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Sample records for allosteric regulation

  1. Allosteric Regulation of Phenylalanine Hydroxylase

    OpenAIRE

    Fitzpatrick, Paul F.

    2011-01-01

    The liver enzyme phenylalanine hydroxylase is responsible for conversion of excess phenylalanine in the diet to tyrosine. Phenylalanine hydroxylase is activated by phenylalanine; this activation is inhibited by the physiological reducing substrate tetrahydrobiopterin. Phosphorylation of Ser16 lowers the concentration of phenylalanine for activation. This review discusses the present understanding of the molecular details of the allosteric regulation of the enzyme.

  2. 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...

  3. Design of an allosterically regulated retroaldolase

    Science.gov (United States)

    Raymond, Elizabeth A; Mack, Korrie L; Yoon, Jennifer H; Moroz, Olesia V; Moroz, Yurii S; Korendovych, Ivan V

    2015-01-01

    We employed a minimalist approach for design of an allosterically controlled retroaldolase. Introduction of a single lysine residue into the nonenzymatic protein calmodulin led to a 15,000-fold increase in the second order rate constant for retroaldol reaction with methodol as a substrate. The resulting catalyst AlleyCatR is active enough for subsequent directed evolution in crude cell bacterial lysates. AlleyCatR's activity is allosterically regulated by Ca2+ ions. No catalysis is observed in the absence of the metal ion. The increase in catalytic activity originates from the hydrophobic interaction of the substrate (∼2000-fold) and the change in the apparent pKa of the active lysine residue. PMID:25516403

  4. 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.

  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. 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...

  7. Binding leverage as a molecular basis for allosteric regulation.

    Directory of Open Access Journals (Sweden)

    Simon Mitternacht

    2011-09-01

    Full Text Available Allosteric regulation involves conformational transitions or fluctuations between a few closely related states, caused by the binding of effector molecules. We introduce a quantity called binding leverage that measures the ability of a binding site to couple to the intrinsic motions of a protein. We use Monte Carlo simulations to generate potential binding sites and either normal modes or pairs of crystal structures to describe relevant motions. We analyze single catalytic domains and multimeric allosteric enzymes with complex regulation. For the majority of the analyzed proteins, we find that both catalytic and allosteric sites have high binding leverage. Furthermore, our analysis of the catabolite activator protein, which is allosteric without conformational change, shows that its regulation involves other types of motion than those modulated at sites with high binding leverage. Our results point to the importance of incorporating dynamic information when predicting functional sites. Because it is possible to calculate binding leverage from a single crystal structure it can be used for characterizing proteins of unknown function and predicting latent allosteric sites in any protein, with implications for drug design.

  8. The structure and allosteric regulation of glutamate dehydrogenase.

    Science.gov (United States)

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2011-09-01

    Glutamate dehydrogenase (GDH) has been extensively studied for more than 50 years. Of particular interest is the fact that, while considered by most to be a 'housekeeping' enzyme, the animal form of GDH is heavily regulated by a wide array of allosteric effectors and exhibits extensive inter-subunit communication. While the chemical mechanism for GDH has remained unchanged through epochs of evolution, it was not clear how or why animals needed to evolve such a finely tuned form of this enzyme. As reviewed here, recent studies have begun to elucidate these issues. Allosteric regulation first appears in the Ciliates and may have arisen to accommodate evolutionary changes in organelle function. The occurrence of allosteric regulation appears to be coincident with the formation of an 'antenna' like feature rising off the tops of the subunits that may be necessary to facilitate regulation. In animals, this regulation further evolved as GDH became integrated into a number of other regulatory pathways. In particular, mutations in GDH that abrogate GTP inhibition result in dangerously high serum levels of insulin and ammonium. Therefore, allosteric regulation of GDH plays an important role in insulin homeostasis. Finally, several compounds have been identified that block GDH-mediated insulin secretion that may be to not only find use in treating these insulin disorders but to kill tumors that require glutamine metabolism for cellular energy.

  9. 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

  10. 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.

  11. 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.

  12. Glutamate dehydrogenase: structure, allosteric regulation, and role in insulin homeostasis.

    Science.gov (United States)

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2014-01-01

    Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine and inhibitors include GTP, palmitoyl CoA, and ATP. Spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds blocked the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.

  13. The structure and allosteric regulation of mammalian glutamate dehydrogenase.

    Science.gov (United States)

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2012-03-15

    Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine, while the most important inhibitors include GTP, palmitoyl CoA, and ATP. Recently, spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds were found to block the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.

  14. 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.

  15. Characteristic features of kynurenine aminotransferase allosterically regulated by (alpha-ketoglutarate in cooperation with kynurenine.

    Directory of Open Access Journals (Sweden)

    Ken Okada

    Full Text Available Kynurenine aminotransferase from Pyrococcus horikoshii OT3 (PhKAT, which is a homodimeric protein, catalyzes the conversion of kynurenine (KYN to kynurenic acid (KYNA. We analyzed the transaminase reaction mechanisms of this protein with pyridoxal-5'-phosphate (PLP, KYN and α-ketoglutaric acid (2OG or oxaloacetic acid (OXA. 2OG significantly inhibited KAT activities in kinetic analyses, suggesting that a KYNA biosynthesis is allosterically regulated by 2OG. Its inhibitions evidently were unlocked by KYN. 2OG and KYN functioned as an inhibitor and activator in response to changes in the concentrations of KYN and 2OG, respectively. The affinities of one subunit for PLP or 2OG were different from that of the other subunit, as confirmed by spectrophotometry and isothermal titration calorimetry, suggesting that the difference of affinities between subunits might play a role in regulations of the KAT reaction. Moreover, we identified two active and allosteric sites in the crystal structure of PhKAT-2OG complexes. The crystal structure of PhKAT in complex with four 2OGs demonstrates that two 2OGs in allosteric sites are effector molecules which inhibit the KYNA productions. Thus, the combined data lead to the conclusion that PhKAT probably is regulated by allosteric control machineries, with 2OG as the allosteric inhibitor.

  16. 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

  17. Allosteric Regulation of the Rotational Speed in a Light-Driven Molecular Motor

    NARCIS (Netherlands)

    Faulkner, Adele; van Leeuwen, Thomas; Feringa, Ben L; Wezenberg, Sander J

    2016-01-01

    The rotational speed of an overcrowded alkene-based molecular rotary motor, having an integrated 4,5-diazafluorenyl coordination motif, can be regulated allosterically via the binding of metal ions. DFT calculations have been used to predict the relative speed of rotation of three different (i.e. zi

  18. Organism-adapted specificity of the allosteric regulation of pyruvate kinase in lactic acid bacteria.

    Directory of Open Access Journals (Sweden)

    Nadine Veith

    Full Text Available Pyruvate kinase (PYK is a critical allosterically regulated enzyme that links glycolysis, the primary energy metabolism, to cellular metabolism. Lactic acid bacteria rely almost exclusively on glycolysis for their energy production under anaerobic conditions, which reinforces the key role of PYK in their metabolism. These organisms are closely related, but have adapted to a huge variety of native environments. They include food-fermenting organisms, important symbionts in the human gut, and antibiotic-resistant pathogens. In contrast to the rather conserved inhibition of PYK by inorganic phosphate, the activation of PYK shows high variability in the type of activating compound between different lactic acid bacteria. System-wide comparative studies of the metabolism of lactic acid bacteria are required to understand the reasons for the diversity of these closely related microorganisms. These require knowledge of the identities of the enzyme modifiers. Here, we predict potential allosteric activators of PYKs from three lactic acid bacteria which are adapted to different native environments. We used protein structure-based molecular modeling and enzyme kinetic modeling to predict and validate potential activators of PYK. Specifically, we compared the electrostatic potential and the binding of phosphate moieties at the allosteric binding sites, and predicted potential allosteric activators by docking. We then made a kinetic model of Lactococcus lactis PYK to relate the activator predictions to the intracellular sugar-phosphate conditions in lactic acid bacteria. This strategy enabled us to predict fructose 1,6-bisphosphate as the sole activator of the Enterococcus faecalis PYK, and to predict that the PYKs from Streptococcus pyogenes and Lactobacillus plantarum show weaker specificity for their allosteric activators, while still having fructose 1,6-bisphosphate play the main activator role in vivo. These differences in the specificity of allosteric

  19. Structural Basis for Allosteric Regulation of GPCRs by Sodium Ions

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Wei; Chun, Eugene; Thompson, Aaron A.; Chubukov, Pavel; Xu, Fei; Katritch, Vsevolod; Han, Gye Won; Roth, Christopher B.; Heitman, Laura H.; IJzerman, Adriaan P.; Cherezov, Vadim; Stevens, Raymond C. (Scripps); (Leiden/Amsterdam); (Receptos)

    2012-08-31

    Pharmacological responses of G protein-coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR structures. We reengineered the human A{sub 2A} adenosine receptor by replacing its third intracellular loop with apocytochrome b{sub 562}RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp{sup 2.50}. Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function.

  20. Engineering allosteric regulation into the hinge region of a circularly permuted TEM-1 beta-lactamase.

    Science.gov (United States)

    Mathieu, Valéry; Fastrez, Jacques; Soumillion, Patrice

    2010-09-01

    In nature, the activity of many enzymes involved in important biochemical pathways is controlled by binding a ligand in a site remote from the active site. The allosteric sites are frequently located in hinge regulatory subunits, in which a conformational change can occur and propagate to the active site. The enzymatic activity is then enhanced or decreased depending on the type of effectors. Many artificial binding sites have been created to engineer an allosteric regulation. Generally, these sites were engineered near the active site in loops or at the surface of contiguous helices or strands but rarely in hinge regions. This work aims at exploring the possibility of regulating a monomeric enzyme whose active site is located at the interface between two domains. We anticipated that binding of a ligand in the hinge region linking the domains would modify their positioning and, consequently, modulate the activity. Here, we describe the design of two mutants in a circularly permuted TEM-1 (cpTEM-1) beta-lactamase. The first one, cpTEM-1-His(3) was created by a rational design. It shows little regulation upon metal ion binding except for a weak activation with Zn(2+). The second one, cpTEM-1-3M-His(2), was selected by a directed evolution strategy. It is allosterically down-regulated by Zn(2+), Ni(2+) and Co(2+) with binding affinities around 300 microM.

  1. Allosteric Regulation of Unidirectional Spring-like Motion of Double-Stranded Helicates.

    Science.gov (United States)

    Suzuki, Yoshimasa; Nakamura, Taiki; Iida, Hiroki; Ousaka, Naoki; Yashima, Eiji

    2016-04-13

    We report the unprecedented allosteric regulation of the extension and contraction motions of double-stranded spiroborate helicates composed of 4,4'-linked 2,2'-bipyridine (bpy) and its N,N'-dioxide units in the middle of ortho-linked tetraphenol strands. NMR and circular dichroism measurements and an X-ray crystallographic analysis along with theoretical calculations revealed that enantiomeric helicates contract and extend upon the binding and release of protons and/or metal ions at the covalently linked two binding bpy or N,N'-dioxide moieties without racemization, respectively, regulated by a cooperative anti-syn conformational change of the two bpy or N,N'-dioxide moieties. These anti-syn conformational changes that occurred at the linkages are amplified into a large-scale molecular motion of the helicates leading to reversible spring-like motions coupled with twisting in one direction in a highly homotropic allosteric fashion. PMID:26910831

  2. The N-terminal domain allosterically regulates cleavage and activation of the epithelial sodium channel.

    Science.gov (United States)

    Kota, Pradeep; Buchner, Ginka; Chakraborty, Hirak; Dang, Yan L; He, Hong; Garcia, Guilherme J M; Kubelka, Jan; Gentzsch, Martina; Stutts, M Jackson; Dokholyan, Nikolay V

    2014-08-15

    The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr(370) in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation.

  3. The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel*

    Science.gov (United States)

    Kota, Pradeep; Buchner, Ginka; Chakraborty, Hirak; Dang, Yan L.; He, Hong; Garcia, Guilherme J. M.; Kubelka, Jan; Gentzsch, Martina; Stutts, M. Jackson; Dokholyan, Nikolay V.

    2014-01-01

    The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr370 in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation. PMID:24973914

  4. Histone tails regulate DNA methylation by allosterically activating de novo methyltransferase

    Institute of Scientific and Technical Information of China (English)

    Bin-Zhong Li; Guo-Liang Xu; Zheng Huang; Qing-Yan Cui; Xue-Hui Song; Lin Du; Albert Jeltsch; Ping Chen; Guohong Li; En Li

    2011-01-01

    Cytosine methylation of genomic DNA controls gene expression and maintains genome stability. How a specific DNA sequence is targeted for methylation by a methyltransferase is largely unknown. Here, we show that histone H3 tails lacking lysine 4 (K4) methylation function as an allosteric activator for methyltransferase Dnmt3a by binding to its plant homeodomain (PHD). In vitro, histone H3 peptides stimulated the methylation activity of Dnmt3a up to 8-fold, in a manner reversely correlated with the level of K4 methylation. The biological significance of allosteric regulation was manifested by molecular modeling and identification of key residues in both the PHD and the catalytic domain of Dnmt3a whose mutations impaired the stimulation of methylation activity by H3 peptides but not the binding of H3 peptides. Significantly, these mutant Dnmt3a proteins were almost inactive in DNA methylation when expressed in mouse embryonic stem cells while their recruitment to genomic targets was unaltered. We therefore propose a two-step mechanism for de novo DNA methylation - first recruitment of the methyltransferase probably assisted by a chromatin- or DNA-binding factor, and then allosteric activation depending on the interaction between Dnmt3a and the histone tails - the latter might serve as a checkpoint for the methylation activity.

  5. Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone.

    Science.gov (United States)

    Steinchen, Wieland; Schuhmacher, Jan S; Altegoer, Florian; Fage, Christopher D; Srinivasan, Vasundara; Linne, Uwe; Marahiel, Mohamed A; Bange, Gert

    2015-10-27

    Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named "(p)ppGpp"] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) from Bacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (SN2) reaction. We show that pppGpp-but not ppGpp-positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles.

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

    Science.gov (United States)

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

    2014-10-27

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

  7. 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.

  8. 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

  9. 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

  10. Molecular kinetics. Ras activation by SOS: allosteric regulation by altered fluctuation dynamics.

    Science.gov (United States)

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

    2014-07-01

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

  11. 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.

  12. 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

  13. 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...

  14. 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

  15. 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...

  16. Bacterial rotary export ATPases are allosterically regulated by the nucleotide second messenger cyclic-di-GMP.

    Science.gov (United States)

    Trampari, Eleftheria; Stevenson, Clare E M; Little, Richard H; Wilhelm, Thomas; Lawson, David M; Malone, Jacob G

    2015-10-01

    The widespread second messenger molecule cyclic di-GMP (cdG) regulates the transition from motile and virulent lifestyles to sessile, biofilm-forming ones in a wide range of bacteria. Many pathogenic and commensal bacterial-host interactions are known to be controlled by cdG signaling. Although the biochemistry of cyclic dinucleotide metabolism is well understood, much remains to be discovered about the downstream signaling pathways that induce bacterial responses upon cdG binding. As part of our ongoing research into the role of cdG signaling in plant-associated Pseudomonas species, we carried out an affinity capture screen for cdG binding proteins in the model organism Pseudomonas fluorescens SBW25. The flagella export AAA+ ATPase FliI was identified as a result of this screen and subsequently shown to bind specifically to the cdG molecule, with a KD in the low micromolar range. The interaction between FliI and cdG appears to be very widespread. In addition to FliI homologs from diverse bacterial species, high affinity binding was also observed for the type III secretion system homolog HrcN and the type VI ATPase ClpB2. The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro. Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits. Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins.

  17. 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

  18. 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.

  19. Controlling allosteric networks in proteins

    Science.gov (United States)

    Dokholyan, Nikolay

    2013-03-01

    We present a novel methodology based on graph theory and discrete molecular dynamics simulations for delineating allosteric pathways in proteins. We use this methodology to uncover the structural mechanisms responsible for coupling of distal sites on proteins and utilize it for allosteric modulation of proteins. We will present examples where inference of allosteric networks and its rewiring allows us to ``rescue'' cystic fibrosis transmembrane conductance regulator (CFTR), a protein associated with fatal genetic disease cystic fibrosis. We also use our methodology to control protein function allosterically. We design a novel protein domain that can be inserted into identified allosteric site of target protein. Using a drug that binds to our domain, we alter the function of the target protein. We successfully tested this methodology in vitro, in living cells and in zebrafish. We further demonstrate transferability of our allosteric modulation methodology to other systems and extend it to become ligh-activatable.

  20. 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.

  1. Allosterically Regulated Phosphatase Activity from Peptide-PNA Conjugates Folded Through Hybridization.

    Science.gov (United States)

    Machida, Takuya; Dutt, Som; Winssinger, Nicolas

    2016-07-18

    The importance of spatial organization in short peptide catalysts is well recognized. We synthesized and screened a library of peptides flanked by peptide nucleic acids (PNAs) such that the peptide would be constrained in a hairpin loop upon hybridization. A screen for phosphatase activity led to the discovery of a catalyst with >25-fold rate acceleration over the linear peptide. We demonstrated that the hybridization-enforced folding of the peptide is necessary for activity, and designed a catalyst that is allosterically controlled using a complementary PNA sequence. PMID:27320214

  2. 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

  3. 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.

  4. 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.

  5. Allosteric Regulation of Serine Protease HtrA2 through Novel Non-Canonical Substrate Binding Pocket

    Science.gov (United States)

    Singh, Nitu; Gadewal, Nikhil; Chaganti, Lalith K.; Sastry, G. Madhavi; Bose, Kakoli

    2013-01-01

    HtrA2, a trimeric proapoptotic serine protease is involved in several diseases including cancer and neurodegenerative disorders. Its unique ability to mediate apoptosis via multiple pathways makes it an important therapeutic target. In HtrA2, C-terminal PDZ domain upon substrate binding regulates its functions through coordinated conformational changes the mechanism of which is yet to be elucidated. Although allostery has been found in some of its homologs, it has not been characterized in HtrA2 so far. Here, with an in silico and biochemical approach we have shown that allostery does regulate HtrA2 activity. Our studies identified a novel non-canonical selective binding pocket in HtrA2 which initiates signal propagation to the distal active site through a complex allosteric mechanism. This non-classical binding pocket is unique among HtrA family proteins and thus unfolds a novel mechanism of regulation of HtrA2 activity and hence apoptosis. PMID:23457469

  6. Allosteric regulation of serine protease HtrA2 through novel non-canonical substrate binding pocket.

    Directory of Open Access Journals (Sweden)

    Pruthvi Raj Bejugam

    Full Text Available HtrA2, a trimeric proapoptotic serine protease is involved in several diseases including cancer and neurodegenerative disorders. Its unique ability to mediate apoptosis via multiple pathways makes it an important therapeutic target. In HtrA2, C-terminal PDZ domain upon substrate binding regulates its functions through coordinated conformational changes the mechanism of which is yet to be elucidated. Although allostery has been found in some of its homologs, it has not been characterized in HtrA2 so far. Here, with an in silico and biochemical approach we have shown that allostery does regulate HtrA2 activity. Our studies identified a novel non-canonical selective binding pocket in HtrA2 which initiates signal propagation to the distal active site through a complex allosteric mechanism. This non-classical binding pocket is unique among HtrA family proteins and thus unfolds a novel mechanism of regulation of HtrA2 activity and hence apoptosis.

  7. 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

  8. 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

  9. GPCR二聚体别构调节的药理学作用%The Pharmacological Effect of Allosteric Regulation at GPCR Heterodimer

    Institute of Scientific and Technical Information of China (English)

    张宁; 陈京; 龚磊; 刘路路; 白波

    2013-01-01

    传统的观点认为G蛋白偶联受体(G protein coupled receptor,GPCR)主要以单体形式存在,其作用是线性的,即配体结合到正位作用位点来引起信号下游传导.但大量事实证明,GPCR也能以同源或异源二聚体的形式存在.在二聚体中,由于配体结合到二聚体中的一个单体而引起对另一个单体的别构调节,从而形成别构位点,使受体的信号途径发生改变,引起一系列功能变化.别构调节剂与传统的激动剂相比有许多优点,因此是重要的GPCR靶标的候选药物.本文就GPCR二聚体的别构调节对受体功能的影响,以及筛选GPCR二聚体别构药物的技术做一简要综述,从而有助于GPCR药物的开发和利用.%Traditionally GPCR can exist as monomer, and its function is linear. The signal is transduced when the ligand binds to the orthosteric site. It has been reported that GPCR can form homodimer or heterodimer. In the dimers, allosteric sites will form when ligand binds to one monomer and then regulates of another monomer. Therefore the signaling pathway and the function of the receptor will be changed. Compared to orthosteric agonists, allosteric modulators have a number of potential advantages, which make allosteric modulators as a drug discovery candidate for GPCR. Herein, we introduce the concept of allosteric regulation at GPCR dimers, and its impact on the function of the receptor. We also introduce the drug screening methods of allosteric regulator, and those will contribute to drug discovery for GPCR.

  10. 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

  11. 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

  12. Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

    Science.gov (United States)

    Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L; Kirk, Kevin L

    2014-07-18

    ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs.

  13. 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

  14. 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...

  15. 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

    crystal X-ray diffraction methods and in dichloromethane solution by means of continuous variation plots and UV-vis spectroscopic titrations. These analyses revealed a 1:1 stoichiometry between the anion-bound TTF-C4Ps and the complexed ftillerenes. The latter guests are bound within the bowl-like cup...... 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...

  16. Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli.

    Science.gov (United States)

    Chen, Lin; Chen, Zhen; Zheng, Ping; Sun, Jibin; Zeng, An-Ping

    2013-04-01

    Biosynthetic threonine deaminase (TD) is a key enzyme for the synthesis of isoleucine which is allosterically inhibited and activated by Ile and Val, respectively. The binding sites of Ile and Val and the mechanism of their regulations in TD are not clear, but essential for a rational design of efficient productive strain(s) for Ile and related amino acids. In this study, structure-based computational approach and site-directed mutagenesis were combined to identify the potential binding sites of Ile and Val in Escherichia coli TD. Our results demonstrated that each regulatory domain of the TD monomer possesses two nonequivalent effector-binding sites. The residues R362, E442, G445, A446, Y369, I460, and S461 only interact with Ile while E347, G350, and F352 are involved not only in the Ile binding but also in the Val binding. By further considering enzyme kinetic data, we propose a concentration-dependent mechanism of the allosteric regulation of TD by Ile and Val. For the construction of Ile overproducing strain, a novel TD mutant with double mutation of F352A/R362F was also created, which showed both higher activity and much stronger resistance to Ile inhibition comparing to those of wild-type enzyme. Overexpression of this mutant TD in E. coli JW3591 significantly increased the production of ketobutyrate and Ile in comparison to the reference strains overexpressing wild-type TD or the catabolic threonine deaminase (TdcB). This work builds a solid basis for the reengineering of TD and related microorganisms for Ile production.

  17. Allosteric regulation of the state of adenylylation of glutamine synthetase in permeabilized cell preparations of Rhodopseudomonas sphaeroides.

    Science.gov (United States)

    Wang, X; Song, H Y

    1989-08-01

    Following a freeze-thaw cycle, and the treatment of Rhodopseudomonas sphaeroides with the nonionic detergent Lubrol PX, the permeabilized cell suspensions can be assayed directly both for the intracellular levels of glutamine synthetase and the state of adenylylation (i.e. the average number n of adenylylated subunits/dodecameric molecules). It seems that all components of the bicycle system are retained if cells grown with low concentrations of ammonia as the sole nitrogen source are used. The value of n was dependent upon the concentration of substrates (ATP, Pi) and allosteric effectors (ATP, glutamine and alpha-ketoglutarate) of adenylytransferase. The value of n affected by UTP, the specific substrate of the uridylyltransferase shows first the evidence that the bicycle cascade control system studied in Escherichia coli may exist in this phototrophic bacterium. PMID:2575389

  18. [Allosteric regulation of glucosamine synthetase activity by naphthoquinone derivatives and ethyl ester of di-(4-oxycumarinyl-3)-acetic acid].

    Science.gov (United States)

    Sharaev, P N; Bogdanov, N G; Sarycheva, I K; Zhukova, E E

    1981-02-01

    The effects of derivatives of naphthoquinone, e.g. 2-methyl-3-phytyl-1,4-naphthoquinone (vitamin K1), 2-methyl-1,4-naphthoquinone (vitamin K3), 3-dihydro-2-methyl-1,4-naphthoquinone-2-sodium sulfonate (vicasol), derivatives of naphthohydroxyquinone, e.g. 2-methyl-1,4-naphthohydroxyquinone 1-monoacetate, 2-methyl-1,4-naphthohydroxyquinone 1,4-diacetate and the oxycumarine derivative di-(4-oxycumarinyl-3)-acetate ethyl ester (pelentan) on the activity of purified glutamine synthetase (EC 5.3.1.19) from rat liver were studied. The enzyme activity was increased under effects of vitamins K1 and K3 and was inhibited by pelentan. The data obtained are indicative of the allosteric effect of these compounds on the enzyme. PMID:7195738

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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;

    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...

  4. Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

    Science.gov (United States)

    Sato, Shogo; Jung, Hunmin; Nakagawa, Tsutomu; Pawlosky, Robert; Takeshima, Tomomi; Lee, Wan-Ru; Sakiyama, Haruhiko; Laxman, Sunil; Wynn, R Max; Tu, Benjamin P; MacMillan, John B; De Brabander, Jef K; Veech, Richard L; Uyeda, Kosaku

    2016-05-13

    The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis. PMID:26984404

  5. Calculated pKa Variations Expose Dynamic Allosteric Communication Networks.

    Science.gov (United States)

    Lang, Eric J M; Heyes, Logan C; Jameson, Geoffrey B; Parker, Emily J

    2016-02-17

    Allosteric regulation of protein function, the process by which binding of an effector molecule provokes a functional response from a distal site, is critical for metabolic pathways. Yet, the way the allosteric signal is communicated remains elusive, especially in dynamic, entropically driven regulation mechanisms for which no major conformational changes are observed. To identify these dynamic allosteric communication networks, we have developed an approach that monitors the pKa variations of ionizable residues over the course of molecular dynamics simulations performed in the presence and absence of an allosteric regulator. As the pKa of ionizable residues depends on their environment, it represents a simple metric to monitor changes in several complex factors induced by binding an allosteric effector. These factors include Coulombic interactions, hydrogen bonding, and solvation, as well as backbone motions and side chain fluctuations. The predictions that can be made with this method concerning the roles of ionizable residues for allosteric communication can then be easily tested experimentally by changing the working pH of the protein or performing single point mutations. To demonstrate the method's validity, we have applied this approach to the subtle dynamic regulation mechanism observed for Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first enzyme of aromatic biosynthesis. We were able to identify key communication pathways linking the allosteric binding site to the active site of the enzyme and to validate these findings experimentally by reestablishing the catalytic activity of allosterically inhibited enzyme via modulation of the working pH, without compromising the binding affinity of the allosteric regulator.

  6. Structural basis for morpheein-type allosteric regulation of Escherichia coli glucosamine-6-phosphate synthase: equilibrium between inactive hexamer and active dimer.

    Science.gov (United States)

    Mouilleron, Stéphane; Badet-Denisot, Marie-Ange; Pecqueur, Ludovic; Madiona, Karine; Assrir, Nadine; Badet, Bernard; Golinelli-Pimpaneau, Béatrice

    2012-10-01

    The amino-terminal cysteine of glucosamine-6-phosphate synthase (GlmS) acts as a nucleophile to release and transfer ammonia from glutamine to fructose 6-phosphate through a channel. The crystal structure of the C1A mutant of Escherichia coli GlmS, solved at 2.5 Å resolution, is organized as a hexamer, where the glutaminase domains adopt an inactive conformation. Although the wild-type enzyme is active as a dimer, size exclusion chromatography, dynamic and quasi-elastic light scattering, native polyacrylamide gel electrophoresis, and ultracentrifugation data show that the dimer is in equilibrium with a hexameric state, in vitro and in cellulo. The previously determined structures of the wild-type enzyme, alone or in complex with glucosamine 6-phosphate, are also consistent with a hexameric assembly that is catalytically inactive because the ammonia channel is not formed. The shift of the equilibrium toward the hexameric form in the presence of cyclic glucosamine 6-phosphate, together with the decrease of the specific activity with increasing enzyme concentration, strongly supports product inhibition through hexamer stabilization. Altogether, our data allow us to propose a morpheein model, in which the active dimer can rearrange into a transiently stable form, which has the propensity to form an inactive hexamer. This would account for a physiologically relevant allosteric regulation of E. coli GlmS. Finally, in addition to cyclic glucose 6-phosphate bound at the active site, the hexameric organization of E. coli GlmS enables the binding of another linear sugar molecule. Targeting this sugar-binding site to stabilize the inactive hexameric state is therefore suggested for the development of specific antibacterial inhibitors.

  7. 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

  8. Mechanistic insight into the conserved allosteric regulation of periplasmic proteolysis by the signaling molecule cyclic-di-GMP.

    Science.gov (United States)

    Chatterjee, Debashree; Cooley, Richard B; Boyd, Chelsea D; Mehl, Ryan A; O'Toole, George A; Sondermann, Holger

    2014-01-01

    Stable surface adhesion of cells is one of the early pivotal steps in bacterial biofilm formation, a prevalent adaptation strategy in response to changing environments. In Pseudomonas fluorescens, this process is regulated by the Lap system and the second messenger cyclic-di-GMP. High cytoplasmic levels of cyclic-di-GMP activate the transmembrane receptor LapD that in turn recruits the periplasmic protease LapG, preventing it from cleaving a cell surface-bound adhesin, thereby promoting cell adhesion. In this study, we elucidate the molecular basis of LapG regulation by LapD and reveal a remarkably sensitive switching mechanism that is controlled by LapD's HAMP domain. LapD appears to act as a coincidence detector, whereby a weak interaction of LapG with LapD transmits a transient outside-in signal that is reinforced only when cyclic-di-GMP levels increase. Given the conservation of key elements of this receptor system in many bacterial species, the results are broadly relevant for cyclic-di-GMP- and HAMP domain-regulated transmembrane signaling.

  9. Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1

    Science.gov (United States)

    Harrison, Joseph S; Cornett, Evan M; Goldfarb, Dennis; DaRosa, Paul A; Li, Zimeng M; Yan, Feng; Dickson, Bradley M; Guo, Angela H; Cantu, Daniel V; Kaustov, Lilia; Brown, Peter J; Arrowsmith, Cheryl H; Erie, Dorothy A; Major, Michael B; Klevit, Rachel E; Krajewski, Krzysztof; Kuhlman, Brian; Strahl, Brian D; Rothbart, Scott B

    2016-01-01

    The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance. DOI: http://dx.doi.org/10.7554/eLife.17101.001 PMID:27595565

  10. Down-regulation of PAR1 activity with a pHLIP-based allosteric antagonist induces cancer cell death.

    Science.gov (United States)

    Burns, Kelly E; Thévenin, Damien

    2015-12-15

    Even though abnormal expression of G protein-coupled receptors (GPCRs) and of their ligands is observed in many cancer cells of various origins, only a few anti-cancer compounds directly act on their signalling. One promising approach to modulate their activity consists of targeting the receptor cytoplasmic surfaces interacting with the associated G-proteins using peptides mimicking the intracellular loops of the receptor. Thus, to be fully effective, the peptide mimics must be selectively targeted to the tumour while sparing healthy tissues, translocated across the cell membrane and stay anchored to the cytoplasmic leaflet of the plasma membrane. In the present study, we introduce a novel way to selectively target and inhibit the activity of a GPCR in cancer cells under acidic conditions, such as those found in solid tumours. We find that the conjugation of a peptide fragment derived from the third intracellular loop (i3) of the protease-activated receptor 1 (PAR1) to a peptide that can selectively target tumours solely based on their acidity [pH(Low) Insertion Peptide (pHLIP)], produces a construct capable of effectively down-regulating PAR1 activity in a concentration- and pH-dependent manner and of inducing a potent cytotoxic effect in a panel of cancer cells that is proportional to the relative level of receptor expression at the cell surface. This strategy not only allows for a more selective targeting and specific intracellular delivery than current approaches, but also offers new possibilities for developing novel anti-cancer drugs targeting GPCRs. PMID:26424552

  11. Detecting Allosteric Networks Using Molecular Dynamics Simulation.

    Science.gov (United States)

    Bowerman, S; Wereszczynski, J

    2016-01-01

    Allosteric networks allow enzymes to transmit information and regulate their catalytic activities over vast distances. In principle, molecular dynamics (MD) simulations can be used to reveal the mechanisms that underlie this phenomenon; in practice, it can be difficult to discern allosteric signals from MD trajectories. Here, we describe how MD simulations can be analyzed to reveal correlated motions and allosteric networks, and provide an example of their use on the coagulation enzyme thrombin. Methods are discussed for calculating residue-pair correlations from atomic fluctuations and mutual information, which can be combined with contact information to identify allosteric networks and to dynamically cluster a system into highly correlated communities. In the case of thrombin, these methods show that binding of the antagonist hirugen significantly alters the enzyme's correlation landscape through a series of pathways between Exosite I and the catalytic core. Results suggest that hirugen binding curtails dynamic diversity and enforces stricter venues of influence, thus reducing the accessibility of thrombin to other molecules. PMID:27497176

  12. Computation of conformational coupling in allosteric proteins.

    Directory of Open Access Journals (Sweden)

    Brian A Kidd

    2009-08-01

    Full Text Available In allosteric regulation, an effector molecule binding a protein at one site induces conformational changes, which alter structure and function at a distant active site. Two key challenges in the computational modeling of allostery are the prediction of the structure of one allosteric state starting from the structure of the other, and elucidating the mechanisms underlying the conformational coupling of the effector and active sites. Here we approach these two challenges using the Rosetta high-resolution structure prediction methodology. We find that the method can recapitulate the relaxation of effector-bound forms of single domain allosteric proteins into the corresponding ligand-free states, particularly when sampling is focused on regions known to change conformation most significantly. Analysis of the coupling between contacting pairs of residues in large ensembles of conformations spread throughout the landscape between and around the two allosteric states suggests that the transitions are built up from blocks of tightly coupled interacting sets of residues that are more loosely coupled to one another.

  13. 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...

  14. 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

  15. 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

  16. Adenine nucleotides as allosteric effectors of pea seed glutamine synthetase.

    Science.gov (United States)

    Knight, T J; Langston-Unkefer, P J

    1988-08-15

    The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observed in the presence of added ADP than the Vmax observed in the absence of ADP. Hill plots of data with ADP present curved upward and crossed the plot with no added ADP. The stoichiometry of adenine nucleotide binding to glutamine synthetase was examined. Two molecules of [gamma-32P]ATP were bound per subunit in the presence of methionine sulfoximine. These ATP molecules were bound at an allosteric site and at the active site. One molecule of either [gamma-32P]ATP or [14C]ADP bound per subunit in the absence of methionine sulfoximine; this nucleotide was bound at an allosteric site. ADP and ATP compete for binding at the allosteric site, although ADP was preferred. ADP binding to the allosteric site proceeded in two kinetic phases. A Vmax value of 1.55 units/mg was measured for glutamine synthetase with one ADP tightly bound per enzyme subunit; a Vmax value of 0.8 unit/mg was measured for enzyme with no adenine nucleotide bound at the allosteric site. The enzyme activation caused by the binding of ADP to the allosteric sites was preceded by a lag phase, the length of which was dependent on the ADP concentration. Enzyme incubated in 10 mM ADP bound approximately 4 mol of ADP/mol of native enzyme before activation was observed; the activation was complete when 7-8 mol of ADP were bound per mol of the octameric, native enzyme. The Km for ATP (2 mM) was not changed by ADP binding to the allosteric sites. ADP was a simple competitive inhibitor (Ki = 0.05 mM) of ATP for glutamine synthetase with eight molecules of ADP tightly bound to the allosteric sites of the octamer. Binding of ATP to the allosteric sites led to marked

  17. Allosteric small-molecule kinase inhibitors

    DEFF Research Database (Denmark)

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

    2015-01-01

    -molecule allosteric inhibitor trametinib in 2013, the progress of more than 10 other allosteric inhibitors in clinical trials, and the emergence of a pipeline of highly selective and potent preclinical molecules, have been reported in the past decade. In this article, we present the current knowledge on allosteric...

  18. 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...

  19. An allosteric photoredox catalyst inspired by photosynthetic machinery.

    Science.gov (United States)

    Lifschitz, Alejo M; Young, Ryan M; Mendez-Arroyo, Jose; Stern, Charlotte L; McGuirk, C Michael; Wasielewski, Michael R; Mirkin, Chad A

    2015-03-30

    Biological photosynthetic machinery allosterically regulate light harvesting via conformational and electronic changes at the antenna protein complexes as a response to specific chemical inputs. Fundamental limitations in current approaches to regulating inorganic light-harvesting mimics prevent their use in catalysis. Here we show that a light-harvesting antenna/reaction centre mimic can be regulated by utilizing a coordination framework incorporating antenna hemilabile ligands and assembled via a high-yielding, modular approach. As in nature, allosteric regulation is afforded by coupling the conformational changes to the disruptions in the electrochemical landscape of the framework upon recognition of specific coordinating analytes. The hemilabile ligands enable switching using remarkably mild and redox-inactive inputs, allowing one to regulate the photoredox catalytic activity of the photosynthetic mimic reversibly and in situ. Thus, we demonstrate that bioinspired regulatory mechanisms can be applied to inorganic light-harvesting arrays displaying switchable catalytic properties and with potential uses in solar energy conversion and photonic devices.

  20. Site-directed mutagenesis of the regulatory domain of Escherichia coli carbamoyl phosphate synthetase identifies crucial residues for allosteric regulation and for transduction of the regulatory signals.

