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Sample records for allosteric communication occurs

  1. 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

  2. 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

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

    PubMed Central

    Guarnera, Enrico; Berezovsky, Igor N.

    2016-01-01

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

  4. 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

  5. Computational Modeling of Allosteric Regulation in the Hsp90 Chaperones: A Statistical Ensemble Analysis of Protein Structure Networks and Allosteric Communications

    PubMed Central

    Blacklock, Kristin; Verkhivker, Gennady M.

    2014-01-01

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

  6. 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.

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

    NASA Astrophysics Data System (ADS)

    Custer, Gregory; Beckett, Dorothy; Matysiak, Silvina

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

  8. The Tryptophan Synthase α2β2 Complex: A Model for Substrate Channeling, Allosteric Communication, and Pyridoxal Phosphate Catalysis

    PubMed Central

    Miles, Edith Wilson

    2013-01-01

    I reflect on my research on pyridoxal phosphate (PLP) enzymes over fifty-five years and on how I combined research with marriage and family. My Ph.D. research with Esmond E. Snell established one aspect of PLP enzyme mechanism. My postdoctoral work first with Hans L. Kornberg and then with Alton Meister characterized the structure and function of another PLP enzyme, l-aspartate β-decarboxylase. My independent research at the National Institutes of Health (NIH) since 1966 has focused on the bacterial tryptophan synthase α2β2 complex. The β subunit catalyzes a number of PLP-dependent reactions. We have characterized these reactions and the allosteric effects of the α subunit. We also used chemical modification to probe enzyme structure and function. Our crystallization of the tryptophan synthase α2β2 complex from Salmonella typhimurium led to the determination of the three-dimensional structure with Craig Hyde and David Davies at NIH in 1988. This landmark structure was the first structure of a multienzyme complex and the first structure revealing an intramolecular tunnel. The structure has provided a basis for exploring mechanisms of catalysis, channeling, and allosteric communication in the tryptophan synthase α2β2 complex. The structure serves as a model for many other multiprotein complexes that are important for biological processes in prokaryotes and eukaryotes. PMID:23426371

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

    PubMed Central

    Stetz, Gabrielle; Verkhivker, Gennady M.

    2017-01-01

    introduced a community-hopping model of allosteric communication. Atomistic reconstruction of signaling pathways in the DnaK structures captured a direction-specific mechanism and molecular details of signal transmission that are fully consistent with the mutagenesis experiments. The results of our study reconciled structural and functional experiments from a network-centric perspective by showing that global properties of the residue interaction networks and coevolutionary signatures may be linked with specificity and diversity of allosteric regulation mechanisms. PMID:28095400

  10. Allosteric communication pathways routed by Ca2+/Mg2+ exchange in GCAP1 selectively switch target regulation modes

    PubMed Central

    Marino, Valerio; Dell’Orco, Daniele

    2016-01-01

    GCAP1 is a neuronal calcium sensor protein that regulates the phototransduction cascade in vertebrates by switching between activator and inhibitor of the target guanylate cyclase (GC) in a Ca2+-dependent manner. We carried out exhaustive molecular dynamics simulations of GCAP1 and determined the intramolecular communication pathways involved in the specific GC activator/inhibitor switch. The switch was found to depend on the Mg2+/Ca2+ loading states of the three EF hands and on the way the information is transferred from each EF hand to specific residues at the GCAP1/GC interface. Post-translational myristoylation is fundamental to mediate long range allosteric interactions including the EF2-EF4 coupling and the communication between EF4 and the GC binding interface. Some hubs in the identified protein network are the target of retinal dystrophy mutations, suggesting that the lack of complete inhibition of GC observed in many cases is likely due to the perturbation of intra/intermolecular communication routes. PMID:27739433

  11. 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.

  12. 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

  13. Variations in clique and community patterns in protein structures during allosteric communication: investigation of dynamically equilibrated structures of methionyl tRNA synthetase complexes.

    PubMed

    Ghosh, Amit; Vishveshwara, Saraswathi

    2008-11-04

    The allosteric concept has played a key role in understanding the biological functions of proteins. The rigidity or plasticity and the conformational population are the two important ideas invoked in explaining the allosteric effect. Although molecular insights have been gained from a large number of structures, a precise assessment of the ligand-induced conformational changes in proteins at different levels, ranging from gross topology to intricate details, remains a challenge. In this study, we have explored the conformational changes in the complexes of methionyl tRNA synthetase (MetRS) through novel network parameters such as cliques and communities, which identify the rigid regions in the protein structure networks (PSNs) constructed from the noncovalent interactions of amino acid side chains. MetRS belongs to the aminoacyl tRNA synthetase (aaRS) family that plays a crucial role in the translation of genetic code. These enzymes are modular with distinct domains from which extensive genetic, kinetic, and structural data are available, highlighting the role of interdomain communication. The network parameters evaluated here on the conformational ensembles of MetRS complexes, generated from molecular dynamics simulations, have enabled us to understand the interdomain communication in detail. Additionally, the characterization of conformational changes in terms of cliques and communities has also become possible, which had eluded conventional analyses. Furthermore, we find that most of the residues participating in cliques and communities are strikingly different from those that take part in long-range communication. The cliques and communities evaluated here for the first time on PSNs have beautifully captured the local geometries in detail within the framework of global topology. Here the allosteric effect is revealed at the residue level via identification of the important residues specific for structural rigidity and functional flexibility in MetRS. This ought

  14. Exploring Molecular Mechanisms of Paradoxical Activation in the BRAF Kinase Dimers: Atomistic Simulations of Conformational Dynamics and Modeling of Allosteric Communication Networks and Signaling Pathways

    PubMed Central

    Tse, Amanda; Verkhivker, Gennady M.

    2016-01-01

    The recent studies have revealed that most BRAF inhibitors can paradoxically induce kinase activation by promoting dimerization and enzyme transactivation. Despite rapidly growing number of structural and functional studies about the BRAF dimer complexes, the molecular basis of paradoxical activation phenomenon is poorly understood and remains largely hypothetical. In this work, we have explored the relationships between inhibitor binding, protein dynamics and allosteric signaling in the BRAF dimers using a network-centric approach. Using this theoretical framework, we have combined molecular dynamics simulations with coevolutionary analysis and modeling of the residue interaction networks to determine molecular determinants of paradoxical activation. We have investigated functional effects produced by paradox inducer inhibitors PLX4720, Dabrafenib, Vemurafenib and a paradox breaker inhibitor PLX7904. Functional dynamics and binding free energy analyses of the BRAF dimer complexes have suggested that negative cooperativity effect and dimer-promoting potential of the inhibitors could be important drivers of paradoxical activation. We have introduced a protein structure network model in which coevolutionary residue dependencies and dynamic maps of residue correlations are integrated in the construction and analysis of the residue interaction networks. The results have shown that coevolutionary residues in the BRAF structures could assemble into independent structural modules and form a global interaction network that may promote dimerization. We have also found that BRAF inhibitors could modulate centrality and communication propensities of global mediating centers in the residue interaction networks. By simulating allosteric communication pathways in the BRAF structures, we have determined that paradox inducer and breaker inhibitors may activate specific signaling routes that correlate with the extent of paradoxical activation. While paradox inducer inhibitors may

  15. Using THz time-scale infrared spectroscopy to examine the role of collective, thermal fluctuations in the formation of myoglobin allosteric communication pathways and ligand specificity.

