenneth A; Wintrode, Patrick L

    2016-05-01

    New nonnucleoside analogs are being developed as part of a multi-drug regimen to treat hepatitis C viral infections. Particularly promising are inhibitors that bind to the surface of the thumb domain of the viral RNA-dependent RNA polymerase (NS5B). Numerous crystal structures have been solved showing small molecule non-nucleoside inhibitors bound to the hepatitis C viral polymerase, but these structures alone do not define the mechanism of inhibition. Our prior kinetic analysis showed that nonnucleoside inhibitors binding to thumb site-2 (NNI2) do not block initiation or elongation of RNA synthesis; rather, they block the transition from the initiation to elongation, which is thought to proceed with significant structural rearrangement of the enzyme-RNA complex. Here we have mapped the effect of three NNI2 inhibitors on the conformational dynamics of the enzyme using hydrogen/deuterium exchange kinetics. All three inhibitors rigidify an extensive allosteric network extending >40 Å from the binding site, thus providing a structural rationale for the observed disruption of the transition from distributive initiation to processive elongation. The two more potent inhibitors also suppress slow cooperative unfolding in the fingers extension-thumb interface and primer grip, which may contribute their stronger inhibition. These results establish that NNI2 inhibitors act through long range allosteric effects, reveal important conformational changes underlying normal polymerase function, and point the way to the design of more effective allosteric inhibitors that exploit this new information. PMID:27006396

  14. Allosteric Modulation of Muscarinic Acetylcholine Receptors

    Directory of Open Access Journals (Sweden)

    Esam E. El-Fakahany

    2010-08-01

    Full Text Available An allosteric modulator is a ligand that binds to an allosteric site on the receptor and changes receptor conformation to produce increase (positive cooperativity or decrease (negative cooperativity in the binding or action of an orthosteric agonist (e.g., acetylcholine. Since the identification of gallamine as the first allosteric modulator of muscarinic receptors in 1976, this unique mode of receptor modulation has been intensively studied by many groups. This review summarizes over 30 years of research on the molecular mechanisms of allosteric interactions of drugs with the receptor and for new allosteric modulators of muscarinic receptors with potential therapeutic use. Identification of positive modulators of acetylcholine binding and function that enhance neurotransmission and the discovery of highly selective allosteric modulators are mile-stones on the way to novel therapeutic agents for the treatment of schizophrenia, Alzheimer’s disease and other disorders involving impaired cognitive function.

  15. Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase Allosterically Block the Transition from Initiation to Elongation.

    Science.gov (United States)

    Li, Jiawen; Johnson, Kenneth A

    2016-05-01

    Replication of the hepatitis C viral genome is catalyzed by the NS5B (nonstructural protein 5B) RNA-dependent RNA polymerase, which is a major target of antiviral drugs currently in the clinic. Prior studies established that initiation of RNA replication could be facilitated by starting with a dinucleotide (pGG). Here we establish conditions for efficient initiation from GTP to form the dinucleotide and subsequent intermediates leading to highly processive elongation, and we examined the effects of four classes of nonnucleoside inhibitors on each step of the reaction. We show that palm site inhibitors block initiation starting from GTP but not when starting from pGG. In addition we show that nonnucleoside inhibitors binding to thumb site-2 (NNI2) lead to the accumulation of abortive intermediates three-five nucleotides in length. Our kinetic analysis shows that NNI2 do not significantly block initiation or elongation of RNA synthesis; rather, they block the transition from initiation to elongation, which is thought to proceed with significant structural rearrangement of the enzyme-RNA complex including displacement of the β-loop from the active site. Direct measurement in single turnover kinetic studies show that pyrophosphate release is faster than the chemistry step, which appears to be rate-limiting during processive synthesis. These results reveal important new details to define the steps involved in initiation and elongation during viral RNA replication, establish the allosteric mechanisms by which NNI2 inhibitors act, and point the way to the design of more effective allosteric inhibitors that exploit this new information. PMID:26851276

  16. Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain

    International Nuclear Information System (INIS)

    The large subunit of Escherichia coli carbamoyl phosphate synthetase is responsible for carbamoyl phosphate synthesis from NH3 and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of [14C]UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradation with [14C]UMP. The [14C]UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. Since the binding sites for IMP and UMP overlap, most probably IMP also binds in this domain. The protection from proteolysis by ornithine suggests that ornithine binds in the same domain. To account for the effects of the allosteric effectors on the binding of ATP, the authors propose a scheme where the two halves of the large subunit form a pseudohomodimer by complementary isologous association, thus placing the NH2 half, which is involved in the binding of the molecule of ATP that yields Pi, close to the regulatory domain

  17. Allosteric effects of R- and S-citalopram on the human 5-HT transporter: evidence for distinct high- and low-affinity binding sites

    DEFF Research Database (Denmark)

    Plenge, Per; Gether, Ulrik; Rasmussen, Søren G

    2007-01-01

    cells, and their 5-HT uptake and uptake inhibitor-binding abilities were studied. The hSERT mutations did not alter affinities for 5-HT or paroxetine, but high-affinity binding of S-citalopram was severely affected, particularly by the I172M, and Y95F/I172M mutations - K(i) respectively 4 nM (wild......-type), 35 nM, 1000 nM, and 17.100 nM (mutants). The allosteric site however, in wild-type hSERT and the three mutants was unaffected by the mutations as attenuation of the dissociation rate of the [(3)H]-paroxetine:hSERT complex in the presence of S-citalopram or paroxetine was the same for wild-type h...

  18. An Allosteric Cross-Talk Between the Activation Loop and the ATP Binding Site Regulates the Activation of Src Kinase

    Science.gov (United States)

    Pucheta-Martínez, Encarna; Saladino, Giorgio; Morando, Maria Agnese; Martinez-Torrecuadrada, Jorge; Lelli, Moreno; Sutto, Ludovico; D’Amelio, Nicola; Gervasio, Francesco Luigi

    2016-04-01

    Phosphorylation of the activation loop is a fundamental step in the activation of most protein kinases. In the case of the Src tyrosine kinase, a prototypical kinase due to its role in cancer and its historic importance, phosphorylation of tyrosine 416 in the activation loop is known to rigidify the structure and contribute to the switch from the inactive to a fully active form. However, whether or not phosphorylation is able per-se to induce a fully active conformation, that efficiently binds ATP and phosphorylates the substrate, is less clear. Here we employ a combination of solution NMR and enhanced-sampling molecular dynamics simulations to fully map the effects of phosphorylation and ATP/ADP cofactor loading on the conformational landscape of Src tyrosine kinase. We find that both phosphorylation and cofactor binding are needed to induce a fully active conformation. What is more, we find a complex interplay between the A-loop and the hinge motion where the phosphorylation of the activation-loop has a significant allosteric effect on the dynamics of the C-lobe.

  19. Computational approaches to detect allosteric pathways in transmembrane molecular machines.

    Science.gov (United States)

    Stolzenberg, Sebastian; Michino, Mayako; LeVine, Michael V; Weinstein, Harel; Shi, Lei

    2016-07-01

    Many of the functions of transmembrane proteins involved in signal processing and transduction across the cell membrane are determined by allosteric couplings that propagate the functional effects well beyond the original site of activation. Data gathered from breakthroughs in biochemistry, crystallography, and single molecule fluorescence have established a rich basis of information for the study of molecular mechanisms in the allosteric couplings of such transmembrane proteins. The mechanistic details of these couplings, many of which have therapeutic implications, however, have only become accessible in synergy with molecular modeling and simulations. Here, we review some recent computational approaches that analyze allosteric coupling networks (ACNs) in transmembrane proteins, and in particular the recently developed Protein Interaction Analyzer (PIA) designed to study ACNs in the structural ensembles sampled by molecular dynamics simulations. The power of these computational approaches in interrogating the functional mechanisms of transmembrane proteins is illustrated with selected examples of recent experimental and computational studies pursued synergistically in the investigation of secondary active transporters and GPCRs. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov. PMID:26806157

  20. Computational fragment-based drug design to explore the hydrophobic sub-pocket of the mitotic kinesin Eg5 allosteric binding site.

    Science.gov (United States)

    Oguievetskaia, Ksenia; Martin-Chanas, Laetitia; Vorotyntsev, Artem; Doppelt-Azeroual, Olivia; Brotel, Xavier; Adcock, Stewart A; de Brevern, Alexandre G; Delfaud, Francois; Moriaud, Fabrice

    2009-08-01

    Eg5, a mitotic kinesin exclusively involved in the formation and function of the mitotic spindle has attracted interest as an anticancer drug target. Eg5 is co-crystallized with several inhibitors bound to its allosteric binding pocket. Each of these occupies a pocket formed by loop 5/helix alpha2 (L5/alpha2). Recently designed inhibitors additionally occupy a hydrophobic pocket of this site. The goal of the present study was to explore this hydrophobic pocket with our MED-SuMo fragment-based protocol, and thus discover novel chemical structures that might bind as inhibitors. The MED-SuMo software is able to compare and superimpose similar interaction surfaces upon the whole protein data bank (PDB). In a fragment-based protocol, MED-SuMo retrieves MED-Portions that encode protein-fragment binding sites and are derived from cross-mining protein-ligand structures with libraries of small molecules. Furthermore we have excluded intra-family MED-Portions derived from Eg5 ligands that occupy the hydrophobic pocket and predicted new potential ligands by hybridization that would fill simultaneously both pockets. Some of the latter having original scaffolds and substituents in the hydrophobic pocket are identified in libraries of synthetically accessible molecules by the MED-Search software. PMID:19533373

  1. Rational engineering of enzyme allosteric regulation through sequence evolution analysis.

    Directory of Open Access Journals (Sweden)

    Jae-Seong Yang

    Full Text Available Control of enzyme allosteric regulation is required to drive metabolic flux toward desired levels. Although the three-dimensional (3D structures of many enzyme-ligand complexes are available, it is still difficult to rationally engineer an allosterically regulatable enzyme without decreasing its catalytic activity. Here, we describe an effective strategy to deregulate the allosteric inhibition of enzymes based on the molecular evolution and physicochemical characteristics of allosteric ligand-binding sites. We found that allosteric sites are evolutionarily variable and comprised of more hydrophobic residues than catalytic sites. We applied our findings to design mutations in selected target residues that deregulate the allosteric activity of fructose-1,6-bisphosphatase (FBPase. Specifically, charged amino acids at less conserved positions were substituted with hydrophobic or neutral amino acids with similar sizes. The engineered proteins successfully diminished the allosteric inhibition of E. coli FBPase without affecting its catalytic efficiency. We expect that our method will aid the rational design of enzyme allosteric regulation strategies and facilitate the control of metabolic flux.

  2. An allosteric synthetic DNA.

    OpenAIRE

    Wu, L.; Curran, J F

    1999-01-01

    Allosteric DNA oligonucleotides are potentially useful diagnostic reagents. Here we develop a model system for the study of allosteric interactions in DNAs. A DNA that binds either Cibacron blue or cholic acid was isolated and partially characterized. Isolation was performed using a multi-stage SELEX. First, short oligos that bind either Cibacron blue or cholic acid were enriched from random oligonucleotide pools. Then, members of the two pools were fused to form longer oligos, which were the...

  3. Allosteric modulation of caspase 3 through mutagenesis

    Directory of Open Access Journals (Sweden)

    Jad Walters

    2012-06-01

    Full Text Available A mutation in the allosteric site of the caspase 3 dimer interface of Val266 to histidine abolishes activity of the enzyme, and models predict that the mutation mimics the action of small molecule allosteric inhibitors by preventing formation of the active site. Mutations were coupled to His266 at two sites in the interface, E124A and Y197C. We present results from X-ray crystallography, enzymatic activity and molecular dynamics simulations for seven proteins, consisting of single, double and triple mutants. The results demonstrate that considering allosteric inhibition of caspase 3 as a shift between discrete ‘off-state’ or ‘on-state’ conformations is insufficient. Although His266 is accommodated in the interface, the structural defects are propagated to the active site through a helix on the protein surface. A more comprehensive view of allosteric regulation of caspase 3 requires the representation of an ensemble of inactive states and shows that subtle structural changes lead to the population of the inactive ensemble.

  4. Role of Arginine 293 and Glutamine 288 in Communication between Catalytic and Allosteric Sites in Yeast Ribonucleotide Reductase

    Energy Technology Data Exchange (ETDEWEB)

    Ahmad, Md. Faiz; Kaushal, Prem Singh; Wan, Qun; Wijerathna, Sanath R.; An, Xiuxiang; Huang, Mingxia; Dealwis, Chris Godfrey (Case Western); (Colorado)

    2012-11-01

    Ribonucleotide reductases (RRs) catalyze the rate-limiting step of de novo deoxynucleotide (dNTP) synthesis. Eukaryotic RRs consist of two proteins, RR1 ({alpha}) that contains the catalytic site and RR2 ({beta}) that houses a diferric-tyrosyl radical essential for ribonucleoside diphosphate reduction. Biochemical analysis has been combined with isothermal titration calorimetry (ITC), X-ray crystallography and yeast genetics to elucidate the roles of two loop 2 mutations R293A and Q288A in Saccharomyces cerevisiae RR1 (ScRR1). These mutations, R293A and Q288A, cause lethality and severe S phase defects, respectively, in cells that use ScRR1 as the sole source of RR1 activity. Compared to the wild-type enzyme activity, R293A and Q288A mutants show 4% and 15%, respectively, for ADP reduction, whereas they are 20% and 23%, respectively, for CDP reduction. ITC data showed that R293A ScRR1 is unable to bind ADP and binds CDP with 2-fold lower affinity compared to wild-type ScRR1. With the Q288A ScRR1 mutant, there is a 6-fold loss of affinity for ADP binding and a 2-fold loss of affinity for CDP compared to the wild type. X-ray structures of R293A ScRR1 complexed with dGTP and AMPPNP-CDP [AMPPNP, adenosine 5-({beta},{gamma}-imido)triphosphate tetralithium salt] reveal that ADP is not bound at the catalytic site, and CDP binds farther from the catalytic site compared to wild type. Our in vivo functional analyses demonstrated that R293A cannot support mitotic growth, whereas Q288A can, albeit with a severe S phase defect. Taken together, our structure, activity, ITC and in vivo data reveal that the arginine 293 and glutamine 288 residues of ScRR1 are crucial in facilitating ADP and CDP substrate selection.

  5. Allosteric modulation of G-protein coupled receptors

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Spalding, Tracy A

    2004-01-01

    The superfamily of G-protein coupled receptors (GPCRs) has more than 1000 members and is the largest family of proteins in the body. GPCRs mediate signalling of stimuli as diverse as light, ions, small molecules, peptides and proteins and are the targets for many pharmaceuticals. Most GPCR ligands...... 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...... 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....

  6. Designing allosteric control into enzymes by chemical rescue of structure

    OpenAIRE

    Deckert, Katelyn; Budiardjo, S. Jimmy; Brunner, Luke C.; Lovell, Scott; Karanicolas, John

    2012-01-01

    Ligand-dependent activity has been engineered into enzymes for purposes ranging from controlling cell morphology to reprogramming cellular signaling pathways. Where these successes have typically fused a naturally allosteric domain to the enzyme of interest, here we instead demonstrate an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruptio...

  7. Crystal structure of the HIV-1 integrase core domain in complex with sucrose reveals details of an allosteric inhibitory binding site

    Energy Technology Data Exchange (ETDEWEB)

    Wielens, Jerome; Headey, Stephen J.; Jeevarajah, Dharshini; Rhodes, David I.; Deadman, John; Chalmers, David K.; Scanlon, Martin J.; Parker, Michael W. (SVIMR-A); (Avea); (Monash IPS)

    2010-04-19

    HIV integrase (IN) is an essential enzyme in HIV replication and an important target for drug design. IN has been shown to interact with a number of cellular and viral proteins during the integration process. Disruption of these important interactions could provide a mechanism for allosteric inhibition of IN. We present the highest resolution crystal structure of the IN core domain to date. We also present a crystal structure of the IN core domain in complex with sucrose which is bound at the dimer interface in a region that has previously been reported to bind integrase inhibitors.

  8. Supramolecular Allosteric Cofacial Porphyrin Complexes

    International Nuclear Information System (INIS)

    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 (RhI or CuI sites) and two cofacially aligned porphyrins (ZnII 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 RhI or CuI 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.

  9. Supramolecular Allosteric Cofacial Porphyrin Complexes

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-04-12

    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{sup I} or Cu{sup I} sites) and two cofacially aligned porphyrins (Zn{sup 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{sup I} or Cu{sup 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

  10. Allosteric Mechanisms in Chaperonin Machines.

    Science.gov (United States)

    Gruber, Ranit; Horovitz, Amnon

    2016-06-01

    Chaperonins are nanomachines that facilitate protein folding by undergoing energy (ATP)-dependent movements that are coordinated in time and space owing to complex allosteric regulation. They consist of two back-to-back stacked oligomeric rings with a cavity at each end where protein substrate folding can take place. Here, we focus on the GroEL/GroES chaperonin system from Escherichia coli and, to a lesser extent, on the more poorly characterized eukaryotic chaperonin CCT/TRiC. We describe their various functional (allosteric) states and how they are affected by substrates and allosteric effectors that include ATP, ADP, nonfolded protein substrates, potassium ions, and GroES (in the case of GroEL). We also discuss the pathways of intra- and inter-ring allosteric communication by which they interconvert and the coupling between allosteric transitions and protein folding reactions. PMID:26726755

  11. Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β- Interface.

    Science.gov (United States)

    Jayakar, Selwyn S; Zhou, Xiaojuan; Savechenkov, Pavel Y; Chiara, David C; Desai, Rooma; Bruzik, Karol S; Miller, Keith W; Cohen, Jonathan B

    2015-09-18

    In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABAARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABAAR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343-19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABAAR responses. In the α1β3γ2 GABAAR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ(+)-β(-) subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β(+)-α(-) subunit interfaces. GABA inhibits S-[(3)H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2-15') in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[(3)H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ(+)-β(-) site, based upon the distance in GABAAR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABAAR function and provide a first demonstration that an intersubunit-binding site in the GABAAR transmembrane domain binds negative and positive allosteric modulators. PMID:26229099

  12. The allosteric regulation of axial/rhombic population in a “Type 1” copper site. Multi-edge x-ray absorption spectroscopic and density functional studies of pseudoazurin

    International Nuclear Information System (INIS)

    The co-existence of “axial” and “rhombic” coordination environments has been demonstrated in a “Type 1” copper site of Pseudoazurin. This observation opens up previously not considered interpretations for the relationship between geometry and electronic structure of the four coordinate copper site. The Met16 variants of pseudoazurin were considered as model systems for investigating the effect of weak interactions from the second coordination sphere. The correlation between geometric and electronic structures of “Type 1” copper site was evaluated by the multi-edge (Cu K-edge and S K-edge) X-ray absorption spectroscopy (XAS) of Met16 variants of pseudoazurin. The co-existing axial and rhombic sites in pseudoazurin were characterized by Cu–ligand distances, effective nuclear charge, and Cu–S(Cys) covalency from XAS. The XAS results were correlated with DFT calculations for investigating the effect of protein environment from the inner-sphere and beyond around the Cu site. The combined experimental and theoretical results support the presence of a close correlation between outer sphere environment and inner sphere coordination environment. This is achieved in pseudoazurin by a previously undisclosed allosteric effect that involves a rearrangement of the protein tertiary structure. (author)

  13. Differentiating a Ligand's Chemical Requirements for Allosteric Interactions from Those for Protein Binding. Phenylalanine Inhibition of Pyruvate Kinase

    International Nuclear Information System (INIS)

    The isoform of pyruvate kinase from brain and muscle of mammals (M1-PYK) is allosterically inhibited by phenylalanine. Initial observations in this model allosteric system indicate that Ala binds competitively with Phe, but elicits a minimal allosteric response. Thus, the allosteric ligand of this system must have requirements for eliciting an allosteric response in addition to the requirements for binding. Phe analogues have been used to dissect what chemical properties of Phe are responsible for eliciting the allosteric response. We first demonstrate that the L-2-aminopropanaldehyde substructure of the amino acid ligand is primarily responsible for binding to M1-PYK. Since the allosteric response to Ala is minimal and linear addition of methyl groups beyond the -carbon increase the magnitude of the allosteric response, we conclude that moieties beyond the -carbon are primarily responsible for allostery. Instead of an all-or-none mechanism of allostery, these findings support the idea that the bulk of the hydrophobic side chain, but not the aromatic nature, is the primary determinant of the magnitude of the observed allosteric inhibition. The use of these results to direct structural studies has resulted in a 1.65 Angstroms structure of M1-PYK with Ala bound. The coordination of Ala in the allosteric amino acid binding site confirms the binding role of the L-2-aminopropanaldehyde substructure of the ligand. Collectively, this study confirms that a ligand can have chemical regions specific for eliciting the allosteric signal in addition to the chemical regions necessary for binding

  14. Activation and Allosteric Modulation of Human μ Opioid Receptor in Molecular Dynamics.

    Science.gov (United States)

    Bartuzi, Damian; Kaczor, Agnieszka A; Matosiuk, Dariusz

    2015-11-23

    Allosteric protein modulation has gained increasing attention in drug design. Its application as a mechanism of action could bring forth safer and more effective medicines. Targeting opioid receptors with allosteric modulators can result in better treatment of pain, depression, and respiratory and immune disorders. In this work we use recent reports on negative modulators of μ opioid receptor as a starting point for identification of allosteric sites and mechanisms of opioid receptor modulation using homology modeling and docking and molecular dynamics studies. An allosteric binding site description is presented. Results suggest a shared binding region for lipophilic allosteric ligands, reveal possible differences in the modulation mechanism between cannabinoids and salvinorin A, and show ambiguous properties of the latter. Also, they emphasize the importance of native-like environment in molecular dynamics simulations and uncover relationships between modulator and orthosteric ligand binding and receptor behavior. Relationships between ligands, transmission switch, and hydrophobic lock are analyzed. PMID:26517559

  15. Allosteric modulators of the hERG K{sup +} channel

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Zhiyi, E-mail: z.yu@lacdr.leidenuniv.nl; Klaasse, Elisabeth, E-mail: elisabethklaasse@hotmail.com; Heitman, Laura H., E-mail: l.h.heitman@lacdr.leidenuniv.nl; IJzerman, Adriaan P., E-mail: ijzerman@lacdr.leidenuniv.nl

    2014-01-01

    Drugs that block the cardiac K{sup +} channel encoded by the human ether-à-go-go gene (hERG) have been associated with QT interval prolongation leading to proarrhythmia, and in some cases, sudden cardiac death. Because of special structural features of the hERG K{sup +} channel, it has become a promiscuous target that interacts with pharmaceuticals of widely varying chemical structures and a reason for concern in the pharmaceutical industry. The structural diversity suggests that multiple binding sites are available on the channel with possible allosteric interactions between them. In the present study, three reference compounds and nine compounds of a previously disclosed series were evaluated for their allosteric effects on the binding of [{sup 3}H]astemizole and [{sup 3}H]dofetilide to the hERG K{sup +} channel. LUF6200 was identified as an allosteric inhibitor in dissociation assays with both radioligands, yielding similar EC{sub 50} values in the low micromolar range. However, potassium ions increased the binding of the two radioligands in a concentration-dependent manner, and their EC{sub 50} values were not significantly different, indicating that potassium ions behaved as allosteric enhancers. Furthermore, addition of potassium ions resulted in a concentration-dependent leftward shift of the LUF6200 response curve, suggesting positive cooperativity and distinct allosteric sites for them. In conclusion, our investigations provide evidence for allosteric modulation of the hERG K{sup +} channel, which is discussed in the light of findings on other ion channels. - Highlights: • Allosteric modulators on the hERG K{sup +} channel were evaluated in binding assays. • LUF6200 was identified as a potent allosteric inhibitor. • Potassium ions were found to behave as allosteric enhancers. • Positive cooperativity and distinct allosteric sites for them were proposed.

  16. Ago-allosteric modulation and other types of allostery in dimeric 7TM receptors

    DEFF Research Database (Denmark)

    Schwartz, Thue W; Holst, Birgitte

    concept for 7TM receptors, it is proposed that the ago-allosteric modulators bind in the orthosteric binding site, but-importantly-in the "other" or allosteric protomer of the dimer. Hereby, they can act both as additive co-agonists, and through intermolecular cooperative effects between the protomers......, they may influence the potency of the endogenous agonist. It is of interest that at least some endogenous agonists can only occupy one protomer of a dimeric 7TM receptor complex at a time and thereby they leave the orthosteric binding site in the allosteric protomer free, potentially for binding of...

  17. 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

    Protein phosphorylation transduces a large set of intracellular signals. One mechanism by which phosphorylation mediates signal transduction is by prompting conformational changes in the target protein or interacting proteins. Previous work described an allosteric site mediating phosphorylation-d...

  18. Recent advance in the discovery of allosteric inhibitors binding to the AMP site of fructose-1, 6-bisphosphatase%果糖-1,6-二磷酸酶AMP变构抑制剂的研究进展

    Institute of Scientific and Technical Information of China (English)

    李占梅; 别建波; 宋宏锐; 徐柏玲

    2011-01-01

    果糖-1,6-二磷酸酶(fructose-1,6-bisphosphatase,FBPase)是肝葡萄糖异生路径中的一个限速酶,催化果糖-1,6-二磷酸水解为果糖-6-磷酸.抑制FBPase的活性,可减少内源性葡萄糖的生成,降低血糖水平,FBPase抑制剂是潜在的新型治疗Ⅱ型糖尿病的药物.本文综述了近年来FBPase一磷酸腺苷(adenosine monophosphate,AMP)变构抑制剂研究的最新进展.%Fructose-1, 6-bisphosphatase (FBPase), a rate-limiting enzyme involved in the pathway of gluconeogenesis, can catalyze the hydrolysis of fructose-1, 6-bisphosphate to fructose-6-phosphate. Upon inhibiting the activity of FBPase, the production of endogenous glucose can be decreased and the level of blood glucose lowered. Therefore, inhibitors of FBPase are expected to be novel potential therapeutics for the treatment of type II diabetes. Recent research efforts were reviewed in the field of developing allosteric inhibitors interacting with the AMP binding site of FBPase.

  19. Allosteric Modulation of Muscarinic Receptors

    Czech Academy of Sciences Publication Activity Database

    Jakubík, Jan; El-Fakahany, E. E.

    New York: Springer, 2016 - (Mysliveček, J.; Jakubík, J.), s. 95-130. (Neuromethods. 107). ISBN 978-1-4939-2857-6 R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : muscarinic receptors * allosteric modulation * radioligand binding functional response Subject RIV: ED - Physiology

  20. Allosteric inhibition of the NS2B-NS3 protease from dengue virus.

    Science.gov (United States)

    Yildiz, Muslum; Ghosh, Sumana; Bell, Jeffrey A; Sherman, Woody; Hardy, Jeanne A

    2013-12-20

    Dengue virus is the flavivirus that causes dengue fever, dengue hemorrhagic disease, and dengue shock syndrome, which are currently increasing in incidence worldwide. Dengue virus protease (NS2B-NS3pro) is essential for dengue virus infection and is thus a target of therapeutic interest. To date, attention has focused on developing active-site inhibitors of NS2B-NS3pro. The flat and charged nature of the NS2B-NS3pro active site may contribute to difficulties in developing inhibitors and suggests that a strategy of identifying allosteric sites may be useful. We report an approach that allowed us to scan the NS2B-NS3pro surface by cysteine mutagenesis and use cysteine reactive probes to identify regions of the protein that are susceptible to allosteric inhibition. This method identified a new allosteric site utilizing a circumscribed panel of just eight cysteine variants and only five cysteine reactive probes. The allosterically sensitive site is centered at Ala125, between the 120s loop and the 150s loop. The crystal structures of WT and modified NS2B-NS3pro demonstrate that the 120s loop is flexible. Our work suggests that binding at this site prevents a conformational rearrangement of the NS2B region of the protein, which is required for activation. Preventing this movement locks the protein into the open, inactive conformation, suggesting that this site may be useful in the future development of therapeutic allosteric inhibitors. PMID:24164286

  1. 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

  2. Allosteric Optical Control of a Class B G-Protein-Coupled Receptor.

    Science.gov (United States)

    Broichhagen, Johannes; Johnston, Natalie R; von Ohlen, Yorrick; Meyer-Berg, Helena; Jones, Ben J; Bloom, Stephen R; Rutter, Guy A; Trauner, Dirk; Hodson, David J

    2016-05-01

    Allosteric regulation promises to open up new therapeutic avenues by increasing drug specificity at G-protein-coupled receptors (GPCRs). However, drug discovery efforts are at present hampered by an inability to precisely control the allosteric site. Herein, we describe the design, synthesis, and testing of PhotoETP, a light-activated positive allosteric modulator of the glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR involved in the maintenance of glucose homeostasis in humans. PhotoETP potentiates Ca(2+) , cAMP, and insulin responses to glucagon-like peptide-1 and its metabolites following illumination of cells with blue light. PhotoETP thus provides a blueprint for the production of small-molecule class B GPCR allosteric photoswitches, and may represent a useful tool for understanding positive cooperativity at the GLP-1R. PMID:27059784

  3. NMR Characterization of Information Flow and Allosteric Communities in the MAP Kinase p38γ

    Science.gov (United States)

    Aoto, Phillip C.; Martin, Bryan T.; Wright, Peter E.

    2016-01-01

    The intramolecular network structure of a protein provides valuable insights into allosteric sites and communication pathways. However, a straightforward method to comprehensively map and characterize these pathways is not currently available. Here we present an approach to characterize intramolecular network structure using NMR chemical shift perturbations. We apply the method to the mitogen activated protein kinase (MAPK) p38γ. p38γ contains allosteric sites that are conserved among eukaryotic kinases as well as unique to the MAPK family. How these regulatory sites communicate with catalytic residues is not well understood. Using our method, we observe and characterize for the first time information flux between regulatory sites through a conserved kinase infrastructure. This network is accessed, reinforced, and broken in various states of p38γ, reflecting the functional state of the protein. We demonstrate that the approach detects critical junctions in the network corresponding to biologically significant allosteric sites and pathways.

  4. NMR Characterization of Information Flow and Allosteric Communities in the MAP Kinase p38γ.

    Science.gov (United States)

    Aoto, Phillip C; Martin, Bryan T; Wright, Peter E

    2016-01-01

    The intramolecular network structure of a protein provides valuable insights into allosteric sites and communication pathways. However, a straightforward method to comprehensively map and characterize these pathways is not currently available. Here we present an approach to characterize intramolecular network structure using NMR chemical shift perturbations. We apply the method to the mitogen activated protein kinase (MAPK) p38γ. p38γ contains allosteric sites that are conserved among eukaryotic kinases as well as unique to the MAPK family. How these regulatory sites communicate with catalytic residues is not well understood. Using our method, we observe and characterize for the first time information flux between regulatory sites through a conserved kinase infrastructure. This network is accessed, reinforced, and broken in various states of p38γ, reflecting the functional state of the protein. We demonstrate that the approach detects critical junctions in the network corresponding to biologically significant allosteric sites and pathways. PMID:27353957

  5. Structure-Based Statistical Mechanical Model Accounts for the Causality and Energetics of Allosteric Communication.

    Science.gov (United States)

    Guarnera, Enrico; Berezovsky, Igor N

    2016-03-01

    Allostery is one of the pervasive mechanisms through which proteins in living systems carry out enzymatic activity, cell signaling, and metabolism control. Effective modeling of the protein function regulation requires a synthesis of the thermodynamic and structural views of allostery. We present here a structure-based statistical mechanical model of allostery, allowing one to observe causality of communication between regulatory and functional sites, and to estimate per residue free energy changes. Based on the consideration of ligand free and ligand bound systems in the context of a harmonic model, corresponding sets of characteristic normal modes are obtained and used as inputs for an allosteric potential. This potential quantifies the mean work exerted on a residue due to the local motion of its neighbors. Subsequently, in a statistical mechanical framework the entropic contribution to allosteric free energy of a residue is directly calculated from the comparison of conformational ensembles in the ligand free and ligand bound systems. As a result, this method provides a systematic approach for analyzing the energetics of allosteric communication based on a single structure. The feasibility of the approach was tested on a variety of allosteric proteins, heterogeneous in terms of size, topology and degree of oligomerization. The allosteric free energy calculations show the diversity of ways and complexity of scenarios existing in the phenomenology of allosteric causality and communication. The presented model is a step forward in developing the computational techniques aimed at detecting allosteric sites and obtaining the discriminative power between agonistic and antagonistic effectors, which are among the major goals in allosteric drug design. PMID:26939022

  6. Allosteric Partial Inhibition of Monomeric Proteases. Sulfated Coumarins Induce Regulation, not just Inhibition, of Thrombin.

    Science.gov (United States)

    Verespy Iii, Stephen; Mehta, Akul Y; Afosah, Daniel; Al-Horani, Rami A; Desai, Umesh R

    2016-01-01

    Allosteric partial inhibition of soluble, monomeric proteases can offer major regulatory advantages, but remains a concept on paper to date; although it has been routinely documented for receptors and oligomeric proteins. Thrombin, a key protease of the coagulation cascade, displays significant conformational plasticity, which presents an attractive opportunity to discover small molecule probes that induce sub-maximal allosteric inhibition. We synthesized a focused library of some 36 sulfated coumarins to discover two agents that display sub-maximal efficacy (~50%), high potency (150-fold). Michaelis-Menten, competitive inhibition, and site-directed mutagenesis studies identified exosite 2 as the site of binding for the most potent sulfated coumarin. Stern-Volmer quenching of active site-labeled fluorophore suggested that the allosteric regulators induce intermediate structural changes in the active site as compared to those that display ~80-100% efficacy. Antithrombin inactivation of thrombin was impaired in the presence of the sulfated coumarins suggesting that allosteric partial inhibition arises from catalytic dysfunction of the active site. Overall, sulfated coumarins represent first-in-class, sub-maximal inhibitors of thrombin. The probes establish the concept of allosteric partial inhibition of soluble, monomeric proteins. This concept may lead to a new class of anticoagulants that are completely devoid of bleeding. PMID:27053426

  7. Chemogenomics of allosteric binding sites in GPCRs

    DEFF Research Database (Denmark)

    Gloriam, David E.

    2013-01-01

    Chemogenomic techniques connect the chemical and biological domains to establish ligand and target relationships not evident from the individual disciplines. Chemogenomics has been applied in lead generation, target classification, focused library design as well as selectivity and polypharmacology...

  8. The Allosteric Switching Mechanism in Bacteriophage MS2

    CERN Document Server

    Perkett, Matthew R

    2015-01-01

    In this article 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 adopt 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 disc...

  9. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs

    Science.gov (United States)

    Dror, Ron O.; Green, Hillary F.; Valant, Celine; Borhani, David W.; Valcourt, James R.; Pan, Albert C.; Arlow, Daniel H.; Canals, Meritxell; Lane, J. Robert; Rahmani, Raphaël; Baell, Jonathan B.; Sexton, Patrick M.; Christopoulos, Arthur; Shaw, David E.

    2013-11-01

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15Å from the classical, `orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  10. Divergent allosteric patterns verify the regulatory paradigm for aspartate transcarbamylase.

    Science.gov (United States)

    Wales, M E; Madison, L L; Glaser, S S; Wild, J R

    1999-12-17

    The native Escherichia coli aspartate transcarbamoylase (ATCase, E.C. 2.1.3.2) provides a classic allosteric model for the feedback inhibition of a biosynthetic pathway by its end products. Both E. coli and Erwinia herbicola possess ATCase holoenzymes which are dodecameric (2(c3):3(r2)) with 311 amino acid residues per catalytic monomer and 153 and 154 amino acid residues per regulatory (r) monomer, respectively. While the quaternary structures of the two enzymes are identical, the primary amino acid sequences have diverged by 14 % in the catalytic polypeptide and 20 % in the regulatory polypeptide. The amino acids proposed to be directly involved in the active site and nucleotide binding site are strictly conserved between the two enzymes; nonetheless, the two enzymes differ in their catalytic and regulatory characteristics. The E. coli enzyme has sigmoidal substrate binding with activation by ATP, and inhibition by CTP, while the E. herbicola enzyme has apparent first order kinetics at low substrate concentrations in the absence of allosteric ligands, no ATP activation and only slight CTP inhibition. In an apparently important and highly conserved characteristic, CTP and UTP impose strong synergistic inhibition on both enzymes. The co-operative binding of aspartate in the E. coli enzyme is correlated with a T-to-R conformational transition which appears to be greatly reduced in the E. herbicola enzyme, although the addition of inhibitory heterotropic ligands (CTP or CTP+UTP) re-establishes co-operative saturation kinetics. Hybrid holoenzymes assembled in vivo with catalytic subunits from E. herbicola and regulatory subunits from E. coli mimick the allosteric response of the native E. coli holoenzyme and exhibit ATP activation. The reverse hybrid, regulatory subunits from E. herbicola and catalytic subunits from E. coli, exhibited no response to ATP. The conserved structure and diverged functional characteristics of the E. herbicola enzyme provides an opportunity

  11. Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain.

    Science.gov (United States)

    Burgess, Selena G; Oleksy, Arkadiusz; Cavazza, Tommaso; Richards, Mark W; Vernos, Isabelle; Matthews, David; Bayliss, Richard

    2016-07-01

    The vast majority of clinically approved protein kinase inhibitors target the ATP-binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, vNAR single domain scaffold, vNAR-D01. Biochemical studies and a crystal structure of the Aurora-A/vNAR-D01 complex show that the vNAR domain overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the vNAR domain stabilizes an inactive conformation, in which the αC-helix is distorted, the canonical Lys-Glu salt bridge is broken and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how single domain antibodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors. PMID:27411893

  12. Detection of allosteric signal transmission by information-theoretic analysis of protein dynamics

    Science.gov (United States)

    Pandini, Alessandro; Fornili, Arianna; Fraternali, Franca; Kleinjung, Jens

    2012-01-01

    Allostery offers a highly specific way to modulate protein function. Therefore, understanding this mechanism is of increasing interest for protein science and drug discovery. However, allosteric signal transmission is difficult to detect experimentally and to model because it is often mediated by local structural changes propagating along multiple pathways. To address this, we developed a method to identify communication pathways by an information-theoretical analysis of molecular dynamics simulations. Signal propagation was described as information exchange through a network of correlated local motions, modeled as transitions between canonical states of protein fragments. The method was used to describe allostery in two-component regulatory systems. In particular, the transmission from the allosteric site to the signaling surface of the receiver domain NtrC was shown to be mediated by a layer of hub residues. The location of hubs preferentially connected to the allosteric site was found in close agreement with key residues experimentally identified as involved in the signal transmission. The comparison with the networks of the homologues CheY and FixJ highlighted similarities in their dynamics. In particular, we showed that a preorganized network of fragment connections between the allosteric and functional sites exists already in the inactive state of all three proteins.—Pandini, A., Fornili, A., Fraternali, F., Kleinjung, J. Detection of allosteric signal transmission by information-theoretic analysis of protein dynamics. PMID:22071506

  13. Allosteric indicator displacement enzyme assay for a cyanogenic glycoside.

    Science.gov (United States)

    Jose, D Amilan; Elstner, Martin; Schiller, Alexander

    2013-10-18

    Indicator displacement assays (IDAs) represent an elegant approach in supramolecular analytical chemistry. Herein, we report a chemical biosensor for the selective detection of the cyanogenic glycoside amygdalin in aqueous solution. The hybrid sensor consists of the enzyme β-glucosidase and a boronic acid appended viologen together with a fluorescent reporter dye. β-Glucosidase degrades the cyanogenic glycoside amygdalin into hydrogen cyanide, glucose, and benzaldehyde. Only the released cyanide binds at the allosteric site of the receptor (boronic acid) thereby inducing changes in the affinity of a formerly bound fluorescent indicator dye at the other side of the receptor. Thus, the sensing probe performs as allosteric indicator displacement assay (AIDA) for cyanide in water. Interference studies with inorganic anions and glucose revealed that cyanide is solely responsible for the change in the fluorescent signal. DFT calculations on a model compound revealed a 1:1 binding ratio of the boronic acid and cyanide ion. The fluorescent enzyme assay for β-glucosidase uses amygdalin as natural substrate and allows measuring Michaelis-Menten kinetics in microtiter plates. The allosteric indicator displacement assay (AIDA) probe can also be used to detect cyanide traces in commercial amygdalin samples. PMID:24123550

  14. 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

  15. 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

  16. Modeling amperometric biosensors based on allosteric enzymes

    Directory of Open Access Journals (Sweden)

    Liutauras Ričkus

    2013-09-01

    Full Text Available Computational modeling of a biosensor with allosteric enzyme layer was investigated in this study. The operation of the biosensor is modeled using non-stationary reaction-diffusion equations. The model involves three regions: the allosteric enzyme layer where the allosteric enzyme reactions as well as then mass transport by diffusion take place, the diffusion region where the mass transport by diffusion and non-enzymatic reactions take place and the convective region in which the analyte concentration is maintained constant. The biosensor response on dependency substrate concentration, cooperativity coefficient and the diffusion layer thickness on the same parameters have been studied.

  17. Optimization of a Dibenzodiazepine Hit to a Potent and Selective Allosteric PAK1 Inhibitor.

    Science.gov (United States)

    Karpov, Alexei S; Amiri, Payman; Bellamacina, Cornelia; Bellance, Marie-Helene; Breitenstein, Werner; Daniel, Dylan; Denay, Regis; Fabbro, Doriano; Fernandez, Cesar; Galuba, Inga; Guerro-Lagasse, Stephanie; Gutmann, Sascha; Hinh, Linda; Jahnke, Wolfgang; Klopp, Julia; Lai, Albert; Lindvall, Mika K; Ma, Sylvia; Möbitz, Henrik; Pecchi, Sabina; Rummel, Gabriele; Shoemaker, Kevin; Trappe, Joerg; Voliva, Charles; Cowan-Jacob, Sandra W; Marzinzik, Andreas L

    2015-07-01

    The discovery of inhibitors targeting novel allosteric kinase sites is very challenging. Such compounds, however, once identified could offer exquisite levels of selectivity across the kinome. Herein we report our structure-based optimization strategy of a dibenzodiazepine hit 1, discovered in a fragment-based screen, yielding highly potent and selective inhibitors of PAK1 such as 2 and 3. Compound 2 was cocrystallized with PAK1 to confirm binding to an allosteric site and to reveal novel key interactions. Compound 3 modulated PAK1 at the cellular level and due to its selectivity enabled valuable research to interrogate biological functions of the PAK1 kinase. PMID:26191365

  18. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    Science.gov (United States)

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-04-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators.

  19. 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.

    2015-01-01

    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.

  20. 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

    2015-01-01

    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. PMID:25600932

  1. 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.

  2. Probing the Sophisticated Synergistic Allosteric Regulation of Aromatic Amino Acid Biosynthesis in Mycobacterium tuberculosis Using ᴅ-Amino Acids

    Science.gov (United States)

    Reichau, Sebastian; Blackmore, Nicola J.; Jiao, Wanting; Parker, Emily J.

    2016-01-01

    Chirality plays a major role in recognition and interaction of biologically important molecules. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of the shikimate pathway, which is responsible for the synthesis of aromatic amino acids in bacteria and plants, and a potential target for the development of antibiotics and herbicides. DAH7PS from Mycobacterium tuberculosis (MtuDAH7PS) displays an unprecedented complexity of allosteric regulation, with three interdependent allosteric binding sites and a ternary allosteric response to combinations of the aromatic amino acids l-Trp, l-Phe and l-Tyr. In order to further investigate the intricacies of this system and identify key residues in the allosteric network of MtuDAH7PS, we studied the interaction of MtuDAH7PS with aromatic amino acids that bear the non-natural d-configuration, and showed that the d-amino acids do not elicit an allosteric response. We investigated the binding mode of d-amino acids using X-ray crystallography, site directed mutagenesis and isothermal titration calorimetry. Key differences in the binding mode were identified: in the Phe site, a hydrogen bond between the amino group of the allosteric ligands to the side chain of Asn175 is not established due to the inverted configuration of the ligands. In the Trp site, d-Trp forms no interaction with the main chain carbonyl group of Thr240 and less favourable interactions with Asn237 when compared to the l-Trp binding mode. Investigation of the MtuDAH7PSN175A variant further supports the hypothesis that the lack of key interactions in the binding mode of the aromatic d-amino acids are responsible for the absence of an allosteric response, which gives further insight into which residues of MtuDAH7PS play a key role in the transduction of the allosteric signal. PMID:27128682

  3. Bioinformatic scaling of allosteric interactions in biomedical isozymes

    Science.gov (United States)

    Phillips, J. C.

    2016-09-01

    Allosteric (long-range) interactions can be surprisingly strong in proteins of biomedical interest. Here we use bioinformatic scaling to connect prior results on nonsteroidal anti-inflammatory drugs to promising new drugs that inhibit cancer cell metabolism. Many parallel features are apparent, which explain how even one amino acid mutation, remote from active sites, can alter medical results. The enzyme twins involved are cyclooxygenase (aspirin) and isocitrate dehydrogenase (IDH). The IDH results are accurate to 1% and are overdetermined by adjusting a single bioinformatic scaling parameter. It appears that the final stage in optimizing protein functionality may involve leveling of the hydrophobic limits of the arms of conformational hydrophilic hinges.

  4. 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

    The metabotropic glutamate receptors have a wide range of modulatory functions in the central nervous system. They are among the most highly pursued drug targets, with relevance for several neurological diseases, and a number of allosteric modulators have entered clinical trials. However, so far...... this has not led to a marketed drug, largely because of the difficulties in achieving subtype-selective compounds with desired properties. Very recently the first crystal structures were published for the transmembrane domain of two metabotropic glutamate receptors in complex with negative allosteric...... 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...

  5. Rational Engineering of Enzyme Allosteric Regulation through Sequence Evolution Analysis

    OpenAIRE

    Jae-Seong Yang; Sang Woo Seo; Sungho Jang; Gyoo Yeol Jung; Sanguk Kim

    2012-01-01

    Control of enzyme allosteric regulation is required to drive metabolic flux toward desired levels. Although the three-dimensional (3D) structures of many enzyme-ligand complexes are available, it is still difficult to rationally engineer an allosterically regulatable enzyme without decreasing its catalytic activity. Here, we describe an effective strategy to deregulate the allosteric inhibition of enzymes based on the molecular evolution and physicochemical characteristics of allosteric ligan...

  6. Structures of quinone binding sites in bc complexes: Functional implications

    International Nuclear Information System (INIS)

    Near-atomic resolution structures are becoming available for the respiratory chain enzyme known as ubiquinol:cytochrome c oxidoreductase or the cytochrome bc1 complex. Here we examine our current structure for the chicken bc1 complex to see what it can tell us about the mode of binding and mechanism of reaction of quinone at the two active sites

  7. Unusual developing sites of dengue vectors and potential epidemiological implications

    Institute of Scientific and Technical Information of China (English)

    Hamady Dieng; Ronald Enrique Morales; Rahman GM Saifur; Abu Hassan Ahmad; MR Che Salmah; Al Thbiani Aziz; Tomomitsu Satho; Fumio Miake; Zairi Jaal; Sazaly Abubakar

    2012-01-01

    Objective: To identify the unusual breeding sites of two dengue vectors, i.e. Aedes albopictus (Ae. albopictus) and Aedes aegypti (Ae. aegypti). Methods: During the second half of 2010, we performed an occasional survey in rural (Teluk Tempoyak) and urban (Gelugor) areas of Penang Island, Malaysia, to identify cryptic breeding sites. Results: In the rural area, we found heterogeneous immature stages of Ae. albopictus in the water bowl of an encaged bird. We also observed Ae. aegypti eggs deposited in the flush tank of a toilet in the urban area. Conclusions:It can be concluded that both breeding patterns can increase contact with hosts (humans and birds) and presumably population densities of Ae. albopictus and Ae. aegypti, thereby potentially boosting the risks for spread and transmission of arboviral diseases.

  8. 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

    The serotonin transporter (SERT) controls synaptic serotonin levels and is the primary target for antidepressants, including selective serotonin reuptake inhibitors (e.g. (S)-citalopram) and tricyclic antidepressants (e.g. clomipramine). In addition to a high affinity binding site, SERT possesses a...... 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...

  9. Orthosteric and Allosteric Regulation in Trypsin-Like Peptidases

    DEFF Research Database (Denmark)

    Kromann-Tofting, Tobias

    2015-01-01

    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 of...... peptidase and allosterically modulate the function of the active site, represents two important activity-regulating mechanisms in trypsin-like serine peptidases. Development of specific orthosteric agents as therapeutics is a challenge due to similar active site topology within the trypsin-like serine...... peptidase. The thesis describes how X-ray crystal structure analysis and biochemical analysis were used to demonstrate new concepts for orthosteric regulation of activity in the trypsin-like serine peptidase urokinase-type plasminogen activator (uPA), studying two types of orthosteric agents, namely cyclic...

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

    International Nuclear Information System (INIS)

    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

  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. 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. PMID:26678378

  14. DF2755A, a novel non-competitive allosteric inhibitor of CXCR1/2, reduces inflammatory and post-operative pain.

    Science.gov (United States)

    Lopes, Alexandre H; Brandolini, Laura; Aramini, Andrea; Bianchini, Gianluca; Silva, Rangel L; Zaperlon, Ana C; Verri, Waldiceu A; Alves-Filho, José C; Cunha, Fernando Q; Teixeira, Mauro M; Allegretti, Marcello; Cunha, Thiago M

    2016-01-01

    The activation of CXCR1/2 has been implicated in the genesis of inflammatory and postoperative pain. Here, we investigated a novel orally acting allosteric inhibitor of CXCR1/2 (DF2755A) and evaluated its antinociceptive effect in several models of inflammatory and post-operatory pain. DF2755A was tested in vitro for efficacy in the chemotaxis assay, selectivity and toxicity. In vivo, C57Bl/6 mice were treated orally with DF2755A and the following experiments were performed: pharmacokinetic profile; inflammatory hyperalgesia models using electronic pressure meter test; neutrophil migration assay assessed by myeloperoxidase assay. DF2755A selectively inhibited neutrophil chemotaxis induced by CXCR1/2 ligands without effect on CXCL8 binding to neutrophils. A single mutation of the allosteric site at CXCR1 abrogated the inhibitory effect of DF2755A on CXCL8-induced chemotaxis. DF2755A given orally was well absorbed (88.2%), and it was able to reduce, in a dose (3-30mg/kg)-dependent manner, inflammatory hyperalgesia induced by carrageenan, LPS and CXCL1/KC as well as neutrophil recruitment and IL-1β production. In addition, DF2755A was able to reduce post-incisional nociception. Therapeutic treatment with DF2755A reduced CFA-induced inflammatory hyperalgesia even when injected intrathecally. The present results indicate that DF2755A is a novel selective allosteric inhibitor of CXCR1/2 with a favorable oral pharmacokinetic profile. Furthermore, the results might suggest that DF2755A might be a candidate of a novel therapeutic option to control inflammatory and post-operative pain. PMID:26592483

  15. Elastic network model of allosteric regulation in protein kinase PDK1

    Directory of Open Access Journals (Sweden)

    Williams Gareth

    2010-05-01

    Full Text Available Abstract Background Structural switches upon binding of phosphorylated moieties underpin many signalling networks. The ligand activation is a form of allosteric modulation of the protein, where the binding site is remote from the structural change in the protein. Recently this structural switch has been elegantly demonstrated with the crystallisation of the activated form of 3-phosphoinositide-dependent protein kinase-1 (PDK1. The purpose of the present work is to determine whether the allosteric coupling in PDK1 emerges at the level of a simple coarse grained model of protein dynamics. Results It is shown here that the allosteric effects of the agonist binding to the small lobe upon the activation loop in the large lobe of PDK1 are explainable within a simple 'ball and spring' elastic network model (ENM of protein dynamics. In particular, the model shows that the bound phospho peptide mimetic fluctuations have a high degree of correlation with the activation loop of PDK1. Conclusions The ENM approach to small molecule activation of proteins may offer a first pass predictive methodology where affinity is encoded in residues remote from the active site, and aid in the design of specific protein agonists that enhance the allosteric coupling and antagonist that repress it.

  16. Rational design of allosteric regulation of homoserine dehydrogenase by a nonnatural inhibitor L-lysine.

    Science.gov (United States)

    Chen, Zhen; Rappert, Sugima; Zeng, An-Ping

    2015-02-20

    Allosteric proteins, which can sense different signals, are interesting biological parts for synthetic biology. In particular, the design of an artificial allosteric enzyme to sense an unnatural signal is both challenging and highly desired, for example, for a precise and dynamical control of fluxes of growth-essential but byproduct pathways in metabolic engineering of industrial microorganisms. In this work, we used homoserine dehydrogenase (HSDH) of Corynebacterium glutamicum, which is naturally allosterically regulated by threonine and isoleucine, as an example to demonstrate the feasibility of reengineering an allosteric enzyme to respond to an unnatural inhibitor L-lysine. For this purpose, the natural threonine binding sites of HSD were first predicted and verified by mutagenesis experiments. The threonine binding sites were then engineered to a lysine binding pocket. The reengineered HSD only responds to lysine inhibition but not to threonine. This is a significant step toward the construction of artificial molecular circuits for dynamic control of growth-essential byproduct formation pathway for lysine biosynthesis. PMID:24344690

  17. 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

  18. The allosteric switching mechanism in bacteriophage MS2

    Science.gov (United States)

    Perkett, Matthew R.; Mirijanian, Dina T.; Hagan, Michael F.

