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Sample records for allosteric binding pocket

  1. Crystal Structure of Human Soluble Adenylate Cyclase Reveals a Distinct, Highly Flexible Allosteric Bicarbonate Binding Pocket

    PubMed Central

    Saalau-Bethell, Susanne M; Berdini, Valerio; Cleasby, Anne; Congreve, Miles; Coyle, Joseph E; Lock, Victoria; Murray, Christopher W; O'Brien, M Alistair; Rich, Sharna J; Sambrook, Tracey; Vinkovic, Mladen; Yon, Jeff R; Jhoti, Harren

    2014-01-01

    Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,β-methylene adenosine 5′-triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar-potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein. PMID:24616449

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

    NASA Astrophysics Data System (ADS)

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

    2009-08-01

    Eg5, a mitotic kinesin exclusively involved in the formation and function of the mitotic spindle has attracted interest as an anticancer drug target. Eg5 is co-crystallized with several inhibitors bound to its allosteric binding pocket. Each of these occupies a pocket formed by loop 5/helix α2 (L5/α2). Recently designed inhibitors additionally occupy a hydrophobic pocket of this site. The goal of the present study was to explore this hydrophobic pocket with our MED-SuMo fragment-based protocol, and thus discover novel chemical structures that might bind as inhibitors. The MED-SuMo software is able to compare and superimpose similar interaction surfaces upon the whole protein data bank (PDB). In a fragment-based protocol, MED-SuMo retrieves MED-Portions that encode protein-fragment binding sites and are derived from cross-mining protein-ligand structures with libraries of small molecules. Furthermore we have excluded intra-family MED-Portions derived from Eg5 ligands that occupy the hydrophobic pocket and predicted new potential ligands by hybridization that would fill simultaneously both pockets. Some of the latter having original scaffolds and substituents in the hydrophobic pocket are identified in libraries of synthetically accessible molecules by the MED-Search software.

  3. Allosteric remodelling of the histone H3 binding pocket in the Pygo2 PHD finger triggered by its binding to the B9L/BCL9 co-factor.

    PubMed

    Miller, Thomas C R; Rutherford, Trevor J; Johnson, Christopher M; Fiedler, Marc; Bienz, Mariann

    2010-09-03

    The Zn-coordinated PHD fingers of Pygopus (Pygo) proteins are critical for beta-catenin-dependent transcriptional switches in normal and malignant tissues. They bind to methylated histone H3 tails, assisted by their BCL9 co-factors whose homology domain 1 (HD1) binds to the rear PHD surface. Although histone-binding residues are identical between the two human Pygo paralogs, we show here that Pygo2 complexes exhibit slightly higher binding affinities for methylated histone H3 tail peptides than Pygo1 complexes. We solved the crystal structure of the Pygo2 PHD-BCL9-2 HD1 complex, which revealed paralog-specific interactions in its PHD-HD1 interface that could contribute indirectly to its elevated affinity for the methylated histone H3 tail. Interestingly, using NMR spectroscopy, we discovered that HD1 binding to PHD triggers an allosteric communication with a conserved isoleucine residue that lines the binding channel for histone H3 threonine 3 (T3), the link between the two adjacent binding pockets accommodating histone H3 alanine 1 and methylated lysine 4, respectively. This modulates the surface of the T3 channel, providing a plausible explanation as to how BCL9 co-factors binding to Pygo PHD fingers impact indirectly on their histone binding affinity. Intriguingly, this allosteric modulation of the T3 channel is propagated through the PHD structural core by a highly conserved tryptophan, the signature residue defining the PHD subclass of Zn fingers, which suggests that other PHD proteins may also be assisted by co-factors in their decoding of modified histone H3 tails.

  4. Allosteric Dynamic Control of Binding

    PubMed Central

    Sumbul, Fidan; Acuner-Ozbabacan, Saliha Ece; Haliloglu, Turkan

    2015-01-01

    Proteins have a highly dynamic nature and there is a complex interrelation between their structural dynamics and binding behavior. By assuming various conformational ensembles, they perform both local and global fluctuations to interact with other proteins in a dynamic infrastructure adapted to functional motion. Here, we show that there is a significant association between allosteric mutations, which lead to high-binding-affinity changes, and the hinge positions of global modes, as revealed by a large-scale statistical analysis of data in the Structural Kinetic and Energetic Database of Mutant Protein Interactions (SKEMPI). We further examined the mechanism of allosteric dynamics by conducting studies on human growth hormone (hGH) and pyrin domain (PYD), and the results show how mutations at the hinge regions could allosterically affect the binding-site dynamics or induce alternative binding modes by modifying the ensemble of accessible conformations. The long-range dissemination of perturbations in local chemistry or physical interactions through an impact on global dynamics can restore the allosteric dynamics. Our findings suggest a mechanism for the coupling of structural dynamics to the modulation of protein interactions, which remains a critical phenomenon in understanding the effect of mutations that lead to functional changes in proteins. PMID:26338442

  5. Allosteric binding sites on muscarinic acetylcholine receptors.

    PubMed

    Wess, Jürgen

    2005-12-01

    In this issue of Molecular Pharmacology, Tränkle et al. (p. 1597) present new findings regarding the existence of a second allosteric site on the M2 muscarinic acetylcholine receptor (M2 mAChR). The M2 mAChR is a prototypic class A G protein-coupled receptor (GPCR) that has proven to be a very useful model system to study the molecular mechanisms involved in the binding of allosteric GPCR ligands. Previous studies have identified several allosteric muscarinic ligands, including the acetylcholinesterase inhibitor tacrine and the bis-pyridinium derivative 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide (Duo3), which, in contrast to conventional allosteric muscarinic ligands, display concentration-effect curves with slope factors >1. By analyzing the interactions of tacrine and Duo3 with other allosteric muscarinic agents predicted to bind to the previously identified ;common' allosteric binding site, Tränkle et al. provide evidence suggesting that two allosteric agents and one orthosteric ligand may be able to bind to the M2 mAChR simultaneously. Moreover, studies with mutant mAChRs indicated that the M2 receptor epitopes involved in the binding of tacrine and Duo3 may not be identical. Molecular modeling and ligand docking studies suggested that the additional allosteric site probably represents a subdomain of the receptor's allosteric binding cleft. Because allosteric binding sites have been found on many other GPCRs and drugs interacting with these sites are thought to have great therapeutic potential, the study by Tränkle et al. should be of considerable general interest.

  6. NMR reveals the allosteric opening and closing of Abelson tyrosine kinase by ATP-site and myristoyl pocket inhibitors

    PubMed Central

    Skora, Lukasz; Mestan, Jürgen; Fabbro, Doriano; Jahnke, Wolfgang; Grzesiek, Stephan

    2013-01-01

    Successful treatment of chronic myelogenous leukemia is based on inhibitors binding to the ATP site of the deregulated breakpoint cluster region (Bcr)–Abelson tyrosine kinase (Abl) fusion protein. Recently, a new type of allosteric inhibitors targeting the Abl myristoyl pocket was shown in preclinical studies to overcome ATP-site inhibitor resistance arising in some patients. Using NMR and small-angle X-ray scattering, we have analyzed the solution conformations of apo Abelson tyrosine kinase (c-Abl) and c-Abl complexes with ATP-site and allosteric inhibitors. Binding of the ATP-site inhibitor imatinib leads to an unexpected open conformation of the multidomain SH3-SH2-kinase c-Abl core, whose relevance is confirmed by cellular assays on Bcr-Abl. The combination of imatinib with the allosteric inhibitor GNF-5 restores the closed, inactivated state. Our data provide detailed insights on the poorly understood combined effect of the two inhibitor types, which is able to overcome drug resistance. PMID:24191057

  7. NMR reveals the allosteric opening and closing of Abelson tyrosine kinase by ATP-site and myristoyl pocket inhibitors.

    PubMed

    Skora, Lukasz; Mestan, Jürgen; Fabbro, Doriano; Jahnke, Wolfgang; Grzesiek, Stephan

    2013-11-19

    Successful treatment of chronic myelogenous leukemia is based on inhibitors binding to the ATP site of the deregulated breakpoint cluster region (Bcr)-Abelson tyrosine kinase (Abl) fusion protein. Recently, a new type of allosteric inhibitors targeting the Abl myristoyl pocket was shown in preclinical studies to overcome ATP-site inhibitor resistance arising in some patients. Using NMR and small-angle X-ray scattering, we have analyzed the solution conformations of apo Abelson tyrosine kinase (c-Abl) and c-Abl complexes with ATP-site and allosteric inhibitors. Binding of the ATP-site inhibitor imatinib leads to an unexpected open conformation of the multidomain SH3-SH2-kinase c-Abl core, whose relevance is confirmed by cellular assays on Bcr-Abl. The combination of imatinib with the allosteric inhibitor GNF-5 restores the closed, inactivated state. Our data provide detailed insights on the poorly understood combined effect of the two inhibitor types, which is able to overcome drug resistance.

  8. Surface-Based Protein Binding Pocket Similarity

    PubMed Central

    Spitzer, Russell; Cleves, Ann E.; Jain, Ajay N.

    2011-01-01

    Protein similarity comparisons may be made on a local or global basis and may consider sequence information or differing levels of structural information. We present a local 3D method that compares protein binding site surfaces in full atomic detail. The approach is based on the morphological similarity method which has been widely applied for global comparison of small molecules. We apply the method to all-by-all comparisons two sets of human protein kinases, a very diverse set of ATP-bound proteins from multiple species, and three heterogeneous benchmark protein binding site data sets. Cases of disagreement between sequence-based similarity and binding site similarity yield informative examples. Where sequence similarity is very low, high pocket similarity can reliably identify important binding motifs. Where sequence similarity is very high, significant differences in pocket similarity are related to ligand binding specificity and similarity. Local protein binding pocket similarity provides qualitatively complementary information to other approaches, and it can yield quantitative information in support of functional annotation. PMID:21769944

  9. Studying the binding interactions of allosteric agonists and antagonists of the CXCR4 receptor.

    PubMed

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

    2015-07-01

    Several examples of allosteric modulators of GPCRs have been reported recently in the literature, but understanding their molecular mechanism presents a new challenge for medicinal chemistry. For the specific case of the cellular receptor CXCR4, it is known that pepducins (lipidated fragments of intracellular GPCR loops) such as ATI-2341 modulate CXCR4 activity agonistically via an allosteric mechanism. Moreover, there are also examples of small organic molecules such as AMD11070 and GSK812397 which may also act as allosteric antagonists. However, incomplete knowledge of the ligand-binding sites has hampered a detailed molecular understanding of how these inhibitors work. Here, we attempt to answer this question by analysing the binding interactions between the CXCR4 receptor and the above-mentioned allosteric modulators. We propose two different allosteric binding sites, one located in the intracellular loops 1, 2 and 3 (ICL1, ICL2 and ICL3) which binds the pepducin agonist ATI-2341, and the other at a subsite of the main extracellular orthosteric binding pocket between extracellular loops 1 and 2 and the N-terminus, which binds the antagonists AMD11070 and GSK812397. Allosteric interactions between the CXCR4 and ATI-2341 were predicted by combining different modeling approaches. First, a rotational blind docking search was applied and the best poses were subsequently refined using flexible docking methods and molecular dynamic simulations. For the AMD11070 and GSK812397 antagonists, the entire CXCR4 protein surface was explored by blind docking in order to define the binding region. A second docking analysis by subsites was then performed to refine the allosteric interactions. Finally, we identified the binding residues that appear to be essential for CXCR4 allosteric modulators.

  10. Ligand Binding to Macromolecules: Allosteric and Sequential Models of Cooperativity.

    ERIC Educational Resources Information Center

    Hess, V. L.; Szabo, Attila

    1979-01-01

    A simple model is described for the binding of ligands to macromolecules. The model is applied to the cooperative binding by hemoglobin and aspartate transcarbamylase. The sequential and allosteric models of cooperative binding are considered. (BB)

  11. Identification of allosteric PIF-pocket ligands for PDK1 using NMR-based fragment screening and 1H-15N TROSY experiments.

    PubMed

    Stockman, Brian J; Kothe, Michael; Kohls, Darcy; Weibley, Laura; Connolly, Brendan J; Sheils, Alissa L; Cao, Qing; Cheng, Alan C; Yang, Lily; Kamath, Ajith V; Ding, Yuan-Hua; Charlton, Maura E

    2009-02-01

    Aberrant activation of the phosphoinositide 3-kinase pathway because of genetic mutations of essential signalling proteins has been associated with human diseases including cancer and diabetes. The pivotal role of 3-phosphoinositide-dependent kinase-1 in the PI3K signalling cascade has made it an attractive target for therapeutic intervention. The N-terminal lobe of the 3-phosphoinositide-dependent kinase-1 catalytic domain contains a docking site which recognizes the non-catalytic C-terminal hydrophobic motifs of certain substrate kinases. The binding of substrate in this so-called PDK1 Interacting Fragment pocket allows interaction with 3-phosphoinositide-dependent kinase-1 and enhanced phosphorylation of downstream kinases. NMR spectroscopy was used to a screen 3-phosphoinositide-dependent kinase-1 domain construct against a library of chemically diverse fragments in order to identify small, ligand-efficient fragments that might interact at either the ATP site or the allosteric PDK1 Interacting Fragment pocket. While majority of the fragment hits were determined to be ATP-site binders, several fragments appeared to interact with the PDK1 Interacting Fragment pocket. Ligand-induced changes in 1H-15N TROSY spectra acquired using uniformly 15N-enriched PDK1 provided evidence to distinguish ATP-site from PDK1 Interacting Fragment-site binding. Caliper assay data and 19F NMR assay data on the PDK1 Interacting Fragment pocket fragments and structurally related compounds identified them as potential allosteric activators of PDK1 function.

  12. Furoates and thenoates inhibit pyruvate dehydrogenase kinase 2 allosterically by binding to its pyruvate regulatory site.

    PubMed

    Masini, Tiziana; Birkaya, Barbara; van Dijk, Simon; Mondal, Milon; Hekelaar, Johan; Jäger, Manuel; Terwisscha van Scheltinga, Anke C; Patel, Mulchand S; Hirsch, Anna K H; Moman, Edelmiro

    2016-01-01

    The last decade has witnessed the reawakening of cancer metabolism as a therapeutic target. In particular, inhibition of pyruvate dehydrogenase kinase (PDK) holds remarkable promise. Dichloroacetic acid (DCA), currently undergoing clinical trials, is a unique PDK inhibitor in which it binds to the allosteric pyruvate site of the enzyme. However, the safety of DCA as a drug is compromised by its neurotoxicity, whereas its usefulness as an investigative tool is limited by the high concentrations required to exert observable effects in cell culture. Herein, we report the identification - by making use of saturation-transfer difference NMR spectroscopy, enzymatic assays and computational methods - of furoate and thenoate derivatives as allosteric pyruvate-site-binding PDK2 inhibitors. This work substantiates the pyruvate regulatory pocket as a druggable target.

  13. Identification of an allosteric binding site for RORγt inhibition

    PubMed Central

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

    2015-01-01

    RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors. PMID:26640126

  14. Free enthalpies of replacing water molecules in protein binding pockets

    NASA Astrophysics Data System (ADS)

    Riniker, Sereina; Barandun, Luzi J.; Diederich, François; Krämer, Oliver; Steffen, Andreas; van Gunsteren, Wilfred F.

    2012-12-01

    Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH3 group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH3 at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design.

  15. Free enthalpies of replacing water molecules in protein binding pockets.

    PubMed

    Riniker, Sereina; Barandun, Luzi J; Diederich, François; Krämer, Oliver; Steffen, Andreas; van Gunsteren, Wilfred F

    2012-12-01

    Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH(3) group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH(3) at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design.

  16. Use of binding enthalpy to drive an allosteric transition.

    PubMed

    Brown, Patrick H; Beckett, Dorothy

    2005-03-01

    The Escherichia coli biotin repressor is an allosteric DNA binding protein and is activated by the small molecule bio-5'-AMP. Binding of this small molecule promotes transcription repression complex assembly between the repressor and the biotin operator of the biotin biosynthetic operon. The ability of the adenylate to activate the assembly process reflects its effect on biotin repressor dimerization. Thus concomitant with small molecule binding the free energy of repressor dimerization becomes more favorable by approximately -4 kcal/mol. The structural, dynamic, and energetic changes in the repressor monomer that accompany allosteric activation are not known. In this work the thermodynamics of binding of four allosteric activators to the repressor have been characterized by isothermal titration calorimetry. While binding of two of the effectors results in relatively modest activation of the dimerization process, binding of the other two small molecules, including the physiological effector, leads to large changes in repressor dimerization energetics. Results of the calorimetric measurements indicate that strong effector binding is accompanied by an enthalpically costly transition in the protein. This transition is "paid for" by the enthalpy that would have otherwise been realized from the formation of noncovalent bonds between the ligand and repressor monomer.

  17. Binding Hydrated Anions with Hydrophobic Pockets.

    PubMed

    Sokkalingam, Punidha; Shraberg, Joshua; Rick, Steven W; Gibb, Bruce C

    2016-01-13

    Using a combination of isothermal titration calorimetry and quantum and molecular dynamics calculations, we demonstrate that relatively soft anions have an affinity for hydrophobic concavity. The results are consistent with the anions remaining partially hydrated upon binding, and suggest a novel strategy for anion recognition.

  18. Allosteric Modulation of Hormone Release from Thyroxine and Corticosteroid-binding Globulins*

    PubMed Central

    Qi, Xiaoqiang; Loiseau, François; Chan, Wee Lee; Yan, Yahui; Wei, Zhenquan; Milroy, Lech-Gustav; Myers, Rebecca M.; Ley, Steven V.; Read, Randy J.; Carrell, Robin W.; Zhou, Aiwu

    2011-01-01

    The release of hormones from thyroxine-binding globulin (TBG) and corticosteroid-binding globulin (CBG) is regulated by movement of the reactive center loop in and out of the β-sheet A of the molecule. To investigate how these changes are transmitted to the hormone-binding site, we developed a sensitive assay using a synthesized thyroxine fluorophore and solved the crystal structures of reactive loop cleaved TBG together with its complexes with thyroxine, the thyroxine fluorophores, furosemide, and mefenamic acid. Cleavage of the reactive loop results in its complete insertion into the β-sheet A and a substantial but incomplete decrease in binding affinity in both TBG and CBG. We show here that the direct interaction between residue Thr342 of the reactive loop and Tyr241 of the hormone binding site contributes to thyroxine binding and release following reactive loop insertion. However, a much larger effect occurs allosterically due to stretching of the connecting loop to the top of the D helix (hD), as confirmed in TBG with shortening of the loop by three residues, making it insensitive to the S-to-R transition. The transmission of the changes in the hD loop to the binding pocket is seen to involve coherent movements in the s2/3B loop linked to the hD loop by Lys243, which is, in turn, linked to the s4/5B loop, flanking the thyroxine-binding site, by Arg378. Overall, the coordinated movements of the reactive loop, hD, and the hormone binding site allow the allosteric regulation of hormone release, as with the modulation demonstrated here in response to changes in temperature. PMID:21325280

  19. Light-activated DNA binding in a designed allosteric protein

    SciTech Connect

    Strickland, Devin; Moffat, Keith; Sosnick, Tobin R.

    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.

  20. Taurine allosterically modulates flunitrazepam binding to synaptic membranes.

    PubMed

    Quinn, M R; Miller, C L

    1992-09-01

    Taurine is hypothesized to exert its inhibitory neuromodulatory effects, in part, by interaction with the GABAA receptor. Although taurine displaces GABA agonist binding to synaptic membranes, its allosteric effects on the benzodiazepine recognition site of the GABAA receptor complex is unsettled. We determined the effects of taurine on [3H]flunitrazepam (Flu) binding to well-washed, frozen-thawed synaptic membranes prepared from rat cortex. Comparative binding studies were conducted at 37 degrees C and on ice (0-4 degrees C). At 37 degrees C taurine increased Flu binding in a concentration dependent way by interaction with a bicuculline sensitive site, similar to GABA. Taurine increased Flu binding by causing a decrease in KD. The maximal effectiveness of taurine on Flu binding could not be increased further by addition of GABA. In contrast, the maximal stimulation of Flu binding by GABA was decreased by addition of taurine to the level attained by taurine alone. These mixed agonist/antagonist effects of taurine are pharmacologically specific and qualify taurine as a partial GABA agonist in this type of allosteric interaction. However, taurine causes opposite effects on Flu binding when measured at 0-4 degrees C: taurine interacts with a bicuculline insensitive site to inhibit Flu binding by increasing the KD. Taurine inhibition of Flu binding is not overcome by increasing concentrations of GABA. Although the mechanism of taurine inhibition of Flu binding at 0-4 degrees C is unclear, it may be an indirect effect of taurine interaction with membrane phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. A threonine turnstile defines a dynamic amphiphilic binding motif in the AAA ATPase p97 allosteric binding site.

    PubMed

    Burnett, James C; Lim, Chaemin; Peyser, Brian D; Samankumara, Lalith P; Kovaliov, Marina; Colombo, Raffaele; Bulfer, Stacie L; LaPorte, Matthew G; Hermone, Ann R; McGrath, Connor F; Arkin, Michelle R; Gussio, Rick; Huryn, Donna M; Wipf, Peter

    2017-03-29

    The turnstile motion of two neighboring threonines sets up a dynamic side chain interplay that can accommodate both polar and apolar ligands in a small molecule allosteric protein binding site. A computational model based on SAR data and both X-ray and cryo-EM structures of the AAA ATPase p97 was used to analyze the effects of paired threonines at the inhibitor site. Specifically, the Thr side chain hydroxyl groups form a hydrogen bonding network that readily accommodates small, highly polar ligand substituents. Conversely, diametric rotation of the χ1 torsion by 150-180° orients the side chain β-methyl groups into the binding cleft, creating a hydrophobic pocket that can accommodate small, apolar substituents. This motif was found to be critical for rationalizing the affinities of a structurally focused set of inhibitors of p97 covering a > 2000-fold variation in potencies, with a preference for either small-highly polar or small-apolar groups. The threonine turnstile motif was further validated by a PDB search that identified analogous binding modes in ligand interactions in PKB, as well as by an analysis of NMR structures demonstrating additional gear-like interactions between adjacent Thr pairs. Combined, these data suggest that the threonine turnstile motif may be a general feature of interest in protein binding pockets.

  2. Computational analysis of negative and positive allosteric modulator binding and function in metabotropic glutamate receptor 5 (in)activation.

    PubMed

    Dalton, James A R; Gómez-Santacana, Xavier; Llebaria, Amadeu; Giraldo, Jesús

    2014-05-27

    Metabotropic glutamate receptors (mGluRs) are high-profile G-protein coupled receptors drug targets because of their involvement in several neurological disease states, and mGluR5 in particular is a subtype whose controlled allosteric modulation, both positive and negative, can potentially be useful for the treatment of schizophrenia and relief of chronic pain, respectively. Here we model mGluR5 with a collection of positive and negative allosteric modulators (PAMs and NAMs) in both active and inactive receptor states, in a manner that is consistent with experimental information, using a specialized protocol that includes homology to increase docking accuracy, and receptor relaxation to generate an individual induced fit with each allosteric modulator. Results implicate two residues in particular for NAM and PAM function: NAM interaction with W785 for receptor inactivation, and NAM/PAM H-bonding with S809 for receptor (in)activation. Models suggest the orientation of the H-bond between allosteric modulator and S809, controlled by PAM/NAM chemistry, influences the position of TM7, which in turn influences the shape of the allosteric site, and potentially the receptor state. NAM-bound and PAM-bound mGluR5 models also reveal that although PAMs and NAMs bind in the same pocket and share similar binding modes, they have distinct effects on the conformation of the receptor. Our models, together with the identification of a possible activation mechanism, may be useful in the rational design of new allosteric modulators for mGluR5.

  3. Development of a radioligand, [(3)H]LY2119620, to probe the human M(2) and M(4) muscarinic receptor allosteric binding sites.

    PubMed

    Schober, Douglas A; Croy, Carrie H; Xiao, Hongling; Christopoulos, Arthur; Felder, Christian C

    2014-07-01

    In this study, we characterized a muscarinic acetylcholine receptor (mAChR) potentiator, LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethoxy]thieno[2,3-b]pyridine-2-carboxamide) as a novel probe of the human M2 and M4 allosteric binding sites. Since the discovery of allosteric binding sites on G protein-coupled receptors, compounds targeting these novel sites have been starting to emerge. For example, LY2033298 (3-amino-5-chloro-6-methoxy-4-methyl-thieno(2,3-b)pyridine-2-carboxylic acid cyclopropylamid) and a derivative of this chemical scaffold, VU152100 (3-amino-N-(4-methoxybenzyl)-4,6-dim​ethylthieno[2,3-b]pyridine carboxamide), bind to the human M4 mAChR allosteric pocket. In the current study, we characterized LY2119620, a compound similar in structure to LY2033298 and binds to the same allosteric site on the human M4 mAChRs. However, LY2119620 also binds to an allosteric site on the human M2 subtype. [(3)H]NMS ([(3)H]N-methylscopolamine) binding experiments confirm that LY2119620 does not compete for the orthosteric binding pocket at any of the five muscarinic receptor subtypes. Dissociation kinetic studies using [(3)H]NMS further support that LY2119620 binds allosterically to the M2 and M4 mAChRs and was positively cooperative with muscarinic orthosteric agonists. To probe directly the allosteric sites on M2 and M4, we radiolabeled LY2119620. Cooperativity binding of [(3)H]LY2119620 with mAChR orthosteric agonists detects significant changes in Bmax values with little change in Kd, suggesting a G protein-dependent process. Furthermore, [(3)H]LY2119620 was displaced by compounds of similar chemical structure but not by previously described mAChR allosteric compounds such as gallamine or WIN 62,577 (17-β-hydroxy-17-α-ethynyl-δ-4-androstano[3,2-b]pyrimido[1,2-a]benzimidazole). Our results therefore demonstrate the development of a radioligand, [(3)H]LY2119620 to probe specifically the human M2 and M4 muscarinic

  4. Inhibiting Helicobacter pylori HtrA protease by addressing a computationally predicted allosteric ligand binding site

    PubMed Central

    Perna, Anna Maria; Reisen, Felix; Schmidt, Thomas P.; Geppert, Tim; Pillong, Max; Weisel, Martin; Hoy, Benjamin; Simister, Philip C.; Feller, Stephan M.; Wessler, Silja; Schneider, Gisbert

    2016-01-01

    Helicobacter pylori is associated with inflammatory diseases and can cause gastric cancer and mucosa-associated lymphoma. One of the bacterium’s key proteins is high temperature requirement A (HpHtrA) protein, an extracellular serine protease that cleaves E-cadherin of gastric epithelial cells, which leads to loss of cell-cell adhesion. Inhibition of HpHtrA may constitute an intervention strategy against H. pylori infection. Guided by the computational prediction of hypothetical ligand binding sites on the surface of HpHtrA, we performed residue mutation experiments that confirmed the functional relevance of an allosteric region. We virtually screened for potential ligands addressing this surface cleft located between the catalytic and PDZ1 domains. Our receptor-based computational method represents protein surface pockets in terms of graph frameworks and retrieves small molecules that satisfy the constraints given by the pocket framework. A new chemical entity was identified that blocked E-cadherin cleavage in vitro by direct binding to HpHtrA, and efficiently blocked pathogen transmigration across the gastric epithelial barrier. A preliminary crystal structure of HpHtrA confirms the validity of a comparative “homology” model of the enzyme, which we used for the computational study. The results of this study demonstrate that addressing orphan protein surface cavities of target macromolecules can lead to new bioactive ligands. PMID:26819700

  5. Exploring the inhibitor binding pocket of respiratory complex I.

    PubMed

    Fendel, Uta; Tocilescu, Maja A; Kerscher, Stefan; Brandt, Ulrich

    2008-01-01

    Numerous hydrophobic and amphipathic compounds including several detergents are known to inhibit the ubiquinone reductase reaction of respiratory chain complex I (proton pumping NADH:ubiquinone oxidoreductase). Guided by the X-ray structure of the peripheral arm of complex I from Thermus thermophilus we have generated a large collection of site-directed mutants in the yeast Yarrowia lipolytica targeting the proposed ubiquinone and inhibitor binding pocket of this huge multiprotein complex at the interface of the 49-kDa and PSST subunits. We could identify a number of residues where mutations changed I(50) values for representatives from all three groups of hydrophobic inhibitors. Many mutations around the domain of the 49-kDa subunit that is homologous to the [NiFe] centre binding region of hydrogenase conferred resistance to DQA (class I/type A) and rotenone (class II/type B) indicating a wider overlap of the binding sites for these two types of inhibitors. In contrast, a region near iron-sulfur cluster N2, where the binding of the n-alkyl-polyoxyethylene-ether detergent C(12)E(8) (type C) was exclusively affected, appeared comparably well separated. Taken together, our data provide structure-based support for the presence of distinct but overlapping binding sites for hydrophobic inhibitors possibly extending into the ubiquinone reduction site of mitochondrial complex I.

  6. Rafoxanide and Closantel Inhibit SPAK and OSR1 Kinases by Binding to a Highly Conserved Allosteric Site on Their C-terminal Domains.

    PubMed

    AlAmri, Mubarak A; Kadri, Hachemi; Alderwick, Luke J; Simpkins, Nigel S; Mehellou, Youcef

    2017-03-31

    SPAK and OSR1 are two protein kinases that have emerged as attractive targets in the discovery of novel antihypertensive agents due to their role in regulating electrolyte balance in vivo. Herein we report the identification of an allosteric pocket on the highly conserved C-terminal domains of these two kinases, which influences their activity. We also show that some known WNK signaling inhibitors bind to this allosteric site. Using in silico screening, we identified the antiparasitic agent rafoxanide as a novel allosteric inhibitor of SPAK and OSR1. Collectively, this work will facilitate the rational design of novel SPAK and OSR1 kinase inhibitors that could be useful antihypertensive agents.

  7. Critical Role of the Secondary Binding Pocket in Modulating the Enzymatic Activity of DUSP5 toward Phosphorylated ERKs.

    PubMed

    Talipov, Marat R; Nayak, Jaladhi; Lepley, Michael; Bongard, Robert D; Sem, Daniel S; Ramchandran, Ramani; Rathore, Rajendra

    2016-11-08

    DUSP5 is an inducible nuclear dual-specificity phosphatase that specifically interacts with and deactivates extracellular signal-regulated kinases ERK1 and ERK2, which are responsible for cell proliferation, differentiation, and survival. The phosphatase domain (PD) of DUSP5 has unique structural features absent from other nuclear DUSPs, such as the presence of a secondary anion-binding site in the proximity of the reaction center and a glutamic acid E264 positioned next to the catalytic cysteine C263, as well as a remote intramolecular disulfide linkage. The overall 400 ns molecular dynamics simulations indicate that the secondary binding site of DUSP5 PD acts as an allosteric regulator of the phosphatase activity of DUSP5. Our studies have identified E264 as a critical constituent of the dual binding pocket, which regulates the catalytic activity of DUSP5 by forming a salt bridge with arginine R269. Molecular dynamics studies showed that initial occupation of the secondary binding pocket leads to the breakage of the salt bridge, which then allows the occupation of the active site. Indeed, biochemical analysis using the pERK assay on mutant E264Q demonstrated that mutation of glutamic acid E264 leads to an increase in the DUSP5 catalytic activity. The role of the secondary binding site in assembling the DUSP5-pERK pre-reactive complex was further demonstrated by molecular dynamics simulations that showed that the remote C197-C219 disulfide linkage controls the structure of the secondary binding pocket based on its redox state (i.e., disulfide/dithiol) and, in turn, the enzymatic activity of DUSP5.

  8. Dewetting-Controlled Binding of Ligands to Hydrophobic Pockets

    PubMed Central

    Setny, P.; Wang, Z.; Cheng, L.-T.; Li, B.; McCammon, J. A.; Dzubiella, J.

    2010-01-01

    We report on a combined atomistic molecular dynamics simulation and implicit solvent analysis of a generic hydrophobic pocket-ligand (host-guest) system. The approaching ligand induces complex wetting-dewetting transitions in the weakly solvated pocket. The transitions lead to bimodal solvent fluctuations which govern magnitude and range of the pocket-ligand attraction. A recently developed implicit water model, based on the minimization of a geometric functional, captures the sensitive aqueous interface response to the concave-convex pocket-ligand configuration semiquantitatively. PMID:19905832

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

    SciTech Connect

    Williams,R.; Holyoak, T.; McDonald, G.; Gui, C.; Fenton, A.

    2006-01-01

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

  10. Identification of the Binding Position of Amilorides in the Quinone Binding Pocket of Mitochondrial Complex I.

    PubMed

    Ito, Takeshi; Murai, Masatoshi; Morisaka, Hironobu; Miyoshi, Hideto

    2015-06-16

    We previously demonstrated that amilorides bind to the quinone binding pocket of bovine mitochondrial complex I, not to the hitherto suspected Na⁺/H⁺ antiporter-like subunits (ND2, ND4, and ND5) [Murai, M., et al. (2015) Biochemistry 54, 2739-2746]. To characterize the binding position of amilorides within the pocket in more detail, we conducted specific chemical labeling [alkynylation (-C≡CH)] of complex I via ligand-directed tosyl (LDT) chemistry using a newly synthesized amide-type amiloride AAT as a LDT chemistry reagent. The inhibitory potency of AAT, in terms of its IC50 value, was markedly higher (∼1000-fold) than that of prototypical guanidine-type amilorides such as commercially available EIPA and benzamil. Detailed proteomic analyses in combination with click chemistry revealed that the chemical labeling occurred at Asp160 of the 49 kDa subunit (49 kDa Asp160). This labeling was significantly suppressed in the presence of an excess amount of other amilorides or ordinary inhibitors such as quinazoline and acetogenin. Taking into consideration the fact that 49 kDa Asp160 was also specifically labeled by LDT chemistry reagents derived from acetogenin [Masuya, T., et al. (2014) Biochemistry 53, 2307-2317, 7816-7823], we found this aspartic acid to elicit very strong nucleophilicity in the local protein environment. The structural features of the quinone binding pocket in bovine complex I are discussed on the basis of this finding.

  11. Proposed Mode of Binding and Action of Positive Allosteric Modulators at Opioid Receptors

    PubMed Central

    2016-01-01

    Available crystal structures of opioid receptors provide a high-resolution picture of ligand binding at the primary (“orthosteric”) site, that is, the site targeted by endogenous ligands. Recently, positive allosteric modulators of opioid receptors have also been discovered, but their modes of binding and action remain unknown. Here, we use a metadynamics-based strategy to efficiently sample the binding process of a recently discovered positive allosteric modulator of the δ-opioid receptor, BMS-986187, in the presence of the orthosteric agonist SNC-80, and with the receptor embedded in an explicit lipid–water environment. The dynamics of BMS-986187 were enhanced by biasing the potential acting on the ligand–receptor distance and ligand–receptor interaction contacts. Representative lowest-energy structures from the reconstructed free-energy landscape revealed two alternative ligand binding poses at an allosteric site delineated by transmembrane (TM) helices TM1, TM2, and TM7, with some participation of TM6. Mutations of amino acid residues at these proposed allosteric sites were found to either affect the binding of BMS-986187 or its ability to modulate the affinity and/or efficacy of SNC-80. Taken together, these combined experimental and computational studies provide the first atomic-level insight into the modulation of opioid receptor binding and signaling by allosteric modulators. PMID:26841170

  12. Docking studies of nickel-peptide deformylase (PDF) inhibitors: exploring the new binding pockets.

    PubMed

    Wang, Qiang; Zhang, Datong; Wang, Jianwu; Cai, Zhengting; Xu, Weiren

    2006-06-20

    The binding modes of a series of known activity inhibitors docking to Peptide deformylase (PDF) have been studied using molecular docking software AutoDock3.0.5. In this study, good correlation (R(2)=0.894) between calculated binding energies and experimental inhibitory activities is obtained. We find that some shallow pockets near the known active pocket are very important which can accommodate the side-chains of the inhibitor. Moreover, a new binding pocket is also explored. All these may provide something useful for designing the potent inhibitors.

  13. Towards the identification of the allosteric Phe-binding site in phenylalanine hydroxylase.

    PubMed

    Carluccio, Carla; Fraternali, Franca; Salvatore, Francesco; Fornili, Arianna; Zagari, Adriana

    2016-01-01

    The enzyme phenylalanine hydroxylase (PAH) is defective in the inherited disorder phenylketonuria. PAH, a tetrameric enzyme, is highly regulated and displays positive cooperativity for its substrate, Phe. Whether Phe binds to an allosteric site is a matter of debate, despite several studies worldwide. To address this issue, we generated a dimeric model for Phe-PAH interactions, by performing molecular docking combined with molecular dynamics simulations on human and rat wild-type sequences and also on a human G46S mutant. Our results suggest that the allosteric Phe-binding site lies at the dimeric interface between the regulatory and the catalytic domains of two adjacent subunits. The structural and dynamical features of the site were characterized in depth and described. Interestingly, our findings provide evidence for lower allosteric Phe-binding ability of the G46S mutant than the human wild-type enzyme. This also explains the disease-causing nature of this mutant.

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

    PubMed Central

    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

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

    PubMed

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

    2015-01-20

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

  16. Crystal structure of the adenosine A2A receptor bound to an antagonist reveals a potential allosteric pocket

    PubMed Central

    Sun, Bingfa; Bachhawat, Priti; Chu, Matthew Ling-Hon; Wood, Martyn; Ceska, Tom; Sands, Zara A.; Mercier, Joel; Lebon, Florence; Kobilka, Tong Sun; Kobilka, Brian K.

    2017-01-01

    The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson’s disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl d-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR–BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A2AR–BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1–bound A2AR–BRIL prevented formation of the lattice observed with the ZM241385–bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson’s disease. PMID:28167788

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

    PubMed Central

    Joseph, Thomas T.

    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

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

    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

  20. Doubling the Size of the Glucocorticoid Receptor Ligand Binding Pocket by Deacylcortivazol

    SciTech Connect

    Suino-Powell, Kelly; Xu, Yong; Zhang, Chenghai; Tao, Yong-guang; Tolbert, W. David; Simons, Jr., S. Stoney; Xu, H. Eric

    2010-03-08

    A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GR LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 {angstrom}{sup 3}, effectively doubling the size of the GR dexamethasone-binding pocket of 540 {angstrom}{sup 3} and yet leaving the structure of the coactivator binding site intact. DAC occupies only {approx}50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.

  1. Characterization of the allosteric anion-binding site of O-acetylserine sulfhydrylase.

    PubMed

    Tai, C H; Burkhard, P; Gani, D; Jenn, T; Johnson, C; Cook, P F

    2001-06-26

    A new crystal structure of the A-isozyme of O-acetylserine sulfhydrylase-A (OASS) with chloride bound to an allosteric site located at the dimer interface has recently been determined [Burkhard, P., Tai, C.-H., Jansonius, J. N., and Cook, P. F. (2000) J. Mol. Biol. 303, 279-286]. Data have been obtained from steady state and presteady-state kinetic studies and from UV-visible spectral studies to characterize the allosteric anion-binding site. Data obtained with chloride and sulfate as inhibitors indicate the following: (i) chloride and sulfate prevent the formation of the external aldimines with L-cysteine or L-serine; (ii) chloride and sulfate increase the external aldimine dissociation constants for O-acetyl-L-serine, L-methionine, and 5-oxo-L-norleucine; (iii) chloride and sulfate bind to the allosteric site in the internal aldimine and alpha-aminoacrylate external aldimine forms of OASS; (iv) sulfate also binds to the active site. Sulfide behaves in a manner identical to chloride and sulfate in preventing the formation of the L-serine external aldimine. The binding of chloride to the allosteric site is pH independent over the pH range 7-9, suggesting no ionizable enzyme side chains ionize over this pH range. Inhibition by sulfide is potent (K(d) is 25 microM at pH 8) suggesting that SH(-) is the physiologic inhibitory species.

  2. Real-Time Ligand Binding Pocket Database Search Using Local Surface Descriptors

    PubMed Central

    Chikhi, Rayan; Sael, Lee; Kihara, Daisuke

    2010-01-01

    Due to the increasing number of structures of unknown function accumulated by ongoing structural genomics projects, there is an urgent need for computational methods for characterizing protein tertiary structures. As functions of many of these proteins are not easily predicted by conventional sequence database searches, a legitimate strategy is to utilize structure information in function characterization. Of a particular interest is prediction of ligand binding to a protein, as ligand molecule recognition is a major part of molecular function of proteins. Predicting whether a ligand molecule binds a protein is a complex problem due to the physical nature of protein-ligand interactions and the flexibility of both binding sites and ligand molecules. However, geometric and physicochemical complementarity is observed between the ligand and its binding site in many cases. Therefore, ligand molecules which bind to a local surface site in a protein can be predicted by finding similar local pockets of known binding ligands in the structure database. Here, we present two representations of ligand binding pockets and utilize them for ligand binding prediction by pocket shape comparison. These representations are based on mapping of surface properties of binding pockets, which are compactly described either by the two dimensional pseudo-Zernike moments or the 3D Zernike descriptors. These compact representations allow a fast real-time pocket searching against a database. Thorough benchmark study employing two different datasets show that our representations are competitive with the other existing methods. Limitations and potentials of the shape-based methods as well as possible improvements are discussed. PMID:20455259

  3. Competitive binding of a benzimidazole to the histone-binding pocket of the Pygo PHD finger.

    PubMed

    Miller, Thomas C R; Rutherford, Trevor J; Birchall, Kristian; Chugh, Jasveen; Fiedler, Marc; Bienz, Mariann

    2014-12-19

    The Pygo-BCL9 complex is a chromatin reader, facilitating β-catenin-mediated oncogenesis, and is thus emerging as a potential therapeutic target for cancer. Its function relies on two ligand-binding surfaces of Pygo's PHD finger that anchor the histone H3 tail methylated at lysine 4 (H3K4me) with assistance from the BCL9 HD1 domain. Here, we report the first use of fragment-based screening by NMR to identify small molecules that block protein-protein interactions by a PHD finger. This led to the discovery of a set of benzothiazoles that bind to a cleft emanating from the PHD-HD1 interface, as defined by X-ray crystallography. Furthermore, we discovered a benzimidazole that docks into the H3K4me specificity pocket and displaces the native H3K4me peptide from the PHD finger. Our study demonstrates the ligandability of the Pygo-BCL9 complex and uncovers a privileged scaffold as a template for future development of lead inhibitors of oncogenesis.

  4. Molecular Docking Studies to Explore Potential Binding Pockets and Inhibitors for Chikungunya Virus Envelope Glycoproteins.

    PubMed

    Nguyen, Phuong T V; Yu, Haibo; Keller, Paul A

    2017-03-11

    The chikungunya virus (CHIKV) envelope glycoproteins are considered important potential targets for anti-CHIKV drug discovery due to their crucial roles in virus attachment and virus entry. In this study, using two available crystal structures of the immature and mature forms of envelope glycoproteins, virtual screenings based on blind dockings and focused dockings were carried out to identify potential binding pockets and hit compounds for the virus. The chemical library database of compounds, NCI Diversity Set II, was used in these docking studies. In addition to reproducing previously reported examples, new binding pockets were identified, e.g., Pocket 2 in the 3N40, and Pocket 2 and Pocket 3 in the 3N42. Convergences in conformational sampling in docking using AutoDock Vina were evaluated. An analysis of docking results was carried out to understand interactions of the envelope glycoproteins complexes. Some key residues for interactions, for example Gly91 and His230, are identified as possessing important roles in the fusion process.

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

    NASA Astrophysics Data System (ADS)

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

    2017-01-01

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

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

    PubMed Central

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

    2017-01-01

    The ubiquitin ligase SCFCdc4 mediates phosphorylation-dependent elimination of numerous substrates by binding one or more Cdc4 phosphodegrons (CPDs). Methyl-based NMR analysis of the Cdc4 WD40 domain demonstrates that Cyclin E, Sic1 and Ash1 degrons have variable effects on the primary Cdc4WD40 binding pocket. Unexpectedly, a Sic1-derived multi-CPD substrate (pSic1) perturbs methyls around a previously documented allosteric binding site for the chemical inhibitor SCF-I2. NMR cross-saturation experiments confirm direct contact between pSic1 and the allosteric pocket. Phosphopeptide affinity measurements reveal negative allosteric communication between the primary CPD and allosteric pockets. Mathematical modelling indicates that the allosteric pocket may enhance ultrasensitivity by tethering pSic1 to Cdc4. These results suggest negative allosteric interaction between two distinct binding pockets on the Cdc4WD40 domain may facilitate dynamic exchange of multiple CPD sites to confer ultrasensitive dependence on substrate phosphorylation. PMID:28045046

  7. Identification of Distant Drug Off-Targets by Direct Superposition of Binding Pocket Surfaces

    PubMed Central

    Schumann, Marcel; Armen, Roger S.

    2013-01-01

    Correctly predicting off-targets for a given molecular structure, which would have the ability to bind a large range of ligands, is both particularly difficult and important if they share no significant sequence or fold similarity with the respective molecular target (“distant off-targets”). A novel approach for identification of off-targets by direct superposition of protein binding pocket surfaces is presented and applied to a set of well-studied and highly relevant drug targets, including representative kinases and nuclear hormone receptors. The entire Protein Data Bank is searched for similar binding pockets and convincing distant off-target candidates were identified that share no significant sequence or fold similarity with the respective target structure. These putative target off-target pairs are further supported by the existence of compounds that bind strongly to both with high topological similarity, and in some cases, literature examples of individual compounds that bind to both. Also, our results clearly show that it is possible for binding pockets to exhibit a striking surface similarity, while the respective off-target shares neither significant sequence nor significant fold similarity with the respective molecular target (“distant off-target”). PMID:24391782

  8. Allosteric models for multimeric proteins: oxygen-linked effector binding in hemocyanin.

    PubMed

    Menze, Michael A; Hellmann, Nadja; Decker, Heinz; Grieshaber, Manfred K

    2005-08-02

    In many crustaceans, changing concentrations of several low molecular weight compounds modulates hemocyanin oxygen binding, resulting in lower or higher oxygen affinities of the pigment. The nonphysiological effector caffeine and the physiological modulator urate, the latter accumulating in the hemolymph of the lobster Homarus vulgaris during hypoxia, increase hemocyanin oxygen affinity and decrease cooperativity of oxygen binding. To derive a model that describes the mechanism of allosteric interaction between hemocyanin and oxygen in the presence of urate or caffeine, studies of oxygen, urate, and caffeine binding to hemocyanin were performed. Exposure of lobster hemocyanin to various pH values between 7.25 and 8.15 resulted in a decrease of p50. In this pH interval, p50 decreases from 95 to 11 Torr without effectors and from 49 to 6 Torr and from 34 to 5 Torr in the presence of 1 mM urate or caffeine, respectively. Thus, the allosteric effects induced by protons and urate or caffeine are coupled. In contrast, isothermal titration calorimetry did not reveal any differences in binding enthalpy (DeltaH degrees ) for urate or caffeine under either normoxic or hypoxic conditions at different pH values. Despite these apparently conflicting results, they can be explained by the nested MWC model if two different types of modulator binding sites are assumed, an allosteric and a nonallosteric type of site. Simulations of in vivo conditions with this model indicate that the naturally occurring modulator urate is physiologically relevant in H. vulgaris only during hypoxic conditions, i.e., either during environmental oxygen limitation or extensive exercise.

  9. RsiteDB: a database of protein binding pockets that interact with RNA nucleotide bases.

    PubMed

    Shulman-Peleg, Alexandra; Nussinov, Ruth; Wolfson, Haim J

    2009-01-01

    We present a new database and an on-line search engine, which store and query the protein binding pockets that interact with single-stranded RNA nucleotide bases. The database consists of a classification of binding sites derived from protein-RNA complexes. Each binding site is assigned to a cluster of similar binding sites in other protein-RNA complexes. Cluster members share similar spatial arrangements of physico-chemical properties, thus can reveal novel similarity between proteins and RNAs with different sequences and folds. The clusters provide 3D consensus binding patterns important for protein-nucleotide recognition. The database search engine allows two types of useful queries: first, given a PDB code of a protein-RNA complex, RsiteDB can detail and classify the properties of the protein binding pockets accommodating extruded RNA nucleotides not involved in local RNA base pairing. Second, given an unbound protein structure, RsiteDB can perform an on-line structural search against the constructed database of 3D consensus binding patterns. Regions similar to known patterns are predicted to serve as binding sites. Alignment of the query to these patterns with their corresponding RNA nucleotides allows making unique predictions of the protein-RNA interactions at the atomic level of detail. This database is accessible at http://bioinfo3d.cs.tau.ac.il/RsiteDB.

  10. The human olfactory receptor 17-40: requisites for fitting into the binding pocket.

    PubMed

    Anselmi, Cecilia; Buonocore, Anna; Centini, Marisanna; Facino, Roberto Maffei; Hatt, Hanns

    2011-06-01

    To gain structural insight on the interactions between odorants and the human olfactory receptor, we did homology modelling of the receptor structure, followed by molecular docking simulation with ligands. Molecular dynamics simulation on the structures resulting from docking served to estimate the binding free energy of the various odorant families. A correlation with the odorous properties of the ligands is proposed. We also investigated which residues were involved in the binding of a set of properly synthesised ligands and which were required for fitting inside the binding pocket. Olfactive stimulation of the olfactory receptor with odorous molecules was also investigated, using calcium imaging or electrophysiological recordings.

  11. Multiscale Monte Carlo Sampling of Protein Sidechains: Application to Binding Pocket Flexibility

    PubMed Central

    Nilmeier, Jerome; Jacobson, Matt

    2008-01-01

    We present a Monte Carlo sidechain sampling procedure and apply it to assessing the flexibility of protein binding pockets. We implemented a multiple “time step” Monte Carlo algorithm to optimize sidechain sampling with a surface generalized Born implicit solvent model. In this approach, certain forces (those due to long-range electrostatics and the implicit solvent model) are updated infrequently, in “outer steps”, while short-range forces (covalent, local nonbonded interactions) are updated at every “inner step”. Two multistep protocols were studied. The first protocol rigorously obeys detailed balance, and the second protocol introduces an approximation to the solvation term that increases the acceptance ratio. The first protocol gives a 10-fold improvement over a protocol that does not use multiple time steps, while the second protocol generates comparable ensembles and gives a 15-fold improvement. A range of 50–200 inner steps per outer step was found to give optimal performance for both protocols. The resultant method is a practical means to assess sidechain flexibility in ligand binding pockets, as we illustrate with proof-of-principle calculations on six proteins: DB3 antibody, thermolysin, estrogen receptor, PPAR-γ, PI3 kinase, and CDK2. The resulting sidechain ensembles of the apo binding sites correlate well with known induced fit conformational changes and provide insights into binding pocket flexibility. PMID:19119325

  12. Allosteric antagonist binding sites in class B GPCRs: corticotropin receptor 1

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Supriyo; Subramanian, Govindan; Hall, Spencer; Lin, Jianping; Laoui, Abdelazize; Vaidehi, Nagarajan

    2010-08-01

    The 41 amino acid neuropeptide, corticotropin-releasing factor (CRF) and its associated receptors CRF1-R and CRF2-R have been targeted for treating stress related disorders. Both CRF1-R and CRF2-R belong to the class B G-protein coupled receptors for which little information is known regarding the small molecule antagonist binding characteristics. However, it has been shown recently that different non-peptide allosteric ligands stabilize different receptor conformations for CRF1-R and hence an understanding of the ligand induced receptor conformational changes is important in the pharmacology of ligand binding. In this study, we modeled the receptor and identified the binding sites of representative small molecule allosteric antagonists for CRF1-R. The predicted binding sites of the investigated compounds are located within the transmembrane (TM) domain encompassing TM helices 3, 5 and 6. The docked compounds show strong interactions with H228 on TM3 and M305 on TM5 that have also been implicated in the binding by site directed mutation studies. H228 forms a hydrogen bond of varied strengths with all the antagonists in this study and this is in agreement with the decreased binding affinity of several compounds with H228F mutation. Also mutating M305 to Ile showed a sharp decrease in the calculated binding energy whereas the binding energy loss on M305 to Leu was less significant. These results are in qualitative agreement with the decrease in binding affinities observed experimentally. We further predicted the conformational changes in CRF1-R induced by the allosteric antagonist NBI-27914. Movement of TM helices 3 and 5 are dominant and generates three degenerate conformational states two of which are separated by an energy barrier from the third, when bound to NBI-27914. Binding of NBI-27914 was predicted to improve the interaction of the ligand with M305 and also enhanced the aromatic stacking between the ligand and F232 on TM3. A virtual ligand screening of 13

  13. Identification of novel allosteric modulator binding sites in NMDA receptors: A molecular modeling study.

    PubMed

    Kane, Lucas T; Costa, Blaise M

    2015-09-01

    The dysfunction of N-methyl-d-Aspartate receptors (NMDARs), a subtype of glutamate receptors, is correlated with schizophrenia, stroke, and many other neuropathological disorders. However, not all NMDAR subtypes equally contribute towards these disorders. Since NMDARs composed of different GluN2 subunits (GluN2A-D) confer varied physiological properties and have different distributions in the brain, pharmacological agents that target NMDARs with specific GluN2 subunits have significant potential for therapeutic applications. In our previous research, we have identified a family of novel allosteric modulators that differentially potentiate and/or inhibit NMDARs of differing GluN2 subunit composition. To further elucidate their molecular mechanisms, in the present study, we have identified four potential binding sites for novel allosteric modulators by performing molecular modeling, docking, and in silico mutations. The molecular determinants of the modulator binding sites (MBS), analysis of particular MBS electrostatics, and the specific loss or gain of binding after mutations have revealed modulators that have strong potential affinities for specific MBS on given subunits and the role of key amino acids in either promoting or obstructing modulator binding. These findings will help design higher affinity GluN2 subunit-selective pharmaceuticals, which are currently unavailable to treat psychiatric and neurological disorders.

  14. Development of purely structure-based pharmacophores for the topoisomerase I-DNA-ligand binding pocket

    NASA Astrophysics Data System (ADS)

    Drwal, Malgorzata N.; Agama, Keli; Pommier, Yves; Griffith, Renate

    2013-12-01

    Purely structure-based pharmacophores (SBPs) are an alternative method to ligand-based approaches and have the advantage of describing the entire interaction capability of a binding pocket. Here, we present the development of SBPs for topoisomerase I, an anticancer target with an unusual ligand binding pocket consisting of protein and DNA atoms. Different approaches to cluster and select pharmacophore features are investigated, including hierarchical clustering and energy calculations. In addition, the performance of SBPs is evaluated retrospectively and compared to the performance of ligand- and complex-based pharmacophores. SBPs emerge as a valid method in virtual screening and a complementary approach to ligand-focussed methods. The study further reveals that the choice of pharmacophore feature clustering and selection methods has a large impact on the virtual screening hit lists. A prospective application of the SBPs in virtual screening reveals that they can be used successfully to identify novel topoisomerase inhibitors.

  15. Spontaneous activation of visual pigments in relation to openness/closedness of chromophore-binding pocket

    PubMed Central

    Yue, Wendy Wing Sze; Frederiksen, Rikard; Ren, Xiaozhi; Luo, Dong-Gen; Yamashita, Takahiro; Shichida, Yoshinori; Cornwall, M Carter; Yau, King-Wai

    2017-01-01

    Visual pigments can be spontaneously activated by internal thermal energy, generating noise that interferes with real-light detection. Recently, we developed a physicochemical theory that successfully predicts the rate of spontaneous activity of representative rod and cone pigments from their peak-absorption wavelength (λmax), with pigments having longer λmax being noisier. Interestingly, cone pigments may generally be ~25 fold noisier than rod pigments of the same λmax, possibly ascribed to an ‘open’ chromophore-binding pocket in cone pigments defined by the capability of chromophore-exchange in darkness. Here, we show in mice that the λmax-dependence of pigment noise could be extended even to a mutant pigment, E122Q-rhodopsin. Moreover, although E122Q-rhodopsin shows some cone-pigment-like characteristics, its noise remained quantitatively predictable by the ‘non-open’ nature of its chromophore-binding pocket as in wild-type rhodopsin. The openness/closedness of the chromophore-binding pocket is potentially a useful indicator of whether a pigment is intended for detecting dim or bright light. DOI: http://dx.doi.org/10.7554/eLife.18492.001 PMID:28186874

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

    NASA Astrophysics Data System (ADS)

    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.

  17. Interactions between Hofmeister anions and the binding pocket of a protein.

    PubMed

    Fox, Jerome M; Kang, Kyungtae; Sherman, Woody; Héroux, Annie; Sastry, G Madhavi; Baghbanzadeh, Mostafa; Lockett, Matthew R; Whitesides, George M

    2015-03-25

    This paper uses the binding pocket of human carbonic anhydrase II (HCAII, EC 4.2.1.1) as a tool to examine the properties of Hofmeister anions that determine (i) where, and how strongly, they associate with concavities on the surfaces of proteins and (ii) how, upon binding, they alter the structure of water within those concavities. Results from X-ray crystallography and isothermal titration calorimetry show that most anions associate with the binding pocket of HCAII by forming inner-sphere ion pairs with the Zn(2+) cofactor. In these ion pairs, the free energy of anion-Zn(2+) association is inversely proportional to the free energetic cost of anion dehydration; this relationship is consistent with the mechanism of ion pair formation suggested by the "law of matching water affinities". Iodide and bromide anions also associate with a hydrophobic declivity in the wall of the binding pocket. Molecular dynamics simulations suggest that anions, upon associating with Zn(2+), trigger rearrangements of water that extend up to 8 Å away from their surfaces. These findings expand the range of interactions previously thought to occur between ions and proteins by suggesting that (i) weakly hydrated anions can bind complementarily shaped hydrophobic declivities, and that (ii) ion-induced rearrangements of water within protein concavities can (in contrast with similar rearrangements in bulk water) extend well beyond the first hydration shells of the ions that trigger them. This study paints a picture of Hofmeister anions as a set of structurally varied ligands that differ in size, shape, and affinity for water and, thus, in their ability to bind to—and to alter the charge and hydration structure of—polar, nonpolar, and topographically complex concavities on the surfaces of proteins.

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

    SciTech Connect

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

    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.