    PubMed

    Woods, K N

    2014-06-28

    In this investigation we use THz time-scale spectroscopy to conduct an initial set of studies on myoglobin with the aim of providing further insight into the global, collective thermal fluctuations in the protein that have been hypothesized to play a prominent role in the dynamic formation of transient ligand channels as well as in shaping the molecular level basis for ligand discrimination. Using the two ligands O2 and CO, we have determined that the perturbation from the heme-ligand complex has a strong influence on the characteristics of the myoglobin collective dynamics that are excited upon binding. Further, the differences detected in the collective protein motions in Mb-O2 compared with those in Mb-CO appear to be intimately tied with the pathways of long-range allosteric communication in the protein, which ultimately determine the trajectories selected by the respective ligands on the path to and from the heme-binding cavity.

  16. Using cholinergic M1 receptor positive allosteric modulators to improve memory via enhancement of brain cholinergic communication.

    PubMed

    Chambon, Caroline; Jatzke, Claudia; Wegener, Nico; Gravius, Andreas; Danysz, Wojciech

    2012-12-15

    Benzylquinolone carboxylic acid (BQCA) is a recently described cholinergic muscarinic M(1) receptor positive allosteric modulator having potential as cognitive enhancer in dementia. The present study focused on the characterisation of BQCA's mode of action in relation to positive effects on memory and side-effects in an animal model. To get insight into this mode of action, in vitro receptor potency/left shift experiments in cells stably expressing the rat's M(1) receptor were performed. They revealed an inflection point value of BQCA corresponding to 306nM, and potentiation of the agonist response up to 47-fold in presence of 10μM of BQCA. In vivo, brain microdialysis showed a maximal brain level of 270nM, 40min after i.p. administration at 10mg/kg. Based on in vitro data obtained with this dose, it can be concluded that BQCA reaches brain levels which should potentiate the agonist response about 4-fold. Behavioural data confirmed that BQCA used at 10mg/kg attenuated scopolamine-induced memory deficit in a spontaneous alternation task. Moreover, BQCA showed no side effect at 10mg/kg and above in spontaneous locomotion and salivation tests. The profile of BQCA observed in the present study displays a clear advantage over the M(1)-M(3) agonist cevimeline. The present data show the therapeutic potential of the M(1) receptor positive allosteric modulator BQCA for the treatment of memory deficits observed in Alzheimer's disease.

  17. 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.

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

    NASA Astrophysics Data System (ADS)

    Negureanu, Lacramioara; Salsbury, Freddie

    2012-02-01

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

  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. Links Between Co-occurring Social-Communication and Hyperactive-Inattentive Trait Trajectories

    PubMed Central

    St. Pourcain, Beate; Mandy, William P.; Heron, Jon; Golding, Jean; Davey Smith, George; Skuse, David H.

    2011-01-01

    Objective There is overlap between an autistic and hyperactive-inattentive symptomatology when studied cross-sectionally. This study is the first to examine the longitudinal pattern of association between social-communication deficits and hyperactive-inattentive symptoms in the general population, from childhood through adolescence. We explored the interrelationship between trajectories of co-occurring symptoms, and sought evidence for shared prenatal/perinatal risk factors. Method Study participants were 5,383 singletons of white ethnicity from the Avon Longitudinal Study of Parents and Children (ALSPAC). Multiple measurements of hyperactive-inattentive traits (Strengths and Difficulties Questionnaire) and autistic social-communication impairment (Social Communication Disorder Checklist) were obtained between 4 and 17 years. Both traits and their trajectories were modeled in parallel using latent class growth analysis (LCGA). Trajectory membership was subsequently investigated with respect to prenatal/perinatal risk factors. Results LCGA analysis revealed two distinct social-communication trajectories (persistently impaired versus low-risk) and four hyperactive-inattentive trait trajectories (persistently impaired, intermediate, childhood-limited and low-risk). Autistic symptoms were more stable than those of attention-deficit/hyperactivity disorder (ADHD) behaviors, which showed greater variability. Trajectories for both traits were strongly but not reciprocally interlinked, such that the majority of children with a persistent hyperactive-inattentive symptomatology also showed persistent social-communication deficits but not vice versa. Shared predictors, especially for trajectories of persistent impairment, were maternal smoking during the first trimester, which included familial effects, and a teenage pregnancy. Conclusions Our longitudinal study reveals that a complex relationship exists between social-communication and hyperactive-inattentive traits. Patterns

  1. 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

  2. Links between Co-Occurring Social-Communication and Hyperactive-Inattentive Trait Trajectories

    ERIC Educational Resources Information Center

    St. Pourcain, Beate; Mandy, William P.; Heron, Jon; Golding, Jean; Smith, George Davey; Skuse, David H.

    2011-01-01

    Objective: There is overlap between an autistic and hyperactive-inattentive symptomatology when studied cross-sectionally. This study is the first to examine the longitudinal pattern of association between social-communication deficits and hyperactive-inattentive symptoms in the general population, from childhood through adolescence. We explored…

  3. 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.

  4. Short communication: Change of naturally occurring benzoic acid during skim milk fermentation by commercial cheese starters.

    PubMed

    Han, Noori; Park, Sun-Young; Kim, Sun-Young; Yoo, Mi-Young; Paik, Hyun-Dong; Lim, Sang-Dong

    2016-11-01

    This study sought to investigate the change of naturally occurring benzoic acid (BA) during skim milk fermentation by 4 kinds of commercial cheese starters used in domestic cheese. The culture was incubated at 3-h intervals for 24h at 30, 35, and 40°C. The BA content during fermentation by Streptococcus thermophilus STB-01 was detected after 12h at all temperatures, sharply increasing at 30°C. In Lactobacillus paracasei LC431, BA was detected after 9h at all temperatures, sharply increasing until 18h and decreasing after 18h at 30 and 35°C. In the case of R707 (consisting of Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris), BA increased from 6h to 15h and decreased after 15h at 40°C. The BA during STB-01 and CHN-11 (1:1; mixture of S. thermophilus, Lc. lactis ssp. lactis, Lc. lactis ssp. cremoris, Lc. lactis ssp. diacetylactis, Leuconostoc mesenteroides ssp. cremoris) fermentation was detected after 3h at 35 and 40°C, sharply increasing up to 12h and decreasing after 15h at 35°C, and after 6h, increasing up to 9h at 30°C. After 3h, it steadily decreased at 40°C. The highest amount of BA was found during the fermentation by R707 at 30°C; 15h with 12.46mg/kg.

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

    PubMed

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

    2013-09-01

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

  6. 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.

  7. Allosteric Motions in Structures of Yeast NAD+-Specific Isocitrate Dehydrogenase

    SciTech Connect

    Taylor,A.; Hu, G.; Hart, P.; McAlister-Henn, L.

    2008-01-01

    Mitochondrial NAD+-specific isocitrate dehydrogenases (IDHs) are key regulators of flux through biosynthetic and oxidative pathways in response to cellular energy levels. Here we present the first structures of a eukaryotic member of this enzyme family, the allosteric, hetero-octameric, NAD+-specific IDH from yeast in three forms: (1) without ligands, (2) with bound analog citrate, and (3) with bound citrate + AMP. The structures reveal the molecular basis for ligand binding to homologous but distinct regulatory and catalytic sites positioned at the interfaces between IDH1 and IDH2 subunits and define pathways of communication between heterodimers and heterotetramers in the hetero-octamer. Disulfide bonds observed at the heterotetrameric interfaces in the unliganded IDH hetero-octamer are reduced in the ligand-bound forms, suggesting a redox regulatory mechanism that may be analogous to the 'on-off' regulation of non-allosteric bacterial IDHs via phosphorylation. The results strongly suggest that eukaryotic IDH enzymes are exquisitely tuned to ensure that allosteric activation occurs only when concentrations of isocitrate are elevated.