    2016-07-01

    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.

  19. Architecture and Co-Evolution of Allosteric Materials

    CERN Document Server

    Yan, Le; Brito, Carolina; Wyart, Matthieu

    2016-01-01

    We introduce a numerical scheme to evolve functional materials that can accomplish a specified mechanical task. In this scheme, the number of solutions, their spatial architectures and the correlations among them can be computed. As an example, we consider an "allosteric" task, which requires the material to respond specifically to a stimulus at a distant active site. We find that functioning materials evolve a less-constrained trumpet-shaped region connecting the stimulus and active sites and that the amplitude of the elastic response varies non-monotonically along the trumpet. As previously shown for some proteins, we find that correlations appearing during evolution alone are sufficient to identify key aspects of this design. Finally, we show that the success of this architecture stems from the emergence of soft edge modes recently found to appear near the surface of marginally connected materials. Overall, our in silico evolution experiment offers a new window to study the relationship between structure, ...

  20. 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…

  1. Mutations in Antibody Fragments Modulate Allosteric Response Via Hydrogen-Bond Network Fluctuations.

    Science.gov (United States)

    Srivastava, Amit; Tracka, Malgorzata B; Uddin, Shahid; Casas-Finet, Jose; Livesay, Dennis R; Jacobs, Donald J

    2016-05-10

    A mechanical perturbation method that locally restricts conformational entropy along the protein backbone is used to identify putative allosteric sites in a series of antibody fragments. The method is based on a distance constraint model that integrates mechanical and thermodynamic viewpoints of protein structure wherein mechanical clamps that mimic substrate or cosolute binding are introduced. Across a set of six single chain-Fv fragments of the anti-lymphotoxin-β receptor antibody, statistically significant responses are obtained by averaging over 10 representative structures sampled from a molecular dynamics simulation. As expected, the introduced clamps locally rigidify the protein, but long-ranged increases in both rigidity and flexibility are also frequently observed. Expanding our analysis to every molecular dynamics frame demonstrates that the allosteric responses are modulated by fluctuations within the hydrogen-bond network where the native ensemble is comprised of conformations that both are, and are not, affected by the perturbation in question. Population shifts induced by the mutations alter the allosteric response by adjusting which hydrogen-bond networks are the most probable. These effects are compared using response maps that track changes across each single chain-Fv fragment, thus providing valuable insight into how sensitive allosteric mechanisms are to mutations. PMID:27166802

  2. Coevolutionary analysis enabled rational deregulation of allosteric enzyme inhibition in Corynebacterium glutamicum for lysine production.

    Science.gov (United States)

    Chen, Zhen; Meyer, Weiqian; Rappert, Sugima; Sun, Jibin; Zeng, An-Ping

    2011-07-01

    Product feedback inhibition of allosteric enzymes is an essential issue for the development of highly efficient microbial strains for bioproduction. Here we used aspartokinase from Corynebacterium glutamicum (CgAK), a key enzyme controlling the biosynthesis of industrially important aspartate family amino acids, as a model to demonstrate a fast and efficient approach to the deregulation of allostery. In the last 50 years many researchers and companies have made considerable efforts to deregulate this enzyme from allosteric inhibition by lysine and threonine. However, only a limited number of positive mutants have been identified so far, almost exclusively by random mutation and selection. In this study, we used statistical coupling analysis of protein sequences, a method based on coevolutionary analysis, to systematically clarify the interaction network within the regulatory domain of CgAK that is essential for allosteric inhibition. A cluster of interconnected residues linking different inhibitors' binding sites as well as other regions of the protein have been identified, including most of the previously reported positions of successful mutations. Beyond these mutation positions, we have created another 14 mutants that can partially or completely desensitize CgAK from allosteric inhibition, as shown by enzyme activity assays. The introduction of only one of the inhibition-insensitive CgAK mutations (here Q298G) into a wild-type C. glutamicum strain by homologous recombination resulted in an accumulation of 58 g/liter L-lysine within 30 h of fed-batch fermentation in a bioreactor. PMID:21531824

  3. Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65.

    Science.gov (United States)

    Huang, Xi-Ping; Karpiak, Joel; Kroeze, Wesley K; Zhu, Hu; Chen, Xin; Moy, Sheryl S; Saddoris, Kara A; Nikolova, Viktoriya D; Farrell, Martilias S; Wang, Sheng; Mangano, Thomas J; Deshpande, Deepak A; Jiang, Alice; Penn, Raymond B; Jin, Jian; Koller, Beverly H; Kenakin, Terry; Shoichet, Brian K; Roth, Bryan L

    2015-11-26

    At least 120 non-olfactory G-protein-coupled receptors in the human genome are 'orphans' for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs. PMID:26550826

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. Coarse-Grained Molecular Simulations of Allosteric Cooperativity

    CERN Document Server

    Nandigrami, Prithviraj

    2015-01-01

    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 calcium 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 is treated implicitly at the mean field level. 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 energy to the open and closed ensembles accurately describe...

  7. 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)

    Equilibrium binding studies with the sigma receptor ligand [3H]1,3-di(2-tolyl)guanidine ([3H]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 [3H]DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of [3H]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 [3H]DTG from site 2, suggesting an association of this binding site with calcium channels

  8. Investigation of allosteric modulation mechanism of metabotropic glutamate receptor 1 by molecular dynamics simulations, free energy and weak interaction analysis

    Science.gov (United States)

    Bai, Qifeng; Yao, Xiaojun

    2016-02-01

    Metabotropic glutamate receptor 1 (mGlu1), which belongs to class C G protein-coupled receptors (GPCRs), can be coupled with G protein to transfer extracellular signal by dimerization and allosteric regulation. Unraveling the dimer packing and allosteric mechanism can be of great help for understanding specific regulatory mechanism and designing more potential negative allosteric modulator (NAM). Here, we report molecular dynamics simulation studies of the modulation mechanism of FITM on the wild type, T815M and Y805A mutants of mGlu1 through weak interaction analysis and free energy calculation. The weak interaction analysis demonstrates that van der Waals (vdW) and hydrogen bonding play an important role on the dimer packing between six cholesterol molecules and mGlu1 as well as the interaction between allosteric sites T815, Y805 and FITM in wild type, T815M and Y805A mutants of mGlu1. Besides, the results of free energy calculations indicate that secondary binding pocket is mainly formed by the residues Thr748, Cys746, Lys811 and Ser735 except for FITM-bound pocket in crystal structure. Our results can not only reveal the dimer packing and allosteric regulation mechanism, but also can supply useful information for the design of potential NAM of mGlu1.

  9. Oviposition Site Selection in the Malayan Giant Frog (Limnonectes blythii) in Singapore:Conservation Implications

    Institute of Scientific and Technical Information of China (English)

    Vishnu Vardhan SRIDHAR; David BICKFORD

    2015-01-01

    Amphibians require speciifc habitats for breeding and loss or degradation of such habitats can negatively affect reproductive success. Oviposition site selection within a habitat is also important as site quality is linked to larval survivorship and metamorphic success. We investigated oviposition site preferences of the stream-breeding frog Limnonectes blythii in Singapore through surveys and habitat measurements of breeding and non-breeding sites (N =30 and 32, respectively). The study species L. blythii is classiifed as Near Threatened (NT) in the IUCN red list and is associated with medium sized forest streams. L. blythii appeared to prefer streams with higher water pH and shallower water depths for oviposition. Our ifndings have implications in conservation management as it provides the baseline for habitat restoration for creation of new and for preserving existing breeding habitat of L. blythii.

  10. Molecular Mechanism of Allosteric Communication in Hsp70 Revealed by Molecular Dynamics Simulations

    OpenAIRE

    Chiappori, Federica; Merelli, Ivan; Colombo, Giorgio; Milanesi, Luciano; Morra, Giulia

    2012-01-01

    Author Summary Allostery, or the capability of proteins to respond to ligand binding events with a variation in structure or dynamics at a distant site, is a common feature for biomolecular function and regulation in a large number of proteins. Intra-protein connections and inter-residue coordinations underlie allosteric mechanisms and react to binding primarily through a finely tuned modulation of motions and structures at the microscopic scale. Hence, all-atom molecular dynamics simulations...

  11. Finger printing of mixed contaminants from former manufactured gas plant (MGP) site soils: Implications to bioremediation.

    Science.gov (United States)

    Thavamani, Palanisami; Megharaj, Mallavarapu; Krishnamurti, G S R; McFarland, Ross; Naidu, Ravi

    2011-01-01

    Contaminants in general do not occur as single chemicals but as mixtures at any contaminated site. Gasworks sites are the typical mixed contaminated sites. These sites are not only subjected to PAH contamination but also varying degrees of heavy metal contamination. Bioremediation in these sites is often hindered by the presence of heavy metals. The co-occurrence of PAHs with heavy metals has not been systematically investigated. Metals are reported to inhibit the general soil microbiological processes. The total concentration of soluble metal in the system includes both free metal ion and complexed forms. Within bioavailable fraction, the most toxic form is the free metal species, which was not addressed well so far in gas works site characterisation. This study underpins the science and importance of metal bioavailability and speciation based site characterisation in mixed contaminated sites. In this study a detailed elemental chemistry of the gas works site soils are discussed using different methods. The PAH contamination was contributed by both low and high molecular weight PAHs. The total PAHs concentration ranged from 335 to 8645 mg/kg. Among most toxic metals Pb was found in high concentration ranging from 88 to 671 mg/kg, Cd 8 to 112 mg/kg and Zn varied from 64 to 488 mg/kg. Thermodynamic chemical equilibrium model VMINTEQ (Ver 2.52) was used to calculate the free metal species in gas works site soils. The percentage free metal species showed a different trend compared to total metal concentrations, free Zn species ranged 18-86%, free Cd was 26-87% and Pb showed lowest free metal percentage (0-17%). The bioavailable metal species and its implications to bioremediation have also been discussed. PMID:20875686

  12. In silico-screening approaches for lead generation: identification of novel allosteric modulators of human-erythrocyte pyruvate kinase.

    Science.gov (United States)

    Tripathi, Ashutosh; Safo, Martin K

    2012-01-01

    Identification of allosteric binding site modulators have gained increased attention lately for their potential to be developed as selective agents with a novel chemotype and targeting perhaps a new and unique binding site with probable fewer side effects. Erythrocyte pyruvate kinase (R-PK) is an important glycolytic enzyme that can be pharmacologically modulated through its allosteric effectors for the treatment of hemolytic anemia, sickle-cell anemia, hypoxia-related diseases, and other disorders arising from erythrocyte PK malfunction. An in-silico screening approach was applied to identify novel allosteric modulators of pyruvate kinase. A small-molecules database of the National Cancer Institute (NCI), was virtually screened based on structure/ligand-based pharmacophore. The virtual screening campaign led to the identification of several compounds with similar pharmacophoric features as fructose-1,6-bisphosphate (FBP), the natural allosteric activator of the kinase. The compounds were subsequently docked into the FBP-binding site using the programs FlexX and GOLD, and their interactions with the protein were analyzed with the energy-scoring function of HINT. Seven promising candidates were obtained from the NCI and subjected to kinetics analysis, which revealed both activators and inhibitors of the R-isozyme of PK (R-PK). PMID:22052500

  13. Allosteric small-molecule kinase inhibitors

    DEFF Research Database (Denmark)

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

    2015-01-01

    Small-molecule kinase inhibitors are invaluable targeted therapeutics for the treatment of various human diseases, especially cancers. While the majority of approved and developed preclinical small-molecule inhibitors are characterized as type I or type II inhibitors that target the ATP......-binding pocket of kinases, the remarkable sequential and structural similarity among ATP pockets renders the selective inhibition of kinases a daunting challenge. Therefore, targeting allosteric pockets of kinases outside the highly conversed ATP pocket has been proposed as a promising alternative to overcome...

  14. Peptide- and proton-driven allosteric clamps catalyze anthrax toxin translocation across membranes.

    Science.gov (United States)

    Das, Debasis; Krantz, Bryan A

    2016-08-23

    Anthrax toxin is an intracellularly acting toxin in which sufficient information is available regarding the structure of its transmembrane channel, allowing for detailed investigation of models of translocation. Anthrax toxin, comprising three proteins-protective antigen (PA), lethal factor (LF), and edema factor-translocates large proteins across membranes. Here we show that the PA translocase channel has a transport function in which its catalytic active sites operate allosterically. We find that the phenylalanine clamp (ϕ-clamp), the known conductance bottleneck in the PA translocase, gates as either a more closed state or a more dilated state. Thermodynamically, the two channel states have >300-fold different binding affinities for an LF-derived peptide. The change in clamp thermodynamics requires distant α-clamp and ϕ-clamp sites. Clamp allostery and translocation are more optimal for LF peptides with uniform stereochemistry, where the least allosteric and least efficiently translocated peptide had a mixed stereochemistry. Overall, the kinetic results are in less agreement with an extended-chain Brownian ratchet model but, instead, are more consistent with an allosteric helix-compression model that is dependent also on substrate peptide coil-to-helix/helix-to-coil cooperativity. PMID:27506790

  15. Coupled Dynamics and Entropic Contribution to the Allosteric Mechanism of Pin1.

    Science.gov (United States)

    Barman, Arghya; Hamelberg, Donald

    2016-08-25

    Allosteric communication in proteins regulates a plethora of downstream processes in subcellular signaling pathways. Describing the effects of cooperative ligand binding on the atomic level is a key to understanding many regulatory processes involving biomolecules. Here, we use microsecond-long molecular dynamics simulations to investigate the allosteric mechanism of Pin1, a potential therapeutic target and a phosphorylated-Ser/Thr dependent peptidyl-prolyl cis-trans isomerase that regulates several subcellular processes and has been implicated in many diseases, including cancer and Alzheimer's. Experimental studies suggest that the catalytic domain and the noncatalytic WW domain are allosterically coupled; however, an atomic level description of the dynamics associated with the interdomain communication is lacking. We show that binding of the substrate to the WW domain is directly coupled to the dynamics of the catalytic domain, causing rearrangement of the residue-residue contact dynamics from the WW domain to the catalytic domain. The binding affinity of the substrate in the catalytic domain is also enhanced upon binding of the substrate to the WW domain. Modulation of the dynamics of the catalytic domain upon binding of the substrate to the WW domain leads to prepayment of the entropic cost of binding the substrate to the catalytic domain. This study shows that Ile 28 at the interfacial region between the catalytic and WW domains is certainly one of the residues responsible for bridging the communication between the two domains. The results complement previous experiments and provide valuable atomistic insights into the role of dynamics and possible entropic contribution to the allosteric mechanism of proteins. PMID:27077947

  16. The N-Terminal Domain of the E. coli PriA Helicase Contains Both the DNA- and the Nucleotide-Binding Sites. Energetics of Domain-DNA Interactions and Allosteric Effect of the Nucleotide Cofactors§

    OpenAIRE

    Szymanski, Michal R.; Bujalowski, Paul J.; Jezewska, Maria J.; Gmyrek, Aleksandra M.; Bujalowski, Wlodzimierz

    2011-01-01

    Functional interactions of the E. coli PriA helicase 181N-terminal domain with the DNA and nucleotide cofactors have been quantitatively examined. The isolated 181N-terminal domain forms a stable dimer in solution, most probably reflecting the involvement of the domain in specific cooperative interactions of the intact PriA protein - dsDNA complex. Only one monomer of the domain dimer binds the DNA, i.e., the dimer has one effective DNA-binding site. Although the total site-size of the dimer ...

  17. Synthesis of a Series of Novel 3,9-Disubstituted Phenanthrenes as Analogues of Known NMDA Receptor Allosteric Modulators

    OpenAIRE

    Irvine, Mark W.; Fang, Guangyu; Eaves, Richard; Mayo-Martin, Maria B.; Burnell, Erica S.; Costa, Blaise M.; Culley, Georgia R.; Volianskis, Arturas; Collingridge, Graham L; Monaghan, Daniel T.; Jane, David E.

    2015-01-01

    9-Substituted phenanthrene-3-carboxylic acids have been reported to have allosteric modulatory activity at the NMDA receptor. This receptor is activated by the excitatory neurotransmitter L-glutamate and has been implicated in a range of neurological disorders such as schizophrenia, epilepsy and chronic pain and neurodegenerative disorders such as Alzheimer’s disease. Herein, the convenient synthesis of a wide range of novel 3,9-disubstituted phenanthrene derivatives starting from a few commo...

  18. Structural basis for drug-induced allosteric changes to human β-cardiac myosin motor activity

    Science.gov (United States)

    Winkelmann, Donald A.; Forgacs, Eva; Miller, Matthew T.; Stock, Ann M.

    2015-08-01

    Omecamtiv Mecarbil (OM) is a small molecule allosteric effector of cardiac myosin that is in clinical trials for treatment of systolic heart failure. A detailed kinetic analysis of cardiac myosin has shown that the drug accelerates phosphate release by shifting the equilibrium of the hydrolysis step towards products, leading to a faster transition from weak to strong actin-bound states. The structure of the human β-cardiac motor domain (cMD) with OM bound reveals a single OM-binding site nestled in a narrow cleft separating two domains of the human cMD where it interacts with the key residues that couple lever arm movement to the nucleotide state. In addition, OM induces allosteric changes in three strands of the β-sheet that provides the communication link between the actin-binding interface and the nucleotide pocket. The OM-binding interactions and allosteric changes form the structural basis for the kinetic and mechanical tuning of cardiac myosin.

  19. An allosteric modulator of HIV-1 protease shows equipotent inhibition of wild-type and drug-resistant proteases.

    Science.gov (United States)

    Ung, Peter M-U; Dunbar, James B; Gestwicki, Jason E; Carlson, Heather A

    2014-08-14

    NMR and MD simulations have demonstrated that the flaps of HIV-1 protease (HIV-1p) adopt a range of conformations that are coupled with its enzymatic activity. Previously, a model was created for an allosteric site located between the flap and the core of HIV-1p, called the Eye site (Biopolymers 2008, 89, 643-652). Here, results from our first study were combined with a ligand-based, lead-hopping method to identify a novel compound (NIT). NIT inhibits HIV-1p, independent of the presence of an active-site inhibitor such as pepstatin A. Assays showed that NIT acts on an allosteric site other than the dimerization interface. MD simulations of the ligand-protein complex show that NIT stably binds in the Eye site and restricts the flaps. That bound state of NIT is consistent with a crystal structure of similar fragments bound in the Eye site (Chem. Biol. Drug Des. 2010, 75, 257-268). Most importantly, NIT is equally potent against wild-type and a multidrug-resistant mutant of HIV-1p, which highlights the promise of allosteric inhibitors circumventing existing clinical resistance. PMID:25062388

  20. Allosteric Regulation by a Critical Membrane

    CERN Document Server

    Kimchi, Ofer; Machta, Benjamin B

    2016-01-01

    Many of the processes that underly neural computation are carried out by ion channels embedded in the plasma membrane, a two-dimensional liquid that surrounds all cells. Recent experiments have demonstrated that this membrane is poised close to a liquid-liquid critical point in the Ising universality class. Here we use both exact and stochastic techniques on the lattice Ising model to explore the ramifications of proximity to criticality for proteins that are allosterically coupled to Ising composition modes. Owing to diverging generalized susceptibilities, such a protein's activity becomes strongly influenced by perturbations that influence the two relevant parameters of the critical point, especially the critical temperature. In addition, the protein's kinetics acquire a range of time scales from its surrounding membrane, naturally leading to non-Markovian dynamics.

  1. 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

  2. A unified framework and an alternative mechanism for allosteric regulation

    CERN Document Server

    Xing, J

    2007-01-01

    Allosteric regulation is an important property for many proteins. Several models have been proposed to explain the allosteric effect, such as the concerted MWC (Monod, Wyman, Changeux) model, the sequential KNF (Koshland, Nemethy, Filmer) model, and recent population shift models. Here we discuss a unified theoretical framework to describe allosteric effects. The existing models appear as special cases of the framework. The theoretical work also reveals an alternative mechanism currently overlooked. Theoretically it is possible that the reactivity of a protein is limited by some internal conformational change step (due to slow effective diffusion along rugged potential surfaces). Effector binding may modify the ruggedness and thus the protein dynamics and reactivity. Compared to conventional models, the new mechanism has less requirements on the mechanical properties of an allosteric protein to propagate mechanical signals over long distances. Thus some signal transduction proteins may adopt the new mechanism...

  3. 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.

  4. 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.

  5. Pathways of allosteric regulation in Hsp70 chaperones

    OpenAIRE

    Kityk, Roman; Vogel, Markus; Schlecht, Rainer; Bukau, Bernd; Mayer, Matthias P

    2015-01-01

    Central to the protein folding activity of Hsp70 chaperones is their ability to interact with protein substrates in an ATP-controlled manner, which relies on allosteric regulation between their nucleotide-binding (NBD) and substrate-binding domains (SBD). Here we dissect this mechanism by analysing mutant variants of the Escherichia coli Hsp70 DnaK blocked at distinct steps of allosteric communication. We show that the SBD inhibits ATPase activity by interacting with the NBD through a highly ...

  6. Positive allosteric action of eburnamonine on cardiac muscarinic acetylcholine receptors.

    Science.gov (United States)

    Proska, J; Tucek, S

    1996-06-01

    It was discovered recently that alcuronium and strychnine (which is a precursor of alcuronium) allosterically increase the affinity of cardiac muscarinic receptors for the antagonist, N-methylscopolamine. We have now investigated the effects of l-eburnamonine and vincamine, which are both closely related to strychnine. In experiments on rat heart atria, l-eburnamonine was found to increase the binding of [3H]N-methylscopolamine with Ehlert's cooperativity coefficient alpha = 0.35, which indicates that the strength of its allosteric action is close to that of alcuronium and strychnine (alpha = 0.31 and 0.44, respectively). However, the affinity of l-eburnamonine for the cardiac muscarinic receptors is lower than the affinities of alcuronium and strychnine (KAR = 22.6 microM, 0.15 microM, and 3.4 microM, respectively). In spite of its extremely close similarity to l-eburnamonine, vincamine has a negative allosteric effect on the binding of [3H]N-methylscopolamine (alpha = 4.1; KAR = 22.8 microM). It is likely that a systematic investigation of the allosteric effects of the analogues of strychnine will not only yield new allosteric effectors on muscarinic receptors, but also clarify the structural features responsible for the direction (positive or negative) of their allosteric effect. PMID:8813554

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

    International Nuclear Information System (INIS)

    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 (D0) 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

  8. Computational Investigation on the Allosteric Modulation of Androgen Receptor

    Institute of Scientific and Technical Information of China (English)

    OU Min-Rui; LI Jun-Qian

    2012-01-01

    Androgens have similar structures with different biological activities. To identify molecular determinants responsible for the activity difference, we have docked six steroidal androgens to the binding site or the surface of androgen receptor by using molecular docking with computational investigation. The energy was calculated respectively based on the QM (quantum mechanics) and MM (molecular mechanics) methods. The result shows that the allosteric modulation of androgen receptor plays an important role in the binding process between androgens and receptor. The open state receptor is less stable than the close state one, but the latter is more favorable for binding with androgens. It is worthy of note that when the androgen receptors binding or without binding with androgen are in close state, they are difficult to return to their open state. This phenomenon is an exception of the well known two-state model theory in which the two states are reversible. Whether the internal of close state androgen receptor has a combination of androgen or not, the androgen receptor surface can be combined with another androgen, and their surface binding energies could be very close. The result is consistent with the experimental observations, but this phenomenon of continuous combination from open state is also an exception of the two-state model theory.

  9. Allosteric modulation of the effect of escitalopram, paroxetine and fluoxetine: in-vitro and in-vivo studies

    DEFF Research Database (Denmark)

    Mansari, Mostafa El; Wiborg, Ove; Mnie-Filali, Ouissame;

    2006-01-01

    escitalopram. This effect was suggested to occur via an allosteric modulation at the level of the 5-HT transporter. Using in-vitro binding assays at membranes from COS-1 cells expressing the human 5-HT transporter (hSERT) and in-vivo electrophysiological and microdialysis techniques in rats, the present study...... was directed at determining whether R-citalopram modifies the action of selective serotonin reuptake inhibitors (SSRIs) known to act on allosteric sites namely escitalopram, and to a lesser extent paroxetine, compared to fluoxetine, which has no affinity for these sites. In-vitro binding studies...... showed that R-citalopram attenuated the association rates of escitalopram and paroxetine to the 5-HT transporter, but had no effect on the association rates of fluoxetine, venlafaxine or sertraline. In the rat dorsal raphe nucleus, R-citalopram (250 microg/kg i.v.) blocked the suppressant effect on...

  10. Identification of an allosteric pocket on human hsp70 reveals a mode of inhibition of this therapeutically important protein.

    Science.gov (United States)

    Rodina, Anna; Patel, Pallav D; Kang, Yanlong; Patel, Yogita; Baaklini, Imad; Wong, Michael J H; Taldone, Tony; Yan, Pengrong; Yang, Chenghua; Maharaj, Ronnie; Gozman, Alexander; Patel, Maulik R; Patel, Hardik J; Chirico, William; Erdjument-Bromage, Hediye; Talele, Tanaji T; Young, Jason C; Chiosis, Gabriela

    2013-12-19

    Hsp70s are important cancer chaperones that act upstream of Hsp90 and exhibit independent anti-apoptotic activities. To develop chemical tools for the study of human Hsp70, we developed a homology model that unveils a previously unknown allosteric site located in the nucleotide binding domain of Hsp70. Combining structure-based design and phenotypic testing, we discovered a previously unknown inhibitor of this site, YK5. In cancer cells, this compound is a potent and selective binder of the cytosolic but not the organellar human Hsp70s and has biological activity partly by interfering with the formation of active oncogenic Hsp70/Hsp90/client protein complexes. YK5 is a small molecule inhibitor rationally designed to interact with an allosteric pocket of Hsp70 and represents a previously unknown chemical tool to investigate cellular mechanisms associated with Hsp70. PMID:24239008

  11. Molecular Basis of Enhanced Activity in Factor VIIa-Trypsin Variants Conveys Insights into Tissue Factor-mediated Allosteric Regulation of Factor VIIa Activity

    DEFF Research Database (Denmark)

    Sorensen, Anders B.; Madsen, Jesper Jonasson; Svensson, L. Anders; Pedersen, Anette A.; Østergaard, Henrik; Overgaard, Michael T.; Olsen, Ole H.; Gandhi, Prafull S

    2016-01-01

    The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic...... properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x......-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215-217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215-217 conformation is...

  12. Selective binding modes and allosteric inhibitory effects of lupane triterpenes on protein tyrosine phosphatase 1B.

    Science.gov (United States)

    Jin, Tiantian; Yu, Haibo; Huang, Xu-Feng

    2016-01-01

    Protein Tyrosine Phosphatase 1B (PTP1B) has been recognized as a promising therapeutic target for treating obesity, diabetes, and certain cancers for over a decade. Previous drug design has focused on inhibitors targeting the active site of PTP1B. However, this has not been successful because the active site is positively charged and conserved among the protein tyrosine phosphatases. Therefore, it is important to develop PTP1B inhibitors with alternative inhibitory strategies. Using computational studies including molecular docking, molecular dynamics simulations, and binding free energy calculations, we found that lupane triterpenes selectively inhibited PTP1B by targeting its more hydrophobic and less conserved allosteric site. These findings were verified using two enzymatic assays. Furthermore, the cell culture studies showed that lupeol and betulinic acid inhibited the PTP1B activity stimulated by TNFα in neurons. Our study indicates that lupane triterpenes are selective PTP1B allosteric inhibitors with significant potential for treating those diseases with elevated PTP1B activity. PMID:26865097

  13. The geoarchaeology of the Sawame site of the Shibushi coastal dune, Kyushu Island, Japan, and implications for Japanese history

    International Nuclear Information System (INIS)

    The Sawame site, a sand-buried Yoyoi village (c. 100 BC-300 AD), is located in the Shibushi coastal dune, southwest Japan. This paper examines, using tephrochronology, the palaeogeography of the Shibushi coastal dune sequence in the Middle to Late Holocene. The excavations at Sawame are introduced, and the location/abandonment factors of this site are considered. From the significance of the Sawame site the implications for Japanese history are discussed, mainly in terms of maritime transfer. (author). 20 refs., 4 figs., 1 tab

  14. 137Cs fallout depth distributions in forest versus field sites: implications for external gamma dose rates

    International Nuclear Information System (INIS)

    The depth profile of 137Cs fallout in soil from atmospheric nuclear weapons tests was measured at neighboring field and forest areas for seven sites in northeastern US. The inferred dose rates in air at 1 m above the ground per unit inventory of 137Cs averaged a factor of 1.8 higher in forest as compared to field areas. Calculations indicate that dose rate in forest areas would be a factor of four higher than that over deeply ploughed land. Based on a limited set of historical measurements made since 1972, it appears that the dose rate per unit inventory in both field and forest areas has more or less stabilized after a sharp decrease following deposition events in the early 1960s. Estimated dose commitments for various land types are compared to the value suggested by UNSCEAR and implications with respect to certain population groups are discussed. The findings may have application in estimating future external doses from deposited 137Cs associated with Chernobyl fallout in Europe. (author)

  15. Study on the Model for Regulation of the Allosteric Enzyme Activity

    Institute of Scientific and Technical Information of China (English)

    LI,Qian-Zhong(李前忠); LUO,Liao-Fu(罗辽复); ZHANG,Li-Rong(张利绒)

    2002-01-01

    The effects of activator molecule and repressive molecule on binding process between allosteric enzyme and substrate are disused by considering the heterotropic effect of the regulating molecule that binds to allosteric enzyme. A model of allosteric enzyme with heterotropic effect is presented. The cooperativity and anticooperativity in the regulation process are studied.

  16. Pathways of allosteric regulation in Hsp70 chaperones.

    Science.gov (United States)

    Kityk, Roman; Vogel, Markus; Schlecht, Rainer; Bukau, Bernd; Mayer, Matthias P

    2015-01-01

    Central to the protein folding activity of Hsp70 chaperones is their ability to interact with protein substrates in an ATP-controlled manner, which relies on allosteric regulation between their nucleotide-binding (NBD) and substrate-binding domains (SBD). Here we dissect this mechanism by analysing mutant variants of the Escherichia coli Hsp70 DnaK blocked at distinct steps of allosteric communication. We show that the SBD inhibits ATPase activity by interacting with the NBD through a highly conserved hydrogen bond network, and define the signal transduction pathway that allows bound substrates to trigger ATP hydrolysis. We identify variants deficient in only one direction of allosteric control and demonstrate that ATP-induced substrate release is more important for chaperone activity than substrate-stimulated ATP hydrolysis. These findings provide evidence of an unexpected dichotomic allostery mechanism in Hsp70 chaperones and provide the basis for a comprehensive mechanical model of allostery in Hsp70s. PMID:26383706

  17. Fructose-1,6-Bisphosphate Is an Allosteric Activator of Pyrophosphate:Fructose-6-Phosphate 1-Phosphotransferase.

    Science.gov (United States)

    Nielsen, T. H.

    1995-05-01

    The activity of highly purified pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) from barley (Hordeum vulgare) leaves was studied under conditions where the catalyzed reaction was allowed to approach equilibrium. The activity of PFP was monitored by determining the changes in the levels of fructose-6-phosphate, orthophosphate, and fructose-1,6-bisphosphate (Fru-1,6-bisP). Under these conditions PFP activity was not dependent on activation by fructose-2,6-bisphosphate (Fru-2,6-bisP). Inclusion of aldolase in the reaction mixture temporarily restored the dependence of PFP on Fru-2,6-bisP. Alternatively, PFP was activated by Fru-1,6-bisP in the presence of aldolase. It is concluded that Fru-1,6-bisP is an allosteric activator of barley PFP, which can substitute for Fru-2,6-bisP as an activator. A significant activation was observed at a concentration of 5 to 25 [mu]M Fru-1,6-bisP, which demonstrates that the allosteric site of barley PFP has a very high affinity for Fru-1,6-bisP. The high affinity for Fru-1,6-bisP at the allosteric site suggests that the observed activation of PFP by Fru-1,6-bisP constitutes a previously unrecognized in vivo regulation mechanism. PMID:12228454

  18. A negative allosteric modulator modulates GABAB-receptor signalling through GB2 subunits.

    Science.gov (United States)

    Sun, Bing; Chen, Linhai; Liu, Lei; Xia, Zhixiong; Pin, Jean-Philippe; Nan, Fajun; Liu, Jianfeng

    2016-03-15

    An γ-aminobutyric acid type B (GABAB)-receptor mediates slow and prolonged synaptic inhibition in the central nervous system, which represents an interesting target for the treatment of various diseases and disorders of the central nervous system. To date, only one activator of the GABAB-receptor, baclofen, is on the market for the treatment of spasticity. Inhibitors of the GABAB-receptor, such as antagonists, show anti-absence seizure activity and pro-cognitive properties. In a search for allosteric compounds of the GABAB-receptor, although several positive allosteric modulators have been developed, it is only recently that the first negative allosteric modulator (NAM), CLH304a (also named Compound 14), has been reported. In the present study, we provide further information on the mechanism of action of CLH304a, and also show the possibility of designing more NAMs, such as CLH391 and CLH393, based on the structure of CLH304a. First we show that CLH304a inhibits native GABAB-receptor activity in cultured cerebellar granular neurons. We then show that CLH304a has inverse agonist properties and non-competitively inhibits the effect of agonists, indicating that it binds at a different site to GABA. The GABAB-receptor is a mandatory heterodimer made of GB1 subunits, in which agonists bind, and GB2 subunits, which activate G-proteins. By using various combinations made up of wild-type and/or mutated GB1 and GB2 subunits, we show that CLH304a acts on the heptahelical domain of GB2 subunits. These data revealed the possibility of designing innovative NAMs acting in the heptahelical domain of the GB2 subunits, offering novel possibilities for therapeutic intervention based on GABAB-receptor inhibition. PMID:26772870

  19. 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.

  20. SEQUENTIAL INVESTMENT IN SITE-SPECIFIC CROP MANAGEMENT UNDER OUTPUT PRICE UNCERTAINTY: IMPLICATIONS FOR NITROGEN POLLUTION CONTROL

    OpenAIRE

    Isik, Murat; Khanna, Madhu; Winter-Nelson, Alex

    2000-01-01

    This paper develops an option value model to examine the extent to which output price uncertainty creates incentives to adopt two interrelated components of site-specific technologies sequentially. It analyzes how the impact of uncertainty on the sequential adoption decision differs across heterogeneous soil conditions, and examines the implications of adoption for nitrogen pollution generation and for the design of a cost-share subsidy policy.

  1. Novel positive allosteric modulators of the human α7 nicotinic acetylcholine receptor.

    Science.gov (United States)

    Arias, Hugo R; Gu, Ruo-Xu; Feuerbach, Dominik; Guo, Bao-Bao; Ye, Yong; Wei, Dong-Qing

    2011-06-14

    The pharmacological activity of a series of novel amide derivatives was characterized on several nicotinic acetylcholine receptors (AChRs). Ca(2+) influx results indicate that these compounds are not agonists of the human (h) α4β2, α3β4, α7, and α1β1γδ AChRs; compounds 2-4 are specific positive allosteric modulators (PAMs) of hα7 AChRs, whereas compounds 1-4, 7, and 12 are noncompetitive antagonists of the other AChRs. Radioligand binding results indicate that PAMs do not inhibit binding of [(3)H]methyllycaconitine but enhance binding of [(3)H]epibatidine to hα7 AChRs, indicating that these compounds do not directly, but allosterically, interact with the hα7 agonist sites. Additional competition binding results indicate that the antagonistic action mediated by these compounds is produced by direct interaction with neither the phencyclidine site in the Torpedo AChR ion channel nor the imipramine and the agonist sites in the hα4β2 and hα3β4 AChRs. Molecular dynamics and docking results suggest that the binding site for compounds 2-4 is mainly located in the inner β-sheet of the hα7-α7 interface, ∼12 Å from the agonist locus. Hydrogen bond interactions between the amide group of the PAMs and the hα7 AChR binding site are found to be critical for their activity. The dual PAM and antagonistic activities elicited by compounds 2-4 might be therapeutically important. PMID:21510634

  2. Metal ion coupled protein folding and allosteric motions

    Science.gov (United States)

    Wang, Wei

    2014-03-01

    Many proteins need the help of cofactors for their successful folding and functioning. Metal ions, i.e., Zn2+, Ca2+, and Mg2+ etc., are typical biological cofactors. Binding of metal ions can reshape the energy landscapes of proteins, thereby modifying the folding and allosteric motions. For example, such binding may make the intrinsically disordered proteins have funneled energy landscapes, consequently, ensures their spontaneous folding. In addition, the binding may activate certain biological processes by inducing related conformational changes of regulation proteins. However, how the local interactions involving the metal ion binding can induce the global conformational motions of proteins remains elusive. Investigating such question requires multiple models with different details, including quantum mechanics, atomistic models, and coarse grained models. In our recent work, we have been developing such multiscale methods which can reasonably model the metal ion binding induced charge transfer, protonation/deprotonation, and large conformational motions of proteins. With such multiscale model, we elucidated the zinc-binding induced folding mechanism of classical zinc finger and the calcium-binding induced dynamic symmetry breaking in the allosteric motions of calmodulin. In addition, we studied the coupling of folding, calcium binding and allosteric motions of calmodulin domains. In this talk, I will introduce the above progresses on the metal ion coupled protein folding and allosteric motions. We thank the finacial support from NSFC and the 973 project.

  3. Biomimetic Design Results in a Potent Allosteric Inhibitor of Dihydrodipicolinate Synthase from Campylobacter jejuni.

    Science.gov (United States)

    Skovpen, Yulia V; Conly, Cuylar J T; Sanders, David A R; Palmer, David R J

    2016-02-17

    Dihydrodipicolinate synthase (DHDPS), an enzyme required for bacterial peptidoglycan biosynthesis, catalyzes the condensation of pyruvate and β-aspartate semialdehyde (ASA) to form a cyclic product which dehydrates to form dihydrodipicolinate. DHDPS has, for several years, been considered a putative target for novel antibiotics. We have designed the first potent inhibitor of this enzyme by mimicking its natural allosteric regulation by lysine, and obtained a crystal structure of the protein-inhibitor complex at 2.2 Å resolution. This novel inhibitor, which we named "bislysine", resembles two lysine molecules linked by an ethylene bridge between the α-carbon atoms. Bislysine is a mixed partial inhibitor with respect to the first substrate, pyruvate, and a noncompetitive partial inhibitor with respect to ASA, and binds to all forms of the enzyme with a Ki near 200 nM, more than 300 times more tightly than lysine. Hill plots show that the inhibition is cooperative, indicating that the allosteric sites are not independent despite being located on opposite sides of the protein tetramer, separated by approximately 50 Å. A mutant enzyme resistant to lysine inhibition, Y110F, is strongly inhibited by this novel inhibitor, suggesting this may be a promising strategy for antibiotic development. PMID:26836694

  4. Mechanisms of allosteric gene regulation by NMR quantification of microsecond-millisecond protein dynamics.

    Science.gov (United States)

    Kleckner, Ian R; Gollnick, Paul; Foster, Mark P

    2012-01-13

    The trp RNA-binding attenuation protein (TRAP) is a paradigmatic allosteric protein that regulates the tryptophan biosynthetic genes associated with the trp operon in bacilli. The ring-shaped 11-mer TRAP is activated for recognition of a specific trp-mRNA target by binding up to 11 tryptophan molecules. To characterize the mechanisms of tryptophan-induced TRAP activation, we have performed methyl relaxation dispersion (MRD) nuclear magnetic resonance (NMR) experiments that probe the time-dependent structure of TRAP in the microsecond-to-millisecond "chemical exchange" time window. We find significant side chain flexibility localized to the RNA and tryptophan binding sites of the apo protein and that these dynamics are dramatically reduced upon ligand binding. Analysis of the MRD NMR data provides insights into the structural nature of transiently populated conformations sampled in solution by apo TRAP. The MRD data are inconsistent with global two-state exchange, indicating that conformational sampling in apo TRAP is asynchronous. These findings imply a temporally heterogeneous population of structures that are incompatible with RNA binding and substantiate the study of TRAP as a paradigm for probing and understanding essential dynamics in allosteric, regulatory proteins. PMID:22115774

  5. 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

    2016-05-01

    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. PMID:27041592

  6. The Structural Basis for Allosteric Inhibition of a Threonine-sensitive Aspartokinase*

    Science.gov (United States)

    Liu, Xuying; Pavlovsky, Alexander G.; Viola, Ronald E.

    2008-01-01

    The commitment step to the aspartate pathway of amino acid biosynthesis is the phosphorylation of aspartic acid catalyzed by aspartokinase (AK). Most microorganisms and plants have multiple forms of this enzyme, and many of these isofunctional enzymes are subject to feedback regulation by the end products of the pathway. However, the archeal species Methanococcus jannaschii has only a single, monofunctional form of AK. The substrate l-aspartate binds to this recombinant enzyme in two different orientations, providing the first structural evidence supporting the relaxed regiospecificity previously observed with several alternative substrates of Escherichia coli AK (Angeles, T. S., Hunsley, J. R., and Viola, R. E. (1992) Biochemistry31 ,799 -8051731937). Binding of the nucleotide substrate triggers significant domain movements that result in a more compact quaternary structure. In contrast, the highly cooperative binding of the allosteric regulator l-threonine to multiple sites on this dimer of dimers leads to an open enzyme structure. A comparison of these structures supports a mechanism for allosteric regulation in which the domain movements induced by threonine binding causes displacement of the substrates from the enzyme, resulting in a relaxed, inactive conformation. PMID:18334478

  7. The Structural Basis for Allosteric Inhibition of a Threonine-sensitive Aspartokinase

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xuying; Pavlovsky, Alexander G.; Viola, Ronald E. (Toledo)

    2008-10-08

    The commitment step to the aspartate pathway of amino acid biosynthesis is the phosphorylation of aspartic acid catalyzed by aspartokinase (AK). Most microorganisms and plants have multiple forms of this enzyme, and many of these isofunctional enzymes are subject to feedback regulation by the end products of the pathway. However, the archeal species Methanococcus jannaschii has only a single, monofunctional form of AK. The substrate l-aspartate binds to this recombinant enzyme in two different orientations, providing the first structural evidence supporting the relaxed regiospecificity previously observed with several alternative substrates of Escherichia coli AK. Binding of the nucleotide substrate triggers significant domain movements that result in a more compact quaternary structure. In contrast, the highly cooperative binding of the allosteric regulator l-threonine to multiple sites on this dimer of dimers leads to an open enzyme structure. A comparison of these structures supports a mechanism for allosteric regulation in which the domain movements induced by threonine binding causes displacement of the substrates from the enzyme, resulting in a relaxed, inactive conformation.

  8. Contrasting Effects of Allosteric and Orthosteric Agonists on M1 Muscarinic Acetylcholine Receptor Internalization and Down-regulation

    OpenAIRE

    Thomas, Rachel L.; Christopher J Langmead; Wood, Martyn D; Challiss, R.A. John

    2009-01-01

    A new class of subtype-selective muscarinic acetylcholine (mACh) receptor agonist that activates the receptor through interaction at a site distinct from the orthosteric acetylcholine binding site has been reported recently. Here, we have compared the effects of orthosteric (oxotremorine-M, arecoline, pilocarpine) and allosteric [4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine (AC-42); 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1)] agonists on M1 mAC...

  9. Identification of an Allosteric Pocket on Human Hsp70 Reveals a Mode of Inhibition of This Therapeutically Important Protein

    OpenAIRE

    Rodina, Anna; Patel, Pallav D.; Kang, Yanlong; Patel, Yogita; Baaklini, Imad; Wong, Michael J. H.; Taldone, Tony; Yan, Pengrong; Yang, Chenghua; Maharaj, Ronnie; Gozman, Alexander; Patel, Maulik R.; Patel, Hardik J.; Chirico, William; Erdjument-Bromage, Hediye

    2013-01-01

    Hsp70s are important cancer chaperones that act upstream of Hsp90 and exhibit independent anti-apoptotic activities. To develop chemical tools for the study of human Hsp70, we developed a homology model that unveils a previously unknown allosteric site located in the nucleotide binding domain of Hsp70. Combining structure-based design and phenotypic testing, we discovered a previously unknown inhibitor of this site, YK5. In cancer cells, this compound is a potent and selective binder of the c...

  10. Virtual Screening and Molecular Dynamics Study of Potential Negative Allosteric Modulators of mGluR1 from Chinese Herbs

    Directory of Open Access Journals (Sweden)

    Ludi Jiang

    2015-07-01

    Full Text Available The metabotropic glutamate subtype 1 (mGluR1, a member of the metabotropic glutamate receptors, is a therapeutic target for neurological disorders. However, due to the lower subtype selectivity of mGluR1 orthosteric compounds, a new targeted strategy, known as allosteric modulators research, is needed for the treatment of mGluR1-related diseases. Recently, the structure of the seven-transmembrane domain (7TMD of mGluR1 has been solved, which reveals the binding site of allosteric modulators and provides an opportunity for future subtype-selectivity drug design. In this study, a series of computer-aided drug design methods were utilized to discover potential mGluR1 negative allosteric modulators (NAMs. Pharmacophore models were constructed based on three different structure types of mGluR1 NAMs. After validation using the built-in parameters and test set, the optimal pharmacophore model of each structure type was selected and utilized as a query to screen the Traditional Chinese Medicine Database (TCMD. Then, three different hit lists of compounds were obtained. Molecular docking was used based on the latest crystal structure of mGluR1-7TMD to further filter these hits. As a compound with high QFIT and LibDock Score was preferred, a total of 30 compounds were retained. MD simulation was utilized to confirm the stability of potential compounds binding. From the computational results, thesinine-4ʹ-O-β-d-glucoside, nigrolineaxanthone-P and nodakenin might exhibit negative allosteric moderating effects on mGluR1. This paper indicates the applicability of molecular simulation technologies for discovering potential natural mGluR1 NAMs from Chinese herbs.

  11. 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; Hudson, Brian D; Kostenis, Evi; Ulven, Trond; Morris, Joanne C; Tränkle, Christian; Tikhonova, Irina G; Adams, David R; Milligan, Graeme

    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...

  12. 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

  13. pH dependence of the kinetic properties of allosteric phosphofructokinase from Escherichia coli.

    Science.gov (United States)

    Deville-Bonne, D; Bourgain, F; Garel, J R

    1991-06-11

    The pH dependence of the activity of the allosteric phosphofructokinase from Escherichia coli has been studied in the pH range from 6 to 9, in the absence or presence of allosteric effectors. The sigmoidal cooperative saturation of phosphofructokinase by fructose 6-phosphate has been analyzed according to the Hill equation, and the following results have been obtained: (i) the apparent affinity for Fru-6P, as measured by the half-saturating concentration, [Fru-6P]0.5, does not change with pH; (ii) the cooperativity, as measured empirically by the Hill coefficient, nH, increases markedly with pH and reaches a value of 5.5-6 at pH 9; (iii) the catalytic rate constant, kcat, is controlled by the ionization of a critical group which has a pK of 7 in the absence of effector and must be deprotonated for phosphofructokinase to be active. The observation that pH affects both the cooperativity and the maximum velocity suggests that the catalytic efficiency of a given active site could be modified by the binding of fructose 6-phosphate to other remote sites. Finding values of the cooperativity coefficient larger than the number of substrate binding sites indicates that slow conformational changes may occur in phosphofructokinase. The cooperative saturation of phosphofructokinase by fructose 6-phosphate appears more complex than described by the classical concerted model at steady state and could involve two slowly interconverting states which differ in both their turnover rate constants and their affinities for fructose 6-phosphate. The presence of GDP shifts the pK of the critical group which controls kcat from 7 to 6.6.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1828369

  14. Lymphoscintigraphy Defines New Lymphatic Pathways from Cutaneous Melanoma Site: Clinical Implications and Surgical Management

    International Nuclear Information System (INIS)

    Sentinel lymph node biopsy is commonly applied as staging procedure of regional lymph nodes in patients with cutaneous melanoma. Dynamic lymphoscintigraphy defines the lymphatic pathways from a primary melanoma site and allows to identify the node receiving lymphatic drainage from the primary tumor, which is the sentinel lymph node. In rare cases, lymphoscintigraphy shows sites of lymphatic drainage in nonclassical basins never described in the past when lymphatic drainage was considered only according to the anatomical proximity of the tumor primary site. These peculiar sentinel nodes, so-called “uncommon/interval” nodes, must be surgically removed because they may contain micrometastatic disease and may be the only site of nodal involvement

  15. Benzothiazole Derivative as a Novel Mycobacterium tuberculosis Shikimate Kinase Inhibitor: Identification and Elucidation of Its Allosteric Mode of Inhibition.

    Science.gov (United States)

    Mehra, Rukmankesh; Rajput, Vikrant Singh; Gupta, Monika; Chib, Reena; Kumar, Amit; Wazir, Priya; Khan, Inshad Ali; Nargotra, Amit

    2016-05-23

    Mycobacterium tuberculosis shikimate kinase (Mtb-SK) is a key enzyme involved in the biosynthesis of aromatic amino acids through the shikimate pathway. Since it is proven to be essential for the survival of the microbe and is absent from mammals, it is a promising target for anti-TB drug discovery. In this study, a combined approach of in silico similarity search and pharmacophore building using already reported inhibitors was used to screen a procured library of 20,000 compounds of the commercially available ChemBridge database. From the in silico screening, 15 hits were identified, and these hits were evaluated in vitro for Mtb-SK enzyme inhibition. Two compounds presented significant enzyme inhibition with IC50 values of 10.69 ± 0.9 and 46.22 ± 1.2 μM. The best hit was then evaluated for the in vitro mode of inhibition where it came out to be an uncompetitive and noncompetitive inhibitor with respect to shikimate (SKM) and ATP, respectively, suggesting its binding at an allosteric site. Potential binding sites of Mtb-SK were identified which confirmed the presence of an allosteric binding pocket apart from the ATP and SKM binding sites. The docking simulations were performed at this pocket in order to find the mode of binding of the best hit in the presence of substrates and the products of the enzymatic reaction. Molecular dynamics (MD) simulations elucidated the probability of inhibitor binding at the allosteric site in the presence of ADP and shikimate-3-phosphate (S-3-P), that is, after the formation of products of the reaction. The inhibitor binding may prevent the release of the product from Mtb-SK, thereby inhibiting its activity. The binding stability and the key residue interactions of the inhibitor to this product complex were also revealed by the MD simulations. Residues ARG43, ILE45, and PHE57 were identified as crucial that were involved in interactions with the best hit. This is the first report of an allosteric binding site of Mtb-SK, which

  16. Allosteric Pathways in the PPARγ-RXRα nuclear receptor complex

    Science.gov (United States)

    Ricci, Clarisse G.; Silveira, Rodrigo L.; Rivalta, Ivan; Batista, Victor S.; Skaf, Munir S.

    2016-01-01

    Understanding the nature of allostery in DNA-nuclear receptor (NR) complexes is of fundamental importance for drug development since NRs regulate the transcription of a myriad of genes in humans and other metazoans. Here, we investigate allostery in the peroxisome proliferator-activated/retinoid X receptor heterodimer. This important NR complex is a target for antidiabetic drugs since it binds to DNA and functions as a transcription factor essential for insulin sensitization and lipid metabolism. We find evidence of interdependent motions of Ω-loops and PPARγ-DNA binding domain with contacts susceptible to conformational changes and mutations, critical for regulating transcriptional functions in response to sequence-dependent DNA dynamics. Statistical network analysis of the correlated motions, observed in molecular dynamics simulations, shows preferential allosteric pathways with convergence centers comprised of polar amino acid residues. These findings are particularly relevant for the design of allosteric modulators of ligand-dependent transcription factors.

  17. Allosterism and Structure in Thermally Activated Transient Receptor Potential Channels.

    Science.gov (United States)

    Diaz-Franulic, Ignacio; Poblete, Horacio; Miño-Galaz, Germán; González, Carlos; Latorre, Ramón

    2016-07-01

    The molecular sensors that mediate temperature changes in living organisms are a large family of proteins known as thermosensitive transient receptor potential (TRP) ion channels. These membrane proteins are polymodal receptors that can be activated by cold or hot temperatures, depending on the channel subtype, voltage, and ligands. The stimuli sensors are allosterically coupled to a pore domain, increasing the probability of finding the channel in its ion conductive conformation. In this review we first discuss the allosteric coupling between the temperature and voltage sensor modules and the pore domain, and then discuss the thermodynamic foundations of thermo-TRP channel activation. We provide a structural overview of the molecular determinants of temperature sensing. We also posit an anisotropic thermal diffusion model that may explain the large temperature sensitivity of TRP channels. Additionally, we examine the effect of several ligands on TRP channel function and the evidence regarding their mechanisms of action. PMID:27297398

  18. Allosteric process of human glucokinase conducive to fight against diabetes

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ More than 200 million people worldwide have diabetes. In China alone, about 60 million people are suffering from the disease.Fortunately, scientists are pushing back its boundaries. For instance, a recent study by CAS researchers may shed new light on the treatment of the disease by making cutting-edge progress on studies of the allosteric process of human glucokinase, which has been published by the latest issue of the Proceedings of National Academy of Sciences.