    Science.gov (United States)

    Fresquet, V; Mora, P; Rochera, L; Ramón-Maiques, S; Rubio, V; Cervera, J

    2000-06-16

    Carbamoyl phosphate (CP), the essential precursor of pyrimidines and arginine, is made in Escherichia coli by a single carbamoyl phosphate synthetase (CPS) consisting of 41.4 and 117.7 kDa subunits, which is feed-back inhibited by UMP and activated by IMP and ornithine. The large subunit catalyzes CP synthesis from ammonia in three steps, and binds the effectors in its 15 kDa C-terminal domain. Fifteen site-directed mutations were introduced in 13 residues of this domain to investigate the mechanism of allosteric modulation by UMP and IMP. Two mutations, K993A and V994A, decreased significantly or abolished enzyme activity, apparently by interfering with the step of carbamate synthesis, and one mutation, T974A, negatively affected ornithine activation. S948A, K954A, T974A, K993A and K993W/H995A abolished or greatly hampered IMP activation and UMP inhibition as well as the binding of both effectors, monitored using photoaffinity labeling and ultracentrifugation binding assays. V994A also decreased significantly IMP and UMP binding. L990A, V991A, H995A, G997A and G1008A had more modest effects or affected more the modulation by and the binding of one than of the other nucleotide. K993W, R1020A, R1021A and K1061A were without substantial effects. The results confirm the independence of the regulatory and catalytic centers, and also confirm functional predictions based on the X-ray structure of an IMP-CPS complex. They prove that the inhibitor UMP and the activator IMP bind in the same site, and exclude that the previously observed binding of ornithine and glutamine in this site were relevant for enzyme activation. K993 and V994 appear to be involved in the transmission of the regulatory signals triggered by UMP and IMP binding. These effectors possibly change the position of K993 and V994, and alter the intermolecular contacts mediated by the regulatory domain. PMID:10843852

  1. Untangling the glutamate dehydrogenase allosteric nightmare.

    Science.gov (United States)

    Smith, Thomas J; Stanley, Charles A

    2008-11-01

    Glutamate dehydrogenase (GDH) is found in all living organisms, but only animal GDH is regulated by a large repertoire of metabolites. More than 50 years of research to better understand the mechanism and role of this allosteric network has been frustrated by its sheer complexity. However, recent studies have begun to tease out how and why this complex behavior evolved. Much of GDH regulation probably occurs by controlling a complex ballet of motion necessary for catalytic turnover and has evolved concomitantly with a long antenna-like feature of the structure of the enzyme. Ciliates, the 'missing link' in GDH evolution, might have created the antenna to accommodate changing organelle functions and was refined in humans to, at least in part, link amino acid catabolism with insulin secretion.

  2. 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.

  3. Allosteric regulation of protein kinase PKCζ by the N-terminal C1 domain and small compounds to the PIF-pocket

    DEFF Research Database (Denmark)

    Lopez-Garcia, Laura A; Schulze, Jörg O; Fröhner, Wolfgang;

    2011-01-01

    Protein kinases are key mediators of cellular signaling, and therefore, their activities are tightly controlled. AGC kinases are regulated by phosphorylation and by N- and C-terminal regions. Here, we studied the molecular mechanism of inhibition of atypical PKCζ and found that the inhibition by ...

  4. Type I and II positive allosteric modulators differentially modulate agonist-induced up-regulation of α7 nicotinic acetylcholine receptors

    DEFF Research Database (Denmark)

    Thomsen, Morten Skøtt; Mikkelsen, Jens D

    2012-01-01

    Long-term treatment with nicotine or selective α7 nicotinic acetylcholine receptor (nAChR) agonists increases the number of α7 nAChRs and this up-regulation may be involved in the mechanism underlying the sustained procognitive effect of these compounds. Here, we investigate the influence of type I...

  5. 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...

  6. 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

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

    DEFF Research Database (Denmark)

    Schwartz, Thue W; Holst, Birgitte

    2007-01-01

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

  8. Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor.

    Science.gov (United States)

    Kahsai, Alem W; Wisler, James W; Lee, Jungmin; Ahn, Seungkirl; Cahill Iii, Thomas J; Dennison, S Moses; Staus, Dean P; Thomsen, Alex R B; Anasti, Kara M; Pani, Biswaranjan; Wingler, Laura M; Desai, Hemant; Bompiani, Kristin M; Strachan, Ryan T; Qin, Xiaoxia; Alam, S Munir; Sullenger, Bruce A; Lefkowitz, Robert J

    2016-09-01

    G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies the ability of 'biased agonists' to activate specific subsets of a given receptor's signaling profile. However, stabilizing distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function through unique, previously unappreciated mechanisms. Here, using a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, the β2-adrenoceptor (β2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs. PMID:27398998

  9. Unraveling structural mechanisms of allosteric drug action.

    Science.gov (United States)

    Nussinov, Ruth; Tsai, Chung-Jung

    2014-05-01

    Orthosteric drugs block the active site to obstruct function; allosteric drugs modify the population of the active state, to modulate function. Available data lead us to propose that allosteric drugs can constitute anchors and drivers. The anchor docks into an allosteric pocket. The conformation with which it interacts is unchanged during the transition between the inactive and active states. The anchor provides the foundation that allows the driver to exert a 'pull' and/or 'push' action that shifts the receptor population from the inactive to the active state. The presence or absence of driver atom in an allosteric drug can exert opposite agonism. We map a strategy for driver identification and expect the allosteric trigger concept to transform agonist/antagonist drug discovery.

  10. Dissection of the conduit for allosteric control of carbamoyl phosphate synthetase by ornithine.

    Science.gov (United States)

    Pierrat, Olivier A; Javid-Majd, Farah; Raushel, Frank M

    2002-04-01

    Ornithine is an allosteric activator of carbamoyl phosphate synthetase (CPS) from Escherichia coli. Nine amino acids in the vicinity of the binding sites for ornithine and potassium were mutated to alanine, glutamine, or lysine. The residues E783, T1042, and T1043 were found to be primarily responsible for the binding of ornithine to CPS, while E783 and E892, located within the carbamate domain of the large subunit, were necessary for the transmission of the allosteric signals to the active site. In the K loop for the binding of the monovalent cation potassium, only E761 was crucial for the exhibition of the allosteric effects of ornithine, UMP, and IMP. The mutations H781K and S792K altered significantly the allosteric properties of ornithine, UMP, and IMP, possibly by modifying the conformation of the K-loop structure. Overall, these mutations affected the allosteric properties of ornithine and IMP more than those of UMP. The mutants S792K and D1041A altered the allosteric regulation by ornithine and IMP in a similar way, suggesting common features in the activation mechanism exhibited by these two effectors. PMID:11913967

  11. 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

  12. 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.

  13. 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.

    2015-01-01

    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. PMID:26558346

  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

    Directory of Open Access Journals (Sweden)

    Francisco Andrés Peralta

    2016-07-01

    Full Text Available 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.

  16. 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

  17. 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.

  18. Chemogenomics of allosteric binding sites in GPCRs

    DEFF Research Database (Denmark)

    Gloriam, David E.

    2013-01-01

    profiling. This review describes recent developments structured into ligand-, target- and combined chemogenomic techniques and applications to allosteric GPCR ligands. It also outlines relative strengths and limitations of these techniques and the impact of the increasing crystallographic data....

  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. PMID:25600932

  20. Coherent conformational degrees of freedom as a structural basis for allosteric communication.

    Directory of Open Access Journals (Sweden)

    Simon Mitternacht

    2011-12-01

    Full Text Available Conformational changes in allosteric regulation can to a large extent be described as motion along one or a few coherent degrees of freedom. The states involved are inherent to the protein, in the sense that they are visited by the protein also in the absence of effector ligands. Previously, we developed the measure binding leverage to find sites where ligand binding can shift the conformational equilibrium of a protein. Binding leverage is calculated for a set of motion vectors representing independent conformational degrees of freedom. In this paper, to analyze allosteric communication between binding sites, we introduce the concept of leverage coupling, based on the assumption that only pairs of sites that couple to the same conformational degrees of freedom can be allosterically connected. We demonstrate how leverage coupling can be used to analyze allosteric communication in a range of enzymes (regulated by both ligand binding and post-translational modifications and huge molecular machines such as chaperones. Leverage coupling can be calculated for any protein structure to analyze both biological and latent catalytic and regulatory sites.

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

    Directory of Open Access Journals (Sweden)

    Julien F Ollivier

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

  2. 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.

  3. 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.

    2016-08-01

    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.

  4. Molecular basis of positive allosteric modulation of GluN2B NMDA receptors by polyamines.

    Science.gov (United States)

    Mony, Laetitia; Zhu, Shujia; Carvalho, Stéphanie; Paoletti, Pierre

    2011-06-17

    NMDA receptors (NMDARs) form glutamate-gated ion channels that have central roles in neuronal communication and plasticity throughout the brain. Dysfunctions of NMDARs are involved in several central nervous system disorders, including stroke, chronic pain and schizophrenia. One hallmark of NMDARs is that their activity can be allosterically regulated by a variety of extracellular small ligands. While much has been learned recently regarding allosteric inhibition of NMDARs, the structural determinants underlying positive allosteric modulation of these receptors remain poorly defined. Here, we show that polyamines, naturally occurring polycations that selectively enhance NMDARs containing the GluN2B subunit, bind at a dimer interface between GluN1 and GluN2B subunit N-terminal domains (NTDs). Polyamines act by shielding negative charges present on GluN1 and GluN2B NTD lower lobes, allowing their close apposition, an effect that in turn prevents NTD clamshell closure. Our work reveals the mechanistic basis for positive allosteric modulation of NMDARs. It provides the first example of an intersubunit binding site in this class of receptors, a discovery that holds promise for future drug interventions.

  5. 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

  6. 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)

  7. Rational design of allosteric-inhibition sites in classical protein tyrosine phosphatases

    Science.gov (United States)

    Chio, Cynthia M.; Yu, Xiaoling; Bishop, Anthony C.

    2015-01-01

    Protein tyrosine phosphatases (PTPs), which catalyze the dephosphorylation of phosphotyrosine in protein substrates, are critical regulators of metazoan cell signaling and have emerged as potential drug targets for a range of human diseases. Strategies for chemically targeting the function of individual PTPs selectively could serve to elucidate the signaling roles of these enzymes and would potentially expedite validation of the therapeutic promise of PTP inhibitors. Here we report a novel strategy for the design of non-natural allosteric-inhibition sites in PTPs; these sites, which can be introduced into target PTPs through protein engineering, serve to sensitize target PTPs to potent and selective inhibition by a biarsenical small molecule. Building on the recent discovery of a naturally occurring cryptic allosteric site in wild-type Src-homology-2 domain containing PTP (Shp2) that can be targeted by biarsenical compounds, we hypothesized that Shp2’s unusual sensitivity to biarsenicals could be strengthened through rational design and that the Shp2-specific site could serve as a blueprint for the introduction of non-natural inhibitor sensitivity in other PTPs. Indeed, we show here that the strategic introduction of a cysteine residue at a position removed from the Shp2 active site can serve to increase the potency and selectivity of the interaction between Shp2’s allosteric site and the biarsenical inhibitor. Moreover, we find that “Shp2-like” allosteric sites can be installed de novo in PTP enzymes that do not possess naturally occurring sensitivity to biarsenical compounds. Using primary-sequence alignments to guide our enzyme engineering, we have successfully introduced allosteric-inhibition sites in four classical PTPs—PTP1B, PTPH-1, FAP-1, and HePTP—from four different PTP subfamilies, suggesting that our sensitization approach can likely be applied widely across the classical PTP family to generate biarsenical-responsive PTPs. PMID:25828055

  8. 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...

  9. Allosteric control in a metalloprotein dramatically alters function.

    Science.gov (United States)

    Baxter, Elizabeth Leigh; Zuris, John A; Wang, Charles; Vo, Phu Luong T; Axelrod, Herbert L; Cohen, Aina E; Paddock, Mark L; Nechushtai, Rachel; Onuchic, Jose N; Jennings, Patricia A

    2013-01-15

    Metalloproteins (MPs) comprise one-third of all known protein structures. This diverse set of proteins contain a plethora of unique inorganic moieties capable of performing chemistry that would otherwise be impossible using only the amino acids found in nature. Most of the well-studied MPs are generally viewed as being very rigid in structure, and it is widely thought that the properties of the metal centers are primarily determined by the small fraction of amino acids that make up the local environment. Here we examine both theoretically and experimentally whether distal regions can influence the metal center in the diabetes drug target mitoNEET. We demonstrate that a loop (L2) 20 Å away from the metal center exerts allosteric control over the cluster binding domain and regulates multiple properties of the metal center. Mutagenesis of L2 results in significant shifts in the redox potential of the [2Fe-2S] cluster and orders of magnitude effects on the rate of [2Fe-2S] cluster transfer to an apo-acceptor protein. These surprising effects occur in the absence of any structural changes. An examination of the native basin dynamics of the protein using all-atom simulations shows that twisting in L2 controls scissoring in the cluster binding domain and results in perturbations to one of the cluster-coordinating histidines. These allosteric effects are in agreement with previous folding simulations that predicted L2 could communicate with residues surrounding the metal center. Our findings suggest that long-range dynamical changes in the protein backbone can have a significant effect on the functional properties of MPs.

  10. 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

  11. Polypharmacology within CXCR4: Multiple binding sites and allosteric behavior

    Science.gov (United States)

    Planesas, Jesús M.; Pérez-Nueno, Violeta I.; Borrell, José I.; Teixidó, Jordi

    2014-10-01

    CXCR4 is a promiscuous receptor, which binds multiple diverse ligands. As usual in promiscuous proteins, CXCR4 has a large binding site, with multiple subsites, and high flexibility. Hence, it is not surprising that it is involved in the phenomenon of allosteric modulation. However, incomplete knowledge of allosteric ligand-binding sites has hampered an in-depth molecular understanding of how these inhibitors work. For example, it is known that lipidated fragments of intracellular GPCR loops, so called pepducins, such as pepducin ATI-2341, modulate CXCR4 activity using an agonist allosteric mechanism. Nevertheless, there are also examples of small organic molecules, such as AMD11070 and GSK812397, which may act as antagonist allosteric modulators. Here, we give new insights into this issue by proposing the binding interactions between the CXCR4 receptor and the above-mentioned allosteric modulators. We propose that CXCR4 has minimum two topographically different allosteric binding sites. One allosteric site would be in the intracellular loop 1 (ICL1) where pepducin ATI-2341 would bind to CXCR4, and the second one, in the extracellular side of CXCR4 in a subsite into the main orthosteric binding pocket, delimited by extracellular loops n° 1, 2, and the N-terminal end, where antagonists AMD11070 and GSK812397 would bind. Prediction of allosteric interactions between CXCR4 and pepducin ATI-2341 were studied first by rotational blind docking to determine the main binding region and a subsequent refinement of the best pose was performed using flexible docking methods and molecular dynamics. For the antagonists AMD11070 and GSK812397, the entire CXCR4 protein surface was explored by blind docking to define the binding region. A second docking analysis by subsites of the identified binding region was performed to refine the allosteric interactions. Finally, we identified the binding residues that appear to be essential for CXCR4 (agonists and antagonists) allosteric

  12. 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

  13. Adenine nucleotides as allosteric effectors of PEA seed glutamine synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Unkefer, P.J.; Knight, T.J.

    1986-05-01

    The energy charge in the plant cell has been proposed as a regulator of glutamine synthetase (GS) activity. The authors have shown that 2.1 moles of ..gamma..(/sup 32/P)-ATP were bound/mole subunits of purified pea seed GS during complete inactivation with methionine sulfoximine. Since GS has one active site per subunit, the second binding site provides the potential for allosteric regulation of GS by adenine nucleotides. The authors have investigated the inhibition of the ATP-dependent synthetic activity by ADP and AMP. ADP and AMP cannot completely inhibit GS; but ATP does overcome the inhibition by ADP and AMP as shown by plots of % inhibition vs inhibitor concentration. This indicates that inhibition of GS by ADP or AMP is not completely due to competitive inhibition. In the absence of ADP or AMP, double reciprocal plots for ATP are linear below 10 mM; however, in the presence of either ADP or AMP these pots are curvilinear downwards. The ratio of Vm/asymptote is less than 1. The Hill number for ATP in the absence of ADP or AMP is 0.93 but decreases with increasing ADP or AMP to a value of 0.28 with 10 mM ADP. These data are consistent with negative cooperativity by ADP and AMP. Thus, as the ADP/ATP or AMP/ATP ratios are increased GS activity decreases. This is consistent with regulation of GS activity by energy charge in planta.

  14. International Union of Basic and Clinical Pharmacology. XC. multisite pharmacology: recommendations for the nomenclature of receptor allosterism and allosteric ligands.

    Science.gov (United States)

    Christopoulos, Arthur; Changeux, Jean-Pierre; Catterall, William A; Fabbro, Doriano; Burris, Thomas P; Cidlowski, John A; Olsen, Richard W; Peters, John A; Neubig, Richard R; Pin, Jean-Philippe; Sexton, Patrick M; Kenakin, Terry P; Ehlert, Frederick J; Spedding, Michael; Langmead, Christopher J

    2014-10-01

    Allosteric interactions play vital roles in metabolic processes and signal transduction and, more recently, have become the focus of numerous pharmacological studies because of the potential for discovering more target-selective chemical probes and therapeutic agents. In addition to classic early studies on enzymes, there are now examples of small molecule allosteric modulators for all superfamilies of receptors encoded by the genome, including ligand- and voltage-gated ion channels, G protein-coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As a consequence, a vast array of pharmacologic behaviors has been ascribed to allosteric ligands that can vary in a target-, ligand-, and cell-/tissue-dependent manner. The current article presents an overview of allostery as applied to receptor families and approaches for detecting and validating allosteric interactions and gives recommendations for the nomenclature of allosteric ligands and their properties.

  15. An allosteric signaling pathway of human 3-phosphoglycerate kinase from force distribution analysis.

    Directory of Open Access Journals (Sweden)

    Zoltan Palmai

    2014-01-01

    Full Text Available 3-Phosphogycerate kinase (PGK is a two domain enzyme, which transfers a phosphate group between its two substrates, 1,3-bisphosphoglycerate bound to the N-domain and ADP bound to the C-domain. Indispensable for the phosphoryl transfer reaction is a large conformational change from an inactive open to an active closed conformation via a hinge motion that should bring substrates into close proximity. The allosteric pathway resulting in the active closed conformation has only been partially uncovered. Using Molecular Dynamics simulations combined with Force Distribution Analysis (FDA, we describe an allosteric pathway, which connects the substrate binding sites to the interdomain hinge region. Glu192 of alpha-helix 7 and Gly394 of loop L14 act as hinge points, at which these two secondary structure elements straighten, thereby moving the substrate-binding domains towards each other. The long-range allosteric pathway regulating hPGK catalytic activity, which is partially validated and can be further tested by mutagenesis, highlights the virtue of monitoring internal forces to reveal signal propagation, even if only minor conformational distortions, such as helix bending, initiate the large functional rearrangement of the macromolecule.

  16. An allosteric signaling pathway of human 3-phosphoglycerate kinase from force distribution analysis.

    Science.gov (United States)

    Palmai, Zoltan; Seifert, Christian; Gräter, Frauke; Balog, Erika

    2014-01-01

    3-Phosphogycerate kinase (PGK) is a two domain enzyme, which transfers a phosphate group between its two substrates, 1,3-bisphosphoglycerate bound to the N-domain and ADP bound to the C-domain. Indispensable for the phosphoryl transfer reaction is a large conformational change from an inactive open to an active closed conformation via a hinge motion that should bring substrates into close proximity. The allosteric pathway resulting in the active closed conformation has only been partially uncovered. Using Molecular Dynamics simulations combined with Force Distribution Analysis (FDA), we describe an allosteric pathway, which connects the substrate binding sites to the interdomain hinge region. Glu192 of alpha-helix 7 and Gly394 of loop L14 act as hinge points, at which these two secondary structure elements straighten, thereby moving the substrate-binding domains towards each other. The long-range allosteric pathway regulating hPGK catalytic activity, which is partially validated and can be further tested by mutagenesis, highlights the virtue of monitoring internal forces to reveal signal propagation, even if only minor conformational distortions, such as helix bending, initiate the large functional rearrangement of the macromolecule.

  17. Reciprocal allosteric modulation of carbon monoxide and warfarin binding to ferrous human serum heme-albumin.

    Directory of Open Access Journals (Sweden)

    Alessio Bocedi

    Full Text Available Human serum albumin (HSA, the most abundant protein in human plasma, could be considered as a prototypic monomeric allosteric protein, since the ligand-dependent conformational adaptability of HSA spreads beyond the immediate proximity of the binding site(s. As a matter of fact, HSA is a major transport protein in the bloodstream and the regulation of the functional allosteric interrelationships between the different binding sites represents a fundamental information for the knowledge of its transport function. Here, kinetics and thermodynamics of the allosteric modulation: (i of carbon monoxide (CO binding to ferrous human serum heme-albumin (HSA-heme-Fe(II by warfarin (WF, and (ii of WF binding to HSA-heme-Fe(II by CO are reported. All data were obtained at pH 7.0 and 25°C. Kinetics of CO and WF binding to the FA1 and FA7 sites of HSA-heme-Fe(II, respectively, follows a multi-exponential behavior (with the same relative percentage for the two ligands. This can be accounted for by the existence of multiple conformations and/or heme-protein axial coordination forms of HSA-heme-Fe(II. The HSA-heme-Fe(II populations have been characterized by resonance Raman spectroscopy, indicating the coexistence of different species characterized by four-, five- and six-coordination of the heme-Fe atom. As a whole, these results suggest that: (i upon CO binding a conformational change of HSA-heme-Fe(II takes place (likely reflecting the displacement of an endogenous ligand by CO, and (ii CO and/or WF binding brings about a ligand-dependent variation of the HSA-heme-Fe(II population distribution of the various coordinating species. The detailed thermodynamic and kinetic analysis here reported allows a quantitative description of the mutual allosteric effect of CO and WF binding to HSA-heme-Fe(II.

  18. An allosteric model for the functional plasticity of olfactory chemoreceptors

    Science.gov (United States)

    Colosimo, Alfredo

    2000-12-01

    A simple allosteric model may describe the relatively (a)specific behaviour of olfactory chemoreceptors (OCs) and their functional plasticity with a minimum number of parameters. Allosteric, heterotropic effectors are suggested as a possible cause of variable responses documented, in particular, in frog OCs. As an immediate spinoff of the continuously increasing amount of structural information available on natural OCs, development of appropriate allosteric models is foreseen to provide plausible molecular mechanisms for their complex functional performance. This may also have implications in the design of artificial olfaction systems.

  19. ETA-receptor antagonists or allosteric modulators?

    DEFF Research Database (Denmark)

    De Mey, Jo G R; Compeer, Matthijs G; Lemkens, Pieter;

    2011-01-01

    The paracrine signaling peptide endothelin-1 (ET1) is involved in cardiovascular diseases, cancer and chronic pain. It acts on class A G-protein-coupled receptors (GPCRs) but displays atypical pharmacology. It binds tightly to ET receptor type A (ET(A)) and causes long-lasting effects. In resista......The paracrine signaling peptide endothelin-1 (ET1) is involved in cardiovascular diseases, cancer and chronic pain. It acts on class A G-protein-coupled receptors (GPCRs) but displays atypical pharmacology. It binds tightly to ET receptor type A (ET(A)) and causes long-lasting effects......(A) and that ERAs and the physiological antagonist allosterically reduce ET(A) functions. Combining the two-state model and the two-domain model of GPCR function and considering receptor activation beyond agonist binding might lead to better anti-endothelinergic drugs. Future studies could lead to compounds...

  20. Various Themes of Myosin Regulation.

    Science.gov (United States)

    Heissler, Sarah M; Sellers, James R

    2016-05-01

    Members of the myosin superfamily are actin-based molecular motors that are indispensable for cellular homeostasis. The vast functional and structural diversity of myosins accounts for the variety and complexity of the underlying allosteric regulatory mechanisms that determine the activation or inhibition of myosin motor activity and enable precise timing and spatial aspects of myosin function at the cellular level. This review focuses on the molecular basis of posttranslational regulation of eukaryotic myosins from different classes across species by allosteric intrinsic and extrinsic effectors. First, we highlight the impact of heavy and light chain phosphorylation. Second, we outline intramolecular regulatory mechanisms such as autoinhibition and subsequent activation. Third, we discuss diverse extramolecular allosteric mechanisms ranging from actin-linked regulatory mechanisms to myosin:cargo interactions. At last, we briefly outline the allosteric regulation of myosins with synthetic compounds.

  1. Allosteric modulators of the hERG K(+) channel: radioligand binding assays reveal allosteric characteristics of dofetilide analogs.

    Science.gov (United States)

    Yu, Zhiyi; Klaasse, Elisabeth; Heitman, Laura H; Ijzerman, Adriaan P

    2014-01-01

    Drugs that block the cardiac K(+) 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(+) 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 [(3)H]astemizole and [(3)H]dofetilide to the hERG K(+) channel. LUF6200 was identified as an allosteric inhibitor in dissociation assays with both radioligands, yielding similar EC50 values in the low micromolar range. However, potassium ions increased the binding of the two radioligands in a concentration-dependent manner, and their EC50 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(+) channel, which is discussed in the light of findings on other ion channels. PMID:24200993

  2. Small-world networks of residue interactions in the Abl kinase complexes with cancer drugs: topology of allosteric communication pathways can determine drug resistance effects.

    Science.gov (United States)

    Tse, A; Verkhivker, G M

    2015-07-01

    The human protein kinases play a fundamental regulatory role in orchestrating functional processes in complex cellular networks. Understanding how conformational equilibrium between functional kinase states can be modulated by ligand binding or mutations is critical for quantifying molecular basis of allosteric regulation and drug resistance. In this work, molecular dynamics simulations of the Abl kinase complexes with cancer drugs (Imatinib and Dasatinib) were combined with structure-based network modeling to characterize dynamics of the residue interaction networks in these systems. The results have demonstrated that structural architecture of kinase complexes can produce a small-world topology of the interaction networks. Our data have indicated that specific Imatinib binding to a small number of highly connected residues could lead to network-bridging effects and allow for efficient allosteric communication, which is mediated by a dominant pathway sensitive to the unphosphorylated Abl state. In contrast, Dasatinib binding to the active kinase form may activate a broader ensemble of allosteric pathways that are less dependent on the phosphorylation status of Abl and provide a better balance between the efficiency and resilience of signaling routes. Our results have unveiled how differences in the residue interaction networks and allosteric communications of the Abl kinase complexes can be directly related to drug resistance effects. This study offers a plausible perspective on how efficiency and robustness of the residue interaction networks and allosteric pathways in kinase structures may be associated with protein responses to drug binding.

  3. Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

    DEFF Research Database (Denmark)

    Nestoras, Konstantinos; Mohammed, Asma Hadi; Schreurs, Ann-Sofie;

    2010-01-01

    The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and...

  4. Allosteric Inhibition of Human Immunodeficiency Virus Integrase

    Science.gov (United States)

    Gupta, Kushol; Brady, Troy; Dyer, Benjamin M.; Malani, Nirav; Hwang, Young; Male, Frances; Nolte, Robert T.; Wang, Liping; Velthuisen, Emile; Jeffrey, Jerry; Van Duyne, Gregory D.; Bushman, Frederic D.

    2014-01-01

    HIV-1 replication in the presence of antiviral agents results in evolution of drug-resistant variants, motivating the search for additional drug classes. Here we report studies of GSK1264, which was identified as a compound that disrupts the interaction between HIV-1 integrase (IN) and the cellular factor lens epithelium-derived growth factor (LEDGF)/p75. GSK1264 displayed potent antiviral activity and was found to bind at the site occupied by LEDGF/p75 on IN by x-ray crystallography. Assays of HIV replication in the presence of GSK1264 showed only modest inhibition of the early infection steps and little effect on integration targeting, which is guided by the LEDGF/p75·IN interaction. In contrast, inhibition of late replication steps was more potent. Particle production was normal, but particles showed reduced infectivity. GSK1264 promoted aggregation of IN and preformed LEDGF/p75·IN complexes, suggesting a mechanism of inhibition. LEDGF/p75 was not displaced from IN during aggregation, indicating trapping of LEDGF/p75 in aggregates. Aggregation assays with truncated IN variants revealed that a construct with catalytic and C-terminal domains of IN only formed an open polymer associated with efficient drug-induced aggregation. These data suggest that the allosteric inhibitors of IN are promising antiviral agents and provide new information on their mechanism of action. PMID:24904063

  5. Allosteric modulators for the treatment of schizophrenia: targeting glutamatergic networks.

    Science.gov (United States)

    Menniti, Frank S; Lindsley, Craig W; Conn, P Jeffrey; Pandit, Jayvardhan; Zagouras, Panayiotis; Volkmann, Robert A

    2013-01-01

    Schizophrenia is a highly debilitating mental disorder which afflicts approximately 1% of the global population. Cognitive and negative deficits account for the lifelong disability associated with schizophrenia, whose symptoms are not effectively addressed by current treatments. New medicines are needed to treat these aspects of the disease. Neurodevelopmental, neuropathological, genetic, and behavioral pharmacological data indicate that schizophrenia stems from a dysfunction of glutamate synaptic transmission, particularly in frontal cortical networks. A number of novel pre- and postsynaptic mechanisms affecting glutamatergic synaptic transmission have emerged as viable targets for schizophrenia. While developing orthosteric glutamatergic agents for these targets has proven extremely difficult, targeting allosteric sites of these targets has emerged as a promising alternative. From a medicinal chemistry perspective, allosteric sites provide an opportunity of finding agents with better drug-like properties and greater target specificity. Furthermore, allosteric modulators are better suited to maintaining the highly precise temporal and spatial aspects of glutamatergic synaptic transmission. Herein, we review neuropathological and genomic/genetic evidence underscoring the importance of glutamate synaptic dysfunction in the etiology of schizophrenia and make a case for allosteric targets for therapeutic intervention. We review progress in identifying allosteric modulators of AMPA receptors, NMDA receptors, and metabotropic glutamate receptors, all with the aim of restoring physiological glutamatergic synaptic transmission. Challenges remain given the complexity of schizophrenia and the difficulty in studying cognition in animals and humans. Nonetheless, important compounds have emerged from these efforts and promising preclinical and variable clinical validation has been achieved.

  6. Molecular Synchronization Waves in Arrays of Allosterically Regulated Enzymes

    CERN Document Server

    Casagrande, Vanessa; Mikhailov, Alexander S

    2007-01-01

    Spatiotemporal pattern formation in a product-activated enzymic reaction at high enzyme concentrations is investigated. Stochastic simulations show that catalytic turnover cycles of individual enzymes can become coherent and that complex wave patterns of molecular synchronization can develop. The analysis based on the mean-field approximation indicates that the observed patterns result from the presence of Hopf and wave bifurcations in the considered system.

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

    Science.gov (United States)

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

    2004-01-01

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

  8. Evolution of allosteric citrate binding sites on 6-phosphofructo-1-kinase.

    Directory of Open Access Journals (Sweden)

    Aleksandra Usenik

    Full Text Available As an important part of metabolism, metabolic flux through the glycolytic pathway is tightly regulated. The most complex control is exerted on 6-phosphofructo-1-kinase (PFK1 level; this control overrules the regulatory role of other allosteric enzymes. Among other effectors, citrate has been reported to play a vital role in the suppression of this enzyme's activity. In eukaryotes, amino acid residues forming the allosteric binding site for citrate are found both on the N- and the C-terminal region of the enzyme. These site has evolved from the phosphoenolpyruvate/ADP binding site of bacterial PFK1 due to the processes of duplication and tandem fusion of prokaryotic ancestor gene followed by the divergence of the catalytic and effector binding sites. Stricter inhibition of the PFK1 enzyme was needed during the evolution of multi-cellular organisms, and the most stringent control of PFK1 by citrate occurs in vertebrates. By substituting a single amino acid (K557R or K617A as a component of the allosteric binding site in the C-terminal region of human muscle type PFK-M with a residue found in the corresponding site of a fungal enzyme, the inhibitory effect of citrate was attenuated. Moreover, the proteins carrying these single mutations enabled growth of E. coli transformants encoding mutated human PFK-M in a glucose-containing medium that did not support the growth of E. coli transformed with native human PFK-M. Substitution of another residue at the citrate-binding site (D591V of human PFK-M resulted in the complete loss of activity. Detailed analyses revealed that the mutated PFK-M subunits formed dimers but were unable to associate into the active tetrameric holoenzyme. These results suggest that stricter control over glycolytic flux developed in metazoans, whose somatic cells are largely characterized by slow proliferation.

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

    DEFF Research Database (Denmark)

    Schwartz, Thue W; Holst, Birgitte

    2006-01-01

    Conventionally, an allosteric modulator is neutral in respect of efficacy and binds to a receptor site distant from the orthosteric site of the endogenous agonist. However, recently compounds being ago-allosteric modulators have been described i.e., compounds acting both as agonists on their own...... 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 exogenous......, allosteric modulators. If the allosteric modulator is an agonist, it is an ago-allosteric modulator; if it is neutral, it is a classical enhancer. Molecular mapping in hetero-dimeric class-C receptors, where the endogenous agonist clearly binds only in one protomer, supports the notion that allosteric...

  10. An allosteric inhibitor of protein arginine methyltransferase 3.

    Science.gov (United States)

    Siarheyeva, Alena; Senisterra, Guillermo; Allali-Hassani, Abdellah; Dong, Aiping; Dobrovetsky, Elena; Wasney, Gregory A; Chau, Irene; Marcellus, Richard; Hajian, Taraneh; Liu, Feng; Korboukh, Ilia; Smil, David; Bolshan, Yuri; Min, Jinrong; Wu, Hong; Zeng, Hong; Loppnau, Peter; Poda, Gennadiy; Griffin, Carly; Aman, Ahmed; Brown, Peter J; Jin, Jian; Al-Awar, Rima; Arrowsmith, Cheryl H; Schapira, Matthieu; Vedadi, Masoud

    2012-08-01

    PRMT3, a protein arginine methyltransferase, has been shown to influence ribosomal biosynthesis by catalyzing the dimethylation of the 40S ribosomal protein S2. Although PRMT3 has been reported to be a cytosolic protein, it has been shown to methylate histone H4 peptide (H4 1-24) in vitro. Here, we report the identification of a PRMT3 inhibitor (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-cyclohexenylethyl)urea; compound 1) with IC50 value of 2.5 μM by screening a library of 16,000 compounds using H4 (1-24) peptide as a substrate. The crystal structure of PRMT3 in complex with compound 1 as well as kinetic analysis reveals an allosteric mechanism of inhibition. Mutating PRMT3 residues within the allosteric site or using compound 1 analogs that disrupt interactions with allosteric site residues both abrogated binding and inhibitory activity. These data demonstrate an allosteric mechanism for inhibition of protein arginine methyltransferases, an emerging class of therapeutic targets.

  11. Allosteric properties of phosphate-activated glutaminase of human liver mitochondria.

    Science.gov (United States)

    Snodgrass, P J; Lund, P

    1984-03-22

    The kinetics of human liver phosphate-activated glutaminase were studied in mitochondria isolated from surgical biopsies. The pH profile and activation by phosphate closely resembled rat liver glutaminase and differed clearly from human or rat kidney mitochondrial glutaminases. The activity responses to glutamine or phosphate were allosteric, showing positive cooperativity, as in the rat liver enzyme. Exogenous 1 mM NH4Cl shifted the glutamine concentration at half-maximal velocity, [Gln]0.5, to lower values without changing Vmax or sigmoidicity. Hill plots showed a parallel shift to the left with NH4Cl and the apparent number of binding sites, nH, was 2-3. 25 mM KHCO3 gave the same effects as NH4Cl on [Gln]0.5, Vmax, sigmoidicity and nH. The combination of the two activators was less than additive. Glutamate did not inhibit. We postulate that liver glutaminase is allosteric in its kinetics because it plays a key role in urea synthesis by regulating provision of glutamate for synthesis of N-acetylglutamate, the obligatory co-factor of carbamoylphosphate synthetase. PMID:6704422

  12. 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

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  14. Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site.

    Science.gov (United States)

    Chen, Hongming; Tucker, Julie; Wang, Xiaotao; Gavine, Paul R; Phillips, Chris; Augustin, Martin A; Schreiner, Patrick; Steinbacher, Stefan; Preston, Marian; Ogg, Derek

    2016-05-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5-inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented.

  15. 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

  16. 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...

  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. Light-activated DNA binding in a designed allosteric protein

    Energy Technology Data Exchange (ETDEWEB)

    Strickland, Devin; Moffat, Keith; Sosnick, Tobin R. (UC)

    2008-09-03

    An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an {alpha}-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical 'allosteric lever arm' is a general scheme for coupling the function of two proteins.

  19. 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.

  20. 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.

  1. 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

  2. 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...

  3. 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...

  4. 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

  5. Identification of the allosteric regulatory site of insulysin.

    Directory of Open Access Journals (Sweden)

    Nicholas Noinaj

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

  6. 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

  7. 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.; Gerrard, Juliet Ann

    2011-06-24

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

  8. 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.

  9. 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

  10. 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 ......Glu allosteric modulator binding modes relates to selective pharmacological actions will be very valuable for rational design of safer drugs....

  11. Advances in NMR Methods To Map Allosteric Sites: From Models to Translation.

    Science.gov (United States)

    Boulton, Stephen; Melacini, Giuseppe

    2016-06-01

    The last five years have witnessed major developments in the understanding of the allosteric phenomenon, broadly defined as coupling between remote molecular sites. Such advances have been driven not only by new theoretical models and pharmacological applications of allostery, but also by progress in the experimental approaches designed to map allosteric sites and transitions. Among these techniques, NMR spectroscopy has played a major role given its unique near-atomic resolution and sensitivity to the dynamics that underlie allosteric couplings. Here, we highlight recent progress in the NMR methods tailored to investigate allostery with the goal of offering an overview of which NMR approaches are best suited for which allosterically relevant questions. The picture of the allosteric "NMR toolbox" is provided starting from one of the simplest models of allostery (i.e., the four-state thermodynamic cycle) and continuing to more complex multistate mechanisms. We also review how such an "NMR toolbox" has assisted the elucidation of the allosteric molecular basis for disease-related mutations and the discovery of novel leads for allosteric drugs. From this overview, it is clear that NMR plays a central role not only in experimentally validating transformative theories of allostery, but also in tapping the full translational potential of allosteric systems. PMID:27111288

  12. 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.

  13. 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

  14. Designing Allosteric Control into Enzymes by Chemical Rescue of Structure

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-08-07

    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 disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. We present two examples, W33G in a {beta}-glycosidase enzyme ({beta}-gly) and W492G in a {beta}-glucuronidase enzyme ({beta}-gluc), in which we engineer indole-dependent activity into enzymes by removing a buried tryptophan side chain that serves as a buttress for the active site architecture. In both cases, we observe a loss of function, and in both cases we find that the subsequent addition of indole can be used to restore activity. Through a detailed analysis of {beta}-gly W33G kinetics, we demonstrate that this rescued enzyme is fully functionally equivalent to the corresponding wild-type enzyme. We then present the apo and indole-bound crystal structures of {beta}-gly W33G, which together establish the structural basis for enzyme inactivation and rescue. Finally, we use this designed switch to modulate {beta}-glycosidase activity in living cells using indole. Disruption and recovery of protein structure may represent a general technique for introducing allosteric control into enzymes, and thus may serve as a starting point for building a variety of bioswitches and sensors.

  15. 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.

  16. 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.

  17. 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.

  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. Allosteric Inhibition of Macrophage Migration Inhibitory Factor Revealed by Ibudilast

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-01-01

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

  20. 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

  1. 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...