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

    PubMed Central

    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

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

    PubMed Central

    2009-01-01

    Background Many neuromuscular blockers act as negative allosteric modulators of muscarinic acetylcholine receptors by decreasing affinity and potency of acetylcholine. The neuromuscular blocker rapacuronium has been shown to have facilitatory effects at muscarinic receptors leading to bronchospasm. We examined the influence of rapacuronium on acetylcholine (ACh) binding to and activation of individual subtypes of muscarinic receptors expressed in Chinese hamster ovary cells to determine its receptor selectivity. Results At equilibrium rapacuronium bound to all subtypes of muscarinic receptors with micromolar affinity (2.7-17 μM) and displayed negative cooperativity with both high- and low-affinity ACh binding states. Rapacuronium accelerated [3H]ACh association with and dissociation from odd-numbered receptor subtypes. With respect to [35S]GTPγS binding rapacuronium alone behaved as an inverse agonist at all subtypes. Rapacuronium concentration-dependently decreased the potency of ACh-induced [35S]GTPγS binding at M2 and M4 receptors. In contrast, 0.1 μM rapacuronium significantly increased ACh potency at M1, M3, and M5 receptors. Kinetic measurements at M3 receptors showed acceleration of the rate of ACh-induced [35S]GTPγS binding by rapacuronium. Conclusions Our data demonstrate a novel dichotomy in rapacuronium effects at odd-numbered muscarinic receptors. Rapacuronium accelerates the rate of ACh binding but decreases its affinity under equilibrium conditions. This results in potentiation of receptor activation at low concentrations of rapacuronium (1 μM) but not at high concentrations (10 μM). These observations highlight the relevance and necessity of performing physiological tests under non-equilibrium conditions in evaluating the functional effects of allosteric modulators at muscarinic receptors. They also provide molecular basis for potentiating M3 receptor-mediated bronchoconstriction. PMID:20038295

  1. Spatial Decomposition of Translational Water-Water Correlation Entropy in Binding Pockets.

    PubMed

    Nguyen, Crystal N; Kurtzman, Tom; Gilson, Michael K

    2016-01-12

    A number of computational tools available today compute the thermodynamic properties of water at surfaces and in binding pockets by using inhomogeneous solvation theory (IST) to analyze explicit-solvent simulations. Such methods enable qualitative spatial mappings of both energy and entropy around a solute of interest and can also be applied quantitatively. However, the entropy estimates of existing methods have, to date, been almost entirely limited to the first-order terms in the IST's entropy expansion. These first-order terms account for localization and orientation of water molecules in the field of the solute but not for the modification of water-water correlations by the solute. Here, we present an extension of the Grid Inhomogeneous Solvation Theory (GIST) approach which accounts for water-water translational correlations. The method involves rewriting the two-point density of water in terms of a conditional density and utilizes the efficient nearest-neighbor entropy estimation approach. Spatial maps of this second order term, for water in and around the synthetic host cucurbit[7]uril and in the binding pocket of the enzyme Factor Xa, reveal mainly negative contributions, indicating solute-induced water-water correlations relative to bulk water; particularly strong signals are obtained for sites at the entrances of cavities or pockets. This second-order term thus enters with the same, negative, sign as the first order translational and orientational terms. Numerical and convergence properties of the methodology are examined.

  2. Spatial Decomposition of Translational Water–Water Correlation Entropy in Binding Pockets

    PubMed Central

    2015-01-01

    A number of computational tools available today compute the thermodynamic properties of water at surfaces and in binding pockets by using inhomogeneous solvation theory (IST) to analyze explicit-solvent simulations. Such methods enable qualitative spatial mappings of both energy and entropy around a solute of interest and can also be applied quantitatively. However, the entropy estimates of existing methods have, to date, been almost entirely limited to the first-order terms in the IST’s entropy expansion. These first-order terms account for localization and orientation of water molecules in the field of the solute but not for the modification of water–water correlations by the solute. Here, we present an extension of the Grid Inhomogeneous Solvation Theory (GIST) approach which accounts for water–water translational correlations. The method involves rewriting the two-point density of water in terms of a conditional density and utilizes the efficient nearest-neighbor entropy estimation approach. Spatial maps of this second order term, for water in and around the synthetic host cucurbit[7]uril and in the binding pocket of the enzyme Factor Xa, reveal mainly negative contributions, indicating solute-induced water–water correlations relative to bulk water; particularly strong signals are obtained for sites at the entrances of cavities or pockets. This second-order term thus enters with the same, negative, sign as the first order translational and orientational terms. Numerical and convergence properties of the methodology are examined. PMID:26636620

  3. Computational approaches for identification of conserved/unique binding pockets in the A chain of ricin

    SciTech Connect

    Ecale Zhou, C L; Zemla, A T; Roe, D; Young, M; Lam, M; Schoeniger, J; Balhorn, R

    2005-01-29

    Specific and sensitive ligand-based protein detection assays that employ antibodies or small molecules such as peptides, aptamers, or other small molecules require that the corresponding surface region of the protein be accessible and that there be minimal cross-reactivity with non-target proteins. To reduce the time and cost of laboratory screening efforts for diagnostic reagents, we developed new methods for evaluating and selecting protein surface regions for ligand targeting. We devised combined structure- and sequence-based methods for identifying 3D epitopes and binding pockets on the surface of the A chain of ricin that are conserved with respect to a set of ricin A chains and unique with respect to other proteins. We (1) used structure alignment software to detect structural deviations and extracted from this analysis the residue-residue correspondence, (2) devised a method to compare corresponding residues across sets of ricin structures and structures of closely related proteins, (3) devised a sequence-based approach to determine residue infrequency in local sequence context, and (4) modified a pocket-finding algorithm to identify surface crevices in close proximity to residues determined to be conserved/unique based on our structure- and sequence-based methods. In applying this combined informatics approach to ricin A we identified a conserved/unique pocket in close proximity (but not overlapping) the active site that is suitable for bi-dentate ligand development. These methods are generally applicable to identification of surface epitopes and binding pockets for development of diagnostic reagents, therapeutics, and vaccines.

  4. Comparison of three classes of human liver alcohol dehydrogenase. Emphasis on different substrate binding pockets.

    PubMed

    Eklund, H; Müller-Wille, P; Horjales, E; Futer, O; Holmquist, B; Vallee, B L; Höög, J O; Kaiser, R; Jörnvall, H

    1990-10-24

    Conformational models of the three characterized classes of mammalian liver alcohol dehydrogenase were constructed using computer graphics based on the known three-dimensional structure of the E subunit of the horse enzyme (class I) and the primary structures of the three human enzyme classes. This correlates the substrate-binding pockets of the class I subunits (alpha, beta and gamma in the human enzyme) with those of the class II and III subunits (pi and chi, respectively) for three enzymes that differ in substrate specificity, inhibition pattern and many other properties. The substrate-binding sites exhibit pronounced differences in both shape and properties. Comparing human class I subunits with those of class II and III subunits there are no less than 8 and 10 replacements, respectively, out of 11 residues in the substrate pocket, while in the human class I isozyme variants, only 1-3 of these 11 positions differ. A single residue, Val294, is conserved throughout. The liver alcohol dehydrogenases, with different substrate-specificity pockets, resemble the patterns of other enzyme families such as the pancreatic serine proteases. The inner part of the substrate cleft in the class II and III enzymes is smaller than in the horse class I enzyme, because both Ser48 and Phe93 are replaced by larger residues, Thr and Tyr, respectively. In class II, the residues in the substrate pocket are larger in about half of the positions. It is rich in aromatic residues, four Phe and one Tyr, making the substrate site distinctly smaller than in the class I subunits. In class III, the inner part of the substrate cleft is narrow but the outer part considerably wider and more polar than in the class I and II enzymes. In addition, Ser (or Thr) and Tyr in class II and III instead of His51 may influence proton abstraction/donation at the active site.

  5. Glutamate Water Gates in the Ion Binding Pocket of Na(+) Bound Na(+), K(+)-ATPase.

    PubMed

    Han, Minwoo; Kopec, Wojciech; Solov'yov, Ilia A; Khandelia, Himanshu

    2017-01-13

    The dynamically changing protonation states of the six acidic amino acid residues in the ion binding pocket of the Na(+), K(+) -ATPase (NKA) during the ion transport cycle are proposed to drive ion binding, release and possibly determine Na(+) or K(+) selectivity. We use molecular dynamics (MD) and density functional theory (DFT) simulations to determine the protonation scheme of the Na(+) bound conformation of NKA. MD simulations of all possible protonation schemes show that the bound Na(+) ions are most stably bound when three or four protons reside in the binding sites, and that Glu954 in site III is always protonated. Glutamic acid residues in the three binding sites act as water gates, and their deprotonation triggers water entry to the binding sites. From DFT calculations of Na(+) binding energies, we conclude that three protons in the binding site are needed to effectively bind Na(+) from water and four are needed to release them in the next step. Protonation of Asp926 in site III will induce Na(+) release, and Glu327, Glu954 and Glu779 are all likely to be protonated in the Na(+) bound occluded conformation. Our data provides key insights into the role of protons in the Na(+) binding and release mechanism of NKA.

  6. Glutamate Water Gates in the Ion Binding Pocket of Na+ Bound Na+, K+-ATPase

    PubMed Central

    Han, Minwoo; Kopec, Wojciech; Solov’yov, Ilia A.; Khandelia, Himanshu

    2017-01-01

    The dynamically changing protonation states of the six acidic amino acid residues in the ion binding pocket of the Na+, K+ -ATPase (NKA) during the ion transport cycle are proposed to drive ion binding, release and possibly determine Na+ or K+ selectivity. We use molecular dynamics (MD) and density functional theory (DFT) simulations to determine the protonation scheme of the Na+ bound conformation of NKA. MD simulations of all possible protonation schemes show that the bound Na+ ions are most stably bound when three or four protons reside in the binding sites, and that Glu954 in site III is always protonated. Glutamic acid residues in the three binding sites act as water gates, and their deprotonation triggers water entry to the binding sites. From DFT calculations of Na+ binding energies, we conclude that three protons in the binding site are needed to effectively bind Na+ from water and four are needed to release them in the next step. Protonation of Asp926 in site III will induce Na+ release, and Glu327, Glu954 and Glu779 are all likely to be protonated in the Na+ bound occluded conformation. Our data provides key insights into the role of protons in the Na+ binding and release mechanism of NKA. PMID:28084301

  7. Allosteric transition and substrate binding are entropy-driven in glucosamine-6-phosphate deaminase from Escherichia coli.

    PubMed

    Bustos-Jaimes, I; Calcagno, M L

    2001-10-15

    Glucosamine-6P-deaminase (EC 3.5.99.6, formerly glucosamine-6-phosphate isomerase, EC 5.3.1.10) from Escherichia coli is an attractive experimental model for the study of allosteric transitions because it is both kinetically and structurally well-known, and follows rapid equilibrium random kinetics, so that the kinetic K(m) values are true thermodynamic equilibrium constants. The enzyme is a typical allosteric K-system activated by N-acetylglucosamine 6-P and displays an allosteric behavior that can be well described by the Monod-Wyman-Changeux model. This thermodynamic study based on the temperature dependence of allosteric parameters derived from this model shows that substrate binding and allosteric transition are both entropy-driven processes in E. coli GlcN6P deaminase. The analysis of this result in the light of the crystallographic structure of the enzyme implicates the active-site lid as the structural motif that could contribute significantly to this entropic component of the allosteric transition because of the remarkable change in its crystallographic B factors.

  8. Evidence for allosteric interactions of antagonist binding to the smoothened receptor.

    PubMed

    Rominger, Cynthia M; Bee, Wei-Lin Tiger; Copeland, Robert A; Davenport, Elizabeth A; Gilmartin, Aidan; Gontarek, Richard; Hornberger, Keith R; Kallal, Lorena A; Lai, Zhihong; Lawrie, Kenneth; Lu, Quinn; McMillan, Lynette; Truong, Maggie; Tummino, Peter J; Turunen, Brandon; Will, Matthew; Zuercher, William J; Rominger, David H

    2009-06-01

    The Smoothened receptor (Smo) mediates hedgehog (Hh) signaling critical for development, cell growth, and migration, as well as stem cell maintenance. Aberrant Hh signaling pathway activation has been implicated in a variety of cancers, and small-molecule antagonists of Smo have entered human clinical trials for the treatment of cancer. Here, we report the biochemical characterization of allosteric interactions of agonists and antagonists for Smo. Binding of two radioligands, [(3)H]3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)-phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.3) (agonist) and [(3)H]cyclopamine (antagonist), was characterized using human Smo expressed in human embryonic kidney 293F membranes. We observed full displacement of [(3)H]cyclopamine by all Smo agonist and antagonist ligands examined. N-[(1E)-(3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene]-4-(phenylmethyl)-1-piperazinamine (SANT-1), an antagonist, did not fully inhibit the binding of [(3)H]SAG-1.3. In a functional cell-based beta-lactamase reporter gene assay, SANT-1 and N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-tris(ethyloxy)-benzamide (SANT-2) fully inhibited 3-chloro-4,7-difluoro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.5)-induced Hh pathway activation. Detailed "Schild-type" radioligand binding analysis with [(3)H]SAG-1.3 revealed that two structurally distinct Smoothened receptor antagonists, SANT-1 and SANT-2, bound in a manner consistent with that of allosteric modulation. Our mechanism of action characterization of radioligand binding to Smo combined with functional data provides a better understanding of small-molecule interactions with Smo and their influence on the Hh pathway.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27139631

  10. A substrate-induced biotin binding pocket in the carboxyltransferase domain of pyruvate carboxylase.

    PubMed

    Lietzan, Adam D; St Maurice, Martin

    2013-07-05

    Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyltransferase domain of PC from Rhizobium etli. Structures of the carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp(590) and Tyr(628) and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes.

  11. HPC Analysis of Multiple Binding Sites Communication and Allosteric Modulations in Drug Design: The HSP Case Study.

    PubMed

    Chiappori, Federica; Milanesi, Luciano; Merelli, Ivan

    2016-01-01

    Allostery is a long-range macromolecular mechanism of internal regulation, in which the binding of a ligand in an allosteric site induces distant conformational changes in a distant portion of the protein, modifying its activity. From the drug design point of view, this mechanism can be exploited to achieve important therapeutic effects, since ligands able to bind allosteric sites may be designed to regulate target proteins. Computational tools are a valid support in this sense, since they allow the characterization of allosteric communications within proteins, which are essential to design modulator ligands. While considering long-range interactions in macromolecules, the principal drug design tool available to researcher is molecular dynamics, and related applications, since it allows the evaluation of conformational changes of a protein bound to a ligand. In particular, all-atoms molecular dynamics is suitable to verify the internal mechanisms that orchestrate allosteric communications, in order to identify key residues and internal pathways that modify the protein behaviour. The problem is that these techniques are heavily time-consuming and computationally intensive, thus high performance computing systems, including parallel computing and GPU-accelerated computations, are necessary to achieve results in a reasonable time. In this review, we will discuss how it is possible to exploit in silico approaches to characterize allosteric modulations and long-range interactions within proteins, describing the case study of the Heat Shock Proteins, a class of chaperons regulated by stress conditions, which is particularly important since it is involved in many cancers and neurodegenerative diseases.

  12. Distinct pose of discodermolide in taxol binding pocket drives a complementary mode of microtubule stabilization.

    PubMed

    Khrapunovich-Baine, Marina; Menon, Vilas; Verdier-Pinard, Pascal; Smith, Amos B; Angeletti, Ruth Hogue; Fiser, Andras; Horwitz, Susan Band; Xiao, Hui

    2009-12-15

    The microtubule cytoskeleton has proven to be an effective target for cancer therapeutics. One class of drugs, known as microtubule stabilizing agents (MSAs), binds to microtubule polymers and stabilizes them against depolymerization. The prototype of this group of drugs, Taxol, is an effective chemotherapeutic agent used extensively in the treatment of human ovarian, breast, and lung carcinomas. Although electron crystallography and photoaffinity labeling experiments determined that the binding site for Taxol is in a hydrophobic pocket in beta-tubulin, little was known about the effects of this drug on the conformation of the entire microtubule. A recent study from our laboratory utilizing hydrogen-deuterium exchange (HDX) in concert with various mass spectrometry (MS) techniques has provided new information on the structure of microtubules upon Taxol binding. In the current study we apply this technique to determine the binding mode and the conformational effects on chicken erythrocyte tubulin (CET) of another MSA, discodermolide, whose synthetic analogues may have potential use in the clinic. We confirmed that, like Taxol, discodermolide binds to the taxane binding pocket in beta-tubulin. However, as opposed to Taxol, which has major interactions with the M-loop, discodermolide orients itself away from this loop and toward the N-terminal H1-S2 loop. Additionally, discodermolide stabilizes microtubules mainly via its effects on interdimer contacts, specifically on the alpha-tubulin side, and to a lesser extent on interprotofilament contacts between adjacent beta-tubulin subunits. Also, our results indicate complementary stabilizing effects of Taxol and discodermolide on the microtubules, which may explain the synergy observed between the two drugs in vivo.

  13. Progress in antiandrogen design targeting hormone binding pocket to circumvent mutation based resistance

    PubMed Central

    Tian, Xiaohong; He, Yang; Zhou, Jinming

    2015-01-01

    Androgen receptor (AR) plays a critical role in the development and progression of prostate cancer (PCa). Current clinically used antiandrogens such as flutamide, bicalutamide, and newly approved enzalutamide mainly target the hormone binding pocket (HBP) of AR. However, over time, drug resistance invariably develops and switches these antiandrogens from antagonist to agonist of the AR. Accumulated evidence indicates that AR mutation is an important cause for the drug resistance. This review will give an overview of the mutation based resistance of the current clinically used antiandrogens and the rational drug design to overcome the resistance, provides a promising strategy for the development of the new generation of antiandrogens targeting HBP. PMID:25852559

  14. Growth suppression by an E2F-binding-defective retinoblastoma protein (RB): contribution from the RB C pocket.

    PubMed

    Whitaker, L L; Su, H; Baskaran, R; Knudsen, E S; Wang, J Y

    1998-07-01

    Growth suppression by the retinoblastoma protein (RB) is dependent on its ability to form complexes with transcription regulators. At least three distinct protein-binding activities have been identified in RB: the large A/B pocket binds E2F, the A/B pocket binds the LXCXE peptide motif, and the C pocket binds the nuclear c-Abl tyrosine kinase. Substitution of Trp for Arg 661 in the B region of RB (mutant 661) inactivates both E2F and LXCXE binding. The tumor suppression function of mutant 661 is not abolished, because this allele predisposes its carriers to retinoblastoma development with a low penetrance. In cell-based assays, 661 is shown to inhibit G1/S progression. This low-penetrance mutant also induces terminal growth arrest with reduced but detectable activity. We have constructed mutations that disrupt C pocket activity. When overproduced, the RB C-terminal fragment did not induce terminal growth arrest but could inhibit G1/S progression, and this activity was abolished by the C-pocket mutations. In full-length RB, the C-pocket mutations reduced but did not abolish RB function. Interestingly, combination of the C-pocket and 661 mutations completely abolished RB's ability to cause an increase in the percentage of cells in G1 and to induce terminal growth arrest. These results suggest that the A/B or C region can induce a prolongation of G1 through mechanisms that are independent of each other. In contrast, long-term growth arrest requires combined activities from both regions of RB. In addition, E2F and LXCXE binding are not the only mechanisms through which RB inhibits cell growth. The C pocket also contributes to RB-mediated growth suppression.

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

    PubMed Central

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

    2016-01-01

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

  16. Computational Assessment of Potassium and Magnesium Ion Binding to a Buried Pocket in GTPase-Associating Center RNA

    PubMed Central

    2016-01-01

    An experimentally well-studied model of RNA tertiary structures is a 58mer rRNA fragment, known as GTPase-associating center (GAC) RNA, in which a highly negative pocket walled by phosphate oxygen atoms is stabilized by a chelated cation. Although such deep pockets with more than one direct phosphate to ion chelation site normally include magnesium, as shown in one GAC crystal structure, another GAC crystal structure and solution experiments suggest potassium at this site. Both crystal structures also depict two magnesium ions directly bound to the phosphate groups comprising this controversial pocket. Here, we used classical molecular dynamics simulations as well as umbrella sampling to investigate the possibility of binding of potassium versus magnesium inside the pocket and to better characterize the chelation of one of the binding magnesium ions outside the pocket. The results support the preference of the pocket to accommodate potassium rather than magnesium and suggest that one of the closely binding magnesium ions can only bind at high magnesium concentrations, such as might be present during crystallization. This work illustrates the complementary utility of molecular modeling approaches with atomic-level detail in resolving discrepancies between conflicting experimental results. PMID:27983843

  17. Allosteric modulation of neurotoxin binding to voltage-sensitive sodium channels by Ptychodiscus brevis toxin 2.

    PubMed

    Sharkey, R G; Jover, E; Couraud, F; Baden, D G; Catterall, W A

    1987-03-01

    The effects of Ptychodiscus brevis toxin 2 (PbTx-2) on the binding of neurotoxins at four different neurotoxin receptor sites on voltage-sensitive sodium channels in rat brain synaptosomes were examined. Binding of saxitoxin at neurotoxin receptor site 1 and Leiurus quinquestriatus alpha-scorpion toxin (LqTx) at neurotoxin receptor site 3 was unaffected. PbTx-2 enhanced binding of batrachotoxinin A 20-alpha-benzoate (BTX-B) to neurotoxin receptor site 2 and Centruroides suffusus suffusus beta-scorpion toxin (CsTx II) to site 4 on sodium channels. These results support the proposal that PbTx-2 and related toxins act at a new receptor site (site 5) that has not been previously analyzed in binding experiments. Half-maximal effects of PbTx-2 were observed in the range of 20-50 nM PbTx-2. The enhancement of BTX-B binding was reduced by depolarization. Saturating concentrations of PbTx-2 reduced KD values for binding of BTX-B and CsTx-II 2.9-fold and 2.6-fold, respectively. The effects of PbTx-2 and LqTx in enhancing BTX-B binding were synergistic. A model involving both preferential binding of BTX-B, PbTx-2, LqTx, and CsTx II to active states of sodium channels and allosteric interactions among the four receptor sites at which these toxins act accommodates these and previous results.

  18. Structural and dynamic studies of the transcription factor ERG reveal DNA binding is allosterically autoinhibited.

    PubMed

    Regan, Michael C; Horanyi, Peter S; Pryor, Edward E; Sarver, Jessica L; Cafiso, David S; Bushweller, John H

    2013-08-13

    The Ets-Related Gene (ERG) belongs to the Ets family of transcription factors and is critically important for maintenance of the hematopoietic stem cell population. A chromosomal translocation observed in the majority of human prostate cancers leads to the aberrant overexpression of ERG. We have identified regions flanking the ERG Ets domain responsible for autoinhibition of DNA binding and solved crystal structures of uninhibited, autoinhibited, and DNA-bound ERG. NMR-based measurements of backbone dynamics show that uninhibited ERG undergoes substantial dynamics on the millisecond-to-microsecond timescale but autoinhibited and DNA-bound ERG do not. We propose a mechanism whereby the allosteric basis of ERG autoinhibition is mediated predominantly by the regulation of Ets-domain dynamics with only modest structural changes.

  19. WDR5 Intearcts with Mixed Lineage Leukemia (MLL) Protein via the Histone H3-binding Pocket

    SciTech Connect

    Song, J.; Kingston, R

    2008-01-01

    WDR5 is a component of the mixed lineage leukemia (MLL) complex, which methylates lysine 4 of histone H3, and was identified as a methylated Lys-4 histone H3-binding protein. Here, we present a crystal structure of WDR5 bound to an MLL peptide. Surprisingly, we find that WDR5 utilizes the same pocket shown to bind histone H3 for this MLL interaction. Furthermore, the WDR5-MLL interaction is disrupted preferentially by mono- and di-methylated Lys-4 histone H3 over unmodified and tri-methylated Lys-4 histone H3. These data implicate a delicate interplay between the effector, WDR5, the catalytic subunit, MLL, and the substrate, histone H3, of the MLL complex. We suggest that the activity of the MLL complex might be regulated through this interplay.

  20. Mapping protein pockets through their potential small-molecule binding volumes: QSCD applied to biological protein structures

    NASA Astrophysics Data System (ADS)

    Mason, Keith; Patel, Nehal M.; Ledel, Aric; Moallemi, Ciamac C.; Wintner, Edward A.

    2004-01-01

    Previously we demonstrated a method, Quantized Surface Complementarity Diversity (QSCD), of defining molecular diversity by measuring shape and functional complementarity of molecules to a basis set of theoretical target surfaces [Wintner E.A. and Moallemi C.C., J. Med. Chem., 43 (2000) 1993]. In this paper we demonstrate a method of mapping actual protein pockets to the same basis set of theoretical target surfaces, thereby allowing categorization of protein pockets by their properties of shape and functionality. The key step in the mapping is a `dissection' algorithm that breaks any protein pocket into a set of potential small molecule binding volumes. It is these binding volumes that are mapped to the basis set of theoretical target surfaces, thus measuring a protein pocket not as a single surface but as a collection of molecular recognition environments.

  1. The same pocket in menin binds both MLL and JUND but has opposite effects on transcription

    SciTech Connect

    Huang, Jing; Gurung, Buddha; Wan, Bingbing; Matkar, Smita; Veniaminova, Natalia A.; Wan, Ke; Merchant, Juanita L.; Hua, Xianxin; Lei, Ming

    2013-04-08

    Menin is a tumour suppressor protein whose loss or inactivation causes multiple endocrine neoplasia 1 (MEN1), a hereditary autosomal dominant tumour syndrome that is characterized by tumorigenesis in multiple endocrine organs. Menin interacts with many proteins and is involved in a variety of cellular processes. Menin binds the JUN family transcription factor JUND and inhibits its transcriptional activity. Several MEN1 missense mutations disrupt the menin-JUND interaction, suggesting a correlation between the tumour-suppressor function of menin and its suppression of JUND-activated transcription. Menin also interacts with mixed lineage leukaemia protein 1 (MLL1), a histone H3 lysine 4 methyltransferase, and functions as an oncogenic cofactor to upregulate gene transcription and promote MLL1-fusion-protein-induced leukaemogenesis. A recent report on the tethering of MLL1 to chromatin binding factor lens epithelium-derived growth factor (LEDGF) by menin indicates that menin is a molecular adaptor coordinating the functions of multiple proteins. Despite its importance, how menin interacts with many distinct partners and regulates their functions remains poorly understood. Here we present the crystal structures of human menin in its free form and in complexes with MLL1 or with JUND, or with an MLL1-LEDGF heterodimer. These structures show that menin contains a deep pocket that binds short peptides of MLL1 or JUND in the same manner, but that it can have opposite effects on transcription. The menin-JUND interaction blocks JUN N-terminal kinase (JNK)-mediated JUND phosphorylation and suppresses JUND-induced transcription. In contrast, menin promotes gene transcription by binding the transcription activator MLL1 through the peptide pocket while still interacting with the chromatin-anchoring protein LEDGF at a distinct surface formed by both menin and MLL1.

  2. Discovery of multiple hidden allosteric sites by combining Markov state models and experiments.

    PubMed

    Bowman, Gregory R; Bolin, Eric R; Hart, Kathryn M; Maguire, Brendan C; Marqusee, Susan

    2015-03-03

    The discovery of drug-like molecules that bind pockets in proteins that are not present in crystallographic structures yet exert allosteric control over activity has generated great interest in designing pharmaceuticals that exploit allosteric effects. However, there have only been a small number of successes, so the therapeutic potential of these pockets--called hidden allosteric sites--remains unclear. One challenge for assessing their utility is that rational drug design approaches require foreknowledge of the target site, but most hidden allosteric sites are only discovered when a small molecule is found to stabilize them. We present a means of decoupling the identification of hidden allosteric sites from the discovery of drugs that bind them by drawing on new developments in Markov state modeling that provide unprecedented access to microsecond- to millisecond-timescale fluctuations of a protein's structure. Visualizing these fluctuations allows us to identify potential hidden allosteric sites, which we then test via thiol labeling experiments. Application of these methods reveals multiple hidden allosteric sites in an important antibiotic target--TEM-1 β-lactamase. This result supports the hypothesis that there are many as yet undiscovered hidden allosteric sites and suggests our methodology can identify such sites, providing a starting point for future drug design efforts. More generally, our results demonstrate the power of using Markov state models to guide experiments.

  3. Specificity of anion-binding in the substrate-pocket ofbacteriorhodopsin

    SciTech Connect

    Facciotti, Marc T.; Cheung, Vincent S.; Lunde, Christopher S.; Rouhani, Shahab; Baliga, Nitin S.; Glaeser, Robert M.

    2003-08-30

    The structure of the D85S mutant of bacteriorhodopsin with a nitrate anion bound in the Schiff-base binding site, and the structure of the anion-free protein have been obtained in the same crystal form. Together with the previously solved structures of this anion pump, in both the anion-free state and bromide-bound state, these new structures provide insight into how this mutant of bacteriorhodopsin is able to bind a variety of different anions in the same binding pocket. The structural analysis reveals that the main structural change that accommodates different anions is the repositioning of the polar side-chain of S85. On the basis of these x-ray crystal structures, the prediction is then made that the D85S/D212N double mutant might bind similar anions and do so over a broader pH range than does the single mutant. Experimental comparison of the dissociation constants, K{sub d}, for a variety of anions confirms this prediction and demonstrates, in addition, that the binding affinity is dramatically improved by the D212N substitution.

  4. A comprehensive ligand based mapping of the σ₂ receptor binding pocket.

    PubMed

    Rhoades, Derek J; Kinder, David H; Mahfouz, Tarek M

    2014-01-01

    The sigma (σ) receptor system consists of at least two major receptor subtypes: σ₁ and σ₂. Several potential therapeutic applications would benefit from structural knowledge of the σ₂ receptor but gaining this knowledge has been hampered by the difficulties associated with its isolation and, thus, characterization. Here, a ligand based approach has been adopted using the program PHASE® and a group of 41 potent and structurally diverse σ₂ ligands to develop several pharmacophore models for different families of σ₂ ligands. These pharmacophores were analyzed to identify the different binding modes to the receptor and were combined together to construct a comprehensive pharmacophore that was used to develop a structural model for the σ₂ binding pocket. A total of six binding modes were identified and could be classified as neutral or charged modes. The results presented here also indicate the significance of hydrophobic interactions to σ₂ binding and the requirement of hydrogen bonding interactions to increase the affinity for this receptor subtype. This work adds breadth to our knowledge of this receptor's binding site, and should contribute significantly to the development of novel selective σ₂ ligands.

  5. A Charge-inverting Mutation in the “Linker” Region of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Alters Agonist Binding and Gating Kinetics Independently of Allosteric Modulators*

    PubMed Central

    Harms, Jonathan E.; Benveniste, Morris; Kessler, Markus; Stone, Leslie M.; Arai, Amy C.; Partin, Kathryn M.

    2014-01-01

    AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728–4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators. PMID:24550387

  6. A charge-inverting mutation in the "linker" region of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors alters agonist binding and gating kinetics independently of allosteric modulators.

    PubMed

    Harms, Jonathan E; Benveniste, Morris; Kessler, Markus; Stone, Leslie M; Arai, Amy C; Partin, Kathryn M

    2014-04-11

    AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728-4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators.

  7. Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin

    SciTech Connect

    Whittle, James R.R.; Zhang, Ruijun; Khurana, Surender; King, Lisa R.; Manischewitz, Jody; Golding, Hana; Dormitzer, Philip R.; Haynes, Barton F.; Walter, Emmanuel B.; Moody, M. Anthony; Kepler, Thomas B.; Liao, Hua-Xin; Harrison, Stephen C.

    2011-09-20

    Seasonal antigenic drift of circulating influenza virus leads to a requirement for frequent changes in vaccine composition, because exposure or vaccination elicits human antibodies with limited cross-neutralization of drifted strains. We describe a human monoclonal antibody, CH65, obtained by isolating rearranged heavy- and light-chain genes from sorted single plasma cells, coming from a subject immunized with the 2007 trivalent influenza vaccine. The crystal structure of a complex of the hemagglutinin (HA) from H1N1 strain A/Solomon Islands/3/2006 with the Fab of CH65 shows that the tip of the CH65 heavy-chain complementarity determining region 3 (CDR3) inserts into the receptor binding pocket on HA1, mimicking in many respects the interaction of the physiological receptor, sialic acid. CH65 neutralizes infectivity of 30 out of 36 H1N1 strains tested. The resistant strains have a single-residue insertion near the rim of the sialic-acid pocket. We conclude that broad neutralization of influenza virus can be achieved by antibodies with contacts that mimic those of the receptor.

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

    PubMed Central

    2016-01-01

    Allosteric drug development holds promise for delivering medicines that are more selective and less toxic than those that target orthosteric sites. To date, the discovery of allosteric binding sites and lead compounds has been mostly serendipitous, achieved through high-throughput screening. Over the past decade, structural data has become more readily available for larger protein systems and more membrane protein classes (e.g., GPCRs and ion channels), which are common allosteric drug targets. In parallel, improved simulation methods now provide better atomistic understanding of the protein dynamics and cooperative motions that are critical to allosteric mechanisms. As a result of these advances, the field of predictive allosteric drug development is now on the cusp of a new era of rational structure-based computational methods. Here, we review algorithms that predict allosteric sites based on sequence data and molecular dynamics simulations, describe tools that assess the druggability of these pockets, and discuss how Markov state models and topology analyses provide insight into the relationship between protein dynamics and allosteric drug binding. In each section, we first provide an overview of the various method classes before describing relevant algorithms and software packages. PMID:27074285

  9. On the functional role of Arg172 in substrate binding and allosteric transition in Escherichia coli glucosamine-6-phosphate deaminase.

    PubMed

    Lucumí-Moreno, Armando; Calcagno, Mario L

    2005-10-01

    Glucosamine-6-phosphate deaminase from Escherichia coli (EC 3.5.99.6) is an allosteric enzyme, activated by N-acetylglucosamine 6-phosphate, which converts glucosamine-6-phosphate into fructose 6-phosphate and ammonia. X-ray crystallographic structural models have showed that Arg172 and Lys208, together with the segment 41-44 of the main chain backbone, are involved in binding the substrate phospho group when the enzyme is in the R activated state. A set of mutants of the enzyme involving the targeted residues were constructed to analyze the role of Arg172 and Lys208 in deaminase allosteric function. The mutant enzymes were characterized by kinetic, chemical, and spectrometric methods, revealing conspicuous changes in their allosteric properties. The study of these mutants indicated that Arg172 which is located in the highly flexible motif 158-187 forming the active site lid has a specific role in binding the substrate to the enzyme in the T state. The possible role of this interaction in the conformational coupling of the active and the allosteric sites is discussed.

  10. Allosteric Model of Maraviroc Binding to CC Chemokine Receptor 5 (CCR5)*

    PubMed Central

    Garcia-Perez, Javier; Rueda, Patricia; Alcami, Jose; Rognan, Didier; Arenzana-Seisdedos, Fernando; Lagane, Bernard; Kellenberger, Esther

    2011-01-01

    Maraviroc is a nonpeptidic small molecule human immunodeficiency virus type 1 (HIV-1) entry inhibitor that has just entered the therapeutic arsenal for the treatment of patients. We recently demonstrated that maraviroc binding to the HIV-1 coreceptor, CC chemokine receptor 5 (CCR5), prevents it from binding the chemokine CCL3 and the viral envelope glycoprotein gp120 by an allosteric mechanism. However, incomplete knowledge of ligand-binding sites and the lack of CCR5 crystal structures have hampered an in-depth molecular understanding of how the inhibitor works. Here, we addressed these issues by combining site-directed mutagenesis (SDM) with homology modeling and docking. Six crystal structures of G-protein-coupled receptors were compared for their suitability for CCR5 modeling. All CCR5 models had equally good geometry, but that built from the recently reported dimeric structure of the other HIV-1 coreceptor CXCR4 bound to the peptide CVX15 (Protein Data Bank code 3OE0) best agreed with the SDM data and discriminated CCR5 from non-CCR5 binders in a virtual screening approach. SDM and automated docking predicted that maraviroc inserts deeply in CCR5 transmembrane cavity where it can occupy three different binding sites, whereas CCL3 and gp120 lie on distinct yet overlapped regions of the CCR5 extracellular loop 2. Data suggesting that the transmembrane cavity remains accessible for maraviroc in CCL3-bound and gp120-bound CCR5 help explain our previous observation that the inhibitor enhances dissociation of preformed ligand-CCR5 complexes. Finally, we identified residues in the predicted CCR5 dimer interface that are mandatory for gp120 binding, suggesting that receptor dimerization might represent a target for new CCR5 entry inhibitors. PMID:21775441

  11. Structural Basis of the Lactate-dependent Allosteric Regulation of Oxygen Binding in Arthropod Hemocyanin*

    PubMed Central

    Hirota, Shun; Tanaka, Naoki; Mičetić, Ivan; Di Muro, Paolo; Nagao, Satoshi; Kitagishi, Hiroaki; Kano, Koji; Magliozzo, Richard S.; Peisach, Jack; Beltramini, Mariano; Bubacco, Luigi

    2010-01-01

    Hemocyanin (Hc) is an oxygen carrier protein in which oxygen binding is regulated by allosteric effectors such as H+ and l-lactate. Isothermal titration calorimetric measurements showed that l-lactate binds to dodecameric and heterohexameric Hc and to the CaeSS3 homohexamer but not to the CaeSS2 monomer. The binding of lactate caused no change in the optical absorption and x-ray absorption spectra of either oxy- or deoxy-Hc, suggesting that no structural rearrangement of the active site occurred. At pH 6.5, the oxygen binding rate constant kobs obtained by flash photolysis showed a significant increase upon addition of l-lactate, whereas l-lactate addition had little effect at pH 8.3. Lactate binding caused a concentration-dependent shift in the interhexameric distances at pH 6.5 based on small angle x-ray scattering measurements. These results show that l-lactate affects oxygen affinity at pH 6.5 by modulating the global structure of Hc without affecting its binuclear copper center (the active site). In contrast to this, the active site structure of deoxy-Hc is affected by changes in pH (Hirota, S., Kawahara, T., Beltramini, M., Di Muro, P., Magliozzo, R. S., Peisach, J., Powers, L. S., Tanaka, N., Nagao, S., and Bubacco, L. (2008) J. Biol. Chem. 283, 31941–31948). Upon addiction of lactate, the kinetic behavior of oxygen rebinding for Hc was heterogeneous under low oxygen concentrations at pH 6.5 due to changes in the T and R state populations, and the equilibrium was found to shift from the T toward the R state with addition of lactate. PMID:20406810

  12. Structural Basis of the Lactate-dependent Allosteric Regulation of Oxygen Binding in Arthropod Hemocyanin

    SciTech Connect

    Hirota, S.; Tanaka, N; Micetic, I; Di Muro, P; Nagao, S; Kitagishi, H; Magliozzo, R; Peisach, J; Beltramini, M; Bubacco, L

    2010-01-01

    Hemocyanin (Hc) is an oxygen carrier protein in which oxygen binding is regulated by allosteric effectors such as H{sup +} and L-lactate. Isothermal titration calorimetric measurements showed that L-lactate binds to dodecameric and heterohexameric Hc and to the CaeSS3 homohexamer but not to the CaeSS2 monomer. The binding of lactate caused no change in the optical absorption and x-ray absorption spectra of either oxy- or deoxy-Hc, suggesting that no structural rearrangement of the active site occurred. At pH 6.5, the oxygen binding rate constant k{sub obs} obtained by flash photolysis showed a significant increase upon addition of L-lactate, whereas L-lactate addition had little effect at pH 8.3. Lactate binding caused a concentration-dependent shift in the interhexameric distances at pH 6.5 based on small angle x-ray scattering measurements. These results show that L-lactate affects oxygen affinity at pH 6.5 by modulating the global structure of Hc without affecting its binuclear copper center (the active site). In contrast to this, the active site structure of deoxy-Hc is affected by changes in pH (Hirota, S., Kawahara, T., Beltramini, M., Di Muro, P., Magliozzo, R. S., Peisach, J., Powers, L. S., Tanaka, N., Nagao, S., and Bubacco, L. (2008) J. Biol. Chem. 283, 31941-31948). Upon addiction of lactate, the kinetic behavior of oxygen rebinding for Hc was heterogeneous under low oxygen concentrations at pH 6.5 due to changes in the T and R state populations, and the equilibrium was found to shift from the T toward the R state with addition of lactate.

  13. Insight into the structural mechanism for PKBα allosteric inhibition by molecular dynamics simulations and free energy calculations.

    PubMed

    Chen, Shi-Feng; Cao, Yang; Han, Shuang; Chen, Jian-Zhong

    2014-03-01

    Protein kinase B (PKB/Akt) is an attractive target for the treatment of tumor. Unlike PKB's ATP-competitive inhibitors, its allosteric inhibitors can maintain PKB's inactive state via its binding in a pocket between PH domain and kinase domain, which specifically inhibit PKB by preventing the phosphorylations of Thr308 and Ser473. In the present studies, MD simulations were performed on three allosteric inhibitors with different inhibitory potencies (IC50) to investigate the interaction modes between the inhibitors and PKBα. MM/GB(PB)SA were further applied to calculate the binding free energies of these inhibitors binding to PKBα. The computed binding free energies were consistent with the ranking of their experimental bioactivities. The key residues of PKBα interacting with the allosteric inhibitor were further discussed by analyzing the different interaction modes of these three inhibitors binding to PKBα and by calculating binding free energy contributions of corresponding residues around the binding pocket. The structural requirements were then summarized for the allosteric inhibitor binding to PKBα. A possible structural mechanism of PKBα inhibition induced by the binding of allosteric inhibitor was formulated. The current studies indicate that there should be an optimum balance between the van der Waals and total electrostatic interactions for further designing of PKBα allosteric inhibitors.

  14. Allosteric communication between DNA-binding and light-responsive domains of diatom class I aureochromes

    PubMed Central

    Banerjee, Ankan; Herman, Elena; Serif, Manuel; Maestre-Reyna, Manuel; Hepp, Sebastian; Pokorny, Richard; Kroth, Peter G.; Essen, Lars-Oliver; Kottke, Tilman

    2016-01-01

    The modular architecture of aureochrome blue light receptors, found in several algal groups including diatoms, is unique by having the LOV-type photoreceptor domain fused to the C-terminus of its putative effector, an N-terminal DNA-binding bZIP module. The structural and functional understanding of aureochromes’ light-dependent signaling mechanism is limited, despite their promise as an optogenetic tool. We show that class I aureochromes 1a and 1c from the diatom Phaeodactylum tricornutum are regulated in a light-independent circadian rhythm. These aureochromes are capable to form functional homo- and heterodimers, which recognize the ACGT core sequence within the canonical ‘aureo box’, TGACGT, in a light-independent manner. The bZIP domain holds a more folded and less flexible but extended conformation in the duplex DNA-bound state. FT-IR spectroscopy in the absence and the presence of DNA shows light-dependent helix unfolding in the LOV domain, which leads to conformational changes in the bZIP region. The solution structure of DNA bound to aureochrome points to a tilted orientation that was further validated by molecular dynamics simulations. We propose that aureochrome signaling relies on an allosteric pathway from LOV to bZIP that results in conformational changes near the bZIP-DNA interface without major effects on the binding affinity. PMID:27179025

  15. Allosteric inhibition of a stem cell RNA-binding protein by an intermediary metabolite

    PubMed Central

    Clingman, Carina C; Deveau, Laura M; Hay, Samantha A; Genga, Ryan M; Shandilya, Shivender MD; Massi, Francesca; Ryder, Sean P

    2014-01-01

    Gene expression and metabolism are coupled at numerous levels. Cells must sense and respond to nutrients in their environment, and specialized cells must synthesize metabolic products required for their function. Pluripotent stem cells have the ability to differentiate into a wide variety of specialized cells. How metabolic state contributes to stem cell differentiation is not understood. In this study, we show that RNA-binding by the stem cell translation regulator Musashi-1 (MSI1) is allosterically inhibited by 18–22 carbon ω-9 monounsaturated fatty acids. The fatty acid binds to the N-terminal RNA Recognition Motif (RRM) and induces a conformational change that prevents RNA association. Musashi proteins are critical for development of the brain, blood, and epithelium. We identify stearoyl-CoA desaturase-1 as a MSI1 target, revealing a feedback loop between ω-9 fatty acid biosynthesis and MSI1 activity. We propose that other RRM proteins could act as metabolite sensors to couple gene expression changes to physiological state. DOI: http://dx.doi.org/10.7554/eLife.02848.001 PMID:24935936

  16. Mutations in adenine-binding pockets enhance catalytic properties of NAD(P)H-dependent enzymes

    PubMed Central

    Cahn, J.K.B.; Baumschlager, A.; Brinkmann-Chen, S.; Arnold, F.H.

    2016-01-01

    NAD(P)H-dependent enzymes are ubiquitous in metabolism and cellular processes and are also of great interest for pharmaceutical and industrial applications. Here, we present a structure-guided enzyme engineering strategy for improving catalytic properties of NAD(P)H-dependent enzymes toward native or native-like reactions using mutations to the enzyme's adenine-binding pocket, distal to the site of catalysis. Screening single-site saturation mutagenesis libraries identified mutations that increased catalytic efficiency up to 10-fold in 7 out of 10 enzymes. The enzymes improved in this study represent three different cofactor-binding folds (Rossmann, DHQS-like, and FAD/NAD binding) and utilize both NADH and NADPH. Structural and biochemical analyses show that the improved activities are accompanied by minimal changes in other properties (cooperativity, thermostability, pH optimum, uncoupling), and initial tests on two enzymes (ScADH6 and EcFucO) show improved functionality in Escherichia coli. PMID:26512129

  17. The Second Extracellular Loop of the Adenosine A1 Receptor Mediates Activity of Allosteric Enhancers

    PubMed Central

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

    2014-01-01

    Allosteric enhancers of the adenosine A1 receptor amplify signaling by orthosteric agonists. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied by an agonist, thereby conferring specificity to stressed or injured tissues that produce adenosine. To explore the mechanism of allosteric enhancer activity, we examined their action on several A1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants, and (4) site-specific mutants. These findings were combined with homology modeling of the A1 receptor and in silico screening of an allosteric enhancer library. The binding modes of known docked allosteric enhancers correlated with the known structure-activity relationship, suggesting that these allosteric enhancers bind to a pocket formed by the second extracellular loop, flanked by residues S150 and M162. We propose a model in which this vestibule controls the entry and efflux of agonists from the orthosteric site and agonist binding elicits a conformational change that enables allosteric enhancer binding. This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists. PMID:24217444

  18. Allosteric FBPase inhibitors gain 10(5) times in potency when simultaneously binding two neighboring AMP sites.

    PubMed

    Hebeisen, Paul; Kuhn, Bernd; Kohler, Philipp; Gubler, Marcel; Huber, Walter; Kitas, Eric; Schott, Brigitte; Benz, Jörg; Joseph, Catherine; Ruf, Armin

    2008-08-15

    Human fructose-1,6-bisphosphatase (FBPase, EC 3.1.3.11) is a key gluconeogenic enzyme, responsible for the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate, and thus presents an opportunity for the development of novel therapeutics focused on lowering the hepatic glucose production in type 2 diabetics. In its active form FBPase exists as a homotetramer and is allosterically regulated by AMP. In an HTS campaign aromatic sulfonylureas have been identified as FBPase inhibitors mimicking AMP. By bridging two adjacent allosteric binding sites using two aromatic sulfonylureas as anchor units and covalently linking them, it was possible to obtain dual binding AMP site inhibitors that exhibit a strong inhibitory effect.

  19. Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding Pocket.

    PubMed

    Forster, Michael; Chaikuad, Apirat; Bauer, Silke M; Holstein, Julia; Robers, Matthew B; Corona, Cesear R; Gehringer, Matthias; Pfaffenrot, Ellen; Ghoreschi, Kamran; Knapp, Stefan; Laufer, Stefan A

    2016-11-17

    Janus kinases (JAKs) are a family of cytoplasmatic tyrosine kinases that are attractive targets for the development of anti-inflammatory drugs given their roles in cytokine signaling. One question regarding JAKs and their inhibitors that remains under intensive debate is whether JAK inhibitors should be isoform selective. Since JAK3 functions are restricted to immune cells, an isoform-selective inhibitor for JAK3 could be especially valuable to achieve clinically more useful and precise effects. However, the high degree of structural conservation makes isoform-selective targeting a challenging task. Here, we present picomolar inhibitors with unprecedented kinome-wide selectivity for JAK3. Selectivity was achieved by concurrent covalent reversible targeting of a JAK3-specific cysteine residue and a ligand-induced binding pocket. We confirmed that in vitro activity and selectivity translate well into the cellular environment and suggest that our inhibitors are powerful tools to elucidate JAK3-specific functions.

  20. How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function.

    PubMed

    Otero, Lisandro H; Rojas-Altuve, Alzoray; Llarrull, Leticia I; Carrasco-López, Cesar; Kumarasiri, Malika; Lastochkin, Elena; Fishovitz, Jennifer; Dawley, Matthew; Hesek, Dusan; Lee, Mijoon; Johnson, Jarrod W; Fisher, Jed F; Chang, Mayland; Mobashery, Shahriar; Hermoso, Juan A

    2013-10-15

    The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the DD-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain--a remarkable 60 Å distant from the DD-transpeptidase active site--discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design.

  1. How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function

    PubMed Central

    Otero, Lisandro H.; Rojas-Altuve, Alzoray; Llarrull, Leticia I.; Carrasco-López, Cesar; Kumarasiri, Malika; Lastochkin, Elena; Fishovitz, Jennifer; Dawley, Matthew; Hesek, Dusan; Lee, Mijoon; Johnson, Jarrod W.; Fisher, Jed F.; Chang, Mayland; Mobashery, Shahriar; Hermoso, Juan A.

    2013-01-01

    The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the dd-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain—a remarkable 60 Å distant from the dd-transpeptidase active site—discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design. PMID:24085846

  2. An Allosteric Pathway Revealed in the Ribosome Binding Stress Factor BipA

    SciTech Connect

    Makanji, H.; deLivron, M; Robinson, V

    2009-01-01

    BipA is a highly conserved prokaryotic GTPase that functions as a master regulator of stress and virulence processes in bacteria. It is a member of the translational factor family of GTPases along with EF-G, IF-2 and LepA. Structural and biochemical data suggest that ribosome binding specificity for each member of this family lies in an effector domain. As with other bacterial GTPases, the ribosome binding and GTPase activities of this protein are tightly coupled. However, the mechanism by which this occurs is still unknown. A series of experiments have been designed to probe structural features of the protein to see if we can pinpoint specific areas of BipA, perhaps even individual residues, which are important to its association with the ribosome. Included in the list are the C-terminal effector domain of the protein, which is distinct to the BipA family of proteins, and amino acid residues in the switch I and II regions of the G domain. Using sucrose density gradients, we have shown that the C-terminal domain is required in order for BipA to bind to the ribosome. Moreover, deletion of this domain increases the GTP hydrolysis rates of the protein, likely through relief of inhibitory contacts. Additional evidence has revealed an allosteric connection between the conformationally flexible switch II region and the C-terminal domain of BipA. Site directed mutagenesis, sucrose gradients and malachite green assays are being used to elucidate the details of this coupling.

  3. Cytochrome c Can Form a Well-Defined Binding Pocket for Hydrocarbons.

    PubMed

    McClelland, Levi J; Steele, Harmen B B; Whitby, Frank G; Mou, Tung-Chung; Holley, David; Ross, J B Alexander; Sprang, Stephen R; Bowler, Bruce E

    2016-12-28

    Cytochrome c can acquire peroxidase activity when it binds to cardiolipin in mitochondrial membranes. The resulting oxygenation of cardiolipin by cytochrome c provides an early signal for the onset of apoptosis. The structure of this enzyme-substrate complex is a matter of considerable debate. We present three structures at 1.7-2.0 Å resolution of a domain-swapped dimer of yeast iso-1-cytochrome c with the detergents, CYMAL-5, CYMAL-6, and ω-undecylenyl-β-d-maltopyranoside, bound in a channel that places the hydrocarbon moieties of these detergents next to the heme. The heme is poised for peroxidase activity with water bound in place of Met80, which serves as the axial heme ligand when cytochrome c functions as an electron carrier. The hydroxyl group of Tyr67 sits 3.6-4.0 Å from the nearest carbon of the detergents, positioned to act as a relay in radical abstraction during peroxidase activity. Docking studies with linoleic acid, the most common fatty acid component of cardiolipin, show that C11 of linoleic acid can sit adjacent to Tyr67 and the heme, consistent with the oxygenation pattern observed in lipidomics studies. The well-defined hydrocarbon binding pocket provides atomic resolution evidence for the extended lipid anchorage model for cytochrome c/cardiolipin binding. Dimer dissociation/association kinetics for yeast versus equine cytochrome c indicate that formation of mammalian cytochrome c dimers in vivo would require catalysis. However, the dimer structure shows that only a modest deformation of monomeric cytochrome c would suffice to form the hydrocarbon binding site occupied by these detergents.

  4. Evolutionary diversification of retinoic acid receptor ligand-binding pocket structure by molecular tinkering

    PubMed Central

    Gutierrez-Mazariegos, Juliana; Nadendla, Eswar Kumar; Studer, Romain A.; Alvarez, Susana; de Lera, Angel R.; Kuraku, Shigehiro; Bourguet, William; Laudet, Vincent

    2016-01-01

    Whole genome duplications (WGDs) have been classically associated with the origin of evolutionary novelties and the so-called duplication–degeneration–complementation model describes the possible fates of genes after duplication. However, how sequence divergence effectively allows functional changes between gene duplicates is still unclear. In the vertebrate lineage, two rounds of WGDs took place, giving rise to paralogous gene copies observed for many gene families. For the retinoic acid receptors (RARs), for example, which are members of the nuclear hormone receptor (NR) superfamily, a unique ancestral gene has been duplicated resulting in three vertebrate paralogues: RARα, RARβ and RARγ. It has previously been shown that this single ancestral RAR was neofunctionalized to give rise to a larger substrate specificity range in the RARs of extant jawed vertebrates (also called gnathostomes). To understand RAR diversification, the members of the cyclostomes (lamprey and hagfish), jawless vertebrates representing the extant sister group of gnathostomes, provide an intermediate situation and thus allow the characterization of the evolutionary steps that shaped RAR ligand-binding properties following the WGDs. In this study, we assessed the ligand-binding specificity of cyclostome RARs and found that their ligand-binding pockets resemble those of gnathostome RARα and RARβ. In contrast, none of the cyclostome receptors studied showed any RARγ-like specificity. Together, our results suggest that cyclostome RARs cover only a portion of the specificity repertoire of the ancestral gnathostome RARs and indicate that the establishment of ligand-binding specificity was a stepwise event. This iterative process thus provides a rare example for the diversification of receptor–ligand interactions of NRs following WGDs. PMID:27069642

  5. Allosteric Coupling between the Intracellular Coupling Helix 4 and Regulatory Sites of the First Nucleotide-binding Domain of CFTR

    PubMed Central

    Dawson, Jennifer E.; Farber, Patrick J.; Forman-Kay, Julie D.

    2013-01-01

    Cystic fibrosis is caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator), leading to folding and processing defects and to chloride channel gating misfunction. CFTR is regulated by ATP binding to its cytoplasmic nucleotide-binding domains, NBD1 and NBD2, and by phosphorylation of the NBD1 regulatory insert (RI) and the regulatory extension (RE)/R region. These regulatory effects are transmitted to the rest of the channel via NBD interactions with intracellular domain coupling helices (CL), particularly CL4. Using a sensitive method for detecting inter-residue correlations between chemical shift changes in NMR spectra, an allosteric network was revealed within NBD1, with a construct lacking RI. The CL4-binding site couples to the RI-deletion site and the C-terminal residues of NBD1 that precede the R region in full-length CFTR. Titration of CL4 peptide into NBD1 perturbs the conformational ensemble in these sites with similar titration patterns observed in F508del, the major CF-causing mutant, and in suppressor mutants F494N, V510D and Q637R NBD1, as well as in a CL4-NBD1 fusion construct. Reciprocally, the C-terminal mutation, Q637R, perturbs dynamics in these three sites. This allosteric network suggests a mechanism synthesizing diverse regulatory NBD1 interactions and provides biophysical evidence for the allosteric coupling required for CFTR function. PMID:24058550

  6. Identification of the putative binding pocket of valerenic acid on GABAA receptors using docking studies and site‐directed mutagenesis

    PubMed Central

    Luger, D; Poli, G; Wieder, M; Stadler, M; Ke, S; Ernst, M; Hohaus, A; Linder, T; Seidel, T; Langer, T; Hering, S

    2015-01-01

    Background and Purpose β2/3‐subunit‐selective modulation of GABAA receptors by valerenic acid (VA) is determined by the presence of transmembrane residue β2/3N265. Currently, it is not known whether β2/3N265 is part of VA's binding pocket or is involved in the transduction pathway of VA's action. The aim of this study was to clarify the localization of VA's binding pocket on GABAA receptors. Experimental Approach Docking and a structure‐based three‐dimensional pharmacophore were employed to identify candidate amino acid residues that are likely to interact with VA. Selected amino acid residues were mutated, and VA‐induced modulation of the resulting GABAA receptors expressed in Xenopus oocytes was analysed. Key Results A binding pocket for VA at the β+/α− interface encompassing amino acid β3N265 was predicted. Mutational analysis of suggested amino acid residues revealed a complete loss of VA's activity on β3M286W channels as well as significantly decreased efficacy and potency of VA on β3N265S and β3F289S receptors. In addition, reduced efficacy of VA‐induced I GABA enhancement was also observed for α1M235W, β3R269A and β3M286A constructs. Conclusions and Implications Our data suggest that amino acid residues β3N265, β3F289, β3M286, β3R269 in the β3 subunit, at or near the etomidate/propofol binding site(s), form part of a VA binding pocket. The identification of the binding pocket for VA is essential for elucidating its pharmacological effects and might also help to develop new selective GABAA receptor ligands. PMID:26375408

  7. Angiotensin type 1 receptor resistance to blockade in the opossum proximal tubule cell due to variations in the binding pocket.

    PubMed

    Nistala, Ravi; Andresen, Bradley T; Pulakat, Lakshmi; Meuth, Alex; Sinak, Catherine; Mandavia, Chirag; Thekkumkara, Thomas; Speth, Robert C; Whaley-Connell, Adam; Sowers, James R

    2013-04-15

    Blockade of the angiotensin (ANG) II receptor type 1 (AT(1)R) with angiotensin receptor blockers (ARBs) is widely used in the treatment of hypertension. However, ARBs are variably effective in reducing blood pressure, likely due, in part, to polymorphisms in the ARB binding pocket of the AT(1)R. Therefore, we need a better understanding of variations/polymorphisms that alter binding of ARBs in heterogeneous patient populations. The opossum proximal tubule cell (OKP) line is commonly used in research to evaluate renal sodium handling and therefore blood pressure. Investigating this issue, we found natural sequence variations in the opossum AT(1)R paralleling those observed in the human AT(1)R. Therefore, we posited that these sequence variations may explain ARB resistance. We demonstrate that OKP cells express AT(1)R mRNA, bind (125)I-labeled ANG II, and exhibit ANG II-induced phosphorylation of Jak2. However, Jak2 phosphorylation is not inhibited by five different ARBs commonly used to treat hypertension. Additionally, nonradioactive ANG II competes (125)I-ANG II efficiently, whereas a 10-fold molar excess of olmesartan and the ANG II receptor type 2 blocker PD-123319 is unable to block (125)I-ANG II binding. In contrast, ANG II binding to OKP cells stably expressing rat AT(1A)Rs, which have a conserved AT(1)R-binding pocket with human AT(1)R, is efficiently inhibited by olmesartan. A novel observation was that resistance to ARB binding to opossum AT(1)Rs correlates with variations from the human receptor at positions 108, 163, 192, and 198 within the ARB-binding pocket. These observations highlight the potential utility of evaluating AT(1)R polymorphisms within the ARB-binding pocket in various hypertensive populations.

  8. Novel selective allosteric and bitopic ligands for the S1P(3) receptor.

    PubMed

    Jo, Euijung; Bhhatarai, Barun; Repetto, Emanuela; Guerrero, Miguel; Riley, Sean; Brown, Steven J; Kohno, Yasushi; Roberts, Edward; Schürer, Stephan C; Rosen, Hugh

    2012-12-21

    Sphingosine 1-phosphate (S1P) is a lysophospholipid signaling molecule that regulates important biological functions, including lymphocyte trafficking and vascular development, by activating G protein-coupled receptors for S1P, namely, S1P(1) through S1P(5). Here, we map the S1P(3) binding pocket with a novel allosteric agonist (CYM-5541), an orthosteric agonist (S1P), and a novel bitopic antagonist (SPM-242). With a combination of site-directed mutagenesis, ligand competition assay, and molecular modeling, we concluded that S1P and CYM-5541 occupy different chemical spaces in the ligand binding pocket of S1P(3). CYM-5541 allowed us to identify an allosteric site where Phe263 is a key gate-keeper residue for its affinity and efficacy. This ligand lacks a polar moiety, and the novel allosteric hydrophobic pocket permits S1P(3) selectivity of CYM-5541 within the highly similar S1P receptor family. However, a novel S1P(3)-selective antagonist, SPM-242, in the S1P(3) pocket occupies the ligand binding spaces of both S1P and CYM-5541, showing its bitopic mode of binding. Therefore, our coordinated approach with biochemical data and molecular modeling, based on our recently published S1P(1) crystal structure data in a highly conserved set of related receptors with a shared ligand, provides a strong basis for the successful optimization of orthosteric, allosteric, and bitopic modulators of S1P(3).