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

    OpenAIRE

    Lucas R. Watterson; M. Foster Olive

    2013-01-01

    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 m...

  20. Allosteric activation of coagulation factor VIIa visualized by hydrogen exchange

    DEFF Research Database (Denmark)

    Rand, Kasper Dyrberg; Jørgensen, Thomas; Olsen, Ole H; Persson, Egon; Jensen, Ole; Stennicke, Henning R; Andersen, Mette

    2006-01-01

    Coagulation factor VIIa (FVIIa) is a serine protease that, after binding to tissue factor (TF), plays a pivotal role in the initiation of blood coagulation. We used hydrogen exchange monitored by mass spectrometry to visualize the details of FVIIa activation by comparing the exchange kinetics of ...... provide novel insights into the cofactor-induced activation of this important protease and reveal the potential for allosteric regulation in the trypsin family of proteases....

  1. The structural basis of ATP as an allosteric modulator.

    OpenAIRE

    Shaoyong Lu; Wenkang Huang; Qi Wang; Qiancheng Shen; Shuai Li; Ruth Nussinov; Jian Zhang

    2014-01-01

    Adenosine-5'-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP...

  2. The Structural Basis of ATP as an Allosteric Modulator

    OpenAIRE

    Lu, Shaoyong; Huang, Wenkang; Wang, Qi; Shen, Qiancheng; Li, Shuai; Nussinov, Ruth; Zhang, Jian

    2014-01-01

    Adenosine-5’-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP...

  3. The implications of episodic nonequilibrium fracture-matrix flow on site suitability and total system performance

    International Nuclear Information System (INIS)

    We apply our work on fracture- and matrix-dominated flow to develop a conceptual model of hydrological flow processes in the unsaturated zone at Yucca Mountain. The possibility of fracture-dominated flow is discussed, and various deductions are made on its impact on natural and total system performance, site characterization activities, and site suitability determination

  4. Multiplicity of carbohydrate-binding sites in -prism fold lectins: occurrence and possible evolutionary implications

    Indian Academy of Sciences (India)

    Alok Sharma; Divya Chandran; Desh D Singh; M Vijayan

    2007-09-01

    The -prism II fold lectins of known structure, all from monocots, invariably have three carbohydrate-binding sites in each subunit/domain. Until recently, -prism I fold lectins of known structure were all from dicots and they exhibited one carbohydrate-binding site per subunit/domain. However, the recently determined structure of the -prism fold I lectin from banana, a monocot, has two very similar carbohydrate-binding sites. This prompted a detailed analysis of all the sequences appropriate for two-lectin folds and which carry one or more relevant carbohydrate-binding motifs. The very recent observation of a -prism I fold lectin, griffithsin, with three binding sites in each domain further confirmed the need for such an analysis. The analysis demonstrates substantial diversity in the number of binding sites unrelated to the taxonomical position of the plant source. However, the number of binding sites and the symmetry within the sequence exhibit reasonable correlation. The distribution of the two families of -prism fold lectins among plants and the number of binding sites in them, appear to suggest that both of them arose through successive gene duplication, fusion and divergent evolution of the same primitive carbohydrate-binding motif involving a Greek key. Analysis with sequences in individual Greek keys as independent units lends further support to this conclusion. It would seem that the preponderance of three carbohydrate-binding sites per domain in monocot lectins, particularly those with the -prism II fold, is related to the role of plant lectins in defence.

  5. Small Molecule-Induced Allosteric Activation of the Vibrio Cholerae RTX Cysteine Protease Domain

    Energy Technology Data Exchange (ETDEWEB)

    Lupardus, P.J.; Shen, A.; Bogyo, M.; Garcia, K.C.

    2009-05-19

    Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP{sub 6}), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP{sub 6}. InsP{sub 6} binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP{sub 6} binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.

  6. Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding

    Science.gov (United States)

    Beveridge, Rebecca; Migas, Lukasz G.; Payne, Karl A. P.; Scrutton, Nigel S.; Leys, David; Barran, Perdita E.

    2016-01-01

    Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1Ubix, the cofactor confers structural stability to the enzyme. IM–MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. HDX-MS reveals that while the dominant structural changes occur proximal to the cofactor-binding site, rearrangements on cofactor binding are evident throughout the protein, predominantly attributable to allosteric conformational tightening, consistent with IM–MS data. PMID:27418477

  7. Allosteric function and dysfunction of the prion protein.

    Science.gov (United States)

    Linden, Rafael; Cordeiro, Yraima; Lima, Luis Mauricio T R

    2012-04-01

    Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases associated with progressive oligo- and multimerization of the prion protein (PrP(C)), its conformational conversion, aggregation and precipitation. We recently proposed that PrP(C) serves as a cell surface scaffold protein for a variety of signaling modules, the effects of which translate into wide-range functional consequences. Here we review evidence for allosteric functions of PrP(C), which constitute a common property of scaffold proteins. The available data suggest that allosteric effects among PrP(C) and its partners are involved in the assembly of multi-component signaling modules at the cell surface, impose upon both physiological and pathological conformational responses of PrP(C), and that allosteric dysfunction of PrP(C) has the potential to entail progressive signal corruption. These properties may be germane both to physiological roles of PrP(C), as well as to the pathogenesis of the TSEs and other degenerative/non-communicable diseases. PMID:21984610

  8. Shear wave velocity mapping of Hat Yai district, southern Thailand: implication for seismic site classification

    International Nuclear Information System (INIS)

    Soil characteristics play an important role in the degree of ground shaking due to local site amplification during an earthquake. The objectives of this work are to study shear wave velocity (Vs) distribution in the near surface, and to develop a seismic site classification map for soil effect characterization and seismic hazard assessment in Hat Yai district, southern Thailand. The Vs determination based on the multichannel analysis of surface waves technique, has been carried out and analyzed at 70 measuring sites throughout the district. On the basis of the weighted-average Vs in the upper 30 m depth (Vs30), a seismic site classification map, based on the National Earthquake Hazards Reduction Program (NEHRP) standard has been developed. It is found that the NEHRP site class in Hat Yai can be classified into four groups in accordance with the value of Vs30 within the range of about 150 to 1160 m s−1. Most parts of the study area are typically classified as site class C and D. Site class C is mostly found within the colluvial and terrace deposits in the western and eastern part of the area, whereas site class D is concentrated in the alluvial sediment of the middle and northern flood plain areas. A small portion of site class B is observed in the western mountain ranges, where there is a thin overburden on the firm rock. There is a remarkably low Vs30 value at only one site, located near the main stream in the northern part of the study area. The results imply that the soil characteristics in the central and northern Hat Yai district pose a medium to high amplification rate with respect to the other regions. Although Vs data alone are insufficient to verify the potential of the amplification of ground shaking, this study provides an initial attempt to understand seismic hazards in the study area. (paper)

  9. The Nature of Allosteric Inhibition in Glutamate Racemase: discovery and characterization of a cryptic inhibitory pocket using atomistic MD simulations and pKa calculations

    OpenAIRE

    Whalen, Katie L.; Tussey, Kenneth B.; Blanke, Steven R.; Spies, M. Ashley

    2011-01-01

    Enzyme inhibition via allostery, in which the ligand binds remotely from the active site, is a poorly understood phenomenon, and represents a significant challenge to structure-based drug design. Dipicolinic acid (DPA), a major component of Bacillus spores, is shown to inhibit glutamate racemase from Bacillus anthracis, a monosubstrate/monoproduct enzyme, in a novel allosteric fashion. Glutamate racemase has long been considered an important drug target for its integral role in bacterial cell...

  10. 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.

  11. Chimpanzee nest distribution and site reuse in a dry habitat: implications for early hominin ranging.

    Science.gov (United States)

    Hernandez-Aguilar, R Adriana

    2009-10-01

    This paper reports on a 20-month study of chimpanzee nesting patterns in Issa, Ugalla, western Tanzania. Ugalla is one of the driest, most open, and seasonal habitats where chimpanzees are found. The methods used were ethoarchaeological, as the chimpanzees were not habituated and behavioural observations were rare. Systematic data on the spatial and temporal distribution of nests are presented. Places with no nests at the beginning of the study, despite being suitable for nesting, were used as controls. Similar to other chimpanzee study sites, nests were highly concentrated in some parts of the landscape. Issa chimpanzees preferred to nest on slopes. They extensively used the woodland vegetation type of their habitat for nesting throughout the annual cycle. Ninety percent of nest sites were used repeatedly throughout the study period, but none of the control places had nests during this period. The results indicate that chimpanzees ranged more widely during the dry season, when food abundance was lowest, food was available mainly in open vegetation types, and when drinking water was restricted to a few sources. Early hominins in similar habitats may have followed the ranging strategy of Issa chimpanzees. As with a previous study, the distribution of nests was spatially similar to archaeological distributions in early hominin sites. Hominin topography and vegetation type preferences may be misrepresented in the archaeological record. Nest sites may have been the antecedents of carcass processing sites. PMID:19744699

  12. Donor site morbidity in oral mucosa graft urethroplasty: implications of tobacco consumption

    Directory of Open Access Journals (Sweden)

    Sankhwar SN

    2009-09-01

    Full Text Available Abstract Background The purpose of this prospective study was to evaluate the donor site morbidity in patients who have undergone oral mucosa graft urethroplasty for stricture of the urethra. The impact of smoking and oral consumption of tobacco and/or paan masala on the donor site was also assessed. This study is probably the first of its kind where the affect of smoking, paan masala and tobacco chewing on the donor site morbidity has been documented. Methods Forty-eight patients suffering from stricture of the urethra underwent oral mucosa graft urethroplasty between July 2005 and December 2007. The patients were divided into two groups (users or non-users based on tobacco consumption and oral hygiene. The donor site was evaluated at frequent intervals for pain, swelling, numbness, bleeding, salivation and tightness of mouth. Results Donor site morbidity was more in users with poor oral hygiene. Pain scores were higher amongst the users and the morbidity persisted longer in the users compared to non-users with good oral hygiene. Conclusion Patients who consume tobacco and have poor oral hygiene should be warned regarding poorer outcomes after oral mucosa graft urethroplasty.

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

    International Nuclear Information System (INIS)

    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

  14. Geo archaeology in Cerro Lutz site, paleoenvironmental and palaeo geographic implications. Province of Entre Rios, Argentina

    International Nuclear Information System (INIS)

    The aim of this paper is to present the geo morphological evolution of the Cerro Lutz archaeological site located in the Lower Par ana Delta, Argentina River (Gualeguaychu Department). This is to add information to the paleogeographic and paleoenvironmental scheme during the occupation of site and add data to refine the regional evolution of deltaic advance against the Parana River.The geomorphology of the delta is composed of landforms resulting from fluvial modeling, coastal ( wave and tidal ) and to a lesser extent wind.They have different relative ages and dating from the late Pleistocene to the present

  15. 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…

  16. 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 accepta

  17. 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…

  18. Structure of N-acetyl-L-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor L-arginine bound.

    Science.gov (United States)

    Zhao, Gengxiang; Haskins, Nantaporn; Jin, Zhongmin; M Allewell, Norma; Tuchman, Mendel; Shi, Dashuang

    2013-08-01

    Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in L-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by L-arginine, although L-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by L-arginine, we have determined the structure of the mmNAGS/K complexed with L-arginine at 2.8 Å resolution. In contrast to the structure of mmNAGS/K in the absence of L-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the L-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when L-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by L-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism. PMID:23850694

  19. Oviposition site selection by the dengue vector Aedes aegypti and its implications for dengue control.

    Directory of Open Access Journals (Sweden)

    Jacklyn Wong

    Full Text Available 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

  20. Allosteric Inhibitors of the NS3 Protease from the Hepatitis C Virus

    Science.gov (United States)

    Abian, Olga; Vega, Sonia; Sancho, Javier; Velazquez-Campoy, Adrian

    2013-01-01

    The nonstructural protein 3 (NS3) from the hepatitis C virus processes the non-structural region of the viral precursor polyprotein in infected hepatic cells. The NS3 protease activity has been considered a target for drug development since its identification two decades ago. Although specific inhibitors have been approved for clinical therapy very recently, resistance-associated mutations have already been reported for those drugs, compromising their long-term efficacy. Therefore, there is an urgent need for new anti-HCV agents with low susceptibility to resistance-associated mutations. Regarding NS3 protease, two strategies have been followed: competitive inhibitors blocking the active site and allosteric inhibitors blocking the binding of the accessory viral protein NS4A. In this work we exploit the intrinsic Zn+2-regulated plasticity of the protease to identify a new type of allosteric inhibitors. In the absence of Zn+2, the NS3 protease adopts a partially-folded inactive conformation. We found ligands binding to the Zn+2-free NS3 protease, trap the inactive protein, and block the viral life cycle. The efficacy of these compounds has been confirmed in replicon cell assays. Importantly, direct calorimetric assays reveal a low impact of known resistance-associated mutations, and enzymatic assays provide a direct evidence of their inhibitory activity. They constitute new low molecular-weight scaffolds for further optimization and provide several advantages: 1) new inhibition mechanism simultaneously blocking substrate and cofactor interactions in a non-competitive fashion, appropriate for combination therapy; 2) low impact of known resistance-associated mutations; 3) inhibition of NS4A binding, thus blocking its several effects on NS3 protease. PMID:23936097

  1. 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

    identifying allosteric leads and their often flat or confusing SAR. The present review will consider the advantages and challenges associated with allosteric GPCR ligands, and examine how the particular properties of these ligands may be exploited to uncover the therapeutic potential for free fatty acid...

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Mehanna, A.S.; Abraham, D.J. (Virginia Commonwealth Univ., Richmond (USA))

    1990-04-24

    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.

  4. 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 solvent source zones, suggesting that back diffusion of 1,4-dioxane mass may be serving as the dominant long-term "secondary source" at many contaminated sites that must be managed using alternative approaches. PMID:27096631

  5. 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.

  6. On the rapid rise of social networking sites: new findings and policy implications

    OpenAIRE

    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 increases the likelihood of new risks to the self, these centring on loss of privacy, bullying, harmful contacts and more. This article reviews recent ...

  7. 2001 Columbia River Recreation Survey -- Implications for Hanford Site Integrated Assessment

    International Nuclear Information System (INIS)

    This report presents the results from the Columbia River Recreation Survey conducted in the summer of 2001. The survey combined on-site personal interviews with parties engaged in river recreation with on-site field observations to develop a picture of summer river recreation on the Columbia. The study area stretched from just below Priest Rapids Dam in the north to McNary Dam in the south, and was divided into four ''Areas'' that correspond to the river areas used by the Groundwater/Vadose Zone Integration Project. This study is part of the Groundwater/Vadose Zone Integration Project and was commissioned specifically to document the current recreation use levels in these areas of the river, and to elicit recreation-related expenditure information from visitors. This information informs economic and environmental models used to measure the economic risk posed by possible, but unlikely, releases of contaminants from the Hanford site into the Columbia River. During the study period, researchers collected 256 survey responses and 396 field observations from recreation sites up and down both shores of the river in the study area. Results presented include analysis of trip duration by river activity, trip frequency, and visitor place of origin. Economics-related results include trip expenditure profiles by activity and place of origin. Data also were collected on fishing effort. Visitors also were asked to indicate what activity or destination substitution they would choose in the hypothetical example that the river could not be accessed. The report also highlights some limitations in the approach. Principally, by doing this research in the summer, the recreational use of the river in the other seasons was not documented. The report provides data that suggest the significance of the other seasons--particularly spring and fall--for salmon and steelhead fishing. This stretch of the Columbia is also well known for waterfowl hunting.

  8. Health-based cleanup goals at hazardous waste sites: Implications for risk management

    International Nuclear Information System (INIS)

    The challenge of setting realistic and well-defined remediation goals at hazardous waste sites is not being met. The majority of remedial alternatives are being selected without adequate evidence of their effectiveness and permanence. Remediation goals are being set that are not technically achievable. To correct this situation, DOE should focus on the fundamental areas of setting priorities in the remediation process and developing and selecting effective and permanent remedial alternatives

  9. Allosteric activation mechanism of the cys-loop receptors

    Institute of Scientific and Technical Information of China (English)

    Yong-chang CHANG; Wen WU; Jian-liang ZHANG; Yao HUANG

    2009-01-01

    Binding of a neurotransmitter to its ionotropic receptor opens a distantly located ion channel, a process termed allosteric activation. Here we review recent advances in the molecular mechanism by which the cys-loop receptors are activated with emphasis on the best studied nicotinic acetylcholine receptors (nAChRs). With a combination of affinity labeling, mutagenesis, electrophysiology, kinetic modeling, electron microscopy (EM), and crystal structure analysis, the allosteric activation mechanism is emerging. Specifically, the binding domain and gating domain are interconnected by an allosteric activation network. Agonist binding induces conformational changes, resulting in the rotation of a β sheet of amino-terminal domain and outward movement of loop 2, loop F, and cys-loop, which are coupled to the M2-M3 linker to pull the channel to open. However, there are still some controversies about the movement of the channel-lining domain M2. Nine angstrom resolution EM structure of a nAChR imaged in the open state suggests that channel opening is the result of rotation of the M2 domain. In contrast, recent crystal structures of bacterial homologues of the cys-loop receptor family in apparently open state have implied an M2 tilting model with pore dilation and quaternary twist of the whole pentameric receptor. An elegant study of the nAChR using protonation scanning of M2 domain supports a similar pore dilation activation mechanism with minimal rotation of M2. This remains to be validated with other approaches including high resolution structure determination of the mammalian cys-loop receptors in the open state.

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

    International Nuclear Information System (INIS)

    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. 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

  12. 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. PMID:24951728

  13. Multi-site characterization of tropical aerosols: Implications for regional radiative forcing

    Science.gov (United States)

    Sumit, Kumar; Devara, P. C. S.; Manoj, M. G.

    2012-03-01

    A land campaign, as a part of the Indian Space Research Organization-Geosphere Biosphere Program (ISRO-GBP), has been organized using a suit of instruments like AERONET (Aerosol Robotic Network) Sun/Sky sunphotometer, Microtops-II (MICROprocessor-controlled Total Ozone Portable Spectrometer), short-wave pyranometer from December 1, 2006 to April 30, 2007, over five locations (Ahmedabad, Pune, Sinhgad, Trivandrum and Gadanki) representing different environments. The dominance of different aerosol types such as biomass burning, urban/industrial pollution, marine origin and desert-dust particles is expected at these five sites. In all locations, significant day-to-day variability in AOD and Ångström exponent is observed. The Ångström exponent exhibits its lowest values over semi-arid region (Ahmedabad) 0.4-0.7, while it is around 1.8 at rural site (Gadanki). The retrieved volume size distributions for Pune, Ahmedabad and Trivandrum are found to be bimodal with varying concentration of each mode. Interesting feature of this observation is, very low coarse-mode volume concentration observed at Trivandrum even though observations were made about 300 m from the coast. The synergy of results from these complementary measurements is reflected in the computed regional aerosol radiative forcing and heating rates. We have used a radiative transfer model (SBDART) to examine the variations of aerosol direct radiative effect (ADRE) and heating rates to give an overall estimation of the effect on climate. The ADRE, over different measurement sites, at short wavelength is found to be negative at the surface in the range of - 18 to - 59 W m - 2 , and TOA forcing values varied from + 0.9 to - 8 W m - 2 .

  14. The implications of LC36 on a mixed site -- Devonport Management Limited

    International Nuclear Information System (INIS)

    DML owns and operates Devonport Royal Dockyard, the largest marine support complex in Western Europe. The company's primary business is refitting and refuelling nuclear submarines, modernising and refitting warships and updating and overhauling naval weapons systems, as well as providing a wide range of design and management services. Consequently, DML's nuclear and non-nuclear activities coexist on the same site and its location, business diversity and history make it unique within the nuclear industry. Within this setting, Licence Condition 36 (LC36) compliance has presented specific management challenges. This paper outlines the company's approach to living with LC36 within a fast-changing, dual-regulated environment. (author)

  15. Monetary implications of Surgical Site Infections Health Services: an Integrative Review

    Directory of Open Access Journals (Sweden)

    Suellen Rodrigues de Oliveira Maier

    2015-11-01

    Full Text Available Background and Objective: In order to meet the costs related to the Surgical Site Infection (SSI and that they were linked, this research guided the scientific literature was developed with the following objective: to perform an integrative review of literature related to expenses and costs of surgical site infections, or surgical wound, to health systems. Methods: We selected 11 scientific articles available in electronic databases of the Medical Literature Analysis and Retrieval System Online (MEDLINE, and Latin American Literature on Health Sciences (LILACS, through the portal of the Virtual Health Library (VHL. It is also one of the research methods used in Evidence Based Practice (EBP. Results: It was observed that SAIs are costly to health services, publications in very differ and used methods not specifically describe how the results of the costs were obtained. Conclusion: Thus, other studies adopt an effective and standardized assessment for the collection and calculation of costs are needed to ensure greater reliability of the information obtained. KEYWORDS: Surgical wound infection.Costs and Cost Analysis. Health service.

  16. 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. PMID:20735254

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

    International Nuclear Information System (INIS)

    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 NO3-/NO2- 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)

  18. Point-charge calculation of quadrupolar parameters for bridging oxygen sites in vitreous silica: Structural implications

    International Nuclear Information System (INIS)

    The quadrupolar coupling constant Cq and the asymmetry parameter η for the bridging oxygen sites in vitreous silica structures derived via classical molecular dynamics simulation have been calculated using the point-charge lattice summation method. The results of these calculations indicate that while Cq is a function of both the Si-O-Si angle and the associated Si-O distances, η depends only on the Si-O-Si angle. The analytical forms of these functional dependences are found to be similar to those reported in previous studies based on ab initio calculations on small clusters. However, the magnitude of Cq is found to decrease with increasing Si-O distance in contrast with a reverse trend obtained in previous ab initio calculations on small clusters. The present results, when combined with the previously reported 17O nuclear magnetic resonance spectroscopic results for bridging oxygen sites in vitreous silica, imply a strong negative correlation between Si-O bond lengths and Si-O-Si bond angles in the glass structure. Such a negative correlation is consistent with the anomalous density variation in silica as well as with energetic considerations

  19. 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)

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

    International Nuclear Information System (INIS)

    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. Structural Analysis of Iac Repressor Bound to Allosteric Effectors

    Energy Technology Data Exchange (ETDEWEB)

    Daber,R.; Stayrook, S.; Rosenberg, A.; Lewis, M.

    2007-01-01

    The lac operon is a model system for understanding how effector molecules regulate transcription and are necessary for allosteric transitions. The crystal structures of the lac repressor bound to inducer and anti-inducer molecules provide a model for how these small molecules can modulate repressor function. The structures of the apo repressor and the repressor bound to effector molecules are compared in atomic detail. All effectors examined here bind to the repressor in the same location and are anchored to the repressor through hydrogen bonds to several hydroxyl groups of the sugar ring. Inducer molecules form a more extensive hydrogen-bonding network compared to anti-inducers and neutral effector molecules. The structures of these effector molecules suggest that the O6 hydroxyl on the galactoside is essential for establishing a water-mediated hydrogen bonding network that bridges the N-terminal and C-terminal sub-domains. The altered hydrogen bonding can account in part for the different structural conformations of the repressor, and is vital for the allosteric transition.

  2. 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.

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

    International Nuclear Information System (INIS)

    A 12 month survey of ichthyoplankton in the cooling water system of Wylfa Power Station and the surrounding 40 km2 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)

  4. 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.

  5. 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.

  6. Lid L11 of the glutamine amidotransferase domain of CTP synthase mediates allosteric GTP activation of glutaminase activity

    DEFF Research Database (Denmark)

    Willemoës, Martin; Mølgaard, Anne; Johansson, Eva;

    2005-01-01

    GTP is an allosteric activator of CTP synthase and acts to increase the k(cat) for the glutamine-dependent CTP synthesis reaction. GTP is suggested, in part, to optimally orient the oxy-anion hole for hydrolysis of glutamine that takes place in the glutamine amidotransferase class I (GATase) domain...... position depending on the presence or absence of glutamine in the glutamine binding site. Displacement or rearrangement of this loop may provide a means for the suggested role of allosteric activation by GTP to optimize the oxy-anion hole for glutamine hydrolysis. Arg359, Gly360 and Glu362 of the...... enzyme behaved like wild-type enzyme. Apart from the G360A enzyme, the results from kinetic analysis of the enzymes altered at position 359 and 360 showed a 10- to 50-fold decrease in GTP activation of glutamine dependent CTP synthesis and concomitant four- to 10-fold increases in K(A) for GTP. The R359M...

  7. Allosteric Activation of Ubiquitin-Specific Proteases by β-Propeller Proteins UAF1 and WDR20.

    Science.gov (United States)

    Li, Heng; Lim, Kah Suan; Kim, Hyungjin; Hinds, Thomas R; Jo, Ukhyun; Mao, Haibin; Weller, Caroline E; Sun, Ji; Chatterjee, Champak; D'Andrea, Alan D; Zheng, Ning

    2016-07-21

    Ubiquitin-specific proteases (USPs) constitute the largest family of deubiquitinating enzymes, whose catalytic competency is often modulated by their binding partners through unknown mechanisms. Here we report on a series of crystallographic and biochemical analyses of an evolutionarily conserved deubiquitinase, USP12, which is activated by two β-propeller proteins, UAF1 and WDR20. Our structures reveal that UAF1 and WDR20 interact with USP12 at two distinct sites far from its catalytic center. Without increasing the substrate affinity of USP12, the two β-propeller proteins potentiate the enzyme through different allosteric mechanisms. UAF1 docks at the distal end of the USP12 Fingers domain and induces a cascade of structural changes that reach a critical ubiquitin-contacting loop adjacent to the catalytic cleft. By contrast, WDR20 anchors at the base of this loop and remotely modulates the catalytic center of the enzyme. Our results provide a mechanistic example for allosteric activation of USPs by their regulatory partners. PMID:27373336

  8. 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.

  9. 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

  10. Engineering an allosteric transcription factor to respond to new ligands.

    Science.gov (United States)

    Taylor, Noah D; Garruss, Alexander S; Moretti, Rocco; Chan, Sum; Arbing, Mark A; Cascio, Duilio; Rogers, Jameson K; Isaacs, Farren J; Kosuri, Sriram; Baker, David; Fields, Stanley; Church, George M; Raman, Srivatsan

    2016-02-01

    Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits. PMID:26689263

  11. Positive allosteric feedback regulation of the stringent response enzyme RelA by its product

    OpenAIRE

    Shyp, Viktoriya; Tankov, Stoyan; Ermakov, Andrey; Kudrin, Pavel; English, Brian P.; Ehrenberg, Måns; Tenson, Tanel; Elf, Johan; Hauryliuk, Vasili

    2012-01-01

    This report identifies a new mechanism of enzyme activation—positive allosteric regulation by the product—in the context of the bacterial stringent response, which is essential for bacterial adaptation to environmental conditions.

  12. Allosteric Mechanism of Pyruvate Kinase from Leishmania mexicana Uses a Rock and Lock Model*

    OpenAIRE

    Morgan, Hugh P.; McNae, Iain W.; Matthew W Nowicki; Hannaert, Véronique; Michels, Paul A M; Fothergill-Gilmore, Linda A.; Walkinshaw, Malcolm D.

    2010-01-01

    Allosteric regulation provides a rate management system for enzymes involved in many cellular processes. Ligand-controlled regulation is easily recognizable, but the underlying molecular mechanisms have remained elusive. We have obtained the first complete series of allosteric structures, in all possible ligated states, for the tetrameric enzyme, pyruvate kinase, from Leishmania mexicana. The transition between inactive T-state and active R-state is accompanied by a simple symmetrical 6° rigi...

  13. Allosteric Partial Inhibition of Monomeric Proteases. Sulfated Coumarins Induce Regulation, not just Inhibition, of Thrombin

    OpenAIRE

    Stephen Verespy III; Mehta, Akul Y.; Daniel Afosah; Al-Horani, Rami A.; Desai, Umesh R.

    2016-01-01

    Allosteric partial inhibition of soluble, monomeric proteases can offer major regulatory advantages, but remains a concept on paper to date; although it has been routinely documented for receptors and oligomeric proteins. Thrombin, a key protease of the coagulation cascade, displays significant conformational plasticity, which presents an attractive opportunity to discover small molecule probes that induce sub-maximal allosteric inhibition. We synthesized a focused library of some 36 sulfated...

  14. Fumarate analogs act as allosteric inhibitors of the human mitochondrial NAD(P+-dependent malic enzyme.

    Directory of Open Access Journals (Sweden)

    Ju-Yi Hsieh

    Full Text Available Human mitochondrial NAD(P+-dependent malic enzyme (m-NAD(P-ME is allosterically activated by the four-carbon trans dicarboxylic acid, fumarate. Previous studies have suggested that the dicarboxylic acid in a trans conformation around the carbon-carbon double bond is required for the allosteric activation of the enzyme. In this paper, the allosteric effects of fumarate analogs on m-NAD(P-ME are investigated. Two fumarate-insensitive mutants, m-NAD(P-ME_R67A/R91A and m-NAD(P-ME_K57S/E59N/K73E/D102S, as well as c-NADP-ME, were used as the negative controls. Among these analogs, mesaconate, trans-aconitate, monomethyl fumarate and monoethyl fumarate were allosteric activators of the enzyme, while oxaloacetate, diethyl oxalacetate, and dimethyl fumarate were found to be allosteric inhibitors of human m-NAD(P-ME. The IC50 value for diethyl oxalacetate was approximately 2.5 mM. This paper suggests that the allosteric inhibitors may impede the conformational change from open form to closed form and therefore inhibit m-NAD(P-ME enzyme activity.

  15. Structural Features of Ion Transport and Allosteric Regulation in Sodium-Calcium Exchanger (NCX) Proteins.

    Science.gov (United States)

    Giladi, Moshe; Tal, Inbal; Khananshvili, Daniel

    2016-01-01

    Na(+)/Ca(2+) exchanger (NCX) proteins extrude Ca(2+) from the cell to maintain cellular homeostasis. Since NCX proteins contribute to numerous physiological and pathophysiological events, their pharmacological targeting has been desired for a long time. This intervention remains challenging owing to our poor understanding of the underlying structure-dynamic mechanisms. Recent structural studies have shed light on the structure-function relationships underlying the ion-transport and allosteric regulation of NCX. The crystal structure of an archaeal NCX (NCX_Mj) along with molecular dynamics simulations and ion flux analyses, have assigned the ion binding sites for 3Na(+) and 1Ca(2+), which are being transported in separate steps. In contrast with NCX_Mj, eukaryotic NCXs contain the regulatory Ca(2+)-binding domains, CBD1 and CBD2, which affect the membrane embedded ion-transport domains over a distance of ~80 Å. The Ca(2+)-dependent regulation is ortholog, isoform, and splice-variant dependent to meet physiological requirements, exhibiting either a positive, negative, or no response to regulatory Ca(2+). The crystal structures of the two-domain (CBD12) tandem have revealed a common mechanism involving a Ca(2+)-driven tethering of CBDs in diverse NCX variants. However, dissociation kinetics of occluded Ca(2+) (entrapped at the two-domain interface) depends on the alternative-splicing segment (at CBD2), thereby representing splicing-dependent dynamic coupling of CBDs. The HDX-MS, SAXS, NMR, FRET, equilibrium (45)Ca(2+) binding and stopped-flow techniques provided insights into the dynamic mechanisms of CBDs. Ca(2+) binding to CBD1 results in a population shift, where more constraint conformational states become highly populated without global conformational changes in the alignment of CBDs. This mechanism is common among NCXs. Recent HDX-MS studies have demonstrated that the apo CBD1 and CBD2 are stabilized by interacting with each other, while Ca(2+) binding to CBD1

  16. Structural basis for allosteric regulation of human ribonucleotide reductase by nucleotide-induced oligomerization

    Energy Technology Data Exchange (ETDEWEB)

    Fairman, James Wesley; Wijerathna, Sanath Ranjan; Ahmad, Md Faiz; Xu, Hai; Nakano, Ryo; Jha, Shalini; Prendergast, Jay; Welin, R. Martin; Flodin, Susanne; Roos, Annette; Nordlund, Pär; Li, Zongli; Walz, Thomas; Dealwis, Chris Godfrey (Case Western); (Harvard-Med); (Karolinska); (Tennessee-K)

    2011-07-25

    Ribonucleotide reductase (RR) is an {alpha}{sub n}{beta}{sub n} (RR1-RR2) complex that maintains balanced dNTP pools by reducing NDPs to dNDPs. RR1 is the catalytic subunit, and RR2 houses the free radical required for catalysis. RR is allosterically regulated by its activator ATP and its inhibitor dATP, which regulate RR activity by inducing oligomerization of RR1. Here, we report the first X-ray structures of human RR1 bound to TTP alone, dATP alone, TTP-GDP, TTP-ATP, and TTP-dATP. These structures provide insights into regulation of RR by ATP or dATP. At physiological dATP concentrations, RR1 forms inactive hexamers. We determined the first X-ray structure of the RR1-dATP hexamer and used single-particle electron microscopy to visualize the {alpha}{sub 6}-{beta}{beta}'-dATP holocomplex. Site-directed mutagenesis and functional assays confirm that hexamerization is a prerequisite for inhibition by dATP. Our data indicate a mechanism for regulating RR activity by dATP-induced oligomerization.

  17. Allosteric effects in binuclear homo- and heterometallic triple-stranded lanthanide podates.

    Science.gov (United States)

    Ryan, Patrick E; Canard, Gabriel; Koeller, Sylvain; Bocquet, Bernard; Piguet, Claude

    2012-09-17

    This work illustrates a simple approach for deciphering and exploiting the various free energy contributions to the global complexation process leading to the binuclear triple-stranded podates [Ln(2)(L9)](6+) (Ln is a trivalent lanthanide). Despite the larger microscopic affinities exhibited by the binding sites for small Ln(3+), the stability constants measured for [Ln(2)(L9)](6+) decrease along the lanthanide series; a phenomenon which can be ascribed to the severe enthalpic penalty accompanying the intramolecular cyclization around small Ln(III), combined with increasing anticooperative allosteric interligand interactions. Altogether, the microscopic thermodynamic characteristics predict β(1,1,1)(La,Lu,L9)/β(1,1,1)(Lu,La,L9) = 145 for the ratio of the formation constants of the target heterobimetallic [LaLu(L9)](6+) and [LuLa(L9)](6+) microspecies, a value in line with the quantitative preparation (>90%) of [LaLu(L9)](6+) at millimolar concentrations. Preliminary NMR titrations indeed confirm the rare thermodynamic programming of a pure heterometallic f-f' complex. PMID:22946598

  18. Virus assembly and maturation: auto-regulation through allosteric molecular switches.

    Science.gov (United States)

    Domitrovic, Tatiana; Movahed, Navid; Bothner, Brian; Matsui, Tsutomu; Wang, Qiu; Doerschuk, Peter C; Johnson, John E

    2013-05-13

    We generalize the concept of allostery from the traditional non-active-site control of enzymes to virus maturation. Virtually, all animal viruses transition from a procapsid noninfectious state to a mature infectious state. The procapsid contains an encoded chemical program that is executed following an environmental cue. We developed an exceptionally accessible virus system for the study of the activators of maturation and the downstream consequences that result in particle stability and infectivity. Nudaurelia capensis omega virus (NωV) is a T=4 icosahedral virus that undergoes a dramatic maturation in which the 490-Å spherical procapsid condenses to a 400-Å icosahedral-shaped capsid with associated specific auto-proteolysis and stabilization. Employing X-ray crystallography, time-resolved electron cryo-microscopy and hydrogen/deuterium exchange as well as biochemistry, it was possible to define the mechanisms of allosteric communication among the four quasi-equivalent subunits in the icosahedral asymmetric unit. These gene products undergo proteolysis at different rates, dependent on quaternary structure environment, while particle stability is conferred globally following only a few local subunit transitions. We show that there is a close similarity between the concepts of tensegrity (associated with geodesic domes and mechanical engineering) and allostery (associated with biochemical control mechanisms). PMID:23485419

  19. Optimization of allosteric MEK inhibitors. Part 2: Taming the sulfamide group balances compound distribution properties.

    Science.gov (United States)

    Hartung, Ingo V; Hammer, Stefanie; Hitchcock, Marion; Neuhaus, Roland; Scholz, Arne; Siemeister, Gerhard; Bohlmann, Rolf; Hillig, Roman C; Pühler, Florian

    2016-01-01

    Recently, we had identified an unexplored pocket adjacent to the known binding site of allosteric MEK inhibitors which allowed us to design highly potent and in vivo efficacious novel inhibitors. We now report that our initial preclinical candidate, featuring a phenoxy side chain with a sulfamide capping group, displayed human carbonic anhydrase off-target activity and species-dependent blood cell accumulation, which prevented us from advancing this candidate further. Since this sulfamide MEK inhibitor displayed an exceptionally favorable PK profile with low brain penetration potential despite being highly oral bioavailable, we elected to keep the sulfamide capping group intact while taming its unwanted off-target activity by optimizing the structural surroundings. Introduction of a neighboring fluorine atom or installation of a methylene linker reduced hCA potency sufficiently, at the cost of MEK target potency. Switching to a higher fluorinated central core reinstated high MEK potency, leading to two new preclinical candidates with long half-lives, high bioavailabilities, low brain penetration potential and convincing efficacy in a K-Ras-mutated A549 xenograft model. PMID:26611920

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

    International Nuclear Information System (INIS)

    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

  1. 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.

  2. Role of a novel PH-kinase domain interface in PKB/Akt regulation: structural mechanism for allosteric inhibition.

    Directory of Open Access Journals (Sweden)

    Véronique Calleja

    2009-01-01

    Full Text Available Protein kinase B (PKB/Akt belongs to the AGC superfamily of related serine/threonine protein kinases. It is a key regulator downstream of various growth factors and hormones and is involved in malignant transformation and chemo-resistance. Full-length PKB protein has not been crystallised, thus studying the molecular mechanisms that are involved in its regulation in relation to its structure have not been simple. Recently, the dynamics between the inactive and active conformer at the molecular level have been described. The maintenance of PKB's inactive state via the interaction of the PH and kinase domains prevents its activation loop to be phosphorylated by its upstream activator, phosphoinositide-dependent protein kinase-1 (PDK1. By using a multidisciplinary approach including molecular modelling, classical biochemical assays, and Förster resonance energy transfer (FRET/two-photon fluorescence lifetime imaging microscopy (FLIM, a detailed model depicting the interaction between the different domains of PKB in its inactive conformation was demonstrated. These findings in turn clarified the molecular mechanism of PKB inhibition by AKT inhibitor VIII (a specific allosteric inhibitor and illustrated at the molecular level its selectivity towards different PKB isoforms. Furthermore, these findings allude to the possible function of the C-terminus in sustaining the inactive conformer of PKB. This study presents essential insights into the quaternary structure of PKB in its inactive conformation. An understanding of PKB structure in relation to its function is critical for elucidating its mode of activation and discovering how to modulate its activity. The molecular mechanism of inhibition of PKB activation by the specific drug AKT inhibitor VIII has critical implications for determining the mechanism of inhibition of other allosteric inhibitors and for opening up opportunities for the design of new generations of modulator drugs.

  3. Steady-state kinetics and analysis of pH dependence on wild-type and a modified allosteric Pseudomonas aeruginosa ornithine carbamoyltransferase containing the replacement of glutamate 105 by alanine.

    Science.gov (United States)

    Tricot, C; Nguyen, V T; Stalon, V

    1993-08-01

    The substitution of alanine for glutamate at position 105 (E105A) of the allosteric ornithine carbamoyltransferase (OTCase) of Pseudomonas aeruginosa abolishes the carbamoylphosphate (CP) cooperativity observed in the wild-type enzyme. A kinetic analysis of [E105A]OTCase was performed in order to determine the mechanism of the reaction. The results of initial velocity and inhibition studies are consistent with an ordered mechanism with CP as the first substrate to add to the enzyme. In addition, similar studies have been made using the wild-type enzyme in the presence of the activator, phosphate. The results are similar to those obtained with [E105A]OTCase indicating that the residue E105 is critical for the allosteric transition of the wild-type enzyme. The activities of the wild-type allosteric OTCase and of [E105A]OTCase have been studied in the pH range 5.8-8.2 in the absence and in the presence of positive and negative effectors. The sigmoid saturation of OTCases by CP has been analyzed according to the Hill equation. At low pH values, CP cooperativity is low in the wild-type enzyme but cooperativity and [S]CP0.5 values increase markedly with pH. For [E105A]OTCase, the saturation by CP is hyperbolic at all pH values; in this modified enzyme, the presence of spermidine, an allosteric inhibitor of the wild-type enzyme, results in an inhibition which induces CP cooperativity. Thus, the ionization of the residue E105 apparently results in a conformational change in the wild-type enzyme which modifies the catalytic site. Since the [E105A] enzyme retains the heterotropic effects of the wild-type enzyme, other structural features are required for the allosteric transition in the wild-type catabolic OTCase. PMID:8102605

  4. The Ascaris suum nicotinic receptor, ACR-16, as a drug target: Four novel negative allosteric modulators from virtual screening.

    Science.gov (United States)

    Zheng, Fudan; Robertson, Alan P; Abongwa, Melanie; Yu, Edward W; Martin, Richard J

    2016-04-01

    Soil-transmitted helminth infections in humans and livestock cause significant debility, reduced productivity and economic losses globally. There are a limited number of effective anthelmintic drugs available for treating helminths infections, and their frequent use has led to the development of resistance in many parasite species. There is an urgent need for novel therapeutic drugs for treating these parasites. We have chosen the ACR-16 nicotinic acetylcholine receptor of Ascaris suum (Asu-ACR-16), as a drug target and have developed three-dimensional models of this transmembrane protein receptor to facilitate the search for new bioactive compounds. Using the human α7 nAChR chimeras and Torpedo marmorata nAChR for homology modeling, we defined orthosteric and allosteric binding sites on the Asu-ACR-16 receptor for virtual screening. We identified four ligands that bind to sites on Asu-ACR-16 and tested their activity using electrophysiological recording from Asu-ACR-16 receptors expressed in Xenopus oocytes. The four ligands were acetylcholine inhibitors (SB-277011-A, IC50, 3.12 ± 1.29 μM; (+)-butaclamol Cl, IC50, 9.85 ± 2.37 μM; fmoc-1, IC50, 10.00 ± 1.38 μM; fmoc-2, IC50, 16.67 ± 1.95 μM) that behaved like negative allosteric modulators. Our work illustrates a structure-based in silico screening method for seeking anthelmintic hits, which can then be tested electrophysiologically for further characterization. PMID:27054065

  5. 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. PMID:26503377

  6. 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

  7. 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

  8. Are AMPA receptor positive allosteric modulators potential pharmacotherapeutics for addiction?

    Science.gov (United States)

    Watterson, Lucas R; Olive, M Foster

    2013-01-01

    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. PMID:24380895

  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. Allosteric modulation of metabotropic glutamate receptors by chloride ions.

    Science.gov (United States)

    Tora, Amélie S; Rovira, Xavier; Dione, Ibrahima; Bertrand, Hugues-Olivier; Brabet, Isabelle; De Koninck, Yves; Doyon, Nicolas; Pin, Jean-Philippe; Acher, Francine; Goudet, Cyril

    2015-10-01

    Metabotropic glutamate receptors (mGluRs) play key roles in the modulation of many synapses. Chloride (Cl(-)) is known to directly bind and regulate the function of different actors of neuronal activity, and several studies have pointed to the possible modulation of mGluRs by Cl(-). Herein, we demonstrate that Cl(-) behaves as a positive allosteric modulator of mGluRs. For example, whereas glutamate potency was 3.08 ± 0.33 μM on metabotropic glutamate (mGlu) 4 receptors in high-Cl(-) buffer, signaling activity was almost abolished in low Cl(-) in cell-based assays. Cl(-) potency was 78.6 ± 3.5 mM. Cl(-) possesses a high positive cooperativity with glutamate (Hill slope ≈6 on mGlu4), meaning that small variations in [Cl(-)] lead to large variations in glutamate action. Using molecular modeling and mutagenesis, we have identified 2 well-conserved Cl(-) binding pockets in the extracellular domain of mGluRs. Moreover, modeling of activity-dependent Cl(-) variations at GABAergic synapses suggests that these variations may be compatible with a dynamic modulation of the most sensitive mGluRs present in these synapses. Taken together, these data reveal a necessary role of Cl(-) for the glutamate activation of many mGluRs. Exploiting Cl(-) binding pockets may yield to the development of innovative regulators of mGluR activity. PMID:26116702

  11. Allosteric receptor activation by the plant peptide hormone phytosulfokine.

    Science.gov (United States)

    Wang, Jizong; Li, Hongju; Han, Zhifu; Zhang, Heqiao; Wang, Tong; Lin, Guangzhong; Chang, Junbiao; Yang, Weicai; Chai, Jijie

    2015-09-10

    Phytosulfokine (PSK) is a disulfated pentapeptide that has a ubiquitous role in plant growth and development. PSK is perceived by its receptor PSKR, a leucine-rich repeat receptor kinase (LRR-RK). The mechanisms underlying the recognition of PSK, the activation of PSKR and the identity of the components downstream of the initial binding remain elusive. Here we report the crystal structures of the extracellular LRR domain of PSKR in free, PSK- and co-receptor-bound forms. The structures reveal that PSK interacts mainly with a β-strand from the island domain of PSKR, forming an anti-β-sheet. The two sulfate moieties of PSK interact directly with PSKR, sensitizing PSKR recognition of PSK. Supported by biochemical, structural and genetic evidence, PSK binding enhances PSKR heterodimerization with the somatic embryogenesis receptor-like kinases (SERKs). However, PSK is not directly involved in PSKR-SERK interaction but stabilizes PSKR island domain for recruitment of a SERK. Our data reveal the structural basis for PSKR recognition of PSK and allosteric activation of PSKR by PSK, opening up new avenues for the design of PSKR-specific small molecules. PMID:26308901

  12. Allosteric coupling from G protein to the agonist-binding pocket in GPCRs.

    Science.gov (United States)

    DeVree, Brian T; Mahoney, Jacob P; Vélez-Ruiz, Gisselle A; Rasmussen, Soren G F; Kuszak, Adam J; Edwald, Elin; Fung, Juan-Jose; Manglik, Aashish; Masureel, Matthieu; Du, Yang; Matt, Rachel A; Pardon, Els; Steyaert, Jan; Kobilka, Brian K; Sunahara, Roger K

    2016-07-01

    G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades. Crystallographic evidence from a prototypic GPCR, the β2-adrenergic receptor (β2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the β2AR stabilizes a ‘closed’ receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR–G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity. PMID:27362234

  13. Positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors.

    Science.gov (United States)

    Jakubík, J; Bacáková, L; El-Fakahany, E E; Tucek, S

    1997-07-01

    It is well known that allosteric modulators of muscarinic acetylcholine receptors can both diminish and increase the affinity of receptors for their antagonists. We investigated whether the allosteric modulators can also increase the affinity of receptors for their agonists. Twelve agonists and five allosteric modulators were tested in experiments on membranes of CHO cells that had been stably transfected with genes for the M1-M4 receptor subtypes. Allosterically induced changes in the affinities for agonists were computed from changes in the ability of a fixed concentration of each agonist to compete with [3H]N-methylscopolamine for the binding to the receptors in the absence and the presence of varying concentrations of allosteric modulators. The effects of allosteric modulators varied greatly depending on the agonists and the subtypes of receptors. The affinity for acetylcholine was augmented by (-)-eburnamonine on the M2 and M4 receptors and by brucine on the M1 and M3 receptors. Brucine also enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pilocarpine, 3-(3-pentylthio-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1- methylpyridine (pentylthio-TZTP), oxotremorine-M, and McN-A-343 on the M1, M3, and M4 receptors, for pentylthio-TZTP on the M2 receptors, and for arecoline on the M3 receptors. (-)-Eburnamonine enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pentylthio-TZTP, pilocarpine, oxotremorine and oxotremorine-M on the M2 receptors and for pilocarpine on the M4 receptors. Vincamine, strychnine, and alcuronium displayed fewer positive allosteric interactions with the agonists, but each allosteric modulator displayed positive cooperativity with at least one agonist on at least one muscarinic receptor subtype. The highest degrees of positive cooperativity were observed between (-)-eburnamonine and pilocarpine and (-)-eburnamonine and oxotremorine-M on the M2 receptors (25- and 7-fold increases in

  14. 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.

  15. Identifying Allosteric Hotspots with Dynamics: Application to Inter- and Intra-species Conservation.

    Science.gov (United States)

    Clarke, Declan; Sethi, Anurag; Li, Shantao; Kumar, Sushant; Chang, Richard W F; Chen, Jieming; Gerstein, Mark

    2016-05-01

    The rapidly growing volume of data being produced by next-generation sequencing initiatives is enabling more in-depth analyses of conservation than previously possible. Deep sequencing is uncovering disease loci and regions under selective constraint, despite the fact that intuitive biophysical reasons for such constraint are sometimes absent. Allostery may often provide the missing explanatory link. We use models of protein conformational change to identify allosteric residues by finding essential surface pockets and information-flow bottlenecks, and we develop a software tool that enables users to perform this analysis on their own proteins of interest. Though fundamentally 3D-structural in nature, our analysis is computationally fast, thereby allowing us to run it across the PDB and to evaluate general properties of predicted allosteric residues. We find that these tend to be conserved over diverse evolutionary time scales. Finally, we highlight examples of allosteric residues that help explain poorly understood disease-associated variants. PMID:27066750

  16. Hemoglobin and the origins of the concept of allosterism.

    Science.gov (United States)

    Edsall, J T

    1980-02-01

    heterotropic interactions. Brief final comments relate to the evolution of the concept of reversible conformational transitions as the basis for both homotropic and heterotropic interactions in allosteric proteins. PMID:6986293

  17. Steric and allosteric effects of fatty acids on the binding of warfarin to human serum albumin revealed by molecular dynamics and free energy calculations.

    Science.gov (United States)

    Fujiwara, Shin-Ichi; Amisaki, Takashi

    2011-01-01

    Human serum albumin (HSA) binds with drugs and fatty acids (FAs). This study was initiated to elucidate the relationship between the warfarin binding affinity of HSA and the positions of bound FA molecules. Molecular dynamics simulations of 11 HSA-warfarin-myristate complexes were performed. HSA-warfarin binding free energy was then calculated for each of the complexes by the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. The results indicated that the magnitude of the binding free energy was smaller in HSA-warfarin complexes that had 4 or more myristate molecules than in complexes with no myristate molecules. The unfavorable effect on the HSA-warfarin binding affinity was caused sterically by the binding of a myristate molecule to the FA binding site closest to the warfarin binding site. On the other hand, the magnitude of HSA-warfarin binding free energy was largest when 3 myristate molecules were bound to the high-affinity sites. The strongest HSA-warfarin binding was attributable to favorable entropic contribution related to larger atomic fluctuations of the amino acid residues at the warfarin binding site. In the binding of 2 myristate molecules to the sites with the highest and second-highest affinities, allosteric modulation that enhanced electrostatic interactions between warfarin and some of the amino acid residues around the warfarin binding site was observed. This study clarified the structural and energetic properties of steric/allosteric effects of FAs on the HSA-warfarin binding affinity and illustrated the approach to analyze protein-ligand interactions in situations such that multiple ligands bind to the other sites of the protein. PMID:21720037

  18. 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

  19. Molecular Motions as a Drug Target: Mechanistic Simulations of Anthrax Toxin Edema Factor Function Led to the Discovery of Novel Allosteric Inhibitors

    Directory of Open Access Journals (Sweden)

    Arnaud Blondel

    2012-07-01

    Full Text Available Edema Factor (EF is a component of Bacillus anthracis toxin essential for virulence. Its adenylyl cyclase activity is induced by complexation with the ubiquitous eukaryotic cellular protein, calmodulin (CaM. EF and its complexes with CaM, nucleotides and/or ions, have been extensively characterized by X-ray crystallography. Those structural data allowed molecular simulations analysis of various aspects of EF action mechanism, including the delineation of EF and CaM domains through their association energetics, the impact of calcium binding on CaM, and the role of catalytic site ions. Furthermore, a transition path connecting the free inactive form to the CaM-complexed active form of EF was built to model the activation mechanism in an attempt to define an inhibition strategy. The cavities at the surface of EF were determined for each path intermediate to identify potential sites where the binding of a ligand could block activation. A non-catalytic cavity (allosteric was found to shrink rapidly at early stages of the path and was chosen to perform virtual screening. Amongst 18 compounds selected in silico and tested in an enzymatic assay, 6 thiophen ureidoacid derivatives formed a new family of EF allosteric inhibitors with IC50 as low as 2 micromolars.