  2. 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, ...

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

    Science.gov (United States)

    Harpsøe, Kasper; Isberg, Vignir; Tehan, Benjamin G.; Weiss, Dahlia; Arsova, Angela; Marshall, Fiona H.; Bräuner-Osborne, Hans; Gloriam, David E.

    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 mGlu allosteric modulator binding modes relates to selective pharmacological actions will be very valuable for rational design of safer drugs. PMID:26359761

  4. 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...

  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

    molecules often act more deeply in an allosteric mode. However, opposed to the well described molecular interaction of allosteric modulators in class C 7-transmembrane helix (7TM) receptors, the interaction in class A, to which the chemokine receptors belong, is more sparsely described. Using the CCR5...... chemokine receptor as a model system, we studied the molecular interaction and conformational interchange required for proper action of various orthosteric chemokines and allosteric small molecules, including the well known CCR5 antagonists TAK-779, SCH-C, and aplaviroc, and four novel CCR5 ago......-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...

  6. 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.

  7. Profiling two indole-2-carboxamides for allosteric modulation of the CB1 receptor.

    Science.gov (United States)

    Ahn, Kwang H; Mahmoud, Mariam M; Samala, Sushma; Lu, Dai; Kendall, Debra A

    2013-03-01

    Allosteric modulation of G-protein coupled receptors (GPCRs) represents a novel approach for fine-tuning GPCR functions. The cannabinoid CB1 receptor, a GPCR associated with the CNS, has been implicated in the treatment of drug addiction, pain, and appetite disorders. We report here the synthesis and pharmacological characterization of two indole-2-carboxamides:5-chloro-3-ethyl-1-methyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ICAM-a) and 5-chloro-3-pentyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ICAM-b). Although both ICAM-a and ICAM-b enhanced CP55, 940 binding, ICAM-b exhibited the strongest positive cooperativity thus far demonstrated for enhancing agonist binding to the CB1 receptor. Although it displayed negative modulatory effects on G-protein coupling to CB1, ICAM-b induced β-arrestin-mediated downstream activation of extracellular signal-regulated kinase (ERK) signaling. These results indicate that this compound represents a novel class of CB1 ligands that produce biased signaling via CB1.

  8. Required allosteric effector site for N-acetylglutamate on carbamoyl-phosphate synthetase I.

    Science.gov (United States)

    McCudden, C R; Powers-Lee, S G

    1996-07-26

    Carbamoyl-phosphate synthetase I (CPSase I) catalyzes the entry and rate-limiting step in the urea cycle, the pathway by which mammals detoxify ammonia. One facet of CPSase I regulation is a requirement for N-acetylglutamate (AGA), which induces an active enzyme conformation and does not participate directly in the chemical reaction. We have utilized labeling with carbodiimide-activated [14C]AGA to identify peptides 120-127, 234-237, 625-630, and 1351-1356 as potentially being near the binding site for AGA. Identification of peptide 1351-1356 confirms the previous demonstration (Rodriquez-Aparicio, L. B., Guadalajara, A. M., and Rubio, V.(1989) Biochemistry 28, 3070-3074) that the C-terminal region is involved in binding AGA. Identification of peptides 120-127 and 234-237 constitutes the first evidence that the N-terminal region of the synthetase is involved in ligand binding. Since peptides 631-638 and 1327-1348 have been identified near the ATP site of CPSase I (Potter, M. D., and Powers-Lee, S. G.(1992) J. Biol. Chem. 267, 2023-2031), the present finding of involvement of peptides 625-630 and 1351-1356 at an "allosteric" activator site was unexpected. The idea that portions of the AGA effector site might be derived from an ancestral glutamine substrate site via a gene duplication and diversification event was considered. PMID:8663466

  9. 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

  10. Allosteric activation of brain hexokinase by magnesium ions and by magnesium ion--adenosine triphosphate complex.

    Science.gov (United States)

    Bachelard, H S

    1971-11-01

    1. Substrate-saturation curves of brain hexokinase for MgATP(2-) were sigmoidal at sub-saturating concentrations of glucose when the Mg(2+)/ATP ratio was maintained at 1:1. Under identical conditions, except that Mg(2+) was present in excess, hyperbolic curves were observed. 2. The number of binding sites (calculated from Hill plots) is 1.8 at a Mg(2+)/ATP ratio 1:1, and 1.0 with excess of Mg(2+). The apparent K(m) for MgATP(2-) is 6.5x10(-4)m at a Mg(2+)/ATP ratio 1:1, and 3.5x10(-4)m with excess of Mg(2+). 3. Interdependence between substrate-binding sites was indicated by the effects of varying the concentration of glucose. The sigmoidality and deviation from Michaelis-Menten kinetics at a Mg(2+)/ATP ratio 1:1 became less pronounced with increasing glucose concentration. Also, although substrate-saturation curves for glucose were hyperbolic when the Mg(2+)/ATP ratio was 1:1, reciprocal plots were non-linear. These were linear with excess of Mg(2+). 4. High concentrations of Mg(2+) (Mg(2+)/ATP ratios above 5:1) were inhibitory. 5. The results are taken to indicate homotropic co-operative binding of MgATP(2-) and that Mg(2+) is an allosteric activator. Possible implications in regulation are discussed.

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

    Directory of Open Access Journals (Sweden)

    Simone ePelliciari

    2015-08-01

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

  12. 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.

  13. An allosteric mechanism for switching between parallel tracks in mammalian sulfur metabolism.

    Directory of Open Access Journals (Sweden)

    Tatyana K Korendyaseva

    2008-05-01

    Full Text Available Methionine (Met is an essential amino acid that is needed for the synthesis of S-adenosylmethionine (AdoMet, the major biological methylating agent. Methionine used for AdoMet synthesis can be replenished via remethylation of homocysteine. Alternatively, homocysteine can be converted to cysteine via the transsulfuration pathway. Aberrations in methionine metabolism are associated with a number of complex diseases, including cancer, anemia, and neurodegenerative diseases. The concentration of methionine in blood and in organs is tightly regulated. Liver plays a key role in buffering blood methionine levels, and an interesting feature of its metabolism is that parallel tracks exist for the synthesis and utilization of AdoMet. To elucidate the molecular mechanism that controls metabolic fluxes in liver methionine metabolism, we have studied the dependencies of AdoMet concentration and methionine consumption rate on methionine concentration in native murine hepatocytes at physiologically relevant concentrations (40-400 microM. We find that both [AdoMet] and methionine consumption rates do not change gradually with an increase in [Met] but rise sharply (approximately 10-fold in the narrow Met interval from 50 to 100 microM. Analysis of our experimental data using a mathematical model reveals that the sharp increase in [AdoMet] and the methionine consumption rate observed within the trigger zone are associated with metabolic switching from methionine conservation to disposal, regulated allosterically by switching between parallel pathways. This regulatory switch is triggered by [Met] and provides a mechanism for stabilization of methionine levels in blood over wide variations in dietary methionine intake.

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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.

  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

    G protein coupled receptors (GPCRs) are the most historically successful therapeutic targets. Despite this success there are many important aspects of GPCR pharmacology and function that have yet to be exploited to their full therapeutic potential. One in particular that has been gaining attention...... in recent times is that of GPCR ligands that bind to allosteric sites on the receptor distinct from the orthosteric site of the endogenous ligand. As therapeutics, allosteric ligands possess many theoretical advantages over their orthosteric counterparts, including more complex modes of action, improved...... safety, more physiologically appropriate responses, better target selectivity, and reduced likelihood of desensitisation and tachyphylaxis. Despite these advantages, the development of allosteric ligands is often difficult from a medicinal chemistry standpoint due to the more complex challenge...

  2. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

    Science.gov (United States)

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2‧-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

  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. PMID:27353957

  4. Synthesis and biological evaluation of negative allosteric modulators of the Kv11.1(hERG) channel.

    Science.gov (United States)

    Yu, Zhiyi; van Veldhoven, Jacobus P D; 't Hart, Ingrid M E; Kopf, Adrian H; Heitman, Laura H; IJzerman, Adriaan P

    2015-12-01

    We synthesized and evaluated a series of compounds for their allosteric modulation at the Kv11.1 (hERG) channel. Most compounds were negative allosteric modulators of [(3)H]dofetilide binding to the channel, in particular 7f, 7h-j and 7p. Compounds 7f and 7p were the most potent negative allosteric modulators amongst all ligands, significantly increasing the dissociation rate of dofetilide in the radioligand kinetic binding assay, while remarkably reducing the affinities of dofetilide and astemizole in a competitive displacement assay. Additionally, both 7f and 7p displayed peculiar displacement characteristics with Hill coefficients significantly distinct from unity as shown by e.g., dofetilide, further indicative of their allosteric effects on dofetilide binding. Our findings in this investigation yielded several promising negative allosteric modulators for future functional and clinical research with respect to their antiarrhythmic propensities, either alone or in combination with known Kv11.1 blockers. PMID:26519929

  5. The therapeutic promise of positive allosteric modulation of nicotinic receptors.

    Science.gov (United States)

    Uteshev, Victor V

    2014-03-15

    In the central nervous system, deficits in cholinergic neurotransmission correlate with decreased attention and cognitive impairment, while stimulation of neuronal nicotinic acetylcholine receptors improves attention, cognitive performance and neuronal resistance to injury as well as produces robust analgesic and anti-inflammatory effects. The rational basis for the therapeutic use of orthosteric agonists and positive allosteric modulators (PAMs) of nicotinic receptors arises from the finding that functional nicotinic receptors are ubiquitously expressed in neuronal and non-neuronal tissues including brain regions highly vulnerable to traumatic and ischemic types of injury (e.g., cortex and hippocampus). Moreover, functional nicotinic receptors do not vanish in age-, disease- and trauma-related neuropathologies, but their expression and/or activation levels decline in a subunit- and brain region-specific manner. Therefore, augmenting the endogenous cholinergic tone by nicotinic agents is possible and may offset neurological impairments associated with cholinergic hypofunction. Importantly, because neuronal damage elevates extracellular levels of choline (a selective agonist of α7 nicotinic acetylcholine receptors) near the site of injury, α7-PAM-based treatments may augment pathology-activated α7-dependent auto-therapies where and when they are most needed (i.e., in the penumbra, post-injury). Thus, nicotinic-PAM-based treatments are expected to augment the endogenous cholinergic tone in a spatially and temporally restricted manner creating the potential for differential efficacy and improved safety as compared to exogenous orthosteric nicotinic agonists that activate nicotinic receptors indiscriminately. In this review, I will summarize the existing trends in therapeutic applications of nicotinic PAMs.

  6. 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

  7. 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

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  9. Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase.

    Science.gov (United States)

    Zhang, Shengnan; Fitzpatrick, Paul F

    2016-04-01

    Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that requires activation by phenylalanine for full activity. The location of the allosteric site for phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25-117) is the regulatory domain of PheH lacking residues 1-24) of the rat enzyme in the presence of phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25-117) were identified as being in the phenylalanine-binding site on the basis of intermolecular NOEs between unlabeled phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25-117) for phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, and H64N mutations into the intact protein increased the concentration of phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH.

  10. Allosteric Indole Amide Inhibitors of p97: Identification of a Novel Probe of the Ubiquitin Pathway.

    Science.gov (United States)

    Alverez, Celeste; Bulfer, Stacie L; Chakrasali, Ramappa; Chimenti, Michael S; Deshaies, Raymond J; Green, Neal; Kelly, Mark; LaPorte, Matthew G; Lewis, Taber S; Liang, Mary; Moore, William J; Neitz, R Jeffrey; Peshkov, Vsevolod A; Walters, Michael A; Zhang, Feng; Arkin, Michelle R; Wipf, Peter; Huryn, Donna M

    2016-02-11

    A high-throughput screen to discover inhibitors of p97 ATPase activity identified an indole amide that bound to an allosteric site of the protein. Medicinal chemistry optimization led to improvements in potency and solubility. Indole amide 3 represents a novel uncompetitive inhibitor with excellent physical and pharmaceutical properties that can be used as a starting point for drug discovery efforts. PMID:26985295

  11. Thermodynamic Analysis of Allosteric and Chelate Cooperativity in Di- and Trivalent Ammonium/Crown-Ether Pseudorotaxanes.

    Science.gov (United States)

    Nowosinski, Karol; von Krbek, Larissa K S; Traulsen, Nora L; Schalley, Christoph A

    2015-10-16

    A detailed thermodynamic analysis of the axle-wheel binding in di- and trivalent secondary ammonium/[24]crown-8 pseudorotaxanes is presented. Isothermal titration calorimetry (ITC) data and double mutant cycle analyses reveal an interesting interplay of positive as well as negative allosteric and positive chelate cooperativity thus providing profound insight into the effects governing multivalent binding in these pseudorotaxanes.

  12. Computational predictions suggest that structural similarity in viral polymerases may lead to comparable allosteric binding sites.

    Science.gov (United States)

    Brown, Jodian A; Espiritu, Marie V; Abraham, Joel; Thorpe, Ian F

    2016-08-15

    The identification of ligand-binding sites is often the first step in drug targeting and design. To date there are numerous computational tools available to predict ligand binding sites. These tools can guide or mitigate the need for experimental methods to identify binding sites, which often require significant resources and time. Here, we evaluate four ligand-binding site predictor (LBSP) tools for their ability to predict allosteric sites within the Hepatitis C Virus (HCV) polymerase. Our results show that the LISE LBSP is able to identify all three target allosteric sites within the HCV polymerase as well as a known allosteric site in the Coxsackievirus polymerase. LISE was then employed to identify novel binding sites within the polymerases of the Dengue, West Nile, and Foot-and-mouth Disease viruses. Our results suggest that all three viral polymerases have putative sites that share structural or chemical similarities with allosteric pockets of the HCV polymerase. Thus, these binding locations may represent an evolutionarily conserved structural feature of several viral polymerases that could be exploited for the development of small molecule therapeutics. PMID:27262620

  13. 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

  14. An Allosteric Receptor by Simultaneous "Casting" and "Molding" in a Dynamic Combinatorial Library

    NARCIS (Netherlands)

    Li, Jianwei; Nowak, Piotr; Otto, Sijbren

    2015-01-01

    Allosteric synthetic receptors are difficult to access by design. Herein we report a dynamic combinatorial strategy towards such systems based on the simultaneous use of two different templates. Through a process of simultaneous casting (the assembly of a library member around a template) and moldin

  15. 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

  16. 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

  17. 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.

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

    DEFF Research Database (Denmark)

    Plenge, Per; Shi, Lei; Beuming, Thijs;

    2012-01-01

    be involved in the allosteric binding in the extracellular vestibule located above the central substrate binding (S1) site. Indeed, mutagenesis of selected residues in the vestibule reduces the allosteric potency of (S)-citalopram and clomipramine. The identified site is further supported by the inhibitory...... effects of Zn(2+) binding in an engineered site and the covalent attachment of benzocaine-methanethiosulfonate to a cysteine introduced in the extracellular vestibule. The data provide a mechanistic explanation for the allosteric action of antidepressants at SERT and suggest that the role of the vestibule...

  19. Moving Beyond Active-Site Detection: MixMD Applied to Allosteric Systems.

    Science.gov (United States)

    Ghanakota, Phani; Carlson, Heather A

    2016-08-25

    Mixed-solvent molecular dynamics (MixMD) is a hotspot-mapping technique that relies on molecular dynamics simulations of proteins in binary solvent mixtures. Previous work on MixMD has established the technique's effectiveness in capturing binding sites of small organic compounds. In this work, we show that MixMD can identify both competitive and allosteric sites on proteins. The MixMD approach embraces full protein flexibility and allows competition between solvent probes and water. Sites preferentially mapped by probe molecules are more likely to be binding hotspots. There are two important requirements for the identification of ligand-binding hotspots: (1) hotspots must be mapped at very high signal-to-noise ratio and (2) the hotspots must be mapped by multiple probe types. We have developed our mapping protocol around acetonitrile, isopropanol, and pyrimidine as probe solvents because they allowed us to capture hydrophilic, hydrophobic, hydrogen-bonding, and aromatic interactions. Charged probes were needed for mapping one target, and we introduce them in this work. In order to demonstrate the robust nature and wide applicability of the technique, a combined total of 5 μs of MixMD was applied across several protein targets known to exhibit allosteric modulation. Most notably, all the protein crystal structures used to initiate our simulations had no allosteric ligands bound, so there was no preorganization of the sites to predispose the simulations to find the allosteric hotspots. The protein test cases were ABL Kinase, Androgen Receptor, CHK1 Kinase, Glucokinase, PDK1 Kinase, Farnesyl Pyrophosphate Synthase, and Protein-Tyrosine Phosphatase 1B. The success of the technique is demonstrated by the fact that the top-four sites solely map the competitive and allosteric sites. Lower-ranked sites consistently map other biologically relevant sites, multimerization interfaces, or crystal-packing interfaces. Lastly, we highlight the importance of including protein

  20. Normal coordinate structural decomposition of the heme distortions of hemoglobin in various quaternary states and bound to allosteric effectors.

    Science.gov (United States)

    Laberge, Monique; Yonetani, Takashi; Fidy, Judit

    2003-01-01

    The distortions of the alpha1, alpha2, beta1, and beta2 hemes of human hemoglobin (HbA) in various quaternary states and as affected by the presence of allosteric effectors was investigated by subjecting CHARMM energy-minimized models to normal coordinate structural decomposition (NSD) analysis. NSD was applied to the individual hemes extracted from the R, T, and R2-state models of HbA and to HbA bound to DPG and to IHP. Overall, NSD results are indicative of characteristic distortions, not only for the hemes of the different HbA quaternary states, but also for the hemes of the HbA models bound to allosteric effectors. Comparing the distortions of the inequivalent alpha and beta hemes in T-state HbA, we show good correlation between NSD and the experimentally observed low-frequency nu52 (Eg) and gamma7 (A2u) modes reported in the literature for alpha and beta HbA hemes while noting substantial differences between these types for B2u and B1u distortions. For the R2 hemes, NSD yields heme distortions that are more comparable to those of the R-state, especially in magnitude. However, the R2 hemes do not exhibit inequivalence of alpha and beta heme distortions, a result that may contribute to an understanding of the functional importance of this state. Relative to T-state heme distortions, NSD results on the effector-bound hemes show that tertiary changes induced in T-state HbA as a result of binding DPG and IHP drastically affect heme distortions. In the alpha hemes extracted from the HbA-DPG model, most noteworthy are the increased wav(x) and wav(y) distortions and enhancement of ruf and dom deformations. In the beta hemes, the wav(y) is the most affected distortion with increase in sad. The NSD results are also different for the hemes of the HbA-IHP model, in that the beta sad and ruf deformations are more enhanced with increase of doming in the alpha hemes. Our results describe the effect of the subtle protein-induced changes on the nonplanarity of the HbA hemes

  1. Allosteric Transitions Direct Protein Tagging by PafA, the Prokaryotic Ubiquitin-like Protein (Pup) Ligase*

    Science.gov (United States)

    Ofer, Naomi; Forer, Nadav; Korman, Maayan; Vishkautzan, Marina; Khalaila, Isam; Gur, Eyal

    2013-01-01

    Protein degradation via prokaryotic ubiquitin-like protein (Pup) tagging is conserved in bacteria belonging to the phyla Actinobacteria and Nitrospira. The physiological role of this novel proteolytic pathway is not yet clear, although in Mycobacterium tuberculosis, the world's most threatening bacterial pathogen, Pup tagging is important for virulence. PafA, the Pup ligase, couples ATP hydrolysis with Pup conjugation to lysine side chains of protein substrates. PafA is the sole Pup ligase in M. tuberculosis and apparently, in other bacteria. Thus, whereas PafA is a key player in the Pup tagging (i.e. pupylation) system, control of its activity and interactions with target protein substrates remain poorly understood. In this study, we examined the mechanism of protein pupylation by PafA in Mycobacterium smegmatis, a model mycobacterial organism. We report that PafA is an allosteric enzyme that binds its target substrates cooperatively and find that PafA allostery is controlled by the binding of target protein substrates, yet is unaffected by Pup binding. Analysis of PafA pupylation using engineered substrates differing in the number of pupylation sites points to PafA acting as a dimer. These findings suggest that protein pupylation can be regulated at the level of PafA allostery. PMID:23471967

  2. Allosteric transitions direct protein tagging by PafA, the prokaryotic ubiquitin-like protein (Pup) ligase.

    Science.gov (United States)

    Ofer, Naomi; Forer, Nadav; Korman, Maayan; Vishkautzan, Marina; Khalaila, Isam; Gur, Eyal

    2013-04-19

    Protein degradation via prokaryotic ubiquitin-like protein (Pup) tagging is conserved in bacteria belonging to the phyla Actinobacteria and Nitrospira. The physiological role of this novel proteolytic pathway is not yet clear, although in Mycobacterium tuberculosis, the world's most threatening bacterial pathogen, Pup tagging is important for virulence. PafA, the Pup ligase, couples ATP hydrolysis with Pup conjugation to lysine side chains of protein substrates. PafA is the sole Pup ligase in M. tuberculosis and apparently, in other bacteria. Thus, whereas PafA is a key player in the Pup tagging (i.e. pupylation) system, control of its activity and interactions with target protein substrates remain poorly understood. In this study, we examined the mechanism of protein pupylation by PafA in Mycobacterium smegmatis, a model mycobacterial organism. We report that PafA is an allosteric enzyme that binds its target substrates cooperatively and find that PafA allostery is controlled by the binding of target protein substrates, yet is unaffected by Pup binding. Analysis of PafA pupylation using engineered substrates differing in the number of pupylation sites points to PafA acting as a dimer. These findings suggest that protein pupylation can be regulated at the level of PafA allostery. PMID:23471967

  3. Allosteric interactions and bifunctionality make the response of glutamine synthetase cascade system of Escherichia coli robust and ultrasensitive.

    Science.gov (United States)

    Mutalik, Vivek K; Shah, Parag; Venkatesh, K V

    2003-07-18

    Glutamine synthetase (GS) regulation in Escherichia coli by reversible covalent modification cycles is a prototype of signal transduction by enzyme cascades. Such enzyme cascades are known to exhibit ultrasensitive response to primary stimuli and act as signal integration systems. Here, we have quantified GS bicyclic cascade based on steady state analysis by evaluating Hill coefficient. We demonstrate that adenylylation of GS with glutamine as input is insensitive to total enzyme concentrations of GS, uridylyltransferase/uridylyl-removing enzyme, regulatory protein PII, and adenylyltransferase/adenylyl-removing enzyme. This robust response of GS adenylylation is also observed for change in system parameters. From numerical analyses, we show that the robust ultrasensitive response of bicyclic cascade is because of allosteric interactions of glutamine and 2-ketoglutarate, bifunctionality of converter enzymes, and closed loop bicyclic cascade structure. By system level quantification of the GS bicyclic cascade, we conclude that such a robust response may help the cell in adapting to different carbon and nitrogen status. PMID:12676964

  4. Allosteric Inhibitory Molecular Recognition of a Photochromic Dye by a Digestive Enzyme: Dihydroindolizine makes α-chymotrypsin Photo-responsive

    Science.gov (United States)

    Bagchi, Damayanti; Ghosh, Abhijit; Singh, Priya; Dutta, Shreyasi; Polley, Nabarun; Althagafi, Ismail. I.; Jassas, Rabab S.; Ahmed, Saleh A.; Pal, Samir Kumar

    2016-09-01

    The structural-functional regulation of enzymes by the administration of an external stimulus such as light could create photo-switches that exhibit unique biotechnological applications. However, molecular recognition of small ligands is a central phenomenon involved in all biological processes. We demonstrate herein that the molecular recognition of a photochromic ligand, dihydroindolizine (DHI), by serine protease α-chymotrypsin (CHT) leads to the photo-control of enzymatic activity. We synthesized and optically characterized the photochromic DHI. Light-induced reversible pyrroline ring opening and a consequent thermal back reaction via 1,5-electrocyclization are responsible for the photochromic behavior. Furthermore, DHI inhibits the enzymatic activity of CHT in a photo-controlled manner. Simultaneous binding of the well-known inhibitors 4-nitrophenyl anthranilate (NPA) or proflavin (PF) in the presence of DHI displays spectral overlap between the emission of CHT-NPA or CHT-PF with the respective absorption of cis or trans DHI. The results suggest an opportunity to explore the binding site of DHI using Förster resonance energy transfer (FRET). Moreover, to more specifically evaluate the DHI binding interactions, we employed molecular docking calculations, which suggested binding near the hydrophobic site of Cys-1-Cys-122 residues. Variations in the electrostatic interactions of the two conformers of DHI adopt unfavorable conformations, leading to the allosteric inhibition of enzymatic activity.

  5. Allosteric activation of SENP1 by SUMO1 β-grasp domain involves a dock-and-coalesce mechanism

    Science.gov (United States)

    Guo, Jingjing; Zhou, Huan-Xiang

    2016-01-01

    Small ubiquitin-related modifiers (SUMOs) are conjugated to proteins to regulate a variety of cellular processes. SENPs are cysteine proteases with a catalytic center located within a channel between two subdomains that catalyzes SUMO C-terminal cleavage for processing of SUMO precursors and de-SUMOylation of target proteins. The β-grasp domain of SUMOs binds to an exosite cleft, and allosterically activates SENPs via an unknown mechanism. Our molecular dynamics simulations showed that binding of the β-grasp domain induces significant conformational and dynamic changes in SENP1, including widening of the exosite cleft and quenching of nanosecond dynamics in all but a distal region. A dock-and-coalesce mechanism emerges for SENP-catalyzed SUMO cleavage: the wedging of the β-grasp domain enables the docking of the proximal portion of the C-terminus and the strengthened cross-channel motional coupling initiates inter-subdomain correlated motions to allow for the distal portion to coalesce around the catalytic center. DOI: http://dx.doi.org/10.7554/eLife.18249.001 PMID:27576863

  6. 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

  7. 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...

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    -citalopram, sertraline,       serotonin and paroxetine. EC50 values for S- and R-citalopram are 3.6 +/-       0.4 microm and 19.4 +/- 2.3 microm, respectively. Fluoxetine, venlafaxine       and duloxetine have no significant effect on the dissociation of       [3H]S-citalopram. Allosteric modulation of dissociation...

  9. Asymmetric processing of a substrate protein in sequential allosteric cycles of AAA+ nanomachines

    Science.gov (United States)

    Kravats, Andrea N.; Tonddast-Navaei, Sam; Bucher, Ryan J.; Stan, George

    2013-09-01

    Essential protein quality control includes mechanisms of substrate protein (SP) unfolding and translocation performed by powerful ring-shaped AAA+ (ATPases associated with various cellular activities) nanomachines. These SP remodeling actions are effected by mechanical forces imparted by AAA+ loops that protrude into the central channel. Sequential intra-ring allosteric motions, which underlie repetitive SP-loop interactions, have been proposed to comprise clockwise (CW), counterclockwise (CCW), or random (R) conformational transitions of individual AAA+ subunits. To probe the effect of these allosteric mechanisms on unfoldase and translocase functions, we perform Langevin dynamics simulations of a coarse-grained model of an all-alpha SP processed by the single-ring ClpY ATPase or by the double-ring p97 ATPase. We find that, in all three allosteric mechanisms, the SP undergoes conformational transitions along a common set of pathways, which reveals that the active work provided by the ClpY machine involves single loop-SP interactions. Nevertheless, the rates and yields of SP unfolding and translocation are controlled by mechanism-dependent loop-SP binding events, as illustrated by faster timescales of SP processing in CW allostery compared with CCW and R allostery. The distinct efficacy of allosteric mechanisms is due to the asymmetric collaboration of adjacent subunits, which involves CW-biased structural motions of AAA+ loops and results in CW-compatible torque applied onto the SP. Additional simulations of mutant ClpY rings, which render a subset of subunits catalytically-defective or reduce their SP binding affinity, reveal that subunit-based conformational transitions play the major role in SP remodeling. Based on these results we predict that the minimally functional AAA+ ring includes three active subunits, only two of which are adjacent.

  10. 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 ...

  11. 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 ...

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  13. 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

  14. Dissecting allosteric effects of activator-coactivator complexes using a covalent small molecule ligand.

    Science.gov (United States)

    Wang, Ningkun; Lodge, Jean M; Fierke, Carol A; Mapp, Anna K

    2014-08-19

    Allosteric binding events play a critical role in the formation and stability of transcriptional activator-coactivator complexes, perhaps in part due to the often intrinsically disordered nature of one or more of the constituent partners. The kinase-inducible domain interacting (KIX) domain of the master coactivator CREB binding protein/p300 is a conformationally dynamic domain that complexes with transcriptional activators at two discrete binding sites in allosteric communication. The complexation of KIX with the transcriptional activation domain of mixed-lineage leukemia protein leads to an enhancement of binding by the activation domain of CREB (phosphorylated kinase-inducible domain of CREB) to the second site. A transient kinetic analysis of the ternary complex formation aided by small molecule ligands that induce positive or negative cooperative binding reveals that positive cooperativity is largely governed by stabilization of the bound complex as indicated by a decrease in koff. Thus, this suggests the increased binding affinity for the second ligand is not due to an allosteric creation of a more favorable binding interface by the first ligand. This is consistent with data from us and from others indicating that the on rates of conformationally dynamic proteins approach the limits of diffusion. In contrast, negative cooperativity is manifested by alterations in both kon and koff, suggesting stabilization of the binary complex.

  15. 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

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

    Science.gov (United States)

    Das, Debasis; Krantz, Bryan A.

    2016-01-01

    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

  17. Compact modeling of allosteric multisite proteins: application to a cell size checkpoint.

    Directory of Open Access Journals (Sweden)

    Germán Enciso

    2014-02-01

    Full Text Available We explore a framework to model the dose response of allosteric multisite phosphorylation proteins using a single auxiliary variable. This reduction can closely replicate the steady state behavior of detailed multisite systems such as the Monod-Wyman-Changeux allosteric model or rule-based models. Optimal ultrasensitivity is obtained when the activation of an allosteric protein by its individual sites is concerted and redundant. The reduction makes this framework useful for modeling and analyzing biochemical systems in practical applications, where several multisite proteins may interact simultaneously. As an application we analyze a newly discovered checkpoint signaling pathway in budding yeast, which has been proposed to measure cell growth by monitoring signals generated at sites of plasma membrane growth. We show that the known components of this pathway can form a robust hysteretic switch. In particular, this system incorporates a signal proportional to bud growth or size, a mechanism to read the signal, and an all-or-none response triggered only when the signal reaches a threshold indicating that sufficient growth has occurred.

  18. Intrinsic Enzyme Dynamics in the Unbound State and Relation to Allosteric Regulation

    OpenAIRE

    Bahar, Ivet; Chennubhotla, Chakra; Tobi, Dror

    2007-01-01

    In recent years, there has been a surge in the number of studies exploring the relationship between proteins’ equilibrium dynamics and structural changes involved in function. An emerging concept, supported by both theory and experiments, is that under native state conditions proteins have an intrinsic ability to sample conformations that meet functional requirements. A typical example is the ability of enzymes to sample open and closed forms, irrespective of substrate, succeeded by the stabi...

  19. Hemoglobin isoform differentiation and allosteric regulation of oxygen binding in the turtle, Trachemys scripta

    DEFF Research Database (Denmark)

    Damsgaard, Christian; Storz, Jay F.; Hoffmann, Federico G.;

    2013-01-01

    When freshwater turtles acclimatize to winter hibernation, there is a gradual transition from aerobic to anaerobic metabolism, which may require adjustments of blood O2 transport before turtles become anoxic. Here, we report the effects of protons, anionic cofactors, and temperature on the O2......-binding properties of isolated hemoglobin (Hb) isoforms, HbA and HbD, in the turtle Trachemys scripta. We determined the primary structures of the constituent subunits of the two Hb isoforms, and we related the measured functional properties to differences in O2 affinity between untreated hemolysates from...... turtles that were acclimated to normoxia and anoxia. Our data show that HbD has a consistently higher O2 affinity compared with HbA, whereas Bohr and temperature effects, as well as thiol reactivity, are similar. Although sequence data show amino acid substitutions at two known β-chain ATP-binding site...

  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. M2 pyruvate kinase provides a mechanism for nutrient sensing and regulation of cell proliferation

    OpenAIRE

    Morgan, Hugh P.; O???Reilly, Francis J.; Wear, Martin A.; O'Neill, Robert; Fothergill-Gilmore, Linda A.; Hupp, Ted; Walkinshaw, Malcolm D.

    2013-01-01

    We show that the M2 isoform of pyruvate kinase (M2PYK) exists in equilibrium between monomers and tetramers regulated by allosteric binding of naturally occurring small-molecule metabolites. Phenylalanine stabilizes an inactive T-state tetrameric conformer and inhibits M2PYK with an IC50 value of 0.24 mM, whereas thyroid hormone (triiodo-l-thyronine, T3) stabilizes an inactive monomeric form of M2PYK with an IC50 of 78 nM. The allosteric activator fructose-1,6-bisphosphate [F16BP, AC50 (conce...

  2. Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1.

    Science.gov (United States)

    Fay, Jonathan F; Farrens, David L

    2015-07-01

    G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB1. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB1-it simultaneously increases agonist binding, decreases G--protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB1 and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB1 structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling.

  3. Allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation.

    Science.gov (United States)

    Wu, Zhuang; Li, Linlang; Zheng, Long-Tai; Xu, Zhihong; Guo, Lin; Zhen, Xuechu

    2015-09-01

    Recent studies have shown that sigma-1 receptor orthodox agonists can inhibit neuroinflammation. SKF83959 (3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine), an atypical dopamine receptor-1 agonist, has been recently identified as a potent allosteric modulator of sigma-1 receptor. Here, we investigated the anti-inflammatory effects of SKF83959 in lipopolysaccharide (LPS)-stimulated BV2 microglia. Our results indicated that SKF83959 significantly suppressed the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), and inhibited the generation of reactive oxygen species. All of these responses were blocked by selective sigma-1 receptor antagonists (BD1047 or BD1063) and by ketoconazole (an inhibitor of enzyme cytochrome c17 to inhibit the synthesis of endogenous dehydroepiandrosterone, DHEA). Additionally, we found that SKF83959 promoted the binding activity of DHEA with sigma-1 receptors, and enhanced the inhibitory effects of DHEA on LPS-induced microglia activation in a synergic manner. Furthermore, in a microglia-conditioned media system, SKF83959 inhibited the cytotoxicity of conditioned medium generated by LPS-activated microglia toward HT-22 neuroblastoma cells. Taken together, our study provides the first evidence that allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation. SKF83959 is a potent allosteric modulator of sigma-1 receptor. Our results indicated that SKF83959 enhanced the activity of endogenous dehydroepiandrosterone (DHEA) in a synergic manner, and inhibited the activation of BV2 microglia and the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS).

  4. 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

  5. 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.

  6. Changes in BQCA Allosteric Modulation of [(3)H]NMS Binding to Human Cortex within Schizophrenia and by Divalent Cations.

    Science.gov (United States)

    Dean, Brian; Hopper, Shaun; Conn, P Jeffrey; Scarr, Elizabeth

    2016-05-01

    Stimulation of the cortical muscarinic M1 receptor (CHRM1) is proposed as a treatment for schizophrenia, a hypothesis testable using CHRM1 allosteric modulators. Allosteric modulators have been shown to change the activity of CHRMs using cloned human CHRMs and CHRM knockout mice but not human CNS, a prerequisite for them working in humans. Here we show in vitro that BQCA, a positive allosteric CHRM1 modulator, brings about the expected change in affinity of the CHRM1 orthosteric site for acetylcholine in human cortex. Moreover, this effect of BQCA is reduced in the cortex of a subset of subjects with schizophrenia, separated into a discrete population because of a profound loss of cortical [(3)H]pirenzepine binding. Surprisingly, there was no change in [(3)H]NMS binding to the cortex from this subset or those with schizophrenia but without a marked loss of cortical CHRM1. Hence, we explored the nature of [(3)H]pirenzepine and [(3)H]NMS binding to human cortex and showed total [(3)H]pirenzepine and [(3)H]NMS binding was reduced by Zn(2+), acetylcholine displacement of [(3)H]NMS binding was enhanced by Mg(2+) and Zn(2+), acetylcholine displacement of [(3)H]pirenzepine was reduced by Mg(2+) and enhanced by Zn(2+), whereas BQCA effects on [(3)H]NMS, but not [(3)H]pirenzepine, binding was enhanced by Mg(2+) and Zn(2+). These data suggest the orthosteric and allosteric sites on CHRMs respond differently to divalent cations and the effects of allosteric modulation of the cortical CHRM1 is reduced in a subset of people with schizophrenia, a finding that may have ramifications for the use of CHRM1 allosteric modulators in the treatment of schizophrenia.

  7. Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor.

    Science.gov (United States)

    Miao, Yinglong; Goldfeld, Dahlia Anne; Moo, Ee Von; Sexton, Patrick M; Christopoulos, Arthur; McCammon, J Andrew; Valant, Celine

    2016-09-20

    Design of ligands that provide receptor selectivity has emerged as a new paradigm for drug discovery of G protein-coupled receptors, and may, for certain families of receptors, only be achieved via identification of chemically diverse allosteric modulators. Here, the extracellular vestibule of the M2 muscarinic acetylcholine receptor (mAChR) is targeted for structure-based design of allosteric modulators. Accelerated molecular dynamics (aMD) simulations were performed to construct structural ensembles that account for the receptor flexibility. Compounds obtained from the National Cancer Institute (NCI) were docked to the receptor ensembles. Retrospective docking of known ligands showed that combining aMD simulations with Glide induced fit docking (IFD) provided much-improved enrichment factors, compared with the Glide virtual screening workflow. Glide IFD was thus applied in receptor ensemble docking, and 38 top-ranked NCI compounds were selected for experimental testing. In [(3)H]N-methylscopolamine radioligand dissociation assays, approximately half of the 38 lead compounds altered the radioligand dissociation rate, a hallmark of allosteric behavior. In further competition binding experiments, we identified 12 compounds with affinity of ≤30 μM. With final functional experiments on six selected compounds, we confirmed four of them as new negative allosteric modulators (NAMs) and one as positive allosteric modulator of agonist-mediated response at the M2 mAChR. Two of the NAMs showed subtype selectivity without significant effect at the M1 and M3 mAChRs. This study demonstrates an unprecedented successful structure-based approach to identify chemically diverse and selective GPCR allosteric modulators with outstanding potential for further structure-activity relationship studies. PMID:27601651

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

    DEFF Research Database (Denmark)

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

    2003-01-01

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

  9. 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...

  10. 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.