  8. 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.

  9. Allosteric control in a metalloprotein dramatically alters function

    PubMed Central

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

    2013-01-01

    Metalloproteins (MPs) comprise one-third of all known protein structures. This diverse set of proteins contain a plethora of unique inorganic moieties capable of performing chemistry that would otherwise be impossible using only the amino acids found in nature. Most of the well-studied MPs are generally viewed as being very rigid in structure, and it is widely thought that the properties of the metal centers are primarily determined by the small fraction of amino acids that make up the local environment. Here we examine both theoretically and experimentally whether distal regions can influence the metal center in the diabetes drug target mitoNEET. We demonstrate that a loop (L2) 20 Å away from the metal center exerts allosteric control over the cluster binding domain and regulates multiple properties of the metal center. Mutagenesis of L2 results in significant shifts in the redox potential of the [2Fe-2S] cluster and orders of magnitude effects on the rate of [2Fe-2S] cluster transfer to an apo-acceptor protein. These surprising effects occur in the absence of any structural changes. An examination of the native basin dynamics of the protein using all-atom simulations shows that twisting in L2 controls scissoring in the cluster binding domain and results in perturbations to one of the cluster-coordinating histidines. These allosteric effects are in agreement with previous folding simulations that predicted L2 could communicate with residues surrounding the metal center. Our findings suggest that long-range dynamical changes in the protein backbone can have a significant effect on the functional properties of MPs. PMID:23271805

  10. Dynamic coupling and allosteric behavior in a non-allosteric protein†

    PubMed Central

    Clarkson, Michael W.; Gilmore, Steven A.; Edgell, Marshall H.; Lee, Andrew L.

    2008-01-01

    Long-range intraprotein interactions give rise to many important protein behaviors. Understanding how energy is transduced through protein structures to either transmit a signal or elicit conformational changes is therefore a current challenge in structural biology. In an effort to understand such linkages, multiple V→A mutations were made in the small globular protein eglin c. The physical responses, as mapped by NMR spin relaxation, residual dipolar couplings (RDCs), and scalar couplings, illustrate that the interior of this non-allosteric protein forms a dynamic network and that local perturbations are transmitted as dynamic and structural changes to distal sites as far as 16 Å away. Two basic types of propagation responses were observed: contiguous pathways of enhanced (attenuated) dynamics with no change in structure; and dispersed (non-contiguous) changes in methyl rotation rates that appear to result from subtle deformation of backbone structure. In addition, energy transmission is found to be unidirectional. In one mutant, an allosteric conformational change of a side chain is seen in the context of a pathway of propagated changes in ps-ns dynamics. The observation of so many long-range interactions in a small, rigid system lends experimental weight to the idea that all well-folded proteins inherently possess allosteric features [Gunasekaran et al. (2004) Proteins 57, 433−443], and that dynamics are a rich source of information for mapping and gaining mechanistic insight into communication pathways in individual proteins. PMID:16784220

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. Communication between the nucleotide binding domains of P-glycoprotein occurs via conformational changes that involve residue 508.

    PubMed

    Gabriel, Mark P; Storm, Janet; Rothnie, Alice; Taylor, Andrew M; Linton, Kenneth J; Kerr, Ian D; Callaghan, Richard

    2003-07-01

    Our aim is to provide molecular understanding of the mechanisms underlying the (i) interaction between the two nucleotide binding domains (NBDs) and (ii) coupling between NBDs and transmembrane domains within P-glycoprotein (Pgp) during a transport cycle. To facilitate this, we have introduced a number of unique cysteine residues at surface exposed positions (E393C, S452C, I500C, N508C, and K578C) in the N-terminal NBD of Pgp, which had previously been engineered to remove endogenous cysteines. Positions of the mutations were designed using a model based on crystallographic features of prokaryotic NBDs. The single cysteine mutants were expressed in insect cells using recombinant baculovirus and the proteins purified by metal affinity chromatography by virtue of a polyhistidine tag. None of the introduced cysteine residues perturbed the function of Pgp as judged by the characteristics of drug stimulated ATP hydrolysis. The role of residues at each of the introduced sites in the catalytic cycle of Pgp was investigated by the effect of covalent conjugation with N-ethyl-maleimide (NEM). All but one mutation (K578C) was accessible to labeling with [(3)H]-NEM. However, perturbation of ATPase activity was only observed for the derivitized N508C isoform. The principle functional manifestation was a marked inhibition of the "basal" rate of ATP hydrolysis. Neither the extent nor potency to which a range of drugs could affect the ATPase activity were altered in the NEM conjugated N508C isoform. The results imply that the accessibility of residue 508, located in the alpha-helical subdomain of NBD1 in Pgp, is altered by the conformational changes that occur during ATP hydrolysis.

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

    PubMed Central

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

    2016-01-01

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

  1. 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

  2. Identification of the Allosteric Regulatory Site of Insulysin

    SciTech Connect

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

    2012-05-25

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

  3. Identification of the Allosteric Regulatory Site of Insulysin

    SciTech Connect

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

    2011-06-24

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

  4. 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.

  5. 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

  6. KINETIC AND STRUCTURAL INVESTIGATIONS INTO THE ALLOSTERIC AND PH EFFECT ON SUBSTRATE SPECIFICITY OF HUMAN EPITHELIAL 15-LIPOXYGENASE-2

    PubMed Central

    Joshi, Netra; Hoobler, Eric K.; Perry, Steven; Diaz, Giovanni; Fox, Brian; Holman, Theodore R.

    2013-01-01

    Lipoxygenases, important enzymes in inflammation, can regulate their substrate specificity by allosteric interactions with its own hydroperoxide products. In the current work, addition of both 13-(S) hydroxy-9Z,11E-octadecadienoic acid (13-(S)-HODE) and 13-(S)-hydroperoxy-6Z,9Z,11E-octadecatrienoic acid (13-(S)-HOTrE) to human epithelial 15-lipoxygenase-2 (15-LOX-2) increases the kcat/KM substrate specificity ratio of arachidonic acid (AA) and (γ)-linolenic acid (GLA) by 4-fold. 13-(S)-HODE achieves this change by activating kcat/KM AA but inhibiting kcat/KM GLA, which indicates that the allosteric structural changes at the active site discriminates between the length and unsaturation differences of AA and GLA to achieve opposite kinetics effects. The substrate specificity ratio is further increased, 11-fold total, by increasing pH, suggesting mechanistic differences between the pH and allosteric effects. Interestingly, the loss of the PLAT domain affects substrate specificity, but does not eliminate the allosteric properties of 15-LOX-2, indicating that the allosteric site is located in the catalytic domain. However, the removal of the PLAT domain does change the magnitude of the allosteric effect. These data suggest that the PLAT domain moderates the communication pathway between the allosteric and catalytic sites, thus affecting substrate specificity. These results are discussed in the context of protein dimerization and other structural changes. PMID:24171444

  7. Communication between Physicians and Family Caregivers about Care at the End of Life: When Do Discussions Occur and What Is Said?

    PubMed Central

    CHERLIN, EMILY; FRIED, TERRI; PRIGERSON, HOLLY G.; SCHULMAN-GREEN, DENA; JOHNSON-HURZELER, ROSEMARY; BRADLEY, ELIZABETH H.