  20. Intramolecular signal transmission in enterobacterial aspartate transcarbamylases II. Engineering co-operativity and allosteric regulation in the aspartate transcarbamylase of Erwinia herbicola.

    Science.gov (United States)

    Cunin, R; Rani, C S; Van Vliet, F; Wild, J R; Wales, M

    1999-12-17

    The aspartate transcarbamylase (ATCase) from Erwinia herbicola differs from the other investigated enterobacterial ATCases by its absence of homotropic co-operativity toward the substrate aspartate and its lack of response to ATP which is an allosteric effector (activator) of this family of enzymes. Nevertheless, the E. herbicola ATCase has the same quaternary structure, two trimers of catalytic chains with three dimers of regulatory chains ((c3)2(r2)3), as other enterobacterial ATCases and shows extensive primary structure conservation. In (c3)2(r2)3 ATCases, the association of the catalytic subunits c3 with the regulatory subunits r2 is responsible for the establishment of positive co-operativity between catalytic sites for the binding of aspartate and it dictates the pattern of allosteric response toward nucleotide effectors. Alignment of the primary sequence of the regulatory polypeptides from the E. herbicola and from the paradigmatic Escherichia coli ATCases reveals major blocks of divergence, corresponding to discrete structural elements in the E. coli enzyme. Chimeric ATCases were constructed by exchanging these blocks of divergent sequence between these two ATCases. It was found that the amino acid composition of the outermost beta-strand of a five-stranded beta-sheet in the effector-binding domain of the regulatory polypeptide is responsible for the lack of co-operativity and response to ATP of the E. herbicola ATCase. A novel structural element involved in allosteric signal recognition and transmission in this family of ATCases was thus identified. PMID:10600394

  1. Role of connecting loop I in catalysis and allosteric regulation of human glucokinase.

    Science.gov (United States)

    Martinez, Juliana A; Larion, Mioara; Conejo, Maria S; Porter, Carol M; Miller, Brian G

    2014-07-01

    Glucokinase (GCK, hexokinase IV) is a monomeric enzyme with a single glucose binding site that displays steady-state kinetic cooperativity, a functional characteristic that affords allosteric regulation of GCK activity. Structural evidence suggests that connecting loop I, comprised of residues 47-71, facilitates cooperativity by dictating the rate and scope of motions between the large and small domains of GCK. Here we investigate the impact of varying the length and amino acid sequence of connecting loop I upon GCK cooperativity. We find that sequential, single amino acid deletions from the C-terminus of connecting loop I cause systematic decreases in cooperativity. Deleting up to two loop residues leaves the kcat value unchanged; however, removing three or more residues reduces kcat by 1000-fold. In contrast, the glucose K0.5 and KD values are unaffected by shortening the connecting loop by up to six residues. Substituting alanine or glycine for proline-66, which adopts a cis conformation in some GCK crystal structures, does not alter cooperativity, indicating that cis/trans isomerization of this loop residue does not govern slow conformational reorganizations linked to hysteresis. Replacing connecting loop I with the corresponding loop sequence from the catalytic domain of the noncooperative isozyme human hexokinase I (HK-I) eliminates cooperativity without impacting the kcat and glucose K0.5 values. Our results indicate that catalytic turnover requires a minimal length of connecting loop I, whereas the loop has little impact upon the binding affinity of GCK for glucose. We propose a model in which the primary structure of connecting loop I affects cooperativity by influencing conformational dynamics, without altering the equilibrium distribution of GCK conformations. PMID:24723372

  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)

    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. Seismic Stratigraphic Correlations Between ODP Sites 742 and 1166: Implications for Depositional Paleoenvironments in Prydz Bay, Antarctica

    Science.gov (United States)

    Erohina, T.; Cooper, A.; Handwerger, D.; Dunbar, R.

    2002-12-01

    Prydz Bay lies at the mouth of the Lambert glacier system and drains approximately 20% of the East Antarctic Ice Sheet. The continental shelf there contains a sediment record of early Cenozoic to late Neogene Antarctic glaciation. New high-resolution seismic reflection data were recorded by the R/V N.B. PALMER in the Prydz Bay basin over the ~40 km between ODP Sites 1166 (Leg 188) and 742 (Leg 119) to link acoustic and lithologic features at the drill sites and help decipher regional paleoenvironments. N. B. PALMER Line 01-1-4 confirmed that Site 1166 drilled an older section than Site 742. The preglacial to glacial unconformity at Site 1166 lies about 50 m below Site 742. The Paleogene units mostly truncate or taper out between the drill sites except for a thick early-glacial fluvial-deltaic sand unit at Site 1166 that may extend laterally to the base of Site 742. A flooding surface lies at the top of this sand unit at Site 1166, and not within the unit as previously reported. Prior correlations of thin Pliocene diatomaceous layers within massive diamictites, based on downhole logging and biostratigraphy at the two sites, cannot be confirmed (or denied) by the seismic-reflection data -- higher resolution seismic data are needed. For the study region, we infer a progression from a pre-glacial setting on a low-relief alluvial fan to glaciomarine and subglacial settings. Late Cretaceous lacustrine and lagoonal environments evolved to a late Eocene broad fluvial channel system or outwash plain. Marine transgression infilled and buried the channel system with glacial deposits that were extensively eroded during Oligocene to late Miocene times. Late Neogene environments were mostly subglacial with episodes of reduced ice and biogenic deposition.

  4. The cyclic di-nucleotide c-di-AMP is an allosteric regulator of metabolic enzyme function

    Science.gov (United States)

    Precit, Mimi; Delince, Matthieu; Pensinger, Daniel; Huynh, TuAnh Ngoc; Jurado, Ashley R.; Goo, Young Ah; Sadilek, Martin; Iavarone, Anthony T.; Sauer, John-Demian; Tong, Liang; Woodward, Joshua J.

    2014-01-01

    SUMMARY Cyclic di-adenosine monophosphate (c-di-AMP) is a broadly conserved second messenger required for bacterial growth and infection. However, the molecular mechanisms of c-di-AMP signaling are still poorly understood. Using a chemical proteomics screen for c-di-AMP interacting proteins in the pathogen Listeria monocytogenes, we identified several broadly conserved protein receptors, including the central metabolic enzyme pyruvate carboxylase (LmPC). Biochemical and crystallographic studies of the LmPC-c-di-AMP interaction revealed a previously unrecognized allosteric regulatory site 25 Å from the active site. Mutations in this site disrupted c-di-AMP binding and affected enzyme catalysis of LmPC as well as PC from pathogenic Enterococcus faecalis. C-di-AMP depletion resulted in altered metabolic activity in L. monocytogenes. Correction of this metabolic imbalance rescued bacterial growth, reduced bacterial lysis, and resulted in enhanced bacterial burdens during infection. These findings greatly expand the c-di-AMP signaling repertoire and reveal a central metabolic regulatory role for a cyclic di-nucleotide. PMID:25215494

  5. Nonlinear excitations match correlated motions unveiled by NMR in proteins: a new perspective on allosteric cross-talk

    International Nuclear Information System (INIS)

    In this paper we propose a novel theoretical framework for interpreting long-range dynamical correlations unveiled in proteins through NMR measurements. The theoretical rationale relies on the hypothesis that correlated motions in proteins may be reconstructed as large-scale, collective modes sustained by long-lived nonlinear vibrations known as discrete breathers (DB) localized at key, hot-spot sites. DBs are spatially localized modes, whose nonlinear nature hinders resonant coupling with the normal modes, thus conferring them long lifetimes as compared to normal modes. DBs have been predicted to exist in proteins, localized at few hot-spot residues typically within the stiffest portions of the structure. We compute DB modes analytically in the framework of the nonlinear network model, showing that the displacement patterns of many DBs localized at key sites match to a remarkable extent the experimentally uncovered correlation blueprint. The computed dispersion relations prove that it is physically possible for some of these DBs to be excited out of thermal fluctuations at room temperature. Based on our calculations, we speculate that transient energy redistribution among the vibrational modes in a protein might favor the emergence of DB-like bursts of long-lived energy at hot-spot sites with lifetimes in the ns range, able to sustain critical, function-encoding correlated motions. More generally, our calculations provide a novel quantitative tool to predict fold-spanning dynamical pathways of correlated residues that may be central to allosteric cross-talk in proteins. (paper)

  6. Site Productivity and Forest Carbon Stocks in the United States: Analysis and Implications for Forest Offset Project Planning

    Directory of Open Access Journals (Sweden)

    James E. Smith

    2012-06-01

    Full Text Available 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 guidance to managers planning land acquisition for forest-based greenhouse gas mitigation projects. Specifically, we summarize two condition class indicators of site productivity within the FIA forest inventory database—physclcd and siteclcd—as they relate to current aboveground live tree carbon stocks. Average carbon density is higher on more productive sites, but compared to the overall variability among sites, the differences are relatively small for all but the highest and lowest site classes. Some minor differences in eastern- versus western-forests were apparent in terms of how carbon on the least productive sites differed from most other forest land over time. Overall results suggest that xeric sites in most regions as well as sites that correspond to the lowest, non-productive classifications of forest land should preferentially not be used forestry-based greenhouse gas mitigation projects, but all other forest areas appear to be suitable.

  7. Papio Cranium from the Hominin-Bearing Site of Malapa: Implications for the Evolution of Modern Baboon Cranial Morphology and South African Plio-Pleistocene Biochronology.

    Directory of Open Access Journals (Sweden)

    Christopher C Gilbert

    Full Text Available A new partial cranium (UW 88-886 of the Plio-Pleistocene baboon Papio angusticeps from Malapa is identified, described and discussed. UW 88-886 represents the only non-hominin primate yet recovered from Malapa and is important both in the context of baboon evolution as well as South African hominin site biochronology. The new specimen may represent the first appearance of modern baboon anatomy and coincides almost perfectly with molecular divergence date estimates for the origin of the modern P. hamadryas radiation. The fact that the Malapa specimen is dated between ~2.026-2.36 million years ago (Ma also has implications for the biochronology of other South African Plio-Pleistocene sites where P. angusticeps is found.

  8. Allosteric transition and binding of small molecule effectors causes curvature change in central β-sheets of selected enzymes.

    Science.gov (United States)

    Tolonen, Ellen; Bueno, Brenda; Kulshreshta, Sanjeev; Cieplak, Piotr; Argáez, Miguel; Velázquez, Leticia; Stec, Boguslaw

    2011-04-01

    A quantitative description of allosteric transition remains a significant science challenge. Many allosteric enzymes contain a central β-sheet in their catalytic domain. When an allosteric protein undergoes the transition between T (tense) and R (relaxed) allosteric states, this central β-sheet undergoes a conformational change. A traditional method of measuring this change, the root mean square deviation (RMSD), appears to be inadequate to describe such changes in meaningful quantitative manner. We designed a novel quantitative method to demonstrate this conformational transition by measuring the change in curvature of the central β-sheet when enzymes transition between allosteric states. The curvature was established by calculating the semiaxes of a 3-D hyperboloid fitted by least squares to the Cα atomic positions of the β-sheet. The two enzymes selected for this study, fructose 1,6-bisphosphatase (FBPase) from pig kidney and aspartate carbamoyltransferase (ATCase) from E. coli, showed while transitioning between the allosteric states (T ⇔ R) a notable change in β-sheet curvature (∼5%) that results in a large lateral shift at the sheet's edge, which is necessary to convey the signal. The results suggest that the β-sheet participates in storing elastic energy associated with the transition. Establishing a tentative link between the energetics of the β-sheet in different allosteric states provides a more objective basis for the naming convention of allosteric states (tense or relaxed), and provides insight into the hysteretic nature of the transition. The approach presented here allows for a better understanding of the internal dynamics of allosteric enzymes by defining the domains that directly participate in the transition. PMID:20602244

  9. An Integrated Approach for Screening and Identification of Positive Allosteric Modulators of N-Methyl-D-Aspartate Receptors.

    Science.gov (United States)

    Jambrina, Enrique; Cerne, Rok; Smith, Emery; Scampavia, Louis; Cuadrado, Maria; Findlay, Jeremy; Krambis, Michael J; Wakulchik, Mark; Chase, Peter; Brunavs, Michael; Burris, Kevin D; Gallagher, Peter; Spicer, Timothy P; Ursu, Daniel

    2016-06-01

    N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an important role in synaptic plasticity and learning and memory formation. Malfunctioning of NMDARs, in particular the reduction in NMDAR activity, is thought to be implicated in major neurological disorders. NMDAR positive allosteric modulators (PAMs) represent potential therapeutic interventions for restoring normal NMDAR function. We report a novel screening approach for identification and characterization of NMDAR-PAMs. The approach combines high-throughput fluorescence imaging with automated electrophysiological recording of glutamate-evoked responses in HEK-293 cells expressing NR1/NR2A NMDAR subunits. Initial high-throughput screening (HTS) of a chemical library containing >810,000 compounds using a calcium flux assay in 1536-well plate format identified a total of 864 NMDAR-PAMs. Concentration response determination in both calcium flux and automated electrophysiological assays found several novel chemical series with EC50 values between 0.49 and 10 µM. A small subset (six series) was selected and analyzed for pharmacological properties, subtype selectivity, mode of action, and activity at native NMDARs. Our approach demonstrates the successful application of HTS functional assays that led to identification of NMDAR-PAMs providing the foundation for further medicinal chemistry work that may lead to novel therapies for treatment of cognitive impairment associated with Alzheimer's disease and schizophrenia. PMID:26838761

  10. The synergistic use of models and observations: understanding the mechanisms behind observed biomass dynamics at 14 Amazonian field sites and the implications for future biomass change

    Science.gov (United States)

    Levine, N. M.; Galbraith, D.; Christoffersen, B. J.; Imbuzeiro, H. A.; Restrepo-Coupe, N.; Malhi, Y.; Saleska, S. R.; Costa, M. H.; Phillips, O.; Andrade, A.; Moorcroft, P. R.

    2011-12-01

    The Amazonian rainforests play a vital role in global water, energy and carbon cycling. The sensitivity of this system to natural and anthropogenic disturbances therefore has important implications for the global climate. Some global models have predicted large-scale forest dieback and the savannization of Amazonia over the next century [Meehl et al., 2007]. While several studies have demonstrated the sensitivity of dynamic global vegetation models to changes in temperature, precipitation, and dry season length [e.g. Galbraith et al., 2010; Good et al., 2011], the ability of these models to accurately reproduce ecosystem dynamics of present-day transitional or low biomass tropical forests has not been demonstrated. A model-data intercomparison was conducted with four state-of-the-art terrestrial ecosystem models to evaluate the ability of these models to accurately represent structure, function, and long-term biomass dynamics over a range of Amazonian ecosystems. Each modeling group conducted a series of simulations for 14 sites including mature forest, transitional forest, savannah, and agricultural/pasture sites. All models were run using standard physical parameters and the same initialization procedure. Model results were compared against forest inventory and dendrometer data in addition to flux tower measurements. While the models compared well against field observations for the mature forest sites, significant differences were observed between predicted and measured ecosystem structure and dynamics for the transitional forest and savannah sites. The length of the dry season and soil sand content were good predictors of model performance. In addition, for the big leaf models, model performance was highest for sites dominated by late successional trees and lowest for sites with predominantly early and mid-successional trees. This study provides insight into tropical forest function and sensitivity to environmental conditions that will aid in predictions of the

  11. Modulation of Pantothenate Kinase 3 Activity by Small Molecules that Interact with the Substrate/Allosteric Regulatory Domain

    Energy Technology Data Exchange (ETDEWEB)

    Leonardi, Roberta; Zhang, Yong-Mei; Yun, Mi-Kyung; Zhou, Ruobing; Zeng, Fu-Yue; Lin, Wenwei; Cui, Jimmy; Chen, Taosheng; Rock, Charles O.; White, Stephen W.; Jackowski, Suzanne (SJCH)

    2010-09-27

    Pantothenate kinase (PanK) catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis. PanK3 is stringently regulated by acetyl-CoA and uses an ordered kinetic mechanism with ATP as the leading substrate. Biochemical analysis of site-directed mutants indicates that pantothenate binds in a tunnel adjacent to the active site that is occupied by the pantothenate moiety of the acetyl-CoA regulator in the PanK3 acetyl-CoA binary complex. A high-throughput screen for PanK3 inhibitors and activators was applied to a bioactive compound library. Thiazolidinediones, sulfonylureas and steroids were inhibitors, and fatty acyl-amides and tamoxifen were activators. The PanK3 activators and inhibitors either stimulated or repressed CoA biosynthesis in HepG2/C3A cells. The flexible allosteric acetyl-CoA regulatory domain of PanK3 also binds the substrates, pantothenate and pantetheine, and small molecule inhibitors and activators to modulate PanK3 activity.

  12. Insights into protein -- DNA interactions, stability and allosteric communications: A computational study of MutS-DNA recognition complexes

    Science.gov (United States)

    Negureanu, Lacramioara; Salsbury, Freddie

    2012-02-01

    DNA mismatch repair proteins (MMR) maintain genetic stability by recognizing and repairing mismatched bases and insertion/deletion loops mistakenly incorporated during DNA replication, and initiate cellular response to certain types of DNA damage. The most abundant MMR mismatch-binding factor in eukaryotes, MutS, recognizes and initiates the repair of base-base mismatches and small insertion/deletions. We performed molecular dynamics simulations on mismatched and damaged MutS-DNA complexes. A comprehensive DNA binding site analysis of relevant conformations shows that MutS proteins recognize the mismatched and platinum cross-linked DNA substrates in significantly different modes. Distinctive conformational changes associated with MutS binding to mismatched and damaged DNA have been identified and they provide insight into the involvement of MMR proteins in DNA-repair and DNA-damage pathways. Stability and allosteric interactions at the heterodimer interface associated with the mismatch and damage recognition step allow for prediction of key residues in MMR cancer-causing mutations. A rigorous hydrogen bonding analysis for ADP molecules at the ATPase binding sites is also presented. A large number of known MMR cancer causing mutations among the residues were found.

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

    Science.gov (United States)

    Lim, Siew Pheng; Noble, Christian Guy; Seh, Cheah Chen; Soh, Tingjin Sherryl; El Sahili, Abbas; Chan, Grace Kar Yarn; Lescar, Julien; Arora, Rishi; Benson, Timothy; Nilar, Shahul; Manjunatha, Ujjini; Wan, Kah Fei; Dong, Hongping; Xie, Xuping; Shi, Pei-Yong; Yokokawa, Fumiaki

    2016-08-01

    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. PMID:27500641

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

    Science.gov (United States)

    Lim, Siew Pheng; Noble, Christian Guy; Seh, Cheah Chen; Soh, Tingjin Sherryl; El Sahili, Abbas; Chan, Grace Kar Yarn; Lescar, Julien; Arora, Rishi; Benson, Timothy; Nilar, Shahul; Manjunatha, Ujjini; Wan, Kah Fei; Dong, Hongping; Xie, Xuping; Yokokawa, Fumiaki

    2016-01-01

    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. PMID:27500641

  15. 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.

  16. 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.

    in tektites and based on the isotope distribution pattern felt that a location in the Song Hong Basin (off Vietnam, South of Gulf of Tonkin (170N; 1070 E) could be a possible impact site. Further, geochemical and Rb-Sr isotopic investigations by Lee et... a hypothetical source crater and suggested Central Cambodia to be a candidate site. This was further refined with the addition of data by Burns and Glass (1989), Lee and Wei (2000), Glass (2003). Glass and Koeberl (2006) in addition...

  17. The Mousterian site of Zafarraya (Andalucia, Spain): dating and implications on the palaeolithic peopling processes of Western Europe

    International Nuclear Information System (INIS)

    The site of Zafarraya, near the NE boundary of the province of Malaga (spain), yielded a typical Mousterian assemblage with extended Levallois debitage and little or no Upper Palaeolithic influence. The human remains unearthed from the lower deposits display an indisputable Neanderthal morphology. Reliable 14C and Th/U dating of the site indicate the persistence of Neanderthals at least after 35 ka BP and of Mousterian industries after 30 ka BP in the south-western extremity of Europe. (authors). 18 refs., 1 tab

  18. An Empirical Study of the Effectiveness of Publicly-Funded "Structured On-Site Training": Implications for Policy and Practice

    Science.gov (United States)

    Gorman, Phil; Moore, Richard; Blake, Daniel; Phillips, Michael G.

    2004-01-01

    This article reports findings from an assessment of the effectiveness of privately administered "structured on-site training" (SOST) programs funded by the California Employment Training Panel. It reports on the characteristics of SOST programs that increased trainees' competitiveness in the internal and external labor markets. In addition, it…

  19. An Exploratory Study of Indian University Students' Use of Social Networking Web Sites: Implications for the Workplace

    Science.gov (United States)

    Agarwal, Shailja; Mital, Monika

    2009-01-01

    Social networking Web sites (SNWs) are online tools that have transformed the virtual encounters of the past that were technical and impersonal to today's virtual socialization that is truly nontechnical, social, and interpersonal. This article presents an exploratory study of Indian University students' use of SNWs. The results indicated that…

  20. 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)

    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

  1. 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)

    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.

  2. 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.

  3. 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

    2013-12-01

    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. PMID:24120673

  4. Targeted reengineering of protein geranylgeranyltransferase type I selectivity functionally implicates active-site residues in protein-substrate recognition.

    Science.gov (United States)

    Gangopadhyay, Soumyashree A; Losito, Erica L; Hougland, James L

    2014-01-21

    Posttranslational modifications are vital for the function of many proteins. Prenylation is one such modification, wherein protein geranylgeranyltransferase type I (GGTase-I) or protein farnesyltransferase (FTase) modify proteins by attaching a 20- or 15-carbon isoprenoid group, respectively, to a cysteine residue near the C-terminus of a target protein. These enzymes require a C-terminal Ca1a2X sequence on their substrates, with the a1, a2, and X residues serving as substrate-recognition elements for FTase and/or GGTase-I. While crystallographic structures of rat GGTase-I show a tightly packed and hydrophobic a2 residue binding pocket, consistent with a preference for moderately sized a2 residues in GGTase-I substrates, the functional impact of enzyme-substrate contacts within this active site remains to be determined. Using site-directed mutagenesis and peptide substrate structure-activity studies, we have identified specific active-site residues within rat GGTase-I involved in substrate recognition and developed novel GGTase-I variants with expanded/altered substrate selectivity. The ability to drastically alter GGTase-I selectivity mirrors similar behavior observed in FTase but employs mutation of a distinct set of structurally homologous active-site residues. Our work demonstrates that tunable selectivity may be a general phenomenon among multispecific enzymes involved in posttranslational modification and raises the possibility of variable substrate selectivity among GGTase-I orthologues from different organisms. Furthermore, the GGTase-I variants developed herein can serve as tools for studying GGTase-I substrate selectivity and the effects of prenylation pathway modifications on specific proteins. PMID:24344934

  5. Results of special monitoring programs at the sites of nuclear facilities: implications for environmental monitoring program (PVRA)

    International Nuclear Information System (INIS)

    In 2008, they occurred in the Spanish plants various events related to the existence on the sites of points or areas of radioactive contamination on the outside of buildings. The events were reported to the CSN in accordance with the provisions of the Instruction CSN IS-10. This presentation brings together the assessments provided, the result of the checks and the consequences of the programs. (Author)

  6. Site Productivity and Forest Carbon Stocks in the United States: Analysis and Implications for Forest Offset Project Planning

    OpenAIRE

    Smith, James E.; Coeli M. Hoover

    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 guidance to managers planning land acquisition for forest-based greenhouse gas mitigation projects. Specifically, we summariz...

  7. Allosteric nucleotide-binding site in the mitochondrial NADH:ubiquinone oxidoreductase (respiratory complex I)

    OpenAIRE

    Grivennikova, Vera G.; Gladyshev, Grigory V.; Vinogradov, Andrei D.

    2011-01-01

    The rotenone-insensitive NADH:hexaammineruthenium III (HAR) oxidoreductase reactions catalyzed by bovine heart and Yarrowia lipolytica submitochondrial particles or purified bovine complex I are stimulated by ATP and other purine nucleotides. The soluble fraction of mammalian complex I (FP) and prokaryotic complex I homolog NDH-1 in Paracoccus denitrificans plasma membrane lack stimulation of their activities by ATP. The stimulation appears as a decrease in apparent Km values for NADH and HAR...

  8. The Role of Distant Mutations and Allosteric Regulation on LovD Active Site Dynamics

    OpenAIRE

    Jiménez-Osés, Gonzalo; Osuna, Sílvia; Gao, Xue; Sawaya, Michael R.; Gilson, Lynne; Collier, Steven J.; Huisman, Gjalt W.; Yeates, Todd O; Tang, Yi; Houk, K. N.

    2014-01-01

    Natural enzymes have evolved to perform their cellular functions under complex selective pressures, which often require their catalytic activities to be regulated by other proteins. We contrasted a natural enzyme, LovD, which acts on a protein-bound (LovF) acyl substrate, with a laboratory-generated variant that was transformed by directed evolution to accept instead a small free acyl thioester, and no longer requires the acyl carrier protein. The resulting 29-mutant variant is 1000-fold more...

  9. Scenario-based modelling of mass transfer mechanisms at a petroleum contaminated field site-numerical implications.

    Science.gov (United States)

    Vasudevan, M; Nambi, Indumathi M; Suresh Kumar, G

    2016-06-15

    Knowledge about distribution of dissolved plumes and their influencing factors is essential for risk assessment and remediation of light non-aqueous phase liquid contamination in groundwater. Present study deals with the applicability of numerical model for simulating various hydro-geological scenarios considering non-uniform source distribution at a petroleum contaminated site in Chennai, India. The complexity associated with the hydrogeology of the site has limited scope for on-site quantification of petroleum pipeline spillage. The change in fuel composition under mass-transfer limited conditions was predicted by simultaneously comparing deviations in aqueous concentrations and activity coefficients (between Raoult's law and analytical approaches). The effects of source migration and weathering on the dissolution of major soluble fractions of petroleum fuel were also studied in relation to the apparent change in their activity coefficients and molar fractions. The model results were compared with field observations and found that field conditions were favourable for biodegradation, especially for the aromatic fraction (benzene and toluene (nearly 95% removal), polycyclic aromatic hydrocarbons (up to 65% removal) and xylene (nearly 45% removal). The results help to differentiate the effect of compositional non-ideality from rate-limited dissolution towards tailing of less soluble compounds (alkanes and trimethylbenzene). Although the effect of non-ideality decreased with distance from the source, the assumption of spatially varying residual saturation could effectively illustrate post-spill scenario by estimating the consequent decrease in mass transfer rate. PMID:27017268

  10. Luminescence ages for three 'Middle Palaeolithic' sites in the Nihewan Basin, northern China, and their archaeological and palaeoenvironmental implications

    Science.gov (United States)

    Guo, Yu-Jie; Li, Bo; Zhang, Jia-Fu; Yuan, Bao-Yin; Xie, Fei; Roberts, Richard Graham

    2016-05-01

    The Nihewan Basin is a key region for studying the Palaeolithic archaeology of East Asia. However, because of the lack of suitable dating methods and representative lithic technologies in this region, the 'Middle Palaeolithic' sites in this basin have been designated based mainly on stratigraphic correlation, which may be unreliable. In this study, three Palaeolithic sites, Motianling, Queergou and Banjingzi, which have been assigned previously to the 'Middle Palaeolithic', are dated based on luminescence dating of K-feldspar grains. Our results show that the cultural layers at Motianling, Queergou and Banjingzi have ages of 315 ± 13, 268 ± 13 and 86 ± 4 ka (corresponding to Marine Isotope Stages 9, 8 and 5), respectively, suggesting that Motianling and Queergou should be assigned to the Lower Palaeolithic, while the age of Banjingzi is consistent with a Middle Palaeolithic attribution. Our results suggest that reassessing the age of 'Middle Palaeolithic' sites in the Nihewan Basin, and elsewhere in North China, is crucial for understanding the presence or absence of the Middle Palaeolithic phase in China. Our dating results also indicate that the Sanggan River developed sometime between about 270 and 86 ka ago.

  11. 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

  12. The use of isomeric testosterone dimers to explore allosteric effects in substrate binding to cytochrome P450 CYP3A4.

    Science.gov (United States)

    Denisov, Ilia G; Mak, Piotr J; Grinkova, Yelena V; Bastien, Dominic; Bérubé, Gervais; Sligar, Stephen G; Kincaid, James R

    2016-05-01

    Cytochrome P450 CYP3A4 is the main drug-metabolizing enzyme in the human liver, being responsible for oxidation of 50% of all pharmaceuticals metabolized by human P450 enzymes. Possessing a large substrate binding pocket, it can simultaneously bind several substrate molecules and often exhibits a complex pattern of drug-drug interactions. In order to better understand structural and functional aspects of binding of multiple substrate molecules to CYP3A4 we used resonance Raman and UV-VIS spectroscopy to document the effects of binding of synthetic testosterone dimers of different configurations, cis-TST2 and trans-TST2. We directly demonstrate that the binding of two steroid molecules, which can assume multiple possible configurations inside the substrate binding pocket of monomeric CYP3A4, can lead to active site structural changes that affect functional properties. Using resonance Raman spectroscopy, we have documented perturbations in the ferric and Fe-CO states by these substrates, and compared these results with effects caused by binding of monomeric TST. While the binding of trans-TST2 yields results similar to those obtained with monomeric TST, the binding of cis-TST2 is much tighter and results in significantly more pronounced conformational changes of the porphyrin side chains and Fe-CO unit. In addition, binding of an additional monomeric TST molecule in the remote allosteric site significantly improves binding affinity and the overall spin shift for CYP3A4 with trans-TST2 dimer bound inside the substrate binding pocket. This result provides the first direct evidence for an allosteric effect of the peripheral binding site at the protein-membrane interface on the functional properties of CYP3A4. PMID:26774838

  13. 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.

  14. 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

    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...

  15. Seven years of observational atmospheric CO2 at a maritime site in northernmost Japan and its implications.

    Science.gov (United States)

    Zhu, Chunmao; Yoshikawa-Inoue, Hisayuki

    2015-08-15

    Surface atmospheric CO2 mixing ratio reflects both natural fluctuation of the carbon cycle and the effect of anthropogenic activities. Long-term observation of atmospheric CO2 forms the basis for model simulations of the carbon cycle both in the straightforward and the inversion ways. Atmospheric CO2 has been measured on Rishiri Island (45.1°N, 141.2°E) in the western North Pacific since May 2006. We report the first 7-year temporal CO2 variations from diurnal to inter-annual scales and the implications on the vegetation phenology. Diurnally, an obvious cycle appeared as a minimum in the afternoon and maximum at midnight in the summer months, caused by local vegetation. Seasonally, the maximum CO2 concentration appeared around the beginning of April, while the minimum appeared around the middle of August. This seasonal variation implied the natural cycle of terrestrial biological activities of the boreal forest, mostly in the east Eurasia. A mean growing season length of ~126 days was estimated. In the period from 2007 to 2012, the peak-to-peak amplitude increased until 2009 and decreased thereafter, with a mean value of 19.7 ppm. Inter-annually, atmospheric CO2 is increasing by a mean growth rate of 2.1 ppm year(-1). The study provides invaluable dataset and useful information to better understand the carbon cycle and its interaction with climate change. PMID:25911544

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

    International Nuclear Information System (INIS)

    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)

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

    International Nuclear Information System (INIS)

    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)

  18. Structural Determinants Defining the Allosteric Inhibition of an Essential Antibiotic Target.

    Science.gov (United States)

    Soares da Costa, Tatiana P; Desbois, Sebastien; Dogovski, Con; Gorman, Michael A; Ketaren, Natalia E; Paxman, Jason J; Siddiqui, Tanzeela; Zammit, Leanne M; Abbott, Belinda M; Robins-Browne, Roy M; Parker, Michael W; Jameson, Geoffrey B; Hall, Nathan E; Panjikar, Santosh; Perugini, Matthew A

    2016-08-01

    Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine biosynthesis pathway of bacteria. The pathway can be regulated by feedback inhibition of DHDPS through the allosteric binding of the end product, lysine. The current dogma states that DHDPS from Gram-negative bacteria are inhibited by lysine but orthologs from Gram-positive species are not. The 1.65-Å resolution structure of the Gram-negative Legionella pneumophila DHDPS and the 1.88-Å resolution structure of the Gram-positive Streptococcus pneumoniae DHDPS bound to lysine, together with comprehensive functional analyses, show that this dogma is incorrect. We subsequently employed our crystallographic data with bioinformatics, mutagenesis, enzyme kinetics, and microscale thermophoresis to reveal that lysine-mediated inhibition is not defined by Gram staining, but by the presence of a His or Glu at position 56 (Escherichia coli numbering). This study has unveiled the molecular determinants defining lysine-mediated allosteric inhibition of bacterial DHDPS. PMID:27427481

  19. 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

  20. mGlu2 Receptor Agonism, but Not Positive Allosteric Modulation, Elicits Rapid Tolerance towards Their Primary Efficacy on Sleep Measures in Rats.

    Directory of Open Access Journals (Sweden)

    Abdallah Ahnaou

    Full Text Available G-protein-coupled receptor (GPCR agonists are known to induce both cellular adaptations resulting in tolerance to therapeutic effects and withdrawal symptoms upon treatment discontinuation. Glutamate neurotransmission is an integral part of sleep-wake mechanisms, which processes have translational relevance for central activity and target engagement. Here, we investigated the efficacy and tolerance potential of the metabotropic glutamate receptors (mGluR2/3 agonist LY354740 versus mGluR2 positive allosteric modulator (PAM JNJ-42153605 on sleep-wake organisation in rats. In vitro, the selectivity and potency of JNJ-42153605 were characterized. In vivo, effects on sleep measures were investigated in rats after once daily oral repeated treatment for 7 days, withdrawal and consecutive re-administration of LY354740 (1-10 mg/kg and JNJ-42153605 (3-30 mg/kg. JNJ-42153605 showed high affinity, potency and selectivity at mGluR2. Binding site analyses and knowledge-based docking confirmed the specificity of JNJ-42153605 at the mGluR2 allosteric binding site. Acute LY354740 and JNJ-42153605 dose-dependently decreased rapid eye movement (REM sleep time and prolonged its onset latency. Sub chronic effects of LY354740 on REM sleep measures disappeared from day 3 onwards, whereas those of JNJ-42153605 were maintained after repeated exposure. LY354740 attenuated REM sleep homeostatic recovery, while this was preserved after JNJ-42153605 administration. JNJ-42153605 enhanced sleep continuity and efficiency, suggesting its potential as an add-on medication for impaired sleep quality during early stages of treatment. Abrupt cessation of JNJ-42153605 did not induce withdrawal phenomena and sleep disturbances, while the initial drug effect was fully reinstated after re-administration. Collectively, long-term treatment with JNJ-42153605 did not induce tolerance phenomena to its primary functional effects on sleep measures, nor adverse effects at withdrawal, while it

  1. RU 24969-induced emesis in the cat - 5-HT1 sites other than 5-HT1A, 5-HT1B or 5-HT1C implicated

    Science.gov (United States)

    Lucot, James B.

    1990-01-01

    RU 24969 was administered s.c. to cats and found to elicit emesis with a maximally effective dose of 1.0 mg/kg 5-Methoxytryptamine was found to have lower efficacy and to produce a higher incidence of nonspecific effects while trifluoromethylphenylpiperizine (TFMPP) was devoid of emetic effects. The emesis elicited by 1.0 mg/kg of RU 24969 was not altered by pretreatment with phentolamine, haloperidol, yohimbine or (-)-propranolol, indicating that catecholamines played no role in this response. The emesis was prevented by metergoline and methysergide but not by ketanserin, cyproheptadine, mesulergine, ICS 205 930, methiothepin, trimethobenzamide or BMY 7378. An indirect argument is presented that implicates a role for 5-HT1D sites. This conclusion must remain tentative until drugs selective for this site are synthesized and tested. The emesis was also prevented by 8-hydroxy-2-(di-n-propylamine)tetralin (8-OH-DPAT), confirming that this drug has a general antiemetic effect in cats.

  2. Targeted Chemical Wedges Reveal the Role of Allosteric DNA Modulation in Protein — DNA Assembly

    OpenAIRE

    Moretti, Rocco; Donato, Leslie J.; Brezinski, Mary L.; Stafford, Ryan L.; Hoff, Helena; Thorson, Jon S.; Dervan, Peter B.; Ansari, Aseem Z.

    2008-01-01

    The cooperative assembly of multiprotein complexes results from allosteric modulations of DNA structure as well as direct intermolecular contacts between proteins. Such cooperative binding plays a critical role in imparting exquisite sequence specificity on the homeobox transcription factor (Hox) family of developmental transcription factors. A well-characterized example includes the interaction of Hox proteins with extradenticle (Exd), a highly conserved DNA binding transcription factor. Alt...

  3. Markov propagation of allosteric effects in biomolecular systems: application to GroEL–GroES

    OpenAIRE

    Chennubhotla, Chakra; Bahar, Ivet

    2006-01-01

    We introduce a novel approach for elucidating the potential pathways of allosteric communication in biomolecular systems. The methodology, based on Markov propagation of ‘information' across the structure, permits us to partition the network of interactions into soft clusters distinguished by their coherent stochastics. Probabilistic participation of residues in these clusters defines the communication patterns inherent to the network architecture. Application to bacterial chaperonin complex ...

  4. Coevolutionary Analysis Enabled Rational Deregulation of Allosteric Enzyme Inhibition in Corynebacterium glutamicum for Lysine Production ▿

    OpenAIRE

    Chen, Zhen; Meyer, Weiqian; Rappert, Sugima; Sun, Jibin; Zeng, An-Ping

    2011-01-01

    Product feedback inhibition of allosteric enzymes is an essential issue for the development of highly efficient microbial strains for bioproduction. Here we used aspartokinase from Corynebacterium glutamicum (CgAK), a key enzyme controlling the biosynthesis of industrially important aspartate family amino acids, as a model to demonstrate a fast and efficient approach to the deregulation of allostery. In the last 50 years many researchers and companies have made considerable efforts to deregul...

  5. TOWARD UNDERSTANDING ALLOSTERIC SIGNALING MECHANISMS IN THE ATPASE DOMAIN OF MOLECULAR CHAPERONES

    OpenAIRE

    Liu, Ying; Bahar, Ivet

    2010-01-01

    The ATPase cycle of the heat shock protein 70 (HSP70) is largely dependent on the ability of its nucleotide binding domain (NBD), also called ATPase domain, to undergo structural changes between its open and closed conformations. We present here a combined study of the Hsp70 NBD sequence, structure and dynamic features to identify the residues that play a crucial role in mediating the allosteric signaling properties of the ATPase domain. Specifically, we identify the residues involved in the ...

  6. Allosteric Regulation of Histidine Kinases by Their Cognate Response Regulator Determines Cell Fate

    OpenAIRE

    Paul, Ralf; Jaeger, Tina; Abel, Sören; Wiederkehr, Irene; Folcher, Marc; Biondi, Emanuele G.; Laub, Michael T.; Jenal, Urs

    2008-01-01

    The two-component phosphorylation network is of critical importance for bacterial growth and physiology. Here, we address plasticity and interconnection of distinct signal transduction pathways within this network. In Caulobacter crescentus antagonistic activities of the PleC phosphatase and DivJ kinase localized at opposite cell poles control the phosphorylation state and subcellular localization of the cell fate determinator protein DivK. We show that DivK functions as an allosteric regulat...

  7. Paleomagnetic results from IODP Expedition 344 Site U1381 and implications for the initial subduction of the Cocos Ridge

    Science.gov (United States)

    Li, Yong-Xiang; Zhao, Xixi; Jovane, Luigi; Petronotis, Katerina; Gong, Zheng; Xie, Siyi

    2016-04-01

    Understanding the processes that govern the strength, nature, and distribution of slip along subduction zones is a fundamental and societally relevant goal of modern earth science. The Costa Rica Seismogenesis Project (CRISP) is specially designed to understand the processes that control nucleation and seismic rupture of large earthquakes at erosional subduction zones. Drilling directly on the Cocos Ridge (CR) during International Ocean Drilling Program (IODP) Expedition 344 discovered a sedimentary hiatus in Site U1381 cores. In this study, we conducted a magnetostratigraphic and rock magnetic study on the Cenozoic sedimentary sequences of site U1381. Anisotropy of magnetic susceptibility data from sediments above and below the hiatus show oblate fabrcis, but the Kmin axes of the AMS data from sediments below the hiatus are more dispersed than those from sediments above the hiatus, implying that formation of hiatus may have affected AMS. Paleomagnetic results of the U1381 core, together with available Ar-Ar dates of ash layers from sediments below the hiatus, allow us to establish a geomagnetic polarity timescale that brackets the hiatus between ca. 9.61 and 1.52 Ma. Analyses of sedimentary records from ODP/IODP cores in the vicinity reveal that the hiatus appears to be regional, spanning the northeastern end of the CR. Also, the hiatus appears to occur only at certain locations. Its regional occurrence at unique locations implies a link to the initial shallow subduction of the Cocos Ridge. The hiatus was probably produced by either bottom current erosion or the CR buckling upon its initial collision with the Middle American trench (MAT). Thus, the initial subduction of the CR must have taken place on or before 1.52 Ma.

  8. Structural Study of a Flexible Active Site Loop in Human Indoleamine 2,3-Dioxygenase and Its Functional Implications.

    Science.gov (United States)

    Álvarez, Lucía; Lewis-Ballester, Ariel; Roitberg, Adrián; Estrin, Darío A; Yeh, Syun-Ru; Marti, Marcelo A; Capece, Luciana

    2016-05-17

    Human indoleamine 2,3-dioxygenase catalyzes the oxidative cleavage of tryptophan to N-formyl kynurenine, the initial and rate-limiting step in the kynurenine pathway. Additionally, this enzyme has been identified as a possible target for cancer therapy. A 20-amino acid protein segment (the JK loop), which connects the J and K helices, was not resolved in the reported hIDO crystal structure. Previous studies have shown that this loop undergoes structural rearrangement upon substrate binding. In this work, we apply a combination of replica exchange molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure and dynamics of this protein region. Our simulations show that the JK loop can be divided into two regions: the first region (JK loop(C)) displays specific and well-defined conformations and is within hydrogen bonding distance of the substrate, while the second region (JK loop(N)) is highly disordered and exposed to the solvent. The peculiar flexible nature of JK loop(N) suggests that it may function as a target for post-translational modifications and/or a mediator for protein-protein interactions. In contrast, hydrogen bonding interactions are observed between the substrate and Thr379 in the highly conserved "GTGG" motif of JK loop(C), thereby anchoring JK loop(C) in a closed conformation, which secures the appropriate substrate binding mode for catalysis. Site-directed mutagenesis experiments confirm the key role of this residue, highlighting the importance of the JK loop(C) conformation in regulating the enzymatic activity. Furthermore, the existence of the partially and totally open conformations in the substrate-free form suggests a role of JK loop(C) in controlling substrate and product dynamics. PMID:27112409

  9. Past climate changes and permafrost depth at the Lake El'gygytgyn site: implications from data and thermal modelling

    Directory of Open Access Journals (Sweden)

    D. Mottaghy

    2012-07-01

    Full Text Available We present results of numerical simulations of the temperature field of the subsurface around and beneath the crater Lake El'gygytgyn in NE Russia, which is subject of an interdisciplinary drilling campaign within the International Continental Drilling Program (ICDP. This study focuses on determining the permafrost depth and the transition between talik and permafrost regimes, both, under steady-state and transient conditions of past climate changes. Thermal properties of the subsurface are deduced from measurements on three representative core samples taken from the quaternary sediments and the underlying impact rock. Further information is derived from the available geophysical logs and literature data. The temperature data from the lake borehole ICDP site 5011-1 down to 400 m depth below lake bottom 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 in the 140 m deep land-based borehole ICDP site 5011-3 allow to determine the mean annual ground surface temperature (GST, as well as its history (GSTH to a certain extent. Although the borehole's depth is by far not sufficient for a complete reconstruction of past temperatures back to the last glacial maximum (LGM, the temperature data and our modelling results show that there is still an influence of the LGM on the thermal regime, and thus on the permafrost depth. Whereas the latter result is obtained from the deeper part of the temperature profile, the rather strong curvature of the temperature data in shallower depths around 30 m can be explained by a comparatively large amplitude of the Little Ice Age (LIA, with a subsequently persistent cool period. Other mechanisms like varying porosity may also have an influence on the temperature profile, however, our modelling studies imply a major contribution from recent climate changes.

  10. Past climate changes and permafrost depth at the Lake El'gygytgyn site: implications from data and thermal modelling

    Science.gov (United States)

    Mottaghy, D.; Schwamborn, G.; Rath, V.

    2012-07-01

    We present results of numerical simulations of the temperature field of the subsurface around and beneath the crater Lake El'gygytgyn in NE Russia, which is subject of an interdisciplinary drilling campaign within the International Continental Drilling Program (ICDP). This study focuses on determining the permafrost depth and the transition between talik and permafrost regimes, both, under steady-state and transient conditions of past climate changes. Thermal properties of the subsurface are deduced from measurements on three representative core samples taken from the quaternary sediments and the underlying impact rock. Further information is derived from the available geophysical logs and literature data. The temperature data from the lake borehole ICDP site 5011-1 down to 400 m depth below lake bottom 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 in the 140 m deep land-based borehole ICDP site 5011-3 allow to determine the mean annual ground surface temperature (GST), as well as its history (GSTH) to a certain extent. Although the borehole's depth is by far not sufficient for a complete reconstruction of past temperatures back to the last glacial maximum (LGM), the temperature data and our modelling results show that there is still an influence of the LGM on the thermal regime, and thus on the permafrost depth. Whereas the latter result is obtained from the deeper part of the temperature profile, the rather strong curvature of the temperature data in shallower depths around 30 m can be explained by a comparatively large amplitude of the Little Ice Age (LIA), with a subsequently persistent cool period. Other mechanisms like varying porosity may also have an influence on the temperature profile, however, our modelling studies imply a major contribution from recent climate changes.

  11. Structural insights into the allosteric effects of 4EBP1 on the eukaryotic translation initiation factor eIF4E

    OpenAIRE

    Siddiqui, Nadeem; Tempel, Wolfram; Nedyalkova, Lucy; Volpon, Laurent; Wernimont, Amy K; Osborne, Michael J.; Park, Hee-won; Borden, Katherine L. B.

    2011-01-01

    The eukaryotic translation initiation factor eIF4E plays key roles in cap dependent translation and mRNA export. These functions rely on binding the 7-methylguanosine moiety (5′cap) to the 5′-end of all mRNAs. eIF4E is regulated by proteins such as eIF4G and eIF4E binding proteins (4EBPs) that bind the dorsal surface of eIF4E, distal to the cap binding site, and modulate cap binding activity. Both proteins increase the affinity of eIF4E for 5′cap. Our understanding of the allosteric effects a...

  12. Thermodynamic basis of site-specific cooperativity.

    Science.gov (United States)

    Di Cera, E

    1994-08-01

    Cooperative phenomena in biological macromolecules arise from the interaction of many distinct subsystems, such as structural domains or binding sites. Cooperative properties of the system as a whole, like protein folding or allosteric transitions, are subject to the restrictions imposed by thermodynamic stability. These restrictions, however, do not apply in the case of individual subsystems open to interactions with the rest of the macromolecule. The site-specific properties of such subsystems can be understood in general thermodynamic terms from those of a multicomponent system under particular conditions. The analogy provides a thermodynamic basis for site-specific cooperativity. PMID:8075382

  13. Experimentally guided structural modeling and dynamics analysis of Hsp90-p53 interactions: allosteric regulation of the Hsp90 chaperone by a client protein.

    Science.gov (United States)

    Blacklock, Kristin; Verkhivker, Gennady M

    2013-11-25

    A fundamental role of the Hsp90 chaperone system in mediating maturation of protein clients is essential for the integrity of signaling pathways involved in cell cycle control and organism development. Molecular characterization of Hsp90 interactions with client proteins is fundamental to understanding the activity of many tumor-inducing signaling proteins and presents an active area of structural and biochemical studies. In this work, we have probed mechanistic aspects of allosteric regulation of Hsp90 by client proteins via a detailed computational study of Hsp90 interactions with the tumor suppressor protein p53. Experimentally guided protein docking and molecular dynamics structural refinement have reconstructed the recognition-competent states of the Hsp90-p53 complexes that are consistent with the NMR studies. Protein structure network analysis has identified critical interacting networks and specific residues responsible for structural integrity and stability of the Hsp90-p53 complexes. Coarse-grained modeling was used to characterize the global dynamics of the regulatory complexes and map p53-induced changes in the conformational equilibrium of Hsp90. The variations in the functional dynamics profiles of the Hsp90-p53 complexes are consistent with the NMR studies and could explain differences in the functional role of the alternative binding sites. Despite the overall similarity of the collective movements and the same global interaction footprint, p53 binding at the C-terminal interaction site of Hsp90 may have a more significant impact on the chaperone dynamics, which is consistent with the stronger allosteric effect of these interactions revealed by the experimental studies. The results suggest that p53-induced modulation of the global dynamics and structurally stable interaction networks can target the regulatory hinge regions and facilitate stabilization of the closed Hsp90 dimer that underlies the fundamental stimulatory effect of the p53 client. PMID

  14. Escherichia coli DnaB Helicase–DnaC Protein Complex: Allosteric Effects of the Nucleotides on the Nucleic Acid Binding and the Kinetic Mechanism of NTP Hydrolysis. 3†

    OpenAIRE

    Roychowdhury, Anasuya; Szymanski, Michal R.; Jezewska, Maria J.; Bujalowski, Wlodzimierz

    2009-01-01

    Allosteric interactions between the DNA- and NTP-binding sites of the Escherichia coli DnaB helicase engaged in the DnaB–DnaC complex and the mechanism of NTP hydrolysis by the complex have been examined using the fluorescence titration, analytical ultracentrifugation, and rapid quench-flow technique. Surprisingly, the ssDNA affinity of the DnaB–DnaC complex is independent of the structure of the phosphate group of the cofactor bound to the helicase. Thus, the DnaC protein eliminates the anta...

  15. Soil characteristics, heavy metal availability and vegetation recovery at a former metallurgical landfill: Implications in risk assessment and site restoration

    International Nuclear Information System (INIS)

    Pedological and botanical characteristics of a former metallurgical landfill were examined to assess the risks of heavy metals mobility and to evaluate remediation feasibility. In addition to very high heavy metals levels (Cu, Cr, Mn, Ni, Pb, Zn), the soil was characterized by a lack of clear horizonation, a relatively high pH, a high mineral and organic carbon contents, a low nitrogen level and a high C/N ratio. A two step sequential extraction showed that heavy metals were poorly labile (i.e. not soluble in diluted CaCl2), indicating that their leaching under natural conditions was probably very low. However, extraction with DTPA generated significant amounts of metals (mainly Pb and Cu), suggesting they were potentially mobilizable. A botanical survey of the area showed a biodiverse plant community (28 species and 11 families), with no obvious toxicity symptoms. Measurements of metal contents in dominant species confirmed that they were closely similar to those reported for species growing in unpolluted environments. Consequently, for an effective site restoration, indigenous species could be well suited to cope with local conditions in a phytostabilization strategy. - Heavy metals at a former metallurgical landfill are not leachable and poorly phytoavailable

  16. Element mobility during pyrite weathering: implications for acid and heavy metal pollution at mining-impacted sites

    Science.gov (United States)

    Lu, Long; Wang, Rucheng; Chen, Fanrong; Xue, Jiyue; Zhang, Peihua; Lu, Jianjun

    2005-11-01

    Based on back scattered electron images and electron micro-probe analysis results, four alteration layers, including a transition layer, a reticulated ferric oxide layer, a nubby ferric oxide layer and a cellular ferric oxide layer, were identified in the naturally weathering products of pyrite. These layers represent a progressive alteration sequence of pyrite under weathering conditions. The cellular ferric oxide layer correlates with the strongest weathering phase and results from the dissolution of nubby ferric oxide by acidic porewater. Leaching coefficient was introduced to better express the response of element mobility to the degree of pyrite weathering. Its variation shows that the mobility of S, Co and Bi is stronger than As, Cu and Zn. Sulfur in pyrite is oxidized to sulfuric acid and sulfate that are basically released into to porewater, and heavy metals Co and Bi are evidently released by acid dissolution. As, Cu and Zn are enriched in ferric oxide by adsorption and by co-precipitation, but they would re-release to the environment via desorption or dissolution when porewater pH becomes low enough. Consequently, Co, Bi, As, Cu and Zn may pose a substantial impact on water quality. Considering that metal mobility and its concentration in mine waste are two important factors influencing heavy metal pollution at mining-impacted sites, Bi and Co are more important pollutants in this case.

  17. Basis for Half-Site Ligand Binding in Yeast NAD+-Specific Isocitrate Dehydrogenase†

    OpenAIRE

    Lin, An-Ping; McAlister-Henn, Lee

    2011-01-01

    Yeast NAD+-specific isocitrate dehydrogenase is an allosterically regulated octameric enzyme composed of four heterodimers of a catalytic IDH2 subunit and a regulatory IDH1 subunit. Despite structural predictions that the enzyme would contain eight isocitrate binding sites, four NAD+ binding sites, and four AMP binding sites, only half of the sites for each ligand are measurable in binding assays. Based on a potential interaction between side chains of Cys-150 residues in IDH2 subunits in eac...