  11. Chemogenomic discovery of allosteric antagonists at the GPRC6A receptor

    DEFF Research Database (Denmark)

    Gloriam, David E.; Wellendorph, Petrine; Johansen, Lars Dan;

    2011-01-01

    and pharmacological character: (1) chemogenomic lead identification through the first, to our knowledge, ligand inference between two different GPCR families, Families A and C; and (2) the discovery of the most selective GPRC6A allosteric antagonists discovered to date. The unprecedented inference of...... pharmacological activity across GPCR families provides proof-of-concept for in silico approaches against Family C targets based on Family A templates, greatly expanding the prospects of successful drug design and discovery. The antagonists were tested against a panel of seven Family A and C G protein-coupled receptors...

  12. 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...

  13. 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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  15. ThermoTRP channels as modular proteins with allosteric gating.

    Science.gov (United States)

    Latorre, Ramon; Brauchi, Sebastian; Orta, Gerardo; Zaelzer, Cristián; Vargas, Guillermo

    2007-01-01

    . Caspani, P.A. Heppenstall, Direct activation of the ion channel TRPA1 by Ca(2+), Nat. Neurosci. 10 (2007) 277-279]. These stimuli include voltage, pH, agonist binding, and temperature. Understanding how each of these distinct physiological signals regulate channel opening will be informative about the mechanical linkages that can act either independently or in concert to influence channel activation. In this paper we show that thermoTRP channel-forming proteins are modular in the sense that certain structure or structures (modules) confer temperature-dependent regulation, whereas others confer voltage-dependent regulation. We also discuss the thermodynamic basis of heat and cold activation in an effort to elucidate what confer to these channels the capability to be gated by temperature directly. PMID:17499848

  16. Regulation of nitrite transport in red blood cells by hemoglobin oxygen fractional saturation

    OpenAIRE

    Vitturi, Dario A; Teng, Xinjun; Toledo, José C.; Matalon, Sadis; Lancaster, Jack R.; Patel, Rakesh P.

    2009-01-01

    Allosteric regulation of nitrite reduction by deoxyhemoglobin has been proposed to mediate nitric oxide (NO) formation during hypoxia. Nitrite is predominantly an anion at physiological pH, raising questions about the mechanism by which it enters the red blood cell (RBC) and whether this is regulated and coupled to deoxyhemoglobin-mediated reduction. We tested the hypothesis that nitrite transport by RBCs is regulated by fractional saturation. Using human RBCs, nitrite consumption was faster ...

  17. A substrate-driven allosteric switch that enhances PDI catalytic activity

    Science.gov (United States)

    Bekendam, Roelof H.; Bendapudi, Pavan K.; Lin, Lin; Nag, Partha P.; Pu, Jun; Kennedy, Daniel R.; Feldenzer, Alexandra; Chiu, Joyce; Cook, Kristina M.; Furie, Bruce; Huang, Mingdong; Hogg, Philip J.; Flaumenhaft, Robert

    2016-01-01

    Protein disulfide isomerase (PDI) is an oxidoreductase essential for folding proteins in the endoplasmic reticulum. The domain structure of PDI is a–b–b′–x–a′, wherein the thioredoxin-like a and a′ domains mediate disulfide bond shuffling and b and b′ domains are substrate binding. The b′ and a′ domains are connected via the x-linker, a 19-amino-acid flexible peptide. Here we identify a class of compounds, termed bepristats, that target the substrate-binding pocket of b′. Bepristats reversibly block substrate binding and inhibit platelet aggregation and thrombus formation in vivo. Ligation of the substrate-binding pocket by bepristats paradoxically enhances catalytic activity of a and a′ by displacing the x-linker, which acts as an allosteric switch to augment reductase activity in the catalytic domains. This substrate-driven allosteric switch is also activated by peptides and proteins and is present in other thiol isomerases. Our results demonstrate a mechanism whereby binding of a substrate to thiol isomerases enhances catalytic activity of remote domains. PMID:27573496

  18. 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

  19. 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.

  20. Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and Orally Efficacious Phosphatase Inhibitor.

    Science.gov (United States)

    Garcia Fortanet, Jorge; Chen, Christine Hiu-Tung; Chen, Ying-Nan P; Chen, Zhouliang; Deng, Zhan; Firestone, Brant; Fekkes, Peter; Fodor, Michelle; Fortin, Pascal D; Fridrich, Cary; Grunenfelder, Denise; Ho, Samuel; Kang, Zhao B; Karki, Rajesh; Kato, Mitsunori; Keen, Nick; LaBonte, Laura R; Larrow, Jay; Lenoir, Francois; Liu, Gang; Liu, Shumei; Lombardo, Franco; Majumdar, Dyuti; Meyer, Matthew J; Palermo, Mark; Perez, Lawrence; Pu, Minying; Ramsey, Timothy; Sellers, William R; Shultz, Michael D; Stams, Travis; Towler, Christopher; Wang, Ping; Williams, Sarah L; Zhang, Ji-Hu; LaMarche, Matthew J

    2016-09-01

    SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein-ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor. PMID:27347692

  1. Discovery of allosteric modulators for GABAA receptors by ligand-directed chemistry.

    Science.gov (United States)

    Yamaura, Kei; Kiyonaka, Shigeki; Numata, Tomohiro; Inoue, Ryuji; Hamachi, Itaru

    2016-10-01

    The fast inhibitory actions of γ-aminobutyric acid (GABA) are mainly mediated by GABAA receptors (GABAARs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABAARs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABAAR ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABAARs. Electrophysiological measurements revealed that one hit, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABAAR-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins. PMID:27526031

  2. 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

  3. Amyloid-β peptides act as allosteric modulators of cholinergic signalling through formation of soluble BAβACs.

    Science.gov (United States)

    Kumar, Rajnish; Nordberg, Agneta; Darreh-Shori, Taher

    2016-01-01

    -β interacts readily in an apolipoprotein-facilitated manner with butyrylcholinesterase, forming highly stable and soluble complexes, BAβACs, which can be separated in their native states by sucrose density gradient technique. Enzymological analyses further evinced that amyloid-β concentration dependently increased the acetylcholine-hydrolyzing capacity of cholinesterases. In silico biomolecular analysis further deciphered the allosteric amino acid fingerprint of the amyloid-β-cholinesterase molecular interaction in formation of BAβACs. In the case of butyrylcholinesterase, the results indicated that amyloid-β interacts with a putative activation site at the mouth of its catalytic tunnel, most likely leading to increased acetylcholine influx into the catalytic site, and thereby increasing the intrinsic catalytic rate of butyrylcholinesterase. In conclusion, at least one of the native physiological functions of amyloid-β is allosteric modulation of the intrinsic catalytic efficiency of cholinesterases, and thereby regulation of synaptic and extrasynaptic cholinergic signalling. High apolipoprotein-E may pathologically alter the biodynamics of this amyloid-β function.

  4. 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;

    2014-01-01

    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...

  5. 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

  6. 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.

  7. 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]...

  8. Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by xmeta, an allosteric partial agonist antibody

    Science.gov (United States)

    XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

  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

    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...

  10. Cellular regulation by protein phosphorylation.

    Science.gov (United States)

    Fischer, Edmond H

    2013-01-11

    A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca(2+) and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert.

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

    Science.gov (United States)

    Al-Horani, Rami A; Karuturi, Rajesh; Lee, Michael; Afosah, Daniel K; Desai, Umesh R

    2016-01-01

    Factor XIIIa (FXIIIa) is a transglutaminase that catalyzes the last step in the coagulation process. Orthostery is the only approach that has been exploited to design FXIIIa inhibitors. Yet, allosteric inhibition of FXIIIa is a paradigm that may offer a key advantage of controlled inhibition over orthosteric inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We reasoned that targeting a collection of basic amino acid residues distant from FXIIIa's active site by using sulfated glycosaminoglycans (GAGs) or non-saccharide GAG mimetics (NSGMs) would lead to the discovery of the first allosteric FXIIIa inhibitors. We tested a library of 22 variably sulfated GAGs and NSGMs against human FXIIIa to discover promising hits. Interestingly, although some GAGs bound to FXIIIa better than NSGMs, no GAG displayed any inhibition. An undecasulfated quercetin analog was found to inhibit FXIIIa with reasonable potency (efficacy of 98%). Michaelis-Menten kinetic studies revealed an allosteric mechanism of inhibition. Fluorescence studies confirmed close correspondence between binding affinity and inhibition potency, as expected for an allosteric process. The inhibitor was reversible and at least 9-fold- and 26-fold selective over two GAG-binding proteins factor Xa (efficacy of 71%) and thrombin, respectively, and at least 27-fold selective over a cysteine protease papain. The inhibitor also inhibited the FXIIIa-mediated polymerization of fibrin in vitro. Overall, our work presents the proof-of-principle that FXIIIa can be allosterically modulated by sulfated non-saccharide agents much smaller than GAGs, which should enable the design of selective and safe anticoagulants. PMID:27467511

  12. Allosteric Modulation of Beta1 Integrin Function Induces Lung Tissue Repair

    Directory of Open Access Journals (Sweden)

    Rehab AlJamal-Naylor

    2012-01-01

    Full Text Available The cellular cytoskeleton, adhesion receptors, extracellular matrix composition, and their spatial distribution are together fundamental in a cell's balanced mechanical sensing of its environment. We show that, in lung injury, extracellular matrix-integrin interactions are altered and this leads to signalling alteration and mechanical missensing. The missensing, secondary to matrix alteration and cell surface receptor alterations, leads to increased cellular stiffness, injury, and death. We have identified a monoclonal antibody against β1 integrin which caused matrix remodelling and enhancement of cell survival. The antibody acts as an allosteric dual agonist/antagonist modulator of β1 integrin. Intriguingly, this antibody reversed both functional and structural tissue injury in an animal model of degenerative disease in lung.

  13. The sweet taste of true synergy: positive allosteric modulation of the human sweet taste receptor.

    Science.gov (United States)

    Servant, Guy; Tachdjian, Catherine; Li, Xiaodong; Karanewsky, Donald S

    2011-11-01

    A diet low in carbohydrates helps to reduce the amount of ingested calories and to maintain a healthy weight. With this in mind, food and beverage companies have reformulated a large number of their products, replacing sugar or high fructose corn syrup with several different types of zero-calorie sweeteners to decrease or even totally eliminate their caloric content. A challenge remains, however, with the level of acceptance of some of these products in the market-place. Many consumers believe that zero-calorie sweeteners simply do not taste like sugar. A recent breakthrough reveals that positive allosteric modulators of the human sweet taste receptor, small molecules that enhance the receptor activity and sweetness perception, could be more effective than other reported taste enhancers at reducing calories in consumer products without compromising on the true taste of sugar. A unique mechanism of action at the receptor level could explain the robust synergy achieved with these new modulators.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. Allosteric inhibition of factor XIa. Sulfated non-saccharide glycosaminoglycan mimetics as promising anticoagulants.

    Science.gov (United States)

    Al-Horani, Rami A; Gailani, David; Desai, Umesh R

    2015-08-01

    Recent development of sulfated non-saccharide glycosaminoglycan mimetics, especially sulfated pentagalloyl glucopyranoside (SPGG), as potent inhibitors of factor XIa (FXIa) (J. Med. Chem. 2013; 56:867-878 and J. Med. Chem. 2014; 57:4805-4818) has led to a strong possibility of developing a new line of factor XIa-based anticoagulants. In fact, SPGG represents the first synthetic, small molecule inhibitor that appears to bind in site remote from the active site. Considering that allosteric inhibition of FXIa is a new mechanism for developing a distinct line of anticoagulants, we have studied SPGG's interaction with FXIa with a goal of evaluating its pre-clinical relevance. Comparative inhibition studies with several glycosaminoglycans revealed the importance of SPGG's non-saccharide backbone. SPGG did not affect the activity of plasma kallikrein, activated protein C and factor XIIIa suggesting that SPGG-based anticoagulation is unlikely to affect other pathways connected with coagulation factors. SPGG's effect on APTT of citrated human plasma was also not dependent on antithrombin or heparin cofactor II. Interestingly, SPGG's anticoagulant potential was diminished by serum albumin as well as factor XI, while it could be reversed by protamine or polybrene, which implies possible avenues for developing antidote strategy. Studies with FXIa mutants indicated that SPGG engages Lys529, Arg530 and Arg532, but not Arg250, Lys252, Lys253 and Lys255. Finally, SPGG competes with unfractionated heparin, but not with polyphosphates and/or glycoprotein Ibα, for binding to FXIa. These studies enhance understanding on the first allosteric inhibitor of FXIa and highlight its value as a promising anticoagulant. PMID:25935648

  20. 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.

  1. Changes of IK,ATP current density and allosteric modulation during chronic atrial fibrillation

    Institute of Scientific and Technical Information of China (English)

    WU Gang; HUANG Cong-xin; TANG Yan-hong; JIANG Hong; WAN Jun; CHEN Hui; XIE Qiang; HUANG Zheng-rong

    2005-01-01

    Background Atrial fibrillation (AF) is the most common supraventricular arrhythmia in clinical practice. Chronic atrial fibrillation (CAF) is associated with ionic remodeling. However, little is known about the activity of ATP-sensitive potassium current (IK,ATP) during CAF. So we studied the changes of IK,ATP density and allosteric modulation of ATP-sensitivity by intracellular pH during CAF.Methods Myocardium samples were obtained from the right auricular appendage of patients with rheumatic heart disease complicated with valvular disease in sinus rhythm (SR) or CAF. There were 14 patients in SR group and 9 patients in CAF group. Single atrial cells were isolated using an enzyme dispersion technique. IK,ATP was recorded using the whole-cell and inside-out configuration of voltage-clamp techniques. In whole-cell model, myocytes of SR and CAF groups were perfused with simulated ischemic solution to elicit IK,ATP. In inside-out configuration, the internal patch membranes were exposed to different ATP concentrations in pH 7.4 and 6.8.Results Under simulated ischemia, IK,ATP current density of CAF group was significantly higher than in SR group [(83.5±10.8) vs. (58.7±8.4) pA/pF, P<0.01]. IK,ATP of the two groups showed ATP concentration-dependent inhibition. The ATP concentration for 50% current inhibition (IC50) for the SR group was significantly different in pH 7.4 and pH 6.8 (24 vs. 74 μmol/L, P<0.01). The IC50 did not change significantly in CAF group when the pH decreased from 7.4 to 6.8.Conclusions During CAF, IK,ATP current density was increased and its allosteric modulation of ATP-sensitivity by intracellular pH was diminished.

  2. 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 ...

  3. Probing molecular mechanisms of the Hsp90 chaperone: biophysical modeling identifies key regulators of functional dynamics.

    Directory of Open Access Journals (Sweden)

    Anshuman Dixit

    Full Text Available Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based "conformational selection" of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected

  4. 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

  5. Allosteric-Activation Mechanism of Bovine Chymosin Revealed by Bias-Exchange Metadynamics and Molecular Dynamics Simulations

    DEFF Research Database (Denmark)

    Ansari, Samiul M; Coletta, Andrea; Skeby, Katrine Kirkeby;

    2016-01-01

    -inhibited conformation in which the side chain of Tyr77 occludes the binding site. On the basis of kinetic, mutagenesis and crystallographic data, it has been widely reported that a HPHPH sequence in the P8-P4 residues of the natural substrate κ-casein acts as the allosteric activator, but the mechanism by which...... to vacate a pocket that may then be occupied by the side chain of Tyr77. The free energy surface for the self-inhibited to open transition is significantly altered by the presence of the HPHPH sequence of κ-casein....... and to compute the free energy surface for the process. The simulations reveal that allosteric activation is initiated by interactions between the HPHPH sequence of κ-casein and a small α-helical region of chymosin (residues 112-116). A small conformational change in the α-helix causes the side chain of Phe114...

  6. 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

  7. 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...

  8. 5-(N, N-Hexamethylene) amiloride is a GABA-A ρ1 receptor positive allosteric modulator.

    Science.gov (United States)

    Snell, Heather D; Gonzales, Eric B

    2016-11-01

    Guanidine compounds act as ion channel modulators. In the case of Cys-loop receptors, the guanidine compound amiloride antagonized the heteromeric GABA-A, glycine, and nicotinic acetylcholine receptors. However, amiloride exhibits characteristics consistent with a positive allosteric modulator for the human GABA-A (hGABA-A) ρ1 receptor. Site-directed mutagenesis revealed that the positive allosteric modulation was influenced by the GABA-A ρ1 second transmembrane domain 15' position, a site implicated in ligand allosteric modulation of Cys-loop receptors. There are a variety of amiloride derivatives that provide opportunities to assess the significance of amiloride functional groups (e.g., the guanidine group, the pyrazine ring, etc.) in the modulation of the GABA-A ρ1 receptor activity. We utilized 3 amiloride derivatives (benzamil, phenamil, and 5-(N, N-Hexamethylene) amiloride) to assess the contribution of these groups toward the potentiation of the GABA-A ρ1 receptor. Benzamil and phenamil failed to potentiate on the wild type GABA-A ρ1 GABA-mediated current while HMA demonstrated efficacy only at the highest concentration studied. The hGABA-A ρ1 (I15'N) mutant receptor activity was potentiated by lower HMA concentrations compared to the wild type receptor. Our findings suggest that an exposed guanidine group on amiloride and amiloride derivatives is critical for modulating the GABA-A ρ1 receptor. The present study provides a conceptual framework for predicting which amiloride derivatives will demonstrate positive allosteric modulation of the GABA-A ρ1 receptor.

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

    DEFF Research Database (Denmark)

    Krintel, Christian; Frydenvang, Karla; Olsen, Lars;

    2012-01-01

    Positive allosteric modulators of the ionotropic glutamate receptor-2 (GluA2) are promising compounds for the treatment of cognitive disorders, e.g. Alzheimer's disease. These modulators bind within the dimer interface of the ligand-binding domain and stabilize the agonist-bound conformation...... by the ethyl substituent of BPAM-97. These results add important information on binding affinities and thermodynamic details, and provide a new tool in development of drugs against cognitive disorders....

  10. Structure-activity relationships of substituted 1H-indole-2-carboxamides as CB1 receptor allosteric modulators.

    Science.gov (United States)

    Nguyen, Thuy; German, Nadezhda; Decker, Ann M; Li, Jun-Xu; Wiley, Jenny L; Thomas, Brian F; Kenakin, Terry P; Zhang, Yanan

    2015-05-01

    A series of substituted 1H-indole-2-carboxamides structurally related to compounds Org27569 (1), Org29647 (2) and Org27759 (3) were synthesized and evaluated for CB1 allosteric modulating activity in calcium mobilization assays. Structure-activity relationship studies showed that the modulation potency of this series at the CB1 receptor was enhanced by the presence of a diethylamino group at the 4-position of the phenyl ring, a chloro or fluoro group at the C5 position and short alkyl groups at the C3 position on the indole ring. The most potent compound (45) had an IC₅₀ value of 79 nM which is ∼2.5 and 10 fold more potent than the parent compounds 3 and 1, respectively. These compounds appeared to be negative allosteric modulators at the CB1 receptor and dose-dependently reduced the Emax of agonist CP55,940. These analogs may provide the basis for further optimization and use of CB1 allosteric modulators.

  11. 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

  12. Novel small-molecule AMP-activated protein kinase allosteric activator with beneficial effects in db/db mice.

    Directory of Open Access Journals (Sweden)

    Li-Na Zhang

    Full Text Available AMP-activated protein kinase (AMPK is an energy sensor of metabolism that is an attractive therapeutic target for type 2 diabetes mellitus and metabolic syndrome. Using a homogeneous scintillation proximity assay (SPA, we identified a new small-molecule AMPK activator, ZLN024, which allosterically stimulated active AMPK heterotrimers and the inactive α1 subunit truncations α1 (1-394 and α1 (1-335 but not α1 (1-312. AMPK activation by ZLN024 requires the pre-phosphorylation of Thr-172 by at least one upstream kinase and protects AMPK Thr-172 against dephosphorylation by PP2Cα. ZLN024 activated AMPK in L6 myotubes and stimulated glucose uptake and fatty acid oxidation without increasing the ADP/ATP ratio. ZLN024 also activated AMPK in primary hepatocytes, decreased fatty acid synthesis and glucose output. Treatment of db/db mice with 15 mg/kg/day ZLN024 improved glucose tolerance; liver tissue weight, triacylglycerol and the total cholesterol content were decreased. The hepatic transcriptional level of G6Pase, FAS and mtGPAT were reduced. The transcription of genes involved in fatty acid oxidation and the mitochondrial biogenesis of muscle tissue were elevated. The ACC phosphorylation was increased in muscle and liver. This study provides a novel allosteric AMPK activator for functional study in vitro and in vivo and demonstrates that AMPK allosteric activators could be a promising therapeutic approach for type 2 diabetes mellitus and metabolic syndrome.

  13. Exploitation of latent protein allostery enables the evolution of novel and divergent MAP kinase regulation

    Science.gov (United States)

    Coyle, Scott M.; Flores, Jonathan; Lim, Wendell A.

    2013-01-01

    SUMMARY Allosteric interactions provide precise spatiotemporal control over signaling proteins, but how allosteric activators and their targets co-evolve is poorly understood. Here, we trace the evolution of two allosteric activator motifs within the yeast scaffold protein Ste5 that specifically target the mating MAP kinase Fus3. One activator (Ste5-VWA) provides pathway insulation and dates to the divergence of Fus3 from its paralog, Kss1; a second activator (Ste5-FBD) that tunes mating behavior is, in contrast, not conserved in most lineages. Surprisingly, both Ste5 activator motifs could regulate MAP kinases that diverged from Fus3 prior to the emergence of Ste5, suggesting that Ste5 activators arose by exploiting latent regulatory features already present in the MAPK ancestor. The magnitude of this latent allosteric potential drifts widely among pre-Ste5 MAP kinases, providing a pool of hidden phenotypic diversity that, when revealed by new activators, could lead to functional divergence and the evolution of distinct signaling behaviors. PMID:23953117

  14. Exploitation of latent allostery enables the evolution of new modes of MAP kinase regulation.

    Science.gov (United States)

    Coyle, Scott M; Flores, Jonathan; Lim, Wendell A

    2013-08-15

    Allosteric interactions provide precise spatiotemporal control over signaling proteins, but how allosteric activators and their targets coevolve is poorly understood. Here, we trace the evolution of two allosteric activator motifs within the yeast scaffold protein Ste5 that specifically target the mating MAP kinase Fus3. One activator (Ste5-VWA) provides pathway insulation and dates to the divergence of Fus3 from its paralog, Kss1; a second activator (Ste5-FBD) that tunes mating behavior is, in contrast, not conserved in most lineages. Surprisingly, both Ste5 activator motifs could regulate MAP kinases that diverged from Fus3 prior to the emergence of Ste5, suggesting that Ste5 activators arose by exploiting latent regulatory features already present in the MAPK ancestor. The magnitude of this latent allosteric potential drifts widely among pre-Ste5 MAP kinases, providing a pool of hidden phenotypic diversity that, when revealed by new activators, could lead to functional divergence and to the evolution of distinct signaling behaviors. PMID:23953117

  15. Structural evolution of differential amino acid effector regulation in plant chorismate mutases.

    Science.gov (United States)

    Westfall, Corey S; Xu, Ang; Jez, Joseph M

    2014-10-10

    Chorismate mutase converts chorismate into prephenate for aromatic amino acid biosynthesis. To understand the molecular basis of allosteric regulation in the plant chorismate mutases, we analyzed the three Arabidopsis thaliana chorismate mutase isoforms (AtCM1-3) and determined the x-ray crystal structures of AtCM1 in complex with phenylalanine and tyrosine. Functional analyses show a wider range of effector control in the Arabidopsis chorismate mutases than previously reported. AtCM1 is activated by tryptophan with phenylalanine and tyrosine acting as negative effectors; however, tryptophan, cysteine, and histidine activate AtCM3. AtCM2 is a nonallosteric form. The crystal structure of AtCM1 in complex with tyrosine and phenylalanine identifies differences in the effector sites of the allosterically regulated yeast enzyme and the other two Arabidopsis isoforms. Site-directed mutagenesis of residues in the effector site reveals key features leading to differential effector regulation in these enzymes. In AtCM1, mutations of Gly-213 abolish allosteric regulation, as observed in AtCM2. A second effector site position, Gly-149 in AtCM1 and Asp-132 in AtCM3, controls amino acid effector specificity in AtCM1 and AtCM3. Comparisons of chorismate mutases from multiple plants suggest that subtle differences in the effector site are conserved in different lineages and may lead to specialized regulation of this branch point enzyme.

  16. Diffusive coupling can discriminate between similar reaction mechanisms in an allosteric enzyme system

    Directory of Open Access Journals (Sweden)

    Nicola Ernesto M

    2010-11-01

    Full Text Available Abstract Background A central question for the understanding of biological reaction networks is how a particular dynamic behavior, such as bistability or oscillations, is realized at the molecular level. So far this question has been mainly addressed in well-mixed reaction systems which are conveniently described by ordinary differential equations. However, much less is known about how molecular details of a reaction mechanism can affect the dynamics in diffusively coupled systems because the resulting partial differential equations are much more difficult to analyze. Results Motivated by recent experiments we compare two closely related mechanisms for the product activation of allosteric enzymes with respect to their ability to induce different types of reaction-diffusion waves and stationary Turing patterns. The analysis is facilitated by mapping each model to an associated complex Ginzburg-Landau equation. We show that a sequential activation mechanism, as implemented in the model of Monod, Wyman and Changeux (MWC, can generate inward rotating spiral waves which were recently observed as glycolytic activity waves in yeast extracts. In contrast, in the limiting case of a simple Hill activation, the formation of inward propagating waves is suppressed by a Turing instability. The occurrence of this unusual wave dynamics is not related to the magnitude of the enzyme cooperativity (as it is true for the occurrence of oscillations, but to the sensitivity with respect to changes of the activator concentration. Also, the MWC mechanism generates wave patterns that are more stable against long wave length perturbations. Conclusions This analysis demonstrates that amplitude equations, which describe the spatio-temporal dynamics near an instability, represent a valuable tool to investigate the molecular effects of reaction mechanisms on pattern formation in spatially extended systems. Using this approach we have shown that the occurrence of inward

  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. Investigating the allosteric reverse signalling of PARP inhibitors with microsecond molecular dynamic simulations and fluorescence anisotropy.

    Science.gov (United States)

    Marchand, Jean-Rémy; Carotti, Andrea; Passeri, Daniela; Filipponi, Paolo; Liscio, Paride; Camaioni, Emidio; Pellicciari, Roberto; Gioiello, Antimo; Macchiarulo, Antonio

    2014-10-01

    The inhibition of the poly(ADP-ribose) polymerase (PARP) family members is a strategy pursued for the development of novel therapeutic agents in a range of diseases, including stroke, cardiac ischemia, cancer, inflammation and diabetes. Even though some PARP-1 inhibitors have advanced to clinical setting for cancer therapy, a great deal of attention is being devoted to understand the polypharmacology of current PARP inhibitors. Besides blocking the catalytic activity, recent works have shown that some PARP inhibitors exhibit a poisoning activity, by trapping the enzyme at damaged sites of DNA and forming cytotoxic complexes. In this study we have used microsecond molecular dynamics to study the allosteric reverse signalling that is at the basis of such an effect. We show that Olaparib, but not Veliparib and HYDAMTIQ, is able to induce a specific conformational drift of the WGR domain of PARP-1, which stabilizes PARP-1/DNA complex through the locking of several salt bridge interactions. Fluorescence anisotropy assays support such a mechanism, providing the first experimental evidence that HYDAMTIQ, a potent PARP inhibitor with neuroprotective properties, is less potent than Olaparib to trap PARP-1/DNA complex.

  19. 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...

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

    Science.gov (United States)

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

    2012-02-01

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

  1. 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.

  2. Discovery and structural characterization of an allosteric inhibitor of bacterial cis-prenyltransferase.

    Science.gov (United States)

    Danley, Dennis E; Baima, Eric T; Mansour, Mahmoud; Fennell, Kimberly F; Chrunyk, Boris A; Mueller, John P; Liu, Shenping; Qiu, Xiayang

    2015-01-01

    Undecaprenyl pyrophosphate synthase (UPPs) is an essential enzyme in a key bacterial cell wall synthesis pathway. It catalyzes the consecutive condensations of isopentenyl pyrophosphate (IPP) groups on to a trans-farnesyl pyrophosphate (FPP) to produce a C55 isoprenoid, undecaprenyl pyrophosphate (UPP). Here we report the discovery and co-crystal structures of a drug-like UPPs inhibitor in complex with Streptococcus pneumoniae UPPs, with and without substrate FPP, at resolutions of 2.2 and 2.1 Å, respectively. The UPPs inhibitor has a low molecular weight (355 Da), but displays potent inhibition of UPP synthesis in vitro (IC50 50 nM) that translates into excellent whole cell antimicrobial activity against pathogenic strains of Streptococcal species (MIC90 0.4 µg mL(-1) ). Interestingly, the inhibitor does not compete with the substrates but rather binds at a site adjacent to the FPP binding site and interacts with the tail of the substrate. Based on the structures, an allosteric inhibition mechanism of UPPs is proposed for this inhibitor. This inhibition mechanism is supported by biochemical and biophysical experiments, and provides a basis for the development of novel antibiotics targeting Streptococcus pneumoniae. PMID:25287857

  3. 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.

  4. 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)

  5. 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

    The human 5-HT transporter (hSERT) has two binding sites for 5-HT and 5-HT uptake inhibitors: the orthosteric high-affinity site and a low-affinity allosteric site. Activation of the allosteric site increases the dissociation half-life for some uptake inhibitors. The objectives of this study were 1......) to identify hSERT mutations that inactivate the high-affinity site without affecting the allosteric site and 2) to observe allosteric effects in which hSERT binds R-citalopram with higher affinity than S-citalopram. Wild-type and mutant (Y95F, I172M, and Y95F/I172M) hSERTs were expressed in COS-7 cells...... 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...

  6. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin

    DEFF Research Database (Denmark)

    Eghorn, Laura Friis; Høstgaard-Jensen, Kirsten; Kongstad, Kenneth Thermann;

    2014-01-01

    γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate...... whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive...... conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed...

  7. Discovery of Novel Thiophene-Based, Thumb Pocket 2 Allosteric Inhibitors of the Hepatitis C NS5B Polymerase with Improved Potency and Physicochemical Profiles.

    Science.gov (United States)

    Court, John J; Poisson, Carl; Ardzinski, Andrzej; Bilimoria, Darius; Chan, Laval; Chandupatla, Kishan; Chauret, Nathalie; Collier, Philip N; Das, Sanjoy Kumar; Denis, Francois; Dorsch, Warren; Iyer, Ganesh; Lauffer, David; L'Heureux, Lucille; Li, Pan; Luisi, Brian S; Mani, Nagraj; Nanthakumar, Suganthi; Nicolas, Olivier; Rao, B Govinda; Ronkin, Steven; Selliah, Subajini; Shawgo, Rebecca S; Tang, Qing; Waal, Nathan D; Yannopoulos, Constantin G; Green, Jeremy

    2016-07-14

    The hepatitis C viral proteins NS3/4A protease, NS5B polymerase, and NS5A are clinically validated targets for direct-acting antiviral therapies. The NS5B polymerase may be inhibited directly through the action of nucleosides or nucleotide analogues or allosterically at a number of well-defined sites. Herein we describe the further development of a series of thiophene carboxylate allosteric inhibitors of NS5B polymerase that act at the thumb pocket 2 site. Lomibuvir (1) is an allosteric HCV NS5B inhibitor that has demonstrated excellent antiviral activity and potential clinical utility in combination with other direct acting antiviral agents. Efforts to further explore and develop this series led to compound 23, a compound with comparable potency and improved physicochemical properties.

  8. Possible regulation of the Salmonella typhimurium histidine operon by adenosine triphosphate phosphoribosyltransferase: large metabolic effects.

    OpenAIRE

    Goitein, R K; Parsons, S. M.

    1980-01-01

    An effort to find growth conditions leading to conditional regulation of the histidine operon of Salmonella typhimurium by the allosteric first enzyme of the pathway, adenosine triphosphate phosphoribosyltransferase (EC 2.4.2.17), is reported. A strain deleting the enzyme, TR3343, behaved simply and predictably under all growth conditions, whereas histidine auxotrophs containing active enzyme behaved in complicated ways dependent upon the location of the histidine pathway lesion. hisE strains...

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    Chao-Yie Yang

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

  11. 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

  12. Positive allosteric modulators of alpha 7 nicotinic acetylcholine receptors reverse ketamine-induced schizophrenia-like deficits in rats.

    Science.gov (United States)

    Nikiforuk, Agnieszka; Kos, Tomasz; Hołuj, Małgorzata; Potasiewicz, Agnieszka; Popik, Piotr

    2016-02-01

    Alpha 7 nicotinic acetylcholine receptors (α7-nAChRs) have generated great interest as targets of new pharmacological treatments for cognitive dysfunction in schizophrenia. One promising recent approach is based on the use of positive allosteric modulators (PAMs) of α7-nAChRs, which demonstrate several advantages over direct agonists. Nevertheless, the efficacy of these newly introduced α7-nAChR agents has not been extensively characterised in animal models of schizophrenia. The aim of the present study was to evaluate the efficacy of type I and II PAMs, N-(5-chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)urea (PNU-120596) and N-(4-chlorophenyl)-[[(4-chlorophenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide (CCMI), respectively, and galantamine, an acetylcholinesterase inhibitor (AChE) that also allosterically modulates nAChRs, against ketamine-induced cognitive deficits and social withdrawal in rats. The orthosteric α7-nAChR agonist octahydro-2-methyl-5-(6-phenyl-3-pyridazinyl)-pyrrolo[3,4-c]pyrrole (A-582941) was used as a positive control. Additionally, the antipsychotic activities of the tested compounds were assessed using the conditioned avoidance response (CAR) test. PNU-120596, CCMI, galantamine and A-582941 reversed ketamine-induced cognitive inflexibility, as assessed in the attentional set-shifting task (ASST). The tested compounds were also effective against ketamine-induced impairment in the novel object recognition task (NORT). PNU-120596, CCMI, and A-582941 ameliorated ketamine-induced social interaction deficits, whereas galantamine was ineffective. Moreover, all tested compounds selectively suppressed the CAR. The positive allosteric modulation of α7-nAChRs demonstrates preclinical efficacy not only against schizophrenia-like cognition impairments but also positive and negative symptoms. Therefore, the use of α7-nAChR PAMs as a potential treatment strategy in schizophrenia is supported.

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

    Directory of Open Access Journals (Sweden)

    M Cecchini

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

  14. Positive allosteric modulators of the human sweet taste receptor enhance sweet taste.

    Science.gov (United States)

    Servant, Guy; Tachdjian, Catherine; Tang, Xiao-Qing; Werner, Sara; Zhang, Feng; Li, Xiaodong; Kamdar, Poonit; Petrovic, Goran; Ditschun, Tanya; Java, Antoniette; Brust, Paul; Brune, Nicole; DuBois, Grant E; Zoller, Mark; Karanewsky, Donald S

    2010-03-01

    To identify molecules that could enhance sweetness perception, we undertook the screening of a compound library using a cell-based assay for the human sweet taste receptor and a panel of selected sweeteners. In one of these screens we found a hit, SE-1, which significantly enhanced the activity of sucralose in the assay. At 50 microM, SE-1 increased the sucralose potency by >20-fold. On the other hand, SE-1 exhibited little or no agonist activity on its own. SE-1 effects were strikingly selective for sucralose. Other popular sweeteners such as aspartame, cyclamate, and saccharin were not enhanced by SE-1 whereas sucrose and neotame potency were increased only by 1.3- to 2.5-fold at 50 microM. Further assay-guided chemical optimization of the initial hit SE-1 led to the discovery of SE-2 and SE-3, selective enhancers of sucralose and sucrose, respectively. SE-2 (50 microM) and SE-3 (200 microM) increased sucralose and sucrose potencies in the assay by 24- and 4.7-fold, respectively. In human taste tests, 100 microM of SE-1 and SE-2 allowed for a reduction of 50% to >80% in the concentration of sucralose, respectively, while maintaining the sweetness intensity, and 100 microM SE-3 allowed for a reduction of 33% in the concentration of sucrose while maintaining the sweetness intensity. These enhancers did not exhibit any sweetness when tasted on their own. Positive allosteric modulators of the human sweet taste receptor could help reduce the caloric content in food and beverages while maintaining the desired taste.

  15. Allosteric activation and contrasting properties of L-serine dehydratase types 1 and 2.

    Science.gov (United States)

    Chen, Shawei; Xu, Xiao Lan; Grant, Gregory A

    2012-07-01

    Bacterial L-serine dehydratases differ from mammalian L- and D-serine dehydratases and bacterial D-serine dehydratases by the presence of an iron-sulfur center rather than a pyridoxyl phosphate prosthetic group. They exist in two forms, types 1 and 2, distinguished by their sequence and oligomeric configuration. Both types contain an ASB domain, and the type 1 enzymes also contain an ACT domain in a tandem arrangement with the ASB domain like that in type 1 D-3-phosphoglycerate dehydrogenases (PGDHs). This investigation reveals striking kinetic differences between L-serine dehydratases from Bacillus subtilis (bsLSD, type 1) and Legionella pneumophila (lpLSD, type 2). lpLSD is activated by monovalent cations and inhibited by monovalent anions. bsLSD is strongly activated by cations, particularly potassium, and shows a mixed response to anions. Flouride is a competitive inhibitor for lpLSD but an apparent activator for bsLSD at low concentrations and an inhibitor at high concentrations. The reaction products, pyruvate and ammonia, also act as activators but to different extents for each type. Pyruvate activation is competitive with L-serine, but activation of the enzyme is not compatible with it simply competing for binding at the active site and suggests the presence of a second, allosteric site. Because activation can be eliminated by higher levels of L-serine, it may be that this second site is actually a second serine binding site. This is consistent with type 1 PGDH in which the ASB domain functions as a second site for substrate binding and activation.

  16. 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.

  17. 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

  18. Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry

    Science.gov (United States)

    McMillan, Kirk; Adler, Marc; Auld, Douglas S.; Baldwin, John J.; Blasko, Eric; Browne, Leslie J.; Chelsky, Daniel; Davey, David; Dolle, Ronald E.; Eagen, Keith A.; Erickson, Shawn; Feldman, Richard I.; Glaser, Charles B.; Mallari, Cornell; Morrissey, Michael M.; Ohlmeyer, Michael H. J.; Pan, Gonghua; Parkinson, John F.; Phillips, Gary B.; Polokoff, Mark A.; Sigal, Nolan H.; Vergona, Ronald; Whitlow, Marc; Young, Tish A.; Devlin, James J.