    2006-01-01

    Background: Few studies have examined physician-family caregiver communication at the end of life, despite the important role families have in end-of-life care decisions. We examined family caregiver reports of physician communication about incurable illness, life expectancy, and hospice; the timing of these discussions; and subsequent family understanding of these issues. Design: Mixed methods study using a closed-ended survey of 206 family caregivers and open-ended, in-depth interviews with 12 additional family caregivers. Setting/Subjects: Two hundred eighteen primary family caregivers of patients with cancer enrolled with hospice between October 1999 and June 2002 Measurements: Family caregiver reports provided at the time of hospice enrollment of physician discussions of incurable illness, life expectancy, and hospice. Results: Many family caregivers reported that a physician never told them the patient’s illness could not be cured (20.8%), never provided life expectancy (40% of those reportedly told illness was incurable), and never discussed using hospice (32.2%). Caregivers reported the first discussion of the illness being incurable and of hospice as a possibility occurred within 1 month of the patient’s death in many cases (23.5% and 41.1%, respectively). In open-ended interviews, however, family caregivers expressed ambivalence about what they wanted to know, and their difficulty comprehending and accepting “bad news” was apparent in both qualitative and quantitative data. Conclusion: Our findings suggest that ineffective communication about end-of-life issues likely results from both physician’s lack of discussion and family caregiver’s difficulty hearing the news. Future studies should examine strategies for optimal physician-family caregiver communication about incurable illness, so that families and patients can begin the physical, emotional, and spiritual work that can lead to acceptance of the irreversible condition. PMID:16351531

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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

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

    PubMed Central

    Chennubhotla, Chakra; Bahar, Ivet

    2006-01-01

    We introduce a novel approach for elucidating the potential pathways of allosteric communication in biomolecular systems. The methodology, based on Markov propagation of ‘information' across the structure, permits us to partition the network of interactions into soft clusters distinguished by their coherent stochastics. Probabilistic participation of residues in these clusters defines the communication patterns inherent to the network architecture. Application to bacterial chaperonin complex GroEL–GroES, an allostery-driven structure, identifies residues engaged in intra- and inter-subunit communication, including those acting as hubs and messengers. A number of residues are distinguished by their high potentials to transmit allosteric signals, including Pro33 and Thr90 at the nucleotide-binding site and Glu461 and Arg197 mediating inter- and intra-ring communication, respectively. We propose two most likely pathways of signal transmission, between nucleotide- and GroES-binding sites across the cis and trans rings, which involve several conserved residues. A striking observation is the opposite direction of information flow within cis and trans rings, consistent with negative inter-ring cooperativity. Comparison with collective modes deduced from normal mode analysis reveals the propensity of global hinge regions to act as messengers in the transmission of allosteric signals. PMID:16820777

  19. 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

  20. 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.

  1. Differential Modulation of Functional Dynamics and Allosteric Interactions in the Hsp90-Cochaperone Complexes with p23 and Aha1: A Computational Study

    PubMed Central

    Blacklock, Kristin; Verkhivker, Gennady M.

    2013-01-01

    Allosteric interactions of the molecular chaperone Hsp90 with a large cohort of cochaperones and client proteins allow for molecular communication and event coupling in signal transduction networks. The integration of cochaperones into the Hsp90 system is driven by the regulatory mechanisms that modulate the progression of the ATPase cycle and control the recruitment of the Hsp90 clientele. In this work, we report the results of computational modeling of allosteric regulation in the Hsp90 complexes with the cochaperones p23 and Aha1. By integrating protein docking, biophysical simulations, modeling of allosteric communications, protein structure network analysis and the energy landscape theory we have investigated dynamics and stability of the Hsp90-p23 and Hsp90-Aha1 interactions in direct comparison with the extensive body of structural and functional experiments. The results have revealed that functional dynamics and allosteric interactions of Hsp90 can be selectively modulated by these cochaperones via specific targeting of the regulatory hinge regions that could restrict collective motions and stabilize specific chaperone conformations. The protein structure network parameters have quantified the effects of cochaperones on conformational stability of the Hsp90 complexes and identified dynamically stable communities of residues that can contribute to the strengthening of allosteric interactions. According to our results, p23-mediated changes in the Hsp90 interactions may provide “molecular brakes” that could slow down an efficient transmission of the inter-domain allosteric signals, consistent with the functional role of p23 in partially inhibiting the ATPase cycle. Unlike p23, Aha1-mediated acceleration of the Hsp90-ATPase cycle may be achieved via modulation of the equilibrium motions that facilitate allosteric changes favoring a closed dimerized form of Hsp90. The results of our study have shown that Aha1 and p23 can modulate the Hsp90-ATPase activity

  2. 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.

  3. MCPath: Monte Carlo path generation approach to predict likely allosteric pathways and functional residues

    PubMed Central

    Kaya, Cihan; Armutlulu, Andac; Ekesan, Solen; Haliloglu, Turkan

    2013-01-01

    Allosteric mechanism of proteins is essential in biomolecular signaling. An important aspect underlying this mechanism is the communication pathways connecting functional residues. Here, a Monte Carlo (MC) path generation approach is proposed and implemented to define likely allosteric pathways through generating an ensemble of maximum probability paths. The protein structure is considered as a network of amino acid residues, and inter-residue interactions are described by an atomistic potential function. PDZ domain structures are presented as case studies. The analysis for bovine rhodopsin and three myosin structures are also provided as supplementary case studies. The suggested pathways and the residues constituting the pathways are maximally probable and mostly agree with the previous studies. Overall, it is demonstrated that the communication pathways could be multiple and intrinsically disposed, and the MC path generation approach provides an effective tool for the prediction of key residues that mediate the allosteric communication in an ensemble of pathways and functionally plausible residues. The MCPath server is available at http://safir.prc.boun.edu.tr/clbet_server. PMID:23742907

  4. 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.

  5. 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.

  6. Communications

    NASA Technical Reports Server (NTRS)

    Stouffer, Donald D.

    1990-01-01

    Communication in its many forms is a critical component for an effective Space Grant Program. Good communication is needed within individual Space Grant College/Consortia, for example between consortium affiliates and the consortium program office. Effective communication between the several programs, NASA Headquarters, and NASA field centers also is required. Further, communication among the above program elements, industry, local and state government, and the public also are necessary for meeting program objectives.

  7. Communication.

    ERIC Educational Resources Information Center

    Hancock, Alan

    An informal introduction to the study of communication deals with the major topics in the field. It presents basic theories of communication and language, reviews how language takes on meaning, explains the stimulus-response and Piaget theories of learning, and presents major theories dealing with communications and society. These theories include…

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. Allosteric pathway identification through network analysis: from molecular dynamics simulations to interactive 2D and 3D graphs.