  9. Auxin-binding pocket of ABP1 is crucial for its gain-of-function cellular and developmental roles.

    PubMed

    Grones, Peter; Chen, Xu; Simon, Sibu; Kaufmann, Walter A; De Rycke, Riet; Nodzyński, Tomasz; Zažímalová, Eva; Friml, Jiří

    2015-08-01

    The plant hormone auxin is a key regulator of plant growth and development. Auxin levels are sensed and interpreted by distinct receptor systems that activate a broad range of cellular responses. The Auxin-Binding Protein1 (ABP1) that has been identified based on its ability to bind auxin with high affinity is a prime candidate for the extracellular receptor responsible for mediating a range of auxin effects, in particular, the fast non-transcriptional ones. Contradictory genetic studies suggested prominent or no importance of ABP1 in many developmental processes. However, how crucial the role of auxin binding to ABP1 is for its functions has not been addressed. Here, we show that the auxin-binding pocket of ABP1 is essential for its gain-of-function cellular and developmental roles. In total, 16 different abp1 mutants were prepared that possessed substitutions in the metal core or in the hydrophobic amino acids of the auxin-binding pocket as well as neutral mutations. Their analysis revealed that an intact auxin-binding pocket is a prerequisite for ABP1 to activate downstream components of the ABP1 signalling pathway, such as Rho of Plants (ROPs) and to mediate the clathrin association with membranes for endocytosis regulation. In planta analyses demonstrated the importance of the auxin binding pocket for all known ABP1-mediated postembryonic developmental processes, including morphology of leaf epidermal cells, root growth and root meristem activity, and vascular tissue differentiation. Taken together, these findings suggest that auxin binding to ABP1 is central to its function, supporting the role of ABP1 as auxin receptor.

  10. Allosteric Regulation in the Ligand Binding Domain of Retinoic Acid Receptorγ

    PubMed Central

    Amal, Ismail; Lutzing, Régis; Stote, Roland H.; Rochette-Egly, Cécile; Rochel, Natacha; Dejaegere, Annick

    2017-01-01

    Retinoic acid (RA) plays key roles in cell differentiation and growth arrest through nuclear retinoic acid receptors (RARs), which are ligand-dependent transcription factors. While the main trigger of RAR activation is the binding of RA, phosphorylation of the receptors has also emerged as an important regulatory signal. Phosphorylation of the RARγ N-terminal domain (NTD) is known to play a functional role in neuronal differentiation. In this work, we investigated the phosphorylation of RARγ ligand binding domain (LBD), and present evidence that the phosphorylation status of the LBD affects the phosphorylation of the NTD region. We solved the X-ray structure of a phospho-mimetic mutant of the LBD (RARγ S371E), which we used in molecular dynamics simulations to characterize the consequences of the S371E mutation on the RARγ structural dynamics. Combined with simulations of the wild-type LBD, we show that the conformational equilibria of LBD salt bridges (notably R387-D340) are affected by the S371E mutation, which likely affects the recruitment of the kinase complex that phosphorylates the NTD. The molecular dynamics simulations also showed that a conservative mutation in this salt bridge (R387K) affects the dynamics of the LBD without inducing large conformational changes. Finally, cellular assays showed that the phosphorylation of the NTD of RARγ is differentially regulated by retinoic acid in RARγWT and in the S371N, S371E and R387K mutants. This multidisciplinary work highlights an allosteric coupling between phosphorylations of the LBD and the NTD of RARγ and supports the importance of structural dynamics involving electrostatic interactions in the regulation of RARs activity. PMID:28125680

  11. Discovery of Allosteric Modulators of Factor XIa by Targeting Hydrophobic Domains Adjacent to its Heparin-Binding Site

    PubMed Central

    Karuturi, Rajesh; Al-Horani, Rami A.; Mehta, Shrenik C.; Gailani, David; Desai, Umesh R.

    2013-01-01

    To discover promising sulfated allosteric modulators (SAMs) of glycosaminoglycan-binding proteins (GBPs), such as human factor XIa (FXIa), we screened a library of 26 synthetic, sulfated quinazolin-4(3H)-ones (QAOs) resulting in the identification of six molecules that reduced the VMAX of substrate hydrolysis without influencing the KM. Mutagenesis of residues of the heparin-binding site of FXIa introduced a nearly 5-fold loss in inhibition potency supporting recognition of an allosteric site. Fluorescence studies showed a sigmoidal binding profile indicating highly cooperative binding. Competition with a positively-charged, heparin-binding polymer did not fully nullify inhibition suggesting importance of hydrophobic forces to binding. This discovery suggest the operation of a dual-element recognition process, which relies on an initial Coulombic attraction of anionic SAMs to the cationic HBS of FXIa that forms a locked complex through tight interaction with an adjacent hydrophobic patch. The dual-element strategy may be widely applicable for discovering SAMs of other GBPs. PMID:23451707

  12. The structure of apo ArnA features an unexpected central binding pocket and provides an explanation for enzymatic cooperativity.

    PubMed

    Fischer, Utz; Hertlein, Simon; Grimm, Clemens

    2015-03-01

    The bacterial protein ArnA is an essential enzyme in the pathway leading to the modification of lipid A with the pentose sugar 4-amino-4-deoxy-L-arabinose. This modification confers resistance to polymyxins, which are antibiotics that are used as a last resort to treat infections with multiple drug-resistant Gram-negative bacteria. ArnA contains two domains with distinct catalytic functions: a dehydrogenase domain and a transformylase domain. The protein forms homohexamers organized as a dimer of trimers. Here, the crystal structure of apo ArnA is presented and compared with its ATP- and UDP-glucuronic acid-bound counterparts. The comparison reveals major structural rearrangements in the dehydrogenase domain that lead to the formation of a previously unobserved binding pocket at the centre of each ArnA trimer in its apo state. In the crystal structure, this pocket is occupied by a DTT molecule. It is shown that formation of the pocket is linked to a cascade of structural rearrangements that emerge from the NAD(+)-binding site. Based on these findings, a small effector molecule is postulated that binds to the central pocket and modulates the catalytic properties of ArnA. Furthermore, the discovered conformational changes provide a mechanistic explanation for the strong cooperative effect recently reported for the ArnA dehydrogenase function.

  13. Ionic contacts at DnaK substrate binding domain involved in the allosteric regulation of lid dynamics.

    PubMed

    Fernández-Sáiz, Vanesa; Moro, Fernando; Arizmendi, Jesus M; Acebrón, Sergio P; Muga, Arturo

    2006-03-17

    To gain further insight into the interactions involved in the allosteric transition of DnaK we have characterized wild-type (wt) protein and three mutants in which ionic interactions at the interface between the two subdomains of the substrate binding domain, and within the lid subdomain have been disrupted. Our data show that ionic contacts, most likely forming an electrically charged network, between the N-terminal region of helix B and an inner loop of the beta-sandwich are involved in maintaining the position of the lid relative to the beta-subdomain in the ADP state but not in the ATP state of the protein. Disruption of the ionic interactions between the C-terminal region of helix B and the outer loops of the beta-sandwich, known as the latch, does not have the same conformational consequences but results equally in an inactive protein. This indicates that a variety of mechanisms can inactivate this complex allosteric machine. Our results identify the ionic contacts at the subdomain and interdomain interfaces that are part of the hinge region involved in the ATP-induced allosteric displacement of the lid away from the peptide binding site. These interactions also stabilize peptide-Hsp70 complexes at physiological (37 degrees C) and stress (42 degrees C) temperatures, a requirement for productive substrate (re)folding.

  14. Structure-guided Development of Specific Pyruvate Dehydrogenase Kinase Inhibitors Targeting the ATP-binding Pocket*

    PubMed Central

    Tso, Shih-Chia; Qi, Xiangbing; Gui, Wen-Jun; Wu, Cheng-Yang; Chuang, Jacinta L.; Wernstedt-Asterholm, Ingrid; Morlock, Lorraine K.; Owens, Kyle R.; Scherer, Philipp E.; Williams, Noelle S.; Tambar, Uttam K.; Wynn, R. Max; Chuang, David T.

    2014-01-01

    Pyruvate dehydrogenase kinase isoforms (PDKs 1–4) negatively regulate activity of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation. PDK isoforms are up-regulated in obesity, diabetes, heart failure, and cancer and are potential therapeutic targets for these important human diseases. Here, we employed a structure-guided design to convert a known Hsp90 inhibitor to a series of highly specific PDK inhibitors, based on structural conservation in the ATP-binding pocket. The key step involved the substitution of a carbonyl group in the parent compound with a sulfonyl in the PDK inhibitors. The final compound of this series, 2-[(2,4-dihydroxyphenyl)sulfonyl]isoindoline-4,6-diol, designated PS10, inhibits all four PDK isoforms with IC50 = 0.8 μm for PDK2. The administration of PS10 (70 mg/kg) to diet-induced obese mice significantly augments pyruvate dehydrogenase complex activity with reduced phosphorylation in different tissues. Prolonged PS10 treatments result in improved glucose tolerance and notably lessened hepatic steatosis in the mouse model. The results support the pharmacological approach of targeting PDK to control both glucose and fat levels in obesity and type 2 diabetes. PMID:24356970

  15. Structures of BmrR-Drug Complexes Reveal a Rigid Multidrug Binding Pocket And Transcription Activation Through Tyrosine Expulsion

    SciTech Connect

    Newberry, K.J.; Huffman, J.L.; Miller, M.C.; Vazquez-Laslop, N.; Neyfakh, A.A.; Brennan, R.G.

    2009-05-22

    BmrR is a member of the MerR family and a multidrug binding transcription factor that up-regulates the expression of the bmr multidrug efflux transporter gene in response to myriad lipophilic cationic compounds. The structural mechanism by which BmrR binds these chemically and structurally different drugs and subsequently activates transcription is poorly understood. Here, we describe the crystal structures of BmrR bound to rhodamine 6G (R6G) or berberine (Ber) and cognate DNA. These structures reveal each drug stacks against multiple aromatic residues with their positive charges most proximal to the carboxylate group of Glu-253 and that, unlike other multidrug binding pockets, that of BmrR is rigid. Substitution of Glu-253 with either alanine (E253A) or glutamine (E253Q) results in unpredictable binding affinities for R6G, Ber, and tetraphenylphosphonium. Moreover, these drug binding studies reveal that the negative charge of Glu-253 is not important for high affinity binding to Ber and tetraphenylphosphonium but plays a more significant, but unpredictable, role in R6G binding. In vitro transcription data show that E253A and E253Q are constitutively active, and structures of the drug-free E253A-DNA and E253Q-DNA complexes support a transcription activation mechanism requiring the expulsion of Tyr-152 from the multidrug binding pocket. In sum, these data delineate the mechanism by which BmrR binds lipophilic, monovalent cationic compounds and suggest the importance of the redundant negative electrostatic nature of this rigid drug binding pocket that can be used to discriminate against molecules that are not substrates of the Bmr multidrug efflux pump.

  16. Structures of BmrR-drug complexes reveal a rigid multidrug binding pocket and transcription activation through tyrosine expulsion.

    PubMed

    Newberry, Kate J; Huffman, Joy L; Miller, Marshall C; Vazquez-Laslop, Nora; Neyfakh, Alex A; Brennan, Richard G

    2008-09-26

    BmrR is a member of the MerR family and a multidrug binding transcription factor that up-regulates the expression of the bmr multidrug efflux transporter gene in response to myriad lipophilic cationic compounds. The structural mechanism by which BmrR binds these chemically and structurally different drugs and subsequently activates transcription is poorly understood. Here, we describe the crystal structures of BmrR bound to rhodamine 6G (R6G) or berberine (Ber) and cognate DNA. These structures reveal each drug stacks against multiple aromatic residues with their positive charges most proximal to the carboxylate group of Glu-253 and that, unlike other multidrug binding pockets, that of BmrR is rigid. Substitution of Glu-253 with either alanine (E253A) or glutamine (E253Q) results in unpredictable binding affinities for R6G, Ber, and tetraphenylphosphonium. Moreover, these drug binding studies reveal that the negative charge of Glu-253 is not important for high affinity binding to Ber and tetraphenylphosphonium but plays a more significant, but unpredictable, role in R6G binding. In vitro transcription data show that E253A and E253Q are constitutively active, and structures of the drug-free E253A-DNA and E253Q-DNA complexes support a transcription activation mechanism requiring the expulsion of Tyr-152 from the multidrug binding pocket. In sum, these data delineate the mechanism by which BmrR binds lipophilic, monovalent cationic compounds and suggest the importance of the redundant negative electrostatic nature of this rigid drug binding pocket that can be used to discriminate against molecules that are not substrates of the Bmr multidrug efflux pump.

  17. A Characteristic Back Support Structure in the Bisphenol A-Binding Pocket in the Human Nuclear Receptor ERRγ

    PubMed Central

    Liu, Xiaohui; Matsushima, Ayami; Shimohigashi, Miki; Shimohigashi, Yasuyuki

    2014-01-01

    The endocrine disruptor bisphenol A (BPA) affects various genes and hormones even at merely physiological levels. We recently demonstrated that BPA binds strongly to human nuclear receptor estrogen-related receptor (ERR) γ and that the phenol-A group of BPA is in a receptacle pocket with essential amino acid residues to provide structural support at the backside. This led BPA to bind to ERRγ in an induced-fit-type binding mode, for example, with a rotated motion of Val313 to support the Tyr326-binding site. A similar binding mechanism appears to occur at the binding site of the BPA phenol-B ring. X-ray crystal analysis of the ERRγ-ligand-binding domain/BPA complex suggested that the ERRγ receptor residues Leu342, Leu345, Asn346, and Ile349 function as intrinsic binding sites of the BPA phenol-B, whereas Leu265, Leu268, Ile310, Val313, Leu324, Tyr330, Lys430, Ala431, and His434 work as structural elements to assist these binding sites. In the present study, by evaluating the mutant receptors replaced by a series of amino acids, we demonstrated that a finely assembled structural network indeed exists around the two adjacent Leu342-Asn346 and Leu345-Ile349 ridges on the same α-helix 7 (H7), constructing a part of the binding pocket structure with back support residues for the BPA phenol-B ring. The results reveal that the double-layer binding sites, namely, the ordinary ligand binding sites and their back support residues, substantiate the strong binding of BPA to ERRγ. When ERRγ-Asn346 was replaced by the corresponding Gly and Tyr in ERRα and ERRβ, respectively, the binding affinity of BPA and even 4-hydroxytamxifen (4-OHT) is much reduced. Asn346 was found to be one of the residues that make ERRγ to be exclusive to BPA. PMID:24978476

  18. A characteristic back support structure in the bisphenol A-binding pocket in the human nuclear receptor ERRγ.

    PubMed

    Liu, Xiaohui; Matsushima, Ayami; Shimohigashi, Miki; Shimohigashi, Yasuyuki

    2014-01-01

    The endocrine disruptor bisphenol A (BPA) affects various genes and hormones even at merely physiological levels. We recently demonstrated that BPA binds strongly to human nuclear receptor estrogen-related receptor (ERR) γ and that the phenol-A group of BPA is in a receptacle pocket with essential amino acid residues to provide structural support at the backside. This led BPA to bind to ERRγ in an induced-fit-type binding mode, for example, with a rotated motion of Val313 to support the Tyr326-binding site. A similar binding mechanism appears to occur at the binding site of the BPA phenol-B ring. X-ray crystal analysis of the ERRγ-ligand-binding domain/BPA complex suggested that the ERRγ receptor residues Leu342, Leu345, Asn346, and Ile349 function as intrinsic binding sites of the BPA phenol-B, whereas Leu265, Leu268, Ile310, Val313, Leu324, Tyr330, Lys430, Ala431, and His434 work as structural elements to assist these binding sites. In the present study, by evaluating the mutant receptors replaced by a series of amino acids, we demonstrated that a finely assembled structural network indeed exists around the two adjacent Leu342-Asn346 and Leu345-Ile349 ridges on the same α-helix 7 (H7), constructing a part of the binding pocket structure with back support residues for the BPA phenol-B ring. The results reveal that the double-layer binding sites, namely, the ordinary ligand binding sites and their back support residues, substantiate the strong binding of BPA to ERRγ. When ERRγ-Asn346 was replaced by the corresponding Gly and Tyr in ERRα and ERRβ, respectively, the binding affinity of BPA and even 4-hydroxytamxifen (4-OHT) is much reduced. Asn346 was found to be one of the residues that make ERRγ to be exclusive to BPA.

  19. Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli

    PubMed Central

    Soparkar, Ketaki; Kinana, Alfred D.; Weeks, Jon W.; Morrison, Keith D.; Nikaido, Hiroshi

    2015-01-01

    ABSTRACT The AcrB protein of Escherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions—Y49S, V127A, V127G, D153E, and G288C—mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions—F453C and L486W—were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain. IMPORTANCE The AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure

  20. Allosteric binding site in a Cys-loop receptor ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine

    PubMed Central

    Nys, Mieke; Wijckmans, Eveline; Farinha, Ana; Yoluk, Özge; Andersson, Magnus; Brams, Marijke; Spurny, Radovan; Peigneur, Steve; Tytgat, Jan; Lindahl, Erik; Ulens, Chris

    2016-01-01

    Pentameric ligand-gated ion channels or Cys-loop receptors are responsible for fast inhibitory or excitatory synaptic transmission. The antipsychotic compound chlorpromazine is a widely used tool to probe the ion channel pore of the nicotinic acetylcholine receptor, which is a prototypical Cys-loop receptor. In this study, we determine the molecular determinants of chlorpromazine binding in the Erwinia ligand-gated ion channel (ELIC). We report the X-ray crystal structures of ELIC in complex with chlorpromazine or its brominated derivative bromopromazine. Unexpectedly, we do not find a chlorpromazine molecule in the channel pore of ELIC, but behind the β8–β9 loop in the extracellular ligand-binding domain. The β8–β9 loop is localized downstream from the neurotransmitter binding site and plays an important role in coupling of ligand binding to channel opening. In combination with electrophysiological recordings from ELIC cysteine mutants and a thiol-reactive derivative of chlorpromazine, we demonstrate that chlorpromazine binding at the β8–β9 loop is responsible for receptor inhibition. We further use molecular-dynamics simulations to support the X-ray data and mutagenesis experiments. Together, these data unveil an allosteric binding site in the extracellular ligand-binding domain of ELIC. Our results extend on previous observations and further substantiate our understanding of a multisite model for allosteric modulation of Cys-loop receptors. PMID:27791038

  1. Access Path to the Ligand Binding Pocket May Play a Role in Xenobiotics Selection by AhR

    PubMed Central

    Szöllősi, Dániel; Erdei, Áron; Gyimesi, Gergely; Magyar, Csaba; Hegedűs, Tamás

    2016-01-01

    Understanding of multidrug binding at the atomic level would facilitate drug design and strategies to modulate drug metabolism, including drug transport, oxidation, and conjugation. Therefore we explored the mechanism of promiscuous binding of small molecules by studying the ligand binding domain, the PAS-B domain of the aryl hydrocarbon receptor (AhR). Because of the low sequence identities of PAS domains to be used for homology modeling, structural features of the widely employed HIF-2α and a more recent suitable template, CLOCK were compared. These structures were used to build AhR PAS-B homology models. We performed molecular dynamics simulations to characterize dynamic properties of the PAS-B domain and the generated conformational ensembles were employed in in silico docking. In order to understand structural and ligand binding features we compared the stability and dynamics of the promiscuous AhR PAS-B to other PAS domains exhibiting specific interactions or no ligand binding function. Our exhaustive in silico binding studies, in which we dock a wide spectrum of ligand molecules to the conformational ensembles, suggest that ligand specificity and selection may be determined not only by the PAS-B domain itself, but also by other parts of AhR and its protein interacting partners. We propose that ligand binding pocket and access channels leading to the pocket play equally important roles in discrimination of endogenous molecules and xenobiotics. PMID:26727491

  2. Assembly-directed antivirals differentially bind quasiequivalent pockets to modify hepatitis B virus capsid tertiary and quaternary structure.

    PubMed

    Katen, Sarah P; Tan, Zhenning; Chirapu, Srinivas Reddy; Finn, M G; Zlotnick, Adam

    2013-08-06

    Hepatitis B virus (HBV) is a major cause of liver disease. Assembly of the HBV capsid is a critical step in virus production and an attractive target for new antiviral therapies. We determined the structure of HBV capsid in complex with AT-130, a member of the phenylpropenamide family of assembly effectors. AT-130 causes tertiary and quaternary structural changes but does not disrupt capsid structure. AT-130 binds a hydrophobic pocket that also accommodates the previously characterized heteroaryldihydropyrimidine compounds but favors a unique quasiequivalent location on the capsid surface. Thus, this pocket is a promiscuous drug-binding site and a likely target for different assembly effectors with a broad range of mechanisms of activity. That AT-130 successfully decreases virus production by increasing capsid assembly rate without disrupting capsid structure delineates a paradigm in antiviral design, that disrupting reaction timing is a viable strategy for assembly effectors of HBV and other viruses.

  3. Allosteric regulation of the glucose:H+ symporter of Lactobacillus brevis: cooperative binding of glucose and HPr(ser-P).

    PubMed Central

    Ye, J J; Saier, M H

    1995-01-01

    Lactobacillus brevis transports glucose and the nonmetabolizable glucose analog 2-deoxyglucose via a proton symport mechanism that is allosterically inhibited by the seryl-phosphorylated derivative of HPr, the small phosphocarrier protein of the phosphotransferase system. We have demonstrate that S46DHPr, a mutant analog of HPr which conformationally resembles HPr(ser-P) but not free HPr, specifically binds to membranes derived from glucose-grown L. brevis cells if and only if a substrate of the glucose permease is also present. PMID:7896720

  4. Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket.

    PubMed

    Albani, Jihad R

    2004-02-25

    Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the Förster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.

  5. Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production.

    PubMed

    Kandasamy, Suresh K; Fukunaga, Ryuya

    2016-12-06

    The enzyme Dicer produces small silencing RNAs such as micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22-24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5'-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5'-monophosphate of a long dsRNA substrate is anchored by the phosphate-binding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing.

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

    PubMed Central

    Ahmed, Ahmed H.; Oswald, Robert E.

    2010-01-01

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

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

    PubMed

    Ahmed, Ahmed H; Oswald, Robert E

    2010-03-11

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

  8. Identification of transmembrane domain 6 & 7 residues that contribute to the binding pocket of the urotensin II receptor.

    PubMed

    Holleran, Brian J; Domazet, Ivana; Beaulieu, Marie-Eve; Yan, Li Ping; Guillemette, Gaétan; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard

    2009-04-15

    Urotensin II (U-II), a cyclic undecapeptide, is the natural ligand of the urotensin II (UT) receptor, a G protein-coupled receptor. In the present study, we used the substituted-cysteine accessibility method to identify specific residues in transmembrane domains (TMDs) six and seven of the rat urotensin II receptor (rUT) that contribute to the formation of the binding pocket of the receptor. Each residue in the R256(6.32)-Q283(6.59) fragment of TMD6 and the A295(7.31)-T321(7.57) fragment of TMD7 was mutated, individually, to a cysteine. The resulting mutants were expressed in COS-7 cells, which were subsequently treated with the positively charged methanethiosulfonate-ethylammonium (MTSEA) or the negatively charged methanethiosulfonate-ethylsulfonate (MTSES) sulfhydryl-specific alkylating agents. MTSEA treatment resulted in a significant reduction in the binding of TMD6 mutants F268C(6.44) and W278C(6.54) and TMD7 mutants L298C(7.34), T302C(7.38), and T303C(7.39) to (125)I-U-II. MTSES treatment resulted in a significant reduction in the binding of two additional mutants, namely L282C(6.58) in TMD6 and Y300C(7.36) in TMD7. These results suggest that specific residues orient themselves within the water-accessible binding pocket of the rUT receptor. This approach, which allowed us to identify key determinants in TMD6 and TMD7 that contribute to the UT receptor binding pocket, enabled us to further refine our homology-based model of how U-II interacts with its cognate receptor.

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

    SciTech Connect

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

    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.

  10. In vitro binding of a radio-labeled positive allosteric modulator for metabotropic glutamate receptor subtype 5.

    PubMed

    Zysk, John R; Spear, Nathan; Fieles, William; Stein, Mark M; Sygowski, Linda S; King, Megan M; Hoesch, Valerie; Hastings, Richard; Brockel, Becky; Do, Mylinh; Ström, Peter; Gadient, Reto; Chhajlani, Vijay; Elmore, Charles S; Maier, Donna L

    2013-03-01

    The positive allosteric modulator (PAM) binding site for metabotropic glutamate receptor subtype 5 (mGlu(5)) lacks a readily available radio-labeled tracer fordetailed structure-activity studies. This communication describes a selective mGlu(5) compound, 7-methyl-2-(4-(pyridin-2-yloxy)benzyl)-5-(pyridin-3-yl)isoindolin-1-one (PBPyl) that binds with high affinity to human mGlu(5) and exhibits functional PAM activity. Analysis of PBPyl by FLIPR revealed an EC(50) of 87 nM with an 89% effect in transfected HEK293 cells and an EC(50) of 81 nM with a 42% effect in rat primary neurons. PBPyl exhibited 5-fold higher functional selectivity for mGlu(5) in a full mGlu receptor panel. Unlabeled PBPyl was tested for specific binding using a liquid chromatography mass spectrometry (LC/MS/MS)-based filtration binding assay and exhibited 40% specific binding in recombinant membranes, a value higher than any candidate compound tested. In competition binding studies with [(3)H]MPEP, the mGlu(5) receptor negative allosteric modulator (NAM), PBPyl exhibited a k(i) value of 34 nM. PBPyl also displaced [(3)H]ABP688, a mGluR(5) receptor NAM, in tissue sections from mouse and rat brain using autoradiography. Areas of specific binding included the frontal cortex, striatum and nucleus accumbens. PBPyl was radiolabeled to a specific activity of 15 Ci/mmol and tested for specific binding in a filter plate format. In recombinant mGlu(5b) membranes, [(3)H] PBPyl exhibited saturable binding with a K(d) value of 18.6 nM. In competition binding experiments, [(3)H] PBPyl was displaced by high affinity mGlu(5) positive and negative modulators. Further tests showed that PBPyl displays less than optimal characteristics as an in vivo tool, including a high volume of distribution and ClogP, making it more suitable as an in vitro compound. However, as a first report of direct binding of an mGlu(5) receptor PAM, this study offers value toward the development of novel PET imaging agents for this important

  11. Structure and function of Plasmodium falciparum malate dehydrogenase: role of critical amino acids in co-substrate binding pocket.

    PubMed

    Pradhan, Anupam; Tripathi, Abhai K; Desai, Prashant V; Mukherjee, Prasenjit K; Avery, Mitchell A; Walker, Larry A; Tekwani, Babu L

    2009-01-01

    The malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our laboratory have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal glycine motif, which forms a characteristic Rossman dinucleotide-binding fold in the co-substrate binding pocket, differentiates PfMDH (GlyXGlyXXGly) from other eukaryotic and prokaryotic malate dehydrogenases (GlyXXGlyXXGly). The amino acids lining the co-substrate binding pocket are completely conserved in MDHs from different species of human, primate and rodent malaria parasites. Based on this knowledge and conserved domains among prokaryotic and eukaryotic MDH, the role of critical amino acids lining the co-substrate binding pocket was analyzed in catalytic functions of PfMDH using site-directed mutagenesis. Insertion of Ala at the 9th or 10th position, which converts the N-terminal GlyXGlyXXGly motif (characteristic of malarial MDH and LDH) to GlyXXGlyXXGly (as in bacterial and eukaryotic MDH), uncoupled regulation of the enzyme through substrate inhibition. The dinucleotide fold GlyXGlyXXGly motif seems not to be responsible for the distinct affinity of PfMDH to 3-acetylpyridine-adenine dinucleotide (APAD, a synthetic analog of NAD), since Ala9 and Ala10 insertion mutants still utilized APADH. The Gln11Met mutation, which converts the signature glycine motif in PfMDH to that of PfLDH, did not change the enzyme function. However, the Gln11Gly mutant showed approximately a 5-fold increase in catalytic activity, and higher susceptibility to inhibition with gossypol. Asn119 and His174 participate in binding of both co-substrate and substrate. The Asn119Gly mutant exhibited approximately a 3-fold decrease in catalytic efficiency, while mutation of His174 to Asn or Ala resulted in an inactive enzyme. These studies provide critical insights into the co

  12. Discovery of benzimidazole-diamide finger loop (Thumb Pocket I) allosteric inhibitors of HCV NS5B polymerase: Implementing parallel synthesis for rapid linker optimization.

    PubMed

    Goulet, Sylvie; Poupart, Marc-André; Gillard, James; Poirier, Martin; Kukolj, George; Beaulieu, Pierre L

    2010-01-01

    Previously described SAR of benzimidazole-based non-nucleoside finger loop (Thumb Pocket I) inhibitors of HCV NS5B polymerase was expanded. Prospecting studies using parallel synthesis techniques allowed the rapid identification of novel cinnamic acid right-hand sides that provide renewed opportunities for further optimization of these inhibitors. Novel diamide derivatives such as 44 exhibited comparable potency (enzymatic and cell-based HCV replicon) as previously described tryptophan-based inhibitors but physicochemical properties (e.g., aqueous solubility and lipophilicity) have been improved, resulting in molecules with reduced off-target liabilities (CYP inhibition) and increased metabolic stability.

  13. A conserved flagellar pocket exposed high mannose moiety is used by African trypanosomes as a host cytokine binding molecule.

    PubMed

    Magez, S; Radwanska, M; Stijlemans, B; Xong, H V; Pays, E; De Baetselier, P

    2001-09-07

    Trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as defense against the host immune system. However, in order to sustain their growth, they need to expose conserved epitopes, allowing host macromolecule binding and receptor-mediated endocytosis. Here we show that Trypanosoma brucei uses the conserved chitobiose-oligomannose (GlcNAc(2)-Man(5-9)) moieties of its VSG as a binding ligand for tumor necrosis factor (TNF), a host cytokine with lectin-like properties. As endocytosis in trypanosomes is restricted to the flagellar pocket, we show that soluble flagellar pocket extracts, and in particular soluble VSG, inhibit the binding of (125)I-TNF to trypanosomes. The interaction between TNF and VSG is confirmed by affinity chromatography, biosensor, and dot-blot affinity measurements, and soluble VSG inhibition of TNF-mediated trypanolysis. In all approaches, removal of N-linked carbohydrates abrogates the TNF-VSG interaction. In addition, synthetic high mannose oligosaccharides can block TNF-VSG interactions, and a VSG glycopeptide carrying the GlcNAc(2)-Man(5-9) moiety is shown to inhibit TNF-mediated trypanosome killing in mixed parasite/macrophage cell cultures. Together, these results support the observation that TNF plays a role in growth control of trypanosomes and, moreover, suggest that, by the use of conserved VSG carbohydrates as lectin-binding epitopes, trypanosomes can limit the necessity to express large numbers of invariant surface exposed receptors.

  14. The structure of the SBP-Tag–streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits

    SciTech Connect

    Barrette-Ng, Isabelle H.; Wu, Sau-Ching; Tjia, Wai-Mui; Wong, Sui-Lam; Ng, Kenneth K. S.

    2013-05-01

    The structure of the SBP-Tag–streptavidin complex reveals a novel mode of peptide recognition in which a single peptide binds simultaneously to biotin-binding pockets from adjacent subunits of streptavidin. The molecular details of peptide recognition suggest how the SBP-Tag can be further modified to become an even more useful tag for a wider range of biotechnological applications. The 38-residue SBP-Tag binds to streptavidin more tightly (K{sub d} ≃ 2.5–4.9 nM) than most if not all other known peptide sequences. Crystallographic analysis at 1.75 Å resolution shows that the SBP-Tag binds to streptavidin in an unprecedented manner by simultaneously interacting with biotin-binding pockets from two separate subunits. An N-terminal HVV peptide sequence (residues 12–14) and a C-terminal HPQ sequence (residues 31–33) form the bulk of the direct interactions between the SBP-Tag and the two biotin-binding pockets. Surprisingly, most of the peptide spanning these two sites (residues 17–28) adopts a regular α-helical structure that projects three leucine side chains into a groove formed at the interface between two streptavidin protomers. The crystal structure shows that residues 1–10 and 35–38 of the original SBP-Tag identified through in vitro selection and deletion analysis do not appear to contact streptavidin and thus may not be important for binding. A 25-residue peptide comprising residues 11–34 (SBP-Tag2) was synthesized and shown using surface plasmon resonance to bind streptavidin with very similar affinity and kinetics when compared with the SBP-Tag. The SBP-Tag2 was also added to the C-terminus of β-lactamase and was shown to be just as effective as the full-length SBP-Tag in affinity purification. These results validate the molecular structure of the SBP-Tag–streptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed.

  15. Allosteric Modulation of Metabotropic Glutamate Receptors: Structural Insights and Therapeutic Potential

    PubMed Central

    Gregory, Karen J.; Dong, Elizabeth N.; Meiler, Jens; Conn, P. Jeffrey

    2010-01-01

    Allosteric modulation of G protein-coupled receptors (GPCRs) represents a novel approach to the development of probes and therapeutics that is expected to enable subtype-specific regulation of central nervous system target receptors. The metabotropic glutamate receptors (mGlus) are class C GPCRs that play important neuromodulatory roles throughout the brain, as such they are attractive targets for therapeutic intervention for a number of psychiatric and neurological disorders including anxiety, depression, Fragile X Syndrome, Parkinson’s disease and schizophrenia. Over the last fifteen years, selective allosteric modulators have been identified for many members of the mGlu family. The vast majority of these allosteric modulators are thought to bind within the transmembrane-spanning domains of the receptors to enhance or inhibit functional responses. A combination of mutagenesis-based studies and pharmacological approaches are beginning to provide a better understanding of mGlu allosteric sites. Collectively, when mapped onto a homology model of the different mGlu subtypes based on the β2-adrenergic receptor, the previous mutagenesis studies suggest commonalities in the location of allosteric sites across different members of the mGlu family. In addition, there is evidence for multiple allosteric binding pockets within the transmembrane region that can interact to modulate one another. In the absence of a class C GPCR crystal structure, this approach has shown promise with respect to the interpretation of mutagenesis data and understanding structure-activity relationships of allosteric modulator pharmacophores. PMID:20637216

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

    PubMed

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

    2009-10-25

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

  17. Conformational Changes in the Activation Loop of Mitochondrial Glutaminase C: A Direct Fluorescence Read-Out that Distinguishes the Binding of Allosteric Inhibitors from Activators.

    PubMed

    Stalnecker, Clint A; Erickson, Jon W; Cerione, Richard A

    2017-02-14

    The first step in glutamine catabolism is catalyzed by the mitochondrial enzyme glutaminase, with a specific isoform, glutaminase C (GAC), being highly expressed in cancer cells. GAC activation requires the formation of homo-tetramers, promoted by anionic allosteric activators such as inorganic phosphate. This leads to the proper orientation of a flexible loop proximal to the dimer-dimer interface that is essential for catalysis (i.e. the activation loop). A major class of allosteric inhibitors of GAC, with the prototype being BPTES (bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide), and the related molecule CB-839, binds to the activation loop and induces the formation of an inactive tetramer (2 inhibitors bound per active tetramer). Here, we describe a direct readout for monitoring the dynamics of the activation loop of GAC in response to these allosteric inhibitors, as well as allosteric activators, through the substitution of phenylalanine at position 327 with tryptophan (F327W). The tryptophan fluorescence of the GAC(F327W) mutant undergoes a marked quenching upon the binding of BPTES or CB-839, yielding titration profiles that make it possible to measure the binding affinities of these inhibitors for the enzyme. Allosteric activators like phosphate induce the opposite effect (i.e. a fluorescence enhancement). These results describe direct read-outs for the binding of the BPTES-class of allosteric inhibitors, as well as for inorganic phosphate and related activators of GAC, which should facilitate screening for additional modulators of this important metabolic enzyme.

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

    PubMed

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

    2014-10-05

    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.

  19. Binding-pocket and lid-region substitutions render human STING sensitive to the species-specific drug DMXAA.

    PubMed

    Gao, Pu; Zillinger, Thomas; Wang, Weiyi; Ascano, Manuel; Dai, Peihong; Hartmann, Gunther; Tuschl, Thomas; Deng, Liang; Barchet, Winfried; Patel, Dinshaw J

    2014-09-25

    The drug DMXAA (5,6-dimethylxanthenone-4-acetic acid) showed therapeutic promise against solid tumors in mouse models but subsequently failed in human clinical trials. DMXAA was later discovered to activate mouse, but not human, STING, an adaptor protein in the cyclic dinucleotide cGAMP-mediated signaling pathway, inducing type I interferon expression. To facilitate the development of compounds that target human STING, we combined structural, biophysical, and cellular assays to study mouse and human chimeric proteins and their interaction with DMXAA. We identified a single substitution (G230I) that enables a DMXAA-induced conformational transition of hSTING from an inactive "open" to an active "closed" state. We also identified a substitution within the binding pocket (Q266I) that cooperates with G230I and the previously identified S162A binding-pocket point substitution, rendering hSTING highly sensitive to DMXAA. These findings should facilitate the reciprocal engineering of DMXAA analogs that bind and stimulate wild-type hSTING and their exploitation for vaccine-adjuvant and anticancer drug development.

  20. Free energy changes and components implicit in the MWC allosteric model for the cooperative oxygen binding of hemoglobin.

    PubMed

    Bucci, Enrico; Pucciarelli, Stefania; Angeletti, Mauro

    2013-06-18

    Hill's plots of oxygen binding isotherms reveal the presence of a transition between two different oxygen affinities at the beginning and end of the isotherm. They correspond to the two conformations anticipated by the MWC model, namely, the T and R conformations at the beginning and end of oxygen binding, when the lower affinity of the T form develops into the higher affinity of the R form. The difference between the binding Gibbs free energy changes of the two affinities (Δ G(L)) is the free energy of binding cooperativity. Notably, Δ G(L) is positive in favor of the T form, which moves to a higher energy level upon oxygen release. Osmotic stress reveals a higher volume/surface ratio of deoxyhemoglobin, with a positive Δ G(W) also in favor of the T form. An increasing protein concentration shifts the isotherms to the right, indicating the formation of intermediate polymeric forms. The enthalpy of the intermediates shows a strong absorption of heat at the third oxygenation step because of polymer formation with quinary, and higher-order, structures. The disassembly of intermediate polymers releases energy with a negative Δ G that compensates and allows the positive values of Δ G(L). High-energy polymers are the barrier preventing the relaxation of the T and R conformations into one another. The MWC allosteric model is the best justification of oxygen binding cooperativity.

  1. Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin.

    PubMed

    Ngo, Kien Xuan; Umeki, Nobuhisa; Kijima, Saku T; Kodera, Noriyuki; Ueno, Hiroaki; Furutani-Umezu, Nozomi; Nakajima, Jun; Noguchi, Taro Q P; Nagasaki, Akira; Tokuraku, Kiyotaka; Uyeda, Taro Q P

    2016-10-20

    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.

  2. Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin

    PubMed Central

    Ngo, Kien Xuan; Umeki, Nobuhisa; Kijima, Saku T.; Kodera, Noriyuki; Ueno, Hiroaki; Furutani-Umezu, Nozomi; Nakajima, Jun; Noguchi, Taro Q. P.; Nagasaki, Akira; Tokuraku, Kiyotaka; Uyeda, Taro Q. P.

    2016-01-01

    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells. PMID:27762277

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

    PubMed Central

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

    2016-01-01

    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

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

    PubMed

    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.

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

    PubMed Central

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

    2013-01-01

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

  6. Structure of Liver Receptor Homolog-1 (NR5A2) with PIP3 hormone bound in the ligand binding pocket.

    PubMed

    Sablin, Elena P; Blind, Raymond D; Uthayaruban, Rubatharshini; Chiu, Hsiu-Ju; Deacon, Ashley M; Das, Debanu; Ingraham, Holly A; Fletterick, Robert J

    2015-12-01

    The nuclear receptor LRH-1 (Liver Receptor Homolog-1, NR5A2) is a transcription factor that regulates gene expression programs critical for many aspects of metabolism and reproduction. Although LRH-1 is able to bind phospholipids, it is still considered an orphan nuclear receptor (NR) with an unknown regulatory hormone. Our prior cellular and structural studies demonstrated that the signaling phosphatidylinositols PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind and regulate SF-1 (Steroidogenic Factor-1, NR5A1), a close homolog of LRH-1. Here, we describe the crystal structure of human LRH-1 ligand binding domain (LBD) bound by PIP3 - the first phospholipid with a head group endogenous to mammals. We show that the phospholipid hormone binds LRH-1 with high affinity, stabilizing the receptor LBD. While the hydrophobic PIP3 tails (C16/C16) are buried inside the LRH-1 ligand binding pocket, the negatively charged PIP3 head group is presented on the receptor surface, similar to the phosphatidylinositol binding mode observed in the PIP3-SF-1 structure. Thus, data presented in this work reinforce our earlier findings demonstrating that signaling phosphatidylinositols regulate the NR5A receptors LRH-1 and SF-1.

  7. Analysis of oxygen binding to Panulirus japonicus hemocyanin. The effect of divalent cations on the allosteric transition.

    PubMed

    Makino, N

    1986-01-02

    The effects of H+ and divalent cations on the O2 equilibrium of hexameric hemocyanin from a spiny lobster, Panulirus japonicus, were examined. The hemocyanin showed the normal Bohr effect. When divalent cations were removed by EDTA treatment, the protein showed a fivefold increase in the O2 affinity and a considerable decrease in the cooperativity. Several cooperativity models were tested for the conformity with the observed O2-binding isotherms by the least-square curve fitting. Among the models examined, the three-state concerted model was found to be most consistent with the results. It was postulated that in the absence of divalent cations deoxyhemocyanin is mainly in the intermediate-affinity state. The arthropod hemocyanins were found to be classifiable into two groups according to their functional responses to the divalent cations. It was suggested that the cations act differently on the allosteric transitions of the two groups of hemocyanins.

  8. Anti-tumor agent calixarene 0118 targets human galectin-1 as an allosteric inhibitor of carbohydrate binding

    PubMed Central

    Dings, Ruud P.M.; Miller, Michelle C.; Nesmelova, Irina; Astorgues-Xerri, Lucile; Kumar, Nigam; Serova, Maria; Chen, Xuimei; Raymond, Eric; Hoye, Thomas R.; Mayo, Kevin H.

    2012-01-01

    Calix[4]arene compound 0118 is an angiostatic agent that inhibits tumor growth in mice. Although 0118 is a topomimetic of galectin-1-targeting angiostatic amphipathic peptide anginex, we had yet to prove that 0118 targets galectin-1. Galectin-1 is involved in pathological disorders like tumor endothelial cell adhesion and migration and therefore presents a relevant target for therapeutic intervention against cancer. Here, 15N-1H HSQC NMR spectroscopy demonstrates that 0118 indeed targets galectin-1 at a site away from the lectin’s carbohydrate binding site, and thereby attenuates lactose binding to the lectin. Flow cytometry and agglutination assays show that 0118 attenuates binding of galectin-1 to cell surface glycans, and the inhibition of cell proliferation by 0118 is found to be correlated with the cellular expression of the lectin. In general, our data indicate that 0118 targets galectin-1 as an allosteric inhibitor of glycan/carbohydrate binding. This work contributes to the clinical development of anti-tumor calixarene compound 0118. PMID:22575017

  9. The Allosteric Site for the Nascent Cell Wall in Penicillin-Binding Protein 2a: An Achilles' Heel of Methicillin-Resistant Staphylococcus aureus.

    PubMed

    Acebrón, Iván; Chang, Mayland; Mobashery, Shahriar; Hermoso, Juan A

    2015-01-01

    The ability to resist the effect of a wide range of antibiotics makes methicillin-resistant Staphylococcus aureus (MRSA) a leading global human pathogen. A key determinant of resistance to β-lactam antibiotics in this organism is penicillin-binding protein 2a (PBP2a), an enzyme that catalyzes the crosslinking reaction between two adjacent peptide stems during the peptidoglycan biosynthesis. The recently published crystal structure of the complex of PBP2a with ceftaroline, a cephalosporin antibiotic that shows efficacy against MRSA, has revealed the allosteric site at 60-Å distance from the transpeptidase domain. Binding of ceftaroline to the allosteric site of PBP2a triggers conformational changes that lead to the opening of the active site from a closed conformation, where a second molecule of ceftaroline binds to give inhibition of the enzyme. The discovery of allostery in MRSA remains the only known example of such regulation of cellwall biosynthesis and represents a new paradigm in fighting MRSA. This review summarizes the present knowledge of the allosteric mechanism, the conformational changes allowing PBP2a catalysis and the means by which some clinical strains have acquired resistance to ceftaroline by disrupting the allosteric mechanism.

  10. eMatchSite: Sequence Order-Independent Structure Alignments of Ligand Binding Pockets in Protein Models

    PubMed Central

    Brylinski, Michal

    2014-01-01

    Detecting similarities between ligand binding sites in the absence of global homology between target proteins has been recognized as one of the critical components of modern drug discovery. Local binding site alignments can be constructed using sequence order-independent techniques, however, to achieve a high accuracy, many current algorithms for binding site comparison require high-quality experimental protein structures, preferably in the bound conformational state. This, in turn, complicates proteome scale applications, where only various quality structure models are available for the majority of gene products. To improve the state-of-the-art, we developed eMatchSite, a new method for constructing sequence order-independent alignments of ligand binding sites in protein models. Large-scale benchmarking calculations using adenine-binding pockets in crystal structures demonstrate that eMatchSite generates accurate alignments for almost three times more protein pairs than SOIPPA. More importantly, eMatchSite offers a high tolerance to structural distortions in ligand binding regions in protein models. For example, the percentage of correctly aligned pairs of adenine-binding sites in weakly homologous protein models is only 4–9% lower than those aligned using crystal structures. This represents a significant improvement over other algorithms, e.g. the performance of eMatchSite in recognizing similar binding sites is 6% and 13% higher than that of SiteEngine using high- and moderate-quality protein models, respectively. Constructing biologically correct alignments using predicted ligand binding sites in protein models opens up the possibility to investigate drug-protein interaction networks for complete proteomes with prospective systems-level applications in polypharmacology and rational drug repositioning. eMatchSite is freely available to the academic community as a web-server and a stand-alone software distribution at http://www.brylinski.org/ematchsite. PMID

  11. A 5′ cytosine binding pocket in Puf3p specifies regulation of mitochondrial mRNAs

    SciTech Connect

    Zhu, Deyu; Stumpf, Craig R.; Krahn, Joseph M.; Wickens, Marvin; Tanaka Hall, Traci M.

    2010-11-03

    A single regulatory protein can control the fate of many mRNAs with related functions. The Puf3 protein of Saccharomyces cerevisiae is exemplary, as it binds and regulates more than 100 mRNAs that encode proteins with mitochondrial function. Here we elucidate the structural basis of that specificity. To do so, we explore the crystal structures of Puf3p complexes with 2 cognate RNAs. The key determinant of Puf3p specificity is an unusual interaction between a distinctive pocket of the protein with an RNA base outside the 'core' PUF-binding site. That interaction dramatically affects binding affinity in vitro and is required for regulation in vivo. The Puf3p structures, combined with those of Puf4p in the same organism, illuminate the structural basis of natural PUF-RNA networks. Yeast Puf3p binds its own RNAs because they possess a -2C and is excluded from those of Puf4p which contain an additional nucleotide in the core-binding site.

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

    NASA Astrophysics Data System (ADS)

    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.

  13. Structural basis for cAMP-mediated allosteric control of the catabolite activator protein.

    PubMed

    Popovych, Nataliya; Tzeng, Shiou-Ru; Tonelli, Marco; Ebright, Richard H; Kalodimos, Charalampos G

    2009-04-28

    The cAMP-mediated allosteric transition in the catabolite activator protein (CAP; also known as the cAMP receptor protein, CRP) is a textbook example of modulation of DNA-binding activity by small-molecule binding. Here we report the structure of CAP in the absence of cAMP, which, together with structures of CAP in the presence of cAMP, defines atomic details of the cAMP-mediated allosteric transition. The structural changes, and their relationship to cAMP binding and DNA binding, are remarkably clear and simple. Binding of cAMP results in a coil-to-helix transition that extends the coiled-coil dimerization interface of CAP by 3 turns of helix and concomitantly causes rotation, by approximately 60 degrees , and translation, by approximately 7 A, of the DNA-binding domains (DBDs) of CAP, positioning the recognition helices in the DBDs in the correct orientation to interact with DNA. The allosteric transition is stabilized further by expulsion of an aromatic residue from the cAMP-binding pocket upon cAMP binding. The results define the structural mechanisms that underlie allosteric control of this prototypic transcriptional regulatory factor and provide an illustrative example of how effector-mediated structural changes can control the activity of regulatory proteins.

  14. Binding to large enzyme pockets: small-molecule inhibitors of trypanothione reductase.

    PubMed

    Persch, Elke; Bryson, Steve; Todoroff, Nickolay K; Eberle, Christian; Thelemann, Jonas; Dirdjaja, Natalie; Kaiser, Marcel; Weber, Maria; Derbani, Hassan; Brun, Reto; Schneider, Gisbert; Pai, Emil F; Krauth-Siegel, R Luise; Diederich, François

    2014-08-01

    The causative agents of the parasitic disease human African trypanosomiasis belong to the family of trypanosomatids. These parasitic protozoa exhibit a unique thiol redox metabolism that is based on the flavoenzyme trypanothione reductase (TR). TR was identified as a potential drug target and features a large active site that allows a multitude of possible ligand orientations, which renders rational structure-based inhibitor design highly challenging. Herein we describe the synthesis, binding properties, and kinetic analysis of a new series of small-molecule inhibitors of TR. The conjunction of biological activities, mutation studies, and virtual ligand docking simulations led to the prediction of a binding mode that was confirmed by crystal structure analysis. The crystal structures revealed that the ligands bind to the hydrophobic wall of the so-called "mepacrine binding site". The binding conformation and potency of the inhibitors varied for TR from Trypanosoma brucei and T. cruzi.

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

    PubMed Central

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

    2014-01-01

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

  16. GPCR crystal structures: Medicinal chemistry in the pocket.

    PubMed

    Shonberg, Jeremy; Kling, Ralf C; Gmeiner, Peter; Löber, Stefan

    2015-07-15

    Recent breakthroughs in GPCR structural biology have significantly increased our understanding of drug action at these therapeutically relevant receptors, and this will undoubtedly lead to the design of better therapeutics. In recent years, crystal structures of GPCRs from classes A, B, C and F have been solved, unveiling a precise snapshot of ligand-receptor interactions. Furthermore, some receptors have been crystallized in different functional states in complex with antagonists, partial agonists, full agonists, biased agonists and allosteric modulators, providing further insight into the mechanisms of ligand-induced GPCR activation. It is now obvious that there is enormous diversity in the size, shape and position of the ligand binding pockets in GPCRs. In this review, we summarise the current state of solved GPCR structures, with a particular focus on ligand-receptor interactions in the binding pocket, and how this can contribute to the design of GPCR ligands with better affinity, subtype selectivity or efficacy.

  17. FUNCTIONAL INSIGHT INTO DEVELOPMENT OF POSITIVE ALLOSTERIC MODULATORS OF AMPA RECEPTORS

    PubMed Central

    Weeks, Autumn M.; Harms, Jonathan E.; Partin, Kathryn M.; Benveniste, Morris

    2014-01-01

    Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) ionotropic glutamate receptors facilitate synaptic plasticity and contribute essentially to learning and memory, properties which make AMPA receptors targets for drug discovery and development. One region at which several different classes of positive allosteric modulators bind lies at the dimer interface between the ligand-binding core of the second, membrane-proximal, extracellular domain of AMPA receptors. This solvent-accessible binding pocket has been the target of drug discovery efforts, leading to the recent delineation of five “subsites” which differentially allow access to modulator moieties, and for which distinct modulator affinities and apparent efficacies are attributed. Here we use the voltage-clamp technique in conjunction with rapid drug application to study the effects of mutants lining subsites “A” and “B” of the allosteric modulator pocket to assess affinity and efficacy of allosteric modulation by cyclothiazide, CX614, CMPDA and CMPDB. A novel analysis of the decay of current produced by the onset of desensitization has allowed us to estimate both affinity and efficacy from single concentrations of modulator. Such an approach may be useful for effective high throughput screening of new target compounds. PMID:24878241

  18. Functional insight into development of positive allosteric modulators of AMPA receptors.

    PubMed

    Weeks, Autumn M; Harms, Jonathan E; Partin, Kathryn M; Benveniste, Morris

    2014-10-01

    Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) ionotropic glutamate receptors facilitate synaptic plasticity and contribute essentially to learning and memory, properties which make AMPA receptors targets for drug discovery and development. One region at which several different classes of positive allosteric modulators bind lies at the dimer interface between the ligand-binding core of the second, membrane-proximal, extracellular domain of AMPA receptors. This solvent-accessible binding pocket has been the target of drug discovery efforts, leading to the recent delineation of five "subsites" which differentially allow access to modulator moieties, and for which distinct modulator affinities and apparent efficacies are attributed. Here we use the voltage-clamp technique in conjunction with rapid drug application to study the effects of mutants lining subsites "A" and "B" of the allosteric modulator pocket to assess affinity and efficacy of allosteric modulation by cyclothiazide, CX614, CMPDA and CMPDB. A novel analysis of the decay of current produced by the onset of desensitization has allowed us to estimate both affinity and efficacy from single concentrations of modulator. Such an approach may be useful for effective high throughput screening of new target compounds.

  19. Analysis of transient and catalytic desosamine-binding pockets in cytochrome P-450 PikC from Streptomyces venezuelae.

    PubMed

    Li, Shengying; Ouellet, Hugues; Sherman, David H; Podust, Larissa M

    2009-02-27

    The cytochrome P-450 PikC from Streptomyces venezuelae exhibits significant substrate tolerance and performs multiple hydroxylation reactions on structurally variant macrolides bearing the deoxyamino sugar desosamine. In previously determined co-crystal structures (Sherman, D. H., Li, S., Yermalitskaya, L. V., Kim, Y., Smith, J. A., Waterman, M. R., and Podust, L. M. (2006) J. Biol. Chem. 281, 26289-26297), the desosamine moiety of the native substrates YC-17 and narbomycin is bound in two distinct buried and surface-exposed binding pockets, mediated by specific interactions between the protonated dimethylamino group and the acidic amino acid residues Asp(50), Glu(85), and Glu(94). Although the Glu(85) and Glu(94) negative charges are essential for maximal catalytic activity of native enzyme, elimination of the surface-exposed negative charge at Asp(50) results in significantly enhanced catalytic activity. Nevertheless, the D50N substitution could not rescue catalytic activity of PikC(E94Q) based on lack of activity in the corresponding double mutant PikC(D50N/E94Q). To address the specific role for each desosamine-binding pocket, we analyzed the x-ray structures of the PikC(D50N) mutant co-crystallized with narbomycin (1.85A resolution) and YC-17 (3.2A resolution). In PikC(D50N), the desosamine moiety of both YC-17 and narbomycin was bound in a catalytically productive "buried site." This finding suggested a two-step substrate binding mechanism, whereby desosamine is recognized in the two subsites to allow the macrolide substrate to sequentially progress toward a catalytically favorable orientation. Collectively, the binding, mutagenesis, kinetic, and x-ray structural data suggest that enhancement of the catalytic activity of PikC(D50N) is due to the facilitated relocation of substrate to the buried site, which has higher binding affinity, as opposed to dissociation in solution from the transient "surface-exposed site."

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

    PubMed Central

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

    2016-01-01

    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. PMID:27063862

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

    NASA Astrophysics Data System (ADS)

    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.

  2. Function of Phe-259 and Thr-314 within the Substrate Binding Pocket of the Juvenile Hormone Esterase of Manduca sexta†

    PubMed Central

    Kamita, Shizuo G.; Wogulis, Mark D.; Law, Christopher S.; Morisseau, Christophe; Tanaka, Hiromasa; Huang, Huazhang; Wilson, David K.; Hammock, Bruce D.

    2013-01-01

    Juvenile hormone (JH) is a key insect developmental hormone that is found at low nanomolar levels in larval insects. The methyl ester of JH is hydrolyzed in many insects by an esterase that shows high specificity for JH. We have previously determined a crystal structure of the JH esterase (JHE) of the tobacco hornworm Manduca sexta (MsJHE) [Wogulis, M., Wheelock, C. E., Kamita, S. G., Hinton, A. C., Whetstone, P. A., Hammock, B. D., and Wilson, D. K. (2006) Biochemistry 45, 4045-4057]. Our molecular modeling indicates that JH fits very tightly within the substrate binding pocket of MsJHE. This tight fit places two non-catalytic amino acid residues, Phe-259 and Thr-314, within the appropriate distance and geometry to potentially interact with the α,β-unsaturated ester and epoxide, respectively, of JH. These residues are highly conserved in numerous biologically active JHEs. Kinetic analyses of mutants of Phe-259 or Thr-314 indicate that these residues contribute to the low KM that MsJHE shows for JH. This low KM, however, comes at the cost of reduced substrate turnover. Neither nucleophilic attack of the resonance stabilized ester by the catalytic serine nor the availability of a water molecule for attack of the acyl-enzyme intermediate appear to be a rate-determining step in the hydrolysis of JH by MsJHE. We hypothesize that the release of the JH acid metabolite from the substrate binding pocket limits the catalytic cycle. Our findings also demonstrate that chemical bond strength does not necessarily correlate with how reactive the bond will be to metabolism. PMID:20307057

  3. Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility, Gating Residues and Multiple Binding Pockets

    PubMed Central

    Palermo, Giulia; Bauer, Inga; Campomanes, Pablo; Cavalli, Andrea; Armirotti, Andrea; Girotto, Stefania; Rothlisberger, Ursula; De Vivo, Marco

    2015-01-01

    The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the “membrane-access” and the “acyl chain-binding” pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH’s mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts. PMID:26111155

  4. Raf kinase inhibitory protein function is regulated via a flexible pocket and novel phosphorylation-dependent mechanism.

    PubMed

    Granovsky, Alexey E; Clark, Matthew C; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

    2009-03-01

    Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.