    2000-01-01

    Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC 1.14.13.39) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC50 values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (Kd ≈ 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor–heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein–protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED50 values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies. PMID:10677491

  19. The anaerobic (Class III) ribonucleotide reductase from Lactococcus lactis : Catalytic properties and allosteric regulation of the pure enzyme system

    NARCIS (Netherlands)

    Torrents, Eduard; Buist, Girbe; Liu, Aimin; Eliasson, Rolf; Kok, Jan; Gibert, Isidre; Gräslund, Astrid; Reichard, Peter

    2000-01-01

    Lactococcus lactis contains an operon with the genes (nrdD and nrdG) for a class III ribonucleotide reductase, Strict anaerobic growth depends on the activity of these genes. Both were sequenced, cloned, and overproduced in Escherichia coli, The corresponding proteins, NrdD and NrdG, were purified c

  20. Allosteric regulation of monocyclic interconvertible enzyme cascade systems: use of Escherichia coli glutamine synthetase as an experimental model.

    Science.gov (United States)

    Rhee, S G; Park, R; Chock, P B; Stadtman, E R

    1978-07-01

    The interconversion of Escherichia coli glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] between its adenylylated and unadenylylated forms has been used to verify the prediction derived from a theoretical analysis of the steady-state functions of a model for a monocyclic interconvertible enzyme cascade system [Stadtman, E. R. & Chock, P. B. (1977) Proc. Natl. Acad. Sci. USA 74, 2761-2770]. Because glutamine and alpha-ketoglutarate are multifunctional effectors and because three active enzyme complexes are involved in both adenylylation and deadenylylation of glutamine synthetase, at least 28 constants are required to describe the glutamine synthetase monocyclic cascade. Of these, 22 constants were determined experimentally and 6 were estimated via computer curve fitting. Despite the complexity, when both adenylylation and deadenylylation reactions are functioning, the number of adenylyl groups bound per mole of enzyme, n, assumes a steady-state level as is predicted by the model. This n value is determined by the mole fraction of P(IIA)-given by ([P(IIA)]/([P(IIA)] + [P(IID)])-and the ratio of glutamine to alpha-ketoglutarate (P(IID) and P(IID) are the unmodified and the uridylylated forms of the P(II) regulatory protein). In the presence of 0.5 mM glutamine and 2 mM alpha-ketoglutarate, the value of n increases as a nearly hyperbolic function in response to increasing mole fractions of P(IIA). When the constant level of alpha-ketoglutarate is gradually increased to 40 muM, the hyperbolic function converts slowly to a parabolic function. When the P(IIA) mole fraction was maintained at 0.6 and alpha-ketoglutarate levels were varied from 1 mM to 4 muM, an 800-fold increase in signal amplification was observed with respect to glutamine activation. In addition, because glutamine activates the adenylylation and inhibits the deadenylylation reaction, a sensitivity index of 2.1 (corresponding to a Hill number of 1.5) was obtained for the variation of n values in response to increasing glutamine concentration. PMID:28522

  1. Thieno[2,3-b]pyridines as negative allosteric modulators of metabotropic GluR5 receptors: Lead optimization.

    Science.gov (United States)

    Nógrádi, Katalin; Wágner, Gábor; Domány, György; Bobok, Amrita; Magdó, Ildikó; Kolok, Sándor; Mikó-Bakk, Mónika L; Vastag, Mónika; Sághy, Katalin; Gyertyán, István; Kóti, János; Gál, Krisztina; Farkas, Sándor; Keserű, György M; Greiner, István; Szombathelyi, Zsolt

    2015-04-15

    An HTS campaign of our corporate compound library, and hit-to lead development resulted in thieno[2,3-b]pyridine derivative leads with mGluR5 negative allosteric modulator effects. During the lead optimization process, our objective was to improve affinity and metabolic stability. Modification of the first two targeted regions resulted in compounds with nanomolar affinity, then optimal substitution of the third region improved metabolic stability. One of the most promising compounds showed excellent in vivo efficacy and is a potential development candidate.

  2. 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...

  3. 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...

  4. 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...

  5. 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

  6. 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.

  7. Targeting AKT with the allosteric AKT inhibitor MK-2206 in non-small cell lung cancer cells with acquired resistance to cetuximab.

    Science.gov (United States)

    Iida, Mari; Brand, Toni M; Campbell, David A; Starr, Megan M; Luthar, Neha; Traynor, Anne M; Wheeler, Deric L

    2013-06-01

    The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for use in oncology. Despite clinical success the majority of patients do not respond to cetuximab and those who initially respond frequently acquire resistance. To understand how tumor cells acquire resistance to cetuximab we developed a model of resistance using the non-small cell lung cancer line NCI-H226. We found that cetuximab-resistant (Ctx (R) ) clones manifested strong activation of EGFR, PI3K/AKT and MAPK. To investigate the role of AKT signaling in cetuximab resistance we analyzed the activation of the AKT pathway effector molecules using a human AKT phospho-antibody array. Strong activation was observed in Ctx (R) clones for several key AKT substrates including c-jun, GSK3β, eIF4E, rpS6, IKKα, IRS-1 and Raf1. Inhibition of AKT signaling by siAKT1/2 or by the allosteric AKT inhibitor MK-2206 resulted in robust inhibition of cell proliferation in all Ctx (R) clones. Moreover, the combinational treatment of cetuximab and MK-2206 resulted in further decreases in proliferation than either drug alone. This combinatorial treatment resulted in decreased activity of both AKT and MAPK thus highlighting the importance of simultaneous pathway inhibition to maximally affect the growth of Ctx (R) cells. Collectively, our findings demonstrate that AKT activation is an important pathway in acquired resistance to cetuximab and suggests that combinatorial therapy directed at both the AKT and EGFR/MAPK pathways may be beneficial in this setting. PMID:23760490

  8. 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...

  9. 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

  10. 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

  11. 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...

  12. Epitope fluctuations in the human papillomavirus are under dynamic allosteric control: a computational evaluation of a new vaccine design strategy.

    Science.gov (United States)

    Singharoy, Abhishek; Polavarapu, Abhigna; Joshi, Harshad; Baik, Mu-Hyun; Ortoleva, Peter

    2013-12-11

    The dynamic properties of the capsid of the human papillomavirus (HPV) type 16 were examined using classical molecular dynamics simulations. By systematically comparing the structural fluctuations of the capsid protein, a strong dynamic allosteric connection between the epitope containing loops and the h4 helix located more than 50 Å away is identified, which was not recognized thus far. Computer simulations show that restricting the structural fluctuations of the h4 helix is key to rigidifying the epitopes, which is thought to be required for eliciting a proper immune response. The allostery identified in the components of the HPV is nonclassical because the mean structure of the epitope carrying loops remains unchanged, but as a result of allosteric effect the structural fluctuations are altered significantly, which in turn changes the biochemical reactivity profile of the epitopes. Exploiting this novel insight, a new vaccine design strategy is proposed wherein a relatively small virus capsid fragment is deposited on a silica nanoparticle in such a way that the fluctuations of the h4 helix are suppressed. The structural and dynamic properties of the epitope carrying loops on this hybrid nanoparticle match the characteristics of epitopes found on the full virus-like particle precisely, suggesting that these nanoparticles may serve as potent, cost-effective, and safe alternatives to traditionally developed vaccines. The structural and dynamic properties of the hybrid nanoparticle are examined in detail to establish the general concepts of the proposed new design. PMID:24199651

  13. Novel Scaffold Identification of mGlu1 Receptor Negative Allosteric Modulators Using a Hierarchical Virtual Screening Approach.

    Science.gov (United States)

    Jang, Jae Wan; Cho, Nam-Chul; Min, Sun-Joon; Cho, Yong Seo; Park, Ki Duk; Seo, Seon Hee; No, Kyoung Tai; Pae, Ae Nim

    2016-02-01

    Metabotropic glutamate receptor 1 (mGluR1) is considered as an attractive drug target for neuropathic pain treatments. The hierarchical virtual screening approach for identifying novel scaffolds of mGluR1 allosteric modulators was performed using a homology model built with the dopamine D3 crystal structure as template. The mGluR1 mutagenesis data, conserved amino acid sequences across class A and class C GPCRs, and previously reported multiple sequence alignments of class C GPCRs to the rhodopsin template, were employed for the sequence alignment to overcome difficulties of model generation with low sequence identity of mGluR1 and dopamine D3. The structures refined by molecular dynamics simulations were employed for docking of Asinex commercial libraries after hierarchical virtual screening with pharmacophore and naïve Bayesian models. Five of 35 compounds experimentally evaluated using a calcium mobilization assay exhibited micromolar activities (IC50) with chemotype novelty that demonstrated the validity of our methods. A hierarchical structure and ligand-based virtual screening approach with homology model of class C GPCR based on dopamine D3 class A GPCR structure was successfully performed and applied to discover novel negative mGluR1 allosteric modulators.

  14. 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

  15. Structure of a small-molecule inhibitor complexed with GlmU from Haemophilus influenzae reveals an allosteric binding site

    Energy Technology Data Exchange (ETDEWEB)

    Mochalkin, Igor; Lightle, Sandra; Narasimhan, Lakshmi; Bornemeier, Dirk; Melnick, Michael; VanderRoest, Steven; McDowell, Laura (Pfizer)

    2008-04-02

    N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential enzyme in aminosugars metabolism and an attractive target for antibiotic drug discovery. GlmU catalyzes the formation of uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), an important precursor in the peptidoglycan and lipopolisaccharide biosynthesis in both Gram-negative and Gram-positive bacteria. Here we disclose a 1.9 {angstrom} resolution crystal structure of a synthetic small-molecule inhibitor of GlmU from Haemophilus influenzae (hiGlmU). The compound was identified through a high-throughput screening (HTS) configured to detect inhibitors that target the uridyltransferase active site of hiGlmU. The original HTS hit exhibited a modest micromolar potency (IC{sub 50} - 18 {mu}M in a racemic mixture) against hiGlmU and no activity against Staphylococcus aureus GlmU (saGlmU). The determined crystal structure indicated that the inhibitor occupies an allosteric site adjacent to the GlcNAc-1-P substrate-binding region. Analysis of the mechanistic model of the uridyltransferase reaction suggests that the binding of this allosteric inhibitor prevents structural rearrangements that are required for the enzymatic reaction, thus providing a basis for structure-guided design of a new class of mechanism-based inhibitors of GlmU.

  16. The regulation of Escherichia coli glutamine synthetase revisited: role of 2-ketoglutarate in the regulation of glutamine synthetase adenylylation state.

    Science.gov (United States)

    Jiang, P; Peliska, J A; Ninfa, A J

    1998-09-15

    The regulation of Escherichia coli glutamine synthetase (GS) by reversible adenylylation has provided one of the classical paradigms for signal transduction by cyclic cascades. Yet, many mechanistic features of this regulation remain to be elucidated. We examined the regulation of GS adenylylation state in a reconstituted system containing GS, adenylyltransferase (ATase), the PII signal transduction protein that controls ATase, and the uridylyltransferase/uridylyl-removing enzyme (UTase/UR), which has a role in regulating PII. In this reconstituted bicyclic cascade system, the adenylylation state of GS was regulated reciprocally by the small molecule effectors 2-ketoglutarate and glutamine at physiological effector concentrations. By examination of the individual regulatory monocycles and comparison to the bicyclic system and existing data, we could deduce that the only sensors of 2-ketoglutarate were PII and PII-UMP. At physiological conditions, we observed that the main role of 2-ketoglutarate in bringing about the deadenylylation of GS was to inhibit GS adenylylation, and this was due to the allosteric regulation of PII activity. Glutamine acted as an allosteric regulator of both ATase and UTase/UR. We also compared the regulation of GS adenylylation state to the regulation of phosphorylation state of the transcription factor NRI (NtrC) in a reconstituted bicyclic system containing NRI, the bifunctional kinase/phosphatase NRII (NtrB), PII, and the UTase/UR. This comparison indicated that, at a fixed 2-ketoglutarate concentration, the regulation of GS adenylylation state by glutamine was sharper and occurred at a higher concentration than did the regulation of NRI phosphorylation. The possible biological implications of this regulatory arrangement are discussed. PMID:9737857

  17. 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

  18. 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

  19. 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;

    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...

  20. 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...

  1. Allosteric modulation and constitutive activity of fusion proteins between the adenosine A1 receptor and different 351Cys-mutated Gi α-subunits

    NARCIS (Netherlands)

    Klaasse, E.; Ligt, R.A.F.de; Roerink, S.F.; Lorenzen, A.; Milligan, G.; Leurs, R.; IJzerman, A.P.

    2004-01-01

    We studied fusion proteins between the human adenosine A1 receptor and different 351Cys-mutated Gi1 α-subunits (A1-Giα) with respect to two important concepts in receptor pharmacology, i.e. allosteric modulation and constitutive activity/inverse agonism. The aim of our study was twofold. We first an

  2. 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

  3. Changes in dynamics upon oligomerization regulate substrate binding and allostery in amino acid kinase family members.

    Directory of Open Access Journals (Sweden)

    Enrique Marcos

    2011-09-01

    Full Text Available Oligomerization is a functional requirement for many proteins. The interfacial interactions and the overall packing geometry of the individual monomers are viewed as important determinants of the thermodynamic stability and allosteric regulation of oligomers. The present study focuses on the role of the interfacial interactions and overall contact topology in the dynamic features acquired in the oligomeric state. To this aim, the collective dynamics of enzymes belonging to the amino acid kinase family both in dimeric and hexameric forms are examined by means of an elastic network model, and the softest collective motions (i.e., lowest frequency or global modes of motions favored by the overall architecture are analyzed. Notably, the lowest-frequency modes accessible to the individual subunits in the absence of multimerization are conserved to a large extent in the oligomer, suggesting that the oligomer takes advantage of the intrinsic dynamics of the individual monomers. At the same time, oligomerization stiffens the interfacial regions of the monomers and confers new cooperative modes that exploit the rigid-body translational and rotational degrees of freedom of the intact monomers. The present study sheds light on the mechanism of cooperative inhibition of hexameric N-acetyl-L-glutamate kinase by arginine and on the allosteric regulation of UMP kinases. It also highlights the significance of the particular quaternary design in selectively determining the oligomer dynamics congruent with required ligand-binding and allosteric activities.

  4. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin.

    Science.gov (United States)

    Eghorn, Laura F; Hoestgaard-Jensen, Kirsten; Kongstad, Kenneth T; Bay, Tina; Higgins, David; Frølund, Bente; Wellendorph, Petrine

    2014-10-01

    γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.

  5. 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

  6. Positive Allosteric Modulation of mGluR5 Accelerates Extinction Learning but Not Relearning Following Methamphetamine Self-Administration.

    Science.gov (United States)

    Kufahl, Peter R; Hood, Lauren E; Nemirovsky, Natali E; Barabas, Piroska; Halstengard, Casey; Villa, Angel; Moore, Elisabeth; Watterson, Lucas R; Olive, M Foster

    2012-01-01

    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 eight daily sessions of short access followed by eight sessions of long access (6 h/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 seven 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 non-existent 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

  7. 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

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

    Science.gov (United States)

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

    2015-10-01

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

  9. 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

  10. Sequence analysis and molecular characterization of Clonorchis sinensis hexokinase, an unusual trimeric 50-kDa glucose-6-phosphate-sensitive allosteric enzyme.

    Directory of Open Access Journals (Sweden)

    Tingjin Chen

    Full Text Available Clonorchiasis, which is induced by the infection of Clonorchis sinensis (C. sinensis, is highly associated with cholangiocarcinoma. Because the available examination, treatment and interrupting transmission provide limited opportunities to prevent infection, it is urgent to develop integrated strategies to prevent and control clonorchiasis. Glycolytic enzymes are crucial molecules for trematode survival and have been targeted for drug development. Hexokinase of C. sinensis (CsHK, the first key regulatory enzyme of the glycolytic pathway, was characterized in this study. The calculated molecular mass (Mr of CsHK was 50.0 kDa. The obtained recombinant CsHK (rCsHK was a homotrimer with an Mr of approximately 164 kDa, as determined using native PAGE and gel filtration. The highest activity was obtained with 50 mM glycine-NaOH at pH 10 and 100 mM Tris-HCl at pH 8.5 and 10. The kinetics of rCsHK has a moderate thermal stability. Compared to that of the corresponding negative control, the enzymatic activity was significantly inhibited by praziquantel (PZQ and anti-rCsHK serum. rCsHK was homotropically and allosterically activated by its substrates, including glucose, mannose, fructose, and ATP. ADP exhibited mixed allosteric effect on rCsHK with respect to ATP, while inorganic pyrophosphate (PPi displayed net allosteric activation with various allosteric systems. Fructose behaved as a dose-dependent V activator with the substrate glucose. Glucose-6-phosphate (G6P displayed net allosteric inhibition on rCsHK with respect to ATP or glucose with various allosteric systems in a dose-independent manner. There were differences in both mRNA and protein levels of CsHK among the life stages of adult worm, metacercaria, excysted metacercaria and egg of C. sinensis, suggesting different energy requirements during different development stages. Our study furthers the understanding of the biological functions of CsHK and supports the need to screen for small

  11. 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

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

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    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.

  13. Computer-aided design of negative allosteric modulators of metabotropic glutamate receptor 5 (mGluR5): Comparative molecular field analysis of aryl ether derivatives.

    Science.gov (United States)

    Selvam, Chelliah; Thilagavathi, Ramasamy; Narasimhan, Balasubramanian; Kumar, Pradeep; Jordan, Brian C; Ranganna, Kasturi

    2016-02-15

    The metabotropic glutamate receptors (mGlu receptors) have emerged as attractive targets for number of neurological and psychiatric disorders. Recently, mGluR5 negative allosteric modulators (NAMs) have gained considerable attention in pharmacological research. Comparative molecular field analysis (CoMFA) was performed on 73 analogs of aryl ether which were reported as mGluR5 NAMs. The study produced a statistically significant model with high correlation coefficient and good predictive abilities.

  14. 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...

  15. Thieno[2,3-b]pyridines as negative allosteric modulators of metabotropic GluR5 receptors: hit-to-lead optimization.

    Science.gov (United States)

    Nógrádi, Katalin; Wágner, Gábor; Domány, György; Bobok, Amrita; Magdó, Ildikó; Kiss, Béla; Kolok, Sándor; Fónagy, Katalin; Gyertyán, István; Háda, Viktor; Kóti, János; Gál, Krisztina; Farkas, Sándor; Keserű, György M; Greiner, István; Szombathelyi, Zsolt

    2014-08-15

    An HTS campaign of our corporate compound library resulted in thieno[2,3-b]pyridines derivative hits with mGluR5 negative allosteric modulator effects. During the hit-to-lead development our objective was to improve affinity, and to keep the ligand efficiency values at an acceptable level. After different modifications of the linker resulted in a 2-sulfonyl-thieno[2,3-b]pyridines derivative, which fulfilled the lead criteria.

  16. 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...

  17. Preclinical pharmacokinetic and toxicological evaluation of MIF-1 peptidomimetic, PAOPA: examining the pharmacology of a selective dopamine D2 receptor allosteric modulator for the treatment of schizophrenia.

    Science.gov (United States)

    Tan, Mattea L; Basu, Dipannita; Kwiecien, Jacek M; Johnson, Rodney L; Mishra, Ram K

    2013-04-01

    Schizophrenia is a mental illness characterized by a breakdown in cognition and emotion. Over the years, drug treatment for this disorder has mainly been compromised of orthosteric ligands that antagonize the active site of the dopamine D2 receptor. However, these drugs are limited in their use and often lead to the development of adverse movement and metabolic side effects. Allosteric modulators are an emerging class of therapeutics with significant advantages over orthosteric ligands, including an improved therapeutic and safety profile. This study investigates our newly developed allosteric modulator, PAOPA, which is a specific modulator of the dopamine D2 receptor. Previous studies have shown PAOPA to attenuate schizophrenia-like behavioral abnormalities in preclinical models. To advance this newly developed allosteric drug from the preclinical to clinical stage, this study examines the pharmacokinetic behavior and toxicological profile of PAOPA. Results from this study prove the effectiveness of PAOPA in reaching the implicated regions of the brain for therapeutic action, particularly the striatum. Pharmacokinetic parameters of PAOPA were found to be comparable to current market antipsychotic drugs. Necropsy and histopathological analyses showed no abnormalities in all examined organs. Acute and chronic treatment of PAOPA indicated no movement abnormalities commonly found with the use of current typical antipsychotic drugs. Moreover, acute and chronic PAOPA treatment revealed no hematological or metabolic abnormalities classically found with the use of atypical antipsychotic drugs. Findings from this study demonstrate a better safety profile of PAOPA, and necessitates the progression of this newly developed therapeutic for the treatment of schizophrenia.

  18. NbIT--a new information theory-based analysis of allosteric mechanisms reveals residues that underlie function in the leucine transporter LeuT.

    Science.gov (United States)

    LeVine, Michael V; Weinstein, Harel

    2014-05-01

    Complex networks of interacting residues and microdomains in the structures of biomolecular systems underlie the reliable propagation of information from an input signal, such as the concentration of a ligand, to sites that generate the appropriate output signal, such as enzymatic activity. This information transduction often carries the signal across relatively large distances at the molecular scale in a form of allostery that is essential for the physiological functions performed by biomolecules. While allosteric behaviors have been documented from experiments and computation, the mechanism of this form of allostery proved difficult to identify at the molecular level. Here, we introduce a novel analysis framework, called N-body Information Theory (NbIT) analysis, which is based on information theory and uses measures of configurational entropy in a biomolecular system to identify microdomains and individual residues that act as (i)-channels for long-distance information sharing between functional sites, and (ii)-coordinators that organize dynamics within functional sites. Application of the new method to molecular dynamics (MD) trajectories of the occluded state of the bacterial leucine transporter LeuT identifies a channel of allosteric coupling between the functionally important intracellular gate and the substrate binding sites known to modulate it. NbIT analysis is shown also to differentiate residues involved primarily in stabilizing the functional sites, from those that contribute to allosteric couplings between sites. NbIT analysis of MD data thus reveals rigorous mechanistic elements of allostery underlying the dynamics of biomolecular systems.

  19. 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

  20. The rational design of specific peptide inhibitor against p38α MAPK at allosteric-site: a therapeutic modality for HNSCC.

    Directory of Open Access Journals (Sweden)

    Kamaldeep Gill

    Full Text Available p38α is a significant target for drug designing against cancer. The overproduction of p38α MAPK promotes tumorigenesis in head and neck squamous cell carcinoma (HNSCC. The ATP binding and an allosteric site referred as DFG are the key sites of the p38α mitogen activated protein kinase (MAPK exploited for the design of inhibitors. This study demonstrated design of peptide inhibitor on the basis of allosteric site using Glide molecular docking software and the biochemical analysis of the best modeled peptide. The best fitted tetrapeptide (FWCS in the allosteric site inhibited the pure recombinant and serum p38α of HNSCC patients by 74 and 72%, respectively. The potency of the peptide was demonstrated by its IC50 (4.6 nM and KD (3.41×10-10 M values, determined by ELISA and by surface plasmon resonance (SPR technology, respectively. The cell viability of oral cancer i.e. KB cell line was reduced in dose dependent manner by 60 and 97% by the treatment of peptide and the IC50 was 600 and 210 µM after 24 and 72 h incubation, respectively. Our result provides an insight for the development of a proficient small peptide as a promising anticancer agent targeting DFG site of p38α kinase.

  1. 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

  2. Serine is a natural ligand and allosteric activator of pyruvate kinase M2

    NARCIS (Netherlands)

    Chaneton, Barbara; Hillmann, Petra; Zheng, Liang; Martin, Agnes C. L.; Maddocks, Oliver D. K.; Chokkathukalam, Achuthanunni; Coyle, Joseph E.; Jankevics, Andris; Holding, Finn P.; Vousden, Karen H.; Frezza, Christian; O'Reilly, Marc; Gottlieb, Eyal

    2012-01-01

    Cancer cells exhibit several unique metabolic phenotypes that are critical for cell growth and proliferation(1). Specifically, they overexpress the M2 isoform of the tightly regulated enzyme pyruvate kinase (PKM2), which controls glycolytic flux, and are highly dependent on de novo biosynthesis of s

  3. Mutations within the putative active site of heterodimeric deoxyguanosine kinase block the allosteric activation of the deoxyadenosine kinase subunit.

    Science.gov (United States)

    Park, Inshik; Ives, David H

    2002-03-31

    Replacement of the Asp-84 residue of the deoxyguanosine kinase subunit of the tandem deoxyadenosine kinase/ deoxyguanosine kinase (dAK/dGK) from Lactobacillus acidophilus R-26 by Ala, Asn, or Glu produced increased Km values for deoxyguanosine on dGK. However, it did not seem to affect the binding of Mg-ATP. The Asp-84 dGK replacements had no apparent effect on the binding of deoxyadenosine by dAK. However, the mutant dGKs were no longer inhibited by dGTP, normally a potent distal endproduct inhibitor of dGK. Moreover, the allosteric activation of dAK activity by dGTP or dGuo was lost in the modified heterodimeric dAK/dGK enzyme. Therefore, it seems very likely that Asp-84 participates in dGuo binding at the active site of the dGK subunit of dAK/dGK from Lactobacillus acidophilus R-26.

  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. 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.

  6. 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

  7. 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.

  8. SUMO: regulating the regulator

    Directory of Open Access Journals (Sweden)

    Bossis Guillaume

    2006-06-01

    Full Text Available Abstract Post-translational modifiers of the SUMO (Small Ubiquitin-related Modifier family have emerged as key regulators of protein function and fate. While the past few years have seen an enormous increase in knowledge on SUMO enzymes, substrates, and consequences of modification, regulation of SUMO conjugation is far from being understood. This brief review will provide an overview on recent advances concerning (i the interplay between sumoylation and other post-translational modifications at the level of individual targets and (ii global regulation of SUMO conjugation and deconjugation.

  9. From Conformational Spread to Allosteric and Cooperative models of E. coli flagellar motor

    CERN Document Server

    Pezzotta, Alberto; Celani, Antonio

    2016-01-01

    Escherichia coli swims using flagella activated by rotary motors. The direction of rotation of the motors is indirectly regulated by the binding of a single messenger protein. The conformational spread model has been shown to accurately describe the equilibrium properties as well as the dynamics of the flagellar motor. In this paper we study this model from an analytic point of view. By exploiting the separation of timescales observed in experiments, we show how to reduce the conformational spread model to a coarse-grained, cooperative binding model. We show that this simplified model reproduces very well the dynamics of the motor switch.

  10. 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.

  11. 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

  12. Targeting the minor pocket of C5aR for the rational design of an oral allosteric inhibitor for inflammatory and neuropathic pain relief

    Science.gov (United States)

    Moriconi, Alessio; Cunha, Thiago M.; Souza, Guilherme R.; Lopes, Alexandre H.; Cunha, Fernando Q.; Carneiro, Victor L.; Pinto, Larissa G.; Brandolini, Laura; Aramini, Andrea; Bizzarri, Cinzia; Bianchini, Gianluca; Beccari, Andrea R.; Fanton, Marco; Bruno, Agostino; Costantino, Gabriele; Bertini, Riccardo; Galliera, Emanuela; Locati, Massimo; Ferreira, Sérgio H.; Teixeira, Mauro M.; Allegretti, Marcello

    2014-01-01

    Chronic pain resulting from inflammatory and neuropathic disorders causes considerable economic and social burden. Pharmacological therapies currently available for certain types of pain are only partially effective and may cause severe adverse side effects. The C5a anaphylatoxin acting on its cognate G protein-coupled receptor (GPCR), C5aR, is a potent pronociceptive mediator in several models of inflammatory and neuropathic pain. Although there has long been interest in the identification of C5aR inhibitors, their development has been complicated, as for many peptidomimetic drugs, mostly by poor drug-like properties. Herein, we report the de novo design of a potent and selective C5aR noncompetitive allosteric inhibitor, DF2593A, guided by the hypothesis that an allosteric site, the “minor pocket,” previously characterized in CXC chemokine receptors-1 and -2, is functionally conserved in the GPCR class. In vitro, DF2593A potently inhibited C5a-induced migration of human and rodent neutrophils. In vivo, oral administration of DF2593A effectively reduced mechanical hyperalgesia in several models of acute and chronic inflammatory and neuropathic pain, without any apparent side effects. Mechanical hyperalgesia after spared nerve injury was also reduced in C5aR−/− mice compared with WT mice. Furthermore, treatment of C5aR−/− mice with DF2593A did not produce any further antinociceptive effect compared with C5aR−/− mice treated with vehicle. The successful medicinal chemistry strategy confirms that a conserved minor pocket is amenable for the rational design of selective inhibitors and the pharmacological results support that the allosteric blockade of the C5aR represents a highly promising therapeutic approach to control chronic inflammatory and neuropathic pain. PMID:25385614

  13. 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

  14. A Cannabinoid CB1 Receptor-Positive Allosteric Modulator Reduces Neuropathic Pain in the Mouse with No Psychoactive Effects.

    Science.gov (United States)

    Ignatowska-Jankowska, Bogna M; Baillie, Gemma L; Kinsey, Steven; Crowe, Molly; Ghosh, Sudeshna; Owens, Robert A; Damaj, Imad M; Poklis, Justin; Wiley, Jenny L; Zanda, Matteo; Zanato, Chiara; Greig, Iain R; Lichtman, Aron H; Ross, Ruth A

    2015-12-01

    The CB1 receptor represents a promising target for the treatment of several disorders including pain-related disease states. However, therapeutic applications of Δ(9)-tetrahydrocannabinol and other CB1 orthosteric receptor agonists remain limited because of psychoactive side effects. Positive allosteric modulators (PAMs) offer an alternative approach to enhance CB1 receptor function for therapeutic gain with the promise of reduced side effects. Here we describe the development of the novel synthetic CB1 PAM, 6-methyl-3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole (ZCZ011), which augments the in vitro and in vivo pharmacological actions of the CB1 orthosteric agonists CP55,940 and N-arachidonoylethanolamine (AEA). ZCZ011 potentiated binding of [(3)H]CP55,940 to the CB1 receptor as well as enhancing AEA-stimulated [(35)S]GTPγS binding in mouse brain membranes and β-arrestin recruitment and ERK phosphorylation in hCB1 cells. In the whole animal, ZCZ011 is brain penetrant, increased the potency of these orthosteric agonists in mouse behavioral assays indicative of cannabimimetic activity, including antinociception, hypothermia, catalepsy, locomotor activity, and in the drug discrimination paradigm. Administration of ZCZ011 alone was devoid of activity in these assays and did not produce a conditioned place preference or aversion, but elicited CB1 receptor-mediated antinociceptive effects in the chronic constriction nerve injury model of neuropathic pain and carrageenan model of inflammatory pain. These data suggest that ZCZ011 acts as a CB1 PAM and provide the first proof of principle that CB1 PAMs offer a promising strategy to treat neuropathic and inflammatory pain with minimal or no cannabimimetic side effects.

  15. 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.

  16. 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.

  17. Allosteric analysis of glucocorticoid receptor-DNA interface induced by cyclic Py-Im polyamide: a molecular dynamics simulation study.

    Directory of Open Access Journals (Sweden)

    Yaru Wang

    Full Text Available BACKGROUND: It has been extensively developed in recent years that cell-permeable small molecules, such as polyamide, can be programmed to disrupt transcription factor-DNA interfaces and can silence aberrant gene expression. For example, cyclic pyrrole-imidazole polyamide that competes with glucocorticoid receptor (GR for binding to glucocorticoid response elements could be expected to affect the DNA dependent binding by interfering with the protein-DNA interface. However, how such small molecules affect the transcription factor-DNA interfaces and gene regulatory pathways through DNA structure distortion is not fully understood so far. METHODOLOGY/PRINCIPAL FINDINGS: In the present work, we have constructed some models, especially the ternary model of polyamides+DNA+GR DNA-binding domain (GRDBD dimer, and carried out molecular dynamics simulations and free energy calculations for them to address how polyamide molecules disrupt the GRDBD and DNA interface when polyamide and protein bind at the same sites on opposite grooves of DNA. CONCLUSIONS/SIGNIFICANCE: We found that the cyclic polyamide binding in minor groove of DNA can induce a large structural perturbation of DNA, i.e. a >4 Å widening of the DNA minor groove and a compression of the major groove by more than 4 Å as compared with the DNA molecule in the GRDBD dimer+DNA complex. Further investigations for the ternary system of polyamides+DNA+GRDBD dimer and the binary system of allosteric DNA+GRDBD dimer revealed that the compression of DNA major groove surface causes GRDBD to move away from the DNA major groove with the initial average distance of ∼4 Å to the final average distance of ∼10 Å during 40 ns simulation course. Therefore, this study straightforward explores how small molecule targeting specific sites in the DNA minor groove disrupts the transcription factor-DNA interface in DNA major groove, and consequently modulates gene expression.

  18. 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.

  19. 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.

  20. Market, Regulation, Market, Regulation

    DEFF Research Database (Denmark)

    Frankel, Christian; Galland, Jean-Pierre

    2015-01-01

    barriers to trade in Europe, realized the free movement of products by organizing progressively several orders of markets and regulation. Based on historical and institutional documents, on technical publications, and on interviews, this article relates how the European Commission and the Member States had......This paper focuses on the European Regulatory system which was settled both for opening the Single Market for products and ensuring the consumers' safety. It claims that the New Approach and Standardization, and the Global Approach to conformity assessment, which suppressed the last technical...... alternatively recourse to markets and to regulations, at the three main levels of the New Approach Directives implementation. The article focuses also more specifically on the Medical Devices sector, not only because this New Approach sector has long been controversial in Europe, and has recently been concerned...

  1. The cholesterol-dependent cytolysin signature motif: a critical element in the allosteric pathway that couples membrane binding to pore assembly.

    Directory of Open Access Journals (Sweden)

    Kelley J Dowd

    Full Text Available The cholesterol-dependent cytolysins (CDCs constitute a family of pore-forming toxins that contribute to the pathogenesis of a large number of Gram-positive bacterial pathogens.The most highly conserved region in the primary structure of the CDCs is the signature undecapeptide sequence (ECTGLAWEWWR. The CDC pore forming mechanism is highly sensitive to changes in its structure, yet its contribution to the molecular mechanism of the CDCs has remained enigmatic. Using a combination of fluorescence spectroscopic methods we provide evidence that shows the undecapeptide motif of the archetype CDC, perfringolysin O (PFO, is a key structural element in the allosteric coupling of the cholesterol-mediated membrane binding in domain 4 (D4 to distal structural changes in domain 3 (D3 that are required for the formation of the oligomeric pore complex. Loss of the undecapeptide function prevents all measurable D3 structural transitions, the intermolecular interaction of membrane bound monomers and the assembly of the oligomeric pore complex. We further show that this pathway does not exist in intermedilysin (ILY, a CDC that exhibits a divergent undecapeptide and that has evolved to use human CD59 rather than cholesterol as its receptor. These studies show for the first time that the undecapeptide of the cholesterol-binding CDCs forms a critical element of the allosteric pathway that controls the assembly of the pore complex.

  2. 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

  3. 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.

  4. 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.

  5. Diarylureas as allosteric modulators of the cannabinoid CB1 receptor: structure-activity relationship studies on 1-(4-chlorophenyl)-3-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]phenyl}urea (PSNCBAM-1).

    Science.gov (United States)

    German, Nadezhda; Decker, Ann M; Gilmour, Brian P; Gay, Elaine A; Wiley, Jenny L; Thomas, Brian F; Zhang, Yanan

    2014-09-25

    The recent discovery of allosteric modulators of the CB1 receptor including PSNCBAM-1 (4) has generated significant interest in CB1 receptor allosteric modulation. Here in the first SAR study on 4, we have designed and synthesized a series of analogs focusing on modifications at two positions. Pharmacological evaluation in calcium mobilization and binding assays revealed the importance of alkyl substitution at the 2-aminopyridine moiety and electron deficient aromatic groups at the 4-chlorophenyl position for activity at the CB1 receptor, resulting in several analogs with comparable potency to 4. These compounds increased the specific binding of [(3)H]CP55,940, in agreement with previous reports. Importantly, 4 and two analogs dose-dependently reduced the Emax of the agonist curve in the CB1 calcium mobilization assays, confirming their negative allosteric modulator characteristics. Given the side effects associated with CB1 receptor orthosteric antagonists, negative allosteric modulators provide an alternative approach to modulate the pharmacologically important CB1 receptor.

  6. 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

  7. 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).

  8. Synthesis, pharmacological and structural characterization, and thermodynamic aspects of GluA2-positive allosteric modulators with a 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide scaffold

    DEFF Research Database (Denmark)

    Nørholm, Ann-Beth; Francotte, Pierre; Olsen, Lars;

    2013-01-01

    Positive allosteric modulators of ionotropic glutamate receptors are potential compounds for treatment of cognitive disorders, e.g., Alzheimer's disease. The modulators bind within the dimer interface of the ligand-binding domain (LBD) and stabilize the agonist-bound conformation, thereby slowing...

  9. 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

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

    Science.gov (United States)

    Ahnaou, Abdallah; Lavreysen, Hilde; Tresadern, Gary; Cid, Jose M; Drinkenburg, Wilhelmus H

    2015-01-01

    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 promoted

  11. The reciprocal regulation of stress hormones and GABAA receptors

    Directory of Open Access Journals (Sweden)

    Istvan eMody

    2012-01-01

    Full Text Available Stress-derived steroid hormones regulate the expression and function of GABAA receptors (GABAARs. Changes in GABAAR subunit expression have been demonstrated under conditions of altered steroid hormone levels, such as stress, as well as following exogenous steroid hormone administration. In addition to the effects of stress-derived steroid hormones on GABAAR subunit expression, stress hormones can also be metabolized to neuroactive derivatives which can alter the function of GABAARs. Neurosteroids allosterically modulate GABAARs at concentrations comparable to those during stress. In addition to the actions of stress-derived steroid hormones on GABAARs, GABAARs reciprocally regulate the production of stress hormones. The stress response is mediated by the hypothalamic-pituitary-adrenal (HPA axis, the activity of which is governed by corticotropin releasing hormone (CRH neurons. The activity of CRH neurons is largely controlled by robust GABAergic inhibition. Recently, it has been demonstrated that CRH neurons are regulated by neurosteroid-sensitive, GABAAR δ subunit-containing receptors representing a novel feedback mechanism onto the HPA axis. Further, it has been demonstrated that neurosteroidogenesis and neurosteroid actions on GABAAR δ subunit-containing receptors on CRH neurons are necessary to mount the physiological response to stress. Here we review the literature describing the effects of steroid hormones on GABAARs as well as the importance of GABAARs in regulating the production of steroid hormones. This review incorporates what we currently know about changes in GABAARs following stress and the role in HPA axis regulation.