    PubMed

    Allain, Ariane; Chauvot de Beauchêne, Isaure; Langenfeld, Florent; Guarracino, Yann; Laine, Elodie; Tchertanov, Luba

    2014-01-01

    Allostery is a universal phenomenon that couples the information induced by a local perturbation (effector) in a protein to spatially distant regulated sites. Such an event can be described in terms of a large scale transmission of information (communication) through a dynamic coupling between structurally rigid (minimally frustrated) and plastic (locally frustrated) clusters of residues. To elaborate a rational description of allosteric coupling, we propose an original approach - MOdular NETwork Analysis (MONETA) - based on the analysis of inter-residue dynamical correlations to localize the propagation of both structural and dynamical effects of a perturbation throughout a protein structure. MONETA uses inter-residue cross-correlations and commute times computed from molecular dynamics simulations and a topological description of a protein to build a modular network representation composed of clusters of residues (dynamic segments) linked together by chains of residues (communication pathways). MONETA provides a brand new direct and simple visualization of protein allosteric communication. A GEPHI module implemented in the MONETA package allows the generation of 2D graphs of the communication network. An interactive PyMOL plugin permits drawing of the communication pathways between chosen protein fragments or residues on a 3D representation. MONETA is a powerful tool for on-the-fly display of communication networks in proteins. We applied MONETA for the analysis of communication pathways (i) between the main regulatory fragments of receptors tyrosine kinases (RTKs), KIT and CSF-1R, in the native and mutated states and (ii) in proteins STAT5 (STAT5a and STAT5b) in the phosphorylated and the unphosphorylated forms. The description of the physical support for allosteric coupling by MONETA allowed a comparison of the mechanisms of (a) constitutive activation induced by equivalent mutations in two RTKs and (b) allosteric regulation in the activated and non

  14. Communicate!

    ERIC Educational Resources Information Center

    Chase, Stuart

    This ten chapter book is designed to provide high school students with an understanding of basic communication processes. The first five chapters include discussions of language development, function, and acquisition in relation to both human and non-human communication. The sixth chapter contains specimen linguistic analyses of speech and…

  15. Communication

    NASA Technical Reports Server (NTRS)

    Griner, James

    2010-01-01

    NASA s communication work for the UAS Command and Control area will build upon work currently being conducted under NASA Recovery Act funds. Communication portions of UAS NextGen ConOps, Stateof- the-Art assessment, and Gap Analysis. Preliminary simulations for UAS CNPC link scalability assessment. Surrogate UAS aircraft upgrades. This work will also leverage FY10 in-guide funding for communication link model development. UAS are currently managed through exceptions and are operating using DoD frequencies for line-of-sight (LOS) and satellite-based communications links, low-power LOS links in amateur bands, or unlicensed Instrument/Scientific/Medical (ISM) frequencies. None of these frequency bands are designated for Safety and Regularity of Flight. No radio-frequency (RF) spectrum has been allocated by the International Telecommunications Union (ITU) specifically for UAS command and control links, for either LOS or Beyond LOS (BLOS) communication.

  16. 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

  17. 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.

  18. 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.

  19. Identification of Allosteric Disulfides from Prestress Analysis

    PubMed Central

    Zhou, Beifei; Baldus, Ilona B.; Li, Wenjin; Edwards, Scott A.; Gräter, Frauke

    2014-01-01

    Disulfide bonds serve to form physical cross-links between residues in protein structures, thereby stabilizing the protein fold. Apart from this purely structural role, they can also be chemically active, participating in redox reactions, and they may even potentially act as allosteric switches controlling protein functions. Specific types of disulfide bonds have been identified in static protein structures from their distinctive pattern of dihedral bond angles, and the allosteric function of such bonds is purported to be related to the torsional strain they store. Using all-atom molecular-dynamics simulations for ∼700 disulfide bonded proteins, we analyzed the intramolecular mechanical forces in 20 classes of disulfide bonds. We found that two particular classes, the −RHStaple and the −/+RHHook disulfides, are indeed more stressed than other disulfide bonds, but the stress is carried primarily by stretching of the S-S bond and bending of the neighboring bond angles, rather than by dihedral torsion. This stress corresponds to a tension force of magnitude ∼200 pN, which is balanced by repulsive van der Waals interactions between the cysteine Cα atoms. We confirm stretching of the S-S bond to be a general feature of the −RHStaples and the −/+RHHooks by analyzing ∼20,000 static protein structures. Given that forced stretching of S-S bonds is known to accelerate their cleavage, we propose that prestress of allosteric disulfide bonds has the potential to alter the reactivity of a disulfide, thereby allowing us to readily switch between functional states. PMID:25099806

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

    PubMed Central

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

    2016-01-01

    The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215–217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215–217 conformation is pivotal to the enhanced activity of the FVIIa variants. We speculate that the allosteric regulation of FVIIa activity by TF binding follows a similar path in conjunction with protease domain N terminus insertion, suggesting a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity. PMID:26694616

  2. Allosteric transitions of the maltose transporter studied by an elastic network model.

    PubMed

    Li, Chun Hua; Yang, Yong Xiao; Su, Ji Guo; Liu, Bin; Tan, Jian Jun; Zhang, Xiao Yi; Wang, Cun Xin

    2014-07-01

    The maltose transporter from Escherichia coli is one of the ATP-binding cassette (ABC) transporters that utilize the energy from ATP hydrolysis to translocate substrates across cellular membranes. Until 2011, three crystal structures have been determined for maltose transporter at different states in the process of transportation. Here, based on these crystal structures, the allosteric pathway from the resting state (inward-facing) to the catalytic intermediate state (outward-facing) is studied by applying an adaptive anisotropic network model. The results suggest that the allosteric transitions proceed in a coupled way. The closing of the nucleotide-binding domains occurs first, and subsequently this conformational change is propagated to the transmembrane domains (TMD) via the EAA and EAS loops, and then to the maltose-binding protein, which facilitates the translocation of the maltose. It is also found that there exist nonrigid-body and asymmetric movements in the TMD. The cytoplasmic gate may only play the role of allosteric propagation during the transition from the pretranslocation to outward-facing states. In addition, the results show that the movment of the helical subdomain towards the RecA-like subdomain mainly occurs in the earlier stages of the transition. These results can provide some insights into the understanding of the mechanism of ABC transporters.

  3. 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

  4. 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.

  5. Communications

    ERIC Educational Resources Information Center

    Bailenson, Jeremy; Buzzanell, Patrice; Deetz, Stanley; Tewksbury, David; Thompson, Robert J.; Turow, Joseph; Bichelmeyer, Barbara; Bishop, M. J.; Gayeski, Diane

    2013-01-01

    Scholars representing the field of communications were asked to identify what they considered to be the most exciting and imaginative work currently being done in their field, as well as how that work might change our understanding. The scholars included Jeremy Bailenson, Patrice Buzzanell, Stanley Deetz, David Tewksbury, Robert J. Thompson, and…

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

    PubMed Central

    Watterson, Lucas R.; Olive, M. Foster

    2013-01-01

    Positive allosteric modulators (PAMs) of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are a diverse class of compounds that increase fast excitatory transmission in the brain. AMPA PAMs have been shown to facilitate long-term potentiation, strengthen communication between various cortical and subcortical regions, and some of these compounds increase the production and release of brain-derived neurotrophic factor (BDNF) in an activity-dependent manner. Through these mechanisms, AMPA PAMs have shown promise as broad spectrum pharmacotherapeutics in preclinical and clinical studies for various neurodegenerative and psychiatric disorders. In recent years, a small collection of preclinical animal studies has also shown that AMPA PAMs may have potential as pharmacotherapeutic adjuncts to extinction-based or cue-exposure therapies for the treatment of drug addiction. The present paper will review this preclinical literature, discuss novel data collected in our laboratory, and recommend future research directions for the possible development of AMPA PAMs as anti-addiction medications. PMID:24380895

  7. Allosteric drugs: the interaction of antitumor compound MKT-077 with human Hsp70 chaperones.

    PubMed

    Rousaki, Aikaterini; Miyata, Yoshinari; Jinwal, Umesh K; Dickey, Chad A; Gestwicki, Jason E; Zuiderweg, Erik R P

    2011-08-19

    Hsp70 (heat shock protein 70 kDa) chaperones are key to cellular protein homeostasis. However, they also have the ability to inhibit tumor apoptosis and contribute to aberrant accumulation of hyperphosphorylated tau in neuronal cells affected by tauopathies, including Alzheimer's disease. Hence, Hsp70 chaperones are increasingly becoming identified as targets for therapeutic intervention in these widely abundant diseases. Hsp70 proteins are allosteric machines and offer, besides classical active-site targets, also opportunities to target the mechanism of allostery. In this work, it is demonstrated that the action of the potent anticancer compound MKT-077 (1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl] pyridinium chloride) occurs through a differential interaction with Hsp70 allosteric states. MKT-077 is therefore an "allosteric drug." Using NMR spectroscopy, we identify the compound's binding site on human HSPA8 (Hsc70). The binding pose is obtained from NMR-restrained docking calculations, subsequently scored by molecular-dynamics-based energy and solvation computations. Suggestions for the improvement of the compound's properties are made on the basis of the binding location and pose.