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

    PubMed

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

    2016-06-15

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

  6. ATP-Binding Pocket-Targeted Suppression of Src and Syk by Luteolin Contributes to Its Anti-Inflammatory Action

    PubMed Central

    Lee, Jeong-Oog; Jeong, Deok; Kim, Mi-Yeon; Cho, Jae Youl

    2015-01-01

    Luteolin is a flavonoid identified as a major anti-inflammatory component of Artemisia asiatica. Numerous reports have demonstrated the ability of luteolin to suppress inflammation in a variety of inflammatory conditions. However, its exact anti-inflammatory mechanism has not been fully elucidated. In the present study, the anti-inflammatory mode of action in activated macrophages of luteolin from Artemisia asiatica was examined by employing immunoblotting analysis, a luciferase reporter gene assay, enzyme assays, and an overexpression strategy. Luteolin dose-dependently inhibited the secretion of nitric oxide (NO) and prostaglandin E2 (PGE2) and diminished the levels of mRNA transcripts of inducible NO synthase (iNOS), tumor necrosis factor- (TNF-) α, and cyclooxygenase-2 (COX-2) in lipopolysaccharide- (LPS-) and pam3CSK-treated macrophage-like RAW264.7 cells without displaying cytotoxicity. Luteolin displayed potent NO-inhibitory activity and also suppressed the nuclear translocation of NF-κB (p65 and p50) via blockade of Src and Syk, but not other mitogen-activated kinases. Overexpression of wild type Src and point mutants thereof, and molecular modelling studies, suggest that the ATP-binding pocket may be the luteolin-binding site in Src. These results strongly suggest that luteolin may exert its anti-inflammatory action by suppressing the NF-κB signaling cascade via blockade of ATP binding in Src and Syk. PMID:26236111

  7. Controlling allosteric networks in proteins

    NASA Astrophysics Data System (ADS)

    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.

  8. The M1 muscarinic receptor allosteric agonists AC-42 and 1-[1'-(2-methylbenzyl)-1,4'-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one bind to a unique site distinct from the acetylcholine orthosteric site.

    PubMed

    Jacobson, Marlene A; Kreatsoulas, Constantine; Pascarella, Danette M; O'Brien, Julie A; Sur, Cyrille

    2010-10-01

    Activation of M1 muscarinic receptors occurs through orthosteric and allosteric binding sites. To identify critical residues, site-directed mutagenesis and chimeric receptors were evaluated in functional calcium mobilization assays to compare orthosteric agonists, acetylcholine and xanomeline, M1 allosteric agonists AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride), TBPB (1-[1'-(2-methylbenzyl)-1,4'-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one), and the clozapine metabolite N-desmethylclozapine. A minimal epitope has been defined for AC-42 that comprises the first 45 amino acids, the third extracellular loop, and seventh transmembrane domain (Mol Pharmacol 61:1297-1302, 2002). Using chimeric M1 and M3 receptor constructs, the AC-42 minimal epitope has been extended to also include transmembrane II. Phe77 was identified as a critical residue for maintenance of AC-42 and TBPB agonist activity. In contrast, the functional activity of N-desmethylclozapine did not require Phe77. To further map the binding site of AC-42, TBPB, and N-desmethylclozapine, point mutations previously reported to affect activities of M1 orthosteric agonists and antagonists were studied. Docking into an M1 receptor homology model revealed that AC-42 and TBPB share a similar binding pocket adjacent to the orthosteric binding site at the opposite face of Trp101. In contrast, the activity of N-desmethylclozapine was generally unaffected by the point mutations studied, and the docking indicated that N-desmethylclozapine bound to a site distinct from AC-42 and TBPB overlapping with the orthosteric site. These results suggest that structurally diverse allosteric agonists AC-42, TBPB, and N-desmethylclozapine may interact with different subsets of residues, supporting the hypothesis that M1 receptor activation can occur through at least three different binding domains.

  9. HIV-1 variants with a single-point mutation in the gp41 pocket region exhibiting different susceptibility to HIV fusion inhibitors with pocket- or membrane-binding domain.

    PubMed

    Lu, Lu; Tong, Pei; Yu, Xiaowen; Pan, Chungen; Zou, Peng; Chen, Ying-Hua; Jiang, Shibo

    2012-12-01

    Enfuvirtide (T20), the first FDA-approved peptide HIV fusion/entry inhibitor derived from the HIV-1 gp41 C-terminal heptad-repeat (CHR) domain, is believed to share a target with C34, another well-characterized CHR-peptide, by interacting with the gp41 N-terminal heptad-repeat (NHR) to form six-helix bundle core. However, our previous studies showed that T20 mainly interacts with the N-terminal region of the NHR (N-NHR) and lipid membranes, while C34 mainly binds to the NHR C-terminal pocket region. But so far, no one has shown that C34 can induce drug-resistance mutation in the gp41 pocket region. In this study, we constructed pseudoviruses in which the Ala at the position of 67 in the gp41 pocket region was substituted with Asp, Gly or Ser, respectively, and found that these mutations rendered the viruses highly resistant to C34, but sensitive to T20. The NHR-peptide N36 with mutations of A67 exhibited reduced anti-HIV-1 activity and decreased α-helicity. The stability of six-helix bundle formed by C34 and N36 with A67 mutations was significantly lower than that formed by C34 and N36 with wild-type sequence. The combination of C34 and T20 resulted in potent synergistic anti-HIV-1 effect against the viruses with mutations in either N- or C-terminal region in NHR. These results suggest that C34 with a pocket-binding domain and T20 containing the N-NHR- and membrane-binding domains inhibit HIV-1 fusion by interacting with different target sites and the combinatorial use of C34 and T20 is expected to be effective against HIV-1 variants resistant to HIV fusion inhibitors.

  10. Probing the ATP-Binding Pocket of Protein Kinase DYRK1A with Benzothiazole Fragment Molecules.

    PubMed

    Rothweiler, Ulli; Stensen, Wenche; Brandsdal, Bjørn Olav; Isaksson, Johan; Leeson, Frederick Alan; Engh, Richard Alan; Svendsen, John S Mjøen

    2016-11-10

    DYRK1A has emerged as a potential target for therapies of Alzheimer's disease using small molecules. On the basis of the observation of selective DYRK1A inhibition by firefly d-luciferin, we have explored static and dynamic structural properties of fragment sized variants of the benzothiazole scaffold with respect to DYRK1A using X-ray crystallography and NMR techniques. The compounds have excellent ligand efficiencies and show a remarkable diversity of binding modes in dynamic equilibrium. Binding geometries are determined in part by interactions often considered "weak", including "orthogonal multipolar" types represented by, for example, F-CO, sulfur-aromatic, and halogen-aromatic interactions, together with hydrogen bonds that are modulated by variation of electron withdrawing groups. These studies show how the benzothiazole scaffold is highly promising for the development of therapeutic DYRK1A inhibitors. In addition, the subtleties of the binding interactions, including dynamics, show how full structural studies are required to fully interpret the essential physical determinants of binding.

  11. A Transient Interaction between the Phosphate Binding Loop and Switch I Contributes to the Allosteric Network between Receptor and Nucleotide in Gαi1*

    PubMed Central

    Thaker, Tarjani M.; Sarwar, Maruf; Preininger, Anita M.; Hamm, Heidi E.; Iverson, T. M.

    2014-01-01

    Receptor-mediated activation of the Gα subunit of heterotrimeric G proteins requires allosteric communication between the receptor binding site and the guanine nucleotide binding site, which are separated by >30 Å. Structural changes in the allosteric network connecting these sites are predicted to be transient in the wild-type Gα subunit, making studies of these connections challenging. In the current work, site-directed mutants that alter the energy barriers between the activation states are used as tools to better understand the transient features of allosteric signaling in the Gα subunit. The observed differences in relative receptor affinity for intact Gαi1 subunits versus C-terminal Gαi1 peptides harboring the K345L mutation are consistent with this mutation modulating the allosteric network in the protein subunit. Measurement of nucleotide exchange rates, affinity for metarhodopsin II, and thermostability suggest that the K345L Gαi1 variant has reduced stability in both the GDP-bound and nucleotide-free states as compared with wild type but similar stability in the GTPγS-bound state. High resolution x-ray crystal structures reveal conformational changes accompanying the destabilization of the GDP-bound state. Of these, the conformation for Switch I was stabilized by an ionic interaction with the phosphate binding loop. Further site-directed mutagenesis suggests that this interaction between Switch I and the phosphate binding loop is important for receptor-mediated nucleotide exchange in the wild-type Gαi1 subunit. PMID:24596087

  12. Fully activated MEK1 exhibits compromised affinity for binding of allosteric inhibitors U0126 and PD0325901.

    PubMed

    Sheth, Payal R; Liu, Yuqi; Hesson, Thomas; Zhao, Jia; Vilenchik, Lev; Liu, Yan-Hui; Mayhood, Todd W; Le, Hung V

    2011-09-20

    Kinases catalyze the transfer of γ-phosphate from ATP to substrate protein residues triggering signaling pathways responsible for a plethora of cellular events. Isolation and production of homogeneous preparations of kinases in their fully active forms is important for accurate in vitro measurements of activity, stability, and ligand binding properties of these proteins. Previous studies have shown that MEK1 can be produced in its active phosphorylated form by coexpression with RAF1 in insect cells. In this study, using activated MEK1 produced by in vitro activation by RAF1 (pMEK1(in vitro)), we demonstrate that the simultaneous expression of RAF1 for production of activated MEK1 does not result in stoichiometric phosphorylation of MEK1. The pMEK1(in vitro) showed higher specific activity toward ERK2 protein substrate compared to the pMEK1 that was activated via coexpression with RAF1 (pMEK1(in situ)). The two pMEK1 preparations showed quantitative differences in the phosphorylation of T-loop residue serine 222 by Western blotting and mass spectrometry. Finally, pMEK1(in vitro) showed marked differences in the ligand binding properties compared to pMEK1(in situ). Contrary to previous findings, pMEK1(in vitro) bound allosteric inhibitors U0126 and PD0325901 with a significantly lower affinity than pMEK1(in situ) as well as its unphosphorylated counterpart (npMEK1) as demonstrated by thermal-shift, AS-MS, and calorimetric studies. The differences in inhibitor binding affinity provide direct evidence that unphosphorylated and RAF1-phosphorylated MEK1 form distinct inhibitor sites.

  13. Investigation of the free energy profiles of amantadine and rimantadine in the AM2 binding pocket.

    PubMed

    Van Nguyen, Hung; Nguyen, Hieu Thanh; Le, Ly Thi

    2016-01-01

    The purpose of this work was to study the mechanism of drug resistance of M2 channel proteins by analyzing the interactions between the drugs amantadine and rimantadine and M2 channel proteins (including the wild type and the three mutants V27A, S31N, and G34A) and the drug binding pathways, by use of a computational approach. Our results showed that multiple drug-binding sites were present in the M2 channel, and the trajectory of the drugs through the M2 channel was determined. A novel method was developed to investigate of free energy profiles of the ligand-protein complexes. Our work provides a new explanation of the large amount of experimental data on drug efficacy.

  14. A conserved NAD(+) binding pocket that regulates protein-protein interactions during aging.

    PubMed

    Li, Jun; Bonkowski, Michael S; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P; Ling, Alvin J Y; Rajman, Luis A; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L; Steegborn, Clemens; Sinclair, David A

    2017-03-24

    DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD(+) (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD(+) to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD(+) concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD(+) Thus, NAD(+) directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging.

  15. Allosteric communication between the nucleotide binding domains of caseinolytic peptidase B.

    PubMed

    Fernández-Higuero, José Ángel; Acebrón, Sergio P; Taneva, Stefka G; Del Castillo, Urko; Moro, Fernando; Muga, Arturo

    2011-07-22

    ClpB is a hexameric chaperone that solubilizes and reactivates protein aggregates in cooperation with the Hsp70/DnaK chaperone system. Each of the identical protein monomers contains two nucleotide binding domains (NBD), whose ATPase activity must be coupled to exert on the substrate the mechanical work required for its reactivation. However, how communication between these sites occurs is at present poorly understood. We have studied herein the affinity of each of the NBDs for nucleotides in WT ClpB and protein variants in which one or both sites are mutated to selectively impair nucleotide binding or hydrolysis. Our data show that the affinity of NBD2 for nucleotides (K(d) = 3-7 μm) is significantly higher than that of NBD1. Interestingly, the affinity of NBD1 depends on nucleotide binding to NBD2. Binding of ATP, but not ADP, to NBD2 increases the affinity of NBD1 (the K(d) decreases from ≈160-300 to 50-60 μm) for the corresponding nucleotide. Moreover, filling of the NBD2 ring with ATP allows the cooperative binding of this nucleotide and substrates to the NBD1 ring. Data also suggest that a minimum of four subunits cooperate to bind and reactivate two different aggregated protein substrates.

  16. Biophysical probes reveal a “compromise” nature of the methyllysine binding pocket in L3MBTL1

    PubMed Central

    Gao, Cen; Herold, J. Martin; Kireev, Dmitri; Wigle, Tim; Norris, Jacqueline L.; Frye, Stephen

    2011-01-01

    Histone lysine methylation (Kme) encodes essential information modulating many biological processes including gene expression and transcriptional regulation. However, the atomic-level recognition mechanisms of methylated histones by their respective adaptor proteins are still elusive. For instance, it is unclear how L3MBTL1, a methyl-lysine histone code reader, recognizes equally well both mono- and di-methyl marks, but ignores unmodified and trimethylated lysine residues. We made use of Molecular Dynamics (MD) and Free Energy Perturbation (FEP) techniques in order to investigate the energetics and dynamics of the methyllysine recognition. Isothermal Titration Calorimetry (ITC) was employed to experimentally validate the computational findings. Both computational and experimental methods were applied to a set of designed “biophysical” probes that mimic the shape of a single lysine residue and reproduce the binding affinities of cognate histone peptides. Our results suggest that, besides forming favorable interactions, the L3MBTL1 binding pocket energetically penalizes both methylation states and has most probably evolved as a “compromise” that non-optimally fit to both mono- and di-methyl-lysine marks. PMID:21428286

  17. Biophysical probes reveal a "compromise" nature of the methyl-lysine binding pocket in L3MBTL1.

    PubMed

    Gao, Cen; Herold, J Martin; Kireev, Dmitri; Wigle, Tim; Norris, Jacqueline L; Frye, Stephen

    2011-04-13

    Histone lysine methylation (Kme) encodes essential information modulating many biological processes including gene expression and transcriptional regulation. However, the atomic-level recognition mechanisms of methylated histones by their respective adaptor proteins are still elusive. For instance, it is unclear how L3MBTL1, a methyl-lysine histone code reader, recognizes equally well both mono- and dimethyl marks but ignores unmodified and trimethylated lysine residues. We made use of molecular dynamics (MD) and free energy perturbation (FEP) techniques in order to investigate the energetics and dynamics of the methyl-lysine recognition. Isothermal titration calorimetry (ITC) was employed to experimentally validate the computational findings. Both computational and experimental methods were applied to a set of designed "biophysical" probes that mimic the shape of a single lysine residue and reproduce the binding affinities of cognate histone peptides. Our results suggest that, besides forming favorable interactions, the L3MBTL1 binding pocket energetically penalizes both methylation states and has most probably evolved as a "compromise" that nonoptimally fits to both mono- and dimethyl-lysine marks.

  18. Fluorescence energy-transfer measurements between the calcium binding site and the specificity pocket of bovine trypsin using lanthanide probes.

    PubMed

    Darnall, D W; Abbott, F; Gomez, J E; Birnbaum, E R

    1976-11-16

    Using fluorescence energy-transfer experiments we have measured the distance between the specificity pocket and the calcium ion binding site of bovine pancreatic trypsin. Proflavin and thionine were used to block the specificity site, whereas various lanthanide ions were substituted for the calcium. It was then possible to choose various donor-acceptor pairs which exhibit suitable energy transfer. We have calculated the distance between proflavin and Nd(III), Pr(III), and Ho(III) to be 10.9, and 10.3, and 10.3 A, respectively. This agrees very well with the value of approximately 10 A we obtained between the methyl protons of p-toluamidine (a competitive inhibitor) and Gd(III) using nuclear magnetic resonance techniques (Abbott, F., Gomez, J.E., Birnbaum, E.R., and Darnall, D.W. (1975), Biochemistry 14, 4935). This is strong evidence that, in solution, the calcium binding site is composed of the side chains of Ser-190 and Asp-194.

  19. Residues remote from the binding pocket control the antagonist selectivity towards the corticotropin-releasing factor receptor-1

    NASA Astrophysics Data System (ADS)

    Sun, Xianqiang; Cheng, Jianxin; Wang, Xu; Tang, Yun; Ågren, Hans; Tu, Yaoquan

    2015-01-01

    The corticotropin releasing factors receptor-1 and receptor-2 (CRF1R and CRF2R) are therapeutic targets for treating neurological diseases. Antagonists targeting CRF1R have been developed for the potential treatment of anxiety disorders and alcohol addiction. It has been found that antagonists targeting CRF1R always show high selectivity, although CRF1R and CRF2R share a very high rate of sequence identity. This has inspired us to study the origin of the selectivity of the antagonists. We have therefore built a homology model for CRF2R and carried out unbiased molecular dynamics and well-tempered metadynamics simulations for systems with the antagonist CP-376395 in CRF1R or CRF2R to address this issue. We found that the side chain of Tyr6.63 forms a hydrogen bond with the residue remote from the binding pocket, which allows Tyr6.63 to adopt different conformations in the two receptors and results in the presence or absence of a bottleneck controlling the antagonist binding to or dissociation from the receptors. The rotameric switch of the side chain of Tyr3566.63 allows the breaking down of the bottleneck and is a perquisite for the dissociation of CP-376395 from CRF1R.

  20. A Novel Allosteric Mechanism of NF-κB Dimerization and DNA Binding Targeted by an Anti-Inflammatory Drug

    PubMed Central

    Ashkenazi, Shaked; Plotnikov, Alexander; Bahat, Anat; Ben-Zeev, Efrat; Warszawski, Shira

    2016-01-01

    The NF-κB family plays key roles in immune and stress responses, and its deregulation contributes to several diseases. Therefore its modulation has become an important therapeutic target. Here, we used a high-throughput screen for small molecules that directly inhibit dimerization of the NF-κB protein p65. One of the identified inhibitors is withaferin A (WFA), a documented anticancer and anti-inflammatory compound. Computational modeling suggests that WFA contacts the dimerization interface on one subunit and surface residues E285 and Q287 on the other. Despite their locations far from the dimerization site, E285 and Q287 substitutions diminished both dimerization and the WFA effect. Further investigation revealed that their effects on dimerization are associated with their proximity to a conserved hydrophobic core domain (HCD) that is crucial for dimerization and DNA binding. Our findings established NF-κB dimerization as a drug target and uncovered an allosteric domain as a target of WFA action. PMID:26830231

  1. Ligand binding pocket of a novel Allatostatin receptor type C of stick insect, Carausius morosus

    PubMed Central

    Duan Sahbaz, Burcin; Sezerman, Osman Ugur; Torun, Hamdi; Birgül Iyison, Necla

    2017-01-01

    Allatostatins (AST) are neuropeptides with variable function ranging from regulation of developmental processes to the feeding behavior in insects. They exert their effects by binding to cognate GPCRs, called Allatostatin receptors (AlstR), which emerge as promising targets for pesticide design. However, AlstRs are rarely studied. This study is the first reported structural study on AlstR-AST interaction. In this work, the first C type AlstR from the stick insect Carausius morosus (CamAlstR-C) was identified and its interaction with type C AST peptide was shown to be physically consistent with the experimental results. The proposed structure of CamAlstR-C revealed a conserved motif within the third extracellular loop, which, together with the N-terminus is essential for ligand binding. In this work, computational studies were combined with molecular and nano-scale approaches in order to introduce an unknown GPCR-ligand system. Consequently, the data obtained provided a reliable target region for future agonist/inverse agonist studies on AlstRs. PMID:28117376

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

    PubMed Central

    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

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

    PubMed

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

    2017-03-07

    Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1(R132H) mutation destabilizes an IDH1 "regulatory segment," which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

  4. Iterative refinement of a binding pocket model: active computational steering of lead optimization.

    PubMed

    Varela, Rocco; Walters, W Patrick; Goldman, Brian B; Jain, Ajay N

    2012-10-25

    Computational approaches for binding affinity prediction are most frequently demonstrated through cross-validation within a series of molecules or through performance shown on a blinded test set. Here, we show how such a system performs in an iterative, temporal lead optimization exercise. A series of gyrase inhibitors with known synthetic order formed the set of molecules that could be selected for "synthesis." Beginning with a small number of molecules, based only on structures and activities, a model was constructed. Compound selection was done computationally, each time making five selections based on confident predictions of high activity and five selections based on a quantitative measure of three-dimensional structural novelty. Compound selection was followed by model refinement using the new data. Iterative computational candidate selection produced rapid improvements in selected compound activity, and incorporation of explicitly novel compounds uncovered much more diverse active inhibitors than strategies lacking active novelty selection.

  5. Successful prediction of substrate-binding pocket in SLC17 transporter sialin.

    PubMed

    Pietrancosta, Nicolas; Anne, Christine; Prescher, Horst; Ruivo, Raquel; Sagné, Corinne; Debacker, Cécile; Bertrand, Hugues-Olivier; Brossmer, Reinhard; Acher, Francine; Gasnier, Bruno

    2012-03-30

    Secondary active transporters from the SLC17 protein family are required for excitatory and purinergic synaptic transmission, sialic acid metabolism, and renal function, and several members are associated with inherited neurological or metabolic diseases. However, molecular tools to investigate their function or correct their genetic defects are limited or absent. Using structure-activity, homology modeling, molecular docking, and mutagenesis studies, we have located the substrate-binding site of sialin (SLC17A5), a lysosomal sialic acid exporter also recently implicated in exocytotic release of aspartate. Human sialin is defective in two inherited sialic acid storage diseases and is responsible for metabolic incorporation of the dietary nonhuman sialic acid N-glycolylneuraminic acid. We built cytosol-open and lumen-open three-dimensional models of sialin based on weak, but significant, sequence similarity with the glycerol-3-phosphate and fucose permeases from Escherichia coli, respectively. Molecular docking of 31 synthetic sialic acid analogues to both models was consistent with inhibition studies. Narrowing the sialic acid-binding site in the cytosol-open state by two phenylalanine to tyrosine mutations abrogated recognition of the most active analogue without impairing neuraminic acid transport. Moreover, a pilot virtual high-throughput screening of the cytosol-open model could identify a pseudopeptide competitive inhibitor showing >100-fold higher affinity than the natural substrate. This validated model of human sialin and sialin-guided models of other SLC17 transporters should pave the way for the identification of inhibitors, glycoengineering tools, pharmacological chaperones, and fluorescent false neurotransmitters targeted to these proteins.

  6. Investigation of the binding pocket of human hematopoietic prostaglandin (PG) D2 synthase (hH-PGDS): a tale of two waters.

    PubMed

    Trujillo, John I; Kiefer, James R; Huang, Wei; Day, Jacqueline E; Moon, Joseph; Jerome, Gina M; Bono, Christine P; Kornmeier, Christine M; Williams, Melanie L; Kuhn, Cyrille; Rennie, Glen R; Wynn, Thomas A; Carron, Christopher P; Thorarensen, Atli

    2012-06-01

    The inhibition of hH-PGDS has been proposed as a potential target for the development of anti-allergic and anti-inflammatory drugs. Herein we describe our investigation of the binding pocket of this important enzyme and our observation that two water molecules bind to our inhibitors and the enzyme. A series of compounds were prepared to the probe the importance of the water molecules in determining the binding affinity of the inhibitors to the enzyme. The study provides insight into the binding requirements for the design of potent hH-PGDS inhibitors.

  7. The nucleotide switch in Cdc42 modulates coupling between the GTPase-binding and allosteric equilibria of Wiskott–Aldrich syndrome protein

    PubMed Central

    Leung, Daisy W.; Rosen, Michael K.

    2005-01-01

    The GTP/GDP nucleotide switch in Ras superfamily GTPases generally involves differential affinity toward downstream effectors, with the GTP-bound state having a higher affinity for effector than the GDP-bound state. We have developed a quantitative model of allosteric regulation of the Wiskott–Aldrich syndrome protein (WASP) by the Rho GTPase Cdc42 to better understand how GTPase binding is coupled to effector activation. The model accurately predicts WASP affinity for Cdc42, activity toward Arp2/3 complex, and activation by Cdc42 as functions of a two-state allosteric equilibrium in WASP. The ratio of GTPase affinities for the inactive and active states of WASP is appreciably larger for Cdc42–GTP than for Cdc42–GDP. The greater ability to distinguish between the two states of WASP makes Cdc42–GTP a full WASP agonist, whereas Cdc42–GDP is only a partial agonist. Thus, the nucleotide switch controls not only the affinity of Cdc42 for its effector but also the efficiency of coupling between the Cdc42-binding and allosteric equilibria in WASP. This effect can ensure high fidelity and specificity in Cdc42 signaling in crowded membrane environments. PMID:15821030

  8. Alternative binding proteins: anticalins - harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities.

    PubMed

    Skerra, Arne

    2008-06-01

    Antibodies are the paradigm for binding proteins, with their hypervariable loop region supported by a structurally rigid framework, thus providing the vast repertoire of antigen-binding sites in the immune system. Lipocalins are another family of proteins that exhibit a binding site with high structural plasticity, which is composed of four peptide loops mounted on a stable beta-barrel scaffold. Using site-directed random mutagenesis and selection via phage display against prescribed molecular targets, it is possible to generate artificial lipocalins with novel ligand specificities, so-called anticalins. Anticalins have been successfully selected both against small hapten-like compounds and against large protein antigens and they usually possess high target affinity and specificity. Their structural analysis has yielded interesting insights into the phenomenon of molecular recognition. Compared with antibodies, they are much smaller, have a simpler molecular architecture (comprising just one polypeptide chain) and they do not require post-translational modification. In addition, anticalins exhibit robust biophysical properties and can easily be produced in microbial expression systems. As their structure-function relationships are well understood, rational engineering of additional features such as site-directed pegylation or fusion with functional effector domains, dimerization modules or even with another anticalin, can be readily achieved. Thus, anticalins offer many applications, not only as reagents for biochemical research but also as a new class of potential drugs for medical therapy.

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

    PubMed Central

    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 positions, we find high ATP affinities for both Hb isoforms, suggesting an alternative and stronger binding site for ATP. The high ATP affinities indicate that, although ATP levels decrease in red blood cells of turtles acclimating to anoxia, the O2 affinity would remain largely unchanged, as confirmed by O2-binding measurements of untreated hemolysates from normoxic and anoxic turtles. Thus, the increase in blood-O2 affinity that accompanies winter acclimation is mainly attributable to a decrease in temperature rather than in concentrations of organic phosphates. This is the first extensive study on freshwater turtle Hb isoforms, providing molecular evidence for adaptive changes in O2 transport associated with acclimation to severe hypoxia. PMID:23986362

  10. Human farnesyl pyrophosphate synthase is allosterically inhibited by its own product

    PubMed Central

    Park, Jaeok; Zielinski, Michal; Magder, Alexandr; Tsantrizos, Youla S.; Berghuis, Albert M.

    2017-01-01

    Farnesyl pyrophosphate synthase (FPPS) is an enzyme of the mevalonate pathway and a well-established therapeutic target. Recent research has focused around a newly identified druggable pocket near the enzyme's active site. Pharmacological exploitation of this pocket is deemed promising; however, its natural biological function, if any, is yet unknown. Here we report that the product of FPPS, farnesyl pyrophosphate (FPP), can bind to this pocket and lock the enzyme in an inactive state. The Kd for this binding is 5–6 μM, within a catalytically relevant range. These results indicate that FPPS activity is sensitive to the product concentration. Kinetic analysis shows that the enzyme is inhibited through FPP accumulation. Having a specific physiological effector, FPPS is a bona fide allosteric enzyme. This allostery offers an exquisite mechanism for controlling prenyl pyrophosphate levels in vivo and thus contributes an additional layer of regulation to the mevalonate pathway. PMID:28098152

  11. A strategy to identify linker-based modules for the allosteric regulation of antibody-antigen binding affinities of different scFvs

    PubMed Central

    Thie, Holger

    2017-01-01

    ABSTRACT Antibody single-chain variable fragments (scFvs) are used in a variety of applications, such as for research, diagnosis and therapy. Essential for these applications is the extraordinary specificity, selectivity and affinity of antibody paratopes, which can also be used for efficient protein purification. However, this use is hampered by the high affinity for the protein to be purified because harsh elution conditions, which may impair folding, integrity or viability of the eluted biomaterials, are typically required. In this study, we developed a strategy to obtain structural elements that provide allosteric modulation of the affinities of different antibody scFvs for their antigen. To identify suitable allosteric modules, a complete set of cyclic permutations of calmodulin variants was generated and tested for modulation of the affinity when substituting the linker between VH and VL. Modulation of affinity induced by addition of different calmodulin-binding peptides at physiologic conditions was demonstrated for 5 of 6 tested scFvs of different specificities and antigens ranging from cell surface proteins to haptens. In addition, a variety of different modulator peptides were tested. Different structural solutions were found in respect of the optimal calmodulin permutation, the optimal peptide and the allosteric effect for scFvs binding to different antigen structures. Significantly, effective linker modules were identified for scFvs with both VH-VL and VL-VH architecture. The results suggest that this approach may offer a rapid, paratope-independent strategy to provide allosteric regulation of affinity for many other antibody scFvs. PMID:28055297

  12. Aromatic amino acid mutagenesis at the substrate binding pocket of Yarrowia lipolytica lipase Lip2 affects its activity and thermostability.

    PubMed

    Wang, Guilong; Liu, Zimin; Xu, Li; Yan, Yunjun

    2014-01-01

    The lipase2 from Yarrowia lipolytica (YLLip2) is a yeast lipase exhibiting high homologous to filamentous fungal lipase family. Though its crystal structure has been resolved, its structure-function relationship has rarely been reported. By contrast, there are two amino acid residues (V94 and I100) with significant difference in the substrate binding pocket of YLLip2; they were subjected to site-directed mutagenesis (SDM) to introduce aromatic amino acid mutations. Two mutants (V94W and I100F) were created. The enzymatic properties of the mutant lipases were detected and compared with the wild-type. The activities of mutant enzymes dropped to some extent towards p-nitrophenyl palmitate (pNPC16) and their optimum temperature was 35°C, which was 5°C lower than that of the wild-type. However, the thermostability of I100F increased 22.44% after incubation for 1 h at 40°C and its optimum substrate shifted from p-nitrophenyl laurate (pNPC12) to p-nitrophenyl caprate (pNPC10). The above results demonstrated that the two substituted amino acid residuals have close relationship with such enzymatic properties as thermostability and substrate selectivity.

  13. Isocyanide binding kinetics to monomeric hemoproteins. A study on the ligand partition between solvent and heme pocket.

    PubMed Central

    Di Iorio, E E; Winterhalter, K H; Giacometti, G M

    1987-01-01

    The kinetics of methyl-, ethyl-, iso-propyl-, and ter-butyl-isocyanide binding to Aplysia limacina myoglobin (distal His----Lys) and the isolated beta chains from hemoglobin Zurich (distal His----Arg) have been investigated by flash photolysis at various temperatures above 0 degrees C. Sperm whale (Physter catodon) myoglobin and the isolated beta chains from normal adult hemoglobin have been used as references. In most reaction systems investigated the apparent extent of photolysis increases with temperature. For sperm whale myoglobin and the normal beta chains the increase is of the same magnitude and not correlated to the type of ligand used. On the contrary, for the two proteins lacking the distal histidine, the phenomenon is dependent on the size of the alkyl side chain of the ligand. The results, analyzed on the basis of the multibarrier model (Austin, R.H., K.W. Beeson, L. Eisenstein, H. Frauenfelder, and I.C. Gunsalus, 1975, Biochemistry, 16:5355-5373), suggest that the partition of the ligand molecules between the solvent and the heme pocket, occurring during the photolysis process, is primarily determined by interactions between the ligand and residues in the heme cavity rather than by diffusion through the protein matrix. PMID:3567310

  14. Structure-Based Design of a Novel SMYD3 Inhibitor that Bridges the SAM-and MEKK2-Binding Pockets.

    PubMed

    Van Aller, Glenn S; Graves, Alan P; Elkins, Patricia A; Bonnette, William G; McDevitt, Patrick J; Zappacosta, Francesca; Annan, Roland S; Dean, Tony W; Su, Dai-Shi; Carpenter, Christopher L; Mohammad, Helai P; Kruger, Ryan G

    2016-05-03

    SMYD3 is a lysine methyltransferase overexpressed in colorectal, breast, prostate, and hepatocellular tumors, and has been implicated as an oncogene in human malignancies. Methylation of MEKK2 by SMYD3 is important for regulation of the MEK/ERK pathway, suggesting the possibility of selectively targeting SMYD3 in RAS-driven cancers. Structural and kinetic characterization of SMYD3 was undertaken leading to a co-crystal structure of SMYD3 with a MEKK2-peptide substrate bound, and the observation that SMYD3 follows a partially processive mechanism. These insights allowed for the design of GSK2807, a potent and selective, SAM-competitive inhibitor of SMYD3 (Ki = 14 nM). A high-resolution crystal structure reveals that GSK2807 bridges the gap between the SAM-binding pocket and the substrate lysine tunnel of SMYD3. Taken together, our data demonstrate that small-molecule inhibitors of SMYD3 can be designed to prevent methylation of MEKK2 and these could have potential use as anticancer therapeutics.

  15. Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

    PubMed Central

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

    2017-01-01

    Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands. PMID:28091608

  16. Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

    NASA Astrophysics Data System (ADS)

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

    2017-01-01

    Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.

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

    PubMed Central

    Bai, Qifeng; Yao, Xiaojun

    2016-01-01

    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. PMID:26887338

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

    NASA Astrophysics Data System (ADS)

    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.

  19. H3K4me3 induces allosteric conformational changes in the DNA-binding and catalytic regions of the V(D)J recombinase

    PubMed Central

    Bettridge, John; Na, Chan Hyun; Desiderio, Stephen

    2017-01-01

    V(D)J recombination is initiated by the recombination-activating gene (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with histone modifications characteristic of active chromatin, including trimethylation of histone H3 at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 stimulates substrate binding and catalysis, which are functions of RAG-1. This has suggested an allosteric mechanism in which information regarding occupancy of the RAG-2 PHD is transmitted to RAG-1. To determine whether the conformational distribution of RAG is altered by H3K4me3, we mapped changes in solvent accessibility of cysteine thiols by differential isotopic chemical footprinting. Binding of H3K4me3 to the RAG-2 PHD induces conformational changes in RAG-1 within a DNA-binding domain and in the ZnH2 domain, which acts as a scaffold for the catalytic center. Thus, engagement of H3K4me3 by the RAG-2 PHD is associated with dynamic conformational changes in RAG-1, consistent with allosteric control by active chromatin. PMID:28174273

  20. Development of Photoactivatable Allosteric Modulators for the Chemokine Receptor CXCR3.

    PubMed

    Admas, Tizita Haimanot; Bernat, Viachaslau; Heinrich, Markus R; Tschammer, Nuska

    2016-03-17

    The CXCR3 receptor, a class A G protein-coupled receptor (GPCR), is involved in the regulation and trafficking of various immune cells. CXCR3 antagonists have been proposed to be beneficial for the treatment of a wide range of disorders including but not limited to inflammatory and autoimmune diseases. The structure-based design of CXCR3 ligands remains, however, hampered by a lack of structural information describing in detail the interactions between an allosteric ligand and the receptor. We designed and synthesized photoactivatable probes for the structural and functional characterization, using photoaffinity labeling followed by mass spectrometry, of the CXCR3 allosteric binding pocket of AMG 487 and RAMX3, two potent and selective CXCR3 negative allosteric modulators. Photoaffinity labeling is a common approach to elucidate binding modes of small-molecule ligands of GPCRs through the aid of photoactivatable probes that convert to extremely reactive intermediates upon photolysis. The photolabile probe N-[({1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-{1-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl}piperidin-4-yl)methyl]acetamide (10) showed significant labeling of the CXCR3 receptor (80%) in a [(3) H]RAMX3 radioligand displacement assay. Compound 10 will serve as an important tool compound for the detailed investigation of the binding pocket of CXCR3 by mass spectrometry.

  1. Mutational analysis in the glycone binding pocket of Dalbergia cochinchinensis β-glucosidase to increase catalytic efficiency toward mannosides.

    PubMed

    Ratananikom, Khakhanang; Choengpanya, Khuanjarat; Tongtubtim, Nusra; Charoenrat, Theppanya; Withers, Stephen G; Kongsaeree, Prachumporn T

    2013-05-24

    Dalcochinase and Abg are glycoside hydrolase family 1 β-glucosidases from Dalbergia cochinchinensis Pierre and Agrobacterium sp., respectively, with 35% sequence identity. However, Abg shows much higher catalytic efficiencies toward a broad range of glycone substrates than dalcochinase does, possibly due to the difference in amino acid residues around their glycone binding pockets. Site-directed mutagenesis was used to replace the amino acid residues of dalcochinase with the corresponding residues of Abg, generating three single mutants, F196H, S251V, and M369E, as well as the corresponding three double mutants and one triple mutant. Among these, the F196H mutant showed increases in catalytic efficiency toward almost all glycoside substrates tested, with the most improved catalytic efficiency being a 3-fold increase for hydrolysis of p-nitrophenyl β-D-mannoside, suggesting a preferred polar residue at this position and consistent with the presence of histidine at this position in two other GH1 glycosidases from barley and rice that prefer β-mannosides. In addition, the M369E mutation resulted in a small increase in catalytic efficiency for cleavage of p-nitrophenyl β-D-galactoside. By contrast, the multiple mutants were up to 8-fold less efficient than the recombinant wild-type dalcochinase, and displayed primarily antagonistic interactions between these residues. Thus, differences in catalytic efficiency between dalcochinase and Abg are therefore not primarily due to differences in the residues that directly contact the substrate, but derive largely from contributions from more remote residues and the overall architecture of the active site.

  2. Characterization of five fatty aldehyde dehydrogenase enzymes from Marinobacter and Acinetobacter: structural insights into the aldehyde binding pocket.

    PubMed

    Bertram, Jonathan H; Mulliner, Kalene M; Shi, Ke; Plunkett, Mary H; Nixon, Peter; Serratore, Nicholas A; Douglas, Christopher J; Aihara, Hideki; Barney, Brett M

    2017-04-07

    Enzymes involved in lipid biosynthesis and metabolism play an important role in energy conversion and storage, and in the function of structural components such as cell membranes. The fatty aldehyde dehydrogenase (FAldDH) plays a central function in the metabolism of lipid intermediates, oxidizing fatty aldehydes to the corresponding fatty acid, and competing with pathways that would further reduce the fatty aldehydes to fatty alcohols or require the fatty aldehydes to produce alkanes. In this report, the genes for four putative FAldDH enzymes from Marinobacter aquaeolei VT8 and an additional enzyme from Acinetobacter baylyi were heterologously expressed in Escherichia coli and shown to display FAldDH activity. Five enzymes (Maqu_0438, Maqu_3316, Maqu_3410, Maqu_3572 and WP_004927398) were found to act on aldehydes ranging from acetaldehyde to hexadecanal, and also acted on the unsaturated long-chain palmitoleyl and oleyl aldehydes. A comparison of the specificity of these enzymes with various aldehydes is presented. Crystallization trials yielded diffraction quality crystals of one particular FAldDH (Maqu_3316) from M. aquaeolei VT8. Crystals were independently treated with both the NAD(+) cofactor and the aldehyde substrate decanal, revealing specific details of the likely substrate binding pocket for this class of enzymes. A likely model for how the catalysis by the enzyme is accomplished is also provided.Importance: This study provides a comparison of multiple enzymes with the ability to oxidize fatty aldehydes to fatty acids, and provides a likely picture of how the fatty aldehyde and NAD(+) is bound to the enzyme to facilitate catalysis. Based on the information obtained from this structural analysis and the comparisons of specificity for the five enzymes that were characterized, correlations may be drawn to the potential roles played by specific residues within the structure.

  3. Avibactam and Class C β-Lactamases: Mechanism of Inhibition, Conservation of the Binding Pocket, and Implications for Resistance

    PubMed Central

    Johnstone, M. R.; Ross, P. L.; McLaughlin, R. E.; Olivier, N. B.

    2014-01-01

    Avibactam is a novel non-β-lactam β-lactamase inhibitor that inhibits a wide range of β-lactamases. These include class A, class C, and some class D enzymes, which erode the activity of β-lactam drugs in multidrug-resistant pathogens like Pseudomonas aeruginosa and Enterobacteriaceae spp. Avibactam is currently in clinical development in combination with the β-lactam antibiotics ceftazidime, ceftaroline fosamil, and aztreonam. Avibactam has the potential to be the first β-lactamase inhibitor that might provide activity against class C-mediated resistance, which represents a growing concern in both hospital- and community-acquired infections. Avibactam has an unusual mechanism of action: it is a covalent inhibitor that acts via ring opening, but in contrast to other currently used β-lactamase inhibitors, this reaction is reversible. Here, we present a high-resolution structure of avibactam bound to a class C β-lactamase, AmpC, from P. aeruginosa that provided insight into the mechanism of both acylation and recyclization in this enzyme class and highlighted the differences observed between class A and class C inhibition. Furthermore, variants resistant to avibactam that identified the residues important for inhibition were isolated. Finally, the structural information was used to predict effective inhibition by sequence analysis and functional studies of class C β-lactamases from a large and diverse set of contemporary clinical isolates (P. aeruginosa and several Enterobacteriaceae spp.) obtained from recent infections to understand any preexisting variability in the binding pocket that might affect inhibition by avibactam. PMID:25022578

  4. Identification of transmembrane domain 1 & 2 residues that contribute to the formation of the ligand-binding pocket of the urotensin-II receptor.

    PubMed

    Sainsily, Xavier; Cabana, Jérôme; Holleran, Brian J; Escher, Emanuel; Lavigne, Pierre; Leduc, Richard

    2014-11-15

    The vasoactive urotensin-II (UII), a cyclic undecapeptide widely distributed in cardiovascular, renal and endocrine systems, specifically binds the UII receptor (UT receptor), a G protein-coupled receptor (GPCR). The involvement of this receptor in numerous pathophysiological conditions including atherosclerosis, heart failure, hypertension, renal impairment and diabetes potentially makes it an interesting therapeutic target. To elucidate how UII binds the UT receptor through the identification of specific residues in transmembrane domains (TM) one (TM1) and two (TM2) that are involved in the formation of the receptor's binding pocket, we used the substituted-cysteine accessibility method (SCAM). Each residue of TM1 (V49((1.30)) to M76((1.57))) and TM2 (V88((2.41)) to H117((2.70))) was mutated, one by one, to a cysteine. The resulting mutants were then expressed in COS-7 cells and subsequently treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA treatment resulted in a significant binding inhibition of (125)I-UII to mutant I54C((1.35)) in TM1 and mutants Y100C((2.53)), S103C((2.56)), F106C((2.59)), I107C((2.60)), T110C((2.63)) and Y111C((2.64)) in TM2. These results identify key structural residues in TM1 and TM2 that participate in the formation of the UT receptor binding pocket. Together with previous SCAM analysis of TM3, TM4, TM5, TM6 and TM7, these results have led us to identify residues within all 7 TMs that participate in UT's binding pocket and have enabled us to propose a model of this receptor's orthosteric binding site.

  5. CORCEMA refinement of the bound ligand conformation within the protein binding pocket in reversibly forming weak complexes using STD-NMR intensities

    NASA Astrophysics Data System (ADS)

    Jayalakshmi, V.; Rama Krishna, N.

    2004-05-01

    We describe an intensity-restrained optimization procedure for refining approximate structures of ligands within the protein binding pockets using STD-NMR intensity data on reversibly forming weak complexes. In this approach, the global minimum for the bound-ligand conformation is obtained by a hybrid structure refinement method involving CORCEMA calculation of intensities and simulated annealing optimization of torsion angles of the bound ligand using STD-NMR intensities as experimental constraints and the NOE R-factor as the pseudo-energy function to be minimized. This method is illustrated using simulated STD data sets for typical carbohydrate and peptide ligands. Our procedure also allows for the optimization of side chain torsion angles of protein residues within the binding pocket. This procedure is useful in refining and improving initial models based on crystallography or computer docking or other algorithms to generate models for the bound ligand (e.g., a lead compound) within the protein binding pocket compatible with solution STD-NMR data. This method may facilitate structure-based drug design efforts.

  6. A structure-guided fragment-based approach for the discovery of allosteric inhibitors targeting the lipophilic binding site of transcription factor EthR.

    PubMed

    Surade, Sachin; Ty, Nancy; Hengrung, Narin; Lechartier, Benoit; Cole, Stewart T; Abell, Chris; Blundell, Tom L

    2014-03-01

    A structure-guided fragment-based approach was used to target the lipophilic allosteric binding site of Mycobacterium tuberculosis EthR. This elongated channel has many hydrophobic residues lining the binding site, with few opportunities for hydrogen bonding. We demonstrate that a fragment-based approach involving the inclusion of flexible fragments in the library leads to an efficient exploration of chemical space, that fragment binding can lead to an extension of the cavity, and that fragments are able to identify hydrogen-bonding opportunities in this hydrophobic environment that are not exploited in Nature. In the present paper, we report the identification of a 1 μM affinity ligand obtained by structure-guided fragment linking.

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

    PubMed Central

    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

  8. Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region

    SciTech Connect

    Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L.; Hood, Molly M.; Lord, John W.; Lu, Wei-Ping; Miller, David F.; Patt, William C.; Smith, Bryan D.; Vogeti, Lakshminarayana; Kaufman, Michael D.; Petillo, Peter A.; Wise, Scott C.; Abendroth, Jan; Chun, Lawrence; Clark, Robin; Feese, Michael; Kim, Hidong; Stewart, Lance; Flynn, Daniel L.

    2012-01-20

    Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.

  9. Pumiliotoxin B binds to a site on the voltage-dependent sodium channel that is allosterically coupled to other binding sites.

    PubMed Central

    Gusovsky, F; Rossignol, D P; McNeal, E T; Daly, J W

    1988-01-01

    Pumiliotoxin B (PTX-B), an alkaloid that has cardiotonic and myotonic activity, increases sodium influx in guinea pig cerebral cortical synaptoneurosomes. In the presence of scorpion venom (Leiurus) or purified alpha-scorpion toxin, the PTX-B-induced sodium influx is enhanced severalfold. PTX-B alone has no effect on sodium flux in N18 neuroblastoma cells but, in the presence of alpha-scorpion toxin, stimulation of sodium influx by PTX-B reaches levels comparable to that attained with the sodium channel activator veratridine. In neuroblastoma LV9 cells, a variant mutant that lacks sodium channels, neither veratridine nor PTX-B induces sodium fluxes in either the presence or absence of alpha-scorpion toxin. In synaptoneurosomes and in N18 cells, the sodium influx induced by the combination of PTX-B and alpha-scorpion toxin is inhibited by tetrodotoxin and local anesthetics. PTX-B does not interact with two of the known toxin sites on the sodium channel, as evidenced by a lack of effect on binding of [3H]saxitoxin or [3H]batrachotoxinin A benzoate to brain synaptoneurosomes. Synergistic effects on sodium influx with alpha-scorpion toxin, beta-scorpion toxin, and brevetoxin indicate that PTX-B does not interact directly with three other toxin sites on the sodium channel. Thus, PTX-B appears to activate sodium influx by interacting with yet another site on the voltage-dependent sodium channel, a site that is coupled allosterically to sites for alpha-scorpion toxin, beta-scorpion toxin, and brevetoxin. PMID:2448797

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

    PubMed

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

    2005-03-01

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

  11. Apolar distal pocket mutants of yeast cytochrome c peroxidase: hydrogen peroxide reactivity and cyanide binding of the TriAla, TriVal, and TriLeu variants.

    PubMed

    Bidwai, Anil K; Meyen, Cassandra; Kilheeney, Heather; Wroblewski, Damian; Vitello, Lidia B; Erman, James E

    2013-01-01

    Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H(2)O(2) compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H(2)O(2) and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H(2)O(2) and HCN while features that inhibit substrate ionization slow the reactions.

  12. Boronic acids as probes for investigation of allosteric modulation of the chemokine receptor CXCR3.

    PubMed

    Bernat, Viachaslau; Admas, Tizita Haimanot; Brox, Regine; Heinemann, Frank W; Tschammer, Nuska

    2014-11-21

    The chemokine receptor CXCR3 is a G protein-coupled receptor, which conveys extracellular signals into cells by changing its conformation upon agonist binding. To facilitate the mechanistic understanding of allosteric modulation of CXCR3, we combined computational modeling with the synthesis of novel chemical tools containing boronic acid moiety, site-directed mutagenesis, and detailed functional characterization. The design of boronic acid derivatives was based on the predictions from homology modeling and docking. The choice of the boronic acid moiety was dictated by its unique ability to interact with proteins in a reversible covalent way, thereby influencing conformational dynamics of target biomolecules. During the synthesis of the library we have developed a novel approach for the purification of drug-like boronic acids. To validate the predicted binding mode and to identify amino acid residues responsible for the transduction of signal through CXCR3, we conducted a site-directed mutagenesis study. With the use of allosteric radioligand RAMX3 we were able to establish the existence of a second allosteric binding pocket in CXCR3, which enables different binding modes of structurally closely related allosteric modulators of CXCR3. We have also identified residues Trp109(2.60) and Lys300(7.35) inside the transmembrane bundle of the receptor as crucial for the regulation of the G protein activation. Furthermore, we report the boronic acid 14 as the first biased negative allosteric modulator of the receptor. Overall, our data demonstrate that boronic acid derivatives represent an outstanding tool for determination of key receptor-ligand interactions and induction of ligand-biased signaling.

  13. Selective Inhibition of Mutant Isocitrate Dehydrogenase 1 (IDH1) via Disruption of a Metal Binding Network by an Allosteric Small Molecule

    PubMed Central

    Deng, Gejing; Shen, Junqing; Yin, Ming; McManus, Jessica; Mathieu, Magali; Gee, Patricia; He, Timothy; Shi, Chaomei; Bedel, Olivier; McLean, Larry R.; Le-Strat, Frank; Zhang, Ying; Marquette, Jean-Pierre; Gao, Qiang; Zhang, Bailin; Rak, Alexey; Hoffmann, Dietmar; Rooney, Eamonn; Vassort, Aurelie; Englaro, Walter; Li, Yi; Patel, Vinod; Adrian, Francisco; Gross, Stefan; Wiederschain, Dmitri; Cheng, Hong; Licht, Stuart

    2015-01-01

    Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg2+. A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme. PMID:25391653

  14. Cross-Neutralising Nanobodies Bind to a Conserved Pocket in the Hemagglutinin Stem Region Identified Using Yeast Display and Deep Mutational Scanning

    PubMed Central

    Gaiotto, Tiziano; Hufton, Simon E.

    2016-01-01

    Cross-neutralising monoclonal antibodies against influenza hemagglutinin (HA) are of considerable interest as both therapeutics and diagnostic tools. We have recently described five different single domain antibodies (nanobodies) which share this cross-neutralising activity and suggest their small size, high stability, and cleft binding properties may present distinct advantages over equivalent conventional antibodies. We have used yeast display in combination with deep mutational scanning to give residue level resolution of positions in the antibody-HA interface which are crucial for binding. In addition, we have mapped positions within HA predicted to have minimal effect on antibody binding when mutated. Our cross-neutralising nanobodies were shown to bind to a highly conserved pocket in the HA2 domain of A(H1N1)pdm09 influenza virus overlapping with the fusion peptide suggesting their mechanism of action is through the inhibition of viral membrane fusion. We also note that the epitope overlaps with that of CR6261 and F10 which are human monoclonal antibodies in clinical development as immunotherapeutics. Although all five nanobodies mapped to the same highly conserved binding pocket we observed differences in the size of the epitope footprint which has implications in comparing the relative genetic barrier each nanobody presents to a rapidly evolving influenza virus. To further refine our epitope map, we have re-created naturally occurring mutations within this HA stem epitope and tested their effect on binding using yeast display. We have shown that a D46N mutation in the HA2 stem domain uniquely interferes with binding of R2b-E8. Further testing of this substitution in the context of full length purified HA from 1918 H1N1 pandemic (Spanish flu), 2009 H1N1 pandemic (swine flu) and highly pathogenic avian influenza H5N1 demonstrated binding which correlated with D46 whereas binding to seasonal H1N1 strains carrying N46 was absent. In addition, our deep sequence

  15. Discovery and Characterization of Allosteric WNK Kinase Inhibitors.

    PubMed

    Yamada, Ken; Zhang, Ji-Hu; Xie, Xiaoling; Reinhardt, Juergen; Xie, Amy Qiongshu; LaSala, Daniel; Kohls, Darcy; Yowe, David; Burdick, Debra; Yoshisue, Hajime; Wakai, Hiromichi; Schmidt, Isabel; Gunawan, Jason; Yasoshima, Kayo; Yue, Q Kimberley; Kato, Mitsunori; Mogi, Muneto; Idamakanti, Neeraja; Kreder, Natasha; Drueckes, Peter; Pandey, Pramod; Kawanami, Toshio; Huang, Waanjeng; Yagi, Yukiko I; Deng, Zhan; Park, Hyi-Man

    2016-12-16

    Protein kinases are known for their highly conserved adenosine triphosphate (ATP)-binding site, rendering the discovery of selective inhibitors a major challenge. In theory, allosteric inhibitors can achieve high selectivity by targeting less conserved regions of the kinases, often with an added benefit of retaining efficacy under high physiological ATP concentration. Although often overlooked in favor of ATP-site directed approaches, performing a screen at high ATP concentration or stringent hit triaging with high ATP concentration offers conceptually simple methods of identifying inhibitors that bind outside the ATP pocket. Here, we applied the latter approach to the With-No-Lysine (K) (WNK) kinases to discover lead molecules for a next-generation antihypertensive that requires a stringent safety profile. This strategy yielded several ATP noncompetitive WNK1-4 kinase inhibitors, the optimization of which enabled cocrystallization with WNK1, revealing an allosteric binding mode consistent with the observed exquisite specificity for WNK1-4 kinases. The optimized compound inhibited rubidium uptake by sodium chloride cotransporter 1 (NKCC1) in HT29 cells, consistent with the reported physiology of WNK kinases in renal electrolyte handling.

  16. High affinity and temperature sensitivity of blood oxygen binding in Pangasianodon hypophthalmus due to lack of chloride-hemoglobin allosteric interaction.

    PubMed

    Damsgaard, Christian; Phuong, Le My; Huong, Do Thi Thanh; Jensen, Frank B; Wang, Tobias; Bayley, Mark

    2015-06-01

    Air-breathing fishes represent interesting organisms in terms of understanding the physiological changes associated with the terrestrialization of vertebrates, and, further, are of great socio-economic importance for aquaculture in Southeast Asia. To understand how environmental factors, such as high temperature, affect O2 transport in air-breathing fishes, this study assessed the effects of temperature on O2 binding of blood and Hb in the economically important air-breathing fish Pangasianodon hypophthalmus. To determine blood O2 binding properties, blood was drawn from resting cannulated fishes and O2 binding curves made at 25°C and 35°C. To determine the allosteric regulation and thermodynamics of Hb O2 binding, Hb was purified, and O2 equilibria were recorded at five temperatures in the absence and presence of ATP and Cl(-). Whole blood had a high O2 affinity (O2 tension at half saturation P50 = 4.6 mmHg at extracellular pH 7.6 and 25°C), a high temperature sensitivity of O2 binding (apparent heat of oxygenation ΔH(app) = -28.3 kcal/mol), and lacked a Root effect. Further, the data on Hb revealed weak ATP binding and a complete lack of Cl(-) binding to Hb, which, in part, explains the high O2 affinity and high temperature sensitivity of blood O2 binding. This study demonstrates how a potent mechanism for increasing O2 affinity is linked to increased temperature sensitivity of O2 transport and provides a basic framework for a better understanding of how hypoxia-adapted species will react to increasing temperatures.

  17. Identification of transmembrane domain 3, 4 & 5 residues that contribute to the formation of the ligand-binding pocket of the urotensin-II receptor.

    PubMed

    Sainsily, Xavier; Cabana, Jérôme; Boulais, Philip E; Holleran, Brian J; Escher, Emanuel; Lavigne, Pierre; Leduc, Richard

    2013-12-01

    Urotensin-II (UII), a cyclic undecapeptide, selectively binds the urotensin-II receptor (UT receptor), a G protein-coupled receptor (GPCR) involved in cardiovascular effects and associated with numerous pathophysiological conditions including hypertension, atherosclerosis, heart failure, pulmonary hypertension and others. In order to identify specific residues in transmembrane domains (TM) three (TM3), four (TM4) and five (TM5) that are involved in the formation of the UT receptor binding pocket, we used the substituted-cysteine accessibility method (SCAM). Each residue in the F118((3.20)) to S146((3.48)) fragment of TM3, the L168((4.44)) to G194((4.70)) fragment of TM4 and the W203((5.30)) to V232((5.59)) fragment of TM5, was mutated, individually, to a cysteine. The resulting mutants were then expressed in COS-7 cells and subsequently treated with the positively charged sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA treatment resulted in a significant reduction in the binding of (125)I-UII to TM3 mutants L126C((3.28)), F127C((3.29)), F131C((3.33)) and M134C((3.36)) and TM4 mutants M184C((4.60)) and I188C((4.64)). No loss of binding was detected following treatment by MTSEA for all TM5 mutants tested. In absence of a crystal structure of UT receptor, these results identify key determinants in TM3, TM4 and TM5 that participate in the formation of the UT receptor binding pocket and has led us to propose a homology model of the UT receptor.

  18. Pathogenicity of the BRCA1 Missense Variant M1775K is Determined by the Disruption of the BRCT Phosphopeptide-Binding Pocket: a Multi-Modal Approach

    SciTech Connect

    Tischkowitz,M.; Hamel, N.; Carvalho, M.; Birrane, G.; Soni, A.; van Beers, E.; Joosse, S.; Wong, N.; Novak, D.; et al

    2008-01-01

    A number of germ-line mutations in the BRCA1 gene confer susceptibility to breast and ovarian cancer. However, it remains difficult to determine whether many single amino-acid (missense) changes in the BRCA1 protein that are frequently detected in the clinical setting are pathologic or not. Here, we used a combination of functional, crystallographic, biophysical, molecular and evolutionary techniques, and classical genetic segregation analysis to demonstrate that the BRCA1 missense variant M1775K is pathogenic. Functional assays in yeast and mammalian cells showed that the BRCA1 BRCT domains carrying the amino-acid change M1775K displayed markedly reduced transcriptional activity, indicating that this variant represents a deleterious mutation. Importantly, the M1775K mutation disrupted the phosphopeptide-binding pocket of the BRCA1 BRCT domains, thereby inhibiting the BRCA1 interaction with the proteins BRIP1 and CtIP, which are involved in DNA damage-induced checkpoint control. These results indicate that the integrity of the BRCT phosphopeptide-binding pocket is critical for the tumor suppression function of BRCA1. Moreover, this study demonstrates that multiple lines of evidence obtained from a combination of functional, structural, molecular and evolutionary techniques, and classical genetic segregation analysis are required to confirm the pathogenicity of rare variants of disease-susceptibility genes and obtain important insights into the underlying pathogenetic mechanisms.