  18. Malaria transmission, infection, and disease at three sites with varied transmission intensity in Uganda: implications for malaria control.

    Science.gov (United States)

    Kamya, Moses R; Arinaitwe, Emmanuel; Wanzira, Humphrey; Katureebe, Agaba; Barusya, Chris; Kigozi, Simon P; Kilama, Maxwell; Tatem, Andrew J; Rosenthal, Philip J; Drakeley, Chris; Lindsay, Steve W; Staedke, Sarah G; Smith, David L; Greenhouse, Bryan; Dorsey, Grant

    2015-05-01

    The intensification of control interventions has led to marked reductions in malaria burden in some settings, but not others. To provide a comprehensive description of malaria epidemiology in Uganda, we conducted surveillance studies over 24 months in 100 houses randomly selected from each of three subcounties: Walukuba (peri-urban), Kihihi (rural), and Nagongera (rural). Annual entomological inoculation rate (aEIR) was estimated from monthly Centers for Disease Control and Prevention (CDC) light trap mosquito collections. Children aged 0.5-10 years were provided long-lasting insecticidal nets (LLINs) and followed for measures of parasite prevalence, anemia and malaria incidence. Estimates of aEIR were 2.8, 32.0, and 310 infectious bites per year, and estimates of parasite prevalence 7.4%, 9.3%, and 28.7% for Walukuba, Kihihi, and Nagongera, respectively. Over the 2-year study, malaria incidence per person-years decreased in Walukuba (0.51 versus 0.31, P = 0.001) and increased in Kihihi (0.97 versus 1.93, P < 0.001) and Nagongera (2.33 versus 3.30, P < 0.001). Of 2,582 episodes of malaria, only 8 (0.3%) met criteria for severe disease. The prevalence of anemia was low and not associated with transmission intensity. In our cohorts, where LLINs and prompt effective treatment were provided, the risk of complicated malaria and anemia was extremely low. However, malaria incidence was high and increased over time at the two rural sites, suggesting improved community-wide coverage of LLIN and additional malaria control interventions are needed in Uganda. PMID:25778501

  19. 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.

  20. 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

  1. Dynamics of allosteric action in multisite protein modification

    CERN Document Server

    Milotti, E; Fabbro, A D; Pellegrina, C D; Chignola, Roberto; Fabbro, Alessio Del; Milotti, Edoardo; Pellegrina, Chiara Dalla

    2006-01-01

    Protein functions in cells may be activated or modified by the attachment of several kinds of chemical groups. While protein phosphorylation, i.e. the attachment of a phosphoryl (PO$_3^-$) group, is the most studied form of protein modification, and is known to regulate the functions of many proteins, protein behavior can also be modified by nitrosylation, acetylation, methylation, etc. A protein can have multiple modification sites, and display some form of transition only when enough sites are modified. In a previous paper we have modeled the generic equilibrium properties of multisite protein modification (R.Chignola, C. Dalla Pellegrina, A. Del Fabbro, E.Milotti, Physica A {\\bf 371}, 463 (2006)) and we have shown that it can account both for sharp, robust thresholds and for information transfer between processes with widely separated timescales. Here we use the same concepts to expand that analysis and describe multisite modification dynamics with a nonlinear differential system. We utilize the differenti...

  2. Targeting PARP-1 allosteric regulation offers therapeutic potential against cancer

    OpenAIRE

    Steffen, Jamin D.; Tholey, Renee M.; Langelier, Marie-France; Planck, Jamie L.; Schiewer, Matthew J.; Lal, Shruti; Bildzukewicz, Nikolai A.; Yeo, Charles J.; Knudsen, Karen E.; Brody, Jonathan R; Pascal, John M.

    2013-01-01

    PARP-1 is a nuclear protein that has important roles in maintenance of genomic integrity. During genotoxic stress, PARP-1 recruits to sites of DNA damage where PARP-1 domain architecture initiates catalytic activation and subsequent poly(ADP-ribose)-dependent DNA repair. PARP-1 inhibition is a promising new way to selectively target cancers harboring DNA repair deficiencies. However, current inhibitors target other PARPs raising important questions concerning long-term off-target effects. Her...

  3. 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

  4. Antidepressant binding site in a bacterial homologue of neurotransmitter transporters.

    Science.gov (United States)

    Singh, Satinder K; Yamashita, Atsuko; Gouaux, Eric

    2007-08-23

    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 A 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 design of

  5. Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

    International Nuclear Information System (INIS)

    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

  6. Mechanism of Positive Allosteric Modulators Acting on AMPA Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Jin,R.; Clark, S.; Weeks, A.; Dudman, J.; Gouaux, E.; Partin, K.

    2005-01-01

    Ligand-gated ion channels involved in the modulation of synaptic strength are the AMPA, kainate, and NMDA glutamate receptors. Small molecules that potentiate AMPA receptor currents relieve cognitive deficits caused by neurodegenerative diseases such as Alzheimer's disease and show promise in the treatment of depression. Previously, there has been limited understanding of the molecular mechanism of action for AMPA receptor potentiators. Here we present cocrystal structures of the glutamate receptor GluR2 S1S2 ligand-binding domain in complex with aniracetam [1-(4-methoxybenzoyl)-2-pyrrolidinone] or CX614 (pyrrolidino-1, 3-oxazino benzo-1, 4-dioxan-10-one), two AMPA receptor potentiators that preferentially slow AMPA receptor deactivation. Both potentiators bind within the dimer interface of the nondesensitized receptor at a common site located on the twofold axis of molecular symmetry. Importantly, the potentiator binding site is adjacent to the 'hinge' in the ligand-binding core 'clamshell' that undergoes conformational rearrangement after glutamate binding. Using rapid solution exchange, patch-clamp electrophysiology experiments, we show that point mutations of residues that interact with potentiators in the cocrystal disrupt potentiator function. We suggest that the potentiators slow deactivation by stabilizing the clamshell in its closed-cleft, glutamate-bound conformation.

  7. Green Tea Polyphenols Control Dysregulated Glutamate Dehydrogenase in Transgenic Mice by Hijacking the ADP Activation Site

    Energy Technology Data Exchange (ETDEWEB)

    Li, Changhong; Li, Ming; Chen, Pan; Narayan, Srinivas; Matschinsky, Franz M.; Bennett, Michael J.; Stanley, Charles A.; Smith, Thomas J. (CH-PA); (UPENN); (Danforth)

    2012-05-09

    Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and, in animals, is extensively regulated by a number of metabolites. Gain of function mutations in GDH that abrogate GTP inhibition cause the hyperinsulinism/hyperammonemia syndrome (HHS), resulting in increased pancreatic {beta}-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. We have previously shown that two of the polyphenols from green tea (epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)) inhibit GDH in vitro and that EGCG blocks GDH-mediated insulin secretion in wild type rat islets. Using structural and site-directed mutagenesis studies, we demonstrate that ECG binds to the same site as the allosteric regulator, ADP. Perifusion assays using pancreatic islets from transgenic mice expressing a human HHS form of GDH demonstrate that the hyperresponse to glutamine caused by dysregulated GDH is blocked by the addition of EGCG. As observed in HHS patients, these transgenic mice are hypersensitive to amino acid feeding, and this is abrogated by oral administration of EGCG prior to challenge. Finally, the low basal blood glucose level in the HHS mouse model is improved upon chronic administration of EGCG. These results suggest that this common natural product or some derivative thereof may prove useful in controlling this genetic disorder. Of broader clinical implication is that other groups have shown that restriction of glutamine catabolism via these GDH inhibitors can be useful in treating various tumors. This HHS transgenic mouse model offers a highly useful means to test these agents in vivo.

  8. Asymmetric allosteric activation of the symmetric ArgR hexamer.

    Science.gov (United States)

    Jin, Lihua; Xue, Wei-Feng; Fukayama, June Wong; Yetter, Jaclyn; Pickering, Michael; Carey, Jannette

    2005-02-11

    Hexameric arginine repressor, ArgR, bound to L-arginine serves both as the master transcriptional repressor/activator at diverse regulons in a wide range of bacteria and as a required cofactor for resolution of ColE1 plasmid multimers. Multifunctional ArgR is thus unusual in possessing features of specific gene regulators, global regulators, and non-specific gene organizers; its closest functional analog is probably CAP, the cyclic AMP receptor/activator protein. Isothermal titration calorimetry, surface plasmon resonance, and proteolysis indicate that binding of a single L-argine [corrected] per ArgR hexamer triggers a global conformation [corrected] change and resets the affinities of the remaining five sites, making them 100-fold weaker. The analysis suggests a novel thermodynamic signature for this mechanism of activation. PMID:15663926

  9. Ultrasensitivity and heavy-metal selectivity of the allosterically modulated MerR transcription complex.

    OpenAIRE

    Ralston, D M; O'Halloran, T V

    1990-01-01

    The MerR metalloregulatory protein is a heavy-metal receptor that functions as the repressor and Hg(II)-responsive transcription activator of the prokaryotic mercury-resistance (mer) genes. We demonstrate that this allosterically modulated regulatory protein is sensitive to HgCl2 concentrations of 1.0 +/- 0.3 x 10(-8) M in the presence of 1.0 x 10(-3) M dithiothreitol for half-maximal induction of transcription of the mer promoter by Escherichia coli RNA polymerase in vitro. Transcription med...

  10. Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors

    Directory of Open Access Journals (Sweden)

    Klein-Seetharaman Judith

    2008-02-01

    Full Text Available Abstract Metabotropic glutamate receptors (mGluRs are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects – enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%, the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%, the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86% and 8/9 (89% for ArgusLab and 10/14 (71% and 7/9 (78% for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by

  11. Ibuprofen impairs allosterically peroxynitrite isomerization by ferric human serum heme-albumin.

    OpenAIRE

    Ascenzi, Paolo; di Masi, Alessandra; Coletta, Massimo; Ciaccio, Chiara; Fanali, Gabriella; Nicoletti, Francesco P; Smulevich, Giulietta; Fasano, Mauro

    2011-01-01

    Human serum albumin (HSA) participates in heme scavenging; in turn, heme endows HSA with myoglobin-like reactivity and spectroscopic properties. Here, the allosteric effect of ibuprofen on peroxynitrite isomerization to NO3− catalyzed by ferric human serum heme-albumin (HSA-heme-Fe(III)) is reported. Data were obtained at 22.0 °C. HSA-heme-Fe(III) catalyzes peroxynitrite isomerization in the absence and presence of CO2; the values of the second order catalytic rate constant (kon) are 4.1 × 10...

  12. Complex pharmacology of novel allosteric free fatty acid 3 receptor ligands

    DEFF Research Database (Denmark)

    Hudson, Brian D; Christiansen, Elisabeth; Murdoch, Hannah; Jenkins, Laura; Hansen, Anders Højgaard; Madsen, Ole; Ulven, Trond; Milligan, Graeme

    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...... chemical series, 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....

  13. A Potential Yeast Actin Allosteric Conduit Dependent on Hydrophobic Core Residues Val-76 and Trp-79*

    OpenAIRE

    Wen, Kuo-Kuang; McKane, Melissa; Stokasimov, Ema; Fields, Jonathon; Rubenstein, Peter A.

    2010-01-01

    Intramolecular allosteric interactions responsible for actin conformational regulation are largely unknown. Previous work demonstrated that replacing yeast actin Val-76 with muscle actin Ile caused decreased nucleotide exchange. Residue 76 abuts Trp-79 in a six-residue linear array beginning with Lys-118 on the surface and ending with His-73 in the nucleotide cleft. To test if altering the degree of packing of these two residues would affect actin dynamics, we constructed V76I, W79F, and W79Y...

  14. Dynamical Allosterism in the Mechanism of Action of DNA Mismatch Repair Protein MutS

    OpenAIRE

    Pieniazek, Susan N.; Hingorani, Manju M.; Beveridge, D.L.

    2011-01-01

    The multidomain protein Thermus aquaticus MutS and its prokaryotic and eukaryotic homologs recognize DNA replication errors and initiate mismatch repair. MutS actions are fueled by ATP binding and hydrolysis, which modulate its interactions with DNA and other proteins in the mismatch-repair pathway. The DNA binding and ATPase activities are allosterically coupled over a distance of ∼70 Å, and the molecular mechanism of coupling has not been clarified. To address this problem, all-atom molecul...

  15. 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.

  16. Chemogenomic discovery of allosteric antagonists at the GPRC6A receptor

    DEFF Research Database (Denmark)

    Gloriam, David E.; Wellendorph, Petrine; Johansen, Lars Dan; Thomsen, Alex Rojas Bie; Phonekeo, Karina; Pedersen, Daniel Sejer; Bräuner-Osborne, Hans

    2011-01-01

    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...

  17. Transplanting a unique allosteric effect from crocodile into human haemoglobin.

    Science.gov (United States)

    Komiyama, N H; Miyazaki, G; Tame, J; Nagai, K

    1995-01-19

    Crocodiles are able to remain under water for more than one hour without surfacing to breathe and often kill their prey by drowning it. How do crocodiles stay under water for a long time? When they hold their breath, bicarbonate ions, the final product of respiration, accumulate and drastically reduce the oxygen affinity of haemoglobin, releasing a large fraction of haemoglobin-bound oxygen into the tissues. We have now located the bicarbonate-ion-binding site at the alpha 1 beta 2-subunit interface by making various human-crocodile chimaeric haemoglobins. Furthermore, we have been able to transplant the bicarbonate effect into human haemoglobin by replacing only a few residues, even though the amino-acid sequence identity between crocodile (Crocodylus niloticus) and human haemoglobins is only 68% for the alpha- and 51% for the beta-subunit. These results indicate that an entirely new function which enables species to adapt to a new environment could evolve in a protein by a relatively small number of amino-acid substitutions in key positions. PMID:7816138

  18. Structural characterization of S100A15 reveals a novel zinc coordination site among S100 proteins and altered surface chemistry with functional implications for receptor binding

    Directory of Open Access Journals (Sweden)

    Murray Jill I

    2012-07-01

    Full Text Available Abstract Background S100 proteins are a family of small, EF-hand containing calcium-binding signaling proteins that are implicated in many cancers. While the majority of human S100 proteins share 25-65% sequence similarity, S100A7 and its recently identified paralog, S100A15, display 93% sequence identity. Intriguingly, however, S100A7 and S100A15 serve distinct roles in inflammatory skin disease; S100A7 signals through the receptor for advanced glycation products (RAGE in a zinc-dependent manner, while S100A15 signals through a yet unidentified G-protein coupled receptor in a zinc-independent manner. Of the seven divergent residues that differentiate S100A7 and S100A15, four cluster in a zinc-binding region and the remaining three localize to a predicted receptor-binding surface. Results To investigate the structural and functional consequences of these divergent clusters, we report the X-ray crystal structures of S100A15 and S100A7D24G, a hybrid variant where the zinc ligand Asp24 of S100A7 has been substituted with the glycine of S100A15, to 1.7 Å and 1.6 Å resolution, respectively. Remarkably, despite replacement of the Asp ligand, zinc binding is retained at the S100A15 dimer interface with distorted tetrahedral geometry and a chloride ion serving as an exogenous fourth ligand. Zinc binding was confirmed using anomalous difference maps and solution binding studies that revealed similar affinities of zinc for S100A15 and S100A7. Additionally, the predicted receptor-binding surface on S100A7 is substantially more basic in S100A15 without incurring structural rearrangement. Conclusions Here we demonstrate that S100A15 retains the ability to coordinate zinc through incorporation of an exogenous ligand resulting in a unique zinc-binding site among S100 proteins. The altered surface chemistry between S100A7 and S100A15 that localizes to the predicted receptor binding site is likely responsible for the differential recognition of distinct

  19. Evaluation of potential implication of membrane estrogen binding sites on ERE-dependent transcriptional activity and intracellular estrogen receptor-alpha regulation in MCF-7 breast cancer cells.

    Science.gov (United States)

    Seo, Hye Sook; Leclercq, Guy

    2002-01-01

    The potential involvement of membrane estrogen binding sites in the induction of ERE-dependent transcriptional activity as well as in the regulation of intracellular estrogen receptor alpha (ER-alpha) level under estradiol (E2) stimulation was investigated. Our approach relied upon the use of two DCC-treated E2-BSA (bovine serum albumin) solutions (E2-6-BSA and E2-17-BSA). The absence of detectable free E2 in these solutions was established. Both E2-BSA conjugates led to a transient dose-dependent stimulation of the expression of ERE-luciferase (LUC) reporter gene in MVLN cells (MCF-7 cells stably transfected with a pVit-tk-LUC reporter plasmid), a property not recorded with free E2, which maintained enhanced transcriptional activity during the whole experiment. A very low concentration of E2 (10 pM) synergistically acted with E2-BSA conjugates. Hence, ERE-dependent transcriptional activity induced by these conjugates appeared to result from their known interactions with membrane estrogen binding sites. Anti-estrogens (AEs: 4-OH-TAM and RU 58,668), which antagonize genomic ER responses, abrogated the luciferase activity induced by E2-BSA conjugates, confirming a potential relationship between membrane-related signals and intracellular ER. Moreover, induction of luciferase was recorded when the cells were exposed to IBMX (3-isobutyl-1-methylxanthine) and cyclic nucleotides (cAMP/cGMP), suggesting the implication of the latter in the signal transduction pathway leading to the expression of the reporter gene. Growth factors (IGF-I, EGF and TGF-alpha) also slightly stimulated luciferase and synergistically acted with 10 pM E2, or 1 microM E2-BSA conjugates, in agreement with the concept of a cross-talk between steroids and peptides acting on the cell membrane. Remarkably, E2-BSA conjugates, IBMX and all investigated growth factors failed to down-regulate intracellular ER in MCF-7 cells, indicating the need for a direct intracellular interaction of the ligand with the

  20. 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.

  1. Engineering and optimization of an allosteric biosensor protein for peroxisome proliferator-activated receptor γ ligands.

    Science.gov (United States)

    Li, Jingjing; Gierach, Izabela; Gillies, Alison R; Warden, Charles D; Wood, David W

    2011-11-15

    The peroxisome proliferator-activated receptor gamma (PPARγ or PPARG) belongs to the nuclear receptor superfamily, and is a potential drug target for a variety of diseases. In this work, we constructed a series of bacterial biosensors for the identification of functional PPARγ ligands. These sensors entail modified Escherichia coli cells carrying a four-domain fusion protein, comprised of the PPARγ ligand binding domain (LBD), an engineered mini-intein domain, the E. coli maltose binding protein (MBD), and a thymidylate synthase (TS) reporter enzyme. E. coli cells expressing this protein exhibit hormone ligand-dependent growth phenotypes. Unlike our published estrogen (ER) and thyroid receptor (TR) biosensors, the canonical PPARγ biosensor cells displayed pronounced growth in the absence of ligand. They were able to distinguish agonists and antagonists, however, even in the absence of agonist. To improve ligand sensitivity of this sensor, we attempted to engineer and optimize linker peptides flanking the PPARγ LBD insertion point. Truncation of the original linkers led to decreased basal growth and significantly enhanced ligand sensitivity of the PPARγ sensor, while substitution of the native linkers with optimized G(4)S (Gly-Gly-Gly-Gly-Ser) linkers further increased the sensitivity. Our studies demonstrate that the properties of linkers, especially the C-terminal linker, greatly influence the efficiency and fidelity of the allosteric signal induced by ligand binding. Our work also suggests an approach to increase allosteric behavior in this multidomain sensor protein, without modification of the functional LBD. PMID:21893405

  2. Allosteric effects of chromophore interaction with dimeric near-infrared fluorescent proteins engineered from bacterial phytochromes.

    Science.gov (United States)

    Stepanenko, Olesya V; Baloban, Mikhail; Bublikov, Grigory S; Shcherbakova, Daria M; Stepanenko, Olga V; Turoverov, Konstantin K; Kuznetsova, Irina M; Verkhusha, Vladislav V

    2016-01-01

    Fluorescent proteins (FPs) engineered from bacterial phytochromes attract attention as probes for in vivo imaging due to their near-infrared (NIR) spectra and use of available in mammalian cells biliverdin (BV) as chromophore. We studied spectral properties of the iRFP670, iRFP682 and iRFP713 proteins and their mutants having Cys residues able to bind BV either in both PAS (Cys15) and GAF (Cys256) domains, in one of these domains, or without these Cys residues. We show that the absorption and fluorescence spectra and the chromophore binding depend on the location of the Cys residues. Compared with NIR FPs in which BV covalently binds to Cys15 or those that incorporate BV noncovalently, the proteins with BV covalently bound to Cys256 have blue-shifted spectra and higher quantum yield. In dimeric NIR FPs without Cys15, the covalent binding of BV to Сys256 in one monomer allosterically inhibits the covalent binding of BV to the other monomer, whereas the presence of Cys15 allosterically promotes BV binding to Cys256 in both monomers. The NIR FPs with both Cys residues have the narrowest blue-shifted spectra and the highest quantum yield. Our analysis resulted in the iRFP713/Val256Cys protein with the highest brightness in mammalian cells among available NIR FPs. PMID:26725513

  3. Identifying paths of allosteric communication in the protein BirA through simulations

    Science.gov (United States)

    Custer, Gregory; Beckett, Dorothy; Matysiak, Silvina

    Biotin ligase/repressor (BirA) is a bifunctional enzyme which adenylates biotin and transfers the product, biotinyl-5'-AMP (bio-5'-AMP) to biotin carboxyl carrier protein (BCCP). In the absence of BCCP, bio-5'-AMP promotes the dimerization of BirA. In dimer form, the BirA.bio-5'-AMP complex is able to bind to the biotin operator and prevents further synthesis of biotin. The bio-5'-AMP binds away from the dimer interface, so it is acting as an allosteric activator. We perform all-atom molecular dynamics simulations with BirA to look at fluctuations within the protein at equilibrium. We simulate apoBirA, liganded BirA, as well as two mutants, M211A and V219A. In agreement with experimental observations, several loops of the protein become stabilized for the liganded BirA when compared to the apo protein. In addition, changes in the dimer interface are observed for the M211A and V219A mutations, which are located in the ligand binding region. Using inter-residue correlation coefficients and pair energies a communication network through the protein is constructed. With this network we have identified paths which have the potential to be important in allosteric activation of BirA. These paths and the methods we use to identify them will be presented.

  4. Allosteric role of the large-scale domain opening in biological catch-binding

    Science.gov (United States)

    Pereverzev, Yuriy V.; Prezhdo, Oleg V.; Sokurenko, Evgeni V.

    2009-05-01

    The proposed model demonstrates the allosteric role of the two-domain region of the receptor protein in the increased lifetimes of biological receptor/ligand bonds subjected to an external force. The interaction between the domains is represented by a bounded potential, containing two minima corresponding to the attached and separated conformations of the two protein domains. The dissociative potential with a single minimum describing receptor/ligand binding fluctuates between deep and shallow states, depending on whether the domains are attached or separated. A number of valuable analytic expressions are derived and are used to interpret experimental data for two catch bonds. The P-selectin/P-selectin-glycoprotein-ligand-1 (PSGL-1) bond is controlled by the interface between the epidermal growth factor (EGF) and lectin domains of P-selectin, and the type 1 fimbrial adhesive protein (FimH)/mannose bond is governed by the interface between the lectin and pilin domains of FimH. Catch-binding occurs in these systems when the external force stretches the receptor proteins and increases the interdomain distance. The allosteric effect is supported by independent measurements, in which the domains are kept separated by attachment of another ligand. The proposed model accurately describes the experimentally observed anomalous behavior of the lifetimes of the P-selectin/PSGL-1 and FimH/mannose complexes as a function of applied force and provides valuable insights into the mechanism of catch-binding.

  5. Enzyme activity and allosteric characteristics of gamma-irradiated solid aspartate transcarbamylase

    International Nuclear Information System (INIS)

    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 D37 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 D37 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 D37 of 2.3 Mrad with doses below the activity maximum. Enzyme lyophilized in the presence of 1 mM CTP has a D37 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

  6. Novel Inhibitors Complexed with Glutamate Dehydrogenase: ALLOSTERIC REGULATION BY CONTROL OF PROTEIN DYNAMICS

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ming; Smith, Christopher J.; Walker, Matthew T.; Smith, Thomas J.; (Danforth)

    2009-12-01

    Mammalian glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate using NAD(P){sup +} as coenzyme. Unlike its counterparts from other animal kingdoms, mammalian GDH is regulated by a host of ligands. The recently discovered hyperinsulinism/hyperammonemia disorder showed that the loss of allosteric inhibition of GDH by GTP causes excessive secretion of insulin. Subsequent studies demonstrated that wild-type and hyperinsulinemia/hyperammonemia forms of GDH are inhibited by the green tea polyphenols, epigallocatechin gallate and epicatechin gallate. This was followed by high throughput studies that identified more stable inhibitors, including hexachlorophene, GW5074, and bithionol. Shown here are the structures of GDH complexed with these three compounds. Hexachlorophene forms a ring around the internal cavity in GDH through aromatic stacking interactions between the drug and GDH as well as between the drug molecules themselves. In contrast, GW5074 and bithionol both bind as pairs of stacked compounds at hexameric 2-fold axes between the dimers of subunits. The internal core of GDH contracts when the catalytic cleft closes during enzymatic turnover. None of the drugs cause conformational changes in the contact residues, but all bind to key interfaces involved in this contraction process. Therefore, it seems likely that the drugs inhibit enzymatic turnover by inhibiting this transition. Indeed, this expansion/contraction process may play a major role in the inter-subunit communication and allosteric regulation observed in GDH.

  7. Allosteric inhibition of glycogen phosphorylase a by the potential antidiabetic drug 3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarbo xylate.

    Science.gov (United States)

    Oikonomakos, N G; Tsitsanou, K E; Zographos, S E; Skamnaki, V T; Goldmann, S; Bischoff, H

    1999-10-01

    The effect of the potential antidiabetic drug (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarbox ylate (W1807) on the catalytic and structural properties of glycogen phosphorylase a has been studied. Glycogen phosphorylase (GP) is an allosteric enzyme whose activity is primarily controlled by reversible phosphorylation of Ser14 of the dephosphorylated enzyme (GPb, less active, predominantly T-state) to form the phosphorylated enzyme (GPa, more active, predominantly R-state). Upon conversion of GPb to GPa, the N-terminal tail (residues 5-22), which carries the Ser14(P), changes its conformation into a distorted 3(10) helix and its contacts from intrasubunit to intersubunit. This alteration causes a series of tertiary and quaternary conformational changes that lead to activation of the enzyme through opening access to the catalytic site. As part of a screening process to identify compounds that might contribute to the regulation of glycogen metabolism in the noninsulin dependent diabetes diseased state, W1807 has been found as the most potent inhibitor of GPb (Ki = 1.6 nM) that binds at the allosteric site of T-state GPb and produces further conformational changes, characteristic of a T'-like state. Kinetics show W1807 is a potent competitive inhibitor of GPa (-AMP) (Ki = 10.8 nM) and of GPa (+1 mM AMP) (Ki = 19.4 microM) with respect to glucose 1-phosphate and acts in synergism with glucose. To elucidate the structural features that contribute to the binding, the structures of GPa in the T-state conformation in complex with glucose and in complex with both glucose and W1807 have been determined at 100 K to 2.0 A and 2.1 A resolution, and refined to crystallographic R-values of 0.179 (R(free) = 0.230) and 0.189 (R(free) = 0.263), respectively. W1807 binds tightly at the allosteric site and induces substantial conformational changes both in the vicinity of the allosteric site and the subunit interface. A disordering of the N

  8. 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...

  9. 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]...

  10. Thermodynamics and structural analysis of positive allosteric modulation of the ionotropic glutamate receptor GluA2

    DEFF Research Database (Denmark)

    Krintel, Christian; Frydenvang, Karla; Olsen, Lars; Kristensen, Maria T; de Barrios, Oriol; Naur, Peter; Francotte, Pierre; Pirotte, Bernard; Gajhede, Michael; Kastrup, Jette Sandholm

    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...

  11. Modulation of Ultrafast Conformational Dynamics in Allosteric Interaction of Gal Repressor Protein with Different Operator DNA Sequences.

    Science.gov (United States)

    Choudhury, Susobhan; Naiya, Gitashri; Singh, Priya; Lemmens, Peter; Roy, Siddhartha; Pal, Samir Kumar

    2016-04-01

    Although all forms of dynamical behaviour of a protein under allosteric interaction with effectors are predicted, little evidence of ultrafast dynamics in the interaction has been reported. Here, we demonstrate the efficacy of a combined approach involving picosecond-resolved FRET and polarisation-gated fluorescence for the exploration of ultrafast dynamics in the allosteric interaction of the Gal repressor (GalR) protein dimer with DNA operator sequences OE and OI . FRET from the single tryptophan residue to a covalently attached probe IAEDANS at a cysteine residue in the C-terminal domain of GalR shows structural perturbation and conformational dynamics during allosteric interaction. Polarisation-gated fluorescence spectroscopy of IAEDANS and another probe (FITC) covalently attached to the operator directly revealed the essential dynamics for cooperativity in the protein-protein interaction. The ultrafast resonance energy transfer from IAEDANS in the protein to FITC also revealed different dynamic flexibility in the allosteric interaction. An attempt was made to correlate the dynamic changes in the protein dimers with OE and OI with the consequent protein-protein interaction (tetramerisation) to form a DNA loop encompassing the promoter segment. PMID:26914958

  12. Molecular Recognition of the Catalytic Zinc(II Ion in MMP-13: Structure-Based Evolution of an Allosteric Inhibitor to Dual Binding Mode Inhibitors with Improved Lipophilic Ligand Efficiencies

    Directory of Open Access Journals (Sweden)

    Thomas Fischer

    2016-03-01

    Full Text Available Matrix metalloproteinases (MMPs are a class of zinc dependent endopeptidases which play a crucial role in a multitude of severe diseases such as cancer and osteoarthritis. We employed MMP-13 as the target enzyme for the structure-based design and synthesis of inhibitors able to recognize the catalytic zinc ion in addition to an allosteric binding site in order to increase the affinity of the ligand. Guided by molecular modeling, we optimized an initial allosteric inhibitor by addition of linker fragments and weak zinc binders for recognition of the catalytic center. Furthermore we improved the lipophilic ligand efficiency (LLE of the initial inhibitor by adding appropriate zinc binding fragments to lower the clogP values of the inhibitors, while maintaining their potency. All synthesized inhibitors showed elevated affinity compared to the initial hit, also most of the novel inhibitors displayed better LLE. Derivatives with carboxylic acids as the zinc binding fragments turned out to be the most potent inhibitors (compound 3 (ZHAWOC5077: IC50 = 134 nM whereas acyl sulfonamides showed the best lipophilic ligand efficiencies (compound 18 (ZHAWOC5135: LLE = 2.91.

  13. Molecular Recognition of the Catalytic Zinc(II) Ion in MMP-13: Structure-Based Evolution of an Allosteric Inhibitor to Dual Binding Mode Inhibitors with Improved Lipophilic Ligand Efficiencies.

    Science.gov (United States)

    Fischer, Thomas; Riedl, Rainer

    2016-01-01

    Matrix metalloproteinases (MMPs) are a class of zinc dependent endopeptidases which play a crucial role in a multitude of severe diseases such as cancer and osteoarthritis. We employed MMP-13 as the target enzyme for the structure-based design and synthesis of inhibitors able to recognize the catalytic zinc ion in addition to an allosteric binding site in order to increase the affinity of the ligand. Guided by molecular modeling, we optimized an initial allosteric inhibitor by addition of linker fragments and weak zinc binders for recognition of the catalytic center. Furthermore we improved the lipophilic ligand efficiency (LLE) of the initial inhibitor by adding appropriate zinc binding fragments to lower the clogP values of the inhibitors, while maintaining their potency. All synthesized inhibitors showed elevated affinity compared to the initial hit, also most of the novel inhibitors displayed better LLE. Derivatives with carboxylic acids as the zinc binding fragments turned out to be the most potent inhibitors (compound 3 (ZHAWOC5077): IC50 = 134 nM) whereas acyl sulfonamides showed the best lipophilic ligand efficiencies (compound 18 (ZHAWOC5135): LLE = 2.91). PMID:26938528

  14. The S-enantiomer of R,S-citalopram, increases inhibitor binding to the human serotonin transporter by an allosteric mechanism. Comparison with other serotonin transporter inhibitors

    DEFF Research Database (Denmark)

    Chen, Fenghua; Larsen, Mads Breum; Sánchez, Connie;

    2005-01-01

    The interaction of the S- and R-enantiomers (escitalopram and R-citalopram) of citalopram, with high- and low-affinity binding sites in COS-1 cell membranes expressing human SERT (hSERT) were investigated. Escitalopram affinity for hSERT and its 5-HT uptake inhibitory potency was in the nanomolar...... range and approximately 40-fold more potent than R-citalopram. Escitalopram considerably stabilised the [3H]-escitalopram/SERT complex via an allosteric effect at a low-affinity binding site. The stereoselectivity between escitalopram and R-citalopram was approximately 3:1 for the [3H]-escitalopram....../hSERT complex. The combined effect of escitalopram and R-citalopram was additive. Paroxetine and sertraline mainly stabilised the [3H]-paroxetine/hSERT complex. Fluoxetine, duloxetine and venlafaxine have only minor effects. 5-HT stabilised the [125I]-RTI-55, [3H]-MADAM, [3H]-paroxetine, [3H]-fluoxetine and [3H...

  15. The S-enantiomer of R, S-citalopram, increases inhibitor binding to the human serotonin transporter by an allosteric mechanism

    DEFF Research Database (Denmark)

    Chen, Fenghua; Larsen, Mads; Sanchez, Connie;

    2005-01-01

    The interaction of the S- and R-enantiomers (escitalopram and R-citalopram) of citalopram, with high- and low-affinity binding sites in COS-1 cell membranes expressing human SERT (hSERT) were investigated. Escitalopram affinity for hSERT and its 5-HT uptake inhibitory potency was in the nanomolar...... range and approximately 40-fold more potent than R-citalopram. Escitalopram considerably stabilised the [3H]-escitalopram/SERT complex via an allosteric effect at a low-affinity binding site. The stereoselectivity between escitalopram and R-citalopram was approximately 3:1 for the [3H]-escitalopram....../hSERT complex. The combined effect of escitalopram and R-citalopram was additive. Paroxetine and sertraline mainly stabilised the [3H]-paroxetine/hSERT complex. Fluoxetine, duloxetine and venlafaxine have only minor effects. 5-HT stabilised the [125I]-RTI-55, [3H]-MADAM, [3H]-paroxetine, [3H]-fluoxetine and [3H...

  16. Identification of the Escherichia coli ADP-glucose synthetase inhibitor binding site(s)

    International Nuclear Information System (INIS)

    The photoaffinity labeling agent 8-azido adenylate (AMP) is an inhibitor site specific probe of the E. coli ADPG synthetase. In the absence of light, 8-azido AMP exhibits the typical reversible allosteric kinetics of the physiological inhibitor AMP. In the presence of light (254 nm), [2-3H]8-azido AMP specifically and covalently incorporates into the enzyme. Photoincorporation is linearly related to loss of catalytic activity up to at least 65% inactivation. The substrate ADP-glucose (ADPG) provides nearly 100% protection from 8-azido AMP photoinactivation, while the substrate AMP provides approximately 50% protection and the inhibitor AMP provides approximately 30% protection. These three adenylate allosteric effects of E. coli ADPG synthetase also protect it from photoincorporation of 8-azido AMP. The reaction site(s) of [2-3H]-azido AMP with the enzyme was identified by reverse phase HPLC isolation and chemical characterization of CNBr and mouse submaxillary arginyl protease generated peptides containing the labeled analog. This site is the same as the major binding region of the substrate site specific probe, 8-azido ADP-[14C]glucose. Conformational analysis of this region predicts that it is a part of a Rossmann fold, the super-secondary structure found in many adenine nucleotide binding proteins. Two minor reaction regions of the enzyme with [2-3H]8-azido AMP were also identified. The three modified peptide regions may be juxtaposed in the enzyme's tertiary structure

  17. An allosteric mechanism inferred from molecular dynamics simulations on phospholamban pentamer in lipid membranes.

    Directory of Open Access Journals (Sweden)

    Peng Lian

    Full Text Available Phospholamban functions as a regulator of Ca(2+ concentration of cardiac muscle cells by triggering the bioactivity of sarcoplasmic reticulum Ca(2+-ATPase. In order to understand its dynamic mechanism in the environment of bilayer surroundings, we performed long time-scale molecular dynamic simulations based on the high-resolution NMR structure of phospholamban pentamer. It was observed from the molecular dynamics trajectory analyses that the conformational transitions between the "bellflower" and "pinwheel" modes were detected for phospholamban. Particularly, the two modes became quite similar to each other after phospholamban was phosphorylated at Ser16. Based on these findings, an allosteric mechanism was proposed to elucidate the dynamic process of phospholamban interacting with Ca(2+-ATPase.

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

    DEFF Research Database (Denmark)

    Trattnig, Sarah M; Harpsøe, Kasper; Thygesen, Sarah B; Rahr, Louise M; Ahring, Philip K; Balle, Thomas; Jensen, Anders A

    2012-01-01

    class of negative allosteric modulators of the 5HT3 receptors (5HT3Rs). PU02 (6[(1naphthylmethyl)thio]9Hpurine) is a potent and selective antagonist displaying IC50 values ~1 µM at 5-HT3Rs and substantially lower activities at other Cys-loop receptors. In an elaborate mutagenesis study of the 5HT3A...... molecular determinants of PU02 activity with minor contributions from Ser292 and Val310, and we propose that the naphthalene group of PU02 docks into the hydrophobic cavity formed by these. Interestingly, specific mutations of Ser248, Thr294 and Gly306 convert PU02 into a complex modulator, potentiating and...

  19. Allosteric inhibitors of plasma membrane Ca2+ pumps: Invention and applications of caloxins

    Institute of Scientific and Technical Information of China (English)

    Jyoti; Pande; M; Szewczyk; Ashok; K; Grover

    2011-01-01

    Plasma membrane Ca2+pumps(PMCA)play a major role in Ca2+homeostasis and signaling by extruding cellular Ca2+with high affinity.PMCA isoforms are encoded by four genes which are expressed differentially in various cell types in normal and disease states.Therefore, PMCA isoform selective inhibitors would aid in delineating their role in physiology and pathophysiology.We are testing the hypothesis that extracellular domains of PMCA can be used as allosteric targets to obtain a novel class of PMCA-specific inhibitors termed caloxins. This review presents the concepts behind the invention of caloxins and our progress in this area.A section is also devoted to the applications of caloxins in literature. We anticipate that isoform-selective caloxins will aid in understanding PMCA physiology in health and disease. With strategies to develop therapeutics from bioactive peptides,caloxins may become clinically useful in car diovascular diseases,neurological disorders,retinopathy,cancer and contraception.

  20. Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones.

    Science.gov (United States)

    Johansson, Renzo; Jonna, Venkateswara Rao; Kumar, Rohit; Nayeri, Niloofar; Lundin, Daniel; Sjöberg, Britt-Marie; Hofer, Anders; Logan, Derek T

    2016-06-01

    Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone. PMID:27133024

  1. Intramolecular allosteric communication in dopamine D2 receptor revealed by evolutionary amino acid covariation.

    Science.gov (United States)

    Sung, Yun-Min; Wilkins, Angela D; Rodriguez, Gustavo J; Wensel, Theodore G; Lichtarge, Olivier

    2016-03-29

    The structural basis of allosteric signaling in G protein-coupled receptors (GPCRs) is important in guiding design of therapeutics and understanding phenotypic consequences of genetic variation. The Evolutionary Trace (ET) algorithm previously proved effective in redesigning receptors to mimic the ligand specificities of functionally distinct homologs. We now expand ET to consider mutual information, with validation in GPCR structure and dopamine D2 receptor (D2R) function. The new algorithm, called ET-MIp, identifies evolutionarily relevant patterns of amino acid covariations. The improved predictions of structural proximity and D2R mutagenesis demonstrate that ET-MIp predicts functional interactions between residue pairs, particularly potency and efficacy of activation by dopamine. Remarkably, although most of the residue pairs chosen for mutagenesis are neither in the binding pocket nor in contact with each other, many exhibited functional interactions, implying at-a-distance coupling. The functional interaction between the coupled pairs correlated best with the evolutionary coupling potential derived from dopamine receptor sequences rather than with broader sets of GPCR sequences. These data suggest that the allosteric communication responsible for dopamine responses is resolved by ET-MIp and best discerned within a short evolutionary distance. Most double mutants restored dopamine response to wild-type levels, also suggesting that tight regulation of the response to dopamine drove the coevolution and intramolecular communications between coupled residues. Our approach provides a general tool to identify evolutionary covariation patterns in small sets of close sequence homologs and to translate them into functional linkages between residues. PMID:26979958

  2. Positive allosteric modulators to peptide GPCRs:a promising class of drugs

    Institute of Scientific and Technical Information of China (English)

    Tamas BARTFAI; Ming-wei WANG

    2013-01-01

    The task of finding selective and stable peptide receptor agonists with low molecular weight,desirable pharmacokinetic properties and penetrable to the blood-brain barrier has proven too difficult for many highly coveted drug targets,including receptors for endothelin,vasoactive intestinal peptide and galanin.These receptors and ligand-gated ion channels activated by structurally simple agonists such as glutamate,glycine and GABA present such a narrow chemical space that the design of subtype-selective molecules capable of distinguishing a dozen of glutamate and GABA receptor subtypes and possessing desirable pharmacokinetic properties has also been problematic.In contrast,the pharmaceutical industry demonstrates a remarkable success in developing 1,4-benzodiazepines,positive allosteric modulators (PMAs) of the GABAA receptor.They were synthesized over 50 years ago and discovered to have anxiolytic potential through an in vivo assay.As exemplified by Librium,Valium and Dormicum,these allosteric ligands of the receptor became the world's first blockbuster drugs.Through molecular manipulation over the past 2 decades,including mutations and knockouts of the endogenous ligands or their receptors,and by in-depth physiological and pharmacological studies,more peptide and glutamate receptors have become well-validated drug targets for which an agonist is sought.In such cases,the pursuit for PAMs has also intensified,and a working paradigm to identify drug candidates that are designed as PAMs has emerged.This review,which focuses on the general principles of finding PAMs of peptide receptors in the 21st century,describes the workflow and some of its resulting compounds such as PAMs of galanin receptor 2 that act as potent anticonvulsant agents.

  3. Assessment of direct gating and allosteric modulatory effects of meprobamate in recombinant GABA(A) receptors.

    Science.gov (United States)

    Kumar, Manish; Dillon, Glenn H

    2016-03-15

    Meprobamate is a schedule IV anxiolytic and the primary metabolite of the muscle relaxant carisoprodol. Meprobamate modulates GABAA (γ-aminobutyric acid Type A) receptors, and has barbiturate-like activity. To gain insight into its actions, we have conducted a series of studies using recombinant GABAA receptors. In αxβzγ2 GABAA receptors (where x=1-6 and z=1-3), the ability to enhance GABA-mediated current was evident for all α subunit isoforms, with the largest effect observed in α5-expressing receptors. Direct gating was present with all α subunits, although attenuated in α3-expressing receptors. Allosteric and direct effects were comparable in α1β1γ2 and α1β2γ2 receptors, whereas allosteric effects were enhanced in α1β2 compared to α1β2γ2 receptors. In "extrasynaptic" (α1β3δ and α4β3δ) receptors, meprobamate enhanced EC20 and saturating GABA currents, and directly activated these receptors. The barbiturate antagonist bemegride attenuated direct effects of meprobamate. Whereas pentobarbital directly gated homomeric β3 receptors, meprobamate did not, and instead blocked the spontaneously open current present in these receptors. In wild type homomeric ρ1 receptors, pentobarbital and meprobamate were ineffective in direct gating; a mutation known to confer sensitivity to pentobarbital did not confer sensitivity to meprobamate. Our results provide insight into the actions of meprobamate and parent therapeutic agents such as carisoprodol. Whereas in general actions of meprobamate were comparable to those of carisoprodol, differential effects of meprobamate at some receptor subtypes suggest potential advantages of meprobamate may be exploited. A re-assessment of previously synthesized meprobamate-related carbamate molecules for myorelaxant and other therapeutic indications is warranted. PMID:26872987

  4. Dual-cavity basket promotes encapsulation in water in an allosteric fashion.

    Science.gov (United States)

    Chen, Shigui; Yamasaki, Makoto; Polen, Shane; Gallucci, Judith; Hadad, Christopher M; Badjić, Jovica D

    2015-09-30

    We prepared dual-cavity basket 1 to carry six (S)-alanine residues at the entrance of its two juxtaposed cavities (289 Å(3)). With the assistance of (1)H NMR spectroscopy and calorimetry, we found that 1 could trap a single molecule of 4 (K1 = 1.45 ± 0.40 × 10(4) M(-1), ITC), akin in size (241 Å(3)) and polar characteristics to nerve agent VX (289 Å(3)). The results of density functional theory calculations (DFT, M06-2X/6-31G*) and experiments ((1)H NMR spectroscopy) suggest that the negative homotropic allosterism arises from the guest forming C-H···π contacts with all three of the aromatic walls of the occupied basket's cavity. In response, the other cavity increases its size and turns rigid to prevent the formation of the ternary complex. A smaller guest 6 (180 Å(3)), akin in size and polar characteristics to soman (186 Å(3)), was also found to bind to dual-cavity 1, although giving both binary [1⊂6] and ternary [1⊂62] complexes (K1 = 7910 M(-1) and K2 = 2374 M(-1), (1)H NMR spectroscopy). In this case, the computational and experimental ((1)H NMR spectroscopy) results suggest that only two aromatic walls of the occupied basket's cavity form C-H···π contacts with the guest to render the singly occupied host flexible enough to undergo additional structural changes necessary for receiving another guest molecule. The structural adaptivity of dual-cavity baskets of type 1 is unique and important for designing multivalent hosts capable of effectively sequestering targeted guests in an allosteric manner to give stable supramolecular polymers. PMID:26348904

  5. Single Enzyme Studies Reveal the Existence of Discrete Functional States for Monomeric Enzymes and How They Are “Selected” upon Allosteric Regulation

    DEFF Research Database (Denmark)

    Hatzakis, Nikos S.; Wei, Li; Jørgensen, Sune Klamer;

    2012-01-01

    Allosteric regulation of enzymatic activity forms the basis for controlling a plethora of vital cellular processes. While the mechanism underlying regulation of multimeric enzymes is generally well understood and proposed to primarily operate via conformational selection, the mechanism underlying...... allosteric regulation of monomeric enzymes is poorly understood. Here we monitored for the first time allosteric regulation of enzymatic activity at the single molecule level. We measured single stochastic catalytic turnovers of a monomeric metabolic enzyme (Thermomyces lanuginosus Lipase) while titrating...... its proximity to a lipid membrane that acts as an allosteric effector. The single molecule measurements revealed the existence of discrete binary functional states that could not be identified in macroscopic measurements due to ensemble averaging. The discrete functional states correlate with the...

  6. Impact of disruption of secondary binding site S2 on dopamine transporter function.

    Science.gov (United States)

    Zhen, Juan; Reith, Maarten E A

    2016-09-01

    The structures of the leucine transporter, drosophila dopamine transporter, and human serotonin transporter show a secondary binding site (designated S2 ) for drugs and substrate in the extracellular vestibule toward the membrane exterior in relation to the primary substrate recognition site (S1 ). The present experiments are aimed at disrupting S2 by mutating Asp476 and Ile159 to Ala. Both mutants displayed a profound decrease in [(3) H]DA uptake compared with wild-type associated with a reduced turnover rate kcat . This was not caused by a conformational bias as the mutants responded to Zn(2+) (10 μM) similarly as WT. The dopamine transporters with either the D476A or I159A mutation both displayed a higher Ki for dopamine for the inhibition of [3H](-)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane binding than did the WT transporter, in accordance with an allosteric interaction between the S1 and S2 sites. The results provide evidence in favor of a general applicability of the two-site allosteric model of the Javitch/Weinstein group from LeuT to dopamine transporter and possibly other monoamine transporters. X-ray structures of transporters closely related to the dopamine (DA) transporter show a secondary binding site S2 in the extracellular vestibule proximal to the primary binding site S1 which is closely linked to one of the Na(+) binding sites. This work examines the relationship between S2 and S1 sites. We found that S2 site impairment severely reduced DA transport and allosterically reduced S1 site affinity for the cocaine analog [(3) H]CFT. Our results are the first to lend direct support for the application of the two-site allosteric model, advanced for bacterial LeuT, to the human DA transporter. The model states that, after binding of the first DA molecule (DA1 ) to the primary S1 site (along with Na(+) ), binding of a second DA (DA2 ) to the S2 site triggers, through an allosteric interaction, the release of DA1 and Na(+) into the cytoplasm. PMID

  7. On the G-Protein-Coupled Receptor Heteromers and Their Allosteric Receptor-Receptor Interactions in the Central Nervous System: Focus on Their Role in Pain Modulation

    OpenAIRE

    Kjell Fuxe; Tarakanov, Alexander O.; Luigi F. Agnati; Alicia Rivera; Kathleen Van Craenenbroeck; Wilber Romero-Fernandez; Dasiel O. Borroto-Escuela

    2013-01-01

    The modulatory role of allosteric receptor-receptor interactions in the pain pathways of the Central Nervous System and the peripheral nociceptors has become of increasing interest. As integrators of nociceptive and antinociceptive wiring and volume transmission signals, with a major role for the opioid receptor heteromers, they likely have an important role in the pain circuits and may be involved in acupuncture. The delta opioid receptor (DOR) exerts an antagonistic allosteric influence on ...

  8. Analysis of Binding Site Hot Spots on the Surface of Ras GTPase

    Energy Technology Data Exchange (ETDEWEB)

    Buhrman, Greg; O; #8242; Connor, Casey; Zerbe, Brandon; Kearney, Bradley M.; Napoleon, Raeanne; Kovrigina, Elizaveta A.; Vajda, Sandor; Kozakov, Dima; Kovrigin, Evgenii L.; Mattos, Carla (NCSU); (MCW); (BU)

    2012-09-17

    We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the 'off' and 'on' allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond the active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.

  9. Development of a highly selective allosteric antagonist radioligand for the type 1 cholecystokinin receptor and elucidation of its molecular basis of binding.

    Science.gov (United States)

    Dong, Maoqing; Vattelana, Ashton M; Lam, Polo C-H; Orry, Andrew J; Abagyan, Ruben; Christopoulos, Arthur; Sexton, Patrick M; Haines, David R; Miller, Laurence J

    2015-01-01

    Understanding the molecular basis of ligand binding to receptors provides insights useful for rational drug design. This work describes development of a new antagonist radioligand of the type 1 cholecystokinin receptor (CCK1R), (2-fluorophenyl)-2,3-dihydro-3-[(3-isoquinolinylcarbonyl)amino]-6-methoxy-2-oxo-l-H-indole-3-propanoate (T-0632), and exploration of the molecular basis of its binding. This radioligand bound specifically with high affinity within an allosteric pocket of CCK1R. T-0632 fully inhibited binding and action of CCK at this receptor, while exhibiting no saturable binding to the closely related type 2 cholecystokinin receptor (CCK2R). Chimeric CCK1R/CCK2R constructs were used to explore the molecular basis of T-0632 binding. Exchanging exonic regions revealed the functional importance of CCK1R exon 3, extending from the bottom of transmembrane segment (TM) 3 to the top of TM5, including portions of the intramembranous pocket as well as the second extracellular loop region (ECL2). However, CCK1R mutants in which each residue facing the pocket was changed to that present in CCK2R had no negative impact on T-0632 binding. Extending the chimeric approach to ECL2 established the importance of its C-terminal region, and site-directed mutagenesis of each nonconserved residue in this region revealed the importance of Ser(208) at the top of TM5. A molecular model of T-0632-occupied CCK1R was consistent with these experimental determinants, also identifying Met(121) in TM3 and Arg(336) in TM6 as important. Although these residues are conserved in CCK2R, mutating them had a distinct impact on the two closely related receptors, suggesting differential orientation. This establishes the molecular basis of binding of a highly selective nonpeptidyl allosteric antagonist of CCK1R, illustrating differences in docking that extend beyond determinants attributable to distinct residues lining the intramembranous pocket in the two receptor subtypes. PMID:25319540

  10. Direct and allosteric inhibition of the FGF2/HSPGs/FGFR1 ternary complex formation by an antiangiogenic, thrombospondin-1-mimic small molecule.