  8. Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control

    PubMed Central

    Huang, Zhi; Shiva, Sruti; Kim-Shapiro, Daniel B.; Patel, Rakesh P.; Ringwood, Lorna A.; Irby, Cynthia E.; Huang, Kris T.; Ho, Chien; Hogg, Neil; Schechter, Alan N.; Gladwin, Mark T.

    2005-01-01

    Hypoxic vasodilation is a fundamental, highly conserved physiological response that requires oxygen and/or pH sensing coupled to vasodilation. While this process was first characterized more than 80 years ago, the precise identity and mechanism of the oxygen sensor and mediators of vasodilation remain uncertain. In support of a possible role for hemoglobin (Hb) as a sensor and effector of hypoxic vasodilation, here we show biochemical evidence that Hb exhibits enzymatic behavior as a nitrite reductase, with maximal NO generation rates occurring near the oxy-to-deoxy (R-to-T) allosteric structural transition of the protein. The observed rate of nitrite reduction by Hb deviates from second-order kinetics, and sigmoidal reaction progress is determined by a balance between 2 opposing chemistries of the heme in the R (oxygenated conformation) and T (deoxygenated conformation) allosteric quaternary structures of the Hb tetramer — the greater reductive potential of deoxyheme in the R state tetramer and the number of unligated deoxyheme sites necessary for nitrite binding, which are more plentiful in the T state tetramer. These opposing chemistries result in a maximal nitrite reduction rate when Hb is 40–60% saturated with oxygen (near the Hb P50), an apparent ideal set point for hypoxia-responsive NO generation. These data suggest that the oxygen sensor for hypoxic vasodilation is determined by Hb oxygen saturation and quaternary structure and that the nitrite reductase activity of Hb generates NO gas under allosteric and pH control. PMID:16041407

  9. 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)

  10. A secreted salivary inositol polyphosphate 5-phosphatase from a blood-feeding insect: allosteric activation by soluble phosphoinositides and phosphatidylserine.

    PubMed

    Andersen, John F; Ribeiro, José M C

    2006-05-02

    Type II inositol polyphosphate 5-phosphatases (IPPs) act on both soluble inositol phosphate and phosphoinositide substrates. In many cases, these enzymes occur as multidomain proteins in which the IPP domain is linked to lipid-binding or additional catalytic domains. Rhodnius prolixus IPPRp exists as an isolated IPP domain which is secreted into the saliva of this blood-feeding insect. It shows selectivity for soluble and lipid substrates having a 1,4,5-trisphosphate substitution pattern while only poorly hydrolyzing substrates containing a D3 phosphate. With soluble diC8 PI(4,5)P(2) as a substrate, sigmoidal kinetics were observed, suggesting the presence of allosteric activation sites. Surprisingly, IPPRp-mediated hydrolysis of PI(4,5)P(2) and PI(3,4,5)P(3) was also stimulated up to 100-fold by diC8 PI(4)P and diC8 phosphatidylserine (PS). The activation kinetics were again sigmoidal, demonstrating that the allosteric sites recognize nonsubstrate phospholipids. Activation was positively cooperative, and analysis by the Hill equation suggests that at least three to four allosteric sites are present. In a vesicular system, hydrolysis of PI(4,5)P(2) followed a surface dilution kinetic model, and as expected, PS was found to be strongly stimulatory. If allosteric activation of type II IPPs by PI(4)P and PS is a widespread feature of the group, it may represent a novel regulatory mechanism for these important enzymes.

  11. Dynamical network of residue–residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation

    PubMed Central

    Doshi, Urmi; Holliday, Michael J.; Eisenmesser, Elan Z.; Hamelberg, Donald

    2016-01-01

    Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue–residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general. PMID:27071107

  12. Dynamical network of residue-residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation.

    PubMed

    Doshi, Urmi; Holliday, Michael J; Eisenmesser, Elan Z; Hamelberg, Donald

    2016-04-26

    Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue-residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general.

  13. The allosteric mechanism of yeast chorismate mutase: a dynamic analysis.

    PubMed

    Kong, Yifei; Ma, Jianpeng; Karplus, Martin; Lipscomb, William N

    2006-02-10

    The effector-regulated allosteric mechanism of yeast chorismate mutase (YCM) was studied by normal mode analysis and targeted molecular dynamics. The normal mode analysis shows that the conformational change between YCM in the R state and in the T state can be represented by a relatively small number of low-frequency modes. This suggests that the transition is coded in the structure and is likely to have a low energetic barrier. Quantitative comparisons (i.e. frequencies) between the low-frequency modes of YCM with and without effectors (modeled structures) reveal that the binding of Trp increases the global flexibility, whereas Tyr decreases global flexibility. The targeted molecular dynamics simulation of substrate analog release from the YCM active site suggests that a series of residues are critical for orienting and "recruiting" the substrate. The simulation led to the switching of a series of substrate-release-coupled salt-bridge partners in the ligand-binding domain; similar changes occur in the transition between YCM R-state and T-state crystal structures. Thus, the normal mode analysis and targeted molecular dynamics results provide evidence that the effectors regulate YCM activity by influencing the global flexibility. The change in flexibility is coupled to the binding of substrate to the T state and release of the product from the R state, respectively.

  14. 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

  15. Ryanodine Receptor Allosteric Coupling and the Dynamics of Calcium Sparks

    PubMed Central

    Groff, Jeffrey R.; Smith, Gregory D.

    2008-01-01

    Puffs and sparks are localized intracellular Ca2+ elevations that arise from the cooperative activity of Ca2+-regulated inositol 1,4,5-trisphosphate receptors and ryanodine receptors clustered at Ca2+ release sites on the surface of the endoplasmic reticulum or the sarcoplasmic reticulum. While the synchronous gating of Ca2+-regulated Ca2+ channels can be mediated entirely though the buffered diffusion of intracellular Ca2+, interprotein allosteric interactions also contribute to the dynamics of ryanodine receptor (RyR) gating and Ca2+ sparks. In this article, Markov chain models of Ca2+ release sites are used to investigate how the statistics of Ca2+ spark generation and termination are related to the coupling of RyRs via local [Ca2+] changes and allosteric interactions. Allosteric interactions are included in a manner that promotes the synchronous gating of channels by stabilizing neighboring closed-closed and/or open-open channel pairs. When the strength of Ca2+-mediated channel coupling is systematically varied (e.g., by changing the Ca2+ buffer concentration), simulations that include synchronizing allosteric interactions often exhibit more robust Ca2+ sparks; however, for some Ca2+ coupling strengths the sparks are less robust. We find no evidence that the distribution of spark durations can be used to distinguish between allosteric interactions that stabilize closed channel pairs, open channel pairs, or both in a balanced fashion. On the other hand, the changes in spark duration, interspark interval, and frequency observed when allosteric interactions that stabilize closed channel pairs are gradually removed from simulations are qualitatively different than the changes observed when open or both closed and open channel pairs are stabilized. Thus, our simulations clarify how changes in spark statistics due to pharmacological washout of the accessory proteins mediating allosteric coupling may indicate the type of synchronizing allosteric interactions exhibited

  16. Allosteric Voltage Gating of Potassium Channels I

    PubMed Central

    Horrigan, Frank T.; Cui, Jianmin; Aldrich, Richard W.