  19. Ligand-binding pocket shape differences between S1P1 and S1P3 determine efficiency of chemical probe identification by uHTS

    PubMed Central

    Schürer, Stephan C.; Brown, Steven J.; Cabrera, Pedro Gonzales; Schaeffer, Marie-Therese; Chapman, Jacqueline; Jo, Euijung; Chase, Peter; Spicer, Tim; Hodder, Peter; Rosen, Hugh

    2008-01-01

    We have studied the Sphingosine 1-phosphate (S1P) receptor system to better understand why certain molecular targets within a closely related family are much more tractable when identifying compelling chemical leads. Five medically important G protein-coupled receptors for S1P regulate heart rate, coronary artery caliber, endothelial barrier integrity, and lymphocyte trafficking. Selective S1P receptor agonist probes would be of great utility to study receptor subtype-specific function. Through systematic screening of the same libraries, we identified novel selective agonists chemotypes for each of the S1P1 and S1P3 receptors. uHTS for S1P1 was more effective than for S1P3, with many selective, low nanomolar hits of proven mechanism emerging for. Receptor structure modeling and ligand docking reveal differences between the receptor binding pockets, which are the basis for sub-type selectivity. Novel selective agonists interact primarily in the hydrophobic pocket of the receptor in the absence of head-group interactions. Chemistry-space and shape-based analysis of the screening libraries in combination with the binding models explain the observed differential hit rates and enhanced efficiency for lead discovery for S1P1 vs. S1P3 in this closely related receptor family. PMID:18590333

  20. Allosteric Modulation of Chemoattractant Receptors

    PubMed Central

    Allegretti, Marcello; Cesta, Maria Candida; Locati, Massimo

    2016-01-01

    Chemoattractants control selective leukocyte homing via interactions with a dedicated family of related G protein-coupled receptor (GPCR). Emerging evidence indicates that the signaling activity of these receptors, as for other GPCR, is influenced by allosteric modulators, which interact with the receptor in a binding site distinct from the binding site of the agonist and modulate the receptor signaling activity in response to the orthosteric ligand. Allosteric modulators have a number of potential advantages over orthosteric agonists/antagonists as therapeutic agents and offer unprecedented opportunities to identify extremely selective drug leads. Here, we resume evidence of allosterism in the context of chemoattractant receptors, discussing in particular its functional impact on functional selectivity and probe/concentration dependence of orthosteric ligands activities. PMID:27199992

  1. The Role of Hydration on the Mechanism of Allosteric Regulation: In Situ Measurements of the Oxygen-Linked Kinetics of Water Binding to Hemoglobin

    PubMed Central

    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

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

    PubMed

    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.

  3. Mutational Analysis of the Binding Pockets of the Diketo Acid Inhibitor L-742,001 in the Influenza Virus PA Endonuclease

    PubMed Central

    Stevaert, Annelies; Dallocchio, Roberto; Dessì, Alessandro; Pala, Nicolino; Rogolino, Dominga; Sechi, Mario

    2013-01-01

    The influenza virus PA endonuclease, which cleaves capped host pre-mRNAs to initiate synthesis of viral mRNA, is a prime target for antiviral therapy. The diketo acid compound L-742,001 was previously identified as a potent inhibitor of the influenza virus endonuclease reaction, but information on its precise binding mode to PA or potential resistance profile is limited. Computer-assisted docking of L-742,001 into the crystal structure of inhibitor-free N-terminal PA (PA-Nter) indicated a binding orientation distinct from that seen in a recent crystallographic study with L-742,001-bound PA-Nter (R. M. DuBois et al., PLoS Pathog. 8:e1002830, 2012). A comprehensive mutational analysis was performed to determine which amino acid changes within the catalytic center of PA or its surrounding hydrophobic pockets alter the antiviral sensitivity to L-742,001 in cell culture. Marked (up to 20-fold) resistance to L-742,001 was observed for the H41A, I120T, and G81F/V/T mutant forms of PA. Two- to 3-fold resistance was seen for the T20A, L42T, and V122T mutants, and the R124Q and Y130A mutants were 3-fold more sensitive to L-742,001. Several mutations situated at noncatalytic sites in PA had no or only marginal impact on the enzymatic functionality of viral ribonucleoprotein complexes reconstituted in cell culture, consistent with the less conserved nature of these PA residues. Our data provide relevant insights into the binding mode of L-742,001 in the PA endonuclease active site. In addition, we predict some potential resistance sites that should be taken into account during optimization of PA endonuclease inhibitors toward tight binding in any of the hydrophobic pockets surrounding the catalytic center of the enzyme. PMID:23824822

  4. Identification of an allosteric modulator of the serotonin transporter with novel mechanism of action.

    PubMed

    Kortagere, Sandhya; Fontana, Andreia Cristina Karklin; Rose, Deja Renée; Mortensen, Ole Valente

    2013-09-01

    Serotonin transporters (SERTs) play an essential role in the termination and regulation of serotonin signaling in the brain. SERT is also the target of antidepressants and psychostimulants. Molecules with novel activities and modes of interaction with regard to SERT function are of great scientific and clinical interest. We explored structural regions outside the putative serotonin translocation pathway to identify potential binding sites for allosteric transporter modulators (ATMs). Mutational studies revealed a pocket of amino acids outside the orthosteric substrate binding sites located in the interface between extracellular loops 1 and 3 that when mutated affect transporter function. Using the structure of the bacterial transporter homolog leucine transporter as a template, we developed a structural model of SERT. We performed molecular dynamics simulations to further characterize the allosteric pocket that was identified by site-directed mutagenesis studies and employed this pocket in a virtual screen for small-molecule modulators of SERT function. In functional transport assays, we found that one of the identified molecules, ATM7, increased the reuptake of serotonin, possibly by facilitating the interaction of serotonin with transport-ready conformations of SERT when concentrations of serotonin were low and rate limiting. In addition, ATM7 potentiates 3,4-methylenedioxy-N-methylamphetamine (MDMA, "Ecstasy")-induced reversed transport by SERT. Taking advantage of a conformationally sensitive residue in transmembrane domain 6, we demonstrate that ATM7 mechanistically stabilizes an outward-facing conformation of SERT. Taken together these observations demonstrate that ATM7 acts through a novel mechanism that involves allosteric modulation of SERT function.

  5. Allosteric modulation of [3H]-CGP39653 binding through the glycine site of the NMDA receptor: further studies in rat and human brain

    PubMed Central

    Mugnaini, Manolo; Meoni, Paolo; Bunnemann, Bernd; Corsi, Mauro; Bowery, Norman G

    2001-01-01

    Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP39653), a selective antagonist at the glutamate site of the NMDA receptor, is modulated by glycine in rat brain tissue. We have further investigated this phenomenon in rodent and human brain by means of receptor binding and quantitative autoradiography techniques.In rat cerebral cortical membranes the glycine antagonist 3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloro-indole-2-carboxylic acid sodium salt (GV150526A) did not change basal [3H]-CGP39653 binding, but competitively reversed the high affinity component of [3H]-CGP39653 binding inhibition by glycine, with a pKB value of 8.38, in line with its affinity for the glycine site (pKi=8.49 vs [3H]-glycine). Glycine (10 μM) significantly decreased [3H]-CGP39653 affinity for the NMDA receptor (with no change in the Bmax), whereas enhanced L-glutamate affinity (P<0.05, paired-samples Student's t-test).In rat brain sections the addition of GV150526A (30 μM) to the incubation medium increased [3H]-CGP39653 binding to 208% of control (average between areas), indicating the presence of endogenous glycine. The enhancement presented significant regional differences (P<0.05, two-way ANOVA), with striatum higher than cerebral cortex (282 and 187% of control, respectively; P<0.05, Fisher's LSD). On the contrary, there was not any significant variation in affinity values of [3H]-CGP39653, L-glutamate, glycine and GV150526A in striatal and cortical membranes. These results confirmed the existence of regionally distinct NMDA receptors subtypes with different glycine/glutamate allosteric modulation.Whole brain autoradiography revealed an uneven distribution of [3H]-CGP39653 binding sites in human brain. High levels of binding were determined in hippocampus and in cingulate, frontoparietal and insular cortex. Intermediate to low levels of binding were found in diencephalic nuclei and basal ganglia. [3H]-CGP39653 binding was increased to 216% of

  6. Binding water clusters to an aromatic-rich hydrophobic pocket: [2.2.2]paracyclophane-(H2O)n, n = 1-5.

    PubMed

    Buchanan, Evan G; Zwier, Timothy S

    2014-09-18

    [2.2.2]Paracylcophane (tricyclophane, TCP) is a macrocycle with three phenyl substituents linked by ethyl bridges (-CH2CH2-) in the para-position, forming an aromatic-rich pocket capable of binding various substituents, including nature's solvent, water. Building on previous work [Buchanan, E. G.; et al. J. Chem. Phys. 2013, 138, 064308] that reported on the ground state conformational preferences of TCP, the focus of the present study is on the infrared and ultraviolet spectroscopy of TCP-(H2O)n clusters with n = 1-5. Resonant two-photon ionization (R2PI) was used to interrogate the mass selected electronic spectrum of the clusters, reporting on the perturbations imposed on the electronic states of TCP as the size of the water clusters bound to it vary in size from n = 1-5. The TCP-(H2O)n S0-S1 origins are shifted to lower frequency from the monomer, indicating an increased binding energy of the water or water network in the excited state. Ground state resonant ion-dip infrared (RIDIR) spectra of TCP-(H2O)n (n = 1-5) clusters were recorded in the OH stretch region, which probes the H-bonded water networks present and the perturbations imposed on them by TCP. The experimental frequencies are compared with harmonic vibrational frequencies calculated using density functional theory (DFT) with the dispersion-corrected functional ωB97X-D and a 6-311+g(d,p) basis set, providing firm assignments for their H-bonding structures. The H2O molecule in TCP-(H2O)1 sits on top of the binding pocket, donating both of its hydrogen atoms to the aromatic-rich interior of the monomer. The antisymmetric stretch fundamental of H2O in the complex is composed of a closely spaced set of transitions that likely reflect contributions from both para- and ortho-forms of H2O due to internal rotation of the H2O in the binding pocket. TCP-(H2O)2 also exists in a single conformational isomer that retains the same double-donor binding motif for the first water molecule, with the second H2O acting

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

    PubMed Central

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

    2015-01-01

    Omecamtiv Mecarbil (OM) is a small molecule allosteric effector of cardiac myosin that is in clinical trials for treatment of systolic heart failure. A detailed kinetic analysis of cardiac myosin has shown that the drug accelerates phosphate release by shifting the equilibrium of the hydrolysis step towards products, leading to a faster transition from weak to strong actin-bound states. The structure of the human β-cardiac motor domain (cMD) with OM bound reveals a single OM-binding site nestled in a narrow cleft separating two domains of the human cMD where it interacts with the key residues that couple lever arm movement to the nucleotide state. In addition, OM induces allosteric changes in three strands of the β-sheet that provides the communication link between the actin-binding interface and the nucleotide pocket. The OM-binding interactions and allosteric changes form the structural basis for the kinetic and mechanical tuning of cardiac myosin. PMID:26246073

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

    PubMed

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

    2015-08-06

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

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

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

    SciTech Connect

    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.

    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.

  11. Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays

    PubMed Central

    Daniels, V; Wood, M; Leclercq, K; Kaminski, R M; Gillard, M

    2013-01-01

    Background and Purpose Synaptic vesicle protein 2A (SV2A) is the specific binding site of the anti-epileptic drug levetiracetam (LEV) and its higher affinity analogue UCB30889. Moreover, the protein has been well validated as a target for anticonvulsant therapy. Here, we report the identification of UCB1244283 acting as a SV2A positive allosteric modulator of UCB30889. Experimental Approach UCB1244283 was characterized in vitro using radioligand binding assays with [3H]UCB30889 on recombinant SV2A expressed in HEK cells and on rat cortex. In vivo, the compound was tested in sound-sensitive mice. Key Results Saturation binding experiments in the presence of UCB1244283 demonstrated a fivefold increase in the affinity of [3H]UCB30889 for human recombinant SV2A, combined with a twofold increase of the total number of binding sites. Similar results were obtained on rat cortex. In competition binding experiments, UCB1244283 potentiated the affinity of UCB30889 while the affinity of LEV remained unchanged. UCB1244283 significantly slowed down both the association and dissociation kinetics of [3H]UCB30889. Following i.c.v. administration in sound-sensitive mice, UCB1244283 showed a clear protective effect against both tonic and clonic convulsions. Conclusions and Implications These results indicate that UCB1244283 can modulate the conformation of SV2A, thereby inducing a higher affinity state for UCB30889. Our results also suggest that the conformation of SV2A per se might be an important determinant of its functioning, especially during epileptic seizures. Therefore, agents that act on the conformation of SV2A might hold great potential in the search for new SV2A-based anticonvulsant therapies. PMID:23530581

  12. Binding of the sphingolipid S1P to hTERT stabilizes telomerase at the nuclear periphery by allosterically mimicking protein phosphorylation.

    PubMed

    Panneer Selvam, Shanmugam; De Palma, Ryan M; Oaks, Joshua J; Oleinik, Natalia; Peterson, Yuri K; Stahelin, Robert V; Skordalakes, Emmanuel; Ponnusamy, Suriyan; Garrett-Mayer, Elizabeth; Smith, Charles D; Ogretmen, Besim

    2015-06-16

    During DNA replication, the enzyme telomerase maintains the ends of chromosomes, called telomeres. Shortened telomeres trigger cell senescence, and cancer cells often have increased telomerase activity to promote their ability to proliferate indefinitely. The catalytic subunit, human telomerase reverse transcriptase (hTERT), is stabilized by phosphorylation. We found that the lysophospholipid sphingosine 1-phosphate (S1P), generated by sphingosine kinase 2 (SK2), bound hTERT at the nuclear periphery in human and mouse fibroblasts. Docking predictions and mutational analyses revealed that binding occurred between a hydroxyl group (C'3-OH) in S1P and Asp(684) in hTERT. Inhibiting or depleting SK2 or mutating the S1P binding site decreased the stability of hTERT in cultured cells and promoted senescence and loss of telomere integrity. S1P binding inhibited the interaction of hTERT with makorin ring finger protein 1 (MKRN1), an E3 ubiquitin ligase that tags hTERT for degradation. Murine Lewis lung carcinoma (LLC) cells formed smaller tumors in mice lacking SK2 than in wild-type mice, and knocking down SK2 in LLC cells before implantation into mice suppressed their growth. Pharmacologically inhibiting SK2 decreased the growth of subcutaneous A549 lung cancer cell-derived xenografts in mice, and expression of wild-type hTERT, but not an S1P-binding mutant, restored tumor growth. Thus, our data suggest that S1P binding to hTERT allosterically mimicks phosphorylation, promoting telomerase stability and hence telomere maintenance, cell proliferation, and tumor growth.

  13. Occupancy of the Zinc-binding Site by Transition Metals Decreases the Substrate Affinity of the Human Dopamine Transporter by an Allosteric Mechanism*

    PubMed Central

    Li, Yang; Mayer, Felix P.; Hasenhuetl, Peter S.; Burtscher, Verena; Schicker, Klaus; Sitte, Harald H.; Freissmuth, Michael; Sandtner, Walter

    2017-01-01

    The human dopamine transporter (DAT) has a tetrahedral Zn2+-binding site. Zn2+-binding sites are also recognized by other first-row transition metals. Excessive accumulation of manganese or of copper can lead to parkinsonism because of dopamine deficiency. Accordingly, we examined the effect of Mn2+, Co2+, Ni2+, and Cu2+ on transport-associated currents through DAT and DAT-H193K, a mutant with a disrupted Zn2+-binding site. All transition metals except Mn2+ modulated the transport cycle of wild-type DAT with affinities in the low micromolar range. In this concentration range, they were devoid of any action on DAT-H193K. The active transition metals reduced the affinity of DAT for dopamine. The affinity shift was most pronounced for Cu2+, followed by Ni2+ and Zn2+ (= Co2+). The extent of the affinity shift and the reciprocal effect of substrate on metal affinity accounted for the different modes of action: Ni2+ and Cu2+ uniformly stimulated and inhibited, respectively, the substrate-induced steady-state currents through DAT. In contrast, Zn2+ elicited biphasic effects on transport, i.e. stimulation at 1 μm and inhibition at 10 μm. A kinetic model that posited preferential binding of transition metal ions to the outward-facing apo state of DAT and a reciprocal interaction of dopamine and transition metals recapitulated all experimental findings. Allosteric activation of DAT via the Zn2+-binding site may be of interest to restore transport in loss-of-function mutants. PMID:28096460

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

    SciTech Connect

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

    2016-12-07

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

  15. Allosteric regulation of helicase core activities of the DEAD-box helicase YxiN by RNA binding to its RNA recognition motif.

    PubMed

    Samatanga, Brighton; Andreou, Alexandra Z; Klostermeier, Dagmar

    2017-01-23

    DEAD-box proteins share a structurally similar core of two RecA-like domains (RecA_N and RecA_C) that contain the conserved motifs for ATP-dependent RNA unwinding. In many DEAD-box proteins the helicase core is flanked by ancillary domains. To understand the regulation of the DEAD-box helicase YxiN by its C-terminal RNA recognition motif (RRM), we investigated the effect of RNA binding to the RRM on its position relative to the core, and on core activities. RRM/RNA complex formation substantially shifts the RRM from a position close to the RecA_C to the proximity of RecA_N, independent of RNA contacts with the core. RNA binding to the RRM is communicated to the core, and stimulates ATP hydrolysis and RNA unwinding. The conformational space of the core depends on the identity of the RRM-bound RNA. Allosteric regulation of core activities by RNA-induced movement of ancillary domains may constitute a general regulatory mechanism of DEAD-box protein activity.

  16. The crystal structure of avian CD1 reveals a smaller, more primordial antigen-binding pocket compared to mammalian CD1

    PubMed Central

    Zajonc, Dirk M.; Striegl, Harald; Dascher, Christopher C.; Wilson, Ian A.

    2008-01-01

    The molecular details of glycolipid presentation by CD1 antigen-presenting molecules are well studied in mammalian systems. However, little is known about how these non-classical MHC class I (MHCI) molecules diverged from the MHC locus to create a more complex, hydrophobic binding groove that binds lipids rather than peptides. To address this fundamental question, we have determined the crystal structure of an avian CD1 (chCD1–2) that shares common ancestry with mammalian CD1 from ≈310 million years ago. The chCD1–2 antigen-binding site consists of a compact, narrow, central hydrophobic groove or pore rather than the more open, 2-pocket architecture observed in mammalian CD1s. Potential antigens then would be restricted in size to single-chain lipids or glycolipids. An endogenous ligand, possibly palmitic acid, serves to illuminate the mode and mechanism of ligand interaction with chCD1–2. The palmitate alkyl chain is inserted into the relatively shallow hydrophobic pore; its carboxyl group emerges at the receptor surface and is stabilized by electrostatic and hydrogen bond interactions with an arginine residue that is conserved in all known CD1 proteins. In addition, other novel features, such as an A′ loop that interrupts and segments the normally long, continuous α1 helix, are unique to chCD1–2 and contribute to the unusually narrow binding groove, thereby limiting its size. Because birds and mammals share a common ancestor, but the rate of evolution is slower in birds than in mammals, the chCD1–2-binding groove probably represents a more primordial CD1-binding groove. PMID:19004781

  17. The N-terminal peptide of mammalian GTP cyclohydrolase I is an autoinhibitory control element and contributes to binding the allosteric regulatory protein GFRP.

    PubMed

    Higgins, Christina E; Gross, Steven S

    2011-04-08

    GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for biosynthesis of tetrahydrobiopterin (BH4), an obligate cofactor for NO synthases and aromatic amino acid hydroxylases. BH4 can limit its own synthesis by triggering decameric GTPCH to assemble in an inhibitory complex with two GTPCH feedback regulatory protein (GFRP) pentamers. Subsequent phenylalanine binding to the GTPCH·GFRP inhibitory complex converts it to a stimulatory complex. An N-terminal inhibitory peptide in GTPCH may also contribute to autoregulation of GTPCH activity, but mechanisms are undefined. To characterize potential regulatory actions of the N-terminal peptide in rat GTPCH, we expressed, purified, and characterized a truncation mutant, devoid of 45 N-terminal amino acids (Δ45-GTPCH) and contrasted its catalytic and GFRP binding properties to wild type GTPCH (wt-GTPCH). Contrary to prior reports, we show that GFRP binds wt-GTPCH in the absence of any small molecule effector, resulting in allosteric stimulation of GTPCH activity: a 20% increase in Vmax, 50% decrease in KmGTP, and increase in Hill coefficient to 1.6, from 1.0. These features of GFRP-stimulated wt-GTPCH activity were phenocopied by Δ45-GTPCH in the absence of bound GFRP. Addition of GFRP to Δ45-GTPCH failed to elicit complex formation or a substantial further increase in GTPCH catalytic activity. Expression of Δ45-GTPCH in HEK-293 cells elicited 3-fold greater BH4 accumulation than an equivalent of wt-GTPCH. Together, results indicate that the N-terminal peptide exerts autoinhibitory control over rat GTPCH and is required for GFRP binding on its own. Displacement of the autoinhibitory peptide provides a molecular mechanism for physiological up-regulation of GTPCH activity.

  18. Structural Asymmetry of Phosphodiesterase-9A and a Unique Pocket for Selective Binding of a Potent Enantiomeric Inhibitor

    PubMed Central

    Huang, Manna; Shao, Yongxian; Hou, Jianying; Cui, Wenjun; Liang, Beibei; Huang, Yingchun; Li, Zhe; Wu, Yinuo; Zhu, Xinhai; Liu, Peiqing

    2015-01-01

    Phosphodiesterase-9 (PDE9) inhibitors have been studied as potential therapeutics for treatment of central nervous system diseases and diabetes. Here, we report the discovery of a new category of PDE9 inhibitors by rational design on the basis of the crystal structures. The best compound, (S)-6-((1-(4-chlorophenyl)ethyl)amino)-1-cyclopentyl-1,5,6,7-tetrahydro-4H-pyrazolo[3,4-day]pyrimidin-4-one [(S)-C33], has an IC50 value of 11 nM against PDE9 and the racemic C33 has bioavailability of 56.5% in the rat pharmacokinetic model. The crystal structures of PDE9 in the complex with racemic C33, (R)-C33, and (S)-C33 reveal subtle conformational asymmetry of two M-loops in the PDE9 dimer and different conformations of two C33 enantiomers. The structures also identified a small hydrophobic pocket that interacts with the tyrosyl tail of (S)-C33 but not with (R)-C33, and is thus possibly useful for improvement of selectivity of PDE9 inhibitors. The asymmetry of the M-loop and the different interactions of the C33 enantiomers imply the necessity to consider the whole PDE9 dimer in the design of inhibitors. PMID:26316540

  19. Pharmacological and molecular characterization of the positive allosteric modulators of metabotropic glutamate receptor 2.

    PubMed

    Lundström, L; Bissantz, C; Beck, J; Dellenbach, M; Woltering, T J; Wichmann, J; Gatti, S

    2017-02-16

    The metabotropic glutamate receptor 2 (mGlu2) plays an important role in the presynaptic control of glutamate release and several mGlu2 positive allosteric modulators (PAMs) have been under assessment for their potential as antipsychotics. The binding mode of mGlu2 PAMs is better characterized in functional terms while few data are available on the relationship between allosteric and orthosteric binding sites. Pharmacological studies characterizing binding and effects of two different chemical series of mGlu2 PAMs are therefore carried out here using the radiolabeled mGlu2 agonist (3)[H]-LY354740 and mGlu2 PAM (3)[H]-2,2,2-TEMPS. A multidimensional approach to the PAM mechanism of action shows that mGlu2 PAMs increase the affinity of (3)[H]-LY354740 for the orthosteric site of mGlu2 as well as the number of (3)[H]-LY354740 binding sites. (3)[H]-2,2,2-TEMPS binding is also enhanced by the presence of LY354740. New residues in the allosteric rat mGlu2 binding pocket are identified to be crucial for the PAMs ligand binding, among these Tyr(3.40) and Asn(5.46). Also of remark, in the described experimental conditions S731A (Ser(5.42)) residue is important only for the mGlu2 PAM LY487379 and not for the compound PAM-1: an example of the structural differences among these mGlu2 PAMs. This study provides a summary of the information generated in the past decade on mGlu2 PAMs adding a detailed molecular investigation of PAM binding mode. Differences among mGlu2 PAM compounds are discussed as well as the mGlu2 regions interacting with mGlu2 PAM and NAM agents and residues driving mGlu2 PAM selectivity.

  20. Pharmacological and molecular characterization of the positive allosteric modulators of metabotropic glutamate receptor 2.

    PubMed

    Lundström, L; Bissantz, C; Beck, J; Dellenbach, M; Woltering, T J; Wichmann, J; Gatti, S

    2016-12-01

    The metabotropic glutamate receptor 2 (mGlu2) plays an important role in the presynaptic control of glutamate release and several mGlu2 positive allosteric modulators (PAMs) have been under assessment for their potential as antipsychotics. The binding mode of mGlu2 PAMs is better characterized in functional terms while few data are available on the relationship between allosteric and orthosteric binding sites. Pharmacological studies characterizing binding and effects of two different chemical series of mGlu2 PAMs are therefore carried out here using the radiolabeled mGlu2 agonist (3)[H]-LY354740 and mGlu2 PAM (3)[H]-2,2,2-TEMPS. A multidimensional approach to the PAM mechanism of action shows that mGlu2 PAMs increase the affinity of (3)[H]-LY354740 for the orthosteric site of mGlu2 as well as the number of (3)[H]-LY354740 binding sites. (3)[H]-2,2,2-TEMPS binding is also enhanced by the presence of LY354740. New residues in the allosteric rat mGlu2 binding pocket are identified to be crucial for the PAMs ligand binding, among these Tyr(3.40) and Asn(5.46). Also of remark, in the described experimental conditions S731A (Ser(5.42)) residue is important only for the mGlu2 PAM LY487379 and not for the compound PAM-1: an example of the structural differences among these mGlu2 PAMs. This study provides a summary of the information generated in the past decade on mGlu2 PAMs adding a detailed molecular investigation of PAM binding mode. Differences among mGlu2 PAM compounds are discussed as well as the mGlu2 regions interacting with mGlu2 PAM and NAM agents and residues driving mGlu2 PAM selectivity.

  1. Azurin as a protein scaffold for a low-coordinate non-heme iron site with a small-molecule binding pocket

    PubMed Central

    McLaughlin, Matthew P.; Retegan, Marius; Bill, Eckhard; Payne, Thomas M.; Shafaat, Hannah S.; Peña, Salvador; Sudhamsu, Jawahar; Ensign, Amy A.; Crane, Brian R.; Neese, Frank; Holland, Patrick L.

    2012-01-01

    The apo-protein of Pseudomonas aeruginosa azurin binds iron(II) to give a 1:1 complex, which has been characterized by electronic absorption, Mössbauer, and NMR spectroscopies, as well as X-ray crystallography and quantum-chemical computations. Despite potential competition by water and other coordinating residues, iron(II) binds tightly to the low-coordinate site. The iron(II) complex does not react with chemical redox agents to undergo oxidation or reduction. Spectroscopically-calibrated quantum-chemical computations show that the complex has high-spin iron(II) in a pseudotetrahedral coordination environment, which features interactions with side chains of two histidines and a cysteine, as well as the C=O of Gly45. In the 5A1 ground state, the dz2 orbital is doubly occupied. Mutation of Met121 to Ala leaves the metal site in a similar environment, but creates a pocket for reversible binding of small anions to the iron(II) center. Specifically, azide forms a high-spin iron(II) complex and cyanide forms a low-spin iron(II) complex. PMID:23167247

  2. Allosteric Modulation of Purine and Pyrimidine Receptors

    PubMed Central

    Jacobson, Kenneth A.; Gao, Zhan-Guo; Göblyös, Anikó; IJzerman, Adriaan P.

    2011-01-01

    Among the purine and pyrimidine receptors, the discovery of small molecular allosteric modulators has been most highly advanced for the A1 and A3 ARs. These AR modulators have allosteric effects that are structurally separated from the orthosteric effects in SAR studies. The benzoylthiophene derivatives tend to act as allosteric agonists, as well as selective positive allosteric modulators (PAMs) of the A1 AR. A 2-amino-3-aroylthiophene derivative T-62 has been under development as a PAM of the A1 AR for the treatment of chronic pain. Several structurally distinct classes of allosteric modulators of the human A3 AR have been reported: 3-(2-pyridinyl)isoquinolines, 2,4-disubstituted quinolines, 1H-imidazo-[4,5-c]quinolin-4-amines, endocannabinoid 2-arachidonylglycerol and the food dye Brilliant Black BN. Site-directed mutagenesis of A1 and A3 ARs has identified residues associated with the allosteric effect, distinct from those that affect orthosteric binding. A few small molecular allosteric modulators have been reported for several of the P2X ligand-gated ion channels and the G protein-coupled P2Y receptor nucleotides. Metal ion modulation of the P2X receptors has been extensively explored. The allosteric approach to modulation of purine and pyrimidine receptors looks promising for development of drugs that are event-specific and site-specific in action. PMID:21586360

  3. Cyanide binding to hexacoordinate cyanobacterial hemoglobins: hydrogen-bonding network and heme pocket rearrangement in ferric H117A Synechocystis hemoglobin.

    PubMed

    Vu, B Christie; Nothnagel, Henry J; Vuletich, David A; Falzone, Christopher J; Lecomte, Juliette T J

    2004-10-05

    The truncated hemoglobin (Hb) from the cyanobacterium Synechocystis sp. PCC 6803 is a bis-histidyl hexacoordinate complex in the absence of exogenous ligands. This protein can form a covalent cross-link between His117 in the H-helix and the heme 2-vinyl group. Cross-linking, the physiological importance of which has not been established, is avoided with the His117Ala substitution. In the present work, H117A Hb was used to explore exogenous ligand binding to the heme group. NMR and thermal denaturation data showed that the replacement was of little consequence to the structural and thermodynamic properties of ferric Synechocystis Hb. It did, however, decelerate the association of cyanide ions with the heme iron. Full complexation required hours, instead of minutes, of incubation at optical and NMR concentrations. At neutral pH and in the presence of excess cyanide, binding occurred with a first-order dependence on cyanide concentration, eliminating distal histidine decoordination as the rate-limiting step. The cyanide complex of the H117A variant was characterized for the conformational changes occurring as the histidine on the distal side, His46 (E10), was displaced. Extensive rearrangement allowed Tyr22 (B10) to insert in the heme pocket and Gln43 (E7) and Gln47 (E11) to come in contact with it. H-bond formation to the bound cyanide was identified in solution with the use of (1)H(2)O/(2)H(2)O mixtures. Cyanide binding also resulted in a change in the ratio of heme orientational isomers, in a likely manifestation of heme environment reshaping. Similar observations were made with the related Synechococcus sp. PCC 7002 H117A Hb, except that cyanide binding was rapid in this protein. In both cases, the (15)N chemical shift of bound cyanide was reminiscent of that in peroxidases and the orientation of the proximal histidine was as in other truncated Hbs. The ensemble of the data provided insight into the structural cooperativity of the heme pocket scaffold and pointed

  4. Ligand-binding pocket bridges DNA-binding and dimerization domains of the urate-responsive MarR homologue MftR from Burkholderia thailandensis.

    PubMed

    Gupta, Ashish; Grove, Anne

    2014-07-15

    Members of the multiple antibiotic resistance regulator (MarR) family often regulate gene activity by responding to a specific ligand. In the absence of ligand, most MarR proteins function as repressors, while ligand binding causes attenuated DNA binding and therefore increased gene expression. Previously, we have shown that urate is a ligand for MftR (major facilitator transport regulator), which is encoded by the soil bacterium Burkholderia thailandensis. We show here that both mftR and the divergently oriented gene mftP encoding a major facilitator transport protein are upregulated in the presence of urate. MftR binds two cognate sites in the mftR-mftP intergenic region with equivalent affinity and sensitivity to urate. Mutagenesis of four conserved residues previously reported to be involved in urate binding to Deinococcus radiodurans HucR and Rhizobium radiobacter PecS significantly reduced protein stability and DNA binding affinity but not ligand binding. These data suggest that residues equivalent to those implicated in ligand binding to HucR and PecS serve structural roles and that MftR relies on distinct residues for ligand binding. MftR exhibits a two-step melting transition suggesting independent unfolding of the dimerization and DNA-binding regions; urate binding or mutations in the predicted ligand-binding sites result in one-step unfolding transitions. We suggest that MftR binds the ligand in a cleft between the DNA-binding lobes and the dimer interface but that the mechanism of ligand-mediated attenuation of DNA binding differs from that proposed for other urate-responsive MarR homologues. Since DNA binding by MftR is attenuated at 37 °C, our data also suggest that MftR responds to both ligand and a thermal upshift by attenuated DNA binding and upregulation of the genes under its control.

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

    NASA Astrophysics Data System (ADS)

    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.

  6. FUNCTIONAL ANALYSIS OF A NOVEL POSITIVE ALLOSTERIC MODULATOR OF AMPA RECEPTORS DERIVED FROM A STRUCTURE-BASED DRUG DESIGN STRATEGY

    PubMed Central

    Harms, Jonathan E.; Benveniste, Morris; Maclean, John K. F.; Partin, Kathryn M.; Jamieson, Craig

    2012-01-01

    Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors facilitate synaptic plasticity and can improve various forms of learning and memory. These modulators show promise as therapeutic agents for the treatment of neurological disorders such as schizophrenia, ADHD, and mental depression. Three classes of positive modulator, the benzamides, the thiadiazides, and the biarylsulfonamides differentially occupy a solvent accessible binding pocket at the interface between the two subunits that form the AMPA receptor ligand-binding pocket. Here, we describe the electrophysiological properties of a new chemotype derived from a structure-based drug design strategy (SBDD), which makes similar receptor interactions compared to previously reported classes of modulator. This pyrazole amide derivative, JAMI1001A, with a promising developability profile, efficaciously modulates AMPA receptor deactivation and desensitization of both flip and flop receptor isoforms. PMID:22735771

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

    PubMed Central

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

    2015-01-01

    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. PMID:26482099

  8. Crystal structures of bovine CD1d reveal altered αGalCer presentation and a restricted A' pocket unable to bind long-chain glycolipids.

    PubMed

    Wang, Jing; Guillaume, Joren; Pauwels, Nora; Van Calenbergh, Serge; Van Rhijn, Ildiko; Zajonc, Dirk M

    2012-01-01

    NKT cells play important roles in immune surveillance. They rapidly respond to pathogens by detecting microbial glycolipids when presented by the non-classical MHC I homolog CD1d. Previously, ruminants were considered to lack NKT cells due to the lack of a functional CD1D gene. However, recent data suggest that cattle express CD1d with unknown function. In an attempt to characterize the function of bovine CD1d, we assessed the lipid binding properties of recombinant Bos taurus CD1d (boCD1d) in vitro. BoCD1d is able to bind glycosphingolipids (GSLs) with fatty acid chain lengths of C₁₈, while GSLs with fatty acids of C₂₄ do not bind. Crystal structures of boCD1d bound to a short-chain C₁₂-di-sulfatide antigen, as well as short-chain C₁₆-αGalCer revealed that the Á pocket of boCD1d is restricted in size compared to that of both mouse and human CD1d, explaining the inability of long chain GSL's to bind to boCD1d. Moreover, while di-sulfatide is presented similarly compared to the presentation of sulfatide by mouse CD1d, αGalCer is presented differently at the cell surface, due to an amino acid Asp151Asn substitution that results in loss of intimate contacts between the αGalCer headgroup and CD1d. The altered αGalCer presentation by boCD1d also explains its lack of cross-activation of mouse iNKT cells and raises the interesting question of the nature and function of bovine lipid-reactive T cells.

  9. Crystal Structures of Bovine CD1d Reveal Altered αGalCer Presentation and a Restricted A’ Pocket Unable to Bind Long-Chain Glycolipids

    PubMed Central

    Wang, Jing; Guillaume, Joren; Pauwels, Nora; Van Calenbergh, Serge; Van Rhijn, Ildiko; Zajonc, Dirk M.

    2012-01-01

    NKT cells play important roles in immune surveillance. They rapidly respond to pathogens by detecting microbial glycolipids when presented by the non-classical MHC I homolog CD1d. Previously, ruminants were considered to lack NKT cells due to the lack of a functional CD1D gene. However, recent data suggest that cattle express CD1d with unknown function. In an attempt to characterize the function of bovine CD1d, we assessed the lipid binding properties of recombinant Bos taurus CD1d (boCD1d) in vitro. BoCD1d is able to bind glycosphingolipids (GSLs) with fatty acid chain lengths of C18, while GSLs with fatty acids of C24 do not bind. Crystal structures of boCD1d bound to a short-chain C12-di-sulfatide antigen, as well as short-chain C16-αGalCer revealed that the Á pocket of boCD1d is restricted in size compared to that of both mouse and human CD1d, explaining the inability of long chain GSL’s to bind to boCD1d. Moreover, while di-sulfatide is presented similarly compared to the presentation of sulfatide by mouse CD1d, αGalCer is presented differently at the cell surface, due to an amino acid Asp151Asn substitution that results in loss of intimate contacts between the αGalCer headgroup and CD1d. The altered αGalCer presentation by boCD1d also explains its lack of cross-activation of mouse iNKT cells and raises the interesting question of the nature and function of bovine lipid-reactive T cells. PMID:23110152

  10. Two types of antibodies are induced by vaccination with A/California/2009 pdm virus: binding near the sialic acid-binding pocket and neutralizing both H1N1 and H5N1 viruses.

    PubMed

    Ohshima, Nobuko; Kubota-Koketsu, Ritsuko; Iba, Yoshitaka; Okuno, Yoshinobu; Kurosawa, Yoshikazu

    2014-01-01

    Many people have a history of catching the flu several times during childhood but no additional flu in adulthood, even without vaccination. We analyzed the total repertoire of antibodies (Abs) against influenza A group 1 viruses induced in such a flu-resistant person after vaccination with 2009 H1N1 pandemic influenza virus. They were classified into two types, with no exceptions. The first type, the products of B cells newly induced through vaccination, binds near the sialic acid-binding pocket. The second type, the products of long-lived memory B cells established before vaccination, utilizes the 1-69 VH gene, binds to the stem of HA, and neutralizes both H1N1 and H5N1 viruses with few exceptions. These observations indicate that the sialic acid-binding pocket and its surrounding region are immunogenically very potent and majority of the B cells whose growth is newly induced by vaccination produce Abs that recognize these regions. However, they play a role in protection against influenza virus infection for a short period since variant viruses that have acquired resistance to these Abs become dominant. On the other hand, although the stem of HA is immunogenically not potent, the second type of B cells eventually becomes dominant. Thus, a selection system should function in forming the repertoire of long-lived memory B cells and the stability of the epitope would greatly affect the fate of the memory cells. Acquisition of the ability to produce Abs that bind to the stable epitope could be a major factor of flu resistance.

  11. The HIV gp41 pocket binding domain enables C-terminal heptad repeat transition from mediating membrane fusion to immune modulation.

    PubMed

    Klug, Yoel A; Kapach, Gal; Rotem, Etai; Dubreuil, Benjamin; Shai, Yechiel

    2016-04-01

    For successful infection and propagation viruses must overcome many obstacles such as the immune system and entry into their host cells. HIV utilizes its trimeric envelope protein gp160, specifically the gp41 subunit, to enter its host cell. During this process, a gp41-central coiled coil is formed from three N- and three C-terminal heptad repeats, termed the six-helix bundle (SHB), which drives membrane fusion. Recently, T-cell suppression has been reported as an additional function for several regions of gp41 by interfering with the T-cell receptor (TCR) signalling cascade. One of these regions encompasses the conserved pocket binding domain (PBD) that is situated in the C-terminal heptad repeat (CHR) and stabilizes SHB formation. This could indicate that the PBD plays a role in T-cell suppression in addition to its role in membrane fusion. To investigate this dual function, we used two independent cell cultures coupled with biophysical techniques. The data reveal that the PBD mediates T-cell suppression by stabilizing a TCR-binding conformation in the membrane. Moreover, we show that the clinically used HIV fusion inhibitor T-20 did not show suppressive abilities, in contrast with the potent fusion inhibitor C34. In addition, by focusing on SHB conformation after its assembly, we shed light on a mechanism by which gp41's function alternates from membrane fusion facilitation to suppression of TCR activation.

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

    PubMed Central

    Fay, Jonathan F.; Farrens, David L.

    2015-01-01

    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. PMID:26100912

  13. Reversibly Bound Chloride in the Atrial Natriuretic Peptide Receptor Hormone Binding Domain: Possible Allosteric Regulation and a Conserved Structural Motif for the Chloride-binding Site

    SciTech Connect

    Ogawa, H.; Qiu, Y; Philo, J; Arakawa, T; Ogata, C; Misono, K

    2010-01-01

    The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP-binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full-length ANPR expressed in CHO cells. ECD without chloride (ECD(-)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. A new X-ray structure of the bromide-bound ECD is essentially identical to that of the chloride-bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP-bound structures, indicating exchangeable and reversible halide binding. Far-UV CD and thermal unfolding data show that ECD(-) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(-) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride-binding site in ANPR are highly conserved among receptor-guanylate cyclases and metabotropic glutamate receptors. The chloride-dependent ANP binding, reversible chloride binding, and the highly conserved chloride-binding site motif suggest a regulatory role for the receptor bound chloride. Chloride-dependent regulation of ANPR may operate in the kidney, modulating ANP-induced natriuresis.

  14. Homology modeling, docking, and molecular dynamics simulation of the receptor GALR2 and its interactions with galanin and a positive allosteric modulator.

    PubMed

    Hui, Wen-Qi; Cheng, Qi; Liu, Tian-Yu; Ouyang, Qin

    2016-04-01

    Galanin receptor type 2 (GALR2) is a class A G-protein-coupled receptor (GPCR), and it has been reported that orthosteric ligands and positive allosteric modulators (PAMs) of GALR2 could potentially be used to treat epilepsy. So far, the X-ray structure of this receptor has not been resolved, and knowledge of the 3D structure of GALR2 may prove informative in attempts to design novel ligands and to explore the mechanism for the allosteric modulation of this receptor. In this study, homology modeling was used to obtain several GALR2 models using known templates. ProSA-web Z-scores and Ramachandran plots as well as pre-screening against a test dataset of known compounds were all utilized to select the best model of GALR2. Molecular dockings of galanin (a peptide) and a nonpeptide ligand were carried out to choose the (GALR2 model)-galanin complex that showed the closest agreement with the corresponding experimental data. Finally, a 50-ns MD simulation was performed to study the interactions between the GALR2 model and the synthetic and endogenous ligands. The results from docking and MD simulation showed that, besides the reported residues, Tyr160(4.60), Ile105(3.32), Ala274(7.35), and Tyr163(ECL2) also appear to play important roles in the binding of galanin. The potential allosteric binding pockets in the GALR2 model were then investigated via MD simulation. The results indicated that the mechanism for the allosteric modulation caused by PAMs is the binding of the PAM at pocket III, which is formed by galanin, ECL2, TM2, TM3, and ECL1; this results in the disruption of the Na(+)-binding site and/or the Na(+) ion pathway, leading to GALR2 agonism.

  15. Quantum Hall conductance and de Haas-van Alphen oscillation in a tight-binding model with electron and hole pockets for (TMTSF) 2NO3

    NASA Astrophysics Data System (ADS)

    Kishigi, Keita; Hasegawa, Yasumasa

    2016-08-01

    Quantized Hall conductance and de Haas-van Alphen (dHvA) oscillation are studied theoretically in the tight-binding model for (TMTSF) 2NO3 , in which there are small pockets of electrons and holes due to the periodic potentials of anion ordering in the a direction. The magnetic field is treated by hoppings as complex numbers due to the phase caused by the vector potential, i.e., Peierls substitution. In realistic values of parameters and the magnetic field, the energy as a function of the magnetic field (Hofstadter butterfly diagram) is obtained. It is shown that the energy levels are broadened and the gaps are closed or almost closed periodically as a function of the inverse magnetic field, which is not seen in the semiclassical theory of the magnetic breakdown. The Hall conductance is quantized with an integer obtained by the Diophantine equation when the chemical potential lies in an energy gap. When electrons or holes are doped in this system, the Hall conductance is quantized in some regions of a magnetic field but it is not quantized in other regions of a magnetic field due to the broadening of the Landau levels. The amplitude of the dHvA oscillation at zero temperature decreases as the magnetic field increases, while it is constant in the semiclassical Lifshitz Kosevich formula.

  16. Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A.

    PubMed

    Banta, Scott; Swanson, Barbara A; Wu, Shan; Jarnagin, Alisha; Anderson, Stephen

    2002-02-01

    The NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductase enzyme is a required component in some novel biosynthetic vitamin C production processes. This enzyme catalyzes the conversion of 2,5-DKG to 2-keto-L-gulonic acid, which is an immediate precursor to L-ascorbic acid. Forty unique site-directed mutations were made at five residues in the cofactor-binding pocket of 2,5-DKG reductase A in an attempt to improve its ability to use NADH as a cofactor. NADH is more stable, less expensive and more prevalent in the cell than is NADPH. To the best of our knowledge, this is the first focused attempt to alter the cofactor specificity of a member of the aldo-keto reductase superfamily by engineering improved activity with NADH into the enzyme. Activity of the mutants with NADH or NADPH was assayed using activity-stained native polyacrylamide gels. Eight of the mutants at three different sites were identified as having improved activity with NADH. These mutants were purified and subjected to a kinetic characterization with NADH as a cofactor. The best mutant obtained, R238H, produced an almost 7-fold improvement in catalysis with NADH compared with the wild-type enzyme. Surprisingly, most of this catalytic improvement appeared to be due to an improvement in the apparent kcat for the reaction rather than a large improvement in the affinity of the enzyme for NADH.

  17. Strength of hydrogen bond network takes crucial roles in the dissociation process of inhibitors from the HIV-1 protease binding pocket.

    PubMed

    Li, Dechang; Ji, Baohua; Hwang, Keh-Chih; Huang, Yonggang

    2011-04-29

    To understand the underlying mechanisms of significant differences in dissociation rate constant among different inhibitors for HIV-1 protease, we performed steered molecular dynamics (SMD) simulations to analyze the entire dissociation processes of inhibitors from the binding pocket of protease at atomistic details. We found that the strength of hydrogen bond network between inhibitor and the protease takes crucial roles in the dissociation process. We showed that the hydrogen bond network in the cyclic urea inhibitors AHA001/XK263 is less stable than that of the approved inhibitor ABT538 because of their large differences in the structures of the networks. In the cyclic urea inhibitor bound complex, the hydrogen bonds often distribute at the flap tips and the active site. In contrast, there are additional accessorial hydrogen bonds formed at the lateral sides of the flaps and the active site in the ABT538 bound complex, which take crucial roles in stabilizing the hydrogen bond network. In addition, the water molecule W301 also plays important roles in stabilizing the hydrogen bond network through its flexible movement by acting as a collision buffer and helping the rebinding of hydrogen bonds at the flap tips. Because of its high stability, the hydrogen bond network of ABT538 complex can work together with the hydrophobic clusters to resist the dissociation, resulting in much lower dissociation rate constant than those of cyclic urea inhibitor complexes. This study may provide useful guidelines for design of novel potent inhibitors with optimized interactions.

  18. An allosteric inhibitor of substrate recognition by the SCF[superscript Cdc4] ubiquitin ligase

    SciTech Connect

    Orlicky, Stephen; Tang, Xiaojing; Neduva, Victor; Elowe, Nadine; Brown, Eric D.; Sicheri, Frank; Tyers, Mike

    2010-09-17

    The specificity of SCF ubiquitin ligase-mediated protein degradation is determined by F-box proteins. We identified a biplanar dicarboxylic acid compound, called SCF-I2, as an inhibitor of substrate recognition by the yeast F-box protein Cdc4 using a fluorescence polarization screen to monitor the displacement of a fluorescein-labeled phosphodegron peptide. SCF-I2 inhibits the binding and ubiquitination of full-length phosphorylated substrates by SCF{sup Cdc4}. A co-crystal structure reveals that SCF-I2 inserts itself between the {beta}-strands of blades 5 and 6 of the WD40 propeller domain of Cdc4 at a site that is 25 {angstrom} away from the substrate binding site. Long-range transmission of SCF-I2 interactions distorts the substrate binding pocket and impedes recognition of key determinants in the Cdc4 phosphodegron. Mutation of the SCF-I2 binding site abrogates its inhibitory effect and explains specificity in the allosteric inhibition mechanism. Mammalian WD40 domain proteins may exhibit similar allosteric responsiveness and hence represent an extensive class of druggable target.

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

    PubMed

    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

  20. Effects of genetic replacements of charged and H-bonding residues in the retinal pocket on Ca2+ binding to deionized bacteriorhodopsin.

    PubMed Central

    Zhang, Y N; el-Sayed, M A; Bonet, M L; Lanyi, J K; Chang, M; Ni, B; Needleman, R

    1993-01-01

    Metal cations are known to be required for proton pumping by bacteriorhodopsin (bR). Previous studies found that bR has two high-affinity and four to six low-affinity Ca(2+)-binding sites. In our efforts to find the location of these Ca2+ sites, the effects of replacing charged (Asp-85, Asp-212, and Arg-82) and H-bonding (Tyr-185) residues in the retinal pocket on the color control and binding affinity of Ca2+ ions in Ca(2+)-regenerated bR were examined. The important results are as follows: (i) The removal of Ca2+ from recombinant bR in which charged residues were replaced by neutral ones shifted the retinal absorption to the blue, opposite to that observed in wild-type bR or in recombinant bR in which the H-bonding residue, Tyr-185, was replaced by a non-H-bonding amino acid (Phe). (ii) Similar to the observation in wild-type bR, the binding of Ca2+ to the second site gave the observed color change in the recombinant bR samples in which charged residues were replaced by neutral ones. (iii) The residue replacements had no effect on the affinity constants of the four to six weakly bound Ca2+. (iv) The two high-affinity sites exhibited reduced affinity with substitutions; while the extent of the reduction depended on the specific substitution, each site was reduced by the same factor for each of the charged residue substitutions but by different factors for the mutant where Tyr-185 was replaced with Phe(Y185F). The above results suggest that the two Ca2+ ions in the two high-affinity sites are within interaction distance with one another and with the charged residues in the retinal pocket. The results further suggest that, while the interaction between Tyr-185 and the high-affinity Ca2+ ions is relatively short range and specific (with more coupling to the Ca2+ ion in the second affinity site), between the charged residues and Ca2+ ions it seems to be of the electrostatic (e.g., ion-ion) long range, nonspecific type. Although neither Asp-85, Asp-212, nor Arg-82 is

  1. Inhibition of muscarinic K+ current in guinea-pig atrial myocytes by PD 81,723, an allosteric enhancer of adenosine binding to A1 receptors

    PubMed Central

    Brandts, B; Bünemann, M; Hluchy, J; Sabin, G V; Pott, L

    1997-01-01

    PD 81,723 has been shown to enhance binding of adenosine to A1 receptors by stabilizing G protein-receptor coupling (‘allosteric enhancement'). Evidence has been provided that in the perfused hearts and isolated atria PD 81,723 causes a sensitization to adenosine via this mechanism. We have studied the effect of PD 81,723 in guinea-pig isolated atrial myocytes by use of whole-cell measurement of the muscarinic K+ current (IK(ACh)) activated by different Gi-coupled receptors (A1, M2, sphingolipid). PD 81,273 caused inhibition of IK(ACh) (IC50≃5 μM) activated by either of the three receptors. Receptor-independent IK(ACh) in cells loaded with GTP-γ-S and background IK(ACh), which contributes to the resting conductance of atrial myocytes, were equally sensitive to PD 81,723. At no combination of concentrations of adenosine and PD 81,723 could an enhancing effect be detected. The compound was active from the outside only. Loading of the cells with PD 81,723 (50 μM) via the patch pipette did not affect either IK(ACh) or its sensitivity to adenosine. We suggest that PD 81,723 acts as an inhibitor of inward rectifying K+ channels; this is supported by the finding that ventricular IK1, which shares a large degree of homology with the proteins (GIRK1/GIRK4) forming IK(ACh) but is not G protein-gated, was also blocked by this compound. It is concluded that the functional effects of PD 81,723 described in the literature are not mediated by the A1 adenosine receptor-Gi-IK(ACh) pathway. PMID:9249260

  2. Structural basis of allosteric and synergistic activation of AMPK by furan-2-phosphonic derivative C2 binding

    PubMed Central

    Langendorf, Christopher G.; Ngoei, Kevin R. W.; Scott, John W.; Ling, Naomi X. Y.; Issa, Sam M. A.; Gorman, Michael A.; Parker, Michael W.; Sakamoto, Kei; Oakhill, Jonathan S.; Kemp, Bruce E.

    2016-01-01

    The metabolic stress-sensing enzyme AMP-activated protein kinase (AMPK) is responsible for regulating metabolism in response to energy supply and demand. Drugs that activate AMPK may be useful in the treatment of metabolic diseases including type 2 diabetes. We have determined the crystal structure of AMPK in complex with its activator 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid (C2), revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results show that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases. PMID:26952388

  3. Allosteric Nanobodies Reveal the Dynamic Range and Diverse Mechanisms of GPCR Activation

    PubMed Central

    Staus, Dean P; Strachan, Ryan T; Manglik, Aashish; Pani, Biswaranjan; Kahsai, Alem W; Kim, Tae Hun; Wingler, Laura M; Ahn, Seungkirl; Chatterjee, Arnab; Masoudi, Ali; Kruse, Andrew C; Pardon, Els; Steyaert, Jan; Weis, William I; Prosser, R. Scott; Kobilka, Brian K; Costa, Tommaso; Lefkowitz, Robert J

    2016-01-01

    G-protein coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signaling effectors such as G proteins and β-arrestins. It is widely appreciated that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (i.e., ‘efficacy’)1. Furthermore, mounting biophysical evidence, primarily using the β-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states2–5. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β2AR conformations using single domain camelid antibodies (nanobodies): a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60)6,7. We show that Nb60 stabilizes a previously unappreciated low affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoproterenol has a 15,000-fold higher affinity for the β2AR in the presence of Nb80 compared to Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the

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

    PubMed Central

    2012-01-01

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

  5. Kinetic analysis of ligand binding to the Ehrlich cell nucleoside transporter: Pharmacological characterization of allosteric interactions with the sup 3 Hnitrobenzylthioinosine binding site

    SciTech Connect

    Hammond, J.R. )

    1991-06-01

    Kinetic analysis of the binding of {sup 3}Hnitrobenzylthioinosine ({sup 3}H NBMPR) to Ehrlich ascites tumor cell plasma membranes was conducted in the presence and absence of a variety of nucleoside transport inhibitors and substrates. The association of {sup 3}H NBMPR with Ehrlich cell membranes occurred in two distinct phases, possibly reflecting functional conformation changes in the {sup 3}HNBMPR binding site/nucleoside transporter complex. Inhibitors of the equilibrium binding of {sup 3}HNBMPR, tested at submaximal inhibitory concentrations, generally decreased the rate of association of {sup 3}HNBMPR, but the magnitude of this effect varied significantly with the agent tested. Adenosine and diazepam had relatively minor effects on the association rate, whereas dipyridamole and mioflazine slowed the rate dramatically. Inhibitors of nucleoside transport also decreased the rate of dissociation of {sup 3}HNBMPR, with an order of potency significantly different from their relative potencies as inhibitors of the equilibrium binding of {sup 3}HNBMPR. Dilazep, dipyridamole, and mioflazine were effective inhibitors of both {sup 3}HNBMPR dissociation and equilibrium binding. The lidoflazine analogue R75231, on the other hand, had no effect on the rate of dissociation of {sup 3}HNBMPR at concentrations below 300 microM, even though it was one of the most potent inhibitors of {sup 3}HNBMPR binding tested (Ki less than 100 nM). In contrast, a series of natural substrates for the nucleoside transport system enhanced the rate of dissociation of {sup 3}HNBMPR with an order of effectiveness that paralleled their relative affinities for the permeant site of the transporter. The most effective enhancers of {sup 3}HNBMPR dissociation, however, were the benzodiazepines diazepam, chlordiazepoxide, and triazolam.

  6. Unexpected Allosteric Network Contributes to LRH-1 Co-regulator Selectivity*

    PubMed Central

    Musille, Paul M.; Kossmann, Bradley R.; Kohn, Jeffrey A.; Ivanov, Ivaylo; Ortlund, Eric A.

    2016-01-01

    Phospholipids (PLs) are unusual signaling hormones sensed by the nuclear receptor liver receptor homolog-1 (LRH-1), which has evolved a novel allosteric pathway to support appropriate interaction with co-regulators depending on ligand status. LRH-1 plays an important role in controlling lipid and cholesterol homeostasis and is a potential target for the treatment of metabolic and neoplastic diseases. Although the prospect of modulating LRH-1 via small molecules is exciting, the molecular mechanism linking PL structure to transcriptional co-regulator preference is unknown. Previous studies showed that binding to an activating PL ligand, such as dilauroylphosphatidylcholine, favors LRH-1's interaction with transcriptional co-activators to up-regulate gene expression. Both crystallographic and solution-based structural studies showed that dilauroylphosphatidylcholine binding drives unanticipated structural fluctuations outside of the canonical activation surface in an alternate activation function (AF) region, encompassing the β-sheet-H6 region of the protein. However, the mechanism by which dynamics in the alternate AF influences co-regulator selectivity remains elusive. Here, we pair x-ray crystallography with molecular modeling to identify an unexpected allosteric network that traverses the protein ligand binding pocket and links these two elements to dictate selectivity. We show that communication between the alternate AF region and classical AF2 is correlated with the strength of the co-regulator interaction. This work offers the first glimpse into the conformational dynamics that drive this unusual PL-mediated nuclear hormone receptor activation. PMID:26553876

  7. The putative pocket protein binding site of Autographa californica nucleopolyhedrovirus BV/ODV-C42 is required for virus-induced nuclear actin polymerization.

    PubMed

    Li, Kun; Wang, Yun; Bai, Huimin; Wang, Qian; Song, Jianhua; Zhou, Yuan; Wu, Chunchen; Chen, Xinwen

    2010-08-01

    Nuclear filamentous actin (F-actin) is essential for nucleocapsid morphogenesis of lepidopteran nucleopolyhedroviruses. Previously, we had demonstrated that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) BV/ODV-C42 (C42) is involved in nuclear actin polymerization by recruiting P78/83, an AcMNPV orf9-encoded N-WASP homology protein that is capable of activating an actin-related-protein 2/3 (Arp2/3) complex to initiate actin polymerization, to the nucleus. To further investigate the role of C42 in virus-induced actin polymerization, the recombinant bacmid vAc(p78/83nls-gfp), with a c42 knockout, p78/83 tagged with a nuclear localization signal coding sequence, and egfp as a reporter gene under the control of the Pp10 promoter, was constructed and transfected to Sf9 cells. In the nuclei of vAc(p78/83nls-gfp)-transfected cells, polymerized F-actin filaments were absent, whereas other actin polymerization elements (i.e., P78/83, G-actin, and Arp2/3 complex) were present. This in vivo evidence indicated that C42 actively participates in the nuclear actin polymerization process as a key element, besides its role in recruiting P78/83 to the nucleus. In order to collect in vitro evidence for the participation of C42 in actin polymerization, an anti-C42 antibody was used to neutralize the viral nucleocapsid, which is capable of initiating actin polymerization in vitro. Both the kinetics of pyrene-actin polymerization and F-actin-specific staining by phalloidin indicated that anti-C42 can significantly attenuate the efficiency of F-actin formation compared to that with control antibodies. Furthermore, we have identified the putative pocket protein binding sequence (PPBS) on C42 that is essential for C42 to exert its function in nuclear actin polymerization.

  8. 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

    PubMed Central

    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

  9. 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.

    PubMed

    Fischer, Thomas; Riedl, Rainer

    2016-03-01

    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).