    Directory of Open Access Journals (Sweden)

    Katiuscia Pagano

    Full Text Available Fibroblast growth factors (FGFs are recognized targets for the development of therapies against angiogenesis-driven diseases, including cancer. The formation of a ternary complex with the transmembrane tyrosine kinase receptors (FGFRs, and heparan sulphate proteoglycans (HSPGs is required for FGF2 pro-angiogenic activity. Here by using a combination of techniques including Nuclear Magnetic Resonance, Molecular Dynamics, Surface Plasmon Resonance and cell-based binding assays we clarify the molecular mechanism of inhibition of an angiostatic small molecule, sm27, mimicking the endogenous inhibitor of angiogenesis, thrombospondin-1. NMR and MD data demonstrate that sm27 engages the heparin-binding site of FGF2 and induces long-range dynamics perturbations along FGF2/FGFR1 interface regions. The functional consequence of the inhibitor binding is an impaired FGF2 interaction with both its receptors, as demonstrated by SPR and cell-based binding assays. We propose that sm27 antiangiogenic activity is based on a twofold-direct and allosteric-mechanism, inhibiting FGF2 binding to both its receptors.

  11. 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

    2016-06-01

    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. PMID:27075672

  12. Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation.

    Science.gov (United States)

    Buller, Andrew R; Brinkmann-Chen, Sabine; Romney, David K; Herger, Michael; Murciano-Calles, Javier; Arnold, Frances H

    2015-11-24

    Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope. PMID:26553994

  13. The positive allosteric GABAB receptor modulator rac-BHFF enhances baclofen-mediated analgesia in neuropathic mice.

    Science.gov (United States)

    Zemoura, Khaled; Ralvenius, William T; Malherbe, Pari; Benke, Dietmar

    2016-09-01

    Neuropathic pain is associated with impaired inhibitory control of spinal dorsal horn neurons, which are involved in processing pain signals. The metabotropic GABAB receptor is an important component of the inhibitory system and is highly expressed in primary nociceptors and intrinsic dorsal horn neurons to control their excitability. Activation of GABAB receptors with the orthosteric agonist baclofen effectively reliefs neuropathic pain but is associated with severe side effects that prevent its widespread application. The recently developed positive allosteric GABAB receptor modulators lack most of these side effects and are therefore promising drugs for the treatment of pain. Here we tested the high affinity positive allosteric modulator rac-BHFF for its ability to relief neuropathic pain induced by chronic constriction of the sciatic nerve in mice. rac-BHFF significantly increased the paw withdrawal threshold to mechanical stimulation in healthy mice, indicating an endogenous GABABergic tone regulating the sensitivity to mechanical stimuli. Surprisingly, rac-BHFF displayed no analgesic activity in neuropathic mice although GABAB receptor expression was not affected in the dorsal horn as shown by quantitative receptor autoradiography. However, activation of spinal GABAB receptors by intrathecal injection of baclofen reduced hyperalgesia and its analgesic effect was considerably potentiated by co-application of rac-BHFF. These results indicate that under conditions of neuropathic pain the GABAergic tone is too low to provide a basis for allosteric modulation of GABAB receptors. However, allosteric modulators would be well suited as an add-on to reduce the dose of baclofen required to achieve analgesia. PMID:27108932

  14. Temperature-dependent effects of cadmium and purine nucleotides on mitochondrial aconitase from a marine ectotherm, Crassostrea virginica: a role of temperature in oxidative stress and allosteric enzyme regulation.

    Science.gov (United States)

    Cherkasov, Anton A; Overton, Robert A; Sokolov, Eugene P; Sokolova, Inna M

    2007-01-01

    Temperature and heavy metals such as cadmium (Cd) are important environmental stressors that can strongly affect mitochondrial function of marine poikilotherms. In this study, we investigated the combined effects of temperature (20 degrees C and 30 degrees C) and Cd stress on production of reactive oxygen species (ROS) and oxidative stress in a marine poikilotherm Crassostrea virginica (the eastern oyster) using mitochondrial aconitase as a sensitive biomarker of oxidative damage. We also assessed potential involvement of mitochondrial uncoupling proteins (UCPs) in antioxidant protection in oyster mitochondria using purine nucleotides (GDP, ATP and ADP) as specific inhibitors, and free fatty acids as stimulators, of UCPs. Our results show that exposure to Cd results in elevated ROS production and oxidative damage as indicated by aconitase inactivation which is particularly pronounced at elevated temperature. Unexpectedly, oyster mitochondrial aconitase was inhibited by physiologically relevant levels of ATP (IC(50)=1.93 and 3.04 mmol l(-1) at 20 degrees C and 30 degrees C, respectively), suggesting that allosteric regulation of aconitase by this nucleotide may be involved in regulation of the tricarboxylic acid flux in oysters. Aconitase was less sensitive to ATP inhibition at 30 degrees C than at 20 degrees C, consistent with the elevated metabolic flux at higher temperatures. ADP and GDP also inhibited mitochondrial aconitase but at the levels well above the physiological concentrations of these nucleotides (6-11 mmol l(-1)). Our study shows expression of at least three UCP isoforms in C. virginica gill tissues but provides no indication that UCPs protect mitochondrial aconitase from oxidative inactivation in oysters. Overall, the results of this study indicate that temperature stress exaggerates toxicity of Cd leading to elevated oxidative stress in mitochondria, which may have important implications for survival of poikilotherms in polluted environments during

  15. Comparing meteorological records between mountainous and valley bottom sites in the upper reaches of the Heihe River, northwestern China: implications for dendroclimatology

    Science.gov (United States)

    Zeng, Qiao; Yang, Bao

    2016-01-01

    Tree-ring records from remote upland areas are widely used in climate reconstructions, but they are typically calibrated by meteorological data from low-lying areas. With the aim of assessing relationships between climatic records from mountainous and valley bottoms, this study compared meteorological records between mountainous sites (lower to upper elevations) and valley bottoms (Qilian (QL) and Yeniugou (YNG)) in the upper reaches of the Heihe River, northwestern China. We found that daily, 5-day, 10-day, and monthly mean air temperatures observed during a 4-year observational period in valley bottoms were strongly correlated (R = 0.90-0.99) to their mountainous site counterparts. Additionally, temperature records from the QL meteorological station shared a higher percentage of variance with each mountainous site compared to YNG. Correlations of precipitation totals between valley bottoms and mountainous sites showed a similar pattern to temperatures. Furthermore, different time series of total rainfall in YNG can explain more variance than those from QL and were also more suitable representations of mountainous sites. Our results confirmed the reliability of utilizing monthly climatic records from valley bottoms to calibrate tree-ring records in mountainous sites. We also caution that when conducting fine-scale microcoring and dendrometer monitoring studies, lower correlations of short-term scale precipitation records between valley bottoms and mountainous sites may introduce unavoidable errors.

  16. The energetics of allosteric regulation of ADP release from myosin heads.

    Science.gov (United States)

    Jackson, Del R; Baker, Josh E

    2009-06-28

    Myosin molecules are involved in a wide range of transport and contractile activities in cells. A single myosin head functions through its ATPase reaction as a force generator and as a mechanosensor, and when two or more myosin heads work together in moving along an actin filament, the interplay between these mechanisms contributes to collective myosin behaviors. For example, the interplay between force-generating and force-sensing mechanisms coordinates the two heads of a myosin V molecule in its hand-over-hand processive stepping along an actin filament. In muscle, it contributes to the Fenn effect and smooth muscle latch. In both examples, a key force-sensing mechanism is the regulation of ADP release via interhead forces that are generated upon actin-myosin binding. Here we present a model describing the mechanism of allosteric regulation of ADP release from myosin heads as a change, DeltaDeltaG(-D), in the standard free energy for ADP release that results from the work, Deltamicro(mech), performed by that myosin head upon ADP release, or DeltaDeltaG(-D) = Deltamicro(mech). We show that this model is consistent with previous measurements for strain-dependent kinetics of ADP release in both myosin V and muscle myosin II. The model makes explicit the energetic cost of accelerating ADP release, showing that acceleration of ADP release during myosin V processivity requires approximately 4 kT of energy whereas the energetic cost for accelerating ADP release in a myosin II-based actin motility assay is only approximately 0.4 kT. The model also predicts that the acceleration of ADP release involves a dissipation of interhead forces. To test this prediction, we use an in vitro motility assay to show that the acceleration of ADP release from both smooth and skeletal muscle myosin II correlates with a decrease in interhead force. Our analyses provide clear energetic constraints for models of the allosteric regulation of ADP release and provide novel, testable insights

  17. 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

  18. Otter (Lontra longicaudis) Spraint and Mucus Depositions: Early Ecological Insights into the Differences in Marking Site Selection and Implications for Monitoring Prey Availability

    OpenAIRE

    Nathan James Roberts; Ryan Matthew Clark; Delyth Williams

    2016-01-01

    Otters are not territorial in the classical sense of marking territory boundaries. Instead they mark key resource areas within the territory with faeces, or spraint, for olfactory communication. There is a high level of site fidelity in otter marking behaviour and the function of scent marks may explain their spatial distribution. Typically marking sites are in proximity to deep water which provides key resource areas for energy-efficient foraging. Occasionally spraint is laden with mucus, wh...

  19. Sanitary Conditions of Food Vending Sites and Food Handling Practices of Street Food Vendors in Benin City, Nigeria: Implication for Food Hygiene and Safety

    OpenAIRE

    Okojie, P. W.; Isah, E C

    2014-01-01

    Objective. To determine the sanitary conditions of vending sites as well as food handling practices of street food vendors in Benin City, Nigeria. Methodology. A descriptive cross-sectional study was done using an observational checklist and researcher-administered questionnaire. 286 randomly selected vending units were surveyed, and their operators interviewed on their food handling practices. Results. A higher proportion, 259 (90.5%), of the observed vending sites appeared clean. The follow...

  20. The earliest well-dated archeological site in the hyper-arid Tarim Basin and its implications for prehistoric human migration and climatic change

    Science.gov (United States)

    Han, WenXia; Yu, LuPeng; Lai, ZhongPing; Madsen, David; Yang, Shengli

    2014-07-01

    The routes and timing of human occupation of the Tibetan Plateau (TP) are crucial for understanding the evolution of Tibetan populations and associated paleoclimatic conditions. Many archeological sites have been found in/around the Tarim Basin, on the northern margin of the Tibetan Plateau. Unfortunately, most of these sites are surface sites and cannot be directly dated. Their ages can only be estimated based on imprecise artifact comparisons. We recently found and dated an archeological site on a terrace along the Keriya River. Our ages indicate that the site was occupied at ~ 7.0-7.6 ka, making it the earliest well-dated archeological site yet identified in the Tarim Basin. This suggests that early human foragers migrated into this region prior to ~ 7.0-7.6 ka during the early to mid-Holocene climatic optimum, which may have provided the impetus for populating the region. We hypothesize that the Keriya River, together with the other rivers originating from the TP, may have served as access routes onto the TP for early human foragers. These rivers may also have served as stepping stones for migration further west into the now hyper-arid regions of the Tarim Basin, leading ultimately to the development of the Silk Road.

  1. Ultrasensitivity and heavy-metal selectivity of the allosterically modulated MerR transcription complex

    International Nuclear Information System (INIS)

    The MerR metalloregulatory protein is a heavy-metal receptor that functions as the repressor and Hg(II)-responsive transcription activator of the prokaryotic mercury-resistance (mer) genes. The authors demonstrate that this allosterically modulated regulatory protein is sensitive to HgCl2 concentrations of 1.0 ± 0.3 x 10-8 M in the presence of 1.0 x 10-3 M dithiothreitol for half-maximal induction of transcription of the mer promoter by Escherichia coli RNA polymerase in vitro. Transcription mediated by MerR increases from 10% to 90% of maximum in response to a 7-fold change in concentration of HgCl2, consistent with a threshold phenomenon known as ultrasensitivity. In addition, MerR exhibits a high degree of selectivity. Cd(II), Zn(II), Ag(I), Au(I), and Au(III) have been found to partially stimulate transcription in the presence of MerR, but concentrations at least two to three orders of magnitude greater than for Hg(II) are required. The molecular basis of the ultrasensitivity and selectivity phenomena are postulated to arise from the unusual topology of the transcription complex and a rare trigonal mercuric ion coordination environment, respectively. This mercuric ion-induced switch is to our knowledge the only known example of ultrasensitivity in a signal-responsive transcription mechanism

  2. GABAB receptor as therapeutic target for drug addiction: from baclofen to positive allosteric modulators

    Directory of Open Access Journals (Sweden)

    Roberta Agabio

    2015-04-01

    Full Text Available The present paper summarizes experimental and clinical data indicating the therapeutic potential of the GABAB receptor agonist, baclofen, in the treatment of alcohol use disorder (AUD and substance use disorder (SUD. Multiple preclinical studies have demonstrated the ability of baclofen to suppress alcohol drinking (including binge- and relapse-like drinking, oral alcohol self-administration, and intravenous self-administration of cocaine, nicotine, amphetamine, methamphetamine, morphine, and heroin in rodents. Some randomized, controlled trials (RCTs and case reports support the efficacy of baclofen in suppressing alcohol consumption, craving for alcohol, and alcohol withdrawal symptomatology in alcohol-dependent patients. Data from RCTs and open studies investigating baclofen efficacy on SUD are currently less conclusive. Interest in testing high doses of baclofen in AUD and SUD treatment has recently emerged. Preclinical research has extended the anti-addictive properties of baclofen to positive allosteric modulators of the GABAB receptor (GABAB PAMs. In light of their more favourable side effect profile (compared to baclofen, GABAB PAMs may represent a major step forward in a GABAB receptor-based pharmacotherapy of AUD and SUD.

  3. An allosteric model of the inositol trisphosphate receptor with nonequilibrium binding

    International Nuclear Information System (INIS)

    The inositol trisphosphate receptor (IPR) is a crucial ion channel that regulates the Ca2+ influx from the endoplasmic reticulum (ER) to the cytoplasm. A thorough study of the IPR channel contributes to a better understanding of calcium oscillations and waves. It has long been observed that the IPR channel is a typical biological system which performs adaptation. However, recent advances on the physical essence of adaptation show that adaptation systems with a negative feedback mechanism, such as the IPR channel, must break detailed balance and always operate out of equilibrium with energy dissipation. Almost all previous IPR models are equilibrium models assuming detailed balance and thus violate the dissipative nature of adaptation. In this article, we constructed a nonequilibrium allosteric model of single IPR channels based on the patch-clamp experimental data obtained from the IPR in the outer membranes of isolated nuclei of the Xenopus oocyte. It turns out that our model reproduces the patch-clamp experimental data reasonably well and produces both the correct steady-state and dynamic properties of the channel. Particularly, our model successfully describes the complicated bimodal [Ca2+] dependence of the mean open duration at high [IP3], a steady-state behavior which fails to be correctly described in previous IPR models. Finally, we used the patch-clamp experimental data to validate that the IPR channel indeed breaks detailed balance and thus is a nonequilibrium system which consumes energy. (paper)

  4. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.

    Science.gov (United States)

    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

    2016-07-01

    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 phosphatase. 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 neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. 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. PMID:27362227

  5. The Metabotropic Glutamate Receptor 4 Positive Allosteric Modulator ADX88178 Inhibits Inflammatory Responses in Primary Microglia.

    Science.gov (United States)

    Ponnazhagan, Ranjani; Harms, Ashley S; Thome, Aaron D; Jurkuvenaite, Asta; Gogliotti, Rocco; Niswender, Colleen M; Conn, P Jeffrey; Standaert, David G

    2016-06-01

    While the specific trigger of Parkinson Disease (PD) in most patients is unknown, considerable evidence suggests that the neuroinflammatory response makes an essential contribution to the neurodegenerative process. Drugs targeting metabotropic glutamate receptors (mGlu receptors), 7 Transmembrane (7TM) spanning/G protein coupled receptors that bind glutamate, are emerging as therapeutic targets for PD and may have anti-inflammatory properties. ADX88178 is novel potent, selective, and brain-penetrant positive allosteric modulator of the mGlu4 which is under evaluation for treatment of PD and other neurological disorders. We used microglia cultured from mouse brain to determine if ADX88178 had direct effects on the inflammatory responses of these cells. We studied both microglia from wild type and Grm4 knock out mice. We found that activation of mGlu4 with ADX88178 attenuated LPS-induced inflammation in primary microglia, leading to a decrease in the expression of TNFα, MHCII, and iNOS, markers of pro-inflammatory responses. These effects were absent in microglia from mice lacking mGlu4. These results demonstrate a cell-autonomous anti-inflammatory effect of ADX88178 mediated mGlu4 activation on microglia, and suggest that this drug or similar activators or potentiators of mGlu4 may have disease-modifying as well as symptomatic effects in PD and other brain disorders with an inflammatory component. PMID:26872456

  6. Comparing allosteric transitions in the domains of calmodulin through coarse-grained simulations

    CERN Document Server

    Nandigrami, Prithviraj

    2015-01-01

    Calmodulin (CaM) is a ubiquitous calcium binding protein consisting of two structurally similar domains with distinct stabilities, binding affinities, and flexibilities. We present coarse grained simulations that suggest the mechanism for the domain's allosteric transitions between the open and closed conformations depend on subtle differences in the folded state topology of the two domains. Throughout a wide temperature range, the simulated transition mechanism of the N-terminal domain (nCaM) follows a two-state transition mechanism while domain opening in the C-terminal domain (cCaM) involves unfolding and refolding of the tertiary structure. The appearance of the unfolded intermediate occurs at a higher temperature in nCaM than it does in cCaM. That is, we find that cCaM unfolds more readily along the transition route than nCaM. Furthermore, unfolding and refolding of the domain significantly slows the domain opening and closing rates of cCaM, a distinct scenario which can potentially influence the mechani...

  7. 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.

  8. 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

    mutational map for agonism but it was not identical with the map for the agonist property of these small-molecule ligands. In molecular models, built over the inactive conformation of rhodopsin, low energy conformations of the nonpeptide agonists could be docked to satisfy many of their mutational hits. It...

  9. NOVEL HETEROCYCLIC RING SYSTEMS DERIVED FROM CARACURINE V AS LIGANDS FOR THE ALLOSTERIC SITE OF MUSCARINIC M 2 RECEPTORS

    OpenAIRE

    Kittisak Sripha

    2003-01-01

    Die vorliegende Arbeit befasst sich mit dem Gebiet allosterischer Modulation des muscarinischen M2 Rezeptors. Allosterische Liganden beeinflussen das Bindungsverhalten eines orthosterischen Liganden (Agonisten oder Antagonisten) an die klassische Bindungsstelle des muscarinischen Rezeptors, indem sie seine Affinität entweder erhöhen(positive Kooperativität) oder erniedrigen (negative Kooperativität). Die allosterische Bindungsstelle befindet sich extrazellulär am Eingang der Rezeptor-Bindungs...

  10. Bimodal TiO2 Contents of Mare Basalts at Apollo and Luna Sites and Implications for TiO2 Derived from Clementine Spectral Reflectance

    Science.gov (United States)

    Gillis, J. J.; Jolliff, B. L.

    2001-01-01

    A revised algorithm to estimate Ti contents of mare regions centered on Apollo and Luna sites shows a bimodal distribution, consistent with mare-basalt sample data. A global TiO2 map shows abundant intermediate TiO2 basalts in western Procellarum. Additional information is contained in the original extended abstract.

  11. 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…

  12. Atmospheric occurrence and gas-particle partitioning of PBDEs at industrial, urban and suburban sites of Thessaloniki, northern Greece: Implications for human health.

    Science.gov (United States)

    Besis, Athanasios; Voutsa, Dimitra; Samara, Constantini

    2016-08-01

    Air samples were collected during the cold and the warm period of the year 2012 and 2013 at three sites in the major Thessaloniki area, northern Greece (urban-industrial, urban-traffic and urban-background) in order to evaluate the occurrence, profiles, seasonal variation and gas/particle partitioning of polybrominated diphenyl ethers (PBDEs). The mean total concentrations of particle phase ∑12PBDE in the cold season were 28.7, 19.5 and 3.87 pg m(-3) at the industrial, urban-traffic and urban-background site, respectively, dropping slightly in the warm season (23.7, 17.5 and 3.14 pg m(-3)), respectively. The corresponding levels of gas-phase ∑12PBDE were 14.4, 7.15 and 4.73 pg m(-3) in the cold season and 21.2, 11.1 and 6.27 pg m(-3) in the warm season, respectively. In all samples, BDE-47 and BDE-99 were the dominant congeners. Absorption of PBDEs in the organic matter of particles appeared to drive their gas/particle partitioning, particularly in the cold season. The estimated average outdoor workday inhalation exposure to ∑12PBDE in the cold and the warm period followed the order: industrial site (288 and 299 pg day(-1)) > urban-traffic site (178 and 191 pg day(-1)) > urban-background site (58 and 63 pg day(-1)). The exposures to BDE-47, BDE-99, BDE-153 and ∑3PBDE via inhalation, for children outdoor worker and seniors were several orders of magnitude lower than their corresponding oral RfD values. PMID:27179330

  13. Implication of site quality on mitochondrial electron transport activity and its interaction with temperature in feral Mya arenaria clams from the Saguenay Fjord

    International Nuclear Information System (INIS)

    The advent of global warming has given rise to questions about the impact of temperature/pollution interactions on the integrity of certain benthic organisms like bivalves. This interaction was examined in intertidal Mya arenaria clams from the Saguenay Fjord using the concepts of cellular energy allocation and temperature-dependent mitochondrial electron transport (METT) activity. Clams were collected at low tide from six sites (two clean, four polluted) for determinations of condition factor (weight/shell length), growth index (age-to-length ratio), gonadal lipids and maturation index, gonad MET at various habitat temperatures, METT, gill xanthine oxidase and gill DNA damage. Condition factor was generally lower at the four polluted sites, with growth index being severely affected at two of them. Gonadal maturation was also significantly dampened at two of the four pollution-impacted sites. Gill xanthine oxidase (purine bases salvage pathway) and DNA strand breaks were significantly increased at most of the polluted sites, confirming pollution-mediated damage in clams. Moreover, MET at 20 deg. C, METT and gonad lipids were significantly induced at the polluted sites. Clam condition factor was negatively correlated with most of the biomarkers for cellular energy allocation (gonadal lipids, MET and METT), but not with gonadal maturation. DNA damage and xanthine oxidase were positively correlated with MET at 20 deg. C and METT. This is the first report of electron transport in mitochondria being more sensitive to incremental temperature increases in clams under pollution stress. The gradual warming of clam habitats would likely worsen the impacts of pollution in feral clam populations

  14. Marte Valles site

    Science.gov (United States)

    Rice, Jim W.

    1994-01-01

    This site is located at 16 deg N, 177 deg W on the flood plains of Marte Valles, which is perhaps the youngest channel system on Mars. The young age of this channel warrants investigation because of climatic implications for fluvial activities in recent geologic time. The paucity of craters makes this an excellent site in terms of safety requirements. Some of the objectives stated previously for the Maja Valles region would also apply to this site (grab bag of rock types, etc.).

  15. Movements and site fidelity of harbour seals (Phoca vitulina) in Kattegat, Denmark, with implications for the epidemiology of the phocine distemper virus

    DEFF Research Database (Denmark)

    Dietz, R.; Teilmann, J.; Andersen, S.M.;

    2013-01-01

    Twenty-seven harbour seals were caught and tagged at the island of Anholt in central Kattegat, Denmark, the epicentre of the phocine distemper virus (PDV) outbreaks in 1988 and 2002 that killed 50–60% of the populations. The satellite tagging shows that harbour seals from Anholt moved widely across...... is larger than for adult seals (1713 km2), showing a strong site fidelity, indicating limited gene flow between haul-out sites. Distances moved and home range sizes increased across autumn, peaked in February–March, and decreased through spring. During the breeding season in spring, all seals were very...... stationary around Anholt. The onset of the PDV epizootics in 1988 and 2002 took place when the Anholt harbour seals congregate on the Island during April. Anholt seal were also documented to have contact with infected seal locations at Hesselø, Læsø, and the Swedish west coast, although this contact takes...

  16. Otter (Lontra longicaudis Spraint and Mucus Depositions: Early Ecological Insights into the Differences in Marking Site Selection and Implications for Monitoring Prey Availability

    Directory of Open Access Journals (Sweden)

    Nathan James Roberts

    2016-04-01

    Full Text Available Otters are not territorial in the classical sense of marking territory boundaries. Instead they mark key resource areas within the territory with faeces, or spraint, for olfactory communication. There is a high level of site fidelity in otter marking behaviour and the function of scent marks may explain their spatial distribution. Typically marking sites are in proximity to deep water which provides key resource areas for energy-efficient foraging. Occasionally spraint is laden with mucus, which may also be deposited in isolation without any faecal material. Any difference in the microhabitat variables predisposing site selection by otters with mucus present and absent in depositions has not yet been quantitatively investigated. This study serves as a primary exploration of these selective processes. Here we show that habitat selection by the Neotropical otter (Lontra longicaudis is different when mucus is deposited at a site compared to when it is absent. The cause of mucus deposition has been suggested in other otter species to indicate reduced prey availability or reproductive state. Depositions here were associated with deep water as in other studies, and temporally, the relative abundance of those with mucus present was highest toward the end of the dry season when prey availability is assumed relatively low. Here we infer how the monitoring of mucus prevalence may be used as a valuable efficient indirect index of the status of otters and their prey. For species whose primary threats include reduced prey availability, such as the Neotropical otter, research attention to these aspects of behavioural ecology are particularly significant in applied conservation. Furthermore, research to validate indirect indices of prey availability and species-habitat interactions may extend to benefit recreational interests and broader human-wildlife conflict mitigation strategies.

  17. Comparison of Cenozoic Faulting at the Savannah River Site to Fault Characteristics of the Atlantic Coast Fault Province: Implications for Fault Capability

    Energy Technology Data Exchange (ETDEWEB)

    Cumbest, R.J.

    2000-11-14

    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.

  18. Identification of noncollagenous sites encoding specific interactions and quaternary assembly of alpha 3 alpha 4 alpha 5(IV) collagen: implications for Alport gene therapy.

    Science.gov (United States)

    Kang, Jeong Suk; Colon, Selene; Hellmark, Thomas; Sado, Yoshikazu; Hudson, Billy G; Borza, Dorin-Bogdan

    2008-12-12

    Defective assembly of alpha 3 alpha 4 alpha 5(IV) collagen in the glomerular basement membrane causes Alport syndrome, a hereditary glomerulonephritis progressing to end-stage kidney failure. Assembly of collagen IV chains into heterotrimeric molecules and networks is driven by their noncollagenous (NC1) domains, but the sites encoding the specificity of these interactions are not known. To identify the sites directing quaternary assembly of alpha 3 alpha 4 alpha 5(IV) collagen, correctly folded NC1 chimeras were produced, and their interactions with other NC1 monomers were evaluated. All alpha1/alpha 5 chimeras containing alpha 5 NC1 residues 188-227 replicated the ability of alpha 5 NC1 to bind to alpha3NC1 and co-assemble into NC1 hexamers. Conversely, substitution of alpha 5 NC1 residues 188-227 by alpha1NC1 abolished these quaternary interactions. The amino-terminal 58 residues of alpha3NC1 encoded binding to alpha 5 NC1, but this interaction was not sufficient for hexamer co-assembly. Because alpha 5 NC1 residues 188-227 are necessary and sufficient for assembly into alpha 3 alpha 4 alpha 5 NC1 hexamers, whereas the immunodominant alloantigenic sites of alpha 5 NC1 do not encode specific quaternary interactions, the findings provide a basis for the rational design of less immunogenic alpha 5(IV) collagen constructs for the gene therapy of X-linked Alport patients. PMID:18930919

  19. In vivo binding of spiperone and N-methylspiperone to dopaminergic and serotonergic sites in the rat brain: Multiple modeling and implications for PET scanning

    International Nuclear Information System (INIS)

    Equilibrium models are derived and applied to in vivo binding of spiperone in the rat brain. The models express the concentration of the ligand in the striatum and frontal cortex as a function of the accumulation in the cerebellum. The models differ with respect to the description of specific binding. Nonlinear regression analysis shows that the in vivo specific binding of 3H-labeled spiperone in the frontal cortex (mainly serotonergic) can be described by a noninteracting sites model, whereas the specific binding in the striatum (mainly dopaminergic) can best be described by models that lead to sigmoid saturation curves. These results were tested and partly confirmed by determining the region-of-interest/cerebellar radioactivity ratio of 11C-labeled N-methylspiperone, with and without pretreatment with haloperidol. The estimated Bmax was 32 fmol/mg wet tissue in the frontal cortex and approximately 90 fmol/mg wet tissue in the striatum. The free plus nonspecific binding of spiperone was similar in the frontal cortex but lower in the striatum than in the cerebellum. The occurrence of sigmoidicity can be best explained by the existence of high-affinity/low-capacity sites in the cerebellum rather than mutual interactions of striatal sites. The consequence of the present analysis for positron emission tomography is that the striatal/cerebellar activity ratio is not an accurate parameter of specific binding features at tracer doses of spiperone or N-methylspiperone

  20. Allosteric Regulation of Fibronectin/α5β1 Interaction by Fibronectin-Binding MSCRAMMs

    Science.gov (United States)

    Liang, Xiaowen; Garcia, Brandon L.; Visai, Livia; Prabhakaran, Sabitha; Meenan, Nicola A. G.; Potts, Jennifer R.; Humphries, Martin J.; Höök, Magnus

    2016-01-01

    Adherence of microbes to host tissues is a hallmark of infectious disease and is often mediated by a class of adhesins termed MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Numerous pathogens express MSCRAMMs that specifically bind the heterodimeric human glycoprotein fibronectin (Fn). In addition to roles in adhesion, Fn-binding MSCRAMMs exploit physiological Fn functions. For example, several pathogens can invade host cells by a mechanism whereby MSCRAMM-bound Fn bridges interaction with α5β1 integrin. Here, we investigate two Fn-binding MSCRAMMs, FnBPA (Staphylococcus aureus) and BBK32 (Borrelia burgdorferi) to probe structure-activity relationships of MSCRAMM-induced Fn/α5β1integrin activation. Circular dichroism, fluorescence resonance energy transfer, and dynamic light scattering techniques uncover a conformational rearrangement of Fn involving domains distant from the MSCRAMM binding site. Surface plasmon resonance experiments demonstrate a significant enhancement of Fn/α5β1 integrin affinity in the presence of FnBPA or BBK32. Detailed kinetic analysis of these interactions reveal that this change in affinity can be attributed solely to an increase in the initial Fn/α5β1 on-rate and that this rate-enhancement is dependent on high-affinity Fn-binding by MSCRAMMs. These data implicate MSCRAMM-induced perturbation of specific intramolecular contacts within the Fn heterodimer resulting in activation by exposing previously cryptic α5β1 interaction motifs. By correlating structural changes in Fn to a direct measurement of increased Fn/α5β1 affinity, this work significantly advances our understanding of the structural basis for the modulation of integrin function by Fn-binding MSCRAMMs. PMID:27434228

  1. Spin exchange monitoring of the strong positive homotropic allosteric binding of a tetraradical by a synthetic receptor in water.

    Science.gov (United States)

    Bardelang, David; Casano, Gilles; Poulhès, Florent; Karoui, Hakim; Filippini, Jessica; Rockenbauer, Antal; Rosas, Roselyne; Monnier, Valérie; Siri, Didier; Gaudel-Siri, Anouk; Ouari, Olivier; Tordo, Paul

    2014-12-17

    The flexible tetranitroxide 4T has been prepared and was shown to exhibit a nine line EPR spectrum in water, characteristic of significant through space spin exchange (J(ij)) between four electron spins interacting with four nitrogen nuclei (J(ij) ≫ a(N)). Addition of CB[8] to 4T decreases dramatically all the Jij couplings, and the nine line spectrum is replaced by the characteristic three line spectrum of a mononitroxide. The supramolecular association between 4T and CB[8] involves a highly cooperative asymmetric complexation by two CB[8] (K1 = 4027 M(-1); K2 = 202,800 M(-1); α = 201) leading to a rigid complex with remote nitroxide moieties. The remarkable enhancement for the affinity of the second CB[8] corresponds to an allosteric interaction energy of ≈13 kJ mol(-1), which is comparable to that of the binding of oxygen by hemoglobin. These results are confirmed by competition and reduction experiments, DFT and molecular dynamics calculations, mass spectrometry, and liquid state NMR of the corresponding reduced complex bearing hydroxylamine moieties. This study shows that suitably designed molecules can generate allosteric complexation with CB[8]. The molecule must (i) carry several recognizable groups for CB[8] and (ii) be folded so that the first binding event reorganizes the molecule (unfold) for a better subsequent recognition. The presence of accessible protonable amines and H-bond donors to fit with the second point are also further stabilizing groups of CB[8] complexation. In these conditions, the spin exchange coupling between four radicals has been efficiently and finely tuned and the resulting allosteric complexation induced a dramatic stabilization enhancement of the included paramagnetic moieties in highly reducing conditions through the formation of the supramolecular 4T@CB[8]2 complex. PMID:25418528

  2. Positive Allosteric Modulator of GABA Lowers BOLD Responses in the Cingulate Cortex.

    Directory of Open Access Journals (Sweden)

    Susanna A Walter

    Full Text Available Knowledge about the neural underpinnings of the negative blood oxygen level dependent (BOLD responses in functional magnetic resonance imaging (fMRI is still limited. We hypothesized that pharmacological GABAergic modulation attenuates BOLD responses, and that blood concentrations of a positive allosteric modulator of GABA correlate inversely with BOLD responses in the cingulate cortex. We investigated whether or not pure task-related negative BOLD responses were co-localized with pharmacologically modulated BOLD responses. Twenty healthy adults received either 5 mg diazepam or placebo in a double blind, randomized design. During fMRI the subjects performed a working memory task. Results showed that BOLD responses in the cingulate cortex were inversely correlated with diazepam blood concentrations; that is, the higher the blood diazepam concentration, the lower the BOLD response. This inverse correlation was most pronounced in the pregenual anterior cingulate cortex and the anterior mid-cingulate cortex. For subjects with diazepam plasma concentration > 0.1 mg/L we observed negative BOLD responses with respect to fixation baseline. There was minor overlap between cingulate regions with task-related negative BOLD responses and regions where the BOLD responses were inversely correlated with diazepam concentration. We interpret that the inverse correlation between the BOLD response and diazepam was caused by GABA-related neural inhibition. Thus, this study supports the hypothesis that GABA attenuates BOLD responses in fMRI. The minimal overlap between task-related negative BOLD responses and responses attenuated by diazepam suggests that these responses might be caused by different mechanisms.

  3. A potential yeast actin allosteric conduit dependent on hydrophobic core residues val-76 and trp-79.

    Science.gov (United States)

    Wen, Kuo-Kuang; McKane, Melissa; Stokasimov, Ema; Fields, Jonathon; Rubenstein, Peter A

    2010-07-01

    Intramolecular allosteric interactions responsible for actin conformational regulation are largely unknown. Previous work demonstrated that replacing yeast actin Val-76 with muscle actin Ile caused decreased nucleotide exchange. Residue 76 abuts Trp-79 in a six-residue linear array beginning with Lys-118 on the surface and ending with His-73 in the nucleotide cleft. To test if altering the degree of packing of these two residues would affect actin dynamics, we constructed V76I, W79F, and W79Y single mutants as well as the Ile-76/Phe-79 and Ile-76/Tyr-79 double mutants. Tyr or Phe should decrease crowding and increase protein flexibility. Subsequent introduction of Ile should restore packing and dampen changes. All mutants showed decreased growth in liquid medium. W79Y alone was severely osmosensitive and exhibited vacuole abnormalities. Both properties were rescued by Ile-76. Phe-79 or Tyr decreased the thermostability of actin and increased its nucleotide exchange rate. These effects, generally greater for Tyr than for Phe, were reversed by introduction of Ile-76. HD exchange showed that the mutations caused propagated conformational changes to all four subdomains. Based on results from phosphate release and light-scattering assays, single mutations affected polymerization in the order of Ile, Phe, and Tyr from least to most. Introduction of Ile-76 partially rescued the polymerization defects caused by either Tyr-79 or Phe-79. Thus, alterations in crowding of the 76-79 residue pair can strongly affect actin conformation and behavior, and these results support the theory that the amino acid array in which they are located may play a central role in actin regulation. PMID:20442407

  4. Identifying allosteric fluctuation transitions between different protein conformational states as applied to Cyclin Dependent Kinase 2

    Directory of Open Access Journals (Sweden)

    Gu Jenny

    2007-02-01

    Full Text Available Abstract Background The mechanisms underlying protein function and associated conformational change are dominated by a series of local entropy fluctuations affecting the global structure yet are mediated by only a few key residues. Transitional Dynamic Analysis (TDA is a new method to detect these changes in local protein flexibility between different conformations arising from, for example, ligand binding. Additionally, Positional Impact Vertex for Entropy Transfer (PIVET uses TDA to identify important residue contact changes that have a large impact on global fluctuation. We demonstrate the utility of these methods for Cyclin-dependent kinase 2 (CDK2, a system with crystal structures of this protein in multiple functionally relevant conformations and experimental data revealing the importance of local fluctuation changes for protein function. Results TDA and PIVET successfully identified select residues that are responsible for conformation specific regional fluctuation in the activation cycle of Cyclin Dependent Kinase 2 (CDK2. The detected local changes in protein flexibility have been experimentally confirmed to be essential for the regulation and function of the kinase. The methodologies also highlighted possible errors in previous molecular dynamic simulations that need to be resolved in order to understand this key player in cell cycle regulation. Finally, the use of entropy compensation as a possible allosteric mechanism for protein function is reported for CDK2. Conclusion The methodologies embodied in TDA and PIVET provide a quick approach to identify local fluctuation change important for protein function and residue contacts that contributes to these changes. Further, these approaches can be used to check for possible errors in protein dynamic simulations and have the potential to facilitate a better understanding of the contribution of entropy to protein allostery and function.

  5. Adaptive response and genomic instability: allosteric response of genome to negative impact

    International Nuclear Information System (INIS)

    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

  6. 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.

  7. 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. PMID:27387797

  8. Geochemical influence of waste trench no. 22T at Chernobyl Pilot Site at the aquifer: Long-term trends, governing processes, and implications for radionuclide migration

    International Nuclear Information System (INIS)

    This article discusses and analyzes data for geochemical monitoring of groundwater, collected in 1998–2008 at the international experimental radioecological study site (Chernobyl Pilot Site) located in the Red Forest radioactive waste dump site in the Chernobyl exclusion zone. Groundwater in the zone of influence of the studied waste trench no. 22T was characterized by a specific geochemistry. Along with a high content of 90Sr (of an order of n × 1000–n × 10,000 Bq/L) groundwater showed elevated concentrations of Ca, K, NO3-, SO42- and of some trace elements (in particular stable Sr), and had more acidic pH values compared to “background” aquifer conditions. The observed water quality changes are apparently related to degradation of organic matter (pine forest remnants, litter, humus containing topsoil layer) buried inside trench no. 22T, which have lead to acidification of groundwater and leaching of cations adsorbed on the exchange complex of soils buried inside the trench. Regime monitoring data for the project period of 1998–2008 shows a progressive decrease of concentrations of the leached ions accompanied by an increase of pH in the chemical plume emerging from the trench. This can be explained by a combined effect of a gradual decrease of the inventory, humification of the original organic matter inside the trench, and of nutrient element uptake by roots of the newly planted pine forest on top of the trench. The identified trends of evolution of geochemical regime favor attenuation of subsurface migration of 90Sr from Red Forest waste dumps due to an increase of the 90Sr distribution coefficients, and stabilization of plumes of contaminated groundwater.

  9. 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.

  10. 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)

    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.

  11. 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...... concept being tested in humans. Prior to assessing the efficacy of GABA-A receptor PAMs in a human volunteer pain model we have compared compounds capable of variously modulating GABA-A receptor function in comparable rat models of capsaicin-induced acute nocifensive flinching behaviour and secondary...

  12. A novel strategy for selection of allosteric ribozymes yields RiboReporter™ sensors for caffeine and aspartame

    OpenAIRE

    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...

  13. Synthesis, Characterization of Heterodinuclear Co-Cu Complex and Its Electrocatalytic Activity towards 02 Reduction: Implications for Cytochrome c Oxidase Active Site Modeling

    Institute of Scientific and Technical Information of China (English)

    卢卫兵; 汪存信; 周晓海; 任建国

    2003-01-01

    A new dinudeating ligand consisting of a tetraphanylporphyrin derivative covalently linked with tris(2-benzimidazylmethyl)-amine and its homodinudear Co-Co and heterodinnelear Co-Cu complexes were synthesized and spectroscopically character-ized. The heterobimetallie cobalt-copper complex bearing three benzimidazole ligands for copper, as cytochrome c oxidase ac-tive site model, was applied to the surface of glassy carbon elec-trode to show electrocatalytie activity for O2 reduction in aque-ous solution at an addity level dose to physiological pH value.The kinetic parameters of this electrocatalytic process were ob-tained.

  14. Late Neogene benthic stable isotope record of ODP Site 999: Implications for Caribbean paleoceanography, organic burial and the Messinian Salinity Crisis

    OpenAIRE

    Bickert, T.; G. Haug; Tiedemann, Ralf

    2004-01-01

    We report on epibenthic foraminiferal δ18O and δ13C and percentage coarse fraction records from Caribbean Ocean Drilling Program (ODP) Site 999 (12°44′N, 78°44′W, water depth 2828 m) spanning the interval from 8.5 to 5.3 Ma. Low epibenthic δ13C values and low amounts of sand-sized particles (mostly foraminifer shells) indicate a poorly ventilated deep Caribbean throughout the late Miocene. At this time the deep Caribbean was dominated by a nutrient-rich and corrosive water mass. A generally c...

  15. Unique Ligand Binding Sites on CXCR4 Probed by a Chemical Biology Approach: Implications for the Design of Selective Human Immunodeficiency Virus Type 1 Inhibitors

    OpenAIRE

    Choi, Won-Tak; Tian, Shaomin; Dong, Chang-Zhi; Kumar, Santosh; Liu, Dongxiang; Madani, Navid; An, Jing; Sodroski, Joseph G.; Huang, Ziwei

    2005-01-01

    The chemokine receptor CXCR4 plays an important role as the receptor for the normal physiological function of stromal cell-derived factor 1α (SDF-1α) and the coreceptor for the entry of human immunodeficiency virus type 1 (HIV-1) into the cell. In a recent work (S. Tian et al., J. Virol. 79:12667-12673, 2005), we found that many residues throughout CXCR4 transmembrane (TM) and extracellular loop 2 domains are specifically involved in interaction with HIV-1 gp120, as most of these sites did no...

  16. Natural Analog CCS Site Characterization Soda Springs, Idaho Implications for the Long-term Fate of Carbon Dioxide Stored in Geologic Environments

    Science.gov (United States)

    McLing, T. L.; Smith, R. W.; Podgorney, R. K.; Taylor, J.

    2009-12-01

    Implementation of commercial scale carbon capture and storage (CCS) requires an understanding of the long-term fate of CO2 sequestered in the subsurface. Critical to the success of large-scale CCS is an understanding of CO2 transport and geochemical process that occur in storage reservoirs, and potentially in the near surface should a leak occur. One way to gain this necessary understanding is to study natural CO2 reservoirs and their interaction with near surface and deep geologic materials. Many of these types of systems are self-contained, storing CO2 in geologic formations for millions of years. However, some of these systems are naturally leaky and may provide an ideal field laboratory to assess the long-term interaction of upward migrating CO2 and the subsurface environment. In addition, these natural analogs sites may provide important insight at time-scales not be available from other sources (such as CCS demonstration projects), critical to the characterization of proposed geologic sequestration reservoirs. The potential applicability of natural CO2 leaks as long-term natural analogs to geologic sequestration has been previously explored (Heath and Mcpherson 2004). An excellent leaky CCS natural analog is located in southeastern Idaho near the community of Soda Springs. At this site CO2 and CO2 charged waters generated at depth migrate upward interacting with the overlying geologic formations and the near surface local ground and surface waters. We hypothesized that the majority of the vertically migrating CO2 and CO2 charged water is prevented from reaching the surface by geochemical interactions with a thoelitic basalt flow that acts as a reactive cap on the reservoir. However, there are some small natural CO2 surface expressions of this system including several carbonated springs and a large CO2 “geyser” that resulted from a well drilled into the pressurized CO2 reservoir. Preliminary results of geochemical modeling based on initial water sampling

  17. Structure of N-acetyl-L-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor L-arginine bound

    OpenAIRE

    Zhao, Gengxiang; Haskins, Nantaporn; Jin, Zhongmin; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang

    2013-01-01

    Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in L-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by L-arginine, although L-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by L-argini...

  18. [(3) H]-L685,458 binding sites are abundant in multiple peripheral organs in rats: implications for safety assessment of putative γ-secretase targeting drugs.

    Science.gov (United States)

    Yang, Zhi-Ying; Li, Jian-Ming; Xiao, Ling; Mou, Lin; Cai, Yan; Huang, He; Luo, Xue-Gang; Yan, Xiao-Xin

    2014-12-01

    γ-Secretase is a multimeric enzyme complex that carries out proteolytic processing to a variety of cellular proteins. It is currently explored as a therapeutic target for Alzheimer's disease (AD) and cancer. Mechanism-based toxicity needs to be thoroughly evaluated for γ-secretase inhibitory and/or modulatory drugs. This study comparatively assessed putative γ-secretase catalytic sites in rat peripheral tissues relative to brain and explored an effort of its pharmacological inhibition on hair regeneration. Using [(3) H]-labelled L685,458, a potent γ-secretase inhibitor, as probe, we found more abundant presence of γ-secretase binding sites in the liver, gastrointestinal tract, hair follicle, pituitary gland, ovary and testis, as compared to the brain. Local application of L658,458 delayed vibrissal regrowth following whisker removal. These results suggest that γ-secretase may execute important biological functions in many peripheral systems, as in the brain. The development of γ-secretase inhibitors/modulators for AD and cancer therapy should include close monitoring of toxicological panels for hepatic, gastrointestinal, endocrinal and reproductive functions. PMID:24861611

  19. Signs of new breeding sites for Peruvian tern, Sternula lorata (Charadriiformes, Laridae at La Libertad, Peru, and its implications for conservation.

    Directory of Open Access Journals (Sweden)

    Amorós, Samuel

    2011-07-01

    Full Text Available Between March and April 2011, 10 study areas in the desert and coastal plains of La Libertad region were evaluated to locate breeding sites of the Peruvian Tern, Sternula lorata. Aproximately 117 km were (systematically walked and in the Pampas of Rio Seco (or Rio Chamán, Pacasmayo and Urricape signs of reproduction of this species were found. These signs included: low flying of Peruvian Terns over the observer, terns flying around the observer, birds flying with fish in their beaks to feed their chicks and the presence of one nest with two inactive eggs in Pampa Urricape. The latter is a fact of particular importance because of this species' fidelity to the nest and/or place of nesting. Also, Pampa Urricape was the site with the largest number of individuals, with a total of 22 located. The problem of habitat loss is affecting Peruvian Tern conservation. In this regard, we observed the practice of different economic activities in the desert which are leading to its modification. Among these activities we observed sugar cane crops, chicken barns, lots for homes, and a wind farm proposal. Finally, we propose recommendations for the conservation of the Peruvian Tern such as the revaluation of the desert ecosystem, the increase of its representation within the National System of Natural Protected Areas by the State, and the development of appropriate policies that include the Integrated Management of the Marine Coastal Zone to enable the sustainable development of these areas.

  20. The role of hydration on the mechanism of allosteric regulation: in situ measurements of the oxygen-linked kinetics of water binding to hemoglobin.

    Science.gov (United States)

    Salvay, Andrés G; Grigera, J Raúl; Colombo, Marcio F

    2003-01-01

    We report here the first direct measurements of changes in protein hydration triggered by a functional binding. This task is achieved by weighing hemoglobin (Hb) and myoglobin films exposed to an atmosphere of 98% relative humidity during oxygenation. The binding of the first oxygen molecules to Hb tetramer triggers a change in protein conformation, which increases binding affinity to the remaining empty sites giving rise to the appearance of cooperative phenomena. Although crystallographic data have evidenced that this structural change increases the protein water-accessible surface area, isobaric osmotic stress experiments in aqueous cosolutions have shown that water binding is linked to Hb oxygenation. Now we show that the differential hydration between fully oxygenated and fully deoxygenated states of these proteins, determined by weighing protein films with a quartz crystal microbalance, agree with the ones determined by osmotic stress in aqueous cosolutions, from the linkage between protein oxygen affinity and water activity. The agreements prove that the changes in water activity brought about by adding osmolytes to the buffer solution shift biochemical equilibrium in proportion to the number of water molecules associated with the reaction. The concomitant kinetics of oxygen and of water binding to Hb have been also determined. The data show that the binding of water molecules to the extra protein surface exposed on the transition from the low-affinity T to the high-affinity R conformations of hemoglobin is the rate-limiting step of Hb cooperative reaction. This evidences that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo. PMID:12524309

  1. AP1- and NF-kappaB-binding sites conserved among mammalian WNT10B orthologs elucidate the TNFalpha-WNT10B signaling loop implicated in carcinogenesis and adipogenesis.

    Science.gov (United States)

    Katoh, Masuko; Katoh, Masaru

    2007-04-01

    WNT signals are context-dependently transduced to canonical and non-canonical signaling cascades. We cloned and characterized wild-type human WNT10B, while another group cloned aberrant human WNT10B with Gly60Asp amino-acid substitution. Proto-oncogene WNT10B is expressed in gastric cancer, pancreatic cancer, breast cancer, esophageal cancer, and cervical cancer. Because WNT10B blocks adipocyte differentiation, coding SNP of WNT10B gene is associated with familial obesity. In 2001, we reported WNT10B upregulation by TNFalpha. Here, comparative integromics analyses on WNT10B orthologs were performed to elucidate the transcriptional mechanism of WNT10B. Chimpanzee WNT10B and cow Wnt10b genes were identified within NW_001223159.1 and AC150975.2 genome sequences, respectively, by using bioinformatics (Techint) and human intelligence (Humint). Chimpanzee WNT10B and cow Wnt10b showed 98.7% and 95.1% total-amino-acid identity with human WNT10B, respectively. N-terminal signal peptide, 24 Cys residues, two Asn-linked glycosylation sites, and Gly60 of human WNT10B were conserved among mammalian WNT10B orthologs. Transcription start site of human WNT10B gene was 106-bp upstream of NM_003394.2 RefSeq 5'-end. Number of GC di-nucleotide repeats just down-stream of WNT10B transcription start site varied among primates and human population. Comparative genomics analyses revealed that double AP1-binding sites in the 5'-flanking promoter region and NF-kappaB-binding site in intron 3 were conserved among human, chimpanzee, cow, mouse, and rat WNT10B orthologs. Because TNFalpha signaling through TNFR1 and TRADD/RIP/TRAF2 complex activates JUN kinase (JNK) and IkappaB kinase (IKK) signaling cascades, conserved AP1- and NF-kappaB-binding sites explain the mechanism of TNFalpha-induced WNT10B upregulation. TNFalpha-WNT10B signaling loop is the negative feedback mechanism of adipogenesis to prevent obesity and metabolic syndrome. On the other hand, TNFalpha-WNT10B signaling loop is

  2. 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

  3. Identification of covalent active site inhibitors of dengue virus protease

    Science.gov (United States)

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  4. Identification of covalent active site inhibitors of dengue virus protease.

    Science.gov (United States)

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  5. Soil-Gas Identification of Environmental Factors Affecting CO2 Concentrations Beneath a Playa Wetland: Implications for Soil-Gas Monitoring at Carbon Storage Sites

    Science.gov (United States)

    Romanak, K.; Bennett, P.