    1999-01-01

    Activation of large conductance Ca2+-activated K+ channels is controlled by both cytoplasmic Ca2+ and membrane potential. To study the mechanism of voltage-dependent gating, we examined mSlo Ca2+-activated K+ currents in excised macropatches from Xenopus oocytes in the virtual absence of Ca2+ (<1 nM). In response to a voltage step, IK activates with an exponential time course, following a brief delay. The delay suggests that rapid transitions precede channel opening. The later exponential time course suggests that activation also involves a slower rate-limiting step. However, the time constant of IK relaxation [τ(IK)] exhibits a complex voltage dependence that is inconsistent with models that contain a single rate limiting step. τ(IK) increases weakly with voltage from −500 to −20 mV, with an equivalent charge (z) of only 0.14 e, and displays a stronger voltage dependence from +30 to +140 mV (z = 0.49 e), which then decreases from +180 to +240 mV (z = −0.29 e). Similarly, the steady state GK–V relationship exhibits a maximum voltage dependence (z = 2 e) from 0 to +100 mV, and is weakly voltage dependent (z ≅ 0.4 e) at more negative voltages, where Po = 10−5–10−6. These results can be understood in terms of a gating scheme where a central transition between a closed and an open conformation is allosterically regulated by the state of four independent and identical voltage sensors. In the absence of Ca2+, this allosteric mechanism results in a gating scheme with five closed (C) and five open (O) states, where the majority of the channel's voltage dependence results from rapid C–C and O–O transitions, whereas the C–O transitions are rate limiting and weakly voltage dependent. These conclusions not only provide a framework for interpreting studies of large conductance Ca2+-activated K+ channel voltage gating, but also have important implications for understanding the mechanism of Ca2+ sensitivity. PMID:10436003

  17. Communications

    NASA Technical Reports Server (NTRS)

    Bonelle, G. J.

    1984-01-01

    Communications in any system is one of the last technologies to be considered, and sometimes it is considered too late to impact the system. This was somewhat the impression on reviewing the NASA budget for two mission scenarios for the space station. However, that budget fortunately was well spent, and the money was spent to get the most benefit per dollar. Another thing that is very often forgotten is that technology is not produced in a vacuum. In fact, in conducting independent research and development (IR&D), the first phase is to define the requirements which must be time phased, becuase very often the conditions will change during the life of the system. From the requirements, a set of architectures that are at least representative of that era are produced. If the exact requirements were not established, at least boundaries are set on the requirements for that architecture. When this is completed, then the technology that is really needed is defined. The major criticism of the work that was presented to the panel is the lack of a firm set of requirements.

  18. Allosteric interactions at adenosine A1 and A3 receptors: new insights into the role of small molecules and receptor dimerization

    PubMed Central

    Hill, Stephen J; May, Lauren T; Kellam, Barrie; Woolard, Jeanette

    2014-01-01

    The purine nucleoside adenosine is present in all cells in tightly regulated concentrations. It is released under a variety of physiological and pathophysiological conditions to facilitate protection and regeneration of tissues. Adenosine acts via specific GPCRs to either stimulate cyclic AMP formation, as exemplified by Gs-protein-coupled adenosine receptors (A2A and A2B), or inhibit AC activity, in the case of Gi/o-coupled adenosine receptors (A1 and A3). Recent advances in our understanding of GPCR structure have provided insights into the conformational changes that occur during receptor activation following binding of agonists to orthosteric (i.e. at the same binding site as an endogenous modulator) and allosteric regulators to allosteric sites (i.e. at a site that is topographically distinct from the endogenous modulator). Binding of drugs to allosteric sites may lead to changes in affinity or efficacy, and affords considerable potential for increased selectivity in new drug development. Herein, we provide an overview of the properties of selective allosteric regulators of the adenosine A1 and A3 receptors, focusing on the impact of receptor dimerization, mechanistic approaches to single-cell ligand-binding kinetics and the effects of A1- and A3-receptor allosteric modulators on in vivo pharmacology. Linked ArticlesThis article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-5 PMID:24024783

  19. Convergent Transmission of RNAi Guide-Target Mismatch Information across Argonaute Internal Allosteric Network

    PubMed Central

    Joseph, Thomas T.; Osman, Roman

    2012-01-01

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

  20. Allosteric Learning Model in English Lesson: Teachers' Views, the Instructions of Curriculum and Course Book, a Sample of Daily Lesson Plan

    ERIC Educational Resources Information Center

    Berkant, Hasan Güner; Baysal, Seda

    2017-01-01

    The changes which occur during the learning process have been explained by many teaching-learning models and theories. One of these models is allosteric learning model (ALM) which was developed by André Giordan in 1989. This model was derived from a biological metaphor related to proteins. The interaction between individual and environment in a…

  1. Allosteric Modulators for the Treatment of Schizophrenia: Targeting Glutamatergic Networks

    PubMed Central

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

    2013-01-01

    Schizophrenia is a highly debilitating mental disorder which afflicts approximately 1% of the global population. Cognitive and negative deficits account for the lifelong disability associated with schizophrenia, whose symptoms are not effectively addressed by current treatments. New medicines are needed to treat these aspects of the disease. Neurodevelopmental, neuropathological, genetic, and behavioral pharmacological data indicate that schizophrenia stems from a dysfunction of glutamate synaptic transmission, particularly in frontal cortical networks. A number of novel pre- and postsynaptic mechanisms affecting glutamatergic synaptic transmission have emerged as viable targets for schizophrenia. While developing orthosteric glutamatergic agents for these targets has proven extremely difficult, targeting allosteric sites of these targets has emerged as a promising alternative. From a medicinal chemistry perspective, allosteric sites provide an opportunity of finding agents with better drug-like properties and greater target specificity. Furthermore, allosteric modulators are better suited to maintaining the highly precise temporal and spatial aspects of glutamatergic synaptic transmission. Herein, we review neuropathological and genomic/genetic evidence underscoring the importance of glutamate synaptic dysfunction in the etiology of schizophrenia and make a case for allosteric targets for therapeutic intervention. We review progress in identifying allosteric modulators of AMPA receptors, NMDA receptors, and metabotropic glutamate receptors, all with the aim of restoring physiological glutamatergic synaptic transmission. Challenges remain given the complexity of schizophrenia and the difficulty in studying cognition in animals and humans. Nonetheless, important compounds have emerged from these efforts and promising preclinical and variable clinical validation has been achieved. PMID:23409764

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

    SciTech Connect

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

    2009-12-01

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

  3. Assembly of the Sos1-Grb2-Gab1 Ternary Signaling Complex Is Under Allosteric Control

    PubMed Central

    McDonald, Caleb B.; Seldeen, Kenneth L.; Deegan, Brian J.; Bhat, Vikas; Farooq, Amjad