  12. Allosteric regulation of the GTP activated and CTP inhibited uracil phosphoribosyltransferase from the thermophilic archaeon Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Arent, Susan; Larsen, Sine;

    2005-01-01

    The upp gene, encoding uracil phosphoribosyltransferase (UPRTase) from the thermoacidophilic archaeon Sulfolobus solfataricus, was cloned and expressed in Escherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH 5.5 when...

  13. Modeling the Contribution of Allosteric Regulation for Flux Control in the Central Carbon Metabolism of E. coli

    DEFF Research Database (Denmark)

    Machado, Daniel; Herrgard, Markus; Rocha, Isabel

    2015-01-01

    Modeling cellular metabolism is fundamental for many biotechnological applications, including drug discovery and rational cell factory design. Central carbon metabolism (CCM) is particularly important as it provides the energy and precursors for other biological processes. However, the complex...

  14. CRYSTAL-STRUCTURE OF DEOXYGENATED LIMULUS-POLYPHEMUS SUBUNIT-II HEMOCYANIN AT 2.18-ANGSTROM RESOLUTION - CLUES FOR A MECHANISM FOR ALLOSTERIC REGULATION

    NARCIS (Netherlands)

    HAZES, B; MAGNUS, KA; BONAVENTURA, C; BONAVENTURA, J; DAUTER, Z; KALK, KH; HOL, WGJ

    1993-01-01

    The crystal structure of Limulus polyphemus subunit type II hemocyanin in the deoxygenated state has been determined to a resolution of 2.18 angstrom. Phase information for this first structure of a cheliceratan hemocyanin was obtained by molecular replacement using the crustacean hemocyanin structu

  15. Targeting α4β2 nAChRs in CNS disorders: Perspectives on positive allosteric modulation as a therapeutic approach

    DEFF Research Database (Denmark)

    Grupe, Morten; Grunnet, Morten; Bastlund, Jesper F.;

    2015-01-01

    , for example schizophrenia and Alzheimer's disease. Additionally, the α4β2 nAChR provides an extra layer of molecular complexity by existing in two different stoichiometries determined by the subunit composition. Such findings have founded the rationale that pharmacological modulation of the α4β2 nAChR may...... characteristics, expression pattern and pharmacological profile. The focus of the present MiniReview is on the heteromeric α4β2 nAChR, as activity at this subtype contributes to cognitive functioning through interactions with multiple neurotransmitter systems and is implicated in various CNS disorders...... be used as a treatment approach in various CNS disorders. As subtype-selective agonists and other cholinergic ligands have only shown limited therapeutic success, the focus of recent drug development endeavours has largely shifted to positive allosteric modulators (PAMs). By potentiating the action...

  16. Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease

    Science.gov (United States)

    den-Haan, Helena; Pérez-Sánchez, Horacio; Del Prete, Rosita; Liguori, Ludovica; Cimmaruta, Chiara; Lukas, Jan; Andreotti, Giuseppina

    2016-01-01

    Personalized therapies are required for Fabry disease due to its large phenotypic spectrum and numerous different genotypes. In principle, missense mutations that do not affect the active site could be rescued with pharmacological chaperones. At present pharmacological chaperones for Fabry disease bind the active site and couple a stabilizing effect, which is required, to an inhibitory effect, which is deleterious. By in silico docking we identified an allosteric hot-spot for ligand binding where a drug-like compound, 2,6-dithiopurine, binds preferentially. 2,6-dithiopurine stabilizes lysosomal alpha-galactosidase in vitro and rescues a mutant that is not responsive to a mono-therapy with previously described pharmacological chaperones, 1-deoxygalactonojirimycin and galactose in a cell based assay. PMID:27788225

  17. Antagonists and the purinergic nerve hypothesis: 2, 2'-pyridylisatogen tosylate (PIT), an allosteric modulator of P2Y receptors. A retrospective on a quarter century of progress.

    Science.gov (United States)

    Spedding, M; Menton, K; Markham, A; Weetman, D F

    2000-07-01

    2,2'-Pyridylisatogen tosylate (PIT) is a selective antagonist of P2Y responses in smooth muscle and does not antagonise the effects of adenosine. Responses to purinergic nerve stimulation are resistant to PIT. PIT is an allosteric modulator of responses to ATP in recombinant P2Y(1) receptors expressed in Xenopus oocytes with potentiation of ATP at low concentrations (0.1-10 microM) and antagonism at higher ones (>10 microM). A radioligand binding profile showed that PIT did not interact with any other receptors, with the exception of low affinity for the adenosine A(1) receptor (pK(i), 5.3). The compound recognises purine sites and then may cause irreversible binding to sulfhydryl groups following prolonged incubation or high concentrations. PIT is a potent spin trapper.

  18. 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

  19. 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

  20. 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

  1. The A128T resistance mutation reveals aberrant protein multimerization as the primary mechanism of action of allosteric HIV-1 integrase inhibitors.

    Science.gov (United States)

    Feng, Lei; Sharma, Amit; Slaughter, Alison; Jena, Nivedita; Koh, Yasuhiro; Shkriabai, Nikolozi; Larue, Ross C; Patel, Pratiq A; Mitsuya, Hiroaki; Kessl, Jacques J; Engelman, Alan; Fuchs, James R; Kvaratskhelia, Mamuka

    2013-05-31

    Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a very promising new class of anti-HIV-1 agents that exhibit a multimodal mechanism of action by allosterically modulating IN multimerization and interfering with IN-lens epithelium-derived growth factor (LEDGF)/p75 binding. Selection of viral strains under ALLINI pressure has revealed an A128T substitution in HIV-1 IN as a primary mechanism of resistance. Here, we elucidated the structural and mechanistic basis for this resistance. The A128T substitution did not affect the hydrogen bonding between ALLINI and IN that mimics the IN-LEDGF/p75 interaction but instead altered the positioning of the inhibitor at the IN dimer interface. Consequently, the A128T substitution had only a minor effect on the ALLINI IC50 values for IN-LEDGF/p75 binding. Instead, ALLINIs markedly altered the multimerization of IN by promoting aberrant higher order WT (but not A128T) IN oligomers. Accordingly, WT IN catalytic activities and HIV-1 replication were potently inhibited by ALLINIs, whereas the A128T substitution in IN resulted in significant resistance to the inhibitors both in vitro and in cell culture assays. The differential multimerization of WT and A128T INs induced by ALLINIs correlated with the differences in infectivity of HIV-1 progeny virions. We conclude that ALLINIs primarily target IN multimerization rather than IN-LEDGF/p75 binding. Our findings provide the structural foundations for developing improved ALLINIs with increased potency and decreased potential to select for drug resistance. PMID:23615903

  2. Cell fate regulation governed by a repurposed bacterial histidine kinase.

    Directory of Open Access Journals (Sweden)

    W Seth Childers

    2014-10-01

    Full Text Available One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK∼P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.

  3. Lipoxygenase regulation in vivo and in vitro by lipid compounds

    Directory of Open Access Journals (Sweden)

    Skaterna T. D.

    2015-06-01

    Full Text Available Lipoxygenases (LOs are known as one of the enzymes of lipid peroxidation. The majority of LOs are soluble enzymes and have affinity to membranes. The enzyme translocation from a cytosol to a membrane surface is one of the stages of regulation of the amount of LO catalysis products in the cell. A sorption to the membrane surface is described for most LOs from plant and animal sources. This review presents the data about regulation of the LO activity by the lipid compounds – both natural and chemically modified. Lipids might regulate the LO activity through: protein-lipid interactions of C2 domain with the membrane, changes in the enzyme affinity, the LOs translocation, allosteric regulation, increase in the selectivity towards substrates. The regulatory effect of active compound on the enzyme activity depends on the lipophilicity of effectors. Considering the LO activity it is necessary to take into account the enzyme microenvironment and its influence on the range of the LO products.

  4. Regulation of the activity of the Bacillus licheniformis A5 glutamine synthetase.

    Science.gov (United States)

    Donohue, T J; Bernlohr, R W

    1981-10-01

    The regulation of glutamine synthetase activity by positive and negative effectors of enzyme activity singularly and in combinations was studied by using a homogeneous enzyme preparation from Bacillus licheniformis A5. Phosphorylribosyl pyrophosphate at concentrations greater than 2mM stimulated glutamine synthetase activity by approximately 70%. The concentration of phosphorylribosyl pyrophosphate required for half-maximal stimulation of enzyme activity was 0.4 mM. Results obtained from studies of fractional inhibition of glutamine synthetase activity were consistent with the presence of one allosteric site for glutamine binding (apparent I0.5, 2.2mM) per active enzyme unit at a glutamate concentration of 50 mM. At a glutamate concentration of 30 mM or less, the data were consistent with the enzyme containing two binding sites for glutamine (one of which was an allosteric site with an apparent I0.5 of 0.4 mM). Bases on an analysis of the response of glutamine synthetase activity to positive and negative effectors in vitro and to the intracellular concentration of these effectors in vivo, the primary modulators of glutamine synthetase activity in B. licheniformis A5 appear to be glutamine and alanine (apparent I0.5, 5.2mM). PMID:6169702

  5. 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

    Serine protease catalytic activity is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. Inhibitory monoclonal antibodies binding to such exosites are potential therapeutics and offer opportunities...

  6. Telomerase Regulation

    OpenAIRE

    Cifuentes-Rojas, Catherine; Dorothy E Shippen

    2011-01-01

    The intimate connection between telomerase regulation and human disease is now well established. The molecular basis for telomerase regulation is highly complex and entails multiple layers of control. While the major target of enzyme regulation is the catalytic subunit TERT, the RNA subunit of telomerase is also implicated in telomerase control. In addition, alterations in gene dosage and alternative isoforms of core telomerase components have been described. Finally, telomerase localization,...

  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.

    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, ...

  8. Regulating Transplants

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Legislation to determine brain death is viewed as essential in controlling the organ transplant industry Organ transplant represents a very sensitive and complicated issue. Experts say the temporary administrative regulations recently promulgated by the Central Government are an important step, but relevant laws and regulations must follow. Among these, the

  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. An allosteric enhancer of M(4) muscarinic acetylcholine receptor function inhibits behavioral and neurochemical effects of cocaine

    DEFF Research Database (Denmark)

    Nielsen, Ditte Dencker; Weikop, Pia; Sørensen, Gunnar;

    2012-01-01

    The mesostriatal dopamine system plays a key role in mediating the reinforcing effects of psychostimulant drugs like cocaine. The muscarinic M(4) acetylcholine receptor subtype is centrally involved in the regulation of dopamine release in striatal areas. Consequently, striatal M(4) receptors could...

  11. NOISE REGULATION

    OpenAIRE

    Cristina Voican; Constantin Stanescu

    2012-01-01

    Noise regulation includes statutes or guidelines relating to sound transmission established by national, state or provincial and municipal levels of government. After the watershed passage of the United States Noise Control Act of 1972, other local and state governments passed further regulations. Although the UK and Japan enacted national laws in 1960 and 1967 respectively, these laws were not at all comprehensive or fully enforceable as to address generally rising ambient noise, enforceable...

  12. Effects of a metabotropic glutamate receptor subtype 7 negative allosteric modulator in the periaqueductal grey on pain responses and rostral ventromedial medulla cell activity in rat.

    Science.gov (United States)

    Palazzo, Enza; Marabese, Ida; Luongo, Livio; Boccella, Serena; Bellini, Giulia; Giordano, Maria Elvira; Rossi, Francesca; Scafuro, Mariantonietta; Novellis, Vito de; Maione, Sabatino

    2013-09-03

    The metabotropic glutamate receptor 7 (mGluR7) negative allosteric modulator, 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP), was locally microinjected into the ventrolateral periaqueductal gray (VL PAG) and the effect on pain responses in formalin and spare nerve injury (SNI) -induced neuropathic pain models was monitored in the rat. The activity of rostral ventromedial medulla (RVM) "pronociceptive" ON and "antinociceptive" OFF cells was also evaluated. Intra-VL PAG MMPIP blocked the first and second phase of nocifensive behaviour in the formalin pain model. MMPIP increased the tail flick latency and simultaneously increased the activity of the OFF cells while inhibiting that of ON cells in rats with SNI of the sciatic nerve. MMPIP failed to modify nociceptive responses and associated RVM ON and OFF cell activity in sham rats. An increase in mGluR7 gene, protein and staining, the latter being associated with vesicular glutamate transporter-positive profiles, has been found in the VL PAG in SNI rats. Blockade of mGluR7 within the VL PAG has an antinociceptive effect in formalin and neuropathic pain models. VL PAG mGluR7 blockade offers a target for dis-inhibiting the VL PAG-RVM pathway and silencing pain in inflammatory and neuropathic pain models.

  13. Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein.

    Directory of Open Access Journals (Sweden)

    Przemysław Karpowicz

    Full Text Available The proteasome is a giant protease responsible for degradation of the majority of cytosolic proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers. However, broadening the use of proteasome-targeting drugs requires new mechanistic approaches to the enzyme's inhibition. In our previous studies we described Tat1 peptide, an allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1 protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized beta-turn conformation at positions 4-5 and 8-9, pointed out by the molecular dynamics modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy studies, supplemented by molecular dynamics simulations. Biological activity tests and structural studies revealed that high flexibility and exposed positive charge are hallmarks of Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8-9 position was necessary to significantly improve the inhibitory potency.

  14. Artificial proteins as allosteric modulators of PDZ3 and SH3 in two-domain constructs: A computational characterization of novel chimeric proteins.

    Science.gov (United States)

    Kirubakaran, Palani; Pfeiferová, Lucie; Boušová, Kristýna; Bednarova, Lucie; Obšilová, Veronika; Vondrášek, Jiří

    2016-10-01

    Artificial multidomain proteins with enhanced structural and functional properties can be utilized in a broad spectrum of applications. The design of chimeric fusion proteins utilizing protein domains or one-domain miniproteins as building blocks is an important advancement for the creation of new biomolecules for biotechnology and medical applications. However, computational studies to describe in detail the dynamics and geometry properties of two-domain constructs made from structurally and functionally different proteins are lacking. Here, we tested an in silico design strategy using all-atom explicit solvent molecular dynamics simulations. The well-characterized PDZ3 and SH3 domains of human zonula occludens (ZO-1) (3TSZ), along with 5 artificial domains and 2 types of molecular linkers, were selected to construct chimeric two-domain molecules. The influence of the artificial domains on the structure and dynamics of the PDZ3 and SH3 domains was determined using a range of analyses. We conclude that the artificial domains can function as allosteric modulators of the PDZ3 and SH3 domains. Proteins 2016; 84:1358-1374. © 2016 Wiley Periodicals, Inc.

  15. 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.

  16. Can a Positive Allosteric Modulation of GABAergic Receptors Improve Motor Symptoms in Patients with Parkinson's Disease? The Potential Role of Zolpidem in the Treatment of Parkinson's Disease.

    Science.gov (United States)

    Daniele, Antonio; Panza, Francesco; Greco, Antonio; Logroscino, Giancarlo; Seripa, Davide

    2016-01-01

    At present, patients with advanced Parkinson's disease (PD) are unsatisfactorily controlled by currently used anti-Parkinsonian dopaminergic drugs. Various studies suggest that therapeutic strategies based on nondopaminergic drugs might be helpful in PD. Zolpidem, an imidazopyridine widely used as sleep inducer, shows high affinity only for GABAA receptors containing the α-1 subunit and facilitates GABAergic neurotransmission through a positive allosteric modulation of GABAA receptors. Various observations, although preliminary, consistently suggest that in PD patients zolpidem may induce beneficial (and sometimes remarkable) effects on motor symptoms even after single doses and may also improve dyskinesias. Since a high density of zolpidem binding sites is in the two main output structures of the basal ganglia which are abnormally overactive in PD (internal globus pallidus, GPi, and substantia nigra pars reticulata, SNr), it was hypothesized that in PD patients zolpidem may induce through GABAA receptors an inhibition of GPi and SNr (and, possibly, of the subthalamic nucleus also), resulting in an increased activity of motor cortical areas (such as supplementary motor area), which may give rise to improvement of motor symptoms of PD. Randomized clinical trials are needed in order to assess the efficacy, safety, and tolerability of zolpidem in treating motor symptoms of PD.

  17. NORM regulations

    Energy Technology Data Exchange (ETDEWEB)

    Gray, P. [ed.

    1997-02-01

    The author reviews the question of regulation for naturally occuring radioactive material (NORM), and the factors that have made this a more prominent concern today. Past practices have been very relaxed, and have often involved very poor records, the involvment of contractors, and the disposition of contaminated equipment back into commercial service. The rationale behind the establishment of regulations is to provide worker protection, to exempt low risk materials, to aid in scrap recycling, to provide direction for remediation and to examine disposal options. The author reviews existing regulations at federal and state levels, impending legislation, and touches on the issue of site remediation and potential liabilities affecting the release of sites contaminated by NORM.

  18. Regulation of transcription and activity of Rhizobium etli glutaminase A.

    Science.gov (United States)

    Huerta-Saquero, Alejandro; Calderón-Flores, Arturo; Díaz-Villaseñor, Andrea; Du Pont, Gisela; Durán, Socorro

    2004-08-01

    The present study determines the regulatory mechanisms that operate on Rhizobium etli glutaminase A. glsA gene expression levels were evaluated under several metabolic conditions by fusions of the glsA gene promoter and the transcriptional reporter cassette uidA2-aad. glsA expression was directly correlated to the glutaminase A activity found under the tested growth conditions, reaching its maximum level in the presence of glutamine and during exponential growth phase. Glutamine induces glsA expression. The influence of allosteric metabolites on glutaminase A activity was also determined. The purified enzyme was inhibited by 2-oxoglutarate and pyruvate, whereas oxaloacetate and glyoxylate modulate it positively. Glutaminase A is not inhibited by glutamate and is activated by ammonium. Glutaminase A participates in an ATP-consuming cycle where glutamine is continually degraded and resynthesized by glutamine synthetase (GS). GS and glutaminase A activities appear simultaneously during bacterial growth under different metabolic conditions and their control mechanisms are not reciprocal. Slight overproduction in glutaminase A expression causes a reduction in growth yield and a dramatic decrease in bacterial growth. We propose a model for regulation of glutaminase A, and discuss its contribution to glutamine cycle regulation. PMID:15279892

  19. 让学习成功--变构模型及其教学应用%Making learning successful:allosteric learning model and its applications in teaching

    Institute of Scientific and Technical Information of China (English)

    裴新宁

    2013-01-01

    Metaphor is pivotal for the construction of a complex theory. From the perspective of allostery of enzyme , learning is ultimately seen as a change occurring on “ active site” of learner’s conceptions. Teachers and mediators can not directly operate the learner’s active site, but who can act on “allosteric site” of learner’s conception system via manipulating the environment that learners interact with so as to trigger some “looseness” or “distortion” (or“deconstruction”) of the conception system and thereby result in a key change (generating new structure and/or new meaning) on the active site. Such a “deconstruction-construction” is a parallel process causing a fundamental learning rather than merely a cognitive process. Using the allosteric learning model, Andre Giordan organically made complex learning elements ( biological, psychological, cultural, emotional, epistemological, etc.) integrated, in particular, incorporated subject’s motivation and epistemology into the mechanism on the process of learning. This model provides a strong explanation for daily learning phenomenon.%隐喻在复杂理论的建构过程中起着关键作用。如果从蛋白质的“变构效应”来理解学习,那么学习归根结底是学习者发生于其概念体系的“活性(概念)基”上的变化;教师及媒介者不可能直接作用到学习者的“活性基”,但可以通过操纵环境(不仅仅是创设环境)作用于概念体系的“变构部位”,引发概念体系结构的“松动”和“变形”(即“解构”),从而导致其“活性基”上的关键变化(新结构和新意义生成)。这样的“解构-建构”并进的根本性学习绝非仅仅是一个认知过程;焦尔当运用变构模型将学习的复杂要素有机地整合起来,特别地,将学习过程与主体认识论及学习动机“契合”在了一起,对于日常学习现象具有较强的解释力。

  20. Effect of S-adenosyl-L-methionine (SAM), an allosteric activator of cystathionine-β-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro.

    Science.gov (United States)

    Módis, Katalin; Coletta, Ciro; Asimakopoulou, Antonia; Szczesny, Bartosz; Chao, Celia; Papapetropoulos, Andreas; Hellmich, Mark R; Szabo, Csaba

    2014-09-15

    Recent data show that colon cancer cells selectively overexpress cystathionine-β-synthase (CBS), which produces hydrogen sulfide (H2S), to maintain cellular bioenergetics, support tumor growth and stimulate angiogenesis and vasorelaxation in the tumor microenvironment. The purpose of the current study was to investigate the effect of the allosteric CBS activator S-adenosyl-L-methionine (SAM) on the proliferation and bioenergetics of the CBS-expressing colon cancer cell line HCT116. The non-transformed, non-tumorigenic colon epithelial cell line NCM356 was used as control. For assessment of cell proliferation, the xCELLigence system was used. Bioenergetic function was measured by Extracellular Flux Analysis. Experiments using human recombinant CBS or HCT116 homogenates complemented the cell-based studies. SAM markedly enhanced CBS-mediated H2S production in vitro, especially when a combination of cysteine and homocysteine was used as substrates. Addition of SAM (0.1-3 mM) to HCT116 cells induced a concentration-dependent increase H2S production. SAM exerted time- and concentration-dependent modulatory effects on cell proliferation. At 0.1-1 mM SAM increased HCT116 proliferation between 0 and 12 h, while the highest SAM concentration (3 mM) inhibited proliferation. Over a longer time period (12-24 h), only the lowest concentration of SAM used (0.1 mM) stimulated cell proliferation; higher SAM concentrations produced a concentration-dependent inhibition. The short-term stimulatory effects of SAM were attenuated by the CBS inhibitor aminooxyacetic acid (AOAA) or by stable silencing of CBS. In contrast, the inhibitory effects of SAM on cell proliferation was unaffected by CBS inhibition or CBS silencing. In contrast to HCT116 cells, the lower rate of proliferation of the low-CBS expressor NCM356 cells was unaffected by SAM. Short-term (1 h) exposure of HCT116 cells to SAM induced a concentration-dependent increase in oxygen consumption and bioenergetic function at 0

  1. Conformational dynamics of cancer-associated MyD88-TIR domain mutant L252P (L265P) allosterically tilts the landscape toward homo-dimerization.

    Science.gov (United States)

    Zhan, Chendi; Qi, Ruxi; Wei, Guanghong; Guven-Maiorov, Emine; Nussinov, Ruth; Ma, Buyong

    2016-09-01

    MyD88 is an essential adaptor protein, which mediates the signaling of the toll-like and interleukin-1 receptors' superfamily. The MyD88 L252P (L265P) mutation has been identified in diffuse large B-cell lymphoma. The identification of this mutation has been a major advance in the diagnosis of patients with aldenstrom macroglobulinemia and related lymphoid neoplasms. Here we used computational methods to characterize the conformational effects of the mutation. Our molecular dynamics simulations revealed that the mutation allosterically quenched the global conformational dynamics of the toll/IL-1R (TIR) domain, and readjusted its salt bridges and dynamic community network. Specifically, the mutation changed the orientation and reduced the fluctuation of α-helix 3, possibly through eliminating/weakening ~8 salt bridges and enhancing the salt bridge D225-K258. Using the energy landscape of the TIR domains of MyD88, we identified two dynamic conformational basins, which correspond to the binding sites used in homo- and hetero-oligomerization, respectively. Our results indicate that the mutation stabilizes the core of the homo-dimer interface of the MyD88-TIR domain, and increases the population of homo-dimer-compatible conformational states in MyD88 family proteins. However, the dampened motion restricts its ability to heterodimerize with other TIR domains, thereby curtailing physiological signaling. In conclusion, the L252P both shifts the landscape toward homo-dimerization and restrains the dynamics of the MyD88-TIR domain, which disfavors its hetero-dimerization with other TIR domains. We further put these observations within the framework of MyD88-mediated cell signaling. PMID:27503954

  2. mGluR5 Positive and Negative Allosteric Modulators Differentially Affect Dendritic Spine Density and Morphology in the Prefrontal Cortex.

    Science.gov (United States)

    LaCrosse, Amber L; Taylor, Sara B; Nemirovsky, Natali E; Gass, Justin T; Olive, Michael F

    2015-01-01

    Positive and negative allosteric modulators (PAMs and NAMs, respectively) of type 5 metabotropic glutamate receptors (mGluR5) are currently being investigated as novel treatments for neuropsychiatric diseases including drug addiction, schizophrenia, and Fragile X syndrome. However, only a handful of studies have examined the effects of mGluR5 PAMs or NAMs on the structural plasticity of dendritic spines in otherwise naïve animals, particularly in brain regions mediating executive function. In the present study, we assessed dendritic spine density and morphology in pyramidal cells of the medial prefrontal cortex (mPFC) after repeated administration of either the prototypical mGluR5 PAM 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5- yl)benzamide (CDPPB, 20 mg/kg), the clinically utilized mGluR5 NAM 1-(3-chlorophenyl)-3-(3-methyl-5-oxo-4Himidazol- 2-yl)urea (fenobam, 20 mg/kg), or vehicle in male Sprague-Dawley rats. Following once daily treatment for 10 consecutive days, coronal brain sections containing the mPFC underwent diolistic labeling and 3D image analysis of dendritic spines. Compared to vehicle treated animals, rats administered fenobam exhibited significant increases in dendritic spine density and the overall frequency of spines with small (0.4 μm). Administration of CDPPB had no discernable effects on dendritic spine density or morphology, and neither CDPPB nor fenobam had any effect on spine length or volume. We conclude that mGluR5 PAMs and NAMs differentially affect mPFC dendritic spine structural plasticity in otherwise naïve animals, and additional studies assessing their effects in combination with cognitive or behavioral tasks are needed. PMID:25921744

  3. Electrostatic and Allosteric Cooperativity in Ion-Pair Binding: A Quantitative and Coupled Experiment-Theory Study with Aryl-Triazole-Ether Macrocycles.

    Science.gov (United States)

    Qiao, Bo; Sengupta, Arkajyoti; Liu, Yun; McDonald, Kevin P; Pink, Maren; Anderson, Joseph R; Raghavachari, Krishnan; Flood, Amar H

    2015-08-01

    Cooperative binding of ion pairs to receptors is crucial for the manipulation of salts, but a comprehensive understanding of cooperativity has been elusive. To this end, we combine experiment and theory to quantify ion-pair binding and to separate allostery from electrostatics to understand their relative contributions. We designed aryl-triazole-ether macrocycles (MC) to be semiflexible, which allows ion pairs (NaX; X = anion) to make contact, and to be monocyclic to simplify analyses. A multiequilibrium model allows us to quantify, for the first time, the experimental cooperativity, α, for the equilibrium MC·Na(+) + MC·X(-) ⇌ MC·NaX + MC, which is associated with contact ion-pair binding of NaI (α = 1300, ΔGα = -18 kJ mol(-1)) and NaClO4 (α = 400, ΔGα = -15 kJ mol(-1)) in 4:1 dichloromethane-acetonitrile. We used accurate energies from density functional theory to deconvolute how the electrostatic effects and the allosteric changes in receptor geometry individually contribute to cooperativity. Computations, using a continuum solvation model (dichloromethane), show that allostery contributes ∼30% to overall positive cooperativity. The calculated trend of electrostatic cooperativity using pairs of spherical ions (NaCl > NaBr > NaI) correlates to experimental observations (NaI > NaClO4). We show that intrinsic ionic size, which dictates charge separation distance in contact ion pairs, controls electrostatic cooperativity. This finding supports the design principle that semiflexible receptors can facilitate optimal electrostatic cooperativity. While Coulomb's law predicts the size-dependent trend, it overestimates electrostatic cooperativity; we suggest that binding of the individual anion and cation to their respective binding sites dilutes their effective charge. This comprehensive understanding is critical for rational designs of ion-pair receptors for the manipulation of salts. PMID:26207611

  4. MMPIP, an mGluR7-selective negative allosteric modulator, alleviates pain and normalizes affective and cognitive behavior in neuropathic mice.

    Science.gov (United States)

    Palazzo, Enza; Romano, Rosaria; Luongo, Livio; Boccella, Serena; De Gregorio, Danilo; Giordano, Maria Elvira; Rossi, Francesca; Marabese, Ida; Scafuro, Maria Antonietta; de Novellis, Vito; Maione, Sabatino

    2015-06-01

    This study investigated the effects of a single administration of 6-(4-methoxyphenyl)-5-methyl-3-pyridinyl-4-isoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP), a negative allosteric modulator (NAM) of metabotropic glutamate receptor 7 (mGluR7), on pain and on affective and cognitive behavior in neuropathic mice. The activity of pyramidal neurons in the prelimbic cortex (PLC), which respond to stimulation of the basolateral amygdala (BLA) with either excitation or inhibition, was also investigated. The spared nerve injury (SNI) of the sciatic nerve induced, 14 days after surgery, thermal hyperalgesia and mechanical allodynia, reduced open-arm choice in the elevated plus-maze, increased time of immobility in the tail suspension, and increased digging and burying in the marble burying test. Cognitive performance was also significantly compromised in the SNI mice. Spared nerve injury induced phenotypic changes on pyramidal neurons of the PLC; excitatory responses increased, whereas inhibitory responses decreased after BLA stimulation. mGluR7 expression, mainly associated with vesicular glutamate transporter, increased in the hippocampus and decreased in the BLA, PLC, and dorsal raphe in SNI mice. MMPIP increased thermal and mechanical thresholds and open-arm choice. It reduced the immobility in the tail suspension test and the number of marbles buried and of digging events in the marble burying test. MMPIP also improved cognitive performance and restored the balance between excitatory and inhibitory responses of PLC neurons in SNI mice. 7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one, XAP044, another selective mGluR7 NAM, reproduced the effects of MMPIP on thermal hyperalgesia, mechanical allodynia, tail suspension, and marble burying test. Altogether, these findings show that mGluR7 NAMs reduce pain responses and affective/cognitive impairments in neuropathic pain conditions.

  5. Differential effects of CSF-1R D802V and KIT D816V homologous mutations on receptor tertiary structure and allosteric communication.

    Directory of Open Access Journals (Sweden)

    Priscila Da Silva Figueiredo Celestino Gomes

    Full Text Available The colony stimulating factor-1 receptor (CSF-1R and the stem cell factor receptor KIT, type III receptor tyrosine kinases (RTKs, are important mediators of signal transduction. The normal functions of these receptors can be compromised by gain-of-function mutations associated with different physiopatological impacts. Whereas KIT D816V/H mutation is a well-characterized oncogenic event and principal cause of systemic mastocytosis, the homologous CSF-1R D802V has not been identified in human cancers. The KIT D816V oncogenic mutation triggers resistance to the RTK inhibitor Imatinib used as first line treatment against chronic myeloid leukemia and gastrointestinal tumors. CSF-1R is also sensitive to Imatinib and this sensitivity is altered by mutation D802V. Previous in silico characterization of the D816V mutation in KIT evidenced that the mutation caused a structure reorganization of the juxtamembrane region (JMR and facilitated its departure from the kinase domain (KD. In this study, we showed that the equivalent CSF-1R D802V mutation does not promote such structural effects on the JMR despite of a reduction on some key H-bonds interactions controlling the JMR binding to the KD. In addition, this mutation disrupts the allosteric communication between two essential regulatory fragments of the receptors, the JMR and the A-loop. Nevertheless, the mutation-induced shift towards an active conformation observed in KIT D816V is not observed in CSF-1R D802V. The distinct impact of equivalent mutation in two homologous RTKs could be associated with the sequence difference between both receptors in the native form, particularly in the JMR region. A local mutation-induced perturbation on the A-loop structure observed in both receptors indicates the stabilization of an inactive non-inhibited form, which Imatinib cannot bind.

  6. The mGluR2 Positive Allosteric Modulator BINA Decreases Cocaine Self-Administration and Cue-Induced Cocaine-Seeking and Counteracts Cocaine-Induced Enhancement of Brain Reward Function in Rats

    OpenAIRE

    Jin, Xinchun; Semenova, Svetlana; Yang, Li; Ardecky, Robert; Sheffler, Douglas J.; Dahl, Russell; Conn, P. Jeffrey; Cosford, Nicholas DP; Markou, Athina

    2010-01-01

    Metabotropic glutamate receptor 2/3 (mGluR2/3) agonists were shown previously to nonselectively decrease both cocaine- and food-maintained responding in rats. mGluR2 positive allosteric modulators (PAMs) may represent improved therapeutic compounds because of their modulatory properties and higher selectivity for mGluR2. We analyzed the effects of the selective, brain penetrant, and systemically active mGluR2 PAM potassium 3′-([(2-cyclopentyl-6-7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy]m...

  7. 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...

  8. Novel Cancer Therapeutics with Allosteric Modulation of the Mitochondrial C-Raf-DAPK Complex by Raf Inhibitor Combination Therapy.

    Science.gov (United States)

    Tsai, Yi-Ta; Chuang, Mei-Jen; Tang, Shou-Hung; Wu, Sheng-Tang; Chen, Yu-Chi; Sun, Guang-Huan; Hsiao, Pei-Wen; Huang, Shih-Ming; Lee, Hwei-Jen; Yu, Cheng-Ping; Ho, Jar-Yi; Lin, Hui-Kuan; Chen, Ming-Rong; Lin, Chung-Chih; Chang, Sun-Yran; Lin, Victor C; Yu, Dah-Shyong; Cha, Tai-Lung

    2015-09-01

    Mitochondria are the powerhouses of cells. Mitochondrial C-Raf is a potential cancer therapeutic target, as it regulates mitochondrial function and is localized to the mitochondria by its N-terminal domain. However, Raf inhibitor monotherapy can induce S338 phosphorylation of C-Raf (pC-Raf(S338)) and impede therapy. This study identified the interaction of C-Raf with S308 phosphorylated DAPK (pDAPK(S308)), which together became colocalized in the mitochondria to facilitate mitochondrial remodeling. Combined use of the Raf inhibitors sorafenib and GW5074 had synergistic anticancer effects in vitro and in vivo, but targeted mitochondrial function, rather than the canonical Raf signaling pathway. C-Raf depletion in knockout MEF(C-Raf-/-) or siRNA knockdown ACHN renal cancer cells abrogated the cytotoxicity of combination therapy. Crystal structure simulation showed that GW5074 bound to C-Raf and induced a C-Raf conformational change that enhanced sorafenib-binding affinity. In the presence of pDAPK(S308), this drug-target interaction compromised the mitochondrial targeting effect of the N-terminal domain of C-Raf, which induced two-hit damages to cancer cells. First, combination therapy facilitated pC-Raf(S338) and pDAPK(S308) translocation from mitochondria to cytoplasm, leading to mitochondrial dysfunction and reactive oxygen species (ROS) generation. Second, ROS facilitated PP2A-mediated dephosphorylation of pDAPK(S308) to DAPK. PP2A then dissociated from the C-Raf-DAPK complex and induced profound cancer cell death. Increased pDAPK(S308) modification was also observed in renal cancer tissues, which correlated with poor disease-free survival and poor overall survival in renal cancer patients. Besides mediating the anticancer effect, pDAPK(S308) may serve as a predictive biomarker for Raf inhibitors combination therapy, suggesting an ideal preclinical model that is worthy of clinical translation.

  9. Nuclear regulation

    International Nuclear Information System (INIS)

    Today, 112 nuclear power plants, 22 facilities that support these plants, 54 reactors used in research, and approximately 23,000 organizations hold licenses from either the Nuclear Regulator Commission or various states to use radioactive material; other facilities are operated by various government agencies. Eventually most of these facilities will be decommissioned, which involves removing the radioactive material and terminating the license. NRC needs to ensure that licensees appropriately decontaminate their facilities because, under current regulations, NRC cannot specifically require additional cleanup once it terminates a license. This paper presents a GAO report on NRC's decommissioning procedures. In two of eight cases GAO reviewed, NRC fully or partially released sites for unrestricted use where radioactive contamination was higher than its guidelines allowed; in the other cases, NRC's information was inadequate or incomplete. Further, NRC lacks information on the types and amounts of radioactive waste buried on-site. At five sites reviewed by GAO, groundwater has been found to be contaminated by radioactive waste

  10. 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.

  11. 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

  12. Discovery of 3-benzyl-1,3-benzoxazine-2,4-dione analogues as allosteric mitogen-activated kinase kinase (MEK) inhibitors and anti-enterovirus 71 (EV71) agents.

    Science.gov (United States)

    Sun, Jing; Niu, Yan; Wang, Chao; Zhang, Hao; Xie, Bingyu; Xu, Fengrong; Jin, Hongwei; Peng, Yihong; Liang, Lei; Xu, Ping

    2016-08-15

    Enterovirus 71 (EV71) is a kind of RNA virus and one of the two causes of Hand, foot and mouth disease (HFMD). Inhibitors that target key components of Ras/Raf/MEK/ERK pathway in host cells could impair replication of EV71. A series of 3-benzyl-1,3-benzoxazine-2,4-diones were designed from a specific MEK inhibitor G8935, by replacing the double bond between C3 and C4 within the coumarin scaffold with amide bond. One compound (9f) showed submicromolar inhibitory activity among the 12 derivatives. Further optimization on 9f led to two active compounds (9k and 9m) with nanomolar bioactivities (55nM and 60nM). The results of enzymatic assays also demonstrated that this series of compounds were allosteric inhibitors of unphosphorylated MEK1. The binding mode of compound 9k was predicted by molecular dynamic simulation and the key interactions were same as published MEK1/2 allosteric inhibitors. In the cell-based assays, compounds 9k and 9m could effectively suppress the ERK1/2 pathway, expression of EV71 VP1, and EV71 induced cytopathic effect (CPE) in rhabdomyosarcoma (RD) cells. PMID:27288186

  13. Discovery of 3-benzyl-1,3-benzoxazine-2,4-dione analogues as allosteric mitogen-activated kinase kinase (MEK) inhibitors and anti-enterovirus 71 (EV71) agents.

    Science.gov (United States)

    Sun, Jing; Niu, Yan; Wang, Chao; Zhang, Hao; Xie, Bingyu; Xu, Fengrong; Jin, Hongwei; Peng, Yihong; Liang, Lei; Xu, Ping

    2016-08-15

    Enterovirus 71 (EV71) is a kind of RNA virus and one of the two causes of Hand, foot and mouth disease (HFMD). Inhibitors that target key components of Ras/Raf/MEK/ERK pathway in host cells could impair replication of EV71. A series of 3-benzyl-1,3-benzoxazine-2,4-diones were designed from a specific MEK inhibitor G8935, by replacing the double bond between C3 and C4 within the coumarin scaffold with amide bond. One compound (9f) showed submicromolar inhibitory activity among the 12 derivatives. Further optimization on 9f led to two active compounds (9k and 9m) with nanomolar bioactivities (55nM and 60nM). The results of enzymatic assays also demonstrated that this series of compounds were allosteric inhibitors of unphosphorylated MEK1. The binding mode of compound 9k was predicted by molecular dynamic simulation and the key interactions were same as published MEK1/2 allosteric inhibitors. In the cell-based assays, compounds 9k and 9m could effectively suppress the ERK1/2 pathway, expression of EV71 VP1, and EV71 induced cytopathic effect (CPE) in rhabdomyosarcoma (RD) cells.