  10. Crystal structure of tert-butyldimethylsilyl-spiroaminooxathioledioxide-thymine (TSAO-T) in complex with HIV-1 reverse transcriptase (RT) redefines the elastic limits of the non-nucleoside inhibitor-binding pocket

    PubMed Central

    Das, Kalyan; Bauman, Joseph D.; Rim, Angela S.; Dharia, Chhaya; Clark, Arthur D.; Camarasa, María-José; Balzarini, Jan; Arnold, Eddy

    2012-01-01

    Tert-butyldimethylsilyl-spiroaminooxathioledioxide (TSAO) compounds have an embedded thymidine-analog backbone; however, TSAO compounds invoke non-nucleoside RT inhibitor (NNRTI) resistance mutations. Our crystal structure of RT:7 (TSAO-T) complex shows that 7 binds inside the NNRTI-binding pocket assuming a “dragon” shape, and interacts extensively with almost all the pocket residues. The structure also explains the structure-activity relationships and resistance data for TSAO compounds. The binding of 7 causes hyper-expansion of the pocket and significant rearrangement of RT subdomains. This non-optimal complex formation is apparently responsible (1) for the lower stability of a RT (p66/p51) dimer and (2) for the lower potency of 7 despite of its extensive interactions with RT. However, the HIV-1 RT:7 structure reveals novel design features, such as (1) interactions with the conserved Tyr183 from the YMDD-motif and (2) a possible way for an NNRTI to reach the polymerase active site that may be exploited in designing new NNRTIs. PMID:21446702

  11. Allosteric Modulation of Metabotropic Glutamate Receptors

    PubMed Central

    Sheffler, Douglas J.; Gregory, Karen J.; Rook, Jerri M.; Conn, P. Jeffrey

    2013-01-01

    The development of receptor subtype-selective ligands by targeting allosteric sites of G protein-coupled receptors (GPCRs) has proven highly successful in recent years. One GPCR family that has greatly benefited from this approach is the metabotropic glutamate receptors (mGlus). These family C GPCRs participate in the neuromodulatory actions of glutamate throughout the CNS, where they play a number of key roles in regulating synaptic transmission and neuronal excitability. A large number of mGlu subtype-selective allosteric modulators have been identified, the majority of which are thought to bind within the transmembrane regions of the receptor. These modulators can either enhance or inhibit mGlu functional responses and, together with mGlu knockout mice, have furthered the establishment of the physiologic roles of many mGlu subtypes. Numerous pharmacological and receptor mutagenesis studies have been aimed at providing a greater mechanistic understanding of the interaction of mGlu allosteric modulators with the receptor, which have revealed evidence for common allosteric binding sites across multiple mGlu subtypes and the presence for multiple allosteric sites within a single mGlu subtype. Recent data have also revealed that mGlu allosteric modulators can display functional selectivity toward particular signal transduction cascades downstream of an individual mGlu subtype. Studies continue to validate the therapeutic utility of mGlu allosteric modulators as a potential therapeutic approach for a number of disorders including anxiety, schizophrenia, Parkinson’s disease, and Fragile X syndrome. PMID:21907906

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

    PubMed Central

    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

  13. Exploitation of a Novel Binding Pocket in Human Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Discovered through X-ray Fragment Screening.

    PubMed

    Woolford, Alison J-A; Pero, Joseph E; Aravapalli, Sridhar; Berdini, Valerio; Coyle, Joseph E; Day, Philip J; Dodson, Andrew M; Grondin, Pascal; Holding, Finn P; Lee, Lydia Y W; Li, Peng; Manas, Eric S; Marino, Joseph; Martin, Agnes C L; McCleland, Brent W; McMenamin, Rachel L; Murray, Christopher W; Neipp, Christopher E; Page, Lee W; Patel, Vipulkumar K; Potvain, Florent; Rich, Sharna; Rivero, Ralph A; Smith, Kirsten; Somers, Donald O; Trottet, Lionel; Velagaleti, Ranganadh; Williams, Glyn; Xie, Ren

    2016-06-09

    Elevated levels of human lipoprotein-associated phospholipase A2 (Lp-PLA2) are associated with cardiovascular disease and dementia. A fragment screen was conducted against Lp-PLA2 in order to identify novel inhibitors. Multiple fragment hits were observed in different regions of the active site, including some hits that bound in a pocket created by movement of a protein side chain (approximately 13 Å from the catalytic residue Ser273). Using structure guided design, we optimized a fragment that bound in this pocket to generate a novel low nanomolar chemotype, which did not interact with the catalytic residues.

  14. Identification of selective agonists and positive allosteric modulators for µ- and δ-opioid receptors from a single high-throughput screen.

    PubMed

    Burford, Neil T; Wehrman, Tom; Bassoni, Daniel; O'Connell, Jonathan; Banks, Martyn; Zhang, Litao; Alt, Andrew

    2014-10-01

    Hetero-oligomeric complexes of G protein-coupled receptors (GPCRs) may represent novel therapeutic targets exhibiting different pharmacology and tissue- or cell-specific site of action compared with receptor monomers or homo-oligomers. An ideal tool for validating this concept pharmacologically would be a hetero-oligomer selective ligand. We set out to develop and execute a 1536-well high-throughput screen of over 1 million compounds to detect potential hetero-oligomer selective ligands using a β-arrestin recruitment assay in U2OS cells coexpressing recombinant µ- and δ-opioid receptors. Hetero-oligomer selective ligands may bind to orthosteric or allosteric sites, and we might anticipate that the formation of hetero-oligomers may provide novel allosteric binding pockets for ligand binding. Therefore, our goal was to execute the screen in such a way as to identify positive allosteric modulators (PAMs) as well as agonists for µ, δ, and hetero-oligomeric receptors. While no hetero-oligomer selective ligands were identified (based on our selection criteria), this single screen did identify numerous µ- and δ-selective agonists and PAMs as well as nonselective agonists and PAMs. To our knowledge, these are the first µ- and δ-opioid receptor PAMs described in the literature.

  15. Detection of multiscale pockets on protein surfaces using mathematical morphology.

    PubMed

    Kawabata, Takeshi

    2010-04-01

    Detection of pockets on protein surfaces is an important step toward finding the binding sites of small molecules. In a previous study, we defined a pocket as a space into which a small spherical probe can enter, but a large probe cannot. The radius of the large probes corresponds to the shallowness of pockets. We showed that each type of binding molecule has a characteristic shallowness distribution. In this study, we introduced fundamental changes to our previous algorithm by using a 3D grid representation of proteins and probes, and the theory of mathematical morphology. We invented an efficient algorithm for calculating deep and shallow pockets (multiscale pockets) simultaneously, using several different sizes of spherical probes (multiscale probes). We implemented our algorithm as a new program, ghecom (grid-based HECOMi finder). The statistics of calculated pockets for the structural dataset showed that our program had a higher performance of detecting binding pockets, than four other popular pocket-finding programs proposed previously. The ghecom also calculates the shallowness of binding ligands, R(inaccess) (minimum radius of inaccessible spherical probes) that can be obtained from the multiscale molecular volume. We showed that each part of the binding molecule had a bias toward a specific range of shallowness. These findings will be useful for predicting the types of molecules that will be most likely to bind putative binding pockets, as well as the configurations of binding molecules. The program ghecom is available through the Web server (http://biunit.naist.jp/ghecom).

  16. 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

    NASA Astrophysics Data System (ADS)

    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

  17. 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.

    PubMed

    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

  18. Structural and Functional Analysis of Two New Positive Allosteric Modulators of GluA2 Desensitization and Deactivation

    PubMed Central

    Timm, David E.; Benveniste, Morris; Weeks, Autumn M.; Nisenbaum, Eric S.

    2011-01-01

    At the dimer interface of the extracellular ligand-binding domain of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors a hydrophilic pocket is formed that is known to interact with two classes of positive allosteric modulators, represented by cyclothiazide and the ampakine 2H,3H,6aH-pyrrolidino(2,1–3′,2′)1,3-oxazino(6′,5′-5,4)benzo(e)1,4-dioxan-10-one (CX614). Here, we present structural and functional data on two new positive allosteric modulators of AMPA receptors, phenyl-1,4-bis-alkylsulfonamide (CMPDA) and phenyl-1,4-bis-carboxythiophene (CMPDB). Crystallographic data show that these compounds bind within the modulator-binding pocket and that substituents of each compound overlap with distinct moieties of cyclothiazide and CX614. The goals of the present study were to determine 1) the degree of modulation by CMPDA and CMPDB of AMPA receptor deactivation and desensitization; 2) whether these compounds are splice isoform-selective; and 3) whether predictions of mechanism of action could be inferred by comparing molecular interactions between the ligand-binding domain and each compound with those of cyclothiazide and CX614. CMPDB was found to be more isoform-selective than would be predicted from initial binding assays. It is noteworthy that these new compounds are both more potent and more effective and may be more clinically relevant than the AMPA receptor modulators described previously. PMID:21543522

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

    PubMed Central

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

    2011-01-01

    Functional interactions of the E. coli PriA helicase 181N-terminal domain with the DNA and nucleotide cofactors have been quantitatively examined. The isolated 181N-terminal domain forms a stable dimer in solution, most probably reflecting the involvement of the domain in specific cooperative interactions of the intact PriA protein - dsDNA complex. Only one monomer of the domain dimer binds the DNA, i.e., the dimer has one effective DNA-binding site. Although the total site-size of the dimer - ssDNA complex is ~13 nucleotides, the DNA-binding subsite engages in direct interactions ~5 nucleotides. A small number of interacting nucleotides indicates that the DNA-binding subsites of the PriA helicase, i.e., the strong subsite on the helicase domain and the weak subsite on the N-terminal domain, are spatially separated in the intact enzyme. Contrary to current views, the subsite has only a slight preference for the 3′-end OH group of the ssDNA and lacks any significant base specificity, although it has a significant dsDNA affinity. Unlike the intact helicase, the DNA-binding subsite of the isolated domain is in an open conformation, indicating the presence of the direct helicase domain - N-terminal domain interactions. The discovery that the 181N-terminal domain possesses a nucleotide-binding site places the allosteric, weak nucleotide-binding site of the intact PriA on the N-terminal domain. The specific ADP effect on the domain DNA-binding subsite indicates that in the intact helicase, the bound ADP not only opens the DNA-binding subsite but also increases its intrinsic DNA affinity. PMID:21888358

  20. Efficient Binding of the NOS1AP C-Terminus to the nNOS PDZ Pocket Requires the Concerted Action of the PDZ Ligand Motif, the Internal ExF Site and Structural Integrity of an Independent Element

    PubMed Central

    Li, Li-Li; Cisek, Katryna; Courtney, Michael J.

    2017-01-01

    Neuronal nitric oxide synthase is widely regarded as an important contributor to a number of disorders of excitable tissues. Recently the adaptor protein NOS1AP has emerged as a contributor to several nNOS-linked conditions. As a consequence, the unexpectedly complex mechanisms of interaction between nNOS and its effector NOS1AP have become a particularly interesting topic from the point of view of both basic research and the potential for therapeutic applications. Here we demonstrate that the concerted action of two previously described motif regions contributing to the interaction of nNOS with NOS1AP, the ExF region and the PDZ ligand motif, efficiently excludes an alternate ligand from the nNOS-PDZ ligand-binding pocket. Moreover, we identify an additional element with a denaturable structure that contributes to interaction of NOS1AP with nNOS. Denaturation does not affect the functions of the individual motifs and results in a relatively mild drop, ∼3-fold, of overall binding affinity of the C-terminal region of NOS1AP for nNOS. However, denaturation selectively prevents the concerted action of the two motifs that normally results in efficient occlusion of the PDZ ligand-binding pocket, and results in 30-fold reduction of competition between NOS1AP and an alternate PDZ ligand. PMID:28360833

  1. Heat Shock Protein 70 Inhibitors. 2. 2,5′-Thiodipyrimidines, 5-(Phenylthio)pyrimidines, 2-(Pyridin-3-ylthio)pyrimidines, and 3-(Phenylthio)pyridines as Reversible Binders to an Allosteric Site on Heat Shock Protein 70

    PubMed Central

    2015-01-01

    The discovery and development of heat shock protein 70 (Hsp70) inhibitors is currently a hot topic in cancer. In the preceding paper in this issue (10.1021/jm401551n), we have described structure–activity relationship studies in the first Hsp70 inhibitor class rationally designed to bind to a novel allosteric pocket located in the N-terminal domain of the protein. These ligands contained an acrylamide to take advantage of an active cysteine embedded in the allosteric pocket and acted as covalent protein modifiers upon binding. Here, we perform chemical modifications around the irreversible inhibitor scaffold to demonstrate that covalent modification is not a requirement for activity within this class of compounds. The study identifies derivative 27c, which mimics the biological effects of the irreversible inhibitors at comparable concentrations. Collectively, the back-to-back manuscripts describe the first pharmacophores that favorably and selectively interact with a never explored pocket in Hsp70 and provide a novel blueprint for a cancer-oriented development of Hsp70-directed ligands. PMID:24548239

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

    PubMed Central

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

    2015-01-01

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

  3. Allosteric modulation of peroxisomal membrane protein recognition by farnesylation of the peroxisomal import receptor PEX19

    PubMed Central

    Emmanouilidis, Leonidas; Schütz, Ulrike; Tripsianes, Konstantinos; Madl, Tobias; Radke, Juliane; Rucktäschel, Robert; Wilmanns, Matthias; Schliebs, Wolfgang; Erdmann, Ralf; Sattler, Michael

    2017-01-01

    The transport of peroxisomal membrane proteins (PMPs) requires the soluble PEX19 protein as chaperone and import receptor. Recognition of cargo PMPs by the C-terminal domain (CTD) of PEX19 is required for peroxisome biogenesis in vivo. Farnesylation at a C-terminal CaaX motif in PEX19 enhances the PMP interaction, but the underlying molecular mechanisms are unknown. Here, we report the NMR-derived structure of the farnesylated human PEX19 CTD, which reveals that the farnesyl moiety is buried in an internal hydrophobic cavity. This induces substantial conformational changes that allosterically reshape the PEX19 surface to form two hydrophobic pockets for the recognition of conserved aromatic/aliphatic side chains in PMPs. Mutations of PEX19 residues that either mediate farnesyl contacts or are directly involved in PMP recognition abolish cargo binding and cannot complement a ΔPEX19 phenotype in human Zellweger patient fibroblasts. Our results demonstrate an allosteric mechanism for the modulation of protein function by farnesylation. PMID:28281558

  4. Solution-state (15)N NMR spectroscopic study of alpha-C-phycocyanin: implications for the structure of the chromophore-binding pocket of the cyanobacterial phytochrome Cph1.

    PubMed

    Hahn, Janina; Kühne, Ronald; Schmieder, Peter

    2007-12-17

    The detailed structure of the chromophore-binding pocket in phytochrome proteins and the structural changes associated with its photocycle are still matters of debate. Insight into the structure and dynamics of the binding pocket has been gained through the comparison of a (15)N NMR spectrum of alpha-C-phycocyanin, which is often used as a model system for the study of phytochromes, with the previously described (15)N NMR spectrum of the cyanobacterial phytochrome Cph1. The former spectrum supports the hypothesis that all four nitrogen atoms of the alpha-C-phycocyanin chromophore are protonated, in analogy with the proposed protonation state for the P(r) and P(fr) forms of Cph1. The spectra show that the chromophores in both proteins exhibit a distinct dynamic behavior, as also indicated by a NOESY spectrum of Cph1. Finally, stereochemical arguments and a Cph1 homology model support the hypothesis that the chromophore in Cph1 is most likely in the ZZZssa conformation in the P(r) form of the protein.

  5. A synthetic peptide targeting the BH4 domain of Bcl-2 induces apoptosis in multiple myeloma and follicular lymphoma cells alone or in combination with agents targeting the BH3-binding pocket of Bcl-2.

    PubMed

    Lavik, Andrew R; Zhong, Fei; Chang, Ming-Jin; Greenberg, Edward; Choudhary, Yuvraj; Smith, Mitchell R; McColl, Karen S; Pink, John; Reu, Frederic J; Matsuyama, Shigemi; Distelhorst, Clark W

    2015-09-29

    Bcl-2 inhibits apoptosis by two distinct mechanisms but only one is targeted to treat Bcl-2-positive malignancies. In this mechanism, the BH1-3 domains of Bcl-2 form a hydrophobic pocket, binding and inhibiting pro-apoptotic proteins, including Bim. In the other mechanism, the BH4 domain mediates interaction of Bcl-2 with inositol 1,4, 5-trisphosphate receptors (IP3Rs), inhibiting pro-apoptotic Ca2+ signals. The current anti-Bcl-2 agents, ABT-263 (Navitoclax) and ABT-199 (Venetoclax), induce apoptosis by displacing pro-apoptotic proteins from the hydrophobic pocket, but do not inhibit Bcl-2-IP3R interaction. Therefore, to target this interaction we developed BIRD-2 (Bcl-2 IP3 Receptor Disruptor-2), a decoy peptide that binds to the BH4 domain, blocking Bcl-2-IP3R interaction and thus inducing Ca2+-mediated apoptosis in chronic lymphocytic leukemia, multiple myeloma, and follicular lymphoma cells, including cells resistant to ABT-263, ABT-199, or the Bruton's tyrosine kinase inhibitor Ibrutinib. Moreover, combining BIRD-2 with ABT-263 or ABT-199 enhances apoptosis induction compared to single agent treatment. Overall, these findings provide strong rationale for developing novel therapeutic agents that mimic the action of BIRD-2 in targeting the BH4 domain of Bcl-2 and disrupting Bcl-2-IP3R interaction.

  6. Conformational landscape of the human immunodeficiency virus type 1 reverse transcriptase non-nucleoside inhibitor binding pocket: lessons for inhibitor design from a cluster analysis of many crystal structures.

    PubMed

    Paris, Kristina A; Haq, Omar; Felts, Anthony K; Das, Kalyan; Arnold, Eddy; Levy, Ronald M

    2009-10-22

    Clustering of 99 available X-ray crystal structures of HIV-1 reverse transcriptase (RT) at the flexible non-nucleoside inhibitor binding pocket (NNIBP) provides information about features of the conformational landscape for binding non-nucleoside inhibitors (NNRTIs), including effects of mutation and crystal forms. The ensemble of NNIBP conformations is separated into eight discrete clusters based primarily on the position of the functionally important primer grip, the displacement of which is believed to be one of the mechanisms of inhibition of RT. Two of these clusters are populated by structures in which the primer grip exhibits novel conformations that differ from the predominant cluster by over 4 A and are induced by the unique inhibitors capravirine and rilpivirine/TMC278. This work identifies a new conformation of the NNIBP that may be used to design NNRTIs. It can also be used to guide more complete exploration of the NNIBP free energy landscape using advanced sampling techniques.

  7. Critical Molecular Determinants of α7 Nicotinic Acetylcholine Receptor Allosteric Activation

    PubMed Central

    Horenstein, Nicole A.; Papke, Roger L.; Kulkarni, Abhijit R.; Chaturbhuj, Ganesh U.; Stokes, Clare; Manther, Khan; Thakur, Ganesh A.

    2016-01-01

    The α7 nicotinic acetylcholine receptors (nAChRs) are uniquely sensitive to selective positive allosteric modulators (PAMs), which increase the efficiency of channel activation to a level greater than that of other nAChRs. Although PAMs must work in concert with “orthosteric” agonists, compounds such as GAT107 ((3aR,4S,9bS)-4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide) have the combined properties of agonists and PAMs (ago-PAM) and produce very effective channel activation (direct allosteric activation (DAA)) by operating at two distinct sites in the absence of added agonist. One site is likely to be the same transmembrane site where PAMs like PNU-120596 function. We show that the other site, required for direct activation, is likely to be solvent-accessible at the extracellular domain vestibule. We identify key attributes of molecules in this family that are able to act at the DAA site through variation at the aryl ring substituent of the tetrahydroquinoline ring system and with two different classes of competitive antagonists of DAA. Analyses of molecular features of effective allosteric agonists allow us to propose a binding model for the DAA site, featuring a largely non-polar pocket accessed from the extracellular vestibule with an important role for Asp-101. This hypothesis is supported with data from site-directed mutants. Future refinement of the model and the characterization of specific GAT107 analogs will allow us to define critical structural elements that can be mapped onto the receptor surface for an improved understanding of this novel way to target α7 nAChR therapeutically. PMID:26742843

  8. Molecular recognition by thrombin. Role of the slow-->fast transition, site-specific ion binding energetics and thermodynamic mapping of structural components.

    PubMed

    Ayala, Y; Di Cera, E

    1994-01-14

    The interaction of thrombin with the potent natural inhibitor hirudin is controlled in a complex fashion by the binding of Na+ and Cl- to the enzyme and allosteric transitions. Binding of hirudin is positively linked to Na+ binding, but is opposed in a competitive fashion by the binding of Cl-. Since Na+ binding induces the slow-->fast transition of thrombin, it follows from linkage principles that hirudin binds to the fast form with higher affinity. Hence, the slow-->fast transition is a key component of molecular recognition of hirudin by thrombin. We propose a three-step mechanism for molecular recognition of hirudin by thrombin, which is also relevant for recognition of fibrinogen and possibly the platelet receptor and thrombomodulin. First, the C-terminal acidic tail of hirudin binds to the fibrinogen recognition site of thrombin displacing one Cl ion from the thrombin surface. Then, the enzyme undergoes a conformational transition that gives rise to increased accessibility of the catalytic pocket to small synthetic substrates through movement of the Trp148 loop. The changes in the catalytic moiety triggered allosterically by binding to the fibrinogen recognition site are linked to the uptake of Na+ and are similar to, if not identical with, those observed in the Na(+)-induced slow-->fast transition. Finally, the compact N-terminal domain is accommodated in the region surrounding the catalytic pocket. Hirudin binding is also used as a probe of site-specific ion-binding interactions of Na+ and Cl- with the enzyme, characterized by cooperativity between the Na+ and Cl- binding domains. The structural components directly involved or linked to Na+ and Cl- binding have been explored in terms of free energy perturbations of the binding of hirudin and a number of ligands. The fibrinogen recognition site stores most of the free energy of coupling with Cl- binding, while regions surrounding the access to the catalytic pocket provide most of the free energy of coupling

  9. Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase

    SciTech Connect

    Nakamura, Akiyoshi; Tamura, Noriko; Yasutake, Yoshiaki

    2015-10-23

    The structure of the HIV-1 reverse transcriptase Q151M mutant was determined at a resolution of 2.6 Å in space group P321. Hepatitis B virus polymerase (HBV Pol) is an important target for anti-HBV drug development; however, its low solubility and stability in vitro has hindered detailed structural studies. Certain nucleotide reverse transcriptase (RT) inhibitors (NRTIs) such as tenofovir and lamivudine can inhibit both HBV Pol and Human immunodeficiency virus 1 (HIV-1) RT, leading to speculation on structural and mechanistic analogies between the deoxynucleotide triphosphate (dNTP)-binding sites of these enzymes. The Q151M mutation in HIV-1 RT, located at the dNTP-binding site, confers resistance to various NRTIs, while maintaining sensitivity to tenofovir and lamivudine. The residue corresponding to Gln151 is strictly conserved as a methionine in HBV Pol. Therefore, the structure of the dNTP-binding pocket of the HIV-1 RT Q151M mutant may reflect that of HBV Pol. Here, the crystal structure of HIV-1 RT Q151M, determined at 2.6 Å resolution, in a new crystal form with space group P321 is presented. Although the structure of HIV-1 RT Q151M superimposes well onto that of HIV-1 RT in a closed conformation, a slight movement of the β-strands (β2–β3) that partially create the dNTP-binding pocket was observed. This movement might be caused by the introduction of the bulky thioether group of Met151. The structure also highlighted the possibility that the hydrogen-bonding network among amino acids and NRTIs is rearranged by the Q151M mutation, leading to a difference in the affinity of NRTIs for HIV-1 RT and HBV Pol.

  10. Allosteric mutants show that PrfA activation is dispensable for vacuole escape but required for efficient spread and Listeria survival in vivo

    PubMed Central

    Deshayes, Caroline; Bielecka, Magdalena K; Cain, Robert J; Scortti, Mariela; de las Heras, Aitor; Pietras, Zbigniew; Luisi, Ben F; Núñez Miguel, Ricardo; Vázquez-Boland, José A

    2012-01-01

    The transcriptional regulator PrfA controls key virulence determinants of the facultative intracellular pathogen Listeria monocytogenes. PrfA-dependent gene expression is strongly induced within host cells. While the basis of this activation is unknown, the structural homology of PrfA with the cAMP receptor protein (Crp) and the finding of constitutively activated PrfA* mutants suggests it may involve ligand-induced allostery. Here, we report the identification of a solvent-accessible cavity within the PrfA N-terminal domain that may accommodate an activating ligand. The pocket occupies a similar position to the cAMP binding site in Crp but lacks the cyclic nucleotide-anchoring motif and has its entrance on the opposite side of the β-barrel. Site-directed mutations in this pocket impaired intracellular PrfA-dependent gene activation without causing extensive structural/functional alterations to PrfA. Two substitutions, L48F and Y63W, almost completely abolished intracellular virulence gene induction and thus displayed the expected phenotype for allosteric activation-deficient PrfA mutations. Neither PrfAallo substitution affected vacuole escape and initial intracellular growth of L. monocytogenes in epithelial cells and macrophages but caused defective cell-to-cell spread and strong attenuation in mice. Our data support the hypothesis that PrfA is allosterically activated during intracellular infection and identify the probable binding site for the effector ligand. They also indicate that PrfA allosteric activation is not required for early intracellular survival but is essential for full Listeria virulence and colonization of host tissues. PMID:22646689

  11. Computational Tools for Allosteric Drug Discovery: Site Identification and Focus Library Design.

    PubMed

    Huang, Wenkang; Nussinov, Ruth; Zhang, Jian

    2017-01-01

    Allostery is an intrinsic phenomenon of biological macromolecules involving regulation and/or signal transduction induced by a ligand binding to an allosteric site distinct from a molecule's active site. Allosteric drugs are currently receiving increased attention in drug discovery because drugs that target allosteric sites can provide important advantages over the corresponding orthosteric drugs including specific subtype selectivity within receptor families. Consequently, targeting allosteric sites, instead of orthosteric sites, can reduce drug-related side effects and toxicity. On the down side, allosteric drug discovery can be more challenging than traditional orthosteric drug discovery due to difficulties associated with determining the locations of allosteric sites and designing drugs based on these sites and the need for the allosteric effects to propagate through the structure, reach the ligand binding site and elicit a conformational change. In this study, we present computational tools ranging from the identification of potential allosteric sites to the design of "allosteric-like" modulator libraries. These tools may be particularly useful for allosteric drug discovery.

  12. New paradigm for allosteric regulation of Escherichia coli aspartate transcarbamoylase.

    PubMed

    Cockrell, Gregory M; Zheng, Yunan; Guo, Wenyue; Peterson, Alexis W; Truong, Jennifer K; Kantrowitz, Evan R

    2013-11-12

    For nearly 60 years, the ATP activation and the CTP inhibition of Escherichia coli aspartate transcarbamoylase (ATCase) has been the textbook example of allosteric regulation. We present kinetic data and five X-ray structures determined in the absence and presence of a Mg(2+) concentration within the physiological range. In the presence of 2 mM divalent cations (Mg(2+), Ca(2+), Zn(2+)), CTP does not significantly inhibit the enzyme, while the allosteric activation by ATP is enhanced. The data suggest that the actual allosteric inhibitor of ATCase in vivo is the combination of CTP, UTP, and a divalent cation, and the actual allosteric activator is a divalent cation with ATP or ATP and GTP. The structural data reveals that two NTPs can bind to each allosteric site with a divalent cation acting as a bridge between the triphosphates. Thus, the regulation of ATCase is far more complex than previously believed and calls many previous studies into question. The X-ray structures reveal that the catalytic chains undergo essentially no alternations; however, several regions of the regulatory chains undergo significant structural changes. Most significant is that the N-terminal region of the regulatory chains exists in different conformations in the allosterically activated and inhibited forms of the enzyme. Here, a new model of allosteric regulation is proposed.

  13. Mapping the binding site pocket of the serotonin 5-Hydroxytryptamine2A receptor. Ser3.36(159) provides a second interaction site for the protonated amine of serotonin but not of lysergic acid diethylamide or bufotenin.

    PubMed

    Almaula, N; Ebersole, B J; Zhang, D; Weinstein, H; Sealfon, S C

    1996-06-21

    Like other amine neurotransmitters that activate G-protein-coupled receptors, 5-hydroxytryptamine (5-HT) binds to the 5-HT2A receptor through the interaction of its cationic primary amino group with the conserved Asp3.32(155) in transmembrane helix 3. Computational experiments with a 5-HT2A receptor model suggest that the same functional group of 5-hydroxytryptamine also forms a hydrogen bond with the side chain of Ser3.36(159), which is adjacent in space to Asp3.32(155). However, other 5-HT2A receptor ligands like lysergic acid diethylamide (LSD), in which the amine nitrogen is embedded in a heterocycle, or N,N-dimethyl 5-HT, in which the side chain is a tertiary amine, are found in the computational simulations to interact with the aspartate but not with the serine, due mainly to steric hindrance. The predicted difference in the interaction of various ligands in the same receptor binding pocket was tested with site-directed mutagenesis of Ser3.36(159) --> Ala and Ser3.36(159) --> Cys. The alanine substitution led to an 18-fold reduction in 5-HT affinity and the cysteine substitution to an intermediate 5-fold decrease. LSD affinity, in contrast, was unaffected by either mutation. N,N-Dimethyl 5-HT affinity was unaffected by the cysteine mutation and had a comparatively small 3-fold decrease in affinity for the alanine mutant. These findings identify a mode of ligand-receptor complexation that involves two receptor side chains interacting with the same functional group of specific serotonergic ligands. This interaction serves to orient the ligands in the binding pocket and may influence the degree of receptor activation.

  14. Heat Shock Protein 70 Inhibitors. 1. 2,5′-Thiodipyrimidine and 5-(Phenylthio)pyrimidine Acrylamides as Irreversible Binders to an Allosteric Site on Heat Shock Protein 70

    PubMed Central

    2015-01-01

    Heat shock protein 70 (Hsp70) is an important emerging cancer target whose inhibition may affect multiple cancer-associated signaling pathways and, moreover, result in significant cancer cell apoptosis. Despite considerable interest from both academia and pharmaceutical companies in the discovery and development of druglike Hsp70 inhibitors, little success has been reported so far. Here we describe structure–activity relationship studies in the first rationally designed Hsp70 inhibitor class that binds to a novel allosteric pocket located in the N-terminal domain of the protein. These 2,5′-thiodipyrimidine and 5-(phenylthio)pyrimidine acrylamides take advantage of an active cysteine embedded in the allosteric pocket to act as covalent protein modifiers upon binding. The study identifies derivatives 17a and 20a, which selectively bind to Hsp70 in cancer cells. Addition of high nanomolar to low micromolar concentrations of these inhibitors to cancer cells leads to a reduction in the steady-state levels of Hsp70-sheltered oncoproteins, an effect associated with inhibition of cancer cell growth and apoptosis. In summary, the described scaffolds represent a viable starting point for the development of druglike Hsp70 inhibitors as novel anticancer therapeutics. PMID:24548207

  15. Sodium binding to hH3R and hH 4R--a molecular modeling study.

    PubMed

    Wittmann, Hans-Joachim; Seifert, Roland; Strasser, Andrea

    2014-08-01

    Several aminergic GPCRs, e.g., the human histamine H3-receptor (hH3R) are sensitive to sodium ions. Based on these experimental results, including site directed mutagenesis studies, a sodium binding pocket near to the highly conserved Asp2.50 was suggested. Recently, in the crystallized adenosine A2A receptor (4EIY), a sodium ion was found in a pocket, coordinated by Asp52, Ser91, and three water molecules. Despite high homology in amino acid sequence between hH3R and hH4R, pharmacological studies revealed that the hH4R is--in contrast to hH3R--not sensitive to sodium ions. In order to obtain a deeper insight onto the differences in sodium sensitivity between hH3R and hH4R, we performed molecular modelling studies, including molecular dynamic simulations and calculation of Gibbs energy of solvation. The results of the modeling studies suggested that the amino acid at position 7.42 influences sodium binding to aminergic GPCRs in different ways. A comparison of the amino acids forming the sodium binding channel between the ligand binding pocket and the sodium binding pocket of all human aminergic GPCRs showed an 80% occurrence of glycine--in contrast to hH3R and hH4R. The Gln7.42 at hH4R disrupts a water chain, connecting the Asp3.32 of the orthosteric binding site and the Asp2.50 of the allosteric binding site. Besides, the oxygen of the glutamine side chain stabilizes the interaction of the sodium ion with the Asp3.32. Thus, the binding of the sodium into the allosteric binding site might be hindered kinetically.

  16. Baclofen and other GABAB receptor agents are allosteric modulators of the CXCL12 chemokine receptor CXCR4.

    PubMed

    Guyon, Alice; Kussrow, Amanda; Olmsted, Ian Roys; Sandoz, Guillaume; Bornhop, Darryl J; Nahon, Jean-Louis

    2013-07-10

    CXCR4, a receptor for the chemokine CXCL12 (stromal-cell derived factor-1α), is a G-protein-coupled receptor (GPCR), expressed in the immune and CNS and integrally involved in various neurological disorders. The GABAB receptor is also a GPCR that mediates metabotropic action of the inhibitory neurotransmitter GABA and is located on neurons and immune cells as well. Using diverse approaches, we report novel interaction between GABAB receptor agents and CXCR4 and demonstrate allosteric binding of these agents to CXCR4. First, both GABAB antagonists and agonists block CXCL12-elicited chemotaxis in human breast cancer cells. Second, a GABAB antagonist blocks the potentiation by CXCL12 of high-threshold Ca(2+) channels in rat neurons. Third, electrophysiology in Xenopus oocytes and human embryonic kidney cell line 293 cells in which we coexpressed rat CXCR4 and the G-protein inward rectifier K(+) (GIRK) channel showed that GABAB antagonist and agonist modified CXCL12-evoked activation of GIRK channels. To investigate whether GABAB ligands bind to CXCR4, we expressed this receptor in heterologous systems lacking GABAB receptors and performed competition binding experiments. Our fluorescent resonance energy transfer experiments suggest that GABAB ligands do not bind CXCR4 at the CXCL12 binding pocket suggesting allosteric modulation, in accordance with our electrophysiology experiments. Finally, using backscattering interferometry and lipoparticles containing only the CXCR4 receptor, we quantified the binding affinity for the GABAB ligands, confirming a direct interaction with the CXCR4 receptor. The effect of GABAergic agents on CXCR4 suggests new therapeutic potentials for neurological and immune diseases.

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

    NASA Astrophysics Data System (ADS)

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

    2014-08-01

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

  18. Apolar distal pocket mutants of yeast cytochrome c peroxidase: Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to the triAla, triVal, and triLeu variants.

    PubMed

    Bidwai, Anil; Ayala, Caitlan; Vitello, Lidia B; Erman, James E

    2015-08-01

    Imidazole binding to three apolar distal heme pocket mutants of yeast cytochrome c peroxidase (CcP) has been investigated between pH4 and 8. The three CcP variants have Arg-48, Trp-51, and His-52 mutated to either all alanine, CcP(triAla), all valine, CcP(triVal), or all leucine residues, CcP(triLeu). The imidazole binding curves for all three mutants are biphasic indicating that each of the mutants exists in at least two conformational states with different affinities for imidazole. At pH7, the high-affinity conformations of the three CcP mutants bind imidazole between 3.8 and 4.7 orders of magnitude stronger than that of wild-type CcP while the low-affinity conformations have binding affinities about 2.5 orders of magnitude larger than wild-type CcP. Imidazole binding to the three CcP mutants is pH dependent with the strongest binding observed at high pH. Apparent pK(a) values for the transition in binding vary between 5.6 and 7.5 for the high-affinity conformations and between 6.2 and 6.8 for the low-affinity conformations of the CcP triple mutants. The kinetics of imidazole binding are also biphasic. The fast phase of imidazole binding to CcP(triAla) and CcP(triLeu) is linearly dependent on the imidazole concentration while the slow phase is independent of imidazole concentration. Both phases of imidazole binding to CcP(triVal) have a hyperbolic dependence on the imidazole concentration. The apparent association rate constants vary between 30 and 170 M(-1)s(-1) while the apparent dissociation rate constants vary between 0.05 and 0.43 s(-1). The CcP triple mutants have higher binding affinities for 1-methylimidazole and 4-nitroimidazole than does wild-type CcP.

  19. Synthesis of new (-)-Bestatin-based inhibitor libraries reveals a novel binding mode in the S1 pocket of the essential malaria M1 metalloaminopeptidase

    PubMed Central

    Velmourougane, Geetha; Harbut, Michael B.; Dalal, Seema; McGowan, Sheena; Oellig, Christine A.; Meinhardt, Nataline; Whisstock, James C.; Klemba, Michael; Greenbaum, Doron C.

    2012-01-01

    The malarial PfA-M1 metallo-aminopeptidase is considered a putative drug target. The natural product dipeptide mimetic, bestatin, is a potent inhibitor of PfA-M1. Herein we present a new, efficient and high-yielding protocol for the synthesis of bestatin derivatives from natural and unnatural N-Boc-D-amino acids. A diverse library of bestatin derivatives was synthesized with variants at the sidechain of either the α-hydroxy-β-amino acid (P1) or the adjacent natural α-amino acid (P1’). Surprisingly, we found that extended aromatic sidechains at the P1 position resulted in potent inhibition against PfA-M1. To understand these data, we determined the X-ray co-crystal structures of PfA-M1 with two derivatives having either a Tyr(OMe) 15 or Tyr(OBzl) 16 at the P1 position and observed substantial inhibitor-induced rearrangement of the primary loop within the PfA-M1 pocket that interacts with the P1 sidechain. Our data provide important insights for the rational design of more potent and selective inhibitors of this enzyme that may eventually lead to new therapies for malaria. PMID:21366301

  20. MHC allele-specific binding of a malaria peptide makes it become promiscuous on fitting a glycine residue into pocket 6.

    PubMed

    Vargas, Luis Eduardo; Parra, Carlos Alberto; Salazar, Luz Mary; Guzmán, Fanny; Pinto, Martha; Patarroyo, Manuel E

    2003-07-18

    Peptide 1585 (EVLYLKPLAGVYRSLKKQLE) has a highly conserved amino-acid sequence located in the Plasmodium falciparum main merozoite surface protein (MSP-1) C-terminal region, required for merozoite entry into human erythrocytes and therefore represents a vaccine candidate for P. falciparum malaria. Original sequence-specific binding to five HLA DRB1* alleles (0101, 0102, 0401, 0701, and 1101) revealed this peptide's specific HLA DRB1*0102 allele binding. This peptide's allele-specific binding to HLA DRB1*0102 took on broader specificity for the DRB1*0101, -0401, and -1101 alleles when lysine was replaced by glycine in position 17 (peptide 5198: EVLYLKPLAGVYRSLKG(17)QLE). Binding of the identified G(10)VYRSLKGQLE(20) C-terminal register to these alleles suggests that peptide promiscuous binding relied on fitting Y(12), L(15), and G(17) into P-1, P-4, and P-6, respectively. The implications of the findings and the future of this synthetic vaccine candidate are discussed.

  1. THE ANTIPSYCHOTIC POTENTIAL OF MUSCARINIC ALLOSTERIC MODULATION

    PubMed Central

    Bridges, Thomas M.; LeBois, Evan P.; Hopkins, Corey R.; Wood, Michael R.; Jones, Carrie K.; Conn, P. Jeffrey; Lindsley, Craig W.

    2016-01-01

    SUMMARY The cholinergic hypothesis of schizophrenia emerged over 50 years ago based on clinical observations with both anticholinergics and pan-muscarinic agonists. Not until the 1990s did the cholinergic hypothesis of schizophrenia receive renewed enthusiasm based on clinical data with xanomeline, a muscarinic acetylcholine receptor M1/M4-preferring orthosteric agonist. In a clinical trial with Alzheimer’s patients, xanomeline not only improved cognitive performance, but also reduced psychotic behaviors. This encouraging data spurred a second clinical trial in schizophrenic patients, wherein xanomeline significantly improved the positive, negative and cognitive symptom clusters. However, the question remained: Was the antipsychotic efficacy due to activation of M1, M4 or both M1/M4? Classical orthosteric ligands lacked the muscarinic receptor subtype selectivity required to address this key question. More recently, functional assays have allowed for the discovery of ligands that bind at allosteric sites, binding sites distinct from the orthosteric (acetylcholine) site, which are structurally less conserved and thereby afford high levels of receptor subtype selectivity. Recently, allosteric ligands, with unprecedented selectivity for either M1 or M4, have been discovered and have demonstrated comparable efficacy to xanomeline in preclinical antipsychotic and cognition models. These data suggest that selective allosteric activation of either M1 or M4 has antipsychotic potential through distinct, yet complimentary mechanisms. PMID:20520852

  2. The 3D Structure of the Binding Pocket of the Human Oxytocin Receptor for Benzoxazine Antagonists, Determined by Molecular Docking, Scoring Functions and 3D-QSAR Methods

    NASA Astrophysics Data System (ADS)

    Jójárt, Balázs; Martinek, Tamás A.; Márki, Árpád

    2005-05-01

    Molecular docking and 3D-QSAR studies were performed to determine the binding mode for a series of benzoxazine oxytocin antagonists taken from the literature. Structural hypotheses were generated by docking the most active molecule to the rigid receptor by means of AutoDock 3.05. The cluster analysis yielded seven possible binding conformations. These structures were refined by using constrained simulated annealing, and the further ligands were aligned in the refined receptor by molecular docking. A good correlation was found between the estimated Δ G bind and the p K i values for complex F. The Connolly-surface analysis, CoMFA and CoMSIA models q CoMFA 2 = 0.653, q CoMSA 2 = 0.630 and r pred,CoMFA 2 = 0.852 , r pred,CoMSIA 2 = 0.815) confirmed the scoring function results. The structural features of the receptor-ligand complex and the CoMFA and CoMSIA fields are in closely connected. These results suggest that receptor-ligand complex F is the most likely binding hypothesis for the studied benzoxazine analogs.

  3. Binding to retinoblastoma pocket domain does not alter the inter-domain flexibility of the J domain of SV40 large T antigen.

    PubMed

    Williams, Christina K; Vaithiyalingam, Sivaraja; Hammel, Michal; Pipas, James; Chazin, Walter J

    2012-02-15

    Simian Virus 40 uses the large T antigen (Tag) to bind and inactivate retinoblastoma tumor suppressor proteins (Rb), which can result in cellular transformation. Tag is a modular protein with four domains connected by flexible linkers. The N-terminal J domain of Tag is necessary for Rb inactivation. Binding of Rb is mediated by an LXCXE consensus motif immediately C-terminal to the J domain. Nuclear magnetic resonance (NMR) and small angle X-ray scattering (SAXS) were used to study the structural dynamics and interaction of Rb with the LXCXE motif, the J domain and a construct (N(260)) extending from the J domain through the origin binding domain (OBD). NMR and SAXS data revealed substantial flexibility between the domains in N(260). Binding of pRb to a construct containing the LXCXE motif and the J domain revealed weak interactions between pRb and the J domain. Analysis of the complex of pRb and N(260) indicated that the OBD is not involved and retains its dynamic independence from the remainder of Tag. These results support a 'chaperone' model in which the J domain of Tag changes its orientation as it acts upon different protein complexes.

  4. An engineered chorismate mutase with allosteric regulation.

    PubMed

    Zhang, Sheng; Wilson, David B; Ganem, Bruce

    2003-07-17

    Besides playing a central role in phenylalanine biosynthesis, the bifunctional P-protein in Eschericia coli provides a unique model system for investigating whether allosteric effects can be engineered into protein catalysts using modular regulatory elements. Previous studies have established that the P-protein contains three distinct domains whose functions are preserved, even when separated: chorismate mutase (residues 1-109), prephenate dehydratase (residues 101-285), and an allosteric domain (residues 286-386) for feedback inhibition by phenylalanine. By deleting the prephenate dehydrase domain, a functional chorismate mutase linked directly to the phenylalanine binding domain has been engineered and overexpressed. This manuscript reports the catalytic properties of the mutase in the absence and presence of phenylalanine.

  5. Allosteric inhibition of HIV-1 integrase activity

    PubMed Central

    Engelman, Alan; Kessl, Jacques J.; Kvaratskhelia, Mamuka

    2013-01-01

    HIV-1 integrase is an important therapeutic target in the fight against HIV/AIDS. Integrase strand transfer inhibitors (INSTIs), which target the enzyme active site, have witnessed clinical success over the past 5 years, but the generation of drug resistance poses challenges to INSTI-based therapies moving forward. Integrase is a dynamic protein, and its ordered multimerization is critical to enzyme activity. The integrase tetramer, bound to viral DNA, interacts with host LEDGF/p75 protein to tether integration to active genes. Allosteric integrase inhibitors (ALLINIs) that compete with LEDGF/p75 for binding to integrase disrupt integrase assembly with viral DNA and allosterically inhibit enzyme function. ALLINIs display steep dose response curves and synergize with INSTIs ex vivo, highlighting this novel inhibitor class for clinical development. PMID:23647983

  6. Pharmacophore screening of the protein data bank for specific binding site chemistry.

    PubMed

    Campagna-Slater, Valérie; Arrowsmith, Andrew G; Zhao, Yong; Schapira, Matthieu

    2010-03-22

    A simple computational approach was developed to screen the Protein Data Bank (PDB) for putative pockets possessing a specific binding site chemistry and geometry. The method employs two commonly used 3D screening technologies, namely identification of cavities in protein structures and pharmacophore screening of chemical libraries. For each protein structure, a pocket finding algorithm is used to extract potential binding sites containing the correct types of residues, which are then stored in a large SDF-formatted virtual library; pharmacophore filters describing the desired binding site chemistry and geometry are then applied to screen this virtual library and identify pockets matching the specified structural chemistry. As an example, this approach was used to screen all human protein structures in the PDB and identify sites having chemistry similar to that of known methyl-lysine binding domains that recognize chromatin methylation marks. The selected genes include known readers of the histone code as well as novel binding pockets that may be involved in epigenetic signaling. Putative allosteric sites were identified on the structures of TP53BP1, L3MBTL3, CHEK1, KDM4A, and CREBBP.

  7. NMR reveals a dynamic allosteric pathway in thrombin

    PubMed Central

    Handley, Lindsey D.; Fuglestad, Brian; Stearns, Kyle; Tonelli, Marco; Fenwick, R. Bryn; Markwick, Phineus R. L.; Komives, Elizabeth A.

    2017-01-01

    Although serine proteases are found ubiquitously in both eukaryotes and prokaryotes, and they comprise the largest of all of the peptidase families, their dynamic motions remain obscure. The backbone dynamics of the coagulation serine protease, apo-thrombin (S195M-thrombin), were compared to the substrate-bound form (PPACK-thrombin). R1, R2, 15N-{1H}NOEs, and relaxation dispersion NMR experiments were measured to capture motions across the ps to ms timescale. The ps-ns motions were not significantly altered upon substrate binding. The relaxation dispersion data revealed that apo-thrombin is highly dynamic, with μs-ms motions throughout the molecule. The region around the N-terminus of the heavy chain, the Na+-binding loop, and the 170 s loop, all of which are implicated in allosteric coupling between effector binding sites and the active site, were dynamic primarily in the apo-form. Most of the loops surrounding the active site become more ordered upon PPACK-binding, but residues in the N-terminal part of the heavy chain, the γ-loop, and anion-binding exosite 1, the main allosteric binding site, retain μs-ms motions. These residues form a dynamic allosteric pathway connecting the active site to the main allosteric site that remains in the substrate-bound form. PMID:28059082

  8. Influence of the conserved disulphide bond, exposed to the putative binding pocket, on the structure and function of the immunoglobulin-like molecular chaperone Caf1M of Yersinia pestis.

    PubMed

    Zav'yalov, V P; Chernovskaya, T V; Chapman, D A; Karlyshev, A V; MacIntyre, S; Zavialov, A V; Vasiliev, A M; Denesyuk, A I; Zav'yalova, G A; Dudich, I V; Korpela, T; Abramov, V M

    1997-06-01

    The Yersinia pestis protein Caf1M is a typical representative of a subfamily of periplasmic molecular chaperones with characteristic structural and functional features, one of which is the location of two conserved cysteine residues close to the putative binding pocket. We show that these residues form a disulphide bond, the reduction and alkylation of which significantly increases the dissociation constant of the Caf1M-Caf1 (where Caf 1 is a polypeptide subunit of the capsule) complex [from a Kd of (4.77+/-0.50)x10(-9) M for the intact protein to one of (3.68+/-0.68)x10(-8) M for the modified protein]. The importance of the disulphide bond for the formation of functional Caf1M in vivo was demonstrated using an Escherichia coli dsbA mutant carrying the Y. pestis f1 operon. In accordance with the CD and fluorescence measurements, the disulphide bond is not important for maintenance of the overall structure of the Caf1M molecule, but would appear to affect the fine structural properties of the subunit binding site. A three-dimensional model of the Caf1M-Caf1 complex was designed based on the published crystal structure of PapD (a chaperone required for Pap pili assembly) complexed with a peptide corresponding to the C-terminus of the papG subunit. In the model the disulphide bond is in close proximity to the invariant Caf1M Arg-23 and Lys-142 residues that are assumed to anchor the C-terminal group of the subunit. The importance of this characteristic disulphide bond for the orchestration of the binding site and subunit binding, as well as for the folding of the protein in vivo, is likely to be a common feature of this subfamily of Caf1M-like chaperones. A possible model for the role of the disulphide bond in Caf1 assembly is discussed.

  9. Structure of an Odorant-Vinding Protein form the Mosquito Aedes aegypti Suggests a Binding Pocket Covered by a pH-Sensitive

    SciTech Connect

    N Leite; R Krogh; W Xu; Y Ishida; J Iulek; W Leal; G Oliva

    2011-12-31

    The yellow fever mosquito, Aedes aegypti, is the primary vector for the viruses that cause yellow fever, mostly in tropical regions of Africa and in parts of South America, and human dengue, which infects 100 million people yearly in the tropics and subtropics. A better understanding of the structural biology of olfactory proteins may pave the way for the development of environmentally-friendly mosquito attractants and repellents, which may ultimately contribute to reduction of mosquito biting and disease transmission. Previously, we isolated and cloned a major, female-enriched odorant-binding protein (OBP) from the yellow fever mosquito, AaegOBP1, which was later inadvertently renamed AaegOBP39. We prepared recombinant samples of AaegOBP1 by using an expression system that allows proper formation of disulfide bridges and generates functional OBPs, which are indistinguishable from native OBPs. We crystallized AaegOBP1 and determined its three-dimensional structure at 1.85 {angstrom} resolution by molecular replacement based on the structure of the malaria mosquito OBP, AgamOBP1, the only mosquito OBP structure known to date. The structure of AaegOBP1 (= AaegOBP39) shares the common fold of insect OBPs with six {alpha}-helices knitted by three disulfide bonds. A long molecule of polyethylene glycol (PEG) was built into the electron-density maps identified in a long tunnel formed by a crystallographic dimer of AaegOBP1. Circular dichroism analysis indicated that delipidated AaegOBP1 undergoes a pH-dependent conformational change, which may lead to release of odorant at low pH (as in the environment in the vicinity of odorant receptors). A C-terminal loop covers the binding cavity and this 'lid' may be opened by disruption of an array of acid-labile hydrogen bonds thus explaining reduced or no binding affinity at low pH.

  10. Functions of key residues in the ligand-binding pocket of vitamin D receptor: Fragment molecular orbital interfragment interaction energy analysis

    NASA Astrophysics Data System (ADS)

    Yamagishi, Kenji; Yamamoto, Keiko; Yamada, Sachiko; Tokiwa, Hiroaki

    2006-03-01

    Fragment molecular orbital-interfragment interaction energy calculations of the vitamin D receptor (VDR)/1α,25-dihydroxyvitamin D 3 complex were utilized to assign functions of key residues of the VDR. Only one residue forms a significant interaction with the corresponding hydroxy group of the ligand, although two residues are located around each hydroxy group. The degradation of binding affinity for derivatives upon removal of a hydroxy group is closely related to the trend in the strength of the hydrogen bonds. Type II hereditary rickets due to an Arg274 point mutation is caused by the lack of the strongest hydrogen bond.

  11. Discovery of Novel Allosteric Effectors Based on the Predicted Allosteric Sites for Escherichia coli D-3-Phosphoglycerate Dehydrogenase

    PubMed Central

    Wang, Qian; Qi, Yifei; Yin, Ning; Lai, Luhua

    2014-01-01

    D-3-phosphoglycerate dehydrogenase (PGDH) from Escherichia coli catalyzes the first critical step in serine biosynthesis, and can be allosterically inhibited by serine. In a previous study, we developed a computational method for allosteric site prediction using a coarse-grained two-state Gō Model and perturbation. Two potential allosteric sites were predicted for E. coli PGDH, one close to the active site and the nucleotide binding site (Site I) and the other near the regulatory domain (Site II). In the present study, we discovered allosteric inhibitors and activators based on site I, using a high-throughput virtual screen, and followed by using surface plasmon resonance (SPR) to eliminate false positives. Compounds 1 and 2 demonstrated a low-concentration activation and high-concentration inhibition phenomenon, with IC50 values of 34.8 and 58.0 µM in enzymatic bioassays, respectively, comparable to that of the endogenous allosteric effector, L-serine. For its activation activity, compound 2 exhibited an AC50 value of 34.7 nM. The novel allosteric site discovered in PGDH was L-serine- and substrate-independent. Enzyme kinetics studies showed that these compounds influenced Km, kcat, and kcat/Km. We have also performed structure-activity relationship studies to discover high potency allosteric effectors. Compound 2-2, an analog of compound 2, showed the best in vitro activity with an IC50 of 22.3 µM. Compounds targeting this site can be used as new chemical probes to study metabolic regulation in E. coli. Our study not only identified a novel allosteric site and effectors for PGDH, but also provided a general strategy for designing new regulators for metabolic enzymes. PMID:24733054

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

    SciTech Connect

    Yu, Zhiyi Klaasse, Elisabeth Heitman, Laura H. IJzerman, Adriaan P.

    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.

  13. Computational and synthetic approaches for developing Lavendustin B derivatives as allosteric inhibitors of HIV-1 integrase

    PubMed Central

    Agharbaoui, Fatima E.; Hoyte, Ashley C.; Ferro, Stefania; Gitto, Rosaria; Buemi, Maria Rosa; Fuchs, James R.; Kvaratskhelia, Mamuka; De Luca, Laura

    2017-01-01

    Through structure-based virtual screening and subsequent activity assays of selected natural products, Lavendustin B was previously identified as an inhibitor of HIV-1 integrase (IN) interaction with its cognate cellular cofactor, lens epithelium-derived growth factor (LEDGF/p75). In order to improve the inhibitory potency we have employed in silico-based approaches. Particularly, a series of new analogues was designed and docked into the LEDGF/p75 binding pocket of HIV-1 IN. To identify promising leads we used the Molecular Mechanics energies combined with the Generalized Born and Surface Area continuum solvation (MM-GBSA) method, molecular dynamics simulations and analysis of hydrogen bond occupancies. On the basis of these studies, six analogues of Lavendustine B, containing the benzylamino-hydroxybenzoic scaffold, were selected for synthesis and structure activity-relationship (SAR) studies. Our results demonstrated a good correlation between computational and experimental data, and all six analogues displayed an improved potency for inhibiting IN binding to LEDGF/p75 in vitro to respect to the parent compound Lavendustin B. Additionally, these analogs show to inhibit weakly LEDGF/p75-independent IN catalytic activity suggesting a multimodal allosteric mechanism of action. Nevertheless, for the synthesized compounds similar profiles for HIV-1 inhibition and cytoxicity were highlighted. Taken together, our studies elucidated the mode of action of Lavendustin B analogs and provided a path for their further development as a new promising class of HIV-1 integrase inhibitors. PMID:27517812

  14. Computational and synthetic approaches for developing Lavendustin B derivatives as allosteric inhibitors of HIV-1 integrase.

    PubMed

    Agharbaoui, Fatima E; Hoyte, Ashley C; Ferro, Stefania; Gitto, Rosaria; Buemi, Maria Rosa; Fuchs, James R; Kvaratskhelia, Mamuka; De Luca, Laura

    2016-11-10

    Through structure-based virtual screening and subsequent activity assays of selected natural products, Lavendustin B was previously identified as an inhibitor of HIV-1 integrase (IN) interaction with its cognate cellular cofactor, lens epithelium-derived growth factor (LEDGF/p75). In order to improve the inhibitory potency we have employed in silico-based approaches. Particularly, a series of new analogues was designed and docked into the LEDGF/p75 binding pocket of HIV-1 IN. To identify promising leads we used the Molecular Mechanics energies combined with the Generalized Born and Surface Area continuum solvation (MM-GBSA) method, molecular dynamics simulations and analysis of hydrogen bond occupancies. On the basis of these studies, six analogues of Lavendustine B, containing the benzylamino-hydroxybenzoic scaffold, were selected for synthesis and structure activity-relationship (SAR) studies. Our results demonstrated a good correlation between computational and experimental data, and all six analogues displayed an improved potency for inhibiting IN binding to LEDGF/p75 in vitro to respect to the parent compound Lavendustin B. Additionally, these analogs show to inhibit weakly LEDGF/p75-independent IN catalytic activity suggesting a multimodal allosteric mechanism of action. Nevertheless, for the synthesized compounds similar profiles for HIV-1 inhibition and cytoxicity were highlighted. Taken together, our studies elucidated the mode of action of Lavendustin B analogs and provided a path for their further development as a new promising class of HIV-1 integrase inhibitors.

  15. Pocket ECG electrode

    NASA Technical Reports Server (NTRS)

    Lund, Gordon F. (Inventor)

    1982-01-01

    A low-noise electrode suited for sensing electrocardiograms when chronically and subcutaneously implanted in a free-ranging subject. The electrode comprises a pocket-shaped electrically conductive member with a single entrance adapted to receive body fluids. The exterior of the member and the entrance region is coated with electrical insulation so that the only electrolyte/electrode interface is within the member remote from artifact-generating tissue. Cloth straps are bonded to the member to permit the electrode to be sutured to tissue and to provide electrical lead flexure relief.

  16. Pocket ECG electrode

    NASA Technical Reports Server (NTRS)

    Lund, G. F. (Inventor)

    1980-01-01

    A low noise electrode suited for sensing electrocardiograms when chronically and subcutaneously implanted in a free ranging subject is described. The electrode comprises a pocket shaped electrically conductive member with a single entrance adapted to receive body fluids. The exterior of the member and the entrance region is coated with electrical insulation so that the only electrolyte/electrode interface is within the member, remote from artifact-generating tissue. Cloth straps are bonded to the member to permit the electrode to be sutured to tissue and to provide electrical lead flexure relief.

  17. NASA Pocket Statistics

    NASA Technical Reports Server (NTRS)

    1994-01-01

    Pocket Statistics is published for the use of NASA managers and their staff. Included herein is Administrative and Organizational information, summaries of Space Flight Activity including the NASA Major Launch Record, and NASA Procurement, Financial, and Manpower data. The NASA Major Launch Record includes all launches of Scout class and larger vehicles. Vehicle and spacecraft development flights are also included in the Major Launch Record. Shuttle missions are counted as one launch and one payload, where free flying payloads are not involved. Satellites deployed from the cargo bay of the Shuttle and placed in a separate orbit or trajectory are counted as an additional payload.