    2009-12-01

    Strategies for identifying and interpreting the effects of environmental factors on near-surface CO2 concentrations are essential to developing accurate monitoring protocols at carbon storage sites. Based on the results of a three-year study of a natural analogue we present, 1) a method for using soil-gas to identify near-surface CO2 cycling, and 2) a framework for developing monitoring protocols and site evaluation for near-surface monitoring. Near-surface CO2 production, consumption, and re-distribution was observed in the vadose-zone of a highly CO2-reactive playa wetland in the Texas High Plains. Atmospheric conditions, organic and inorganic soil carbon, subsurface pressure, water flux, and surface and groundwater chemistry were compared to real-time background measurements of CO2, CH4, O2+Ar, and N2 from depths up to 45 feet. Carbon isotopes and spatially and temporally variable concentrations of CO2 ≤ 17%, CH4 ≤ 2%, and O2 from 21-0% indicate CO2 and CH4 are produced by microbes. Molar gas ratios of O2 and CO2 distinguish between oxidation of organic matter (CH2O + O2 → CO2 + H2O), CH4 oxidation (CH4 + 2O2 → CO2 + 2H2O), and potentially acetate fermentation (CH3COOH → CH4 + CO2). O2 consumption and distribution is regulated by water flux that supplies dissolved organics to microbes at depth and regulates oxygen supply by blocking vertical permeability and atmospheric gas exchange. A surface flux experiment indicates that when playa floors are dry, subsurface wetting fronts from rain events or previous ponding periods block vertical permeability resulting in surface flux measurements that do not represent subsurface conditions. Samples with CO2+O2 78% identify dissolution of CO2 and carbonate minerals into recharging groundwater resulting in loss of pore pressure and chemically-induced advection of atmosphere into pores. Inverse geochemical reaction modeling (PHREEQC) of playa surface water and perched groundwater in high PCO2 zones support

  6. Natural radioactivity levels and the radiological health implications of tailing enriched soil and sediment samples around two mining sites in Southwest Nigeria

    International Nuclear Information System (INIS)

    The activity concentrations of natural radionuclides in tailing enriched soil and sediment samples collected from two mining sites in southwest Nigeria are reported. The samples were analyzed by gamma spectrometry with low background NaI (Tl) detector. The activity concentrations of 40K, 226Ra and 232Th in all the measured samples varied from 249.66 to 1459.25 Bq/kg, 7.62 to 50.31 Bq/kg and 12.68 to 234.18 Bq/kg, respectively in soil samples. For sediment samples, the values varied from 241.86 to 1590.40 Bq/kg, 9.86 to 74.8 Bq/kg and 15.47 to 145.46 Bq/kg for 40K, 226Ra and 232Th, respectively. The results show that the mean activity concentrations of the radionuclides in soil and sediment of the study area are higher than their world-wide average crustal values. The mean concentration of 232Th is >6 times higher than that of 226Ra in soil samples from Ijero mining site. This shows that 232Th is slightly enhanced in the soil from this location than 226Ra. In order to evaluate the radiological hazards of the natural radioactivity, the radium equivalent activity, external hazard index, absorbed gamma dose rates and the annual effective dose rates were determined. All these hazard indices have mean values, which are within their acceptable limits but higher than their world average values. (author)

  7. The propagation of binding interactions to remote sites in proteins: Analysis of the binding of the monoclonal antibody D1.3 to lysozyme

    OpenAIRE

    Freire, Ernesto

    1999-01-01

    The interaction of a ligand with a protein occurs at a local site (the binding site) and involves only a few residues; however, the effects of that interaction are often propagated to remote locations. The chain of events initiated by binding provides the basis for fundamental biological phenomena such as allosterism, signal transduction, and structural-stability modification. In this paper, a structure-based statistical thermodynamic approach is presented and used...

  8. Campylobacter jejuni adenosine triphosphate phosphoribosyltransferase is an active hexamer that is allosterically controlled by the twisting of a regulatory tail.

    Science.gov (United States)

    Mittelstädt, Gerd; Moggré, Gert-Jan; Panjikar, Santosh; Nazmi, Ali Reza; Parker, Emily J

    2016-08-01

    Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first committed step of the histidine biosynthesis in plants and microorganisms. Here, we present the functional and structural characterization of the ATP-PRT from the pathogenic ε-proteobacteria Campylobacter jejuni (CjeATP-PRT). This enzyme is a member of the long form (HisGL ) ATP-PRT and is allosterically inhibited by histidine, which binds to a remote regulatory domain, and competitively inhibited by AMP. In the crystalline form, CjeATP-PRT was found to adopt two distinctly different hexameric conformations, with an open homohexameric structure observed in the presence of substrate ATP, and a more compact closed form present when inhibitor histidine is bound. CjeATP-PRT was observed to adopt only a hexameric quaternary structure in solution, contradicting previous hypotheses favoring an allosteric mechanism driven by an oligomer equilibrium. Instead, this study supports the conclusion that the ATP-PRT long form hexamer is the active species; the tightening of this structure in response to remote histidine binding results in an inhibited enzyme. PMID:27191057

  9. 2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition.

    Science.gov (United States)

    Banerjee, Soojay; Bartesaghi, Alberto; Merk, Alan; Rao, Prashant; Bulfer, Stacie L; Yan, Yongzhao; Green, Neal; Mroczkowski, Barbara; Neitz, R Jeffrey; Wipf, Peter; Falconieri, Veronica; Deshaies, Raymond J; Milne, Jacqueline L S; Huryn, Donna; Arkin, Michelle; Subramaniam, Sriram

    2016-02-19

    p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo-electron microscopy (cryo-EM) structures for adenosine diphosphate (ADP)-bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 and 2.4 angstroms, respectively. We also report cryo-EM structures (at resolutions of ~3.3, 3.2, and 3.3 angstroms, respectively) for three distinct, coexisting functional states of p97 with occupancies of zero, one, or two molecules of adenosine 5'-O-(3-thiotriphosphate) (ATPγS) per protomer. A large corkscrew-like change in molecular architecture, coupled with upward displacement of the N-terminal domain, is observed only when ATPγS is bound to both the D1 and D2 domains of the protomer. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small-molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between the D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function. PMID:26822609

  10. An Autoregulatory Mechanism Imposes Allosteric Control on the V(DJ Recombinase by Histone H3 Methylation

    Directory of Open Access Journals (Sweden)

    Chao Lu

    2015-01-01

    Full Text Available V(DJ recombination is initiated by a specialized transposase consisting of the subunits RAG-1 and RAG-2. The susceptibility of gene segments to DNA cleavage by the V(DJ recombinase is correlated with epigenetic modifications characteristic of active chromatin, including trimethylation of histone H3 on lysine 4 (H3K4me3. Engagement of H3K4me3 by a plant homeodomain (PHD in RAG-2 promotes recombination in vivo and stimulates DNA cleavage by RAG in vitro. We now show that H3K4me3 acts allosterically at the PHD finger to relieve autoinhibition imposed by a separate domain within RAG-2. Disruption of this autoinhibitory domain was associated with constitutive increases in recombination frequency, DNA cleavage activity, substrate binding affinity, and catalytic rate, thus mimicking the stimulatory effects of H3K4me3. Our observations support a model in which allosteric control of RAG is enforced by an autoinhibitory domain whose action is relieved by engagement of active chromatin.

  11. Structural insights into Ca(2+)-activated long-range allosteric channel gating of RyR1.

    Science.gov (United States)

    Wei, Risheng; Wang, Xue; Zhang, Yan; Mukherjee, Saptarshi; Zhang, Lei; Chen, Qiang; Huang, Xinrui; Jing, Shan; Liu, Congcong; Li, Shuang; Wang, Guangyu; Xu, Yaofang; Zhu, Sujie; Williams, Alan J; Sun, Fei; Yin, Chang-Cheng

    2016-09-01

    Ryanodine receptors (RyRs) are a class of giant ion channels with molecular mass over 2.2 mega-Daltons. These channels mediate calcium signaling in a variety of cells. Since more than 80% of the RyR protein is folded into the cytoplasmic assembly and the remaining residues form the transmembrane domain, it has been hypothesized that the activation and regulation of RyR channels occur through an as yet uncharacterized long-range allosteric mechanism. Here we report the characterization of a Ca(2+)-activated open-state RyR1 structure by cryo-electron microscopy. The structure has an overall resolution of 4.9 Å and a resolution of 4.2 Å for the core region. In comparison with the previously determined apo/closed-state structure, we observed long-range allosteric gating of the channel upon Ca(2+) activation. In-depth structural analyses elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism of RyR1. Our work not only provides structural insights into the molecular mechanisms of channel gating and regulation of RyRs, but also sheds light on structural basis for channel-gating and ion selectivity mechanisms for the six-transmembrane-helix cation channel family. PMID:27573175

  12. The M1 Muscarinic Positive Allosteric Modulator PQCA Improves Performance on Translatable Tests of Memory and Attention in Rhesus Monkeys.

    Science.gov (United States)

    Lange, Henry S; Cannon, Christopher E; Drott, Jason T; Kuduk, Scott D; Uslaner, Jason M

    2015-12-01

    Improved treatment of Alzheimer disease (AD) is a significant unmet medical need that is becoming even more critical given the rise in the number of patients and the substantial economic burden. The current standards of care, acetylcholinesterase inhibitors (AChEIs), are hindered by gastrointestinal side effects owing to their nonselective activation of muscarinic and nicotinic receptors. Recently, the highly selective M1 positive allosteric modulator PQCA (1-((4-cyano-4-(pyridine-2-yl)piperidin-1-yl)methyl-4-oxo-4 H-quinolizine-3-carboxylic acid) has been demonstrated to improve cognition in a variety of rodent and nonhuman primate cognition models without producing significant gastrointestinal side effects. Here we describe the effect of PQCA and the AChEI donepezil on two clinically relevant and highly translatable touchscreen cognition tasks in nonhuman primates: paired-associates learning (PAL) and the continuous-performance task (CPT). Blockade of muscarinic signaling by scopolamine produced significant impairments in both PAL and CPT. PQCA and donepezil attenuated the scopolamine deficits in both tasks, and the action of these two compounds was similar in magnitude. In addition, the combination of subeffective doses of PQCA and donepezil enhanced PAL performance. These results further suggest that M1-positive allosteric modulators, either as monotherapy or as an add-on to current standards of care, have potential to reduce the cognitive deficits associated with AD. PMID:26446308

  13. Luminescence dating of gravity deposits on Site C0006 and C0007 of IODP Exp.316 and its implications for large earthquake recurrences in Nankai Trough, Japan

    Science.gov (United States)

    Jiang, T.; Li, S.; Li, C.; Xie, X.; Li, B.; Ren, J.

    2010-12-01

    The Nankai Trough has a 1,300-year record of generating large earthquakes (Mw>8) with a recurrence time of ~180-200 yr, but unfortunately, we haven’t known the frequency of earthquakes before that. Results from expeditions of NanTroSEIZE Stage 1 IODP 314/315/316 recovered many cycles of turbidites since late Pleistocene. Due to unique backgrounds, they are suggested little other triggers except for past large earthquakes. Sites C0006 and C0007 are near to deep Nankai Trough so that most of earthquake-triggered deposits have been drilled into in the two sites. Our purpose is to investigate earthquakes recurrences via these earthquake-triggered event deposits, including turbidites, debris, slope failures and so on. In order to dating those gravity sediments, considering of the possibility of little microfossils at somewhere, the exact location of event-beds and applicable ranges of dating methods, we detected luminescence dating for sandstones in them to reinforce the high resolution age profile. The results show that the ages of sediments get older with depth. Combined with the number of gravity cycles distinguished on XCT scan images, the ages from C0007C1H1 samples indicate that the mean large earthquake interval should be 360 yr during the past 160 ka and it is more long from C0006D1H1 samples during the past 100 ka. Futhermore, a facies transition surface from trench to slope is identified in section 3 of C0006D1H1, where the age of event sediments is about 80 ka. The borehole is 1460 m away from the present edge of Nankai Trough in the crossing seismic line, which implies that the subduction rate is about 1.8 cm/yr during the past 80ka. Compared with present subduction rate with ~4.5 cm/yr and large earthquake recurrences of 180-200 yr, the Philippine Sea plate is subducting fast and the large earthquake is more frequent. If some of event deposits are not triggered by earthquake shaking, the conclusion is the same. For the future work to confirm it, more

  14. 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

    Directory of Open Access Journals (Sweden)

    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

  15. Grain-size records at ODP Site 1146 from the northern South China Sea: Implications on the East Asian monsoon evolution since 20 Ma

    Institute of Scientific and Technical Information of China (English)

    WAN; ShiMing; LI; AnChun; Jan-Berend; W.; STUUT; XU; FangJian

    2007-01-01

    273 samples from Ocean Drilling Program (ODP) Site 1146 in the northern South China Sea (SCS) were analyzed for grain-size distributions using grain-size class vs. standard deviation method and end-member modeling algorithm (EMMA) in order to investigate the evolution of the East Asian monsoon since about 20 Ma. 10-19 μm/1.3-2.4 μm, the ratio of two grain-size populations with the highest variability through time was used to indicate East Asian winter monsoon intensity relative to summer monsoon. The mass accumulation rate of the coarsest end member EM1 (eolian), resulting from EMMA, can be used as a proxy of winter monsoon strength and Asian inland aridity, and the ratio of EM1/(EM2+EM3) as a proxy of winter monsoon intensity relative to summer monsoon. The combined proxies show that a profound enhancement of East Asian winter monsoon strength and winter monsoon intensity relative to summer monsoon occurred at about 8 Ma, and it is possible that the summer monsoon simultaneously intensified with winter monsoon at 3 Ma. Our results are well consistent with the previous studies in loess, eolian deposion in the Pacifc, radiolarians and planktonic foraminifera in the SCS. The phased uplift of the Himalaya-Tibetan Plateau may have played a significant role in strengthening the Asian monsoon at 8 Ma and 3 Ma.

  16. 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)

    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

  17. Analysis of K-net and Kik-net data: implications for ground motion prediction - acceleration time histories, response spectra and nonlinear site response

    International Nuclear Information System (INIS)

    This thesis intends to characterize ground motion during earthquake. This work is based on two Japanese networks. It deals with databases of shallow events, depth less than 25 km, with magnitude between 4.0 and 7.3. The analysis of K-net allows to compute a spectral ground motion prediction equation and to review the shape of the Eurocode 8 design spectra. We show the larger amplification at short period for Japanese data and bring in light the soil amplification that takes place at large period. In addition, we develop a new empirical model for simulating synthetic stochastic nonstationary acceleration time histories. By specifying magnitude, distance and site effect, this model allows to produce many time histories, that a seismic event is liable to produce at the place of interest. Furthermore, the study of near-field borehole records of the Kik-net allows to explore the validity domain of predictive equations and to explain what occurs by extrapolating ground motion predictions. Finally, we show that nonlinearity reduces the dispersion of ground motion at the surface. (author)

  18. 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.

  19. Coupled-processes in the Callovo-Oxfordian shales at the Bure site: implications for the transports of water and solutes

    International Nuclear Information System (INIS)

    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

  20. The Exosporium of B.cereus Contains a Binding Site for gC1qR/p33: Implication in Spore Attachment and/or Entry.

    Energy Technology Data Exchange (ETDEWEB)

    GHEBREHIWET,B.; TANTRAL, L.; TITMUS, M.A.; PANESSA-WARREN, B.J.; TORTORA, G.T.; WONG, S.S.; WARREN, J.B.

    2008-01-01

    B. cereus, is a member of a genus of aerobic, gram-positive, spore-forming rod-like bacilli, which includes the deadly, B. anthracis. Preliminary experiments have shown that gC1qR binds to B.cereus spores that have been attached to microtiter plates. The present studies were therefore undertaken, to examine if cell surface gC1qR plays a role in B.cereus spore attachment and/or entry. Monolayers of human colon carcinoma (Caco-2) and lung cells were grown to confluency on 6 mm coverslips in shell vials with gentle swirling in a shaker incubator. Then, 2 {micro}l of a suspension of strain SB460 B.cereus spores (3x10{sup 8}/ml, in sterile water), were added and incubated (1-4 h; 36{sup 0} C) in the presence or absence of anti-gC1qR mAb-carbon nanoloops. Examination of these cells by EM revealed that: (1) When B. cereus endospores contacted the apical Caco-2 cell surface, or lung cells, gClqR was simultaneously detectable, indicating upregulation of the molecule. (2) In areas showing spore contact with the cell surface, gClqR expression was often adjacent to the spores in association with microvilli (Caco-2 cells) or cytoskeletal projections (lung cells). (3) Furthermore, the exosporia of the activated and germinating spores were often decorated with mAb-nanoloops. These observations were further corroborated by experiments in which B.cereus spores were readily taken up by monocytes and neutrophils, and this uptake was partially inhibited by mAb 60.11, which recognizes the C1q binding site on gC1qR. Taken together, the data suggest a role, for gC1qR at least in the initial stages of spore attachment and/or entry.

  1. Radionuclide contents in industrial raw minerals and soil samples from mining sites and their radiological implications to the population in Akwaibom State Nigeria

    International Nuclear Information System (INIS)

    About 51 Samples of domestically produced raw minerals used for different industrial applications and soil samples at their mining sites have been collected and analyzed for their natural radionuclide concentration levels. The method of gamma-ray spectroscopy was used in the analysis of 226Ra, 232Th and 40K in these samples. The range of activity concentration of radionuclides in the industrial raw minerals were 226Ra(17.55±1.63 - 80.99±2.61), 232Th(7.64±0.77 - 23.94±0.92) and 40K(63.22±3.43 - 503.90±5.69)Bqkg-1 respectively. The obtained mean absorbed dose rate for the collected soil samples ranged from (3.51±0.25 - 23.37±3.79)nGyh-1. These results, along with the results of the estimated annual effective dose rates, radium equivalent (Raeq), external hazard index (Hex), internal hazard index(Hex) and representative of gamma index(Iγr ) are presented in this paper. The results obtained were below the internationally accepted safe limits. It suffices to say therefore that the analysed samples could be used in the local industries in the area where these minerals are component materials in their produced products or as building materials. Also the mining activities of these minerals in the area have not significantly affected the natural radiation dose levels in the area hence the resulting dose to population is therefore considered low.

  2. A novel dualistic profile of an allosteric AMPA receptor modulator identified through studies on recombinant receptors, mouse hippocampal synapses and crystal structures

    DEFF Research Database (Denmark)

    Christiansen, G B; Harbak, Barbara; Hede, S E;

    2015-01-01

    Positive allosteric modulators (PAMs) of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors receive increasing interest as therapeutic drugs and have long served as important experimental tools in the study of the molecular mechanisms underlying glutamate-mediated neurotra...

  3. Changes of cooperativity between N-methylscopolamine and allosteric modulators alcuronium and gallamine induced by mutations of external loops of muscarinic M(3) receptors

    Czech Academy of Sciences Publication Activity Database

    Krejčí, Alena; Tuček, Stanislav

    2001-01-01

    Roč. 60, č. 4 (2001), s. 761-767. ISSN 0026-895X R&D Projects: GA ČR GA309/99/0214 Institutional research plan: CEZ:AV0Z5011922 Keywords : muscarinic receptors * allosteric modulators Subject RIV: FH - Neurology Impact factor: 5.297, year: 2001

  4. The tertiary origin of the allosteric activation of E. coli glucosamine-6-phosphate deaminase studied by sol-gel nanoencapsulation of its T conformer.

    Directory of Open Access Journals (Sweden)

    Sergio Zonszein

    Full Text Available The role of tertiary conformational changes associated to ligand binding was explored using the allosteric enzyme glucosamine-6-phosphate (GlcN6P deaminase from Escherichia coli (EcGNPDA as an experimental model. This is an enzyme of amino sugar catabolism that deaminates GlcN6P, giving fructose 6-phosphate and ammonia, and is allosterically activated by N-acetylglucosamine 6-phosphate (GlcNAc6P. We resorted to the nanoencapsulation of this enzyme in wet silica sol-gels for studying the role of intrasubunit local mobility in its allosteric activation under the suppression of quaternary transition. The gel-trapped enzyme lost its characteristic homotropic cooperativity while keeping its catalytic properties and the allosteric activation by GlcNAc6P. The nanoencapsulation keeps the enzyme in the T quaternary conformation, making possible the study of its allosteric activation under a condition that is not possible to attain in a soluble phase. The involved local transition was slowed down by nanoencapsulation, thus easing the fluorometric analysis of its relaxation kinetics, which revealed an induced-fit mechanism. The absence of cooperativity produced allosterically activated transitory states displaying velocity against substrate concentration curves with apparent negative cooperativity, due to the simultaneous presence of subunits with different substrate affinities. Reaction kinetics experiments performed at different tertiary conformational relaxation times also reveal the sequential nature of the allosteric activation. We assumed as a minimal model the existence of two tertiary states, t and r, of low and high affinity, respectively, for the substrate and the activator. By fitting the velocity-substrate curves as a linear combination of two hyperbolic functions with Kt and Kr as KM values, we obtained comparable values to those reported for the quaternary conformers in solution fitted to MWC model. These results are discussed in the

  5. Combustion of Organic Molecules by the Thermal Decomposition of Perchlorate Salts: Implications for Organics at the Mars Phoenix Scout Landing Site

    Science.gov (United States)

    Ming, D.W.; Morris, R.V.; Niles, B.; Lauer, H.V.; Archer, P.D.; Sutter, B.; Boynton, W.V.; Golden, D.C.

    2009-01-01

    The Mars 2007 Phoenix Scout Mission successfully landed on May 25, 2008 and operated on the northern plains of Mars for 150 sols. The primary mission objective was to study the history of water and evaluate the potential for past and present habitability in Martian arctic ice-rich soil [1]. Phoenix landed near 68 N latitude on polygonal terrain created by ice layers that are a few centimeters under loose soil materials. The Phoenix Mission is assessing the potential for habitability by searching for organic molecules in the ice or icy soils at the landing site. Organic molecules are necessary building blocks for life, although their presence in the ice or soil does not indicate life itself. Phoenix searched for organic molecules by heating soil/ice samples in the Thermal and Evolved-Gas Analyzer (TEGA, [2]). TEGA consists of 8 differential scanning calorimeter (DSC) ovens integrated with a magnetic-sector mass spectrometer with a mass range of 2-140 daltons [2]. Endothermic and exothermic reactions are recorded by the TEGA DSC as samples are heated from ambient to 1000 C. Evolved gases, including any organic molecules and their fragments, are simultaneously measured by the mass spectrometer during heating. Phoenix TEGA data are still under analysis; however, no organic fragments have been identified to date in the evolved gas analysis (EGA). The MECA Wet Chemistry Lab (WCL) discovered a perchlorate salt in the Phoenix soils and a mass 32 peak evolved between 325 and 625 C for one surface sample dubbed Baby Bear [3]. The mass 32 peak is attributed to evolved O2 generated during the thermal decomposition of the perchlorate salt. Perchlorates are very strong oxidizers when heated, so it is possible that organic fragments evolved in the temperature range of 300-600 C were combusted by the O2 released during the thermal decomposition of the perchlorate salt. The byproduct of the combustion of organic molecules is CO2. There is a prominent release of CO2 between 200

  6. Correlations between Cassini VIMS spectra and RADAR SAR images: Implications for Titan's surface composition and the character of the Huygens Probe Landing Site

    Science.gov (United States)

    Soderblom, L.A.; Kirk, R.L.; Lunine, J.I.; Anderson, J.A.; Baines, K.H.; Barnes, J.W.; Barrett, J.M.; Brown, R.H.; Buratti, B.J.; Clark, R.N.; Cruikshank, D.P.; Elachi, C.; Janssen, M.A.; Jaumann, R.; Karkoschka, E.; Le Mouélic, Stéphane; Lopes, R.M.; Lorenz, R.D.; McCord, T.B.; Nicholson, P.D.; Radebaugh, J.; Rizk, B.; Sotin, C.; Stofan, E.R.; Sucharski, T.L.; Tomasko, M.G.; Wall, S.D.

    2007-01-01

    nearest occurrence of dunes visible in the RADAR SAR images. Fluvial/pluvial processes, every few centuries or millennia, must be cleansing the dark floors of the incised channels and scouring the dark plains at the Huygens landing site both imaged by Descent Imager/Spectral Radiometer (DISR). ?? 2007 Elsevier Ltd. All rights reserved.

  7. Characterization of traffic-related air pollutant metrics at four schools in El Paso, Texas, USA: Implications for exposure assessment and siting schools in urban areas

    Science.gov (United States)

    Raysoni, Amit U.; Stock, Thomas H.; Sarnat, Jeremy A.; Montoya Sosa, Teresa; Ebelt Sarnat, Stefanie; Holguin, Fernando; Greenwald, Roby; Johnson, Brent; Li, Wen-Whai

    2013-12-01

    by the measurements from a centralized monitoring site.

  8. Small-Molecule Inhibitors of the LEDGF/p75 Binding Site of Integrase Block HIV Replication and Modulate Integrase Multimerization

    Science.gov (United States)

    Christ, Frauke; Shaw, Stephen; Demeulemeester, Jonas; Desimmie, Belete A.; Marchand, Arnaud; Butler, Scott; Smets, Wim; Chaltin, Patrick; Westby, Mike

    2012-01-01

    Targeting the HIV integrase (HIV IN) is a clinically validated approach for designing novel anti-HIV therapies. We have previously described the discovery of a novel class of integration inhibitors, 2-(quinolin-3-yl)acetic acid derivatives, blocking HIV replication at a low micromolar concentration through binding in the LEDGF/p75 binding pocket of HIV integrase, hence referred to as LEDGINs. Here we report the detailed characterization of their mode of action. The design of novel and more potent analogues with nanomolar activity enabled full virological evaluation and a profound mechanistic study. As allosteric inhibitors, LEDGINs bind to the LEDGF/p75 binding pocket in integrase, thereby blocking the interaction with LEDGF/p75 and interfering indirectly with the catalytic activity of integrase. Detailed mechanism-of-action studies reveal that the allosteric mode of inhibition is likely caused by an effect on HIV-1 integrase oligomerization. The multimodal inhibition by LEDGINs results in a block in HIV integration and in a replication deficiency of progeny virus. The allosteric nature of LEDGINs leads to synergy in combination with the clinically approved active site HIV IN strand transfer inhibitor (INSTI) raltegravir, and cross-resistance profiling proves the distinct mode of action of LEDGINs and INSTIs. The allosteric nature of inhibition and compatibility with INSTIs underline an interest in further (clinical) development of LEDGINs. PMID:22664975

  9. Pumiliotoxin B binds to a site on the voltage-dependent sodium channel that is allosterically coupled to other binding sites.

    OpenAIRE

    Gusovsky, F; Rossignol, D P; McNeal, E T; Daly, J W

    1988-01-01

    Pumiliotoxin B (PTX-B), an alkaloid that has cardiotonic and myotonic activity, increases sodium influx in guinea pig cerebral cortical synaptoneurosomes. In the presence of scorpion venom (Leiurus) or purified alpha-scorpion toxin, the PTX-B-induced sodium influx is enhanced severalfold. PTX-B alone has no effect on sodium flux in N18 neuroblastoma cells but, in the presence of alpha-scorpion toxin, stimulation of sodium influx by PTX-B reaches levels comparable to that attained with the sod...

  10. Allosterism in human complement component 5a ((h)C5a): a damper of C5a receptor (C5aR) signaling.

    Science.gov (United States)

    Rana, Soumendra; Sahoo, Amita Rani; Majhi, Bharat Kumar

    2016-06-01

    The phenomena of allosterism continues to advance the field of drug discovery, by illuminating gainful insights for many key processes, related to the structure-function relationships in proteins and enzymes, including the transmembrane G-protein coupled receptors (GPCRs), both in normal as well as in the disease states. However, allosterism is completely unexplored in the native protein ligands, especially when a small covalent change significantly modulates the pharmacology of the protein ligands toward the signaling axes of the GPCRs. One such example is the human C5a ((h)C5a), the potent cationic anaphylatoxin that engages C5aR and C5L2 to elicit numerous immunological and non-immunological responses in humans. From the recently available structure-function data, it is clear that unlike the mouse C5a ((m)C5a), the (h)C5a displays conformational heterogeneity. However, the molecular basis of such conformational heterogeneity, otherwise allosterism in (h)C5a and its precise contribution toward the overall C5aR signaling is not known. This study attempts to decipher the functional role of allosterism in (h)C5a, by exploring the inherent conformational dynamics in (m)C5a, (h)C5a and in its point mutants, including the proteolytic mutant des-Arg(74)-(h)C5a. Prima facie, the comparative molecular dynamics study, over total 500 ns, identifies Arg(74)-Tyr(23) and Arg(37)-Phe(51) "cation-π" pairs as the molecular "allosteric switches" on (h)C5a that potentially functions as a damper of C5aR signaling. PMID:26212097

  11. Inversion of allosteric effect of arginine on N-acetylglutamate synthase, a molecular marker for evolution of tetrapods

    Directory of Open Access Journals (Sweden)

    Cabrera-Luque Juan

    2008-09-01

    Full Text Available Abstract Background The efficient conversion of ammonia, a potent neurotoxin, into non-toxic metabolites was an essential adaptation that allowed animals to move from the aquatic to terrestrial biosphere. The urea cycle converts ammonia into urea in mammals, amphibians, turtles, snails, worms and many aquatic animals and requires N-acetylglutamate (NAG, an essential allosteric activator of carbamylphosphate synthetase I (CPSI in mammals and amphibians, and carbamylphosphate synthetase III (CPSIII in fish and invertebrates. NAG-dependent CPSI and CPSIII catalyze the formation of carbamylphosphate in the first and rate limiting step of ureagenesis. NAG is produced enzymatically by N-acetylglutamate synthase (NAGS, which is also found in bacteria and plants as the first enzyme of arginine biosynthesis. Arginine is an allosteric inhibitor of microbial and plant NAGS, and allosteric activator of mammalian NAGS. Results Information from mutagenesis studies of E. coli and P. aeruginosa NAGS was combined with structural information from the related bacterial N-acetylglutamate kinases to identify four residues in mammalian NAGS that interact with arginine. Substitutions of these four residues were engineered in mouse NAGS and into the vertebrate-like N-acetylglutamate synthase-kinase (NAGS-K of Xanthomonas campestris, which is inhibited by arginine. All mutations resulted in arginine losing the ability to activate mouse NAGS, and inhibit X. campestris NAGS-K. To examine at what point in evolution inversion of arginine effect on NAGS occur, we cloned NAGS from fish and frogs and examined the arginine response of their corresponding proteins. Fish NAGS were partially inhibited by arginine and frog NAGS were activated by arginine. Conclusion Difference in arginine effect on bacterial and mammalian NAGS most likely stems from the difference in the type of conformational change triggered by arginine binding to these proteins. The change from arginine

  12. Ion-Regulated Allosteric Binding of Fullerenes (C-60 and C-70) by Tetrathiafulvalene-Calix[4]pyrroles

    DEFF Research Database (Denmark)

    Davis, C. M.; Lim, J. M.; Larsen, K. R.;

    2014-01-01

    The effect of ionic species on the binding of fullerenes (C-60 and C-70) by tetrathiafulvalene-calix[4]pyrrole (TTF-C4P) receptors and the nature of the resulting supramolecular complexes (TTF-C4P + fullerene + halide anion + tetraalkylammonium cation) was studied in the solid state through single...... the C4P in a ball-and-socket binding mode. The interactions between the TTF-C4P receptors and the fullerene guests are highly influenced by both the nature of halide anions and their counter tetraalkylammonium cations. Three halides (F-, Cl-, and Br-) were studied. All three potentiate the binding of...... the two test fullerenes by inducing a conformational change from the 1,3-alternate to the cone conformer of the TTF-C4Ps, thus acting as positive heterotropic allosteric effectors. For a particular halide anion, the choice of tetraalkylammonium salts serves to modulate the strength of the TTF-C4P...

  13. Thermodynamic Characterization of New Positive Allosteric Modulators Binding to the Glutamate Receptor A2 Ligand-Binding Domain

    DEFF Research Database (Denmark)

    Nørholm, Ann-Beth; Francotte, Pierre; Goffin, Eric; Botez, Iuliana; Danober, Laurence; Lestage, Pierre; Pirotte, Bernard; Kastrup, Jette Sandholm Jensen; Olsen, Lars; Oostenbrink, Chris

    2014-01-01

    5a (5-F) and 5b (6-F) are entropy driven. For 5d (8-F), both quantities were equal in size. Thermodynamic integration (TI) and one-step perturbation (OSP) were used to calculate the relative binding affinity of the modulators. The OSP calculations had a higher predictive power than those from TI......Positive allosteric modulation of the ionotropic glutamate receptor GluA2 presents a potential treatment of cognitive disorders, for example, Alzheimer's disease. In the present study, we describe the synthesis, pharmacology, and thermodynamic studies of a series of monofluoro-substituted 3......,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides. Measurements of ligand binding by isothermal titration calorimetry (ITC) showed similar binding affinities for the modulator series at the GluA2 LBD but differences in the thermodynamic driving forces. Binding of 5c (7-F) and 6 (no-F) is enthalpy driven, and...

  14. In vitro and in vivo characterization of a novel negative allosteric modulator of neuronal nAChRs

    OpenAIRE

    Abdrakhmanova, Galya R.; Blough, Bruce E.; Nesloney, Carey; Navarro, Hernán A.; Damaj, M. Imad; Carroll, F. Ivy

    2010-01-01

    In this study, we compared the in vitro and in vivo neuronal nicotinic acetylcholine receptor (nAChR) properties of 1,2,3,3a,4,8b-hexahydro-2-benzyl-6-N,N-dimethylamino-1-methylindeno[1,2,-b]pyrrole (HDMP, 4) to that of negative allosteric modulator (NAM), PCP. Patch-clamp experiments showed that HDMP exhibited an inhibitory functional activity at α7 nAChRs with an IC50 of 0.07 μM, and was 357- and 414-fold less potent at α4β2 and α3β4 nAChRs, with IC50s of 25.1 and 29.0 μM, respectively. Con...

  15. Site of anticonvulsant action on sodium channels: autoradiographic and electrophysiological studies in rat brain

    International Nuclear Information System (INIS)

    The anticonvulsants phenytoin and carbamazepine interact allosterically with the batrachotoxin binding site of sodium channels. In the present study, we demonstrate an autoradiographic technique to localize the batrachotoxin binding site on sodium channels in rat brain using [3H]batrachotoxinin-A 20-alpha-benzoate (BTX-B). Binding of [3H]BTX-B to brain sections is dependent on potentiating allosteric interactions with scorpion venom and is displaced by BTX-B (Kd approximately 200 nM), aconitine, veratridine, and phenytoin with the same rank order of potencies as described in brain synaptosomes. The maximum number of [3H]BTX-B binding sites in forebrain sections also agrees with biochemical determinations. Autoradiographic localizations indicate that [3H]BTX-B binding sites are not restricted to cell bodies and axons but are present in synaptic zones throughout the brain. For example, a particularly dense concentration of these sites in the substantia nigra is associated with afferent terminals of the striatonigral projection. By contrast, myelinated structures possess much lower densities of binding sites. In addition, we present electrophysiological evidence that synaptic transmission, as opposed to axonal conduction, is preferentially sensitive to the action of aconitine and veratridine. Finally, the synaptic block produced by these sodium channel activators is inhibited by phenytoin and carbamazepine at therapeutic anticonvulsant concentrations

  16. A Novel α2/α4 Subtype-selective Positive Allosteric Modulator of Nicotinic Acetylcholine Receptors Acting from the C-tail of an α Subunit.

    Science.gov (United States)

    Wang, Jingyi; Kuryatov, Alexander; Jin, Zhuang; Norleans, Jack; Kamenecka, Theodore M; Kenny, Paul J; Lindstrom, Jon

    2015-11-27

    Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChR) are important therapeutic candidates as well as valuable research tools. We identified a novel type II PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which both increases activation and reactivates desensitized nAChRs. This compound increases acetylcholine-evoked responses of α2* and α4* nAChRs but is without effect on α3* or α6* nAChRs (* indicates the presence of other nAChR subunits). Br-BPTC acts from the C-terminal extracellular sequences of α4 subunits, which is also a PAM site for steroid hormone estrogens such as 17β-estradiol. Br-PBTC is much more potent than estrogens. Like 17β-estradiol, the non-steroid Br-PBTC only requires one α4 subunit to potentiate nAChR function, and its potentiation is stronger with more α4 subunits. This feature enables Br-BPTC to potentiate activation of (α4β2)(α6β2)β3 but not (α6β2)2β3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different α6* nAChR subtypes. Besides activation, Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short term desensitized nAChRs that have at least two α4 subunits but not those with only one. Three α4 subunits were required for Br-BPTC to reactivate long term desensitized nAChRs. These data suggest that higher PAM occupancy promotes channel opening more efficiently and overcomes short and long term desensitization. This C-terminal extracellular domain could be a target for developing subtype or state-selective drugs for nAChRs. PMID:26432642

  17. Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1 Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor.

    Directory of Open Access Journals (Sweden)

    Ekaterina N Lyukmanova

    Full Text Available SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1 differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM of human oral keratinocytes (Het-1A cells. Application of mecamylamine and atropine,--non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM. It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1 did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the

  18. A combination of temsirolimus, an allosteric mTOR inhibitor, with clofarabine as a new therapeutic option for patients with acute myeloid leukemia

    OpenAIRE

    Chiarini, Francesca; Lonetti, Annalisa; Teti, Gabriella; Orsini, Ester; Bressanin, Daniela; Cappellini, Alessandra; Ricci, Francesca; Tazzari, Pier Luigi; Ognibene, Andrea; Falconi, Mirella; Pagliaro, Pasqualepaolo; Iacobucci, Ilaria; Martinelli, Giovanni; Amadori, Sergio; McCubrey, James A.

    2012-01-01

    Signaling through the phosphatidylinositol 3-kinase (PI3K) pathway and its downstream effectors, Akt and mechanistic target of rapamycin (mTOR), is aberrantly activated in acute myeloid leukemia (AML) patients, where it contributes to leukemic cell proliferation, survival, and drug-resistance. Thus, inhibiting mTOR signaling in AML blasts could enhance their sensitivity to cytotoxic agents. Preclinical data also suggest that allosteric mTOR inhibition with rapamycin impaired leukemia initiati...

  19. Analysis of K-net and Kik-net data: implications for ground motion prediction - acceleration time histories, response spectra and nonlinear site response; Analyse des donnees accelerometriques de K-net et Kik-net: implications pour la prediction du mouvement sismique - accelerogrammes et spectres de reponse - et la prise en compte des effets de site non-lineaire

    Energy Technology Data Exchange (ETDEWEB)

    Pousse, G

    2005-10-15

    This thesis intends to characterize ground motion during earthquake. This work is based on two Japanese networks. It deals with databases of shallow events, depth less than 25 km, with magnitude between 4.0 and 7.3. The analysis of K-net allows to compute a spectral ground motion prediction equation and to review the shape of the Eurocode 8 design spectra. We show the larger amplification at short period for Japanese data and bring in light the soil amplification that takes place at large period. In addition, we develop a new empirical model for simulating synthetic stochastic nonstationary acceleration time histories. By specifying magnitude, distance and site effect, this model allows to produce many time histories, that a seismic event is liable to produce at the place of interest. Furthermore, the study of near-field borehole records of the Kik-net allows to explore the validity domain of predictive equations and to explain what occurs by extrapolating ground motion predictions. Finally, we show that nonlinearity reduces the dispersion of ground motion at the surface. (author)

  20. Millisecond dynamics in the allosteric enzyme imidazole glycerol phosphate synthase (IGPS) from Thermotoga maritima

    International Nuclear Information System (INIS)

    IGPS is a 51 kDa heterodimeric enzyme comprised of two proteins, HisH and HisF, that catalyze the hydrolysis of glutamine to produce NH3 in the HisH active site and the cyclization of ammonia with N'- [(5'-phosphoribulosyl)formimino] -5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in HisF to produce imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamide ribotide (AICAR). Binding of PRFAR and IGP stimulates glutaminase activity in the HisH enzyme over 5,000 and 100-fold, respectively, despite the active sites being >25 A apart. The details of this long-range protein communication process were investigated by solution NMR spectroscopy and CPMG relaxation dispersion experiments. Formation of the heterodimer enzyme results in a reduction in millisecond motions in HisF that extend throughout the protein. Binding of lGP results in an increase in protein-wide millisecond dynamics evidenced as severe NMR line broadening and elevated Rex values. Together, these data demonstrate a grouping of flexible residues that link the HisF active site with the protein interface to which HisH binds and provide a model for the path of communication between the IGPS active sites

  1. Feline injection site sarcomas

    OpenAIRE

    Nóbrega, C.; Mesquita, Jr.; Cruz, R; C. Coelho; Esteves, F.; Mega, A. C.; Santos, C.; Vala, Helena

    2016-01-01

    Feline injection site sarcoma (FISS), formerly known as Vaccine Associated Sarcoma (VAS) is a rare, but life-threatening disease. The incidence estimates have varied from 1 case of FISS per 1,000-10,000 cats vaccinated in North America and between 1 per 5,000-12,500 vaccination visits in United Kingdom. It has been primarily associated with vaccine administration, but several other injectable materials/substances have been implicated as aetiologic agents, namely lufenuron, penicilin, meti...

  2. Rescue of deficient amygdala tonic γ-aminobutyric acidergic currents in the Fmr(-/y) mouse model of fragile X syndrome by a novel γ-aminobutyric acid type A receptor-positive allosteric modulator.

    Science.gov (United States)

    Martin, Brandon S; Martinez-Botella, Gabriel; Loya, Carlos M; Salituro, Francesco G; Robichaud, Albert J; Huntsman, Molly M; Ackley, Mike A; Doherty, James J; Corbin, Joshua G

    2016-06-01

    Alterations in the ratio of excitatory to inhibitory transmission are emerging as a common component of many nervous system disorders, including autism spectrum disorders (ASDs). Tonic γ-aminobutyric acidergic (GABAergic) transmission provided by peri- and extrasynaptic GABA type A (GABAA ) receptors powerfully controls neuronal excitability and plasticity and, therefore, provides a rational therapeutic target for normalizing hyperexcitable networks across a variety of disorders, including ASDs. Our previous studies revealed tonic GABAergic deficits in principal excitatory neurons in the basolateral amygdala (BLA) in the Fmr1(-/y) knockout (KO) mouse model fragile X syndrome. To correct amygdala deficits in tonic GABAergic neurotransmission in Fmr1(-/y) KO mice, we developed a novel positive allosteric modulator of GABAA receptors, SGE-872, based on endogenously active neurosteroids. This study shows that SGE-872 is nearly as potent and twice as efficacious for positively modulating GABAA receptors as its parent molecule, allopregnanolone. Furthermore, at submicromolar concentrations (≤1 μM), SGE-872 is selective for tonic, extrasynaptic α4β3δ-containing GABAA receptors over typical synaptic α1β2γ2 receptors. We further find that SGE-872 strikingly rescues the tonic GABAergic transmission deficit in principal excitatory neurons in the Fmr1(-/y) KO BLA, a structure heavily implicated in the neuropathology of ASDs. Therefore, the potent and selective action of SGE-872 on tonic GABAA receptors containing α4 subunits may represent a novel and highly useful therapeutic avenue for ASDs and related disorders involving hyperexcitability of neuronal networks. © 2015 Wiley Periodicals, Inc. PMID:26308557

  3. Negative Allosteric Modulators Selective for The NR2B Subtype of The NMDA Receptor Impair Cognition in Multiple Domains.

    Science.gov (United States)

    Weed, Michael R; Bookbinder, Mark; Polino, Joseph; Keavy, Deborah; Cardinal, Rudolf N; Simmermacher-Mayer, Jean; Cometa, Fu-ni L; King, Dalton; Thangathirupathy, Srinivasan; Macor, John E; Bristow, Linda J

    2016-01-01

    Antidepressant activity of N-methyl-D-aspartate (NMDA) receptor antagonists and negative allosteric modulators (NAMs) has led to increased investigation of their behavioral pharmacology. NMDA antagonists, such as ketamine, impair cognition in multiple species and in multiple cognitive domains. However, studies with NR2B subtype-selective NAMs have reported mixed results in rodents including increased impulsivity, no effect on cognition, impairment or even improvement of some cognitive tasks. To date, the effects of NR2B-selective NAMs on cognitive tests have not been reported in nonhuman primates. The current study evaluated two selective NR2B NAMs, CP101,606 and BMT-108908, along with the nonselective NMDA antagonists, ketamine and AZD6765, in the nonhuman primate Cambridge Neuropsychological Test Automated Battery (CANTAB) list-based delayed match to sample (list-DMS) task. Ketamine and the two NMDA NR2B NAMs produced selective impairments in memory in the list-DMS task. AZD6765 impaired performance in a non-specific manner. In a separate cohort, CP101,606 impaired performance of the nonhuman primate CANTAB visuo-spatial Paired Associates Learning (vsPAL) task with a selective impairment at more difficult conditions. The results of these studies clearly show that systemic administration of a selective NR2B NAM can cause transient cognitive impairment in multiple cognitive domains. PMID:26105137

  4. The Central domain of RyR1 is the transducer for long-range allosteric gating of channel opening.

    Science.gov (United States)

    Bai, Xiao-Chen; Yan, Zhen; Wu, Jianping; Li, Zhangqiang; Yan, Nieng

    2016-09-01

    The ryanodine receptors (RyRs) are intracellular calcium channels responsible for rapid release of Ca(2+) from the sarcoplasmic/endoplasmic reticulum (SR/ER) to the cytoplasm, which is essential for the excitation-contraction (E-C) coupling of cardiac and skeletal muscles. The near-atomic resolution structure of closed RyR1 revealed the molecular details of this colossal channel, while the long-range allosteric gating mechanism awaits elucidation. Here, we report the cryo-EM structures of rabbit RyR1 in three closed conformations at about 4 Å resolution and an open state at 5.7 Å. Comparison of the closed RyR1 structures shows a breathing motion of the cytoplasmic platform, while the channel domain and its contiguous Central domain remain nearly unchanged. Comparison of the open and closed structures shows a dilation of the S6 tetrahelical bundle at the cytoplasmic gate that leads to channel opening. During the pore opening, the cytoplasmic "O-ring" motif of the channel domain and the U-motif of the Central domain exhibit coupled motion, while the Central domain undergoes domain-wise displacement. These structural analyses provide important insight into the E-C coupling in skeletal muscles and identify the Central domain as the transducer that couples the conformational changes of the cytoplasmic platform to the gating of the central pore. PMID:27468892

  5. Structural Mechanisms of Peptide Recognition and Allosteric Modulation of Gene Regulation by the RRNPP Family of Quorum-Sensing Regulators.

    Science.gov (United States)

    Do, Hackwon; Kumaraswami, Muthiah

    2016-07-17

    The members of RRNPP family of bacterial regulators sense population density-specific secreted oligopeptides and modulate the expression of genes involved in cellular processes, such as sporulation, competence, virulence, biofilm formation, conjugative plasmid transfer and antibiotic resistance. Signaling by RRNPP regulators include several steps: generation and secretion of the signaling oligopeptides, re-internalization of the signaling molecules into the cytoplasm, signal sensing by the cytosolic RRNPP regulators, signal-specific allosteric structural changes in the regulators, and interaction of the regulators with their respective regulatory target and gene regulation. The recently determined structures of the RRNPP regulators provide insight into the mechanistic aspects for several steps in this signaling circuit. In this review, we discuss the structural principles underlying peptide specificity, regulatory target recognition, and ligand-induced allostery in RRNPP regulators and its impact on gene regulation. Despite the conserved tertiary structure of these regulators, structural analyses revealed unexpected diversity in the mechanism of activation and molecular strategies that couple the peptide-induced allostery to gene regulation. Although these structural studies provide a sophisticated understanding of gene regulation by RRNPP regulators, much needs to be learned regarding the target DNA binding by yet-to-be characterized RNPP regulators and the several aspects of signaling by Rgg regulators. PMID:27283781

  6. Effects of alpha-7 nicotinic acetylcholine receptor positive allosteric modulator on lipopolysaccharide-induced neuroinflammatory pain in mice.

    Science.gov (United States)

    Abbas, Muzaffar; Rahman, Shafiqur

    2016-07-15

    Evidence indicates that microglial activation contributes to the pathophysiology and maintenance of neuroinflammatory pain involving central nervous system alpha-7 nicotinic acetylcholine receptors. The objective of the present study was to determine the effects of 3a,4,5,9b-Tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS), an alpha-7 nicotinic acetylcholine receptor positive allosteric modulator (PAM), on tactile allodynia and thermal hyperalgesia following lipopolysaccharide (LPS)-induced microglial activation in hippocampus, a neuroinflammatory pain model in mice. In addition, we examined the effects of TQS on microglial activation marker, an ionized calcium-binding adapter molecule 1 (Iba-1), in the hippocampus may be associated with neuroinflammatory pain. Pretreatment of TQS (4mg/kg) significantly reduced LPS (1mg/kg)-induced tactile allodynia and thermal hyperalgesia. Moreover, pretreatment of methyllycaconitine (3mg/kg) significantly reversed TQS-induced antiallodynic and antihyperalgesic responses indicating the involvement of alpha-7 nicotinic acetylcholine receptor. Pretreatment of TQS significantly decreased LPS-induced increased in hippocampal Iba-1 expression. Overall, these results suggest that TQS reduces LPS-induced neuroinflammatory pain like symptoms via modulating microglial activation likely in the hippocampus and/or other brain region by targeting alpha-7 nicotinic acetylcholine receptor. Therefore, alpha-7 nicotinic acetylcholine receptor PAM such as TQS could be a potential drug candidate for the treatment of neuroinflammatory pain. PMID:27154173

  7. Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate.

    Science.gov (United States)

    Gelato, Kathy A; Tauber, Maria; Ong, Michelle S; Winter, Stefan; Hiragami-Hamada, Kyoko; Sindlinger, Julia; Lemak, Alexander; Bultsma, Yvette; Houliston, Scott; Schwarzer, Dirk; Divecha, Nullin; Arrowsmith, Cheryl H; Fischle, Wolfgang

    2014-06-19

    UHRF1 is a multidomain protein crucially linking histone H3 modification states and DNA methylation. While the interaction properties of its specific domains are well characterized, little is known about the regulation of these functionalities. We show that UHRF1 exists in distinct active states, binding either unmodified H3 or the H3 lysine 9 trimethylation (H3K9me3) modification. A polybasic region (PBR) in the C terminus blocks interaction of a tandem tudor domain (TTD) with H3K9me3 by occupying an essential peptide-binding groove. In this state the plant homeodomain (PHD) mediates interaction with the extreme N terminus of the unmodified H3 tail. Binding of the phosphatidylinositol phosphate PI5P to the PBR of UHRF1 results in a conformational rearrangement of the domains, allowing the TTD to bind H3K9me3. Our results define an allosteric mechanism controlling heterochromatin association of an essential regulatory protein of epigenetic states and identify a functional role for enigmatic nuclear phosphatidylinositol phosphates. PMID:24813945

  8. Contaminated sites

    International Nuclear Information System (INIS)

    The term 'Contaminated sites' refers to soil and groundwater contamination caused by local sources such as landfills or industrial sites. As of July 2002, there were in Austria 2,372 sites registered as potentially contaminated sites, from them: 165 sites required remediation, for 55 sites non remedial measures were necessary and to date 65 sites were remediated with a cost of 700,000 M Euro. An overview about funding of remedial measures, estimation of the extent of the problem (remediation requirements, chlorinated hydrocarbons accidents), deficits (lack of legal harmonization, slow implementation of remedial measures, etc.) is presented. Table 1. (nevyjel)

  9. Mechanism of Allosteric Inhibition of N-Acetyl-L-glutamate Synthase by L-Arginine

    Energy Technology Data Exchange (ETDEWEB)

    Min, Li; Jin, Zhongmin; Caldovic, Ljubica; Morizono, Hiroki; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang (GUW); (Maryland); (GWU); (Georgia)

    2010-01-07

    N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in L-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by L-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with L-arginine bound and in the active R-state complexed with CoA and L-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold but lacking kinase activity and an N-acetyltransferase (NAT) domain homologous to other GCN5-related transferases. Binding of L-arginine to the AAK domain induces a global conformational change that increases the diameter of the hexamer by {approx}10 {angstrom} and decreases its height by {approx}20{angstrom}. AAK dimers move 5{angstrom} outward along their 2-fold axes, and their tilt relative to the plane of the hexamer decreases by {approx}4{sup o}. The NAT domains rotate {approx}109{sup o} relative to AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on different subunits also change. Local motions of several loops at the L-arginine-binding site enable the protein to close around the bound ligand, whereas several loops at the NAT active site become disordered, markedly reducing enzymatic specific activity.

  10. Mechanism of allosteric inhibition of N-acetyl-L-glutamate synthase by L-arginine.

    Science.gov (United States)

    Min, Li; Jin, Zhongmin; Caldovic, Ljubica; Morizono, Hiroki; Allewell, Norma M; Tuchman, Mendel; Shi, Dashuang

    2009-02-20

    N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in l-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by l-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with l-arginine bound and in the active R-state complexed with CoA and l-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold but lacking kinase activity and an N-acetyltransferase (NAT) domain homologous to other GCN5-related transferases. Binding of l-arginine to the AAK domain induces a global conformational change that increases the diameter of the hexamer by approximately 10 A and decreases its height by approximately 20A(.) AAK dimers move 5A outward along their 2-fold axes, and their tilt relative to the plane of the hexamer decreases by approximately 4 degrees . The NAT domains rotate approximately 109 degrees relative to AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on different subunits also change. Local motions of several loops at the l-arginine-binding site enable the protein to close around the bound ligand, whereas several loops at the NAT active site become disordered, markedly reducing enzymatic specific activity. PMID:19095660

  11. Experimental conditions can obscure the second high-affinity site in LeuT.

    Science.gov (United States)

    Quick, Matthias; Shi, Lei; Zehnpfennig, Britta; Weinstein, Harel; Javitch, Jonathan A

    2012-02-01

    Neurotransmitter:Na(+) symporters (NSSs), the targets of antidepressants and psychostimulants, recapture neurotransmitters from the synapse in a Na(+)-dependent symport mechanism. The crystal structure of the NSS homolog LeuT from Aquifex aeolicus revealed one leucine substrate in an occluded, centrally located (S1) binding site next to two Na(+) ions. Computational studies combined with binding and flux experiments identified a second substrate (S2) site and a molecular mechanism of Na(+)-substrate symport that depends upon the allosteric interaction of substrate molecules in the two high-affinity sites. Here we show that the S2 site, which has not yet been identified by crystallographic approaches, can be blocked during preparation of detergent-solubilized LeuT, thereby obscuring its crucial role in Na(+)-coupled symport. This finding points to the need for caution in selecting experimental environments in which the properties and mechanistic features of membrane proteins can be delineated. PMID:22245968

  12. Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay.