  14. Apo-ghrelin receptor (apo-GHSR1a Regulates Dopamine Signaling in the Brain

    Directory of Open Access Journals (Sweden)

    Andras eKern

    2014-08-01

    Full Text Available The orexigenic peptide hormone ghrelin is synthesized in the stomach and its receptor growth hormone secretagogue receptor (GHSR1a is expressed mainly in the central nervous system (CNS. In this review we confine our discussion to the physiological role of GHSR1a in the brain. Paradoxically, despite broad expression of GHSR1a in the CNS, other than trace amounts in the hypothalamus, ghrelin is undetectable in the brain. In our efforts to elucidate the function of the ligand-free ghrelin receptor (apo-GHSR1a we identified subsets of neurons that co-express GHSR1a and dopamine receptors. In this review we focus on interactions between apo-GHSR1a and dopamine-2 receptor (DRD2 and formation of GHSR1a:DRD2 heteromers in hypothalamic neurons that regulate appetite, and discuss implications for the treatment of Prader-Willi syndrome. GHSR1a antagonists of distinct chemical structures, a quinazolinone and a triazole, respectively enhance and inhibit dopamine signaling through GHSR1a:DRD2 heteromers by an allosteric mechanism. This finding illustrates a potential strategy for designing the next generation of drugs for treating eating disorders as well as psychiatric disorders caused by abnormal dopamine signaling. Treatment with a GHSR1a antagonist that enhances dopamine/DRD2 activity in GHSR1a:DRD2 expressing hypothalamic neurons has the potential to inhibit the uncontrollable hyperphagia associated with Prader-Willi syndrome. DRD2 antagonists are prescribed for treating schizophrenia, but these block dopamine signaling in all DRD2 expressing neurons and are associated with adverse side effects, including enhanced appetite and excessive weight gain. A GHSR1a antagonist of structural class that allosterically blocks dopamine/DRD2 action in GHSR1a:DRD2 expressing neurons would have no effect on neurons expressing DRD2 alone; therefore, the side effects of DRD2 antagonists would potentially be reduced thereby enhancing patient compliance.

  15. 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

  16. An acetylcholine alpha7 positive allosteric modulator rescues a schizophrenia-associated brain endophenotype in the 15q13.3 microdeletion, encompassing CHRNA7.

    Science.gov (United States)

    Gass, Natalia; Weber-Fahr, Wolfgang; Sartorius, Alexander; Becker, Robert; Didriksen, Michael; Stensbøl, Tine Bryan; Bastlund, Jesper Frank; Meyer-Lindenberg, Andreas; Schwarz, Adam J

    2016-07-01

    The 15q13.3 microdeletion copy number variation is strongly associated with schizophrenia and epilepsy. The CHRNA7 gene, encoding nicotinic acetylcholine alpha 7 receptors (nAChA7Rs), is hypothesized to be one of the main genes in this deletion causing the neuropsychiatric phenotype. Here we used a recently developed 15q13.3 microdeletion mouse model to explore whether an established schizophrenia-associated connectivity phenotype is replicated in a murine model, and whether positive modulation of nAChA7 receptor might pharmacologically normalize the connectivity patterns. Resting-state fMRI data were acquired from male mice carrying a hemizygous 15q13.3 microdeletion (N=9) and from wild-type mice (N=9). To study the connectivity profile of 15q13.3 mice and test the effect of nAChA7 positive allosteric modulation, the 15q13.3 mice underwent two imaging sessions, one week apart, receiving a single intraperitoneal injection of either 15mg/kg Lu AF58801 or saline. The control group comprised wild-type mice treated with saline. We performed seed-based functional connectivity analysis to delineate aberrant connectivity patterns associated with the deletion (15q13.3 mice (saline treatment) versus wild-type mice (saline treatment)) and their modulation by Lu AF58801 (15q13.3 mice (Lu AF58801 treatment) versus 15q13.3 mice (saline treatment)). Compared to wild-type mice, 15q13.3 mice evidenced a predominant hyperconnectivity pattern. The main effect of Lu AF58801 was a normalization of elevated functional connectivity between prefrontal and frontal, hippocampal, striatal, thalamic and auditory regions. The strongest effects were observed in brain regions expressing nAChA7Rs, namely hippocampus, cerebral cortex and thalamus. These effects may underlie the antiepileptic, pro-cognitive and auditory gating deficit-reversal effects of nAChA7R stimulation. PMID:27061851

  17. Enhancement of social novelty discrimination by positive allosteric modulators at metabotropic glutamate 5 receptors: adolescent administration prevents adult-onset deficits induced by neonatal treatment with phencyclidine.

    Science.gov (United States)

    Clifton, Nicholas E; Morisot, Nadège; Girardon, Sylvie; Millan, Mark J; Loiseau, Florence

    2013-02-01

    Metabotropic glutamate-5 receptors (mGluR5), which physically and functionally interact with N-methyl-D-Aspartate (NMDA) receptors, likewise control cognitive processes and have been proposed as targets for novel classes of antipsychotic agent. Since social cognition is impaired in schizophrenia and disrupted by NMDA receptor antagonists like dizocilpine, we evaluated its potential modulation by mGluR5. Acute administration (0.63-40 mg/kg) of the mGluR5 positive allosteric modulators (PAMs), 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) and ADX47273, reversed a delay-induced impairment in social novelty discrimination (SND) in adult rats. The action of CDPPB was blocked by the mGluR5 antagonist, 2-methyl-6-(phenylethynyl)-pyridine (2.5-10 mg/kg), and was also expressed upon microinjection into frontal cortex (0.63-10 μg/side), but not striatum. Supporting an interrelationship between mGluR5 and NMDA receptors, enhancement of SND by CDPPB was blocked by dizocilpine (0.08 mg/kg) while, reciprocally, dizocilpine-induced impairment in SND was attenuated by CDPPB (10 mg/kg). The SND deficit elicited by post-natal administration of phencyclidine (10 mg/kg, days 7-11) was reversed by CDPPB or ADX47273 in adults at week 8. This phencyclidine-induced impairment in cognition emerged in adult rats from week 7 on, and chronic, pre-symptomatic treatment of adolescent rats with CDPPB over weeks 5-6 (10 mg/kg per day) prevented the appearance of SND deficits in adults until at least week 13. In conclusion, as evaluated by a SND procedure, mGluR5 PAMs promote social cognition via actions expressed in interaction with NMDA receptors and exerted in frontal cortex. MGluR5 PAMs not only reverse but also (when given during adolescence) prevent the emergence of cognitive impairment associated with a developmental model of schizophrenia.

  18. α4 nicotinic acetylcholine receptor modulated by galantamine on nigrostriatal terminals regulates dopamine receptor-mediated rotational behavior.

    Science.gov (United States)

    Inden, Masatoshi; Takata, Kazuyuki; Yanagisawa, Daijiro; Ashihara, Eishi; Tooyama, Ikuo; Shimohama, Shun; Kitamura, Yoshihisa

    2016-03-01

    Galantamine, an acetylcholine esterase (AChE) inhibitor used to treat dementia symptoms, also acts as an allosteric potentiating ligand (APL) at nicotinic acetylcholine receptors (nAChRs). This study was designed to evaluate the allosteric effect of galantamine on nAChR regulation of nigrostrial dopaminergic neuronal function in the hemiparkinsonian rat model established by unilateral nigral 6-hydroxydopamine (6-OHDA) injection. Methamphetamine, a dopamine releaser, induced ipsilateral rotation, whereas dopamine agonists apomorphine (a non-selective dopamine receptor agonist), SKF38393 (a selective dopamine D1 receptor agonist), and quinpirole (a selective dopamine D2 receptor agonist) induced contralateral rotation. When 6-OHDA-injected rats were co-treated with nomifensine, a dopamine transporter inhibitor, a more pronounced and a remarkable effect of nicotine and galantamine was observed. Under these conditions, the combination of nomifensine with nicotine or galantamine induced the ipsilateral rotation similar to the methamphetamine-induced rotational behavior, indicating that nicotine and galantamine also induce dopamine release from striatal terminals. Both nicotine- and galantamine-induced rotations were significantly blocked by flupenthixol (an antagonist of both D1 and D2 dopamine receptors) and mecamylamine (an antagonist of nAChRs), suggesting that galantamine modulation of nAChRs on striatal dopaminergic terminals regulates dopamine receptor-mediated movement. Immunohistochemical staining showed that α4 nAChRs were highly expressed on striatal dopaminergic terminals, while no α7 nAChRs were detected. Pretreatment with the α4 nAChR antagonist dihydroxy-β-erythroidine significantly inhibited nicotine- and galantamine-induced rotational behaviors, whereas pretreatment with the α7 nAChR antagonist methyllycaconitine was ineffective. Moreover, the α4 nAChR agonist ABT-418 induced ipsilateral rotation, while the α7 nAChR agonist PNU282987 had no

  19. The magic spot: a ppGpp binding site on E. coli RNA polymerase responsible for regulation of transcription initiation.

    Science.gov (United States)

    Ross, Wilma; Vrentas, Catherine E; Sanchez-Vazquez, Patricia; Gaal, Tamas; Gourse, Richard L

    2013-05-01

    The global regulatory nucleotide ppGpp ("magic spot") regulates transcription from a large subset of Escherichia coli promoters, illustrating how small molecules can control gene expression promoter-specifically by interacting with RNA polymerase (RNAP) without binding to DNA. However, ppGpp's target site on RNAP, and therefore its mechanism of action, has remained unclear. We report here a binding site for ppGpp on E. coli RNAP, identified by crosslinking, protease mapping, and analysis of mutant RNAPs that fail to respond to ppGpp. A strain with a mutant ppGpp binding site displays properties characteristic of cells defective for ppGpp synthesis. The binding site is at an interface of two RNAP subunits, ω and β', and its position suggests an allosteric mechanism of action involving restriction of motion between two mobile RNAP modules. Identification of the binding site allows prediction of bacterial species in which ppGpp exerts its effects by targeting RNAP.

  20. Positive Allosteric Modulators of 2-Amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic Acid Receptors Belonging to 4-Cyclopropyl-3,4-dihydro-2H-1,2,4-pyridothiadiazine Dioxides and Diversely Chloro-Substituted 4-Cyclopropyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-Dioxides

    DEFF Research Database (Denmark)

    Francotte, Pierre; Nørholm, Ann-Beth; Deva, Taru;

    2014-01-01

    Two 4-ethyl-substituted pyridothiadiazine dioxides belonging to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor positive allosteric modulators were cocrystallized with the GluA2 ligand binding domain in order to decipher the impact of the position of the nitrogen atom on thei...

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

    Science.gov (United States)

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

    2014-04-01

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

  2. Structure based discovery of small molecules to regulate the activity of human insulin degrading enzyme.

    Directory of Open Access Journals (Sweden)

    Bilal Çakir

    Full Text Available BACKGROUND: Insulin-degrading enzyme (IDE is an allosteric Zn(+2 metalloprotease involved in the degradation of many peptides including amyloid-β, and insulin that play key roles in Alzheimer's disease (AD and type 2 diabetes mellitus (T2DM, respectively. Therefore, the use of therapeutic agents that regulate the activity of IDE would be a viable approach towards generating pharmaceutical treatments for these diseases. Crystal structure of IDE revealed that N-terminal has an exosite which is ∼30 Å away from the catalytic region and serves as a regulation site by orientation of the substrates of IDE to the catalytic site. It is possible to find small molecules that bind to the exosite of IDE and enhance its proteolytic activity towards different substrates. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we applied structure based drug design method combined with experimental methods to discover four novel molecules that enhance the activity of human IDE. The novel compounds, designated as D3, D4, D6, and D10 enhanced IDE mediated proteolysis of substrate V, insulin and amyloid-β, while enhanced degradation profiles were obtained towards substrate V and insulin in the presence of D10 only. CONCLUSION/SIGNIFICANCE: This paper describes the first examples of a computer-aided discovery of IDE regulators, showing that in vitro and in vivo activation of this important enzyme with small molecules is possible.

  3. Structure Based Discovery of Small Molecules to Regulate the Activity of Human Insulin Degrading Enzyme

    Science.gov (United States)

    Çakir, Bilal; Dağliyan, Onur; Dağyildiz, Ezgi; Bariş, İbrahim; Kavakli, Ibrahim Halil; Kizilel, Seda; Türkay, Metin

    2012-01-01

    Background Insulin-degrading enzyme (IDE) is an allosteric Zn+2 metalloprotease involved in the degradation of many peptides including amyloid-β, and insulin that play key roles in Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM), respectively. Therefore, the use of therapeutic agents that regulate the activity of IDE would be a viable approach towards generating pharmaceutical treatments for these diseases. Crystal structure of IDE revealed that N-terminal has an exosite which is ∼30 Å away from the catalytic region and serves as a regulation site by orientation of the substrates of IDE to the catalytic site. It is possible to find small molecules that bind to the exosite of IDE and enhance its proteolytic activity towards different substrates. Methodology/Principal Findings In this study, we applied structure based drug design method combined with experimental methods to discover four novel molecules that enhance the activity of human IDE. The novel compounds, designated as D3, D4, D6, and D10 enhanced IDE mediated proteolysis of substrate V, insulin and amyloid-β, while enhanced degradation profiles were obtained towards substrate V and insulin in the presence of D10 only. Conclusion/Significance This paper describes the first examples of a computer-aided discovery of IDE regulators, showing that in vitro and in vivo activation of this important enzyme with small molecules is possible. PMID:22355395

  4. The Effectors and Sensory Sites of Formaldehyde-responsive Regulator FrmR and Metal-sensing Variant *

    Science.gov (United States)

    Osman, Deenah; Piergentili, Cecilia; Chen, Junjun; Sayer, Lucy N.; Usón, Isabel; Huggins, Thomas G.; Robinson, Nigel J.; Pohl, Ehmke

    2016-01-01

    The DUF156 family of DNA-binding transcriptional regulators includes metal sensors that respond to cobalt and/or nickel (RcnR, InrS) or copper (CsoR) plus CstR, which responds to persulfide, and formaldehyde-responsive FrmR. Unexpectedly, the allosteric mechanism of FrmR from Salmonella enterica serovar Typhimurium is triggered by metals in vitro, and variant FrmRE64H gains responsiveness to Zn(II) and cobalt in vivo. Here we establish that the allosteric mechanism of FrmR is triggered directly by formaldehyde in vitro. Sensitivity to formaldehyde requires a cysteine (Cys35 in FrmR) conserved in all DUF156 proteins. A crystal structure of metal- and formaldehyde-sensing FrmRE64H reveals that an FrmR-specific amino-terminal Pro2 is proximal to Cys35, and these residues form the deduced formaldehyde-sensing site. Evidence is presented that implies that residues spatially close to the conserved cysteine tune the sensitivities of DUF156 proteins above or below critical thresholds for different effectors, generating the semblance of specificity within cells. Relative to FrmR, RcnR is less responsive to formaldehyde in vitro, and RcnR does not sense formaldehyde in vivo, but reciprocal mutations FrmRP2S and RcnRS2P, respectively, impair and enhance formaldehyde reactivity in vitro. Formaldehyde detoxification by FrmA requires S-(hydroxymethyl)glutathione, yet glutathione inhibits formaldehyde detection by FrmR in vivo and in vitro. Quantifying the number of FrmR molecules per cell and modeling formaldehyde modification as a function of [formaldehyde] demonstrates that FrmR reactivity is optimized such that FrmR is modified and frmRA is derepressed at lower [formaldehyde] than required to generate S-(hydroxymethyl)glutathione. Expression of FrmA is thereby coordinated with the accumulation of its substrate. PMID:27474740

  5. Cancer metabolism meets systems biology: Pyruvate kinase isoform PKM2 is a metabolic master regulator

    Directory of Open Access Journals (Sweden)

    Fabian V Filipp

    2013-01-01

    Full Text Available Pyruvate kinase activity is controlled by a tightly woven regulatory network. The oncofetal isoform of pyruvate kinase (PKM2 is a master regulator of cancer metabolism. PKM2 engages in parallel, feed-forward, positive and negative feedback control contributing to cancer progression. Besides its metabolic role, non-metabolic functions of PKM2 as protein kinase and transcriptional coactivator for c-MYC and hypoxia-inducible factor 1-alpha are essential for epidermal growth factor receptor activation-induced tumorigenesis. These biochemical activities are controlled by a shift in the oligomeric state of PKM2 that includes acetylation, oxidation, phosphorylation, prolyl hydroxylation and sumoylation. Metabolically active PKM2 tetramer is allosterically regulated and responds to nutritional and stress signals. Metabolically inactive PKM2 dimer is imported into the nucleus and can function as protein kinase stimulating transcription. A systems biology approach to PKM2 at the genome, transcriptome, proteome, metabolome and fluxome level reveals how differences in biomolecular structure translate into a global rewiring of cancer metabolism. Cancer systems biology takes us beyond the Warburg effect, opening unprecedented therapeutic opportunities.

  6. Small molecule regulation of self-association and catalytic activity in a supramolecular coordination complex.

    Science.gov (United States)

    McGuirk, C Michael; Stern, Charlotte L; Mirkin, Chad A

    2014-03-26

    Herein, we report the synthesis and characterization of the first weak-link approach (WLA) supramolecular construct that employs the small molecule regulation of intermolecular hydrogen bonding interactions for the in situ control of catalytic activity. A biaryl urea group, prone to self-aggregation, was functionalized with a phosphinoalkyl thioether (P,S) hemilabile moiety and incorporated into a homoligated Pt(II) tweezer WLA complex. This urea-containing construct, which has been characterized by a single crystal X-ray diffraction study, can be switched in situ from a rigid fully closed state to a flexible semiopen state via Cl(-) induced changes in the coordination mode at the Pt(II) structural node. FT-IR and (1)H NMR spectroscopy studies were used to demonstrate that while extensive urea self-association persists in the flexible semiopen complex, these interactions are deterred in the rigid, fully closed complex because of geometric and steric restraints. Consequently, the urea moieties in the fully closed complex are able to catalyze a Diels-Alder reaction between cyclopentadiene and methyl vinyl ketone to generate 2-acetyl-5-norbornene. The free urea ligand and the semiopen complex show no such activity. The successful incorporation and regulation of a hydrogen bond donating catalyst in a WLA construct open the doors to a vast and rapidly growing catalogue of allosteric catalysts for applications in the detection and amplification of organic analytes.

  7. Argininosuccinate synthetase regulates hepatic AMPK linking protein catabolism and ureagenesis to hepatic lipid metabolism.

    Science.gov (United States)

    Madiraju, Anila K; Alves, Tiago; Zhao, Xiaojian; Cline, Gary W; Zhang, Dongyan; Bhanot, Sanjay; Samuel, Varman T; Kibbey, Richard G; Shulman, Gerald I

    2016-06-14

    A key sensor of cellular energy status, AMP-activated protein kinase (AMPK), interacts allosterically with AMP to maintain an active state. When active, AMPK triggers a metabolic switch, decreasing the activity of anabolic pathways and enhancing catabolic processes such as lipid oxidation to restore the energy balance. Unlike oxidative tissues, in which AMP is generated from adenylate kinase during states of high energy demand, the ornithine cycle enzyme argininosuccinate synthetase (ASS) is a principle site of AMP generation in the liver. Here we show that ASS regulates hepatic AMPK, revealing a central role for ureagenesis flux in the regulation of metabolism via AMPK. Treatment of primary rat hepatocytes with amino acids increased gluconeogenesis and ureagenesis and, despite nutrient excess, induced both AMPK and acetyl-CoA carboxylase (ACC) phosphorylation. Antisense oligonucleotide knockdown of hepatic ASS1 expression in vivo decreased liver AMPK activation, phosphorylation of ACC, and plasma β-hydroxybutyrate concentrations. Taken together these studies demonstrate that increased amino acid flux can activate AMPK through increased AMP generated by ASS, thus providing a novel link between protein catabolism, ureagenesis, and hepatic lipid metabolism. PMID:27247419

  8. Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role

    Directory of Open Access Journals (Sweden)

    Sluse F.E.

    1998-01-01

    Full Text Available Plants and some other organisms including protists possess a complex branched respiratory network in their mitochondria. Some pathways of this network are not energy-conserving and allow sites of energy conservation to be bypassed, leading to a decrease of the energy yield in the cells. It is a challenge to understand the regulation of the partitioning of electrons between the various energy-dissipating and -conserving pathways. This review is focused on the oxidase side of the respiratory chain that presents a cyanide-resistant energy-dissipating alternative oxidase (AOX besides the cytochrome pathway. The known structural properties of AOX are described including transmembrane topology, dimerization, and active sites. Regulation of the alternative oxidase activity is presented in detail because of its complexity. The alternative oxidase activity is dependent on substrate availability: total ubiquinone concentration and its redox state in the membrane and O2 concentration in the cell. The alternative oxidase activity can be long-term regulated (gene expression or short-term (post-translational modification, allosteric activation regulated. Electron distribution (partitioning between the alternative and cytochrome pathways during steady-state respiration is a crucial measurement to quantitatively analyze the effects of the various levels of regulation of the alternative oxidase. Three approaches are described with their specific domain of application and limitations: kinetic approach, oxygen isotope differential discrimination, and ADP/O method (thermokinetic approach. Lastly, the role of the alternative oxidase in non-thermogenic tissues is discussed in relation to the energy metabolism balance of the cell (supply in reducing equivalents/demand in energy and carbon and with harmful reactive oxygen species formation.

  9. Metabolic regulation during sport events: factual interpretations and inadequate allegations

    Directory of Open Access Journals (Sweden)

    Jacques Remy Poortmans

    2013-09-01

    Full Text Available Different fuels are available to generate ATP for muscle activities during sport events. Glycogen from striated muscles and liver stores may be converted to lactic acid or almost completely oxidized to carbon dioxide (CO2, triacylglycerol within the muscle itself and fatty acids from adipose tissue could be converted to CO2 in acting muscles, some free amino acids can be released within the muscle itself and from intestinal stores to sustain the amount of ATP generation indispensable for muscle contraction. All single biochemical reactions, but one, need one or several enzymes to activate the conversion of a substrate into a product. The energy transformation in biochemical reactions is led by application of so-called free energy. Reversible and non-reversible reactions within a metabolic pathway are dependent on specific enzymes near or far from equilibrium. Allosteric enzymes are regulatory enzymes that provide the direction in the pathway. A regulatory enzyme is either activated or inhibited by small regulators (ligands. A reversible substrate cycle between A and B is catalyzed by two enzymes with different fluxes. The need of ATP production for muscle contraction is under the leadership of regulatory enzymes and available substrate stores. The improvement of adapted metabolic reactions under sport training depends on the appropriate increase of regulatory enzymes within the glycolytic and oxidative pathways. The amount of some specific enzymes is increased by training in order to improve the maximum activity of the metabolic pathway. Unfortunately, several publications do not precisely implicate the appropriate enzyme(s to explain or reject the adaptation induced by the training schedule. A few examples will illustrate the factual interpretation and the inadequate allegation.

  10. Regulating Rho GTPases and their regulators.

    Science.gov (United States)

    Hodge, Richard G; Ridley, Anne J

    2016-08-01

    Rho GTPases regulate cytoskeletal and cell adhesion dynamics and thereby coordinate a wide range of cellular processes, including cell migration, cell polarity and cell cycle progression. Most Rho GTPases cycle between a GTP-bound active conformation and a GDP-bound inactive conformation to regulate their ability to activate effector proteins and to elicit cellular responses. However, it has become apparent that Rho GTPases are regulated by post-translational modifications and the formation of specific protein complexes, in addition to GTP-GDP cycling. The canonical regulators of Rho GTPases - guanine nucleotide exchange factors, GTPase-activating proteins and guanine nucleotide dissociation inhibitors - are regulated similarly, creating a complex network of interactions to determine the precise spatiotemporal activation of Rho GTPases. PMID:27301673

  11. Advances in the research of tageting DFG-out allosteric binding site of inactive kinases%靶向非活性激酶DFG-out变构结合位点的研究进展

    Institute of Scientific and Technical Information of China (English)

    彭文; 张小猛; 张仓; 王芳; 尤启冬

    2012-01-01

    目前大多数激酶抑制剂是通过模拟ATP的结构,以识别激酶的活性构象来竞争性结合于ATP结合位点,从而抑制激酶的自磷酸化和下游的信号传导.然而,最近人们对已上市药物甲磺酸伊马替尼、尼罗替尼及对甲苯磺酸索拉非尼的晶体结构研究发现,在非活性激酶中ATP结合位点的相邻位置存在着第二个能与激酶抑制剂结合的位点——DFG-out变构结合位点.该位点的发现为以蛋白激酶为靶标的小分子激酶抑制剂的设计与开发指明了新的方向,成为抗肿瘤研究领域的新热点之一.因此,本文对非活性激酶的DFG-out变构结合位点的发现、非活性激酶与其抑制剂的结合方式及处于临床研究阶段的非活性激酶抑制剂进行了综述.%Up to nowadays, a majority of kinase inhibitors identify the activity conformation of protein ki-nase to integrate competitively with ATP binding site by simulating the structure of ATP. In this way, kinase inhibitors can inhibit kinase autophosphorylation and restrain signal transduction of downstream. However, the crystal structures of imatinib mesylate, nilotnib and sorafenib tosylate have revealed a secondary binding site adjacent to the ATP binding site, which is also bound by kinase inhibitors, known as the DFG-out allosteric binding site, in the inactive conformation of protein kinase. The discovery of the site has pointed out a new direction for the design and development of small molecule kinase inhibitors, which lakes protein kinase as a target. This becomes a new hotspot in antineoplastic research field. In this paper, we reviewed the discovery and inhibitors of the DFG-out allosteric binding site, and the binding mode between inactive kinases, as well as the inactive kinase inhibitors in clinical studies.

  12. Positive allosteric modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors differentially modulates the behavioural effects of citalopram in mouse models of antidepressant and anxiolytic action.

    Science.gov (United States)

    Fitzpatrick, Ciarán M; Larsen, Maria; Madsen, Louise H; Caballero-Puntiverio, Maitane; Pickering, Darryl S; Clausen, Rasmus P; Andreasen, Jesper T

    2016-09-01

    Drugs that increase monoamine neurotransmission are effective in both anxiety and depression. The therapeutic effects of monoamine-based antidepressant drugs may involve indirect effects on neurotransmission through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPAR). Thus, chronic antidepressant treatment increases AMPAR-mediated neurotransmission and AMPAR-positive allosteric modulators have shown antidepressant-like efficacy in rodents. Here, the effect of enhanced AMPAR neurotransmission on the antidepressant-like and anxiolytic-like actions of the selective serotonin reuptake inhibitor citalopram (0-10 mg/kg) was investigated in mice using the AMPAR-positive allosteric modulator LY451646 (0-3 mg/kg). Antidepressant-like effects were assessed using the forced-swim test (FST), whereas anxiolytic-like effects were tested using the elevated zero maze (EZM) and the marble burying test. LY451646 (3 mg/kg) increased swim distance in the FST and a subactive dose of LY451646 (1 mg/kg) enhanced the effect of citalopram in the FST. In the EZM, LY451646 (3 mg/kg) did not show anxiogenic effects alone, but blocked the anxiolytic-like action of citalopram in the EZM, as reflected by an increase in the latency to enter the open areas and a decrease in the number of entries and time spent in the open areas in citalopram-treated mice. In the marble burying test, LY451646 (3 mg/kg) showed no effect alone, but significantly attenuated the anxiolytic-like effect of citalopram (1.25-2.5 mg/kg) by increasing the number of marbles buried in citalopram-treated mice. These results suggest that AMPAR neurotransmission plays opposite roles in anxiety and depression as AMPAR potentiation facilitated the antidepressant-like effects of citalopram while attenuating its anxiolytic-like effect. These findings have ramifications in the search for AMPAR-based novel anxiolytic and antidepressant treatments. PMID:27341500

  13. Domain organization, catalysis and regulation of eukaryotic cystathionine beta-synthases.

    Directory of Open Access Journals (Sweden)

    Tomas Majtan

    Full Text Available Cystathionine beta-synthase (CBS is a key regulator of sulfur amino acid metabolism diverting homocysteine, a toxic intermediate of the methionine cycle, via the transsulfuration pathway to the biosynthesis of cysteine. Although the pathway itself is well conserved among eukaryotes, properties of eukaryotic CBS enzymes vary greatly. Here we present a side-by-side biochemical and biophysical comparison of human (hCBS, fruit fly (dCBS and yeast (yCBS enzymes. Preparation and characterization of the full-length and truncated enzymes, lacking the regulatory domains, suggested that eukaryotic CBS exists in one of at least two significantly different conformations impacting the enzyme's catalytic activity, oligomeric status and regulation. Truncation of hCBS and yCBS, but not dCBS, resulted in enzyme activation and formation of dimers compared to native tetramers. The dCBS and yCBS are not regulated by the allosteric activator of hCBS, S-adenosylmethionine (AdoMet; however, they have significantly higher specific activities in the canonical as well as alternative reactions compared to hCBS. Unlike yCBS, the heme-containing dCBS and hCBS showed increased thermal stability and retention of the enzyme's catalytic activity. The mass-spectrometry analysis and isothermal titration calorimetry showed clear presence and binding of AdoMet to yCBS and hCBS, but not dCBS. However, the role of AdoMet binding to yCBS remains unclear, unlike its role in hCBS. This study provides valuable information for understanding the complexity of the domain organization, catalytic specificity and regulation among eukaryotic CBS enzymes.

  14. Light- and metabolism-related regulation of the chloroplast ATP synthase has distinct mechanisms and functions.

    Science.gov (United States)

    Kohzuma, Kaori; Dal Bosco, Cristina; Meurer, Jörg; Kramer, David M

    2013-05-01

    The chloroplast CF0-CF1-ATP synthase (ATP synthase) is activated in the light and inactivated in the dark by thioredoxin-mediated redox modulation of a disulfide bridge on its γ subunit. The activity of the ATP synthase is also fine-tuned during steady-state photosynthesis in response to metabolic changes, e.g. altering CO2 levels to adjust the thylakoid proton gradient and thus the regulation of light harvesting and electron transfer. The mechanism of this fine-tuning is unknown. We test here the possibility that it also involves redox modulation. We found that modifying the Arabidopsis thaliana γ subunit by mutating three highly conserved acidic amino acids, D211V, E212L, and E226L, resulted in a mutant, termed mothra, in which ATP synthase which lacked light-dark regulation had relatively small effects on maximal activity in vivo. In situ equilibrium redox titrations and thiol redox-sensitive labeling studies showed that the γ subunit disulfide/sulfhydryl couple in the modified ATP synthase has a more reducing redox potential and thus remains predominantly oxidized under physiological conditions, implying that the highly conserved acidic residues in the γ subunit influence thiol redox potential. In contrast to its altered light-dark regulation, mothra retained wild-type fine-tuning of ATP synthase activity in response to changes in ambient CO2 concentrations, indicating that the light-dark- and metabolic-related regulation occur through different mechanisms, possibly via small molecule allosteric effectors or covalent modification.

  15. Trout Stream Special Regulations

    Data.gov (United States)

    Minnesota Department of Natural Resources — This layer shows Minnesota trout streams that have a special regulation as described in the 2006 Minnesota Fishing Regulations. Road crossings were determined using...

  16. Regulation of Genetic Tests

    Science.gov (United States)

    ... advertised. The Commission has the authority to regulate advertising that delivers health-related information to consumers to ensure that it is not false or misleading. Top of page FDA Regulation and ...

  17. GlgS, described previously as a glycogen synthesis control protein, negatively regulates motility and biofilm formation in Escherichia coli.

    Science.gov (United States)

    Rahimpour, Mehdi; Montero, Manuel; Almagro, Goizeder; Viale, Alejandro M; Sevilla, Ángel; Cánovas, Manuel; Muñoz, Francisco J; Baroja-Fernández, Edurne; Bahaji, Abdellatif; Eydallin, Gustavo; Dose, Hitomi; Takeuchi, Rikiya; Mori, Hirotada; Pozueta-Romero, Javier

    2013-06-15

    Escherichia coli glycogen metabolism involves the regulation of glgBXCAP operon expression and allosteric control of the GlgC [ADPG (ADP-glucose) pyrophosphorylase]-mediated catalysis of ATP and G1P (glucose-1-phosphate) to ADPG linked to glycogen biosynthesis. E. coli glycogen metabolism is also affected by glgS. Though the precise function of the protein it encodes is unknown, its deficiency causes both reduced glycogen content and enhanced levels of the GlgC-negative allosteric regulator AMP. The transcriptomic analyses carried out in the present study revealed that, compared with their isogenic BW25113 wild-type strain, glgS-null (ΔglgS) mutants have increased expression of the operons involved in the synthesis of type 1 fimbriae adhesins, flagella and nucleotides. In agreement, ΔglgS cells were hyperflagellated and hyperfimbriated, and displayed elevated swarming motility; these phenotypes all reverted to the wild-type by ectopic glgS expression. Also, ΔglgS cells accumulated high colanic acid content and displayed increased ability to form biofilms on polystyrene surfaces. F-driven conjugation based on large-scale interaction studies of glgS with all the non-essential genes of E. coli showed that deletion of purine biosynthesis genes complement the glycogen-deficient, high motility and high biofilm content phenotypes of ΔglgS cells. Overall the results of the present study indicate that glycogen deficiency in ΔglgS cells can be ascribed to high flagellar propulsion and high exopolysaccharide and purine nucleotides biosynthetic activities competing with GlgC for the same ATP and G1P pools. Supporting this proposal, glycogen-less ΔglgC cells displayed an elevated swarming motility, and accumulated high levels of colanic acid and biofilm. Furthermore, glgC overexpression reverted the glycogen-deficient, high swarming motility, high colanic acid and high biofilm content phenotypes of ΔglgS cells to the wild-type. As on the basis of the present study Glg

  18. General Theories of Regulation

    NARCIS (Netherlands)

    Hertog, J.A. den

    1999-01-01

    This chapter makes a distinction between three types of theories of regulation: public interest theories, the Chicago theory of regulation and the public choice theories. The Chicago theory is mainly directed at the explanation of economic regulation; public interest theories and public choice theor

  19. Hepcidin: regulation of the master iron regulator

    OpenAIRE

    2015-01-01

    Iron, an essential nutrient, is required for many diverse biological processes. The absence of a defined pathway to excrete excess iron makes it essential for the body to regulate the amount of iron absorbed; a deficiency could lead to iron deficiency and an excess to iron overload and associated disorders such as anaemia and haemochromatosis respectively. This regulation is mediated by the iron-regulatory hormone hepcidin. Hepcidin binds to the only known iron export protein, ferroportin (FP...

  20. Targeting AKT with the allosteric AKT inhibitor MK-2206 in non-small cell lung cancer cells with acquired resistance to cetuximab

    OpenAIRE

    Iida, Mari; Brand, Toni M; Campbell, David A; Starr, Megan M.; Luthar, Neha; Traynor, Anne M.; Wheeler, Deric L

    2013-01-01

    The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for use in oncology. Despite clinical success the majority of patients do not respond to cetuximab and those who initially respond frequently acquire resistance. To understand how tumor cells acquire resistance to cetuximab we developed a model of resistance using the non-small cell lung cancer line NCI-H226. We found th...

  1. Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor

    OpenAIRE

    Lyukmanova, Ekaterina N.; Mikhail A Shulepko; Denis Kudryavtsev; Bychkov, Maxim L.; Kulbatskii, Dmitrii S.; Kasheverov, Igor E.; Astapova, Maria V.; Feofanov, Alexey V; Thomsen, Morten S.; Jens D Mikkelsen; Shenkarev, Zakhar O.; Tsetlin, Victor I.; Dolgikh, Dmitry A.; Mikhail P Kirpichnikov

    2016-01-01

    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 ...

  2. 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.

  3. Views of the regulators

    International Nuclear Information System (INIS)

    In dealing with a challenging problem in occupational exposure the nuclear regulator in South Africa concluded that the involvement of stake holders was critical. Valuable lessons were learnt in the process. These related to co-operation amongst regulators, the involvement of regulators in addressing occupational exposure problems, the training of workers by the regulator and the need for technical training of the workers. In general, it was also learnt that regulators should establish mechanisms to measure and continuously improve the satisfaction of their stake holders. (author)

  4. A Redox 2-Cys Mechanism Regulates the Catalytic Activity of Divergent Cyclophilins1[W

    Science.gov (United States)

    Campos, Bruna Medéia; Sforça, Mauricio Luis; Ambrosio, Andre Luis Berteli; Domingues, Mariane Noronha; Brasil de Souza, Tatiana de Arruda Campos; Barbosa, João Alexandre Ribeiro Gonçalvez; Leme, Adriana Franco Paes; Perez, Carlos Alberto; Whittaker, Sara Britt-Marie; Murakami, Mario Tyago; Zeri, Ana Carolina de Matos; Benedetti, Celso Eduardo

    2013-01-01

    The citrus (Citrus sinensis) cyclophilin CsCyp is a target of the Xanthomonas citri transcription activator-like effector PthA, required to elicit cankers on citrus. CsCyp binds the citrus thioredoxin CsTdx and the carboxyl-terminal domain of RNA polymerase II and is a divergent cyclophilin that carries the additional loop KSGKPLH, invariable cysteine (Cys) residues Cys-40 and Cys-168, and the conserved glutamate (Glu) Glu-83. Despite the suggested roles in ATP and metal binding, the functions of these unique structural elements remain unknown. Here, we show that the conserved Cys residues form a disulfide bond that inactivates the enzyme, whereas Glu-83, which belongs to the catalytic loop and is also critical for enzyme activity, is anchored to the divergent loop to maintain the active site open. In addition, we demonstrate that Cys-40 and Cys-168 are required for the interaction with CsTdx and that CsCyp binds the citrus carboxyl-terminal domain of RNA polymerase II YSPSAP repeat. Our data support a model where formation of the Cys-40-Cys-168 disulfide bond induces a conformational change that disrupts the interaction of the divergent and catalytic loops, via Glu-83, causing the active site to close. This suggests a new type of allosteric regulation in divergent cyclophilins, involving disulfide bond formation and a loop-displacement mechanism. PMID:23709667

  5. Nuclear safety regulations

    International Nuclear Information System (INIS)

    The Nuclear Safety Regulations for Nuclear Installations and Nuclear Safety Codes for Nuclear Pressure Retaining Components were issued by the NNSA in 1995. The Atomic Act and Regulations on the Safety Regulation for Transportation of Radioactive Materials have been finished and submitted to the State Council in 1995. At the same time the NNSA organized a revised collection of regulations on nuclear safety in both Chinese and English, titled 'The Collection of Regulations on Nuclear Safety of the People's Republic of China'. To enhance the implementation of newly issued nuclear safety regulations, the NNSA conducted seven times of propagating activities in relation to the regulations for nuclear pressure retaining components and research reactors design and operating in 1995

  6. Crystal Structures of the Reduced, Sulfenic Acid, and Mixed Disulfide Forms of SarZ, a Redox Active Global Regulator in Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Poor, Catherine B.; Chen, Peng R.; Duguid, Erica; Rice, Phoebe A.; He, Chuan; (UC)

    2010-01-20

    SarZ is a global transcriptional regulator that uses a single cysteine residue, Cys{sup 13}, to sense peroxide stress and control metabolic switching and virulence in Staphylococcus aureus. SarZ belongs to the single-cysteine class of OhrR-MgrA proteins that play key roles in oxidative resistance and virulence regulation in various bacteria. We present the crystal structures of the reduced form, sulfenic acid form, and mixed disulfide form of SarZ. Both the sulfenic acid and mixed disulfide forms are structurally characterized for the first time for this class of proteins. The Cys{sup 13} sulfenic acid modification is stabilized through two hydrogen bonds with surrounding residues, and the overall DNA-binding conformation is retained. A further reaction of the Cys{sup 13} sulfenic acid with an external thiol leads to formation of a mixed disulfide bond, which results in an allosteric change in the DNA-binding domains, disrupting DNA binding. Thus, the crystal structures of SarZ in three different states provide molecular level pictures delineating the mechanism by which this class of redox active regulators undergoes activation. These structures help to understand redox-mediated virulence regulation in S. aureus and activation of the MarR family proteins in general.