  18. NASA Pocket Statistics

    NASA Technical Reports Server (NTRS)

    1995-01-01

    NASA Pocket Statistics is published for the use of NASA managers and their staff. Included herein is Administrative and Organizational information, summaries of Space Flight Activity including the NASA Major Launch Record, and NASA Procurement, Financial, and Manpower data. The NASA Major Launch Record includes all launches of Scout class and larger vehicles. Vehicle and spacecraft development flights are also included in the Major Launch Record. Shuttle missions are counted as one launch and one payload, where free flying payloads are not involved. Satellites deployed from the cargo bay of the Shuttle and placed in a separate orbit or trajectory are counted as an additional payload.

  19. NASA Pocket Statistics

    NASA Technical Reports Server (NTRS)

    1996-01-01

    This booklet of pocket statistics includes the 1996 NASA Major Launch Record, NASA Procurement, Financial, and Workforce data. The NASA Major Launch Record includes all launches of Scout class and larger vehicles. Vehicle and spacecraft development flights are also included in the Major Luanch Record. Shuttle missions are counted as one launch and one payload, where free flying payloads are not involved. Satellites deployed from the cargo bay of the Shuttle and placed in a separate orbit or trajectory are counted as an additional payload.

  20. Targeting allosteric disulphide bonds in cancer.

    PubMed

    Hogg, Philip J

    2013-06-01

    Protein action in nature is generally controlled by the amount of protein produced and by chemical modification of the protein, and both are often perturbed in cancer. The amino acid side chains and the peptide and disulphide bonds that bind the polypeptide backbone can be post-translationally modified. Post-translational cleavage or the formation of disulphide bonds are now being identified in cancer-related proteins and it is timely to consider how these allosteric bonds could be targeted for new therapies.

  1. Total Syntheses and Initial Evaluation of [Ψ[C(=S)NH]Tpg4]vancomycin, [Ψ[C(=NH)NH]Tpg4]vancomycin, [Ψ[CH2NH]Tpg4]vancomycin and their (4-Chlorobiphenyl)methyl Derivatives: Synergistic Binding Pocket and Peripheral Modifications for the Glycopeptide Antibiotics

    PubMed Central

    Okano, Akinori; Nakayama, Atsushi; Wu, Kejia; Lindsey, Erick A.; Schammel, Alex W.; Feng, Yiqing; Collins, Karen C.

    2015-01-01

    Full details of studies are disclosed on the total synthesis of binding pocket analogues of vancomycin, bearing the peripheral L-vancosaminyl-1,2-D-glucosyl disaccharide, that contain changes to a key single atom in the residue 4 amide (residue 4 carbonyl O → S, NH, H2) designed to directly address the underlying molecular basis of resistance to vancomycin. Also disclosed are studies piloting the late stage transformations conducted on the synthetically more accessible C-terminus hydroxymethyl aglycon derivatives and full details of the peripheral chlorobiphenyl functionalization of all the binding pocket modified vancomycin analogues designed for dual D-Ala-D-Ala/D-Ala-D-Lac binding are reported. Their collective assessment indicate that combined binding pocket and chlorobiphenyl peripherally modified analogues exhibit a remarkable spectrum of antimicrobial activity (VSSA, MRSA, VanA and VanB VRE) and impressive potencies against both vancomycin-sensitive and vancomycin-resistant bacteria (MICs = 0.06–0.005 μg/mL and 0.5–0.06 μg/mL for the amidine and methylene analogues, respectively) and likely benefit from two independent and synergistic mechanisms of action, only one of which is dependent on D-Ala-D-Ala/D-Ala-D-Lac binding. Such analogues are likely to display especially durable antibiotic activity not prone to rapidly acquired clinical resistance. PMID:25750995

  2. Bacterial Rotary Export ATPases Are Allosterically Regulated by the Nucleotide Second Messenger Cyclic-di-GMP*

    PubMed Central

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

    2015-01-01

    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. PMID:26265469

  3. Insights into an original pocket-ligand pair classification: a promising tool for ligand profile prediction.

    PubMed

    Pérot, Stéphanie; Regad, Leslie; Reynès, Christelle; Spérandio, Olivier; Miteva, Maria A; Villoutreix, Bruno O; Camproux, Anne-Claude

    2013-01-01

    Pockets are today at the cornerstones of modern drug discovery projects and at the crossroad of several research fields, from structural biology to mathematical modeling. Being able to predict if a small molecule could bind to one or more protein targets or if a protein could bind to some given ligands is very useful for drug discovery endeavors, anticipation of binding to off- and anti-targets. To date, several studies explore such questions from chemogenomic approach to reverse docking methods. Most of these studies have been performed either from the viewpoint of ligands or targets. However it seems valuable to use information from both ligands and target binding pockets. Hence, we present a multivariate approach relating ligand properties with protein pocket properties from the analysis of known ligand-protein interactions. We explored and optimized the pocket-ligand pair space by combining pocket and ligand descriptors using Principal Component Analysis and developed a classification engine on this paired space, revealing five main clusters of pocket-ligand pairs sharing specific and similar structural or physico-chemical properties. These pocket-ligand pair clusters highlight correspondences between pocket and ligand topological and physico-chemical properties and capture relevant information with respect to protein-ligand interactions. Based on these pocket-ligand correspondences, a protocol of prediction of clusters sharing similarity in terms of recognition characteristics is developed for a given pocket-ligand complex and gives high performances. It is then extended to cluster prediction for a given pocket in order to acquire knowledge about its expected ligand profile or to cluster prediction for a given ligand in order to acquire knowledge about its expected pocket profile. This prediction approach shows promising results and could contribute to predict some ligand properties critical for binding to a given pocket, and conversely, some key pocket

  4. Influence of the conserved disulphide bond, exposed to the putative binding pocket, on the structure and function of the immunoglobulin-like molecular chaperone Caf1M of Yersinia pestis.

    PubMed Central

    Zav'yalov, V P; Chernovskaya, T V; Chapman, D A; Karlyshev, A V; MacIntyre, S; Zavialov, A V; Vasiliev, A M; Denesyuk, A I; Zav'yalova, G A; Dudich, I V; Korpela, T; Abramov, V M

    1997-01-01

    The Yersinia pestis protein Caf1M is a typical representative of a subfamily of periplasmic molecular chaperones with characteristic structural and functional features, one of which is the location of two conserved cysteine residues close to the putative binding pocket. We show that these residues form a disulphide bond, the reduction and alkylation of which significantly increases the dissociation constant of the Caf1M-Caf1 (where Caf 1 is a polypeptide subunit of the capsule) complex [from a Kd of (4.77+/-0.50)x10(-9) M for the intact protein to one of (3.68+/-0.68)x10(-8) M for the modified protein]. The importance of the disulphide bond for the formation of functional Caf1M in vivo was demonstrated using an Escherichia coli dsbA mutant carrying the Y. pestis f1 operon. In accordance with the CD and fluorescence measurements, the disulphide bond is not important for maintenance of the overall structure of the Caf1M molecule, but would appear to affect the fine structural properties of the subunit binding site. A three-dimensional model of the Caf1M-Caf1 complex was designed based on the published crystal structure of PapD (a chaperone required for Pap pili assembly) complexed with a peptide corresponding to the C-terminus of the papG subunit. In the model the disulphide bond is in close proximity to the invariant Caf1M Arg-23 and Lys-142 residues that are assumed to anchor the C-terminal group of the subunit. The importance of this characteristic disulphide bond for the orchestration of the binding site and subunit binding, as well as for the folding of the protein in vivo, is likely to be a common feature of this subfamily of Caf1M-like chaperones. A possible model for the role of the disulphide bond in Caf1 assembly is discussed. PMID:9182720

  5. Allosteric mechanisms can be distinguished using structural mass spectrometry

    PubMed Central

    Dyachenko, Andrey; Gruber, Ranit; Shimon, Liat; Horovitz, Amnon; Sharon, Michal

    2013-01-01

    The activity of many proteins, including metabolic enzymes, molecular machines, and ion channels, is often regulated by conformational changes that are induced or stabilized by ligand binding. In cases of multimeric proteins, such allosteric regulation has often been described by the concerted Monod–Wyman–Changeux and sequential Koshland–Némethy–Filmer classic models of cooperativity. Despite the important functional implications of the mechanism of cooperativity, it has been impossible in many cases to distinguish between these various allosteric models using ensemble measurements of ligand binding in bulk protein solutions. Here, we demonstrate that structural MS offers a way to break this impasse by providing the full distribution of ligand-bound states of a protein complex. Given this distribution, it is possible to determine all the binding constants of a ligand to a highly multimeric cooperative system, and thereby infer its allosteric mechanism. Our approach to the dissection of allosteric mechanisms relies on advances in MS—which provide the required resolution of ligand-bound states—and in data analysis. We validated our approach using the well-characterized Escherichia coli chaperone GroEL, a double-heptameric ring containing 14 ATP binding sites, which has become a paradigm for molecular machines. The values of the 14 binding constants of ATP to GroEL were determined, and the ATP-loading pathway of the chaperone was characterized. The methodology and analyses presented here are directly applicable to numerous other cooperative systems and are therefore expected to promote further research on allosteric systems. PMID:23589876

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

    PubMed Central

    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

  7. A novel allosteric mechanism in the cysteine peptidase cathepsin K discovered by computational methods

    NASA Astrophysics Data System (ADS)

    Novinec, Marko; Korenč, Matevž; Caflisch, Amedeo; Ranganathan, Rama; Lenarčič, Brigita; Baici, Antonio

    2014-02-01

    Allosteric modifiers have the potential to fine-tune enzyme activity. Therefore, targeting allosteric sites is gaining increasing recognition as a strategy in drug design. Here we report the use of computational methods for the discovery of the first small-molecule allosteric inhibitor of the collagenolytic cysteine peptidase cathepsin K, a major target for the treatment of osteoporosis. The molecule NSC13345 is identified by high-throughput docking of compound libraries to surface sites on the peptidase that are connected to the active site by an evolutionarily conserved network of residues (protein sector). The crystal structure of the complex shows that NSC13345 binds to a novel allosteric site on cathepsin K. The compound acts as a hyperbolic mixed modifier in the presence of a synthetic substrate, it completely inhibits collagen degradation and has good selectivity for cathepsin K over related enzymes. Altogether, these properties qualify our methodology and NSC13345 as promising candidates for allosteric drug design.

  8. The corneal pocket assay.

    PubMed

    Ziche, Marina; Morbidelli, Lucia

    2015-01-01

    The cornea in most species is physiologically avascular, and thus this assay allows the measurement of newly formed vessels. The continuous monitoring of neovascular growth in the same animal allows the evaluation of drugs acting as suppressors or stimulators of angiogenesis. Under anesthesia a micropocket is produced in the cornea thickness and the angiogenesis stimulus (tumor tissue, cell suspension, growth factor) is placed into the pocket in order to induce vascular outgrowth from the limbal capillaries. Neovascular development and progression can be modified by the presence of locally released or applied inhibitory factors or by systemic treatments. In this chapter the experimental details of the avascular cornea assay, the technical challenges, and advantages and disadvantages in different species are discussed. Protocols for local drug treatment and tissue sampling for histology and pharmacokinetic profile are reported.

  9. Pocket neutron REM meter

    SciTech Connect

    Quam, W.; Del Duca, T.; Plake, W.; Graves, G.; DeVore, T.; Warren, J.

    1982-01-01

    This paper describes a pocket-calculator-sized, neutron-sensitive, REM-responding personnel dosimeter that uses three tissue-equivalent cylindrical proportional counters as neutron-sensitive detectors. These are conventionally called Linear Energy Transfer (LET) counters. Miniaturized hybrid circuits are used for the linear pulse handling electronics, followed by a 256-channel ADC. A CMOS microprocessor is used to calculate REM exposure from the basic rads-tissue data supplied by the LET counters and also to provide timing and display functions. The instrument is used to continuously accumulate time in hours since reset, total counts accumulated, rads-tissue, and REM. At any time the user can display any one of these items or a channel number (an aid in calibration). The instrument provides such data with a precision of +- 3% for a total exposure of 1 mREM over 8 hours.

  10. Pocket neutron REM meter

    SciTech Connect

    Quam, W.; Del Duca, T.; Plake, W.; Graves, G.; DeVore, T.; Warren, J.

    1982-01-01

    This paper describes a pocket-calculator-sized, neutron-sensitive, REM-responding personnel dosimeter that uses three tissue-equivalent cylindrical proportional counters as neutron-sensitive detectors. These are conventionally called Linear Energy Transfer (LET) counters. Miniaturized hybrid circuits are used for the linear pulse handling electronics, followed by a 256-channel ADC. A CMOS microprocessor is used to calculate REM exposure from the basic rads-tissue data supplied by the LET counters and also to provide timing and display functions. The instrument is used to continuously accumulate time in hours since reset, total counts accumulated, rads-tissue, and REM. The user can display any one of these items or a channel number (an aid in calibration) at any time. Such data are provided with a precision of +- 3% for a total exposure of 1 mREM over eight hours.

  11. Mimicking the Regulation Step of Fe-Monooxygenases: Allosteric Modulation of Fe(IV) -Oxo Formation by Guest Binding in a Dinuclear Zn(II) -Fe(II) Calix[6]arene-Based Funnel Complex.

    PubMed

    Ségaud, Nathalie; De Thomasson, Constance; Daverat, Caroline; Sénéchal-David, Katell; Dos Santos, Amandine; Steinmetz, Vincent; Maître, Philippe; Rebilly, Jean-Noël; Banse, Frédéric; Reinaud, Olivia

    2017-02-24

    A heteroditopic ligand associated with a calix[6]arene scaffold bearing a tris(imidazole) coordinating site at its small rim and an amine/pyridine ligand at its large rim has been prepared, and its regioselective coordination to Zn(II) at the small rim and Fe(II) in the amine/pyridine ligand has been achieved. The heterodinuclear complex obtained displays an overall cone conformation capped by the tris(imidazole)Zn(II) moiety and bears a non-heme Fe(II) complex at its base. Each of the metal centers exhibits one labile position, allowing the coordination inside the cavity of a guest alkylamine at Zn(II) and the generation of reaction intermediates (Fe(III) (OOH) and Fe(IV) O) at the large rim. A dependence between the chain length of the encapsulated alkylamine and the distribution of Fe(III) (OOH) intermediates and Fe(III) (OMe) is observed. In addition, it is shown that the generation of the Fe(IV) O intermediate is enhanced by addition of the alkylamine guest. Hence, this supramolecular system gathers the three levels of reactivity control encountered in oxidoreductases: i) control of the Fe(II) redox properties through its first coordination sphere, allowing us to generate high valent reactive species; ii) control of guest binding through a hydrophobic funnel that drives its alkyl chain next to the reactive iron complex, thus mimicking the binding pocket of natural systems; iii) guest-modulated reactivity of the Fe(II) center towards oxidants.

  12. Extracellular Loop 2 of the Free Fatty Acid Receptor 2 Mediates Allosterism of a Phenylacetamide Ago-Allosteric ModulatorS⃞

    PubMed Central

    Smith, Nicola J.; Ward, Richard J.; Stoddart, Leigh A.; Hudson, Brian D.; Kostenis, Evi; Ulven, Trond; Morris, Joanne C.; Tränkle, Christian; Tikhonova, Irina G.; Adams, David R.

    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 molecular mechanisms mediating the activity of 4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide (4-CMTB), a recently described phenylacetamide allosteric agonist and allosteric modulator of endogenous ligand function at human FFA2, by combining our previous knowledge of the orthosteric binding site with targeted examination of 4-CMTB structure-activity relationships and mutagenesis and chimeric receptor generation. Here we show that 4-CMTB is a selective agonist for FFA2 that binds to a site distinct from the orthosteric site of the receptor. Ligand structure-activity relationship studies indicated that the N-thiazolyl amide is likely to provide hydrogen bond donor/acceptor interactions with the receptor. Substitution at Leu173 or the exchange of the entire extracellular loop 2 of FFA2 with that of FFA3 was sufficient to reduce or ablate, respectively, allosteric communication between the endogenous and allosteric agonists. Thus, we conclude that extracellular loop 2 of human FFA2 is required for transduction of cooperative signaling between the orthosteric and an as-yet-undefined allosteric binding site of the FFA2 receptor that is occupied by 4-CMTB. PMID:21498659

  13. Inhibitory Mechanism of an Allosteric Antibody Targeting the Glucagon Receptor*

    PubMed Central

    Mukund, Susmith; Shang, Yonglei; Clarke, Holly J.; Madjidi, Azadeh; Corn, Jacob E.; Kates, Lance; Kolumam, Ganesh; Chiang, Vicky; Luis, Elizabeth; Murray, Jeremy; Zhang, Yingnan; Hötzel, Isidro; Koth, Christopher M.; Allan, Bernard B.

    2013-01-01

    Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases. PMID:24189067

  14. Inhibitory mechanism of an allosteric antibody targeting the glucagon receptor.

    PubMed

    Mukund, Susmith; Shang, Yonglei; Clarke, Holly J; Madjidi, Azadeh; Corn, Jacob E; Kates, Lance; Kolumam, Ganesh; Chiang, Vicky; Luis, Elizabeth; Murray, Jeremy; Zhang, Yingnan; Hötzel, Isidro; Koth, Christopher M; Allan, Bernard B

    2013-12-13

    Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases.

  15. Molecular dynamics approach to probe the allosteric inhibition of PTP1B by chlorogenic and cichoric acid.

    PubMed

    Baskaran, Sarath Kumar; Goswami, Nabajyoti; Selvaraj, Sudhagar; Muthusamy, Velusamy Shanmuganathan; Lakshmi, Baddireddi Subhadra

    2012-08-27

    Protein tyrosine phosphatase 1B (PTP1B), a major negative regulator of the insulin and leptin signaling pathway, is a potential target for therapeutic intervention against diabetes and obesity. The recent discovery of an allosteric site in PTP1B has created an alternate strategy in the development of PTP1B targeted therapy. The current study investigates the molecular interactions between the allosteric site of PTP1B with two caffeoyl derivatives, chlorogenic acid (CGA) and cichoric acid (CHA), using computational strategies. Molecular docking analysis with CGA and CHA at the allosteric site of PTP1B were performed and the resulting protein-ligand complexes used for molecular dynamics simulation studies for a time scale of 10 ns. Results show stable binding of CGA and CHA at the allosteric site of PTP1B. The flexibility of the WPD loop was observed to be constrained by CGA and CHA in the open (inactive), providing molecular mechanism of allosteric inhibition. The allosteric inhibition of CGA and CHA of PTP1B was shown to be favorable due to no restriction by the α-7 helix in the binding of CGA and CHA at the allosteric binding site. In conclusion, our results exhibit an inhibitory pattern of CGA and CHA against PTP1B through potent binding at the allosteric site.

  16. An efficient perturbation method to predict the functionally key sites of glutamine binding protein.

    PubMed

    Lv, Dashuai; Wang, Cunxin; Li, Chunhua; Tan, Jianjun; Zhang, Xiaoyi

    2017-04-01

    Glutamine-Binding Protein (GlnBP) of Escherichia coli, an important member of the periplasmic binding protein family, is responsible for the first step in the active transport of glutamine across the cytoplasmic membrane. In this work, the functionally key regulation sites of GlnBP were identified by utilizing a perturbation method proposed by our group, in which the residues whose perturbations markedly change the binding free energy between GlnBP and glutamine are considered to be functionally key residues. The results show that besides the substrate binding sites, some other residues distant from the binding pocket, including the ones in the hinge regions between the two domains, the front- and back- door channels and the exposed region, are important for the function of glutamine binding and transport. The predicted results are well consistent with the theoretical and experimental data, which indicates that our method is an effective approach to identify the key residues important for both ligand binding and long-range allosteric signal transmission. This work can provide some insights into the function performance of GlnBP and the physical mechanism of its allosteric regulation.

  17. APoc: large-scale identification of similar protein pockets

    PubMed Central

    Gao, Mu; Skolnick, Jeffrey

    2013-01-01

    Motivation: Most proteins interact with small-molecule ligands such as metabolites or drug compounds. Over the past several decades, many of these interactions have been captured in high-resolution atomic structures. From a geometric point of view, most interaction sites for grasping these small-molecule ligands, as revealed in these structures, form concave shapes, or ‘pockets’, on the protein’s surface. An efficient method for comparing these pockets could greatly assist the classification of ligand-binding sites, prediction of protein molecular function and design of novel drug compounds. Results: We introduce a computational method, APoc (Alignment of Pockets), for the large-scale, sequence order-independent, structural comparison of protein pockets. A scoring function, the Pocket Similarity Score (PS-score), is derived to measure the level of similarity between pockets. Statistical models are used to estimate the significance of the PS-score based on millions of comparisons of randomly related pockets. APoc is a general robust method that may be applied to pockets identified by various approaches, such as ligand-binding sites as observed in experimental complex structures, or predicted pockets identified by a pocket-detection method. Finally, we curate large benchmark datasets to evaluate the performance of APoc and present interesting examples to demonstrate the usefulness of the method. We also demonstrate that APoc has better performance than the geometric hashing-based method SiteEngine. Availability and implementation: The APoc software package including the source code is freely available at http://cssb.biology.gatech.edu/APoc. Contact: skolnick@gatech.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23335017

  18. Differences in Allosteric Communication Pipelines in the Inactive and Active States of a GPCR

    PubMed Central

    Bhattacharya, Supriyo; Vaidehi, Nagarajan

    2014-01-01

    G-protein-coupled receptors (GPCRs) are membrane proteins that allosterically transduce the signal of ligand binding in the extracellular (EC) domain to couple to proteins in the intracellular (IC) domain. However, the complete pathway of allosteric communication from the EC to the IC domain, including the role of individual amino acids in the pathway is not known. Using the correlation in torsion angle movements calculated from microseconds-long molecular-dynamics simulations, we elucidated the allosteric pathways in three different conformational states of β2-adrenergic receptor (β2AR): 1), the inverse-agonist-bound inactive state; 2), the agonist-bound intermediate state; and (3), the agonist- and G-protein-bound fully active state. The inactive state is less dynamic compared with the intermediate and active states, showing dense clusters of allosteric pathways (allosteric pipelines) connecting the EC with the IC domain. The allosteric pipelines from the EC domain to the IC domain are weakened in the intermediate state, thus decoupling the EC domain from the IC domain and making the receptor more dynamic compared with the other states. Also, the orthosteric ligand-binding site becomes the initiator region for allosteric communication in the intermediate state. This finding agrees with the paradigm that the nature of the agonist governs the specific signaling state of the receptor. These results provide an understanding of the mechanism of allosteric communication in class A GPCRs. In addition, our analysis shows that mutations that affect the ligand efficacy, but not the binding affinity, are located in the allosteric pipelines. This clarifies the role of such mutations, which has hitherto been unexplained. PMID:25028884

  19. Allosteric mechanisms of nuclear receptors: insights from computational simulations.

    PubMed

    Mackinnon, Jonathan A G; Gallastegui, Nerea; Osguthorpe, David J; Hagler, Arnold T; Estébanez-Perpiñá, Eva

    2014-08-05

    The traditional structural view of allostery defines this key regulatory mechanism as the ability of one conformational event (allosteric site) to initiate another in a separate location (active site). In recent years computational simulations conducted to understand how this phenomenon occurs in nuclear receptors (NRs) has gained significant traction. These results have yield insights into allosteric changes and communication mechanisms that underpin ligand binding, coactivator binding site formation, post-translational modifications, and oncogenic mutations. Moreover, substantial efforts have been made in understanding the dynamic processes involved in ligand binding and coregulator recruitment to different NR conformations in order to predict cell/tissue-selective pharmacological outcomes of drugs. They also have improved the accuracy of in silico screening protocols so that nowadays they are becoming part of optimisation protocols for novel therapeutics. Here we summarise the important contributions that computational simulations have made towards understanding the structure/function relationships of NRs and how these can be exploited for rational drug design.

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

    PubMed

    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

    2013-11-14

    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 15 Å 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.

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

    NASA Astrophysics Data System (ADS)

    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.

  2. A Random Forest Model for Predicting Allosteric and Functional Sites on Proteins.

    PubMed

    Chen, Ava S-Y; Westwood, Nicholas J; Brear, Paul; Rogers, Graeme W; Mavridis, Lazaros; Mitchell, John B O

    2016-04-01

    We created a computational method to identify allosteric sites using a machine learning method trained and tested on protein structures containing bound ligand molecules. The Random Forest machine learning approach was adopted to build our three-way predictive model. Based on descriptors collated for each ligand and binding site, the classification model allows us to assign protein cavities as allosteric, regular or orthosteric, and hence to identify allosteric sites. 43 structural descriptors per complex were derived and were used to characterize individual protein-ligand binding sites belonging to the three classes, allosteric, regular and orthosteric. We carried out a separate validation on a further unseen set of protein structures containing the ligand 2-(N-cyclohexylamino) ethane sulfonic acid (CHES).

  3. Biased signalling and allosteric machines: new vistas and challenges for drug discovery.

    PubMed

    Kenakin, Terry P

    2012-03-01

    Seven transmembrane receptors (7TMRs) are nature's prototype allosteric proteins made to bind molecules at one location to subsequently change their shape to affect the binding of another molecule at another location. This paper attempts to describe the divergent 7TMR behaviours (i.e. third party allostery, receptor oligomerization, biased agonism) observed in pharmacology in terms of a homogeneous group of allosteric behaviours. By considering the bodies involved as a vector defined by a modulator, conduit and guest, these activities can all be described by a simple model of functional allostery made up of the Ehlert allosteric model and the Black/Leff operational model. It will be shown how this model yields parameters that can be used to characterize the activity of any ligand or protein producing effect through allosteric interaction with a 7TMR.

  4. Strain analysis of protein structures and low dimensionality of mechanical allosteric couplings.

    PubMed

    Mitchell, Michael R; Tlusty, Tsvi; Leibler, Stanislas

    2016-10-04

    In many proteins, especially allosteric proteins that communicate regulatory states from allosteric to active sites, structural deformations are functionally important. To understand these deformations, dynamical experiments are ideal but challenging. Using static structural information, although more limited than dynamical analysis, is much more accessible. Underused for protein analysis, strain is the natural quantity for studying local deformations. We calculate strain tensor fields for proteins deformed by ligands or thermal fluctuations using crystal and NMR structure ensembles. Strains-primarily shears-show deformations around binding sites. These deformations can be induced solely by ligand binding at distant allosteric sites. Shears reveal quasi-2D paths of mechanical coupling between allosteric and active sites that may constitute a widespread mechanism of allostery. We argue that strain-particularly shear-is the most appropriate quantity for analysis of local protein deformations. This analysis can reveal mechanical and biological properties of many proteins.

  5. Crystal structure of Sulfolobus acidocaldarius aspartate carbamoyltransferase in complex with its allosteric activator CTP.

    PubMed

    De Vos, Dirk; Xu, Ying; Aerts, Tony; Van Petegem, Filip; Van Beeumen, Jozef J

    2008-07-18

    Aspartate carbamoyltransferase (ATCase) is a paradigm for allosteric regulation of enzyme activity. B-class ATCases display very similar homotropic allosteric behaviour, but differ extensively in their heterotropic patterns. The ATCase from the thermoacidophilic archaeon Sulfolobus acidocaldarius, for example, is strongly activated by its metabolic pathway's end product CTP, in contrast with Escherichia coli ATCase which is inhibited by CTP. To investigate the structural basis of this property, we have solved the crystal structure of the S. acidocaldarius enzyme in complex with CTP. Structure comparison reveals that effector binding does not induce similar large-scale conformational changes as observed for the E. coli ATCase. However, shifts in sedimentation coefficients upon binding of the bi-substrate analogue PALA show the existence of structurally distinct allosteric states. This suggests that the so-called "Nucleotide-Perturbation model" for explaining heterotropic allosteric behaviour, which is based on global conformational strain, is not a general mechanism of B-class ATCases.

  6. ASBench: benchmarking sets for allosteric discovery.

    PubMed

    Huang, Wenkang; Wang, Guanqiao; Shen, Qiancheng; Liu, Xinyi; Lu, Shaoyong; Geng, Lv; Huang, Zhimin; Zhang, Jian

    2015-08-01

    Allostery allows for the fine-tuning of protein function. Targeting allosteric sites is gaining increasing recognition as a novel strategy in drug design. The key challenge in the discovery of allosteric sites has strongly motivated the development of computational methods and thus high-quality, publicly accessible standard data have become indispensable. Here, we report benchmarking data for experimentally determined allosteric sites through a complex process, including a 'Core set' with 235 unique allosteric sites and a 'Core-Diversity set' with 147 structurally diverse allosteric sites. These benchmarking sets can be exploited to develop efficient computational methods to predict unknown allosteric sites in proteins and reveal unique allosteric ligand-protein interactions to guide allosteric drug design.

  7. Hepatitis B Virus Capsids Have Diverse Structural Responses to Small-Molecule Ligands Bound to the Heteroaryldihydropyrimidine Pocket

    PubMed Central

    Venkatakrishnan, Balasubramanian; Katen, Sarah P.; Francis, Samson; Chirapu, Srinivas; Finn, M. G.

    2016-01-01

    ABSTRACT Though the hepatitis B virus (HBV) core protein is an important participant in many aspects of the viral life cycle, its best-characterized activity is self-assembly into 240-monomer capsids. Small molecules that target core protein (core protein allosteric modulators [CpAMs]) represent a promising antiviral strategy. To better understand the structural basis of the CpAM mechanism, we determined the crystal structure of the HBV capsid in complex with HAP18. HAP18 accelerates assembly, increases protein-protein association more than 100-fold, and induces assembly of nonicosahedral macrostructures. In a preformed capsid, HAP18 is found at quasiequivalent subunit-subunit interfaces. In a detailed comparison to the two other extant CpAM structures, we find that the HAP18-capsid structure presents a paradox. Whereas the two other structures expanded the capsid diameter by up to 10 Å, HAP18 caused only minor changes in quaternary structure and actually decreased the capsid diameter by ∼3 Å. These results indicate that CpAMs do not have a single allosteric effect on capsid structure. We suggest that HBV capsids present an ensemble of states that can be trapped by CpAMs, indicating a more complex basis for antiviral drug design. IMPORTANCE Hepatitis B virus core protein has multiple roles in the viral life cycle—assembly, compartment for reverse transcription, intracellular trafficking, and nuclear functions—making it an attractive antiviral target. Core protein allosteric modulators (CpAMs) are an experimental class of antivirals that bind core protein. The most recognized CpAM activity is that they accelerate core protein assembly and strengthen interactions between subunits. In this study, we observe that the CpAM-binding pocket has multiple conformations. We compare structures of capsids cocrystallized with different CpAMs and find that they also affect quaternary structure in different ways. These results suggest that the capsid “breathes” and is

  8. Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme).

    PubMed

    Hung, Hui-Chih; Kuo, Meng-Wei; Chang, Gu-Gang; Liu, Guang-Yaw

    2005-11-15

    Human mitochondrial NAD(P)+-dependent malate dehydrogenase (decarboxylating) (malic enzyme) can be specifically and allosterically activated by fumarate. X-ray crystal structures have revealed conformational changes in the enzyme in the absence and in the presence of fumarate. Previous studies have indicated that fumarate is bound to the allosteric pocket via Arg67 and Arg91. Mutation of these residues almost abolishes the activating effect of fumarate. However, these amino acid residues are conserved in some enzymes that are not activated by fumarate, suggesting that there may be additional factors controlling the activation mechanism. In the present study, we tried to delineate the detailed molecular mechanism of activation of the enzyme by fumarate. Site-directed mutagenesis was used to replace Asp102, which is one of the charged amino acids in the fumarate binding pocket and is not conserved in other decarboxylating malate dehydrogenases. In order to explore the charge effect of this residue, Asp102 was replaced by alanine, glutamate or lysine. Our experimental data clearly indicate the importance of Asp102 for activation by fumarate. Mutation of Asp102 to Ala or Lys significantly attenuated the activating effect of fumarate on the enzyme. Kinetic parameters indicate that the effect of fumarate was mainly to decrease the K(m) values for malate, Mg2+ and NAD+, but it did not notably elevate kcat. The apparent substrate K(m) values were reduced by increasing concentrations of fumarate. Furthermore, the greatest effect of fumarate activation was apparent at low malate, Mg2+ or NAD+ concentrations. The K(act) values were reduced with increasing concentrations of malate, Mg2+ and NAD+. The Asp102 mutants, however, are much less sensitive to regulation by fumarate. Mutation of Asp102 leads to the desensitization of the co-operative effect between fumarate and substrates of the enzyme.

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

    PubMed Central

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

    2015-01-01

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

  10. Allosteric Inhibitors Have Distinct Effects, but Also Common Modes of Action, in the HCV Polymerase

    PubMed Central

    Davis, Brittny C.; Brown, Jodian A.; Thorpe, Ian F.

    2015-01-01

    The RNA-dependent RNA polymerase from the Hepatitis C Virus (gene product NS5B) is a validated drug target because of its critical role in genome replication. There are at least four distinct allosteric sites on the polymerase to which several small molecule inhibitors bind. In addition, numerous crystal structures have been solved with different allosteric inhibitors bound to the polymerase. However, the molecular mechanisms by which these small molecules inhibit the enzyme have not been fully elucidated. There is evidence that allosteric inhibitors alter the intrinsic motions and distribution of conformations sampled by the enzyme. In this study we use molecular dynamics simulations to understand the structural and dynamic changes that result when inhibitors are bound at three different allosteric binding sites on the enzyme. We observe that ligand binding at each site alters the structure and dynamics of NS5B in a distinct manner. Nonetheless, our studies also highlight commonalities in the mechanisms of action of the different inhibitors. Each inhibitor alters the conformational states sampled by the enzyme, either by rigidifying the enzyme and preventing transitions between functional conformational states or by destabilizing the enzyme and preventing functionally relevant conformations from being adequately sampled. By illuminating the molecular mechanisms of allosteric inhibition, these studies delineate the intrinsic functional properties of the enzyme and pave the way for designing novel and more effective polymerase inhibitors. This information may also be important to understand how allosteric regulation occurs in related viral polymerases and other enzymes. PMID:25863069

  11. Structure-based discovery of the first allosteric inhibitors of cyclin-dependent kinase 2.

    PubMed

    Rastelli, Giulio; Anighoro, Andrew; Chripkova, Martina; Carrassa, Laura; Broggini, Massimo

    2014-01-01

    Allosteric targeting of protein kinases via displacement of the structural αC helix with type III allosteric inhibitors is currently gaining a foothold in drug discovery. Recently, the first crystal structure of CDK2 with an open allosteric pocket adjacent to the αC helix has been described, prospecting new opportunities to design more selective inhibitors, but the structure has not yet been exploited for the structure-based design of type III allosteric inhibitors. In this work we report the results of a virtual screening campaign that resulted in the discovery of the first-in-class type III allosteric ligands of CDK2. Using a combination of docking and post-docking analyses made with our tool BEAR, 7 allosteric ligands (hit rate of 20%) with micromolar affinity for CDK2 were identified, some of them inhibiting the growth of breast cancer cell lines in the micromolar range. Competition experiments performed in the presence of the ATP-competitive inhibitor staurosporine confirmed that the 7 ligands are truly allosteric, in agreement with their design. Of these, compound 2 bound CDK2 with an EC50 value of 3 μM and inhibited the proliferation of MDA-MB231 and ZR-75-1 breast cancer cells with IC50 values of approximately 20 μM, while compound 4 had an EC50 value of 71 μM and IC50 values around 4 μM. Remarkably, the most potent compound 4 was able to selectively inhibit CDK2-mediated Retinoblastoma phosphorylation, confirming that its mechanism of action is fully compatible with a selective inhibition of CDK2 phosphorylation in cells. Finally, hit expansion through analog search of the most potent inhibitor 4 revealed an additional ligand 4g with similar in vitro potency on breast cancer cells.

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

    NASA Astrophysics Data System (ADS)

    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.

  13. [G-protein-coupled receptors targeting: the allosteric approach].

    PubMed

    Sebag, Julien A; Pantel, Jacques

    2012-10-01

    G-protein-coupled receptors (GPCR) are a major family of drug targets. Essentially all drugs targeting these receptors on the market compete with the endogenous ligand (agonists or antagonists) for binding the receptor. Recently, non-competitive compounds binding to distinct sites from the cognate ligand were documented in various classes of these receptors. These compounds, called allosteric modulators, generally endowed of a better selectivity are able to modulate specifically the endogenous signaling of the receptor. To better understand the promising potential of this class of GPCRs targeting compounds, this review highlights the properties of allosteric modulators, the strategies used to identify them and the challenges associated with the development of these compounds.

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

    PubMed Central

    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. PMID:26823026

  15. Allosteric Modulation: An Alternate Approach Targeting the Cannabinoid CB1 Receptor.

    PubMed

    Nguyen, Thuy; Li, Jun-Xu; Thomas, Brian F; Wiley, Jenny L; Kenakin, Terry P; Zhang, Yanan

    2016-11-23

    The cannabinoid CB1 receptor is a G protein coupled receptor and plays an important role in many biological processes and physiological functions. A variety of CB1 receptor agonists and antagonists, including endocannabinoids, phytocannabinoids, and synthetic cannabinoids, have been discovered or developed over the past 20 years. In 2005, it was discovered that the CB1 receptor contains allosteric site(s) that can be recognized by small molecules or allosteric modulators. A number of CB1 receptor allosteric modulators, both positive and negative, have since been reported and importantly, they display pharmacological characteristics that are distinct from those of orthosteric agonists and antagonists. Given the psychoactive effects commonly associated with CB1 receptor agonists and antagonists/inverse agonists, allosteric modulation may offer an alternate approach to attain potential therapeutic benefits while avoiding inherent side effects of orthosteric ligands. This review details the complex pharmacological profiles of these allosteric modulators, their structure-activity relationships, and efforts in elucidating binding modes and mechanisms of actions of reported CB1 allosteric modulators. The ultimate development of CB1 receptor allosteric ligands could potentially lead to improved therapies for CB1-mediated neurological disorders.

  16. Prepaying the entropic cost for allosteric regulation in KIX.

    PubMed

    Law, Sean M; Gagnon, Jessica K; Mapp, Anna K; Brooks, Charles L

    2014-08-19

    The kinase-inducible domain interacting (KIX) domain of the CREB binding protein (CBP) is capable of simultaneously binding two intrinsically disordered transcription factors, such as the mixed-lineage leukemia (MLL) and c-Myb peptides, at isolated interaction sites. In vitro, the affinity for binding c-Myb is approximately doubled when KIX is in complex with MLL, which suggests a positive cooperative binding mechanism, and the affinity for MLL is also slightly increased when KIX is first bound by c-Myb. Expanding the scope of recent NMR and computational studies, we explore the allosteric mechanism at a detailed molecular level that directly connects the microscopic structural dynamics to the macroscopic shift in binding affinities. To this end, we have performed molecular dynamics simulations of free KIX, KIX-c-Myb, MLL-KIX, and MLL-KIX-c-Myb using a topology-based Gō-like model. Our results capture an increase in affinity for the peptide in the allosteric site when KIX is prebound by a complementary effector and both peptides follow an effector-independent folding-and-binding mechanism. More importantly, we discover that MLL binding lowers the entropic cost for c-Myb binding, and vice versa, by stabilizing the L12-G2 loop and the C-terminal region of the α3 helix on KIX. This work demonstrates the importance of entropy in allosteric signaling between promiscuous molecular recognition sites and can inform the rational design of small molecule stabilizers to target important regions of conformationally dynamic proteins.

  17. Allosteric regulation of phenylalanine hydroxylase.

    PubMed

    Fitzpatrick, Paul F

    2012-03-15

    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.

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

    PubMed

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-07-02

    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.

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

    PubMed Central

    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

  20. Structural Dynamics of the Heterodimeric ABC Transporter TM287/288 Induced by ATP and Substrate Binding.

    PubMed

    Furuta, Tadaomi; Sato, Yukiko; Sakurai, Minoru

    2016-12-06

    TM287/288 is a heterodimeric ATP-binding cassette (ABC) transporter, which harnesses the energy of ATP binding and hydrolysis at the nucleotide-binding domains (NBDs) to transport a wide variety of molecules through the transmembrane domains (TMDs) by alternating inward- and outward-facing conformations. Here, we conducted multiple 100 ns molecular dynamics simulations of TM287/288 in different ATP- and substrate-bound states to elucidate the effects of ATP and substrate binding. As a result, the binding of two ATP molecules to the NBDs induced the formation of the consensus ATP-binding pocket (ABP2) or the NBD dimerization, whereas these processes did not occur in the presence of a single ATP molecule or when the protein was in its apo state. Moreover, binding of the substrate to the TMDs enhanced the formation of ABP2 through allosteric TMD-NBD communication. Furthermore, in the apo state, α-helical subdomains of the NBDs approached each other, acquiring a conformation with core half-pockets exposed to the solvent, appropriate for ATP binding. We propose a "core-exposed" model for this novel conformation found in the apo state of ABC transporters. These findings provide important insights into the structural dynamics of ABC transporters.

  1. Motion of proximal histidine and structural allosteric transition in soluble guanylate cyclase.

    PubMed

    Yoo, Byung-Kuk; Lamarre, Isabelle; Martin, Jean-Louis; Rappaport, Fabrice; Negrerie, Michel

    2015-04-07

    We investigated the changes of heme coordination in purified soluble guanylate cyclase (sGC) by time-resolved spectroscopy in a time range encompassing 11 orders of magnitude (from 1 ps to 0.2 s). After dissociation, NO either recombines geminately to the 4-coordinate (4c) heme (τG1 = 7.5 ps; 97 ± 1% of the population) or exits the heme pocket (3 ± 1%). The proximal His rebinds to the 4c heme with a 70-ps time constant. Then, NO is distributed in two approximately equal populations (1.5%). One geminately rebinds to the 5c heme (τG2 = 6.5 ns), whereas the other diffuses out to the solution, from where it rebinds bimolecularly (τ = 50 μs with [NO] = 200 μM) forming a 6c heme with a diffusion-limited rate constant of 2 × 10(8) M(-1)⋅s(-1). In both cases, the rebinding of NO induces the cleavage of the Fe-His bond that can be observed as an individual reaction step. Saliently, the time constant of bond cleavage differs depending on whether NO binds geminately or from solution (τ5C1 = 0.66 μs and τ5C2 = 10 ms, respectively). Because the same event occurs with rates separated by four orders of magnitude, this measurement implies that sGC is in different structural states in both cases, having different strain exerted on the Fe-His bond. We show here that this structural allosteric transition takes place in the range 1-50 μs. In this context, the detection of NO binding to the proximal side of sGC heme is discussed.

  2. Motion of proximal histidine and structural allosteric transition in soluble guanylate cyclase

    PubMed Central

    Yoo, Byung-Kuk; Lamarre, Isabelle; Martin, Jean-Louis; Rappaport, Fabrice; Negrerie, Michel

    2015-01-01

    We investigated the changes of heme coordination in purified soluble guanylate cyclase (sGC) by time-resolved spectroscopy in a time range encompassing 11 orders of magnitude (from 1 ps to 0.2 s). After dissociation, NO either recombines geminately to the 4-coordinate (4c) heme (τG1 = 7.5 ps; 97 ± 1% of the population) or exits the heme pocket (3 ± 1%). The proximal His rebinds to the 4c heme with a 70-ps time constant. Then, NO is distributed in two approximately equal populations (1.5%). One geminately rebinds to the 5c heme (τG2 = 6.5 ns), whereas the other diffuses out to the solution, from where it rebinds bimolecularly (τ = 50 μs with [NO] = 200 μM) forming a 6c heme with a diffusion-limited rate constant of 2 × 108 M−1⋅s−1. In both cases, the rebinding of NO induces the cleavage of the Fe-His bond that can be observed as an individual reaction step. Saliently, the time constant of bond cleavage differs depending on whether NO binds geminately or from solution (τ5C1 = 0.66 μs and τ5C2 = 10 ms, respectively). Because the same event occurs with rates separated by four orders of magnitude, this measurement implies that sGC is in different structural states in both cases, having different strain exerted on the Fe-His bond. We show here that this structural allosteric transition takes place in the range 1–50 μs. In this context, the detection of NO binding to the proximal side of sGC heme is discussed. PMID:25831539

  3. Novel Electrophilic and Photoaffinity Covalent Probes for Mapping the Cannabinoid 1 Receptor Allosteric Site(s)

    PubMed Central

    2015-01-01

    Undesirable side effects associated with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R), a tractable target for treating several pathologies affecting humans, have greatly limited their translational potential. Recent discovery of CB1R negative allosteric modulators (NAMs) has renewed interest in CB1R by offering a potentially safer therapeutic avenue. To elucidate the CB1R allosteric binding motif and thereby facilitate rational drug discovery, we report the synthesis and biochemical characterization of first covalent ligands designed to bind irreversibly to the CB1R allosteric site. Either an electrophilic or a photoactivatable group was introduced at key positions of two classical CB1R NAMs: Org27569 (1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays, did not exhibit inverse agonism, and behaved as a robust positive allosteric modulator of binding of orthosteric agonist CP55,940. This novel covalent probe can serve as a useful tool for characterizing CB1R allosteric ligand-binding motifs. PMID:26529344

  4. Hydrophobic pocket targeting probes for enteroviruses

    NASA Astrophysics Data System (ADS)

    Martikainen, Mari; Salorinne, Kirsi; Lahtinen, Tanja; Malola, Sami; Permi, Perttu; Häkkinen, Hannu; Marjomäki, Varpu

    2015-10-01

    Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron microscopes. The probe mildly stabilized the virus particle by increasing the melting temperature by 1-3 degrees, and caused a delay in the uncoating of the virus in the cellular endosomes, but could not however inhibit the receptor binding, cellular entry or infectivity of the virus. The hydrophobic pocket binding moiety of the probe was shown to bind to echovirus 1 particle by STD and tr-NOESY NMR methods. Furthermore, binding to echovirus 1 and Coxsackievirus A9, and to a lesser extent to Coxsackie virus B3 was verified by using a gold nanocluster labeled probe by TEM analysis. Molecular modelling suggested that the probe fits the hydrophobic pockets of EV1 and CVA9, but not of CVB3 as expected, correlating well with the variations in the infectivity and stability of the virus particles. EV1 conjugated to the fluorescent dye labeled probe was efficiently internalized into the cells. The virus-fluorescent probe conjugate accumulated in the cytoplasmic endosomes and caused infection starting from 6 hours onwards. Remarkably, before and during the time of replication, the fluorescent probe was seen to leak from the virus-positive endosomes and thus separate from the capsid proteins that were left in the endosomes. These results suggest that, like the physiological hydrophobic content

  5. Allosteric regulation of rhomboid intramembrane proteolysis.

    PubMed

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

    2014-09-01

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

  6. The allosteric switching mechanism in bacteriophage MS2

    NASA Astrophysics Data System (ADS)

    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.

  7. Coarse-grained molecular simulations of allosteric cooperativity

    NASA Astrophysics Data System (ADS)

    Nandigrami, Prithviraj; Portman, John J.

    2016-03-01

    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 Ca2+ ions to each domain of Calmodulin (CaM) through simulations of a simple coarse-grained model. In this model, the protein's conformational transitions between open and closed conformational ensembles are simulated explicitly and ligand binding and unbinding are treated implicitly within the grand canonical ensemble. Ligand binding is cooperative because the binding sites are coupled through a shift in the dominant conformational ensemble upon binding. The classic Monod-Wyman-Changeux model of allostery with appropriate binding free energies to the open and closed ensembles accurately describes the simulated binding thermodynamics. The simulations predict that the two domains of CaM have distinct binding affinity and cooperativity. In particular, the C-terminal domain binds Ca2+ with higher affinity and greater cooperativity than the N-terminal domain. From a structural point of view, the affinity of an individual binding loop depends sensitively on the loop's structural compatibility with the ligand in the bound ensemble, as well as the conformational flexibility of the binding site in the unbound ensemble.

  8. Discovery of a Negative Allosteric Modulator of GABAB Receptors

    PubMed Central

    2014-01-01

    Initialized from the scaffold of CGP7930, an allosteric agonist of GABAB receptors, a series of noncompetitive antagonists were discovered. Among these compounds, compounds 3, 6, and 14 decreased agonist GABA-induced maximal effect of IP3 production in HEK293 cells overexpressing GABAB receptors and Gqi9 proteins without changing the EC50. Compounds 3, 6, and 14 not only inhibited agonist baclofen-induced ERK1/2 phosphorylation but also blocked CGP7930-induced ERK1/2 phosphorylation in HEK293 cells overexpressing GABAB receptors. The results suggested that compounds 3, 6, and 14 are negative allosteric modulators of GABAB receptors. The representative compound 14 decreased GABA-induced IP3 production with IC50 of 37.9 μM and had no effect on other GPCR Class C members such as mGluR1, mGluR2, and mGluR5. Finally, we showed that compound 14 did not bind to the orthosteric binding sites of GABAB receptors, demonstrating that compound 14 negatively modulated GABAB receptors activity as a negative allosteric modulator. PMID:25050158

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

    PubMed

    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.

  10. Conformationally Selective RNA Aptamers Allosterically Modulate the β2-Adrenoceptor

    PubMed Central

    Kahsai, Alem W.; Wisler, James W.; Lee, Jungmin; Ahn, Seungkirl; Cahill, 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.

    2016-01-01

    G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies how “biased agonists” activate specific subsets of a given receptor’s signaling profile. However, stabilization of 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 via unique, previously unappreciated mechanisms. Here, utilizing 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, 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

  11. Ensemble Properties of Network Rigidity Reveal Allosteric Mechanisms

    PubMed Central

    Jacobs, Donald J.; Livesay, Dennis R.; Mottonen, James M.; Vorov, Oleg K.; Istomin, Andrei Y.; Verma, Deeptak

    2015-01-01

    The distance constraint model (DCM) is a unique computational modeling paradigm that integrates mechanical and thermodynamic descriptions of macromolecular structure. That is, network rigidity calculations are used to account for nonadditivity within entropy components, thus restoring the utility of free energy decomposition. The DCM outputs a large number of structural characterizations that collectively allow for quantified stability/flexibility relationships (QSFR) to be identified. In this review, we describe the theoretical underpinnings of the DCM and introduce several common QSFR metrics. Application of the DCM across protein families highlights the sensitivity within the set of protein structure residue-to-residue couplings. Further, we have developed a perturbation method to identify putative allosteric sites, where large changes in QSFR upon rigidification (mimicking ligand-binding) detect sites likely to invoke allosteric changes. PMID:22052496

  12. Allosteric modulation of glycine receptors is more efficacious for partial rather than full agonists.

    PubMed

    Bíró, Tímea; Maksay, Gábor

    2004-06-01

    Allosteric modulation of [3H]strychnine binding to glycine receptors (GlyRs) was examined in synaptosomal membranes of rat spinal cord. An allosteric model enabled us to determine the cooperativity factors of the allosteric agents with [3H]strychnine and glycine bindings (alpha and beta, respectively). We modified the allosteric model with a slope factor because the slope values of the displacement curves of partial agonists (beta-alanine, taurine and gamma-aminobutyric acid) were beyond unity. The slope factor was reduced only by 100 microM propofol. Further, propofol showed positive cooperativity (beta < 1) stronger with taurine than with glycine. The extent of the positive cooperativity of propofol was nearly independent from the potencies and structures of partial agonists. The steroidal alphaxalone and minaxolone also potentiated taurine better than glycine. Alphaxalone exerted weak negative cooperativity with [3H]strychnine binding. Displacement by taurine is attenuated by granisetron and m-chlorophenylbiguanide representing negative cooperativity (beta > 1) greater than with glycine. The results suggest a developmental role of elevated perinatal levels of taurine and neurosteroids as well as a better allosteric modulation of decreased agonist efficacies for impaired glycine receptor-ionophores.

  13. Ribonuclease H/DNA Polymerase HIV-1 Reverse Transcriptase Dual Inhibitor: Mechanistic Studies on the Allosteric Mode of Action of Isatin-Based Compound RMNC6

    PubMed Central

    Corona, Angela; Meleddu, Rita; Esposito, Francesca; Distinto, Simona; Bianco, Giulia; Masaoka, Takashi; Maccioni, Elias; Menéndez-Arias, Luis; Alcaro, Stefano; Le Grice, Stuart F. J.; Tramontano, Enzo

    2016-01-01

    The DNA polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) are needed for the replication of the viral genome and are validated drug targets. However, there are no approved drugs inhibiting RNase H and the efficiency of DNA polymerase inhibitors can be diminished by the presence of drug resistance mutations. In this context, drugs inhibiting both activities could represent a significant advance towards better anti-HIV therapies. We report on the mechanisms of allosteric inhibition of a newly synthesized isatin-based compound designated as RMNC6 that showed IC50 values of 1.4 and 9.8 μM on HIV-1 RT-associated RNase H and polymerase activities, respectively. Blind docking studies predict that RMNC6 could bind two different pockets in the RT: one in the DNA polymerase domain (partially overlapping the non-nucleoside RT inhibitor [NNRTI] binding pocket), and a second one close to the RNase H active site. Enzymatic studies showed that RMNC6 interferes with efavirenz (an approved NNRTI) in its binding to the RT polymerase domain, although NNRTI resistance-associated mutations such as K103N, Y181C and Y188L had a minor impact on RT susceptibility to RMNC6. In addition, despite being naturally resistant to NNRTIs, the polymerase activity of HIV-1 group O RT was efficiently inhibited by RMNC6. The compound was also an inhibitor of the RNase H activity of wild-type HIV-1 group O RT, although we observed a 6.5-fold increase in the IC50 in comparison with the prototypic HIV-1 group M subtype B enzyme. Mutagenesis studies showed that RT RNase H domain residues Asn474 and Tyr501, and in a lesser extent Ala502 and Ala508, are critical for RMNC6 inhibition of the endonuclease activity of the RT, without affecting its DNA polymerization activity. Our results show that RMNC6 acts as a dual inhibitor with allosteric sites in the DNA polymerase and the RNase H domains of HIV-1 RT. PMID:26800261

  14. Ribonuclease H/DNA Polymerase HIV-1 Reverse Transcriptase Dual Inhibitor: Mechanistic Studies on the Allosteric Mode of Action of Isatin-Based Compound RMNC6.

    PubMed

    Corona, Angela; Meleddu, Rita; Esposito, Francesca; Distinto, Simona; Bianco, Giulia; Masaoka, Takashi; Maccioni, Elias; Menéndez-Arias, Luis; Alcaro, Stefano; Le Grice, Stuart F J; Tramontano, Enzo

    2016-01-01

    The DNA polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) are needed for the replication of the viral genome and are validated drug targets. However, there are no approved drugs inhibiting RNase H and the efficiency of DNA polymerase inhibitors can be diminished by the presence of drug resistance mutations. In this context, drugs inhibiting both activities could represent a significant advance towards better anti-HIV therapies. We report on the mechanisms of allosteric inhibition of a newly synthesized isatin-based compound designated as RMNC6 that showed IC50 values of 1.4 and 9.8 μM on HIV-1 RT-associated RNase H and polymerase activities, respectively. Blind docking studies predict that RMNC6 could bind two different pockets in the RT: one in the DNA polymerase domain (partially overlapping the non-nucleoside RT inhibitor [NNRTI] binding pocket), and a second one close to the RNase H active site. Enzymatic studies showed that RMNC6 interferes with efavirenz (an approved NNRTI) in its binding to the RT polymerase domain, although NNRTI resistance-associated mutations such as K103N, Y181C and Y188L had a minor impact on RT susceptibility to RMNC6. In addition, despite being naturally resistant to NNRTIs, the polymerase activity of HIV-1 group O RT was efficiently inhibited by RMNC6. The compound was also an inhibitor of the RNase H activity of wild-type HIV-1 group O RT, although we observed a 6.5-fold increase in the IC50 in comparison with the prototypic HIV-1 group M subtype B enzyme. Mutagenesis studies showed that RT RNase H domain residues Asn474 and Tyr501, and in a lesser extent Ala502 and Ala508, are critical for RMNC6 inhibition of the endonuclease activity of the RT, without affecting its DNA polymerization activity. Our results show that RMNC6 acts as a dual inhibitor with allosteric sites in the DNA polymerase and the RNase H domains of HIV-1 RT.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  17. Monitoring allostery in D2O: a necessary control in studies using hydrogen/deuterium exchange to characterize allosteric regulation.

    PubMed

    Prasannan, Charulata B; Artigues, Antonio; Fenton, Aron W

    2011-08-01

    There is currently a renewed focus aimed at understanding allosteric mechanisms at atomic resolution. This current interest seeks to understand how both changes in protein conformations and changes in protein dynamics contribute to relaying an allosteric signal between two ligand binding sites on a protein (e.g., active and allosteric sites). Both nuclear magnetic resonance (NMR), by monitoring protein dynamics directly, and hydrogen/deuterium exchange, by monitoring solvent accessibility of backbone amides, offer insights into protein dynamics. Unfortunately, many allosteric proteins exceed the size limitations of standard NMR techniques. Although hydrogen/deuterium exchange as detected by mass spectrometry (H/DX-MS) offers an alternative evaluation method, any application of hydrogen/deuterium exchange requires that the property being measured functions in both H(2)O and D(2)O. Due to the promising future H/DX-MS has in the evaluation of allosteric mechanisms in large proteins, we demonstrate an evaluation of allosteric regulation in D(2)O. Exemplified using phenylalanine inhibition of rabbit muscle pyruvate kinase, we find that binding of the inhibitor is greatly reduced in D(2)O, but the effector continues to elicit an allosteric response.

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

    PubMed Central

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

    2016-01-01

    G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gαi. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function. PMID:26703464

  19. Tuning the endocannabinoid system: allosteric modulators of the CB1 receptor.

    PubMed

    Ross, R A

    2007-11-01

    Cannabinoid CB1 receptor antagonists are novel therapeutics with potential for the treatment of a number of conditions including obesity, nicotine addition and metabolic syndrome. In 2005, Price et al. demonstrated that the cannabinoid CB1 receptor contains an allosteric-binding site which binds synthetic small molecules. In this issue of the British Journal of Pharmacology, Horswill et al. have extended these observations. They demonstrate that a structurally similar small molecule allosterically modulates the cannabinoid CB1 receptor and reduces body weight and food intake in an acute feeding model. Allosteric modulation now contends as a new strategy in the therapeutic exploitation of cannabinoid receptors that may offer certain advantages over the more familiar small molecules targeting the orthosteric site.

  20. The interplay between effector binding and allostery in an engineered protein switch.

    PubMed

    Choi, Jay H; Xiong, Tina; Ostermeier, Marc

    2016-09-01

    The protein design rules for engineering allosteric regulation are not well understood. A fundamental understanding of the determinants of ligand binding in an allosteric context could facilitate the design and construction of versatile protein switches and biosensors. Here, we conducted extensive in vitro and in vivo characterization of the effects of 285 unique point mutations at 15 residues in the maltose-binding pocket of the maltose-activated β-lactamase MBP317-347. MBP317-347 is an allosteric enzyme formed by the insertion of TEM-1 β-lactamase into the E. coli maltose binding protein (MBP). We find that the maltose-dependent resistance to ampicillin conferred to the cells by the MBP317-347 switch gene (the switch phenotype) is very robust to mutations, with most mutations slightly improving the switch phenotype. We identified 15 mutations that improved switch performance from twofold to 22-fold, primarily by decreasing the catalytic activity in the absence of maltose, perhaps by disrupting interactions that cause a small fraction of MBP in solution to exist in a partially closed state in the absence of maltose. Other notable mutations include K15D and K15H that increased maltose affinity 30-fold and Y155K and Y155R that compromised switching by diminishing the ability of maltose to increase catalytic activity. The data also provided insights into normal MBP physiology, as select mutations at D14, W62, and F156 retained high maltose affinity but abolished the switch's ability to substitute for MBP in the transport of maltose into the cell. The results reveal the complex relationship between ligand binding and allostery in this engineered switch.