    Science.gov (United States)

    Pantel, Jacques; Williams, Savannah Y; Mi, Dehui; Sebag, Julien; Corbin, Jackie D; Weaver, C David; Cone, Roger D

    2011-06-11

    The melanocortin MC(4) receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC(4) receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC(4) receptor, we created HEK293 cell lines coexpressing the human melanocortin MC(4) receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate that this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z'=0.50). A pilot screen run on the Microsource Spectrum compound library (n=2000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: β(2)AR agonists - the β(2)AR being endogenously expressed in HEK293 cells, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of a HTS for allosteric modulators for a Gs protein coupled receptor. PMID:21296065

  13. Yellow fluorescent protein-based assay to measure GABA(A channel activation and allosteric modulation in CHO-K1 cells.

    Directory of Open Access Journals (Sweden)

    Teres Johansson

    Full Text Available The γ-aminobutyric acid A (GABA(A ion channels are important drug targets for treatment of neurological and psychiatric disorders. Finding GABA(A channel subtype selective allosteric modulators could lead to new improved treatments. However, the progress in this area has been obstructed by the challenging task of developing functional assays to support screening efforts and the generation of cells expressing functional GABA(A ion channels with the desired subtype composition. To address these challenges, we developed a yellow fluorescent protein (YFP-based assay to be able to study allosteric modulation of the GABA(A ion channel using cryopreserved, transiently transfected, assay-ready cells. We show for the first time how the MaxCyte STX electroporation instrument can be used to generate CHO-K1 cells expressing functional GABA(A α2β3γ2 along with a halide sensing YFP-H148Q/I152L (YFP-GABA(A2 cells. As a basis for a cell-based assay capable of detecting allosteric modulators, experiments with antagonist, ion channel blocker and modulators were used to verify GABA(A subunit composition and functionality. We found that the I(- concentration used in the YFP assay affected both basal quench of YFP and potency of GABA. For the first time the assay was used to study modulation of GABA with 7 known modulators where statistical analysis showed that the assay can distinguish modulatory pEC50 differences of 0.15. In conclusion, the YFP assay proved to be a robust, reproducible and inexpensive assay. These data provide evidence that the assay is suitable for high throughput screening (HTS and could be used to discover novel modulators acting on GABA(A ion channels.

  14. Selective Allosteric Inhibition of MMP9 Is Efficacious in Preclinical Models of Ulcerative Colitis and Colorectal Cancer.

    Directory of Open Access Journals (Sweden)

    Derek C Marshall

    Full Text Available Expression of matrix metalloproteinase 9 (MMP9 is elevated in a variety of inflammatory and oncology indications, including ulcerative colitis and colorectal cancer. MMP9 is a downstream effector and an upstream mediator of pathways involved in growth and inflammation, and has long been viewed as a promising therapeutic target. However, previous efforts to target matrix metalloproteinases (MMPs, including MMP9, have utilized broad-spectrum or semi-selective inhibitors. While some of these drugs showed signs of efficacy in patients, all MMP-targeted inhibitors have been hampered by dose-limiting toxicity or insufficient clinical benefit, likely due to their lack of specificity. Here, we show that selective inhibition of MMP9 did not induce musculoskeletal syndrome (a characteristic toxicity of pan-MMP inhibitors in a rat model, but did reduce disease severity in a dextran sodium sulfate-induced mouse model of ulcerative colitis. We also found that MMP9 inhibition decreased tumor growth and metastases incidence in a surgical orthotopic xenograft model of colorectal carcinoma, and that inhibition of either tumor- or stroma-derived MMP9 was sufficient to reduce primary tumor growth. Collectively, these data suggest that selective MMP9 inhibition is a promising therapeutic strategy for treatment of inflammatory and oncology indications in which MMP9 is upregulated and is associated with disease pathology, such as ulcerative colitis and colorectal cancer. In addition, we report the development of a potent and highly selective allosteric MMP9 inhibitor, the humanized monoclonal antibody GS-5745, which can be used to evaluate the therapeutic potential of MMP9 inhibition in patients.

  15. A Monoclonal Antibody (MCPR3-7) Interfering with the Activity of Proteinase 3 by an Allosteric Mechanism*

    Science.gov (United States)

    Hinkofer, Lisa C.; Seidel, Susanne A. I.; Korkmaz, Brice; Silva, Francisco; Hummel, Amber M.; Braun, Dieter; Jenne, Dieter E.; Specks, Ulrich

    2013-01-01

    Proteinase 3 (PR3) is an abundant serine protease of neutrophil granules and a major target of autoantibodies (PR3 anti-neutrophil cytoplasmic antibodies) in granulomatosis with polyangiitis. Some of the PR3 synthesized by promyelocytes in the bone marrow escapes the targeting to granules and occurs on the plasma membrane of naive and primed neutrophils. This membrane-associated PR3 antigen may represent pro-PR3, mature PR3, or both forms. To discriminate between mature PR3 and its inactive zymogen, which have different conformations, we generated and identified a monoclonal antibody called MCPR3-7. It bound much better to pro-PR3 than to mature PR3. This monoclonal antibody greatly reduced the catalytic activity of mature PR3 toward extended peptide substrates. Using diverse techniques and multiple recombinant PR3 variants, we characterized its binding properties and found that MCPR3-7 preferentially bound to the so-called activation domain of the zymogen and changed the conformation of mature PR3, resulting in impaired catalysis and inactivation by α1-proteinase inhibitor (α1-antitrypsin). Noncovalent as well as covalent complexation between PR3 and α1-proteinase inhibitor was delayed in the presence of MCPR3-7, but cleavage of certain thioester and paranitroanilide substrates with small residues in the P1 position was not inhibited. We conclude that MCPR3-7 reduces PR3 activity by an allosteric mechanism affecting the S1′ pocket and further prime side interactions with substrates. In addition, MCPR3-7 prevents binding of PR3 to cellular membranes. Inhibitory antibodies targeting the activation domain of PR3 could be exploited as highly selective inhibitors of PR3, scavengers, and clearers of the PR3 autoantigen in granulomatosis with polyangiitis. PMID:23902773

  16. Hydrogen-exchange labeling study of the allosteric R-state to T-state equilibrium in methemoglobin

    Science.gov (United States)

    McKinnie, R. E.; Englander, J. J.; Englander, S. W.

    1991-12-01

    Hydrogen-exchange labeling methods can be used to identify functionally important changes at positions all through a protein structure, can monitor the effect at these positions of structure changes anywhere in the protein, and can quantify these effects in terms of change in structural-stabilization free energy. These methods were used to study effects at two widely separated positions in human methemoglobin (metHb). The results show that the observed changes in hydrogen-exchange behavior reflect changes in the global R-state to T-state equilibrium, and specifically that stabilizing salt links at the α-chain N-terminus and the β-chain C-terminus are reformed in the R-T transition. The strong allosteric effector, inositol hexaphosphate (IHP), switches R-state methemoglobin to the T-state, but achieves a T/R equilibrium constant of only ≈ 3 (at pH=6.5, 0°C). Addition of the weaker effector, bezafibrate (Bzf), promotes this transition by an additional 0.7 kcal (T/R shifts to ≈ 12). Bzf alone is insufficient to cause the transition, indicating that R/T is 10 or more in stripped metHb under these conditions. However, R/T is small enough, not more than 103, to be reversed by the differential (T versus R) binding energy of IHP. The R-T transition caused by IHP and Bzf acting together can be reversed by some covalent modifications that sever the stabilizing salt links at the chain termini and thus favor transition back to the R-state.

  17. Selective Allosteric Antagonists for the G Protein-Coupled Receptor GPRC6A Based on the 2-Phenylindole Privileged Structure Scaffold

    DEFF Research Database (Denmark)

    Johansson, Henrik; Boesgaard, Michael Worch; Nørskov-Lauritsen, Lenea; Larsen, Inna; Kuhne, Sebastiaan; Gloriam, David E; Bräuner-Osborne, Hans; Sejer Pedersen, Daniel

    2015-01-01

    G protein-coupled receptors (GPCRs) represent a biological target class of fundamental importance in drug therapy. The GPRC6A receptor is a newly deorphanized class C GPCR that we recently reported for the first allosteric antagonists based on the 2-arylindole privileged structure scaffold (e.g., 1......, and 34b as antagonists at the GPRC6A receptor in the low micromolar range and show that 7 and 34b display >9-fold selectivity for the GPRC6A receptor over related GPCRs, making 7 and 34b the most potent and selective antagonists for the GPRC6A receptor reported to date....

  18. An Allosteric Interaction Links USP7 to Deubiquitination and Chromatin Targeting of UHRF1

    Directory of Open Access Journals (Sweden)

    Zhi-Min Zhang

    2015-09-01

    Full Text Available The protein stability and chromatin functions of UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1 are regulated in a cell-cycle-dependent manner. We report a structural characterization of the complex between UHRF1 and the deubiquitinase USP7. The first two UBL domains of USP7 bind to the polybasic region (PBR of UHRF1, and this interaction is required for the USP7-mediated deubiquitination of UHRF1. Importantly, we find that the USP7-binding site of the UHRF1 PBR overlaps with the region engaging in an intramolecular interaction with the N-terminal tandem Tudor domain (TTD. We show that the USP7-UHRF1 interaction perturbs the TTD-PBR interaction of UHRF1, thereby shifting the conformation of UHRF1 from a TTD-“occluded” state to a state open for multivalent histone binding. Consistently, introduction of a USP7-interaction-defective mutation to UHRF1 significantly reduces its chromatin association. Together, these results link USP7 interaction to the dynamic deubiquitination and chromatin association of UHRF1.

  19. Modeling spatial correlation of DNA deformations: Allosteric effects of DNA protein binding

    Science.gov (United States)

    Xu, Xinliang; Cao, Jianshu; Hao Ge Collaboration; X. Sunney Xie Collaboration

    2013-03-01

    We report a study of DNA deformations by a coarse grained mechanical model. Recent single molecule experimental studies show that when DNA molecule is deformed by its binding to a protein, the binding affinity of a second protein at distance L away from the first binding site is altered. To explain this observation, the relaxation of deformation along the DNA chain is examined. Our method predicts a general exponentially decaying behavior for differenct deformation modes. As an example, inter-helical distance deformation is studied in details, and is found to decay at a previously unknown lengthscale of 10 base pairs as a result of the balance between inter and intra DNA strand energy. This lengthscale is in good agreement with the said single molecule experimental observation. This model of local deformation relaxation helps us better understand many important issues in DNA such as the enhanced flexibility of DNA at short lengthscales and DNA repair mechanism inside cells. Biodynamic Optical Imaging Center, Peking University

  20. Site organization and site arrangement

    International Nuclear Information System (INIS)

    The present paper deals with criteria for the choice of a production unit or power plant site, the organization and development of a site in terms of its particular characteristics and takes into account personnel considerations in site organizations as well as the problem of integrating the architecture into the environment. (RW)

  1. The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor

    Directory of Open Access Journals (Sweden)

    Kratochwil Nicole A

    2007-10-01

    Full Text Available Abstract Background Differences in sweet taste perception among species depend on structural variations of the sweet taste receptor. The commercially used isovanillyl sweetener neohesperidin dihydrochalcone activates the human but not the rat sweet receptor TAS1R2+TAS1R3. Analysis of interspecies combinations and chimeras of rat and human TAS1R2+TAS1R3 suggested that the heptahelical domain of human TAS1R3 is crucial for the activation of the sweet receptor by neohesperidin dihydrochalcone. Results By mutational analysis combined with functional studies and molecular modeling we identified a set of different amino acid residues within the heptahelical domain of human TAS1R3 that forms the neohesperidin dihydrochalcone binding pocket. Sixteen amino acid residues in the transmembrane domains 2 to 7 and one in the extracellular loop 2 of hTAS1R3 influenced the receptor's response to neohesperidin dihydrochalcone. Some of these seventeen residues are also part of the binding sites for the sweetener cyclamate or the sweet taste inhibitor lactisole. In line with this observation, lactisole inhibited activation of the sweet receptor by neohesperidin dihydrochalcone and cyclamate competitively, whereas receptor activation by aspartame, a sweetener known to bind to the N-terminal domain of TAS1R2, was allosterically inhibited. Seven of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are thought to play a role in the binding of allosteric modulators of other class C GPCRs, further supporting our model of the neohesperidin dihydrochalcone pharmacophore. Conclusion From our data we conclude that we identified the neohesperidin dihydrochalcone binding site at the human sweet taste receptor, which overlaps with those for the sweetener cyclamate and the sweet taste inhibitor lactisole. This readily delivers a molecular explanation of our finding that lactisole is a competitive inhibitor of the receptor

  2. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J. (Abbott)

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  3. The active site of oxidative phosphorylation and the origin of hyperhomocysteinemia in aging and dementia.

    Science.gov (United States)

    McCully, Kilmer S

    2015-01-01

    The active site of oxidative phosphorylation and adenosine triphosphate (ATP) synthesis in mitochondria is proposed to consist of two molecules of thioretinamide bound to cobalamin, forming thioretinaco, complexed with ozone, oxygen, nicotinamide adenine dinucleotide. and inorganic phosphate, TR2CoO3O2NAD(+)H2PO4(-). Reduction of the pyridinium nitrogen of the nicotinamide group by an electron from electron transport complexes initiates polymerization of phosphate with adenosine diphosphate, yielding nicotinamide riboside and ATP bound to thioretinaco ozonide oxygen. A second electron reduces oxygen to hydroperoxyl radical, releasing ATP from the active site. A proton gradient is created within F1F0 ATPase complexes of mitochondria by reaction of protons with reduced nicotinamide riboside and with hydroperoxyl radical, yielding reduced nicotinamide riboside and hydroperoxide. The hyperhomocysteinemia of aging and dementia is attributed to decreased synthesis of adenosyl methionine by thioretinaco ozonide and ATP, causing decreased allosteric activation of cystathionine synthase and decreased allosteric inhibition of methylenetetrahydrofolate reductase and resulting in dysregulation of methionine metabolism. PMID:25887881

  4. Site operations

    International Nuclear Information System (INIS)

    This chapter is a discussion of the management and operations practices used at the Barnwell Waste Management Facility in Barnwell, SC. The following topics are discussed: (1) Waste receiving and inspection, including manifest and certificates of compliance, radiological surveys, disposition of nonconforming items, and decontamination and disposition of secondary waste streams; (2) Waste disposal, including Title 10 CFR 61 requirements, disposal area evaluations, shipment offloading, container emplacement, and radiation protection; (3) Trench closure, including trench backfilling, trench capping, and permanent markers; (4) Site maintenance and stabilization, including trench maintenance, surface water management, and site closure activities; (5) Site monitoring programs, including operational monitoring, and environmental monitoring program; (6) Personnel training and qualifications, including basic training program, safety training program, special skills training, and physical qualifications; (7) Records management, including waste records, personnel training records, personnel dosimetry records, site monitoring records, trench qualification and construction records, and site drawings and stabilization records; (8) Site security; (9) Emergency response plans; and (10) Quality assurance

  5. The insect repellent N,N-diethyl-m-toluamide (DEET) induces angiogenesis via allosteric modulation of the M3 muscarinic receptor in endothelial cells.

    Science.gov (United States)

    Legeay, Samuel; Clere, Nicolas; Hilairet, Grégory; Do, Quoc-Tuan; Bernard, Philippe; Quignard, Jean-François; Apaire-Marchais, Véronique; Lapied, Bruno; Faure, Sébastien

    2016-01-01

    The insect repellent N,N-diethyl-m-toluamide (DEET) has been reported to inhibit AChE (acetylcholinesterase) and to possess potential carcinogenic properties with excessive vascularization. In the present paper, we demonstrate that DEET specifically stimulates endothelial cells that promote angiogenesis which increases tumor growth. DEET activates cellular processes that lead to angiogenesis including proliferation, migration and adhesion. This is associated with an enhancement of NO production and VEGF expression in endothelial cells. M3 silencing or the use of a pharmacological M3 inhibitor abrogates all of these effects which reveals that DEET-induced angiogenesis is M3 sensitive. The experiments involving calcium signals in both endothelial and HEK cells overexpressing M3 receptors, as well as binding and docking studies demonstrate that DEET acts as an allosteric modulator of the M3 receptor. In addition, DEET inhibited AChE which increased acetylcholine bioavailability and binding to M3 receptors and also strengthened proangiogenic effects by an allosteric modulation. PMID:27345502

  6. Positive Allosteric Modulators of Type 5 Metabotropic Glutamate Receptors (mGluR5 and Their Therapeutic Potential for the Treatment of CNS Disorders

    Directory of Open Access Journals (Sweden)

    Richard M. Cleva

    2011-03-01

    Full Text Available Studies utilizing selective pharmacological antagonists or targeted gene deletion have demonstrated thattype 5 metabotropic glutamate receptors (mGluR5 are critical mediators and potential therapeutic targets for the treatment of numerous disorders of the central nervous system (CNS, including depression, anxiety, drug addiction, chronic pain, Fragile X syndrome, Parkinson’s disease, and gastroesophageal reflux disease. However, in recent years, the development of positive allosteric modulators (PAMs of the mGluR5 receptor have revealed that allosteric activation of this receptor may also be of potential therapeutic benefit for the treatment of other CNS disorders, including schizophrenia, cognitive deficits associated with chronic drug use, and deficits in extinction learning. Here we summarize the discovery and characterization of various mGluR5 PAMs, with an emphasis on those that are systemically active. We will also review animal studies showing that these molecules have potential efficacy as novel antipsychotic agents. Finally, we will summarize findings that suggest that mGluR5 PAMs have pro-cognitive effects such as the ability toenhance synaptic plasticity, improve performance in various learning and memory tasks, including extinction of drug-seeking behavior, and reverse cognitive deficits produced by chronic drug use.

  7. Site investigations

    International Nuclear Information System (INIS)

    For the construction of nuclear power stations, comprehensive site investigations are required to assure the adequacy and suitability of the site under consideration. The site investigations cover mainly the following matters: 1) hydrology; 2) geology; 3) seismology; 4) meteorology. Site investigations for nuclear power stations are carried out in stages in increasing detail and to an appreciable depth in order to assure the soundness of the project, and, in particular, to determine all measures required to assure the safety of the nuclear power station and the protection of the population against radiation exposure. (orig./RW)

  8. pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins.

    Directory of Open Access Journals (Sweden)

    Priyanka Prakash

    2015-10-01

    Full Text Available Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation.

  9. Allosteric regulation of 6-phosphofructo-1-kinase activity of fat body and flight muscle from the bloodsucking bug Rhodnius prolixus

    Directory of Open Access Journals (Sweden)

    Gutemberg G. Alves

    2007-03-01

    Full Text Available 6-phosphofructo-1-kinase (phosphofructokinase; PFK activity from Rhodnius prolixus, a haematophagous insect which is usually a poor flyer, was measured and compared in two metabolically active tissues - flight muscle and fat body. The activity of this important regulatory glycolytic enzyme was much more pronounced in muscle (15.1 ± 1.4 U/mg than in fat body extracts (3.6±0.4 U/mg, although the latter presented higher levels of enzyme per protein content, as measured by western-blotting. Muscle extracts are more responsible than fat body to ATP and fructose 6-phosphate, both substrates of PFK. Allosteric regulation exerted by different effectors such as ADP, AMP and fructose 2,6-phosphate presented a singular pattern for each tissue. Optimal pH (8.0-8.5 and sensitivity to pH variation was very similar, and citrate was unable to inhibit PFK activity in both extracts. Our results suggest the existence of a particular PFK activity for each tissue, with regulatory patterns that are consistent with their physiological roles.A atividade da fosfofrutocinase (PFK de Rodnius prolixus, um inseto hematófago, o qual vôa somente pequenas distâncias, foi medida e comparada em dois tecidos metabolicamente ativos - músculo de asa e corpo gorduroso. A atividade desta importante enzima glicolítica regulatória foi muito mais pronunciada em músculo de asa (15,1 ±1,4 U/mg do que em extrato de corpo gorduroso (3,6 ±0,4 U/mg embora este último tenha apresentado níveis mais altos da enzima por quantidade de proteína, como medido por western-blotting. Extratos de músculo foram mais responsivos do que corpo gorduroso para ATP e frutose-6-fosfato, ambos substratos da PFK. A regulação alostérica exercida por diferentes efetores tais como ADP, AMP, frutose-2,6-bisfosfato apresentou um padrão singular para cada tecido. O pH ótimo (8,0-8,5 e a sensibilidade a variações de pH, foram muito similares e o citrato foi incapaz de inibir a atividade da PFK em

  10. 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; Jørgensen, Thomas J D; Ostergaard, Henrik; Jensen, Ole N; Stennicke, Henning R; Persson, Egon

    2008-01-01

    region rather than imitate the TF-induced effect. Hydrogen exchange analysis of the FVIIa(M306D) variant, which was unresponsive to stimulation by TF, correlated widespread reductions in exchange to the single mutation in the TF-binding region. These results reveal the delicate interplay between key...

  11. Site assessment

    DEFF Research Database (Denmark)

    Vesth, Allan; Gómez Arranz, Paula

    This report describes the site assessment of given position in a given site, for a wind turbine with a well-defined hub height and rotor diameter. The analysis is carried out in accordance to IEC 61400-12-1 [1], and both an obstacle assessment and a terrain assessment are performed....

  12. 3-(Imidazolyl methyl)-3-aza-bicyclo[3.1.0]hexan-6-yl)methyl ethers: a novel series of mGluR2 positive allosteric modulators.

    Science.gov (United States)

    Zhang, Lei; Rogers, Bruce N; Duplantier, Allen J; McHardy, Stanley F; Efremov, Ivan; Berke, Helen; Qian, Weimin; Zhang, Andy Q; Maklad, Noha; Candler, John; Doran, Angela C; Lazzaro, John T; Ganong, Alan H

    2008-10-15

    The synthesis and structure-activity relationship (SAR) of a novel series of 3-(imidazolyl methyl)-3-aza-bicyclo[3.1.0]hexan-6-yl)methyl ethers, derived from a high throughput screening (HTS), are described. Subsequent optimization led to identification of potent, metabolically stable and orally available mGluR2 positive allosteric modulators (PAMs). PMID:18812259

  13. Citizen participation in power plant siting

    Energy Technology Data Exchange (ETDEWEB)

    Ducsik, D.W.

    1981-04-01

    With continuing public concern for environmental consequences in the siting of large power plants, direct citizen participation in site selection is increasingly suggested as a means of resolving conflict. The relative merits of open planning strategy are discussed. Six basic concerns utility companies often cite about the practical implications of collaboration with environmentalists and other groups are explained.

  14. eBay Law: The Legal Implications of the C2C Electronic Commerce Model

    OpenAIRE

    Guadamuz, Andres

    2003-01-01

    This paper attempts to address some of the legal implications of the popular Consumer-to-Consumer electronic commerce model, in particular the implications of the successful and popular auctions site eBay.

  15. Site selection

    International Nuclear Information System (INIS)

    The conditions and criteria for selecting a site for a nuclear weapons test at the Nevada Test Site are summarized. Factors considered are: (1) scheduling of drill rigs, (2) scheduling of site preparation (dirt work, auger hole, surface casing, cementing), (3) schedule of event (when are drill hole data needed), (4) depth range of proposed W.P., (5) geologic structure (faults, Pz contact, etc.), (6) stratigraphy (alluvium, location of Grouse Canyon Tuff, etc.), (7) material properties (particularly montmorillonite and CO2 content), (8) water table depth, (9) potential drilling problems (caving), (10) adjacent collapse craters and chimneys, (11) adjacent expended but uncollapsed sites, (12) adjacent post-shot or other small diameter holes, (13) adjacent stockpile emplacement holes, (14) adjacent planned events (including LANL), (15) projected needs of Test Program for various DOB's and operational separations, and (16) optimal use of NTS real estate

  16. Revised tuning of Ocean Drilling Program Site 964 and KC01B (Mediterranean) and implications for the delta 0-18, tephra, calcareous nannofossil, and geomagnetic reversal chronologies of the past 1.1 Myr

    NARCIS (Netherlands)

    Lourens, L.J.

    2004-01-01

    High-resolution color reflectance records of KC01 and KC01B (Calabrian Ridge, Ionian Sea) are presented and compared with a modified spliced high-resolution color reflectance record of Ocean Drilling Program (ODP) Site 964. This comparison revealed that KC01B is characterized by intensive deformatio

  17. High affinity and temperature sensitivity of blood oxygen binding in Pangasianodon hypophthalmus due to lack of chloride-hemoglobin allosteric interaction

    DEFF Research Database (Denmark)

    Damsgaard, Christian; Phuong, Le My; Huong, Do Thi Thanh; Jensen, Frank Bo; Wang, Tobias; Bayley, Mark

    2015-01-01

    Air-breathing fishes represent interesting organisms in terms of understanding the physiological changes associated with the terrestrialization of vertebrates, and, further, are of great socio-economic importance for aquaculture in Southeast Asia. To understand how environmental factors, such as...... high temperature, affect O2 transport in air-breathing fishes, this study assessed the effects of temperature on O2 binding of blood and Hb in the economically important air-breathing fish Pangasianodon hypophthalmus. To determine blood O2 binding properties, blood was drawn from resting cannulated...... fishes and O2 binding curves made at 25°C and 35°C. To determine the allosteric regulation and thermodynamics of Hb O2 binding, Hb was purified, and O2 equilibria were recorded at five temperatures in the absence and presence of ATP and Cl-. Whole blood had a high O2 affinity (O2 tension at half...

  18. Regulation of transcription attenuation and translation initiation by allosteric control of an RNA-binding protein: the Bacillus subtilis TRAP protein.

    Science.gov (United States)

    Babitzke, Paul

    2004-04-01

    Tryptophan allosterically controls the 11-subunit trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis. When activated by tryptophan, TRAP binds to multiple trinucleotide repeats in target transcripts. TRAP is responsible for the decision to terminate transcription in the leader region of the trpEDCFBA operon or to allow transcription to proceed into the structural genes. TRAP also regulates translation of trpE by promoting formation of an RNA structure that prevents ribosome binding. In addition, bound TRAP regulates translation initiation of pabA, trpP and ycbK by directly blocking ribosome binding. The anti-TRAP protein inhibits TRAP activity by competing with RNA for the RNA binding surface of TRAP. PMID:15063849

  19. Chronic caffeine or theophylline exposure reduces gamma-aminobutyric acid/benzodiazepine receptor site interactions.

    Science.gov (United States)

    Roca, D J; Schiller, G D; Farb, D H

    1988-05-01

    Methylxanthines, such as caffeine and theophylline, are adenosine receptor antagonists that exert dramatic effects upon the behavior of vertebrate animals by increasing attentiveness, anxiety, and convulsive activity. Benzodiazepines, such as flunitrazepam, generally exert behavioral effects that are opposite to those of methylxanthines. We report the finding that chronic exposure of embryonic brain neurons to caffeine or theophylline reduces the ability of gamma-aminobutyric acid (GABA) to potentiate the binding of [3H]flunitrazepam to the GABA/benzodiazepine receptor. This theophylline-induced "uncoupling" of GABA- and benzodiazepine-binding site allosteric interactions is blocked by chloroadenosine, an adenosine receptor agonist, indicating that the chronic effects of theophylline are mediated by a site that resembles an adenosine receptor. We speculate that adverse central nervous system effects of long-term exposure to methylxanthines such as in caffeine-containing beverages or theophylline-containing medications may be exerted by a cell-mediated modification of the GABAA receptor. PMID:2835648

  20. An mGlu5-Positive Allosteric Modulator Rescues the Neuroplasticity Deficits in a Genetic Model of NMDA Receptor Hypofunction in Schizophrenia.

    Science.gov (United States)

    Balu, Darrick T; Li, Yan; Takagi, Shunsuke; Presti, Kendall Taylor; Ramikie, Teniel S; Rook, Jerri M; Jones, Carrie K; Lindsley, Craig W; Conn, P Jeffrey; Bolshakov, Vadim Y; Coyle, Joseph T

    2016-07-01

    There is substantial evidence that NMDA receptor (NMDAR) hypofunction contributes to the pathophysiology of schizophrenia (SCZ). A recent large-scale genome-wide association study identified serine racemase (SR), the enzyme that produces the NMDAR co-agonist D-serine, as a risk gene for SCZ. Serine racemase knockout (SR-/-) mice, which lack D-serine, exhibit many of the neurochemical and behavioral abnormalities observed in SCZ. Metabotropic glutamate receptor 5 (mGlu5)-positive allosteric modulators (PAMs) are currently being developed to treat cognitive dysfunction. We used in vitro electrophysiology to determine whether the mGlu5 PAM VU0409551 directly enhances NMDAR function in hippocampal slices from adult male SR-/- mice. We administered VU0409551 systemically for 5 days to adult male wild-type C57BL/6 animals to determine the optimal dose to test in SR-/- mice. We used western blot analyses and trace-fear conditioning to determine whether 5 days of VU0409551 treatment could reverse the neuroplasticity and learning deficits, respectively, in SR-/- mice. We show that VU0409551 enhances NMDAR function and rescues long-term potentiation in hippocampal slices obtained from SR-/- mice. Systemic treatment with VU0409551 (10 and 30 mg/kg) to wild-type mice causes a dose-dependent increase in the Akt/GS3Kα/β signaling pathway, which is reduced in SR-/- mice and in SCZ. Furthermore, the administration of VU0409551 to SR-/- mice reverses their deficits in several neuroplasticity signaling pathways and improves their contextual fear memory. These results support positive allosteric modulation of mGlu5, particularly with VU0409551, as a viable mechanism to reverse the deficits in NMDAR function, synaptic plasticity, and memory that are known to be impaired in SCZ. PMID:26741285

  1. Allosteric interactions between the oxytocin receptor and the β2-adrenergic receptor in the modulation of ERK1/2 activation are mediated by heterodimerization.

    Science.gov (United States)

    Wrzal, Paulina K; Devost, Dominic; Pétrin, Darlaine; Goupil, Eugénie; Iorio-Morin, Christian; Laporte, Stéphane A; Zingg, Hans H; Hébert, Terence E

    2012-01-01

    The oxytocin receptor (OTR) and the β(2)-adrenergic receptor (β(2)AR) are key regulators of uterine contraction. These two receptors are targets of tocolytic agents used to inhibit pre-term labor. Our recent study on the nature of OTR- and β(2)AR-mediated ERK1/2 activation in human hTERT-C3 myometrial cells suggested the presence of an OTR/β(2)AR hetero-oligomeric complex (see companion article). The goal of this study was to investigate potential allosteric interactions between OTR and β(2)AR and establish the nature of the interactions between these receptors in myometrial cells. We found that OTR-mediated ERK1/2 activation was attenuated significantly when cells were pretreated with the β(2)AR agonist isoproterenol or two antagonists, propranolol or timolol. In contrast, pretreatment of cells with a third β(2)AR antagonist, atenolol resulted in an increase in OTR-mediated ERK1/2 activation. Similarly, β(2)AR-mediated ERK1/2 activation was strongly attenuated by pretreatment with the OTR antagonists, atosiban and OTA. Physical interactions between OTR and β(2)AR were demonstrated using co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and protein-fragment complementation (PCA) assays in HEK 293 cells, the latter experiments indicating the interactions between the two receptors were direct. Our analyses suggest physical interactions between OTR and β(2)AR in the context of a new heterodimer pair lie at the heart of the allosteric effects. PMID:21963428

  2. Site Restoration

    Energy Technology Data Exchange (ETDEWEB)

    Noynaert, L.; Bruggeman, A.; Cornelissen, R.; Massaut, V.; Rahier, A

    2001-04-01

    The objectives, the programme, and the achievements of the Site Restoration Department of SCK-CEN in 2000 are summarised. Main activities include the decommissioning of the BR3 PWR-reactor as well as other clean-up activities, projects on waste minimisation and activities related to the management of decommissioning projects. The department provides consultancy and services to external organisations.

  3. Site Construction.

    Science.gov (United States)

    Richardson, Eric C.

    1996-01-01

    Presents a guide to planning and building a Web site, with an emphasis on setting up a Web server. Discussion includes hiring a consultant, contracts and payment, assembly of teams, training, development of a business plan, registration of domain name, purchase of hardware and software, local area networks, and types of Internet connection. (JKP)

  4. Site Restoration

    International Nuclear Information System (INIS)

    The objectives, the programme, and the achievements of the Site Restoration Department of SCK-CEN in 2000 are summarised. Main activities include the decommissioning of the BR3 PWR-reactor as well as other clean-up activities, projects on waste minimisation and activities related to the management of decommissioning projects. The department provides consultancy and services to external organisations

  5. Identification of the gC1qR sites for the HIV-1 viral envelope protein gp41 and the HCV core protein: Implications in viral-specific pathogenesis and therapy.

    Science.gov (United States)

    Pednekar, Lina; Valentino, Alisa; Ji, Yan; Tumma, Nithin; Valentino, Christopher; Kadoor, Adarsh; Hosszu, Kinga K; Ramadass, Mahalakshmi; Kew, Richard R; Kishore, Uday; Peerschke, Ellinor I B; Ghebrehiwet, Berhane

    2016-06-01

    A substantial body of evidence accumulated over the past 20 years supports the concept that gC1qR is a major pathogen-associated pattern recognition receptor (PRR). This conclusion is based on the fact that, a wide range of bacterial and viral ligands are able to exploit gC1qR to either suppress the host's immune response and thus enhance their survival, or to gain access into cells to initiate disease. Of the extensive array of viral ligands that have affinity for gC1qR, the HIV-1 envelope glycoprotein gp41, and the core protein of hepatitis C virus (HCV) are of major interest as they are known to contribute to the high morbidity and mortality caused by these pathogens. While the HCV core protein binds gC1qR and suppresses T cell proliferation resulting in a significantly diminished immune response, the gp41 employs gC1qR to induce the surface expression of the NK cell ligand, NKp44L, on uninfected CD4(+) T cells, thereby rendering them susceptible to autologous destruction by NKp44 receptor expressing NK cells. Because of the potential for the design of peptide-based or antibody-based therapeutic options, the present studies were undertaken to define the gC1qR interaction sites for these pathogen-associated molecular ligands. Employing a solid phase microplate-binding assay, we examined the binding of each viral ligand to wild type gC1qR and 11 gC1qR deletion mutants. The results obtained from these studies have identified two major HCV core protein sites on a domain of gC1qR comprising of residues 144-148 and 196-202. Domain 196-202 in turn, is located in the last half of the larger gC1qR segment encoded by exons IV-VI (residues 159-282), which was proposed previously to contain the site for HCV core protein. The major gC1qR site for gp41 on the other hand, was found to be in a highly conserved region encoded by exon IV and comprises of residues 174-180. Interestingly, gC1qR residues 174-180 also constitute the cell surface-binding site for soluble gC1qR (sgC1q

  6. Solubilization and characterization of haloperidol-sensitive (+)-[3H]SKF-10,047 binding sites (sigma sites) from rat liver membranes

    International Nuclear Information System (INIS)

    The zwitterionic detergent 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS) produced optimal solubilization of (+)-[3H]SKF-10,047 binding sites from rat liver membranes at a concentration of 0.2%, well below the critical micellular concentration of the detergent. The pharmacological selectivity of the liver (+)-[3H]SKF-10,047 binding sites corresponds to that of sigma sites from rat and guinea pig brain. When the affinities of 18 different drugs at (+)-[3H]SKF-10,047 binding sites in membranes and solubilized preparations were compared, a correlation coefficient of 0.99 and a slope of 1.03 were obtained, indicating that the pharmacological selectivity of rat liver sigma sites is retained after solubilization. In addition, the binding of 20 nM [3H]progesterone to solubilized rat liver preparations was found to exhibit a pharmacological selectivity appropriate for sigma sites. A stimulatory effect of phenytoin on (+)-[3H]SKF-10,047 binding to sigma sites persisted after solubilization. When the solubilized preparation was subjected to molecular sizing chromatography, a single peak exhibiting specific (+)-[3H]SKF-10,047 binding was obtained. The binding activity of this peak was stimulated symmetrically when assays were performed in the presence of 300 microM phenytoin. The molecular weight of the CHAPS-solubilized sigma site complex was estimated to be 450,000 daltons. After solubilization with CHAPS, rat liver sigma sites were enriched to 12 pmol/mg of protein. The present results demonstrate a successful solubilization of sigma sites from rat liver membranes and provide direct evidence that the gonadal steroid progesterone binds to sigma sites. The results also suggest that the anticonvulsant phenytoin binds to an associated allosteric site on the sigma site complex

  7. Repetitive genome elements in a European corn borer, Ostrinia nubilalis, bacterial artificial chromosome library were indicated by bacterial artificial chromosome end sequencing and development of sequence tag site markers: implications for lepidopteran genomic research.

    Science.gov (United States)

    Coates, Brad S; Sumerford, Douglas V; Hellmich, Richard L; Lewis, Leslie C

    2009-01-01

    The European corn borer, Ostrinia nubilalis, is a serious pest of food, fiber, and biofuel crops in Europe, North America, and Asia and a model system for insect olfaction and speciation. A bacterial artificial chromosome library constructed for O. nubilalis contains 36 864 clones with an estimated average insert size of >or=120 kb and genome coverage of 8.8-fold. Screening OnB1 clones comprising approximately 2.76 genome equivalents determined the physical position of 24 sequence tag site markers, including markers linked to ecologically important and Bacillus thuringiensis toxin resistance traits. OnB1 bacterial artificial chromosome end sequence reads (GenBank dbGSS accessions ET217010 to ET217273) showed homology to annotated genes or expressed sequence tags and identified repetitive genome elements, O. nubilalis miniature subterminal inverted repeat transposable elements (OnMITE01 and OnMITE02), and ezi-like long interspersed nuclear elements. Mobility of OnMITE01 was demonstrated by the presence or absence in O. nubilalis of introns at two different loci. A (GTCT)n tetranucleotide repeat at the 5' ends of OnMITE01 and OnMITE02 are evidence for transposon-mediated movement of lepidopteran microsatellite loci. The number of repetitive elements in lepidopteran genomes will affect genome assembly and marker development. Single-locus sequence tag site markers described here have downstream application for integration within linkage maps and comparative genomic studies. PMID:19132072

  8. Heritage sites

    Czech Academy of Sciences Publication Activity Database

    Drdácký, Tomáš

    Prague : Institute of theoretical and applied mechanics AS CR, v.v.i, 2011 - (Drdácký, M.; Binda, L.; Hennen, I.; Kőpp, C.; Lanza, L.), s. 138-141 ISBN 978-80-86246-37-6 Grant ostatní: evropská komise(XE) CHEF-SSPI-044251 Institutional research plan: CEZ:AV0Z20710524 Keywords : flood * heritage sites * cultural heritage Subject RIV: AL - Art, Architecture, Cultural Heritage

  9. Allosteric inhibition of glycogen phosphorylase a by the potential antidiabetic drug 3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarbo xylate.

    OpenAIRE

    Oikonomakos, N. G.; Tsitsanou, K. E.; Zographos, S. E.; Skamnaki, V. T.; Goldmann, S.; Bischoff, H

    1999-01-01

    The effect of the potential antidiabetic drug (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarbox ylate (W1807) on the catalytic and structural properties of glycogen phosphorylase a has been studied. Glycogen phosphorylase (GP) is an allosteric enzyme whose activity is primarily controlled by reversible phosphorylation of Ser14 of the dephosphorylated enzyme (GPb, less active, predominantly T-state) to form the phosphorylated enzyme (GPa, more active, ...

  10. Determination of OM/OC ratios and specific attenuation coefficients (SAC in ambient fine PM at a rural site in southern Ontario: implications for emission sources, particle aging, and radiative forcing

    Directory of Open Access Journals (Sweden)

    T. W. Chan

    2009-07-01

    Full Text Available Ambient particulate matter (PM samples were collected on quartz filters at a rural site in southern Ontario during intensive studies in 2005 and 2007. The concentrations of organic carbon (OC, pyrolysis organic carbon (POC, and elemental carbon (EC were determined by thermal analysis. These results were compared to the organic aerosol mass concentration (OM measured by an Aerodyne Aerosol Mass Spectrometer (AMS and to the particle absorption coefficient (b_asp obtained from a Radiance Research Particle Soot Absorption Photometer (PSAP. The total organic mass to organic carbon ratios (OM/OC and specific attenuation coefficients (SAC were also derived. According to the results, the POC mass is proportional to the approximated oxygen mass in the aerosols and OM/OC ratios can be estimated directly from thermal measurements. The study also suggests that the air masses from the south, with relatively low OC/EC ratios, high EC, sulphate contents and OM/OC ratios, were originated from urban and industrial emissions and subsequently experienced photo-oxidations in the atmosphere, implying that the oxygenated organics could come from both primary and secondary sources. Whereas the air masses from the north, with relatively high OC/EC ratios, low EC, sulphate contents and OM/OC ratios, were dominant by the background clean air with relatively larger contributions from biogenic emissions.

    The mean SAC derived from the 2005 and 2007 studies are 4.9 m2 g−1 and 3.8 m2 g−1, respectively. When POC mass approaching zero (i.e. the impact of atmospheric aging is minimized, the SAC for primary emitted soot is estimated to be 5.8 m2 g−1 and 6.3 m2 g−1 for the northern and southern air masses, respectively, supported by the corresponding values when particulate sulphate concentration approaches zero. A decreasing trend in the SAC value with atmospheric aging

  11. Découverte de termitières fossiles dans les sites à Vertébrés du Pliocène tchadien: description, identification et implications paléoécologiquesDiscovery of Pliocene fossilised termitaries in Chadian vertebate levels: description, identification and palaeoecological implications.

    Science.gov (United States)

    Schuster, Mathieu; Duringer, Philippe; Nel, André; Brunet, Michel; Vignaud, Patrick; Taïsso Mackaye, Hassan

    2000-07-01

    Some sandstony bioconstructions in the shape of a flattened ball have been found in the Chadian Pliocene. These balls have a diameter of about 40 cm and have an ellipsoidal shape. They are entirely built of centimetric partition-walls. These structures are interpreted as in situ fossilised subterranean termitaries. The presence of termitaries brings new elements to the palaeoecological interpretations of Chadian Australopithecine sites, especially in terms of climate and environmental stability.

  12. Purification and characterization of recombinant sugarcane sucrose phosphate synthase expressed in E. coli and insect Sf9 cells: an importance of the N-terminal domain for an allosteric regulatory property.

    Science.gov (United States)

    Sawitri, Widhi Dyah; Narita, Hirotaka; Ishizaka-Ikeda, Etsuko; Sugiharto, Bambang; Hase, Toshiharu; Nakagawa, Atsushi

    2016-06-01

    Sucrose phosphate synthase (SPS) catalyses the transfer of glycosyl group of uridine diphosphate glucose to fructose-6-phosphate to form sucrose-6-phosphate. Plant SPS plays a key role in photosynthetic carbon metabolisms, which activity is modulated by an allosteric activator glucose-6-phosphate (G6P). We produced recombinant sugarcane SPS using Escherichia coli and Sf9 insect cells to investigate its structure-function relationship. When expressed in E. coli, two forms of SPS with different sizes appeared; the larger was comparable in size with the authentic plant enzyme and the shorter was trimmed the N-terminal 20 kDa region off. In the insect cells, only enzyme with the authentic size was produced. We purified the trimmed SPS and the full size enzyme from insect cells and found their enzymatic properties differed significantly; the full size enzyme was activated allosterically by G6P, while the trimmed one showed a high activity even without G6P. We further introduced a series of N-terminal truncations up to 171 residue and found G6P-independent activity was enhanced by the truncation. These combined results indicated that the N-terminal region of sugarcane SPS is crucial for the allosteric regulation by G6P and may function like a suppressor domain for the enzyme activity. PMID:26826371

  13. An analysis of Apollo lunar soil samples 12070,889, 12030,187, and 12070,891: Basaltic diversity at the Apollo 12 landing site and implications for classification of small-sized lunar samples

    Science.gov (United States)

    Alexander, Louise; Snape, Joshua F.; Joy, Katherine H.; Downes, Hilary; Crawford, Ian A.

    2016-07-01

    Lunar mare basalts provide insights into the compositional diversity of the Moon's interior. Basalt fragments from the lunar regolith can potentially sample lava flows from regions of the Moon not previously visited, thus, increasing our understanding of lunar geological evolution. As part of a study of basaltic diversity at the Apollo 12 landing site, detailed petrological and geochemical data are provided here for 13 basaltic chips. In addition to bulk chemistry, we have analyzed the major, minor, and trace element chemistry of mineral phases which highlight differences between basalt groups. Where samples contain olivine, the equilibrium parent melt magnesium number (Mg#; atomic Mg/[Mg + Fe]) can be calculated to estimate parent melt composition. Ilmenite and plagioclase chemistry can also determine differences between basalt groups. We conclude that samples of approximately 1-2 mm in size can be categorized provided that appropriate mineral phases (olivine, plagioclase, and ilmenite) are present. Where samples are fine-grained (grain size fines) from future sample return missions to investigate lava flow diversity and petrological significance.

  14. An analysis of Apollo lunar soil samples 12070,889, 12030,187, and 12070,891: Basaltic diversity at the Apollo 12 landing site and implications for classification of small-sized lunar samples

    Science.gov (United States)

    Alexander, Louise; Snape, Joshua F.; Joy, Katherine H.; Downes, Hilary; Crawford, Ian A.

    2016-09-01

    Lunar mare basalts provide insights into the compositional diversity of the Moon's interior. Basalt fragments from the lunar regolith can potentially sample lava flows from regions of the Moon not previously visited, thus, increasing our understanding of lunar geological evolution. As part of a study of basaltic diversity at the Apollo 12 landing site, detailed petrological and geochemical data are provided here for 13 basaltic chips. In addition to bulk chemistry, we have analyzed the major, minor, and trace element chemistry of mineral phases which highlight differences between basalt groups. Where samples contain olivine, the equilibrium parent melt magnesium number (Mg#; atomic Mg/[Mg + Fe]) can be calculated to estimate parent melt composition. Ilmenite and plagioclase chemistry can also determine differences between basalt groups. We conclude that samples of approximately 1-2 mm in size can be categorized provided that appropriate mineral phases (olivine, plagioclase, and ilmenite) are present. Where samples are fine-grained (grain size <0.3 mm), a "paired samples t-test" can provide a statistical comparison between a particular sample and known lunar basalts. Of the fragments analyzed here, three are found to belong to each of the previously identified olivine and ilmenite basalt suites, four to the pigeonite basalt suite, one is an olivine cumulate, and two could not be categorized because of their coarse grain sizes and lack of appropriate mineral phases. Our approach introduces methods that can be used to investigate small sample sizes (i.e., fines) from future sample return missions to investigate lava flow diversity and petrological significance.

  15. Cocaine self-administration differentially affects allosteric A2A-D2 receptor-receptor interactions in the striatum. Relevance for cocaine use disorder.

    Science.gov (United States)

    Pintsuk, Julia; Borroto-Escuela, Dasiel O; Pomierny, Bartosz; Wydra, Karolina; Zaniewska, Magdalena; Filip, Malgorzata; Fuxe, Kjell

    2016-05-01

    In the current study behavioral and biochemical experiments were performed to study changes in the allosteric A2AR-D2R interactions in the ventral and dorsal striatum after cocaine self-administration versus corresponding yoked saline control. By using ex vivo [(3)H]-raclopride/quinpirole competition experiments, the effects of the A2AR agonist CGS 21680 (100nM) on the KiH and KiL values of the D2-like receptor (D2-likeR) were determined. One major result was a significant reduction in the D2-likeR agonist high affinity state observed with CGS 21680 after cocaine self-administration in the ventral striatum compared with the yoked saline group. The results therefore support the hypothesis that A2AR agonists can at least in part counteract the motivational actions of cocaine. This action is mediated via the D2-likeR by targeting the A2AR protomer of A2AR-D2-likeR heteroreceptor complexes in the ventral striatum, which leads to the reduction of D2-likeR protomer recognition through the allosteric receptor-receptor interaction. In contrast, in the dorsal striatum the CGS 21680-induced antagonistic modulation in the D2-likeR agonist high affinity state was abolished after cocaine self-administration versus the yoked saline group probably due to a local dysfunction/disruption of the A2AR-D2-likeR heteroreceptor complexes. Such a change in the dorsal striatum in cocaine self-administration can contribute to the development of either locomotor sensitization, habit-forming learning and/or the compulsive drug seeking by enhanced D2-likeR protomer signaling. Potential differences in the composition and stoichiometry of the A2AR-D2R heteroreceptor complexes, including differential recruitment of sigma 1 receptor, in the ventral and dorsal striatum may explain the differential regional changes observed in the A2A-D2-likeR interactions after cocaine self-administration. PMID:26987369

  16. Identification of covalent active site inhibitors of dengue virus protease

    Directory of Open Access Journals (Sweden)

    Koh-Stenta X

    2015-12-01

    Full Text Available Xiaoying Koh-Stenta,1 Joma Joy,1 Si Fang Wang,1 Perlyn Zekui Kwek,1 John Liang Kuan Wee,1 Kah Fei Wan,2 Shovanlal Gayen,1 Angela Shuyi Chen,1 CongBao Kang,1 May Ann Lee,1 Anders Poulsen,1 Subhash G Vasudevan,3 Jeffrey Hill,1 Kassoum Nacro11Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR, Singapore; 2Novartis Institute for Tropical Diseases, Singapore; 3Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, SingaporeAbstract: Dengue virus (DENV protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described.Keywords: flavivirus protease, small molecule optimization, covalent inhibitor, active site binding, pyrazole ester derivatives

  17. Site Restoration

    International Nuclear Information System (INIS)

    The objectives, the programme, and the achievements of SCK-CEN's Site Restoration Department for 2001 are described. Main activities include the decommissioning of the BR3 PWR-reactor as well as other clean-up activities, projects on waste minimisation and the management of spent fuel and the flow of dismantled materials and the recycling of materials from decommissioning activities based on the smelting of metallic materials in specialised foundries. The department provides consultancy and services to external organisations and performs R and D on new techniques including processes for the treatment of various waste components including the reprocessing of spent fuel, the treatment of tritium, the treatment of liquid alkali metals into cabonates through oxidation, the treatment of radioactive organic waste and the reconditioning of bituminised waste products

  18. Site Restoration

    Energy Technology Data Exchange (ETDEWEB)

    Noynaert, L.; Bruggeman, A.; Cornelissen, R.; Massaut, V.; Rahier, A

    2002-04-01

    The objectives, the programme, and the achievements of SCK-CEN's Site Restoration Department for 2001 are described. Main activities include the decommissioning of the BR3 PWR-reactor as well as other clean-up activities, projects on waste minimisation and the management of spent fuel and the flow of dismantled materials and the recycling of materials from decommissioning activities based on the smelting of metallic materials in specialised foundries. The department provides consultancy and services to external organisations and performs R and D on new techniques including processes for the treatment of various waste components including the reprocessing of spent fuel, the treatment of tritium, the treatment of liquid alkali metals into cabonates through oxidation, the treatment of radioactive organic waste and the reconditioning of bituminised waste products.

  19. The environmental implications of landfill gas control

    International Nuclear Information System (INIS)

    The paper reviews the implications for landfill gas control of the Environmental Protection Bill in relation to proposed, existing and closed sites. If the Bill is enacted in its present form these changes will have far reaching implications on the waste management industry and especially those involved in landfill gas monitoring and control. The paper describes the requirements for the management of landfill gas both on and around landfill sites before, during and after the cessation of waste disposal operations. It describes the duties of Waste Regulation Authorities (WRAs) under the Bill in relation to landfill gas including their duties in relation to closed sites. The paper concludes that when the WRAs fulfill these duties the risk of further incidents occurring with landfill gas will be significantly reduced. (author)

  20. Mochovce site

    International Nuclear Information System (INIS)

    In Mochovce site the construction of four units of WWER 440 NPP with V-213 type of reactor is being carried out. The financing of Mochovce units completion was resolved in April 1996. The completion work commenced at the construction site under leadership of SKODA Prague, the general supplier. The completion work on building part and tests of constructional electric distributions and lightning constructors started. The revisions in technological part were finished, and final protocols from revisions are the basis for starting of completion work. The assembly of transport container anchorage,ventilation system in hermetic areas and hermetic coverage of pools for stored spent nuclear fuel is being carried out. The pre-completion tests of instrumentation and control of ventilation systems, individual dosimetric control in medical station, and tests of nuclear programme according to commissioning and assembling work schedule at the equipment for physical protection of the NPP area started. Inspection activities at Mochovce were performed in accordance with inspection plan for 1996. Evaluation of routine inspections was performed by means of quarterly protocols. Main findings from the inspections performed in Mochovce were in the following areas: (a) deficiencies in the knowledge of the respective regulation and conditions from the Resolution of the state regulatory body, concerning selected employees; (b) training of the selected employees; (c) aim of the measures imposes by inspectors is to eliminate deficiencies in preparation of programmes for pre-completion and completion testing. NRA SR assessment activities at Mochovce NPP were focused mainly on approving and inspecting of design modification to approving programmes for pre-completion and completion testing of system s and equipment and on approving quality assurance programmes. The suggestions of international missions, which reviewed Mochovce safety in the years, were taken into consideration in the programme