  7. The development of regulations

    International Nuclear Information System (INIS)

    In October 2002, The Act on Protection Against Ionising Radiation and Nuclear Safety which regulates all aspects of protection against ionising radiation and nuclear safety entered into force in Slovenia. The Slovenian government and its responsible ministries shall issue several governmental and ministerial regulations to support the above - mentioned act. The Slovenian Nuclear Safety Administration (SNSA) which acts within the Ministry of the Environment, Spatial Planing and Energy takes an active part in drafting the regulations which are defined in the act. Due to a very comprehensive and pretentious task, that is to be completed in a relatively short period of time, taking into consideration the involvement of stakeholders and all competent ministries, the SNSA within the Quality Management System developed a special procedure that insures the systematic approach to the preparation of regulations. The article will briefly represent the process that: defines the preparation, development, harmonisation, review, approval and issue of regulations and uniforms the format of developed regulations. (author)

  8. TOWARD MORE EFFECTIVE REGULATION

    Energy Technology Data Exchange (ETDEWEB)

    J. GRAF

    2000-06-01

    This paper proposes a model relationship between the operator engaged in a hazardous activity, the regulator of that activity, and the general public. The roles and responsibilities of each entity are described in a way that allows effective communication flow. The role of the regulator is developed using the steam boiler as an example of a hazard subject to regulation; however, the model applies to any regulated activity. In this model the safety analyst has the extremely important role of communicating sometimes difficult technical information to the regulator in a way that the regulator can provide credible assurance to the general public as to the adequacy of the control of the hazardous activity. The conclusion asserts that acceptance of the model, understanding of the roles and responsibilities and definition of who communicates what information to whom will mitigate frustration on the part of each of the three entities.

  9. Nuclear safety regulations

    International Nuclear Information System (INIS)

    The enactment of nuclear safety regulations in 1996 is mainly focused on the preparation of related regulations, and safety guides for nuclear materials control, the reprocessing installations of spent fuels, the treatment and disposal for radioactive waste. The NNSA also assists the departments concerned of the State Council for modification on the 'Atomic Energy Act' (draft) and the' Regulations on the Safety Supervision and Control of Radioactive Materials Transportation' (draft)

  10. Emotional regulation and friendship

    OpenAIRE

    Zaccagnini, J.L.; Ruiz-Aranda, D.

    2013-01-01

    Previous literature has been shown that emotional regulation facilitates the establishment and maintenance of social relations (Dodge Garber, 1991; Saarni, 1999). The objective of the present study was to analyze the influence of emotional regulation (Gross y John, 2003) in positive friendship (Berscheid, 2003), specifically at the level of intimacy with friends. In addition, we examined the mediating role of positive emotions in the relationship between the emotional regulation and the leve...

  11. Accounting Regulation in Ukraine

    OpenAIRE

    Hora, Michal; Chyzevska, Ludmila

    2013-01-01

    The aim of the paper is to evaluate the regulation and organization of accounting in Ukraine under the changes in the national economic system development and impact of IFRS implementation. The system of legal regulation of accounting in Ukraine is presented by five levels, each comprised of a number of corresponding subjects of regulation and documents. Typical Chart of Accounts is evidence of the continental accounting model in Ukraine. The accounting standards provide freedom of choice as ...

  12. Antipsychotic-like Effects of M4 Positive Allosteric Modulators Are Mediated by CB2 Receptor-Dependent Inhibition of Dopamine Release.

    Science.gov (United States)

    Foster, Daniel J; Wilson, Jermaine M; Remke, Daniel H; Mahmood, M Suhaib; Uddin, M Jashim; Wess, Jürgen; Patel, Sachin; Marnett, Lawrence J; Niswender, Colleen M; Jones, Carrie K; Xiang, Zixiu; Lindsley, Craig W; Rook, Jerri M; Conn, P Jeffrey

    2016-09-21

    Muscarinic receptors represent a promising therapeutic target for schizophrenia, but the mechanisms underlying the antipsychotic efficacy of muscarinic modulators are not well understood. Here, we report that activation of M4 receptors on striatal spiny projection neurons results in a novel form of dopaminergic regulation resulting in a sustained depression of striatal dopamine release that is observed more than 30 min after removal of the muscarinic receptor agonist. Furthermore, both the M4-mediated sustained inhibition of dopamine release and the antipsychotic-like efficacy of M4 activators were found to require intact signaling through CB2 cannabinoid receptors. These findings highlight a novel mechanism by which striatal cholinergic and cannabinoid signaling leads to sustained reductions in dopaminergic transmission and concurrent behavioral effects predictive of antipsychotic efficacy. PMID:27618677

  13. 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

  14. Transfer of noncovalent chiral information along an optically inactive helical peptide chain: allosteric control of asymmetry of the C-terminal site by external molecule that binds to the N-terminal site.

    Science.gov (United States)

    Ousaka, Naoki; Inai, Yoshihito

    2009-02-20

    This study aims at demonstrating end-to-end transfer of noncovalent chiral information along a peptide chain. The domino-type induction of helical sense is proven by using achiral peptides 1-m of bis-chromophoric sequence with different chain lengths: H-(Aib-Delta(Z)Phe)(m)-(Aib-Delta(Z)Bip)(2)-Aib-OCH(3) [m = 2, 4, and 6; Aib = alpha-aminoisobutyric acid; Delta(Z)Phe = (Z)-alpha,beta-didehydrophenylalanine; Delta(Z)Bip = (Z)-beta-(4,4'-biphenyl)-alpha,beta-didehydroalanine]. They all showed the tendency to adopt a 3(10)-helix. Whereas peptide 1-m originally shows no circular dichroism (CD) signals, marked CD signals were induced at around 270-320 nm based on both the beta-aryl didehydroresidues by chiral Boc-proline (Boc = tert-butoxycarbonyl). The observed CD spectra were interpreted on the basis of the exciton chirality method and theoretical CD simulation of several helical conformations that were energy-minimized. The experimental and theoretical CD analysis reveals that Boc-l-proline induces the preference for a right-handed helicity in the whole chain of 1-m. Such noncovalent chiral induction was not observed in the corresponding N-terminally protected 1-m. Obviously, helicity induction in 1-m originates from the binding of Boc-proline to the N-terminal site. In the 17-mer (1-6), the information of helix sense reaches the 16th residue from the N-terminus. We have monitored precise transfer of noncovalent chiral stimulus along a helical peptide chain. The present study also proposes a primitive allosteric model of a single protein-mimicking backbone. Here chiral molecule binding the N-terminal site of 1-6 controls the chiroptical signals and helical sense of the C-terminal site about 30 A away.

  15. Plant Growth Regulators.

    Science.gov (United States)

    Nickell, Louis G.

    1978-01-01

    Describes the effect of "plant growth regulators" on plants, such as controlling the flowering, fruit development, plant size, and increasing crop yields. Provides a list of plant growth regulators which includes their chemical, common, and trade names, as well as their different use(s). (GA)

  16. Mortgage market regulation: Europe

    NARCIS (Netherlands)

    M.B. Aalbers

    2012-01-01

    Despite several European Union (EU) initiatives, there is only limited pan-European mortgage market regulation. The EU strategy can be characterised as one of parallel liberalisation and consolidation. This article highlights the key differences in regulation among European mortgage markets. Mortgag

  17. Emotion-regulation choice

    NARCIS (Netherlands)

    Sheppes, Gal; Scheibe, Susanne; Suri, Gaurav; Gross, James J.

    2011-01-01

    Despite centuries of speculation about how to manage negative emotions, little is actually known about which emotion-regulation strategies people choose to use when confronted with negative situations of varying intensity. On the basis of a new process conception of emotion regulation, we hypothesiz

  18. Benchmarking and Regulation

    DEFF Research Database (Denmark)

    Agrell, Per J.; Bogetoft, Peter

    nchmarking methods, and in particular Data Envelopment Analysis (DEA), have become well-established and informative tools for economic regulation. DEA is now routinely used by European regulators to set reasonable revenue caps for energy transmission and distribution system operators. The applica......nchmarking methods, and in particular Data Envelopment Analysis (DEA), have become well-established and informative tools for economic regulation. DEA is now routinely used by European regulators to set reasonable revenue caps for energy transmission and distribution system operators....... The application of benchmarking in regulation, however, requires specific steps in terms of data validation, model specification and outlier detection that are not systematically documented in open publications, leading to discussions about regulatory stability and economic feasibility of these techniques...

  19. Regulation of megakaryocytopoiesis.

    Science.gov (United States)

    Caen, J P; Han, Z C; Bellucci, S; Alemany, M

    1999-09-01

    After 35 years of research, a physiological regulator of platelet production has been identified and the recombinant protein is available. With the discovery of thrombopoietin (TPO), its potential use in a wide variety of clinical megakaryocytic and platelet disorders has been expected and clinical trials have been undertaken. To date, the reported encouraging pre-clinical studies indicate that, as with erythropoietin or G-CSF, minimal toxicity can be expected. A potential limiting side-effect of TPO could be the induction of thrombosis. Nevertheless, it is too early to know whether this cytokine will be of major therapeutic importance for patients with life-threatening thrombocytopenia, such as patients undergoing bone marrow transplantation or subjected to a high dose of chemotherapy. Several experimental and clinical studies are still needed to determine the efficacy of TPO in the prevention or the amelioration of bleeding, which is the ultimate goal for the appropriate use of cytokines with haemostatic benefit. Basic and clinical studies on regulators of megakaryocytopoiesis have rapidly progressed. Now, there is no doubt that some of these regulators are effective in correcting haematopoietic disorders of various aetiologies. Studies on negative regulators not only are important to understand the regulation of megakaryocytopoiesis in normal and pathological states but also have a potential clinical application. Some of these regulators have been shown to be effective in the treatment of essential thrombocythaemia and other myeloproliferative disorders. Platelet factor 4 (PF4) and some other chemokines are also capable of protecting progenitor cells from the cytotoxicity of chemotherapeutic drugs. However, detailed investigations are still required to determine the precise mechanism(s) of action of these regulators and to establish the optimal clinical protocols of negative regulators alone or in association with positive regulators for the treatment of various

  20. Reciprocal regulation as a source of ultrasensitivity in two-component systems with a bifunctional sensor kinase.

    Directory of Open Access Journals (Sweden)

    Ronny Straube

    2014-05-01

    Full Text Available Two-component signal transduction systems, where the phosphorylation state of a regulator protein is modulated by a sensor kinase, are common in bacteria and other microbes. In many of these systems, the sensor kinase is bifunctional catalyzing both, the phosphorylation and the dephosphorylation of the regulator protein in response to input signals. Previous studies have shown that systems with a bifunctional enzyme can adjust the phosphorylation level of the regulator protein independently of the total protein concentrations--a property known as concentration robustness. Here, I argue that two-component systems with a bifunctional enzyme may also exhibit ultrasensitivity if the input signal reciprocally affects multiple activities of the sensor kinase. To this end, I consider the case where an allosteric effector inhibits autophosphorylation and, concomitantly, activates the enzyme's phosphatase activity, as observed experimentally in the PhoQ/PhoP and NRII/NRI systems. A theoretical analysis reveals two operating regimes under steady state conditions depending on the effector affinity: If the affinity is low the system produces a graded response with respect to input signals and exhibits stimulus-dependent concentration robustness--consistent with previous experiments. In contrast, a high-affinity effector may generate ultrasensitivity by a similar mechanism as phosphorylation-dephosphorylation cycles with distinct converter enzymes. The occurrence of ultrasensitivity requires saturation of the sensor kinase's phosphatase activity, but is restricted to low effector concentrations, which suggests that this mode of operation might be employed for the detection and amplification of low abundant input signals. Interestingly, the same mechanism also applies to covalent modification cycles with a bifunctional converter enzyme, which suggests that reciprocal regulation, as a mechanism to generate ultrasensitivity, is not restricted to two

  1. Electrical installations and regulations

    CERN Document Server

    Whitfield, J F

    1966-01-01

    Electrical Installations and Regulations focuses on the regulations that apply to electrical installations and the reasons for them. Topics covered range from electrical science to alternating and direct current supplies, as well as equipment for providing protection against excess current. Cables, wiring systems, and final subcircuits are also considered, along with earthing, discharge lighting, and testing and inspection.Comprised of 12 chapters, this book begins with an overview of electrical installation work, traits of a good electrician, and the regulations governing installations. The r

  2. The power of regulation

    International Nuclear Information System (INIS)

    Slides accompanying a presentation at The Power of Change Conference in Vancouver, BC in April 1995 about regulations affecting the power industry were presented. Issues addressed included customer choice, incentive regulation changes (price-caps, revenue sharing and pricing flexibility), the reactions of Canadian industry to regulatory changes, and anticipated reactions of the financial markets to changes in regulations. The potential effects of competition and changes that will create competition were discussed. The level of readiness of Canadian financial, ownership and regulatory bodies was discussed. The needs and expectations of investors from a new regulatory regime were quesstimated. Possible alternatives to the present regulatory framework were suggested

  3. Sport Fishing Regulations

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — The regulations for sport fishing on St. Vincent National Wildlife Refuge are outlined in this document. Fishing is only permitted from sunrise to sunset, and only...

  4. Regulating deregulated energy markets

    International Nuclear Information System (INIS)

    The North American gas and electricity markets are fast evolving, and regulators are currently faced with a host of issues such as market-based rates, unbundling, stranded costs, open access, and incentive regulation are surfacing as a result of deregulation. The regulatory environment in Ontario was reviewed by the author. Deregulated markets rule, from commodities to gas and electricity. Additionally, there is an evolution of traditional utility regulation. A look at deregulated markets revealed that there are regulations on boundary conditions on the deregulated market. Under the Ontario Energy Board (OEB), all generators, transmitters, distributors, and retailers of electricity must be licensed. The standard supply service (SSS) offered by electricity distributors and system gas which is still being sold by natural gas distributors continues to be regulated by OEB. One issue that was addressed was separation for revenues and costs of the utility's purchase and sale of gas business, at least for accounting purposes. The next issue discussed was cost of system gas and SSS, followed by timely signals and prudent incurred costs. Historical benefits were reviewed, such as historical commitments to low-cost electricity. Pooling transportation costs, transmission pricing continued, market-based rates, unbundling, stranded costs, open access, incentive regulation/ performance based regulation (PBR) were all discussed. Price cap on PBR, both partial and comprehensive were looked at. A requirement to review guidelines on cost of capital and an application to extend blanket approval provisions for gas storage were discussed, as they are amongst some of the challenges of the future. Other challenges include revised rules and practice and procedure; practice directions for cost awards, appeals, and other functions; confidentiality guidelines; and refinements to the role of and approaches to alternative dispute resolution. The future role of regulators was examined in light

  5. Optimal Regulation of Auditing

    OpenAIRE

    Pagano, Marco; Immordino, Giovanni

    2007-01-01

    We study regulation of the auditing profession in a model where audit quality is unobservable and enforcing regulation is costly. The optimal audit standard falls short of the first-best audit quality, and is increasing in the riskiness of firms and in the amount of funding they seek. The model can encompass collusion between clients and auditors, arising from the joint provision of auditing and consulting services: deflecting collusion requires less ambitious standards. Finally, banning the ...

  6. Financial Regulation Going Forward

    OpenAIRE

    Franklin Allen; Elena Carletti

    2010-01-01

    The financial sector is heavily regulated in order to prevent financial crises. The recent crisis showed how ineffective this regulation and other types of government intervention were in achieving this aim. We argue that the crisis was primarily caused by housing price bubbles. These occurred because of too loose monetary policies and the easy availability of credit resulting from the build up of large foreign exchange reserves by Asian central banks. A number of regulatory reforms are sugge...

  7. Regulating energy industries

    International Nuclear Information System (INIS)

    The concept of sector-based regulation takes on significant importance in the context of market liberalization. The overall aim is to conciliate, in the considered sector, fair competition with the achievement of public service missions. However, the nature of the authority in charge of this regulation is not prone to harmonized clauses, even in Europe. For electricity for example, the 96/92/CE directive of 19 December 1996 concerning common rules for the inner electricity market, does not state this and Germany, which has not designed any sector-based regulator, applies the general procedure of litigation settlement by the equivalent of the competition Council. In France, the law Nr 2000-108 of 10 February 2000 defines the CRE (Electricity Regulation Commission) is article 28 as including six members, three of which are appointed by Government and the three others respectively by each of the presidents of the parliamentary assemblies. Many other countries have made the same choice. However, the scope of the missions given to these specialized authorities varies considerably according to the country. At European level, what are the different models of organisation of sector-based regulation in the energy field? How are the new regulators organised in relation with the competition authorities? Will the new models converge on the medium term or on the long term? Must we anticipate the creation of European regulation authorities to rule the problems concerning several national markets? What can we learn from the recent electricity crisis in California? To try and answer these questions, Mr Michel Matheu presented a European comparative study and before the debate started, Mr Pierre Couveinhes suggested a reflection on the practical implications of the analyses carried out. (authors)

  8. Nuclear regulation in transition

    International Nuclear Information System (INIS)

    The current state of nuclear regulations in the USA is examined. Since Three Mile Island the regulation of the nuclear power industry has been undergoing a noticeable transition. It will be argued here that the transition is characterized by two indicia. First, the primary focus of state and federal regulators has been on the financial aspects of the industry: this is best seen in the context of decisions allocating the costs of nuclear plant cancellations. Second, decisionmaking power has been decentralized: although the regulatory history of nuclear power demonstrates the tradition of centralized decisionmaking power (i.e., formerly the primary decisionmaking body was the Atomic Energy Commission), now States share decisionmaking power with the Nuclear Regulatory Commission. In Section 1 a brief legislative history of nuclear regulation is presented to establish the assertion that nuclear regulation, both de jure and de facto, was centralized. Next, Section 2 canvasses recent United States Supreme Court opinions regarding nuclear regulation. The Court frequently acts as policymaker through the consequences of its opinions, if not by its intent. In the area of nuclear policymaking, the Court has paid allegiance recently both to the tradition of centralization and to the movement toward decentralization. This dualism is reflected in other federal court decisions as well which will be briefly mentioned. Continuing the analysis of Federal regulation, Section 3 examines the current reform efforts of the NRC. Section 4 presents an examination of State responses to nuclear plant cancellations. In this section, State administrative agency and court decisions will be examined and recent State legislation will be discussed. (author)

  9. Worldwide regulations for mycotoxins.

    Science.gov (United States)

    van Egmond, Hans P

    2002-01-01

    Since the discovery of the aflatoxins in the 1960s, regulations have been established in many countries to protect the consumer from the harmful effects of mycotoxins that may contaminate foodstuffs. Various factors play a role in the decision-making process of setting limits for mycotoxins. These include scientific factors such as the availability of toxicological data, survey data, knowledge about the distribution of mycotoxins in commodities, and analytical methodology. Economical and political factors such as commercial interests and sufficiency of food supply have their impact as well. International enquiry's on existing mycotoxin legislation in foodstuffs and animal feedstuffs have been carried out several times in the 1980s and 1990s and details about tolerances, legal basis, responsible authorities, official protocols of analysis and sampling have been published. Recently a comprehensive update on worldwide regulations was published as FAO Food and Nutrition Paper 64. It appeared that at least 77 countries now have specific regulations for mycotoxins, 13 countries are known to have no specific regulations, whereas no data are available for about 50 countries, many of them in Africa. Over the years, a large diversity in tolerance levels for mycotoxins has remained. Some free trade zones (EU, MERCOSUR) are in the process of harmonizing the limits and regulations for mycotoxins in their respective member states, but it is not likely that worldwide harmonized limits for mycotoxins will soon be within reach.

  10. Radiation emitting devices regulations

    International Nuclear Information System (INIS)

    The Radiation Emitting Devices Regulations are the regulations referred to in the Radiation Emitting Devices Act and relate to the operation of devices. They include standards of design and construction, standards of functioning, warning symbol specifications in addition to information relating to the seizure and detention of machines failing to comply with the regulations. The radiation emitting devices consist of the following: television receivers, extra-oral dental x-ray equipment, microwave ovens, baggage inspection x-ray devices, demonstration--type gas discharge devices, photofluorographic x-ray equipment, laser scanners, demonstration lasers, low energy electron microscopes, high intensity mercury vapour discharge lamps, sunlamps, diagnostic x-ray equipment, ultrasound therapy devices, x-ray diffraction equipment, cabinet x-ray equipment and therapeutic x-ray equipment

  11. ANTICIPATING AND REGULATING BIOSYSTEM

    Directory of Open Access Journals (Sweden)

    Ion Iorga Siman

    2010-06-01

    Full Text Available Regulating biosystems closely related to human beings are structures still difficult to understand.Numerous intimate processes taking place in these systems, even their actual constitution, are insufficiently decoded, and that they have populated the world long before man invented the first regulator, appears not to have contributed much to their knowledge. This work is intended to highlight what regulating biosystems are.There is no secret that somatic muscles perform control operations which no act of moving would be possible without. All actions are the result of dynamic controlled processes adjusted to strict control laws. By treating them very seriously may lead to knowledge of processes occurring in complex systems

  12. Staff rules and regulations

    CERN Document Server

    HR Department

    2007-01-01

    The 11th edition of the Staff Rules and Regulations, dated 1 January 2007, adopted by the Council and the Finance Committee in December 2006, is currently being distributed to departmental secretariats. The Staff Rules and Regulations, together with a summary of the main modifications made, will be available, as from next week, on the Human Resources Department's intranet site: http://cern.ch/hr-web/internal/admin_services/rules/default.asp The main changes made to the Staff Rules and Regulations stem from the five-yearly review of employment conditions of members of the personnel. The changes notably relate to: the categories of members of the personnel (e.g. removal of the local staff category); the careers structure and the merit recognition system; the non-residence, installation and re-installation allowances; the definition of family, family allowances and family-related leave; recognition of partnerships; education fees. The administrative circulars, some of which are being revised following the ...

  13. Regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase: product inhibition, cooperativity, and magnesium activation.

    Science.gov (United States)

    Hazra, Suratna; Henderson, J Nathan; Liles, Kevin; Hilton, Matthew T; Wachter, Rebekka M

    2015-10-01

    In many photosynthetic organisms, tight-binding Rubisco inhibitors are released by the motor protein Rubisco activase (Rca). In higher plants, Rca plays a pivotal role in regulating CO2 fixation. Here, the ATPase activity of 0.005 mm tobacco Rca was monitored under steady-state conditions, and global curve fitting was utilized to extract kinetic constants. The kcat was best fit by 22.3 ± 4.9 min(-1), the Km for ATP by 0.104 ± 0.024 mm, and the Ki for ADP by 0.037 ± 0.007 mm. Without ADP, the Hill coefficient for ATP hydrolysis was extracted to be 1.0 ± 0.1, indicating noncooperative behavior of homo-oligomeric Rca assemblies. However, the addition of ADP was shown to introduce positive cooperativity between two or more subunits (Hill coefficient 1.9 ± 0.2), allowing for regulation via the prevailing ATP/ADP ratio. ADP-mediated activation was not observed, although larger amounts led to competitive product inhibition of hydrolytic activity. The catalytic efficiency increased 8.4-fold upon cooperative binding of a second magnesium ion (Hill coefficient 2.5 ± 0.5), suggesting at least three conformational states (ATP-bound, ADP-bound, and empty) within assemblies containing an average of about six subunits. The addition of excess Rubisco (24:1, L8S8/Rca6) and crowding agents did not modify catalytic rates. However, high magnesium provided for thermal Rca stabilization. We propose that magnesium mediates the formation of closed hexameric toroids capable of high turnover rates and amenable to allosteric regulation. We suggest that in vivo, the Rca hydrolytic activity is tuned by fluctuating [Mg(2+)] in response to changes in available light.

  14. Environmentally regulated aerospace coatings

    Science.gov (United States)

    Morris, Virginia L.

    1995-01-01

    Aerospace coatings represent a complex technology which must meet stringent performance requirements in the protection of aerospace vehicles. Topcoats and primers are used, primarily, to protect the structural elements of the air vehicle from exposure to and subsequent degradation by environmental elements. There are also many coatings which perform special functions, i.e., chafing resistance, rain erosion resistance, radiation and electric effects, fuel tank coatings, maskants, wire and fastener coatings. The scheduled promulgation of federal environmental regulations for aerospace manufacture and rework materials and processes will regulate the emissions of photochemically reactive precursors to smog and air toxics. Aerospace organizations will be required to identify, qualify and implement less polluting materials. The elimination of ozone depleting chemicals (ODC's) and implementation of pollution prevention requirements are added constraints which must be addressed concurrently. The broad categories of operations affected are the manufacture, operation, maintenance, and repair of military, commercial, general aviation, and space vehicles. The federal aerospace regulations were developed around the precept that technology had to be available to support the reduction of organic and air toxic emissions, i.e., the regulations cannot be technology forcing. In many cases, the regulations which are currently in effect in the South Coast Air Quality Management District (SCAQMD), located in Southern California, were used as the baseline for the federal regulations. This paper addresses strategies used by Southern California aerospace organizations to cope with these regulatory impacts on aerospace productions programs. All of these regulatory changes are scheduled for implementation in 1993 and 1994, with varying compliance dates established.

  15. Regulation as Rhetoric

    DEFF Research Database (Denmark)

    Boll, Karen; Györy, Csaba

    This paper analyses the way regulatory agencies strategically use public ‘rhetoric’ and ‘management of appearance’ to strengthen their regulation. It reports a comparative study of the Securities and Exchange Commission (SEC) which is the US federal securities regulator and the Danish Tax and...... environment, these two agencies apply strategies that appear to be strikingly similar, and these similarities are worth investigating not despite, but exactly because of the differing political and social environment. We track recent shifts in organizational practice at these two agencies and argue that both...

  16. Nuclear regulations and environment

    International Nuclear Information System (INIS)

    After an historical overview of the nuclear regulation system in Argentina a description is made of the country's Nuclear Regulatory Authority (ARN) and of its regulation and control functions. Its organic structure is also outlined. A detailed report is given of the environmental monitoring activities in the sites of the operating Argentine nuclear power plants as well as those of the nuclear research centres. A special reference is made of the monitoring of the relevant uranium mining districts in Argentina. The radon determination in houses of several regions of the country is also mentioned

  17. Regulated underground storage tanks

    International Nuclear Information System (INIS)

    This guidance package is designed to assist DOE Field operations by providing thorough guidance on the underground storage tank (UST) regulations. [40 CFR 280]. The guidance uses tables, flowcharts, and checklists to provide a ''roadmap'' for DOE staff who are responsible for supervising UST operations. This package is tailored to address the issues facing DOE facilities. DOE staff should use this guidance as: An overview of the regulations for UST installation and operation; a comprehensive step-by-step guidance for the process of owning and operating an UST, from installation to closure; and a quick, ready-reference guide for any specific topic concerning UST ownership or operation

  18. Volume Regulation in Epithelia

    DEFF Research Database (Denmark)

    Larsen, Erik Hviid; Hoffmann, Else Kay

    2016-01-01

    We review studies on regulatory volume decrease (RVD) and regulatory volume increase (RVI) of major ion and water transporting vertebrate epithelia. The rate of RVD and RVI is faster in cells of high osmotic permeability like amphibian gallbladder and mammalian proximal tubule as compared...... to amphibian skin and mammalian cortical collecting tubule of low and intermediate osmotic permeability. Crosstalk between entrance and exit mechanisms interferes with volume regulation both at aniso-osmotic and iso-osmotic volume perturbations. It has been proposed that cell volume regulation is an intrinsic...

  19. Cyberplagiarism in University Regulations

    Directory of Open Access Journals (Sweden)

    Santiago Cavanillas

    2008-12-01

    Full Text Available The article examines the legal framework for plagiarism, and its twofold nature of illicit appropriation (from the author of the plagiarized work and fraud (with regard to the target audience of the plagiarism. Based on these premises, academic cyberplagiarism is analysed as a form of plagiarism carried out using electronic tools in the university setting. The question of responsibility (who can regulate the legal consequences of plagiarism? before and after the Ley Orgánica de Universidades (organic law on universities, LOU is studied, as is the disciplinary handling of cyberplagiarism with the limited regulations currently in place at universities.

  20. Regulation, Competition, and Information

    OpenAIRE

    Mian Muhammad Javed

    2002-01-01

    You know it is very hard after the Governor, State Bank, to make a presentation but I will try to do it in a very mundane way. You know the Regulatory Bodies specially in the Economic Sector in recent times. There has been a sort of resurgence, leaving aside the regulation of the financial sector, which has been doing very well. Our old memory of regulation is not so pleasant. Long ago, there used to be a transport Authority which used to dole out “Route Permits” as political favours, and the...

  1. The Impact of Regulating Social Science Research with Biomedical Regulations

    Science.gov (United States)

    Durosinmi, Brenda Braxton

    2011-01-01

    The Impact of Regulating Social Science Research with Biomedical Regulations Since 1974 Federal regulations have governed the use of human subjects in biomedical and social science research. The regulations are known as the Federal Policy for the Protection of Human Subjects, and often referred to as the "Common Rule" because 18 Federal…

  2. Crystal structure of Escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets.

    Science.gov (United States)

    Endrizzi, James A; Kim, Hanseong; Anderson, Paul M; Baldwin, Enoch P

    2004-06-01

    Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-A resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 glutamine amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer-tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 A between the glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the glutamine hydrolysis active site, providing an entry point for exogenous ammonia. Guanine nucleotide binding sites of structurally related GTPases superimpose on this cleft, providing insights into allosteric regulation by GTP. Mutations that confer nucleoside drug resistance and release CTP inhibition map to a pocket that neighbors the UTP-binding site and can accommodate a pyrimidine ring. Its location suggests that competitive feedback inhibition is affected via a distinct product/drug binding site that overlaps the substrate triphosphate binding site. Overall, the E. coli structure provides a framework for homology modeling of other CTPSs and structure-based design of anti-CTPS therapeutics. PMID:15157079

  3. Nuclear safety regulations

    International Nuclear Information System (INIS)

    The Departmental Rules and The Safety Guides were issued by the NNSA in 1998. The NNSA performed the activities of propagation and implementation of nuclear safety regulations at QTNPP in order to improve the nuclear safety culture of operating organization and construct and contract organizations

  4. Situated bio-regulation

    DEFF Research Database (Denmark)

    Prainsack, Barbara; Wahlberg, Ayo

    2013-01-01

    Several years ago, both authors engaged in research into bioscience and biomedical regulation in Asian countries. One of us (BP) explored why the regulatory and discursive embedding of human embryonic stem cell in Israel was much more permissive than elsewhere. The other author (AW) sought to und...

  5. Regulating the Internet

    Science.gov (United States)

    Anderson, Byron

    2007-01-01

    The Internet's breakthrough to primetime usage beginning in the mid-1990s evolved in an era of openness. Unfettered access seemed key to Internet development. An important foundation for the 1996 Telecommunications Act was the theory that the telecom industry would work best if it were free of government regulation, a guiding principle that has…

  6. Regulating multiple externalities

    DEFF Research Database (Denmark)

    Waldo, Staffan; Jensen, Frank; Nielsen, Max;

    2016-01-01

    instruments. However, solving the open-access externality problem also affects CO2 emissions. By using a bio-economic model covering Iceland, Norway, Denmark, Sweden, and the Faroe Islands, it is shown that regulations of the open-access externality problem have a large effect on both economic performance...

  7. Jordan Corporate Governance Regulations

    OpenAIRE

    Jordan Institute of Directors

    2013-01-01

    As the importance of Corporate Governance increases, an awareness and understanding of the different relevant regulations becomes of paramount value. The importance and value of Corporate Governance is not the core of this publication. The publication is built around the premise that Corporate Governance is important and increasingly becoming of significant importance for growth, continued...

  8. Reconceptualizing Civil Regulation

    DEFF Research Database (Denmark)

    Galang, Roberto Martin; Castello, Itziar

    2011-01-01

    and environmental standards; but also that local, small and medium companies play a key role in the development of Asian civil regulation. We call this second finding the “CSR importation trap”. Our findings are supported by evidence on the limitations in the interchangeable properties of business and governments...

  9. Legislation and regulation

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-09-01

    This document presents the fulfilling of the Brazilian obligations under the Convention on Nuclear Safety. The Chapter 3 of the document contains some details about the Brazilian legislation and regulation, the legislative and regulatory framework, regulatory body and responsibility of the license holder.

  10. Vehicle recycling regulations

    DEFF Research Database (Denmark)

    Smink, Carla

    2007-01-01

    The number of end-of-life vehicles (ELVs) in the EU is increasing continously. Around 75 percent of an ELV are recyclable metals. The forecast growth in the number of ELVs calls for regulation that aims to minimise the environmental impact of a car. Using Denmark as an example, this article...

  11. Volume Regulated Channels

    DEFF Research Database (Denmark)

    Klausen, Thomas Kjær

    expressed ICl, swell will decrease proliferation in several cell types, including Ehrlich cells. A differentiated expression of ICl, swell in the cell cycle has been described in different cell types indicating a regulating role. In Ehrlich Lettré ascites (ELA) cells we suggest the differentiated expression...

  12. Regulation of cholesterol homeostasis

    NARCIS (Netherlands)

    van der Wulp, Mariette Y. M.; Verkade, Henkjan J.; Groen, Albert K.

    2013-01-01

    Hypercholesterolemia is an important risk factor for cardiovascular disease. It is caused by a disturbed balance between cholesterol secretion into the blood versus uptake. The pathways involved are regulated via a complex interplay of enzymes, transport proteins, transcription factors and non-codin

  13. International telecommunications market regulation

    OpenAIRE

    Productivity Commission

    2001-01-01

    On 23 February 1999 the Assistant Treasurer referred international telecommunic-ations market regulation for inquiry and report within six months. The report focused on the reform of international telecommunications markets, in particular on the payment arrangements between providers of international telecommunications services.

  14. Regulation of Energy Balance.

    Science.gov (United States)

    Bray, George A.

    1985-01-01

    Explains relationships between energy intake and expenditure focusing on the cellular, chemical and neural mechanisms involved in regulation of energy balance. Information is referenced specifically to conditions of obesity. (Physicians may earn continuing education credit by completing an appended test). (ML)

  15. Regulating the private security industry

    CERN Document Server

    Percy, Sarah

    2002-01-01

    The under-regulation of the private security industry has increasingly become a topic of media and academic interest. This Adelphi Paper enters the debate by explaining why the industry requires further regulation, and what is wrong with the current system. It begins by briefly defining the industry and explaining the need for more effective regulation, before analysing three types of regulation: domestic, international and informal (including self-regulation).

  16. Markets, religion, regulation

    DEFF Research Database (Denmark)

    Fischer, Johan

    2016-01-01

    of regulation, certification and standardization on a global scale. Building on research on global kosher (a Hebrew term meaning “fit” or “proper”), halal (an Arabic word that literally means “permissible” or “lawful”) and Hindu vegetarianism this paper argues that these economies or markets to a large extent...... are conditioned by and themselves condition forms of transnational governmentality, that is, new and often overlapping practices of government and grassroots politics. I explore religious economies and markets at three interrelated levels of the social scale: state and non-state regulation, the marketplace......Most recent scholarship on moral economies or religious markets argues for the compatibility of economies/markets and religious practices in particular national or regional contexts. However, over the last couple of decades or so religious markets have entered a new phase characterized by new forms...

  17. Regulation of Meiotic Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  18. POTENT REGULATORS OF METABOLISM

    OpenAIRE

    Brun, Pierre-Jacques; Yang, Kryscilla Jian Zhang; Lee, Seung-Ah; Yuen, Jason J.; Blaner, William S.

    2012-01-01

    Retinoids (vitamin A and its analogs) are highly potent regulators of cell differentiation, cell proliferation, and apoptosis. Because of these activities, retinoids have been most extensively studied in the contexts of embryonic development and of proliferative diseases, especially cancer and skin disease. Recently, there has been considerable new research interest focused on gaining understanding of the roles that retinoids and/or retinoid-related proteins may have in the development of met...

  19. Beyond State Regulation

    OpenAIRE

    Madanat, Philip; Pies, Judith

    2011-01-01

    Holding the news media accountable has traditionally been a task of the state in Jordan. Media laws and regulations are numerous and do not leave too much space for self-regulatory practices on a national basis. The Jordan Press Association (JPA) is the core of so-called established media accountability institutions. It conducted a law-like code of ethics in 2003 and runs ombuds committees (currently three) dealing with mishaps of the media to prevent journalists from legal liability. Thou...

  20. Fibronectin Mechanobiology Regulates Tumorigenesis

    OpenAIRE

    Wang, Karin; Seo, Bo Ri; Fischbach, Claudia; Gourdon, Delphine

    2015-01-01

    Fibronectin (Fn) is an essential extracellular matrix (ECM) glycoprotein involved in both physiological and pathological processes. The structure–function relationship of Fn has been and is still being studied, as changes in its molecular structure are integral in regulating (or dysregulating) its biological activities via its cell, matrix component, and growth factor binding sites. Fn comprises three types of repeating modules; among them, FnIII modules are mechanically unstable domains that...