  1. Signal peptides are allosteric activators of the protein translocase

    PubMed Central

    Gouridis, Giorgos; Karamanou, Spyridoula; Gelis, Ioannis; Kalodimos, Charalampos G.; Economou, Anastassios

    2010-01-01

    Extra-cytoplasmic polypeptides are usually synthesized as “preproteins” carrying aminoterminal, cleavable signal peptides1 and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA2,3. Most proteins destined for the periplasm and beyond are exported post-translationally by SecA2,3. Preprotein targeting to SecA is thought to involve signal peptides4 and chaperones like SecB5,6. Here we reveal that signal peptides have a novel role beyond targeting: they are essential allosteric activators of the translocase. Upon docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, “triggering” that drives the translocase to a lower activation energy state; then “trapping” that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus and, finally, “secretion” during which trapped mature domains undergo multiple turnovers of translocation in segments7. A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases. PMID:19924216

  2. CGP7930: a positive allosteric modulator of the GABAB receptor.

    PubMed

    Adams, C L; Lawrence, A J

    2007-01-01

    CGP7930 (3-(3',5'-Di-tert-butyl-4'-hydroxy)phenyl-2,2-dimethylpropanol) is a positive allosteric modulator of the metabotropic GABAB receptor. CGP7930 has been found to modulate the GABAB receptor in the open, or high affinity, state increasing agonist affinity for the receptor and signal transduction efficacy following agonist stimulation. The GABAB heteromeric subunit B2, involved in signal transduction but not ligand binding, seems to be the site of action of CGP7930 and similar allosteric modulators. When administered alone in naïve animals, CGP7930 acts as an anxiolytic in rodents without other overt behavioral effects and has also been demonstrated to reduce self-administration of nicotine, cocaine, or alcohol in rodents, suggesting that "fine tuning" of the GABAB receptor by positive allosteric modulators may be able to regulate abuse of these drugs. Baclofen, the GABAB agonist, is currently finding use in treating addiction and various other disorders, but this can result in off-target effects and tolerance. CGP7930 when co-administered with baclofen enhances its potency, which could in theory minimize deleterious effects. Further study of CGP7930 is required, but this compound, and others like it, holds potential in a clinical setting.

  3. A novel allosteric inhibitor of macrophage migration inhibitory factor (MIF).

    PubMed

    Bai, Fengwei; Asojo, Oluwatoyin A; Cirillo, Pier; Ciustea, Mihai; Ledizet, Michel; Aristoff, Paul A; Leng, Lin; Koski, Raymond A; Powell, Thomas J; Bucala, Richard; Anthony, Karen G

    2012-08-31

    Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.

  4. CB(1) receptor allosteric modulators display both agonist and signaling pathway specificity.

    PubMed

    Baillie, Gemma L; Horswill, James G; Anavi-Goffer, Sharon; Reggio, Patricia H; Bolognini, Daniele; Abood, Mary E; McAllister, Sean; Strange, Phillip G; Stephens, Gary J; Pertwee, Roger G; Ross, Ruth A

    2013-02-01

    We have previously identified allosteric modulators of the cannabinoid CB(1) receptor (Org 27569, PSNCBAM-1) that display a contradictory pharmacological profile: increasing the specific binding of the CB(1) receptor agonist [(3)H]CP55940 but producing a decrease in CB(1) receptor agonist efficacy. Here we investigated the effect one or both compounds in a broad range of signaling endpoints linked to CB(1) receptor activation. We assessed the effect of these compounds on CB(1) receptor agonist-induced [(35)S]GTPγS binding, inhibition, and stimulation of forskolin-stimulated cAMP production, phosphorylation of extracellular signal-regulated kinases (ERK), and β-arrestin recruitment. We also investigated the effect of these allosteric modulators on CB(1) agonist binding kinetics. Both compounds display ligand dependence, being significantly more potent as modulators of CP55940 signaling as compared with WIN55212 and having little effect on [(3)H]WIN55212 binding. Org 27569 displays biased antagonism whereby it inhibits: agonist-induced guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding, simulation (Gα(s)-mediated), and inhibition (Gα(i)-mediated) of cAMP production and β-arrestin recruitment. In contrast, it acts as an enhancer of agonist-induced ERK phosphorylation. Alone, the compound can act also as an allosteric agonist, increasing cAMP production and ERK phosphorylation. We find that in both saturation and kinetic-binding experiments, the Org 27569 and PSNCBAM-1 appeared to influence only orthosteric ligand maximum occupancy rather than affinity. The data indicate that the allosteric modulators share a common mechanism whereby they increase available high-affinity CB(1) agonist binding sites. The receptor conformation stabilized by the allosterics appears to induce signaling and also selectively traffics orthosteric agonist signaling via the ERK phosphorylation pathway.

  5. Allosteric properties of PH domains in Arf regulatory proteins.

    PubMed

    Roy, Neeladri Sekhar; Yohe, Marielle E; Randazzo, Paul A; Gruschus, James M

    2016-01-01

    Pleckstrin Homology (PH) domains bind phospholipids and proteins. They are critical regulatory elements of a number enzymes including guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) for Ras-superfamily guanine nucleotide binding proteins such as ADP-ribosylation factors (Arfs). Recent studies have indicated that many PH domains may bind more than one ligand cooperatively. Here we discuss the molecular basis of PH domain-dependent allosteric behavior of 2 ADP-ribosylation factor exchange factors, Grp1 and Brag2, cooperative binding of ligands to the PH domains of Grp1 and the Arf GTPase-activating protein, ASAP1, and the consequences for activity of the associated catalytic domains.

  6. Identification of overlapping but differential binding sites for the high-affinity CXCR3 antagonists NBI-74330 and VUF11211.

    PubMed

    Scholten, Danny J; Roumen, Luc; Wijtmans, Maikel; Verkade-Vreeker, Marlies C A; Custers, Hans; Lai, Michael; de Hooge, Daniela; Canals, Meritxell; de Esch, Iwan J P; Smit, Martine J; de Graaf, Chris; Leurs, Rob

    2014-01-01

    CXC chemokine receptor CXCR3 and/or its main three ligands CXCL9, CXCL10, and CXCL11 are highly upregulated in a variety of diseases. As such, considerable efforts have been made to develop small-molecule receptor CXCR3 antagonists, yielding distinct chemical classes of antagonists blocking binding and/or function of CXCR3 chemokines. Although it is suggested that these compounds bind in an allosteric fashion, thus far no evidence has been provided regarding the molecular details of their interaction with CXCR3. Using site-directed mutagenesis complemented with in silico homology modeling, we report the binding modes of two high-affinity CXCR3 antagonists of distinct chemotypes: VUF11211 [(S)-5-chloro-6-(4-(1-(4-chlorobenzyl)piperidin-4-yl)-3-ethylpiperazin-1-yl)-N-ethylnicotinamide] (piperazinyl-piperidine) with a rigid elongated structure containing two basic groups and NBI-74330 [(R)-N-(1-(3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)ethyl)-2-(4-fluoro-3-(trifluoromethyl)phenyl)-N-(pyridin-3-ylmethyl)acetamide] (8-azaquinazolinone) without any basic group. Here we show that NBI-74330 is anchored in the transmembrane minor pocket lined by helices 2 (W2.60, D2.63), 3 (F3.32), and 7 (S7.39, Y7.43), whereas VUF11211 extends from the minor pocket into the major pocket of the transmembrane domains, located between residues in helices 1 (Y1.39), 2 (W2.60), 3 (F3.32), 4 (D4.60), 6 (Y6.51), and 7 (S7.39, Y7.43). Mutation of these residues did not affect CXCL11 binding significantly, confirming the allosteric nature of the interaction of these small molecules with CXCR3. Moreover, the model derived from our in silico-guided studies fits well with the already published structure-activity relationship data on these ligands. Altogether, in this study, we show overlapping, yet different binding sites for two high-affinity CXCR3 antagonists, which offer new opportunities for the structure-based design of allosteric modulators for CXCR3.

  7. Characterization of the novel positive allosteric modulator, LY2119620, at the muscarinic M(2) and M(4) receptors.

    PubMed

    Croy, Carrie H; Schober, Douglas A; Xiao, Hongling; Quets, Anne; Christopoulos, Arthur; Felder, Christian C

    2014-07-01

    The M(4) receptor is a compelling therapeutic target, as this receptor modulates neural circuits dysregulated in schizophrenia, and there is clinical evidence that muscarinic agonists possess both antipsychotic and procognitive efficacy. Recent efforts have shifted toward allosteric ligands to maximize receptor selectivity and manipulate endogenous cholinergic and dopaminergic signaling. In this study, we present the pharmacological characterization of LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethoxy] thieno[2,3-b]pyridine-2-carboxamide), a M(2)/M(4) receptor-selective positive allosteric modulator (PAM), chemically evolved from hits identified through a M4 allosteric functional screen. Although unsuitable as a therapeutic due to M(2) receptor cross-reactivity and, thus, potential cardiovascular liability, LY2119620 surpassed previous congeners in potency and PAM activity and broadens research capabilities through its development into a radiotracer. Characterization of LY2119620 revealed evidence of probe dependence in both binding and functional assays. Guanosine 5'-[γ-(35)S]-triphosphate assays displayed differential potentiation depending on the orthosteric-allosteric pairing, with the largest cooperativity observed for oxotremorine M (Oxo-M) LY2119620. Further [(3)H]Oxo-M saturation binding, including studies with guanosine-5'-[(β,γ)-imido]triphosphate, suggests that both the orthosteric and allosteric ligands can alter the population of receptors in the active G protein-coupled state. Additionally, this work expands the characterization of the orthosteric agonist, iperoxo, at the M(4) receptor, and demonstrates that an allosteric ligand can positively modulate the binding and functional efficacy of this high efficacy ligand. Ultimately, it was the M(2) receptor pharmacology and PAM activity with iperoxo that made LY2119620 the most suitable allosteric partner for the M(2) active-state structure recently solved

  8. Allosteric modulation of ATP-gated P2X receptor channels

    PubMed Central

    Coddou, Claudio; Stojilkovic, Stanko S.; Huidobro-Toro, J. Pablo

    2013-01-01

    Seven mammalian purinergic receptor subunits, denoted P2X1 to P2X7, and several spliced forms of these subunits have been cloned. When heterologously expressed, these cDNAs encode ATP-gated non-selective cation channels organized as trimers. All activated receptors produce cell depolarization and promote Ca2+ influx through their pores and indirectly by activating voltage-gated calcium channels. However, the biophysical and pharmacological properties of these receptors differ considerably, and the majority of these subunits are also capable of forming heterotrimers with other members of the P2X receptor family, which confers further different properties. These channels have three ATP binding domains, presumably located between neighboring subunits, and occupancy of at least two binding sites is needed for their activation. In addition to the orthosteric binding sites for ATP, these receptors have additional allosteric sites that modulate the agonist action at receptors, including sites for trace metals, protons, neurosteroids, reactive oxygen species and phosphoinositides. The allosteric regulation of P2X receptors is frequently receptor-specific and could be a useful tool to identify P2X members in native tissues and their roles in signaling. The focus of this review is on common and receptor-specific allosteric modulation of P2X receptors and the molecular base accounting for allosteric binding sites. PMID:21639805

  9. NASA Pocket Statistics: 1997 Edition

    NASA Technical Reports Server (NTRS)

    1997-01-01

    POCKET STATISTICS is published by the NATIONAL AERONAUTICS AND SPACE ADMINISTRATION (NASA). Included in each edition is Administrative and Organizational information, summaries of Space Flight Activity including the NASA Major Launch Record, Aeronautics and Space Transportation and NASA Procurement, Financial and Workforce data. The NASA Major Launch Record includes all launches of Scout class and larger vehicles. Vehicle and spacecraft development flights are also included in the Major Launch Record. Shuttle missions are counted as one launch and one payload, where free flying payloads are not involved. All Satellites deployed from the cargo bay of the Shuttle and placed in a separate orbit or trajectory are counted as an additional payload.

  10. Modulation of hemoglobin dynamics by an allosteric effector

    PubMed Central

    Maccarini, Marco; Fouquet, Peter; Ho, Nancy T.; Ho, Chien; Makowski, Lee

    2017-01-01

    Abstract Hemoglobin (Hb) is an extensively studied paradigm of proteins that alter their function in response to allosteric effectors. Models of its action have been used as prototypes for structure‐function relationships in many proteins, and models for the molecular basis of its function have been deeply studied and extensively argued. Recent reports suggest that dynamics may play an important role in its function. Relatively little is known about the slow, correlated motions of hemoglobin subunits in various structural states because experimental and computational strategies for their characterization are challenging. Allosteric effectors such as inositol hexaphosphate (IHP) bind to both deoxy‐Hb and HbCO, albeit at different sites, leading to a lowered oxygen affinity. The manner in which these effectors impact oxygen binding is unclear and may involve changes in structure, dynamics or both. Here we use neutron spin echo measurements accompanied by wide‐angle X‐ray scattering to show that binding of IHP to HbCO results in an increase in the rate of coordinated motions of Hb subunits relative to one another with little if any change in large scale structure. This increase of large‐scale dynamics seems to be coupled with a decrease in the average magnitude of higher frequency modes of individual residues. These observations indicate that enhanced dynamic motions contribute to the functional changes induced by IHP and suggest that they may be responsible for the lowered oxygen affinity triggered by these effectors. PMID:27977887

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

    PubMed Central

    2015-01-01

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

  12. Regulatory network of the allosteric ATP inhibition of E. coli phosphofructokinase-2 studied by hybrid dimers.

    PubMed

    Villalobos, Pablo; Soto, Francisco; Baez, Mauricio; Babul, Jorge

    2016-01-01

    We have proposed an allosteric ATP inhibition mechanism of Pfk-2 determining the structure of different forms of the enzyme together with a kinetic enzyme analysis. Here we complement the mechanism by using hybrid oligomers of the homodimeric enzyme to get insights about the allosteric communication pathways between the same sites or different ones located in different subunits. Kinetic analysis of the hybrid enzymes indicate that homotropic interactions between allosteric sites for ATP or between substrate sites for fructose-6-P have a minor effect on the enzymatic inhibition induced by ATP. In fact, the sigmoid response for fructose-6-P observed at elevated ATP concentrations can be eliminated even though the enzymatic inhibition is still operative. Nevertheless, leverage coupling analysis supports heterotropic interactions between the allosteric ATP and fructose-6-P binding occurring between and within each subunit.

  13. Cyclic di-GMP allosterically inhibits the CRP-like protein (Clp) of Xanthomonas axonopodis pv. citri.

    PubMed

    Leduc, Jason L; Roberts, Gary P

    2009-11-01

    The protein Clp from Xanthomonas axonopodis pv. citri regulates pathogenesis and is a member of the CRP (cyclic AMP receptor protein) superfamily. We show that unlike the DNA-binding activity of other members of this family, the DNA-binding activity of Clp is allosterically inhibited by its effector and that cyclic di-GMP serves as that effector at physiological concentrations.

  14. Sulfated Pentagalloylglucoside is a Potent, Allosteric, and Selective Inhibitor of Factor XIa

    PubMed Central

    Al-Horani, Rami A.; Ponnusamy, Pooja; Mehta, Akul Y.; Gailani, David; Desai, Umesh R.

    2013-01-01

    Inhibition of factor XIa (FXIa) is a novel paradigm for developing anticoagulants without major bleeding consequences. We present the discovery of sulfated pentagalloylglucoside (6) as a highly selective inhibitor of human FXIa. Biochemical screening of a focused library led to the identification of 6, a sulfated aromatic mimetic of heparin. Inhibitor 6 displayed a potency of 551 nM against FXIa, which was at least 200-fold more selective than other relevant enzymes. It also prevented activation of factor IX and prolonged human plasma and whole blood clotting. Inhibitor 6 reduced VMAX of FXIa hydrolysis of chromogenic substrate without affecting the KM suggesting an allosteric mechanism. Competitive studies showed that 6 bound in the heparin-binding site of FXIa. No allosteric small molecule has been discovered to date that exhibits equivalent potency against FXIa. Inhibitor 6 is expected to open up a major route to allosteric FXIa anticoagulants with clinical relevance. PMID:23316863

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

    PubMed Central

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

    2017-01-01

    Exploitation of drug–drug synergism and allostery could yield superior therapies by capitalizing on the immensely diverse, but highly specific, potential associated with the biological macromolecular landscape. Here we describe a drug–drug synergy mediated by allosteric cross-talk in chromatin, whereby the binding of one drug alters the activity of the second. We found two unrelated drugs, RAPTA-T and auranofin, that yield a synergistic activity in killing cancer cells, which coincides with a substantially greater number of chromatin adducts formed by one of the compounds when adducts from the other agent are also present. We show that this occurs through an allosteric mechanism within the nucleosome, whereby defined histone adducts of one drug promote reaction of the other drug at a distant, specific histone site. This opens up possibilities for epigenetic targeting and suggests that allosteric modulation in nucleosomes may have biological relevance and potential for therapeutic interventions. PMID:28358030

  16. SAR studies on carboxylic acid series M(1) selective positive allosteric modulators (PAMs).

    PubMed

    Kuduk, Scott D; Beshore, Douglas C

    2014-01-01

    There is mounting evidence from preclinical and early proof-of-concept studies suggesting that selective modulation of the M1 muscarinic receptor is efficacious in cognitive models of Alzheimer's disease (AD). A number of nonselective M1 muscarinic agonists have previously shown positive effects on cognitive function in AD patients, but were limited due to cholinergic adverse events thought to be mediated by pan activation of the M2 to M5 sub-types. Thus, there is a need to identify selective activators of the M1 receptor to evaluate their potential in cognitive disorders. One strategy to confer selectivity for M1 is the identification of allosteric agonists or positive allosteric modulators, which would target an allosteric site on the M1 receptor rather than the highly conserved orthosteric acetylcholine binding site. BQCA has been identified as a highly selective carboxylic acid M1 PAM and this review focuses on an extensive lead optimization campaign undertaken on this compound.

  17. Structure-based drug design: exploring the proper filling of apolar pockets at enzyme active sites.

    PubMed

    Zürcher, Martina; Diederich, François

    2008-06-20

    The proper filling of apolar pockets at enzyme active sites is central for increasing binding activity and selectivity of hits and leads in medicinal chemistry. In our structure-based design approach toward the generation of potent enzyme inhibitors, we encountered a variety of challenges in gaining suitable binding affinity from the occupation of such pockets. We summarize them here for the first time. A fluorine scan of tricyclic thrombin inhibitors led to the discovery of favorable orthogonal dipolar C-F...CO interactions. Efficient cation-pi interactions were established in the S4 pocket of factor Xa, another serine protease from the blood coagulation cascade. Changing from mono- to bisubstrate inhibitors of catechol O-methyltransferase, a target in the L-Dopa-based treatment of Parkinson's disease, enabled the full exploitation of a previously unexplored hydrophobic pocket. Conformational preorganization of a pocket at an enzyme active site is crucial for harvesting binding affinity. This is demonstrated for two enzymes from the nonmevalonate pathway of isoprenoid biosynthesis, IspE and IspF, which are pursued as antimalarial targets. Disrupting crystallographically defined water networks on the way into a pocket might cost all of the binding free enthalpy gained from its occupation, as revealed in studies with tRNA-guanine transglycosylase, a target against shigellosis. Investigations of the active site of plasmepsin II, another antimalarial target, showed that principles for proper apolar cavity filling, originally developed for synthetic host-guest systems, are also applicable to enzyme environments.

  18. An allosteric model for ribonuclease.

    PubMed Central

    Walker, E J; Ralston, G B; Darvey, I G

    1975-01-01

    Data from two assay systems show that the kinetics of the hydrolysis of cytidine 2':3'-cyclic monophosphate by bovine pancreatic RNAase (ribonuclease) is not consistent with conventional models. An allosteric model involving a substrate-dependent change in the equilibrium between two enzyme conformations is proposed. Such a model gives rise to a calculated curve of velocity versus substrate concentration which fits the experimental data. The model is also consistent with the results of an examination of the tryptic digestion of RNAase. Substrate analogues are able to protect RNAase against hydrolysis by trypsin and the percentage of RNAase activity which remains after digestion increases sigmoidally as the analogue concentration is increased. The model also explains the pattern seen in the Km values quoted in the literature and is consistent with strong physical evidence for a ligand-induced conformational change for RNAase reported in the literature. PMID:1167152

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

    PubMed Central

    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 (<500 nM) and high selectivity for thrombin (>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

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

    PubMed

    Ollivier, Julien F; Shahrezaei, Vahid; Swain, Peter S

    2010-11-04

    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.

  1. Mechanisms of Allosteric Activation and Inhibition of the Deoxyribonucleoside Triphosphate Triphosphohydrolase from Enterococcus faecalis*♦

    PubMed Central

    Vorontsov, Ivan I.; Wu, Ying; DeLucia, Maria; Minasov, George; Mehrens, Jennifer; Shuvalova, Ludmilla; Anderson, Wayne F.; Ahn, Jinwoo

    2014-01-01

    EF1143 from Enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase), converting dNTPs into the deoxyribonucleosides and triphosphate. The dNTPase activity of EF1143 is regulated by canonical dNTPs, which simultaneously act as substrates and activity modulators. Previous crystal structures of apo-EF1143 and the protein bound to both dGTP and dATP suggested allosteric regulation of its enzymatic activity by dGTP binding at four identical allosteric sites. However, whether and how other canonical dNTPs regulate the enzyme activity was not defined. Here, we present the crystal structure of EF1143 in complex with dGTP and dTTP. The new structure reveals that the tetrameric EF1143 contains four additional secondary allosteric sites adjacent to the previously identified dGTP-binding primary regulatory sites. Structural and enzyme kinetic studies indicate that dGTP binding to the first allosteric site, with nanomolar affinity, is a prerequisite for substrate docking and hydrolysis. Then, the presence of a particular dNTP in the second site either enhances or inhibits the dNTPase activity of EF1143. Our results provide the first mechanistic insight into dNTP-mediated regulation of dNTPase activity. PMID:24338016

  2. Supramolecular Allosteric Cofacial Porphyrin Complexes

    SciTech Connect

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

    2008-04-12

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

  3. Multisite Binding of a General Anesthetic to the Prokaryotic Pentameric Erwinia chrysanthemi Ligand-gated Ion Channel (ELIC)*

    PubMed Central

    Spurny, Radovan; Billen, Bert; Howard, Rebecca J.; Brams, Marijke; Debaveye, Sarah; Price, Kerry L.; Weston, David A.; Strelkov, Sergei V.; Tytgat, Jan; Bertrand, Sonia; Bertrand, Daniel; Lummis, Sarah C. R.; Ulens, Chris

    2013-01-01

    Pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine, glycine, γ-aminobutyric acid GABAA/C receptors, and the Gloeobacter violaceus ligand-gated ion channel (GLIC), are receptors that contain multiple allosteric binding sites for a variety of therapeutics, including general anesthetics. Here, we report the x-ray crystal structure of the Erwinia chrysanthemi ligand-gated ion channel (ELIC) in complex with a derivative of chloroform, which reveals important features of anesthetic recognition, involving multiple binding at three different sites. One site is located in the channel pore and equates with a noncompetitive inhibitor site found in many pLGICs. A second transmembrane site is novel and is located in the lower part of the transmembrane domain, at an interface formed between adjacent subunits. A third site is also novel and is located in the extracellular domain in a hydrophobic pocket between the β7–β10 strands. Together, these results extend our understanding of pLGIC modulation and reveal several specific binding interactions that may contribute to modulator recognition, further substantiating a multisite model of allosteric modulation in this family of ion channels. PMID:23364792

  4. Dancing through Life: Molecular Dynamics Simulations and Network-Centric Modeling of Allosteric Mechanisms in Hsp70 and Hsp110 Chaperone Proteins

    PubMed Central

    Stetz, Gabrielle; Verkhivker, Gennady M.

    2015-01-01

    Hsp70 and Hsp110 chaperones play an important role in regulating cellular processes that involve protein folding and stabilization, which are essential for the integrity of signaling networks. Although many aspects of allosteric regulatory mechanisms in Hsp70 and Hsp110 chaperones have been extensively studied and significantly advanced in recent experimental studies, the atomistic picture of signal propagation and energetics of dynamics-based communication still remain unresolved. In this work, we have combined molecular dynamics simulations and protein stability analysis of the chaperone structures with the network modeling of residue interaction networks to characterize molecular determinants of allosteric mechanisms. We have shown that allosteric mechanisms of Hsp70 and Hsp110 chaperones may be primarily determined by nucleotide-induced redistribution of local conformational ensembles in the inter-domain regions and the substrate binding domain. Conformational dynamics and energetics of the peptide substrate binding with the Hsp70 structures has been analyzed using free energy calculations, revealing allosteric hotspots that control negative cooperativity between regulatory sites. The results have indicated that cooperative interactions may promote a population-shift mechanism in Hsp70, in which functional residues are organized in a broad and robust allosteric network that can link the nucleotide-binding site and the substrate-binding regions. A smaller allosteric network in Hsp110 structures may elicit an entropy-driven allostery that occurs in the absence of global structural changes. We have found that global mediating residues with high network centrality may be organized in stable local communities that are indispensable for structural stability and efficient allosteric communications. The network-centric analysis of allosteric interactions has also established that centrality of functional residues could correlate with their sensitivity to mutations

  5. Investigating Metabotropic Glutamate Receptor 5 Allosteric Modulator Cooperativity, Affinity, and Agonism: Enriching Structure-Function Studies and Structure-Activity Relationships

    PubMed Central

    Gregory, Karen J.; Noetzel, Meredith J.; Rook, Jerri M.; Vinson, Paige N.; Stauffer, Shaun R.; Rodriguez, Alice L.; Emmitte, Kyle A.; Zhou, Ya; Chun, Aspen C.; Felts, Andrew S.; Chauder, Brian A.; Lindsley, Craig W.; Niswender, Colleen M.

    2012-01-01

    Drug discovery programs increasingly are focusing on allosteric modulators as a means to modify the activity of G protein-coupled receptor (GPCR) targets. Allosteric binding sites are topographically distinct from the endogenous ligand (orthosteric) binding site, which allows for co-occupation of a single receptor with the endogenous ligand and an allosteric modulator that can alter receptor pharmacological characteristics. Negative allosteric modulators (NAMs) inhibit and positive allosteric modulators (PAMs) enhance the affinity and/or efficacy of orthosteric agonists. Established approaches for estimation of affinity and efficacy values for orthosteric ligands are not appropriate for allosteric modulators, and this presents challenges for fully understanding the actions of novel modulators of GPCRs. Metabotropic glutamate receptor 5 (mGlu5) is a family C GPCR for which a large array of allosteric modulators have been identified. We took advantage of the many tools for probing allosteric sites on mGlu5 to validate an operational model of allosterism that allows quantitative estimation of modulator affinity and cooperativity values. Affinity estimates derived from functional assays fit well with affinities measured in radioligand binding experiments for both PAMs and NAMs with diverse chemical scaffolds and varying degrees of cooperativity. We observed modulation bias for PAMs when we compared mGlu5-mediated Ca2+ mobilization and extracellular signal-regulated kinase 1/2 phosphorylation data. Furthermore, we used this model to quantify the effects of mutations that reduce binding or potentiation by PAMs. This model can be applied to PAM and NAM potency curves in combination with maximal fold-shift data to derive reliable estimates of modulator affinities. PMID:22863693

  6. What induces pocket openings on protein surface patches involved in protein-protein interactions?

    NASA Astrophysics Data System (ADS)

    Eyrisch, Susanne; Helms, Volkhard

    2009-02-01

    We previously showed for the proteins BCL-XL, IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

  7. Molecular mechanism of the allosteric regulation of the αγ heterodimer of human NAD-dependent isocitrate dehydrogenase

    PubMed Central

    Ma, Tengfei; Peng, Yingjie; Huang, Wei; Ding, Jianping

    2017-01-01

    Human NAD-dependent isocitrate dehydrogenase catalyzes the decarboxylation of isocitrate (ICT) into α-ketoglutarate in the Krebs cycle. It exists as the α2βγ heterotetramer composed of the αβ and αγ heterodimers. Previously, we have demonstrated biochemically that the α2βγ heterotetramer and αγ heterodimer can be allosterically activated by citrate (CIT) and ADP. In this work, we report the crystal structures of the αγ heterodimer with the γ subunit bound without or with different activators. Structural analyses show that CIT, ADP and Mg2+ bind adjacent to each other at the allosteric site. The CIT binding induces conformational changes at the allosteric site, which are transmitted to the active site through the heterodimer interface, leading to stabilization of the ICT binding at the active site and thus activation of the enzyme. The ADP binding induces no further conformational changes but enhances the CIT binding through Mg2+-mediated interactions, yielding a synergistic activation effect. ICT can also bind to the CIT-binding subsite, which induces similar conformational changes but exhibits a weaker activation effect. The functional roles of the key residues are verified by mutagenesis, kinetic and structural studies. Our structural and functional data together reveal the molecular mechanism of the allosteric regulation of the αγ heterodimer. PMID:28098230

  8. The Rational Design of Allosteric Interactions in a Monomeric Protein and its Applications to the Construction of Biosensors

    NASA Astrophysics Data System (ADS)

    Marvin, J. S.; Corcoran, E. E.; Hattangadi, N. A.; Zhang, J. V.; Gere, S. A.; Hellinga, H. W.

    1997-04-01

    Rational protein design is an emerging approach for testing general theories of structure and function. The ability to manipulate function rationally also offers the possibility of creating new proteins of biotechnological value. Here we use the design approach to test the current understanding of the structural principles of allosteric interactions in proteins and demonstrate how a simple allosteric system can form the basis for the construction of a generic biosensor molecular engineering system. We have identified regions in Escherichia coli maltose-binding protein that are predicted to be allosterically linked to its maltose-binding site. Environmentally sensitive fluorophores were covalently attached to unique thiols introduced by cysteine mutations at specific sites within these regions. The fluorescence of such conjugates changes cooperatively with respect to maltose binding, as predicted. Spatial separation of the binding site and reporter groups allows the intrinsic properties of each to be manipulated independently. Provided allosteric linkage is maintained, ligand binding can therefore be altered without affecting transduction of the binding event by fluorescence. To demonstrate applicability to biosensor technology, we have introduced a series of point mutations in the maltose-binding site that lower the affinity of the protein for its ligand. These mutant proteins have been combined in a composite biosensor capable of measuring substrate concentration within 5% accuracy over a concentration range spanning five orders of magnitude.

  9. Allosteric "beta-blocker" isolated from a DNA-encoded small molecule library.

    PubMed

    Ahn, Seungkirl; Kahsai, Alem W; Pani, Biswaranjan; Wang, Qin-Ting; Zhao, Shuai; Wall, Alissa L; Strachan, Ryan T; Staus, Dean P; Wingler, Laura M; Sun, Lillian D; Sinnaeve, Justine; Choi, Minjung; Cho, Ted; Xu, Thomas T; Hansen, Gwenn M; Burnett, Michael B; Lamerdin, Jane E; Bassoni, Daniel L; Gavino, Bryant J; Husemoen, Gitte; Olsen, Eva K; Franch, Thomas; Costanzi, Stefano; Chen, Xin; Lefkowitz, Robert J

    2017-02-14

    The β2-adrenergic receptor (β2AR) has been a model system for understanding regulatory mechanisms of G-protein-coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known β-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human β2AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the β2AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the β2AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the β2AR. In cell-signaling studies, 15 inhibits cAMP production through the β2AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits β-arrestin recruitment to the activated β2AR. This study presents an allosteric small-molecule ligand for the β2AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects.

  10. Allosteric “beta-blocker” isolated from a DNA-encoded small molecule library

    PubMed Central

    Ahn, Seungkirl; Kahsai, Alem W.; Pani, Biswaranjan; Wang, Qin-Ting; Zhao, Shuai; Wall, Alissa L.; Strachan, Ryan T.; Staus, Dean P.; Wingler, Laura M.; Sun, Lillian D.; Sinnaeve, Justine; Choi, Minjung; Cho, Ted; Xu, Thomas T.; Hansen, Gwenn M.; Burnett, Michael B.; Lamerdin, Jane E.; Bassoni, Daniel L.; Gavino, Bryant J.; Husemoen, Gitte; Olsen, Eva K.; Franch, Thomas; Costanzi, Stefano; Chen, Xin; Lefkowitz, Robert J.

    2017-01-01

    The β2-adrenergic receptor (β2AR) has been a model system for understanding regulatory mechanisms of G-protein–coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known β-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human β2AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the β2AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the β2AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the β2AR. In cell-signaling studies, 15 inhibits cAMP production through the β2AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits β-arrestin recruitment to the activated β2AR. This study presents an allosteric small-molecule ligand for the β2AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects. PMID:28130548

  11. Internalization of the chemokine receptor CCR4 can be evoked by orthosteric and allosteric receptor antagonists.

    PubMed

    Ajram, Laura; Begg, Malcolm; Slack, Robert; Cryan, Jenni; Hall, David; Hodgson, Simon; Ford, Alison; Barnes, Ashley; Swieboda, Dawid; Mousnier, Aurelie; Solari, Roberto

    2014-04-15

    The chemokine receptor CCR4 has at least two natural agonist ligands, MDC (CCL22) and TARC (CCL17) which bind to the same orthosteric site with a similar affinity. Both ligands are known to evoke chemotaxis of CCR4-bearing T cells and also elicit CCR4 receptor internalization. A series of small molecule allosteric antagonists have been described which displace the agonist ligand, and inhibit chemotaxis. The aim of this study was to determine which cellular coupling pathways are involved in internalization, and if antagonists binding to the CCR4 receptor could themselves evoke receptor internalization. CCL22 binding coupled CCR4 efficiently to β-arrestin and stimulated GTPγS binding however CCL17 did not couple to β-arrestin and only partially stimulated GTPγS binding. CCL22 potently induced internalization of almost all cell surface CCR4, while CCL17 showed only weak effects. We describe four small molecule antagonists that were demonstrated to bind to two distinct allosteric sites on the CCR4 receptor, and while both classes inhibited agonist ligand binding and chemotaxis, one of the allosteric sites also evoked receptor internalization. Furthermore, we also characterize an N-terminally truncated version of CCL22 which acts as a competitive antagonist at the orthosteric site, and surprisingly also evokes receptor internalization without demonstrating any agonist activity. Collectively this study demonstrates that orthosteric and allosteric antagonists of the CCR4 receptor are capable of evoking receptor internalization, providing a novel strategy for drug discovery against this class of target.

  12. Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G.

    PubMed

    Papadopoulos, Evangelos; Jenni, Simon; Kabha, Eihab; Takrouri, Khuloud J; Yi, Tingfang; Salvi, Nicola; Luna, Rafael E; Gavathiotis, Evripidis; Mahalingam, Poornachandran; Arthanari, Haribabu; Rodriguez-Mias, Ricard; Yefidoff-Freedman, Revital; Aktas, Bertal H; Chorev, Michael; Halperin, Jose A; Wagner, Gerhard

    2014-08-05

    The interaction of the eukaryotic translation initiation factor eIF4E with the initiation factor eIF4G recruits the 40S ribosomal particle to the 5' end of mRNAs, facilitates scanning to the AUG start codon, and is crucial for eukaryotic translation of nearly all genes. Efficient recruitment of the 40S particle is particularly important for translation of mRNAs encoding oncoproteins and growth-promoting factors, which often harbor complex 5' UTRs and require efficient initiation. Thus, inhibiting the eIF4E/eIF4G interaction has emerged as a previously unpursued route for developing anticancer agents. Indeed, we discovered small-molecule inhibitors of this eIF4E/eIF4G interaction (4EGIs) that inhibit translation initiation both in vitro and in vivo and were used successfully in numerous cancer-biology and neurobiology studies. However, their detailed molecular mechanism of action has remained elusive. Here, we show that the eIF4E/eIF4G inhibitor 4EGI-1 acts allosterically by binding to a site on eIF4E distant from the eIF4G binding epitope. Data from NMR mapping and high-resolution crystal structures are congruent with this mechanism, where 4EGI-1 attaches to a hydrophobic pocket of eIF4E between β-sheet2 (L60-T68) and α-helix1 (E69-N77), causing localized conformational changes mainly in the H78-L85 region. It acts by unfolding a short 310-helix (S82-L85) while extending α-helix1 by one turn (H78-S82). This unusual helix rearrangement has not been seen in any previous eIF4E structure and reveals elements of an allosteric inhibition mechanism leading to the dislocation of eIF4G from eIF4E.

  13. Crystal structure of human phosphoribosylpyrophosphate synthetase 1 reveals a novel allosteric site.

    PubMed

    Li, Sheng; Lu, Yongcheng; Peng, Baozhen; Ding, Jianping

    2007-01-01

    PRPP (phosphoribosylpyrophosphate) is an important metabolite essential for nucleotide synthesis and PRS (PRPP synthetase) catalyses synthesis of PRPP from R5P (ribose 5-phosphate) and ATP. The enzymatic activity of PRS is regulated by phosphate ions, divalent metal cations and ADP. In the present study we report the crystal structures of recombinant human PRS1 in complexes with SO4(2-) ions alone and with ATP, Cd2+ and SO4(2-) ions respectively. The AMP moiety of ATP binds at the ATP-binding site, and a Cd2+ ion binds at the active site and in a position to interact with the beta- and gamma-phosphates of ATP. A SO4(2-) ion, an analogue of the activator phosphate, was found to bind at both the R5P-binding site and the allosteric site defined previously. In addi-tion, an extra SO4(2-) binds at a site at the dimer interface between the ATP-binding site and the allosteric site. Binding of this SO4(2-) stabilizes the conformation of the flexible loop at the active site, leading to the formation of the active, open conformation which is essential for binding of ATP and initiation of the catalytic reaction. This is the first time that structural stabilization at the active site caused by binding of an activator has been observed. Structural and biochemical data show that mutations of some residues at this site influence the binding of SO4(2-) and affect the enzymatic activity. The results in the present paper suggest that this new SO4(2-)-binding site is a second allosteric site to regulate the enzymatic activity which might also exist in other eukaryotic PRSs (except plant PRSs of class II), but not in bacterial PRSs.

  14. Single-chain antibody-fragment M6P-1 possesses a mannose 6-phosphate monosaccharide-specific binding pocket that distinguishes N-glycan phosphorylation in a branch-specific manner†

    PubMed Central

    Blackler, Ryan J; Evans, Dylan W; Smith, David F; Cummings, Richard D; Brooks, Cory L; Braulke, Thomas; Liu, Xinyu; Evans, Stephen V; Müller-Loennies, Sven

    2016-01-01

    The acquisition of mannose 6-phosphate (Man6P) on N-linked glycans of lysosomal enzymes is a structural requirement for their transport from the Golgi apparatus to lysosomes mediated by the mannose 6-phosphate receptors, 300 kDa cation-independent mannose 6-phosphate receptor (MPR300) and 46 kDa cation-dependent mannose 6-phosphate receptor (MPR46). Here we report that the single-chain variable domain (scFv) M6P-1 is a unique antibody fragment with specificity for Man6P monosaccharide that, through an array-screening approach against a number of phosphorylated N-glycans, is shown to bind mono- and diphosphorylated Man6 and Man7 glycans that contain terminal αMan6P(1 → 2)αMan(1 → 3)αMan. In contrast to MPR300, scFv M6P-1 does not bind phosphodiesters, monophosphorylated Man8 or mono- or diphosphorylated Man9 structures. Single crystal X-ray diffraction analysis to 2.7 Å resolution of Fv M6P-1 in complex with Man6P reveals that specificity and affinity is achieved via multiple hydrogen bonds to the mannose ring and two salt bridges to the phosphate moiety. In common with both MPRs, loss of binding was observed for scFv M6P-1 at pH values below the second pKa of Man6P (pKa = 6.1). The structures of Fv M6P-1 and the MPRs suggest that the change of the ionization state of Man6P is the main driving force for the loss of binding at acidic lysosomal pH (e.g. lysosome pH ∼ 4.6), which provides justification for the evolution of a lysosomal enzyme transport pathway based on Man6P recognition. PMID:26503547

  15. Global Low Frequency Protein Motions in Long-Range Allosteric Signaling

    NASA Astrophysics Data System (ADS)

    McLeish, Tom; Rogers, Thomas; Townsend, Philip; Burnell, David; Pohl, Ehmke; Wilson, Mark; Cann, Martin; Richards, Shane; Jones, Matthew

    2015-03-01

    We present a foundational theory for how allostery can occur as a function of low frequency dynamics without a change in protein structure. Elastic inhomogeneities allow entropic ``signalling at a distance.'' Remarkably, many globular proteins display just this class of elastic structure, in particular those that support allosteric binding of substrates (long-range co-operative effects between the binding sites of small molecules). Through multi-scale modelling of global normal modes we demonstrate negative co-operativity between the two cAMP ligands without change to the mean structure. Crucially, the value of the co-operativity is itself controlled by the interactions around a set of third allosteric ``control sites.'' The theory makes key experimental predictions, validated by analysis of variant proteins by a combination of structural biology and isothermal calorimetry. A quantitative description of allostery as a free energy landscape revealed a protein ``design space'' that identified the key inter- and intramolecular regulatory parameters that frame CRP/FNR family allostery. Furthermore, by analyzing naturally occurring CAP variants from diverse species, we demonstrate an evolutionary selection pressure to conserve residues crucial for allosteric control. The methodology establishes the means to engineer allosteric mechanisms that are driven by low frequency dynamics.

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

    PubMed

    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.

  17. Probing binding sites and mechanisms of action of an I(Ks) activator by computations and experiments.

    PubMed

    Xu, Yu; Wang, Yuhong; Zhang, Mei; Jiang, Min; Rosenhouse-Dantsker, Avia; Wassenaar, Tsjerk; Tseng, Gea-Ny

    2015-01-06

    The slow delayed rectifier (IKs) channel is composed of the KCNQ1 channel and KCNE1 auxiliary subunit, and functions to repolarize action potentials in the human heart. IKs activators may provide therapeutic efficacy for treating long QT syndromes. Here, we show that a new KCNQ1 activator, ML277, can enhance IKs amplitude in adult guinea pig and canine ventricular myocytes. We probe its binding site and mechanism of action by computational analysis based on our recently reported KCNQ1 and KCNQ1/KCNE1 3D models, followed by experimental validation. Results from a pocket analysis and docking exercise suggest that ML277 binds to a side pocket in KCNQ1 and the KCNE1-free side pocket of KCNQ1/KCNE1. Molecular-dynamics (MD) simulations based on the most favorable channel/ML277 docking configurations reveal a well-defined ML277 binding space surrounded by the S2-S3 loop and S4-S5 helix on the intracellular side, and by S4-S6 transmembrane helices on the lateral sides. A detailed analysis of MD trajectories suggests two mechanisms of ML277 action. First, ML277 restricts the conformational dynamics of the KCNQ1 pore, optimizing K(+) ion coordination in the selectivity filter and increasing current amplitudes. Second, ML277 binding induces global motions in the channel, including regions critical for KCNQ1 gating transitions. We conclude that ML277 activates IKs by binding to an intersubunit space and allosterically influencing pore conductance and gating transitions. KCNE1 association protects KCNQ1 from an arrhythmogenic (constitutive current-inducing) effect of ML277, but does not preclude its current-enhancing effect.

  18. Salvinorin A: allosteric interactions at the mu-opioid receptor.

    PubMed

    Rothman, Richard B; Murphy, Daniel L; Xu, Heng; Godin, Jonathan A; Dersch, Christina M; Partilla, John S; Tidgewell, Kevin; Schmidt, Matthew; Prisinzano, Thomas E

    2007-02-01

    Salvinorin A [(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(acetyloxy)-2-(3-furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2h-naphtho[2,1-c]pyran-7-carboxylic acid methyl ester] is a hallucinogenic kappa-opioid receptor agonist that lacks the usual basic nitrogen atom present in other known opioid ligands. Our first published studies indicated that Salvinorin A weakly inhibited mu-receptor binding, and subsequent experiments revealed that Salvinorin A partially inhibited mu-receptor binding. Therefore, we hypothesized that Salvinorin A allosterically modulates mu-receptor binding. To test this hypothesis, we used Chinese hamster ovary cells expressing the cloned human opioid receptor. Salvinorin A partially inhibited [(3)H]Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) (0.5, 2.0, and 8.0 nM) binding with E(MAX) values of 78.6, 72.1, and 45.7%, respectively, and EC(50) values of 955, 1124, and 4527 nM, respectively. Salvinorin A also partially inhibited [(3)H]diprenorphine (0.02, 0.1, and 0.5 nM) binding with E(MAX) values of 86.2, 64, and 33.6%, respectively, and EC(50) values of 1231, 866, and 3078 nM, respectively. Saturation binding studies with [(3)H]DAMGO showed that Salvinorin A (10 and 30 microM) decreased the mu-receptor B(max) and increased the K(d) in a dose-dependent nonlinear manner. Saturation binding studies with [(3)H]diprenorphine showed that Salvinorin A (10 and 40 microM) decreased the mu-receptor B(max) and increased the K(d) in a dose-dependent nonlinear manner. Similar findings were observed in rat brain with [(3)H]DAMGO. Kinetic experiments demonstrated that Salvinorin A altered the dissociation kinetics of both [(3)H]DAMGO and [(3)H]diprenorphine binding to mu receptors. Furthermore, Salvinorin A acted as an uncompetitive inhibitor of DAMGO-stimulated guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding. Viewed collectively, these data support the hypothesis that Salvinorin A allosterically modulates the mu-opioid receptor.

  19. Principles of Ligand Binding within a Completely Buried Cavity in HIF2[alpha] PAS-B

    SciTech Connect

    Key, Jason; Scheuermann, Thomas H.; Anderson, Peter C.; Daggett, Valerie; Gardner, Kevin H.

    2010-04-19

    Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors responsible for the metazoan hypoxia response and promote tumor growth, metastasis, and resistance to cancer treatment. The C-terminal Per-ARNT-Sim (PAS) domain of HIF2{alpha} (HIF2{alpha} PAS-B) contains a preformed solvent-inaccessible cavity that binds artificial ligands that allosterically perturb the formation of the HIF heterodimer. To better understand how small molecules bind within this domain, we examined the structures and equilibrium and transition-state thermodynamics of HIF2{alpha} PAS-B with several artificial ligands using isothermal titration calorimetry, NMR exchange spectroscopy, and X-ray crystallography. Rapid association rates reveal that ligand binding is not dependent upon a slow conformational change in the protein to permit ligand access, despite the closed conformation observed in the NMR and crystal structures. Compensating enthalpic and entropic contributions to the thermodynamic barrier for ligand binding suggest a binding-competent transition state characterized by increased structural disorder. Finally, molecular dynamics simulations reveal conversion between open and closed conformations of the protein and pathways of ligand entry into the binding pocket.

  20. Generating "fragment-based virtual library" using pocket similarity search of ligand-receptor complexes.

    PubMed

    Khashan, Raed S

    2015-01-01

    As the number of available ligand-receptor complexes is increasing, researchers are becoming more dedicated to mine these complexes to aid in the drug design and development process. We present free software which is developed as a tool for performing similarity search across ligand-receptor complexes for identifying binding pockets which are similar to that of a target receptor. The search is based on 3D-geometric and chemical similarity of the atoms forming the binding pocket. For each match identified, the ligand's fragment(s) corresponding to that binding pocket are extracted, thus forming a virtual library of fragments (FragVLib) that is useful for structure-based drug design. The program provides a very useful tool to explore available databases.

  1. Equilibrated atomic models of outward-facing P-glycoprotein and effect of ATP binding on structural dynamics.

    PubMed

    Pan, Lurong; Aller, Stephen G

    2015-01-20

    P-glycoprotein (Pgp) is an ATP-binding cassette (ABC) transporter that alternates between inward- and outward-facing conformations to capture and force substrates out of cells like a peristaltic pump. The high degree of similarity in outward-facing structures across evolution of ABC transporters allowed construction of a high-confidence outward-facing Pgp atomic model based on crystal structures of outward-facing Sav1866 and inward-facing Pgp. The model adhered to previous experimentally determined secondary- and tertiary- configurations during all-atom molecular dynamics simulations in the presence or absence of MgATP. Three long lasting (>100 ns) meta-stable states were apparent in the presence of MgATP revealing new insights into alternating access. The two ATP-binding pockets are highly asymmetric resulting in differential control of overall structural dynamics and allosteric regulation of the drug-binding pocket. Equilibrated Pgp has a considerably different electrostatic profile compared to Sav1866 that implicates significant kinetic and thermodynamic differences in transport mechanisms.

  2. Elucidation of direct competition and allosteric modulation of small-molecular-weight protein ligands using surface plasmon resonance methods.

    PubMed

    Huber, Walter; Sinopoli, Alessandro; Kohler, Josiane; Hug, Melanie; Ruf, Armin; Huber, Sylwia

    2015-08-01

    The present work introduces a surface plasmon resonance-based method for the discrimination of direct competition and allosteric effects that occur in ternary systems comprising a receptor protein and two small-molecular-weight ligands that bind to it. Fatty acid binding protein 4, fructose-1,6-bisphosphatase and human serum albumin were used as model receptor molecules to demonstrate the performance of the method. For each of the receptor molecules, pairs of ligand molecules were selected for which either direct competition or an allosteric effect had already been determined by other methods. The method of discrimination introduced here is based on the surface plasmon resonance responses observed at equilibrium when an immobilized receptor protein is brought into contact with binary mixtures of interacting ligands. These experimentally determined responses are compared with the responses calculated using a theoretical model that considers both direct competition and allosteric ligand interaction modes. This study demonstrates that the allosteric ternary complex model, which enables calculation of the fractional occupancy of the protein by each ligand in such ternary systems, is well suited for the theoretical calculation of these types of responses. For all of the ternary systems considered in this work, the experimental and calculated responses in the chosen concentration ratio range were identical within a five-σ confidence interval when the calculations considered the correct interaction mode of the ligands (direct competition or different types of allosteric regulation), and in case of allosteric modulation, also the correct strength of this effect. This study also demonstrates that the allosteric ternary complex model-based calculations are well suited to predict the ideal concentration ratio range or even single concentration ratios that can serve as hot spots for discrimination, and such hot spots can drastically reduce the numbers of measurements needed

  3. Structural Analysis of Iac Repressor Bound to Allosteric Effectors

    SciTech Connect

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

    2007-01-01

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

  4. Multimodal mechanism of action of allosteric HIV-1 integrase inhibitors

    PubMed Central

    Jurado, Kellie Ann; Engelman, Alan

    2013-01-01

    Integrase (IN) is required for lentivirus replication and is a proven drug target for the prevention of AIDS in HIV-1 infected patients. While clinical strand transfer inhibitors disarm the IN active site, allosteric inhibition of enzyme activity through the disruption of IN-IN protein interfaces holds great therapeutic potential. A promising class of allosteric IN inhibitors (ALLINIs), 2-(quinolin-3-yl) acetic acid derivatives, engage the IN catalytic core domain dimerization interface at the binding site for the host integration co-factor LEDGF/p75. ALLINIs promote IN multimerization and, independent of LEDGF/p75 protein, block the formation of the active IN-DNA complex, as well as inhibit the IN-LEDGF/p75 interaction in vitro. Yet, rather unexpectedly, the full inhibitory effect of these compounds is exerted during the late phase of HIV-1 replication. ALLINIs impair particle core maturation as well as reverse transcription and integration during the subsequent round of virus infection. Recapitulating the pleiotropic phenotypes observed with numerous IN mutant viruses, ALLINIs provide insight into underlying aspects of IN biology that extend beyond its catalytic activity. Therefore, in addition to the potential to expand our repertoire of HIV-1 antiretrovirals, ALLINIs afford important structural probes to dissect the multifaceted nature of the IN protein throughout the course of HIV-1 replication. PMID:24274067

  5. Allosteric activation of ADAMTS13 by von Willebrand factor.

    PubMed

    Muia, Joshua; Zhu, Jian; Gupta, Garima; Haberichter, Sandra L; Friedman, Kenneth D; Feys, Hendrik B; Deforche, Louis; Vanhoorelbeke, Karen; Westfield, Lisa A; Roth, Robyn; Tolia, Niraj Harish; Heuser, John E; Sadler, J Evan

    2014-12-30

    The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) within endovascular platelet aggregates, and ADAMTS13 deficiency causes fatal microvascular thrombosis. The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains of ADAMTS13 recognize a cryptic site in VWF that is exposed by tensile force. Another seven T and two complement C1r/C1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains of uncertain function are C-terminal to the MDTCS domains. We find that the distal T8-CUB2 domains markedly inhibit substrate cleavage, and binding of VWF or monoclonal antibodies to distal ADAMTS13 domains relieves this autoinhibition. Small angle X-ray scattering data indicate that distal T-CUB domains interact with proximal MDTCS domains. Thus, ADAMTS13 is regulated by substrate-induced allosteric activation, which may optimize VWF cleavage under fluid shear stress in vivo. Distal domains of other ADAMTS proteases may have similar allosteric properties.

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

    SciTech Connect

    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.

  7. Fluorescence characterization of the hydrophobic pocket of cyclophilin B.

    PubMed

    Albani, J R; Carpentier, M; Lansiaux, C

    2008-01-01

    Human cyclophilin B is a monomeric protein that contains two tryptophan residues, Trp104 and 128. Trp128-residue belongs to the binding site of cyclosporin A and is the homologous of Trp 121 in CyPA, while Trp104 residue belongs to the hydrophobic pocket. In the present work, we studied the dynamics of Trp residue(s) of cyclophilin B and of the CyPB(w128A) mutant and of TNS-mutant complex. Our results showed that Trp-104 and TNS show restricted motions within their environments and that energy transfer between the two fluorophores is occurring.

  8. Development of a photoactivatable allosteric ligand for the m1 muscarinic acetylcholine receptor.

    PubMed

    Davie, Briana J; Sexton, Patrick M; Capuano, Ben; Christopoulos, Arthur; Scammells, Peter J

    2014-10-15

    The field of G protein-coupled receptor drug discovery has benefited greatly from the structural and functional insights afforded by photoactivatable ligands. One G protein-coupled receptor subfamily for which photoactivatable ligands have been developed is the muscarinic acetylcholine receptor family, though, to date, all such ligands have been designed to target the orthosteric (endogenous ligand) binding site of these receptors. Herein we report the synthesis and pharmacological investigation of a novel photoaffinity label, MIPS1455 (4), designed to bind irreversibly to an allosteric site of the M1 muscarinic acetylcholine receptor; a target of therapeutic interest for the treatment of cognitive deficits. MIPS1455 may be a valuable molecular tool for further investigating allosteric interactions at this receptor.

  9. Computer Simulations of the Retinoid X Receptor: Conformational Dynamics and Allosteric Networks.

    PubMed

    van der Vaart, Arjan; Lorkowski, Alexander; Ma, Ning; Gray, Geoffrey M

    2017-01-01

    As the heterodimerization partner for a large number of nuclear receptors, the retinoid X receptor (RXR) is important for a large and diverse set of biochemical pathways. Activation and regulation of RXR heterodimers is achieved by complex allosteric mechanisms, which involve the binding of ligands, DNA, coactivators and corepressors, and entail large and subtle conformational motions. Complementing experiments, computer simulations have provided detailed insights into the origins of the allostery by investigating the changes in structure, motion, and interactions upon dimerization, ligand and cofactor binding. This review will summarize a number of simulation studies that have furthered the understanding of the conformational dynamics and the allosteric activation and control of RXR complexes. While the review focuses on the RXR and RXR heterodimers, relevant simulation studies of other nuclear receptors will be discussed as well.

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

    PubMed

    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.

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

    PubMed Central

    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

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

    PubMed Central

    2017-01-01

    It has recently been proposed by Gunasakaran et al. that allostery may be an intrinsic property of all proteins. Here, we develop a computational method that can determine and quantify allosteric activity in any given protein. Based on Schreiber's transfer entropy formulation, our approach leads to an information transfer landscape for the protein that shows the presence of entropy sinks and sources and explains how pairs of residues communicate with each other using entropy transfer. The model can identify the residues that drive the fluctuations of others. We apply the model to Ubiquitin, whose allosteric activity has not been emphasized until recently, and show that there are indeed systematic pathways of entropy and information transfer between residues that correlate well with the activities of the protein. We use 600 nanosecond molecular dynamics trajectories for Ubiquitin and its complex with human polymerase iota and evaluate entropy transfer between all pairs of residues of Ubiquitin and quantify the binding susceptibility changes upon complex formation. We explain the complex formation propensities of Ubiquitin in terms of entropy transfer. Important residues taking part in allosteric communication in Ubiquitin predicted by our approach are in agreement with results of NMR relaxation dispersion experiments. Finally, we show that time delayed correlation of fluctuations of two interacting residues possesses an intrinsic causality that tells which residue controls the interaction and which one is controlled. Our work shows that time delayed correlations, entropy transfer and causality are the required new concepts for explaining allosteric communication in proteins. PMID:28095404

  13. Allosteric Inhibition via R-state Destabilization in ATP Sulfurylase from Penicillium chrysogenum

    SciTech Connect

    MacRae, I. J.

    2002-01-01

    The structure of the cooperative hexameric enzyme ATP sulfurylase from Penicillium chrysogenum bound to its allosteric inhibitor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), was determined to 2.6 {angstrom} resolution. This structure represents the low substrate-affinity T-state conformation of the enzyme. Comparison with the high substrate-affinity R-state structure reveals that a large rotational rearrangement of domains occurs as a result of the R-to-T transition. The rearrangement is accompanied by the 17 {angstrom} movement of a 10-residue loop out of the active site region, resulting in an open, product release-like structure of the catalytic domain. Binding of PAPS is proposed to induce the allosteric transition by destabilizing an R-state-specific salt linkage between Asp 111 in an N-terminal domain of one subunit and Arg 515 in the allosteric domain of a trans-triad subunit. Disrupting this salt linkage by site-directed mutagenesis induces cooperative inhibition behavior in the absence of an allosteric effector, confirming the role of these two residues.

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

    NASA Astrophysics Data System (ADS)

    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.

  15. Controlling the rate of organic reactions: rational design of allosteric Diels-Alderase ribozymes

    PubMed Central

    Amontov, Sergey; Jäschke, Andres

    2006-01-01

    Allosteric mechanisms are widely used in nature to control the rates of enzymatic reactions, but little is known about RNA catalysts controlled by these principles. The only natural allosteric ribozyme reported to date catalyzes an RNA cleavage reaction, and so do almost all artificial systems. RNA has, however, been shown to accelerate a much wider range of chemical reactions. Here we report that RNA catalysts for organic reactions can be put under the stringent control of effector molecules by straight-forward rational design. This approach uses known RNA sequences with catalytic and ligand-binding properties, and exploits weakly conserved sequence elements and available structural information to induce the formation of alternative, catalytically inactive structures. The potential and general applicability is demonstrated by the design of three different systems in which the rate of a catalytic carbon–carbon bond forming reaction is positively regulated up to 2100-fold by theophylline, tobramycin and a specific mRNA sequence, respectively. Although smaller in size than a tRNA, all three ribozymes show typical features of allosteric metabolic enzymes, namely high rate acceleration and tight allosteric regulation. Not only do these findings demonstrate RNA's power as a catalyst, but also highlight on RNA's capabilities as signaling components in regulatory networks. PMID:16990253

  16. Controlling the rate of organic reactions: rational design of allosteric Diels-Alderase ribozymes.

    PubMed

    Amontov, Sergey; Jäschke, Andres

    2006-01-01

    Allosteric mechanisms are widely used in nature to control the rates of enzymatic reactions, but little is known about RNA catalysts controlled by these princi