    2009-01-01

    Allostery has evolved as a form of local communication between interacting protein partners allowing them to quickly sense changes in their immediate vicinity in response to external cues. Herein, using isothermal titration calorimetry (ITC) in conjunction with circular dichroism (CD) and macromolecular modeling (MM), we show that the binding of Grb2 adaptor — a key signaling molecule involved in the activation of Ras GTPase — to its downstream partners Sos1 guanine nucleotide exchange factor and Gab1 docker is under tight allosteric regulation. Specifically, our findings reveal that the binding of one molecule of Sos1 to the nSH3 domain allosterically induces a conformational change within Grb2 such that the loading of a second molecule of Sos1 onto the cSH3 domain is blocked and, in so doing, allows Gab1 access to the cSH3 domain in an exclusively non-competitive manner to generate the Sos1-Grb2-Gab1 ternary signaling complex. PMID:20005866

  4. Novel inhibitors complexed with glutamate dehydrogenase: allosteric regulation by control of protein dynamics.

    PubMed

    Li, Ming; Smith, Christopher J; Walker, Matthew T; Smith, Thomas J

    2009-08-21

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

  5. 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

  6. Allosteric dynamics of SAMHD1 studied by molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Patra, K. K.; Bhattacharya, A.; Bhattacharya, S.

    2016-10-01

    SAMHD1 is a human cellular enzyme that blocks HIV-1 infection in myeloid cells and non-cycling CD4+T cells. The enzyme is an allosterically regulated triphosphohydrolase that modulates the level of cellular dNTP. The virus restriction is attributed to the lowering of the pool of dNTP in the cell to a point where reverse-transcription is impaired. Mutations in SAMHD1 are also implicated in Aicardi-Goutieres syndrome. A mechanistic understanding of the allosteric activation of the enzyme is still elusive. We have performed molecular dynamics simulations to examine the allosteric site dynamics of the protein and to examine the connection between the stability of the tetrameric complex and the Allosite occupancy.

  7. Physical limits on computation by assemblies of allosteric proteins

    NASA Astrophysics Data System (ADS)

    Robinson, John

    2009-03-01

    Assemblies of allosteric proteins are the principle information processing devices in biology. Using the Ca^2+-sensitive cardiac regulatory assembly as a paradigm for Brownian computation, we examine how system complexity and system resetting impose physical limits on computation. Nearest-neighbor-limited interactions among assembly components constrains the topology of the system's macrostate free energy landscape and produces degenerate transition probabilities. As a result, signaling fidelity and deactivation kinetics can not be simultaneously optimized. This imposes an upper limit on the rate of information processing by assemblies of allosteric proteins that couple to a single ligand type.

  8. Physical Limits on Computation by Assemblies of Allosteric Proteins

    NASA Astrophysics Data System (ADS)

    Robinson, John M.

    2008-10-01

    Assemblies of allosteric proteins are the principle information processing devices in biology. Using the Ca2+-sensitive cardiac regulatory assembly as a paradigm for Brownian computation, I examine how system complexity and system resetting impose physical limits on computation. Nearest-neighbor-limited interactions among assembly components constrain the topology of the system’s macrostate free energy landscape and produce degenerate transition probabilities. As a result, signaling fidelity and deactivation kinetics cannot be simultaneously optimized. This imposes an upper limit on the rate of information processing by assemblies of allosteric proteins that couple to a single ligand type.

  9. Disruption of integrin-fibronectin complexes by allosteric but not ligand-mimetic inhibitors.

    PubMed

    Mould, A Paul; Craig, Susan E; Byron, Sarah K; Humphries, Martin J; Jowitt, Thomas A

    2014-12-15

    Failure of Arg-Gly-Asp (RGD)-based inhibitors to reverse integrin-ligand binding has been reported, but the prevalence of this phenomenon among integrin heterodimers is currently unknown. In the present study we have investigated the interaction of four different RGD-binding integrins (α5β1, αVβ1, αVβ3 and αVβ6) with fibronectin (FN) using surface plasmon resonance. The ability of inhibitors to reverse ligand binding was assessed by their capacity to increase the dissociation rate of pre-formed integrin-FN complexes. For all four receptors we showed that RGD-based inhibitors (such as cilengitide) were completely unable to increase the dissociation rate. Formation of the non-reversible state occurred very rapidly and did not rely on the time-dependent formation of a high-affinity state of the integrin, or the integrin leg regions. In contrast with RGD-based inhibitors, Ca2+ (but not Mg2+) was able to greatly increase the dissociation rate of integrin-FN complexes, with a half-maximal response at ~0.4 mM Ca2+ for αVβ3-FN. The effect of Ca2+ was overcome by co-addition of Mn2+, but not Mg2+. A stimulatory anti-β1 monoclonal antibody (mAb) abrogated the effect of Ca2+ on α5β1-FN complexes; conversely, a function-blocking mAb mimicked the effect of Ca2+. These results imply that Ca2+ acts allosterically, probably through binding to the adjacent metal-ion-dependent adhesion site (ADMIDAS), and that the α1 helix in the β subunit I domain is the key element affected by allosteric modulators. The data suggest an explanation for the limited clinical efficacy of RGD-based integrin antagonists, and we propose that allosteric antagonists could prove to be of greater therapeutic benefit.

  10. 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

  11. Biased allosteric modulation at the CaS receptor engendered by structurally diverse calcimimetics

    PubMed Central

    Cook, A E; Mistry, S N; Gregory, K J; Furness, S G B; Sexton, P M; Scammells, P J; Conigrave, A D; Christopoulos, A; Leach, K

    2015-01-01

    BACKGROUND AND PURPOSE Clinical use of cinacalcet in hyperparathyroidism is complicated by its tendency to induce hypocalcaemia, arising partly from activation of calcium-sensing receptors (CaS receptors) in the thyroid and stimulation of calcitonin release. CaS receptor allosteric modulators that selectively bias signalling towards pathways that mediate desired effects [e.g. parathyroid hormone (PTH) suppression] rather than those mediating undesirable effects (e.g. elevated serum calcitonin), may offer better therapies. EXPERIMENTAL APPROACH We characterized the ligand-biased profile of novel calcimimetics in HEK293 cells stably expressing human CaS receptors, by monitoring intracellular calcium (Ca2+i) mobilization, inositol phosphate (IP)1 accumulation, ERK1/2 phosphorylation (pERK1/2) and receptor expression. KEY RESULTS Phenylalkylamine calcimimetics were biased towards allosteric modulation of Ca2+i mobilization and IP1 accumulation. S,R-calcimimetic B was biased only towards IP1 accumulation. R,R-calcimimetic B and AC-265347 were biased towards IP1 accumulation and pERK1/2. Nor-calcimimetic B was unbiased. In contrast to phenylalkylamines and calcimimetic B analogues, AC-265347 did not promote trafficking of a loss-of-expression, naturally occurring, CaS receptor mutation (G670E). CONCLUSIONS AND IMPLICATIONS The ability of R,R-calcimimetic B and AC-265347 to bias signalling towards pERK1/2 and IP1 accumulation may explain their suppression of PTH levels in vivo at concentrations that have no effect on serum calcitonin levels. The demonstration that AC-265347 promotes CaS receptor receptor signalling, but not trafficking reveals a novel profile of ligand-biased modulation at CaS receptors The identification of allosteric modulators that bias CaS receptor signalling towards distinct intracellular pathways provides an opportunity to develop desirable biased signalling profiles in vivo for mediating selective physiological responses. PMID:25220431

  12. 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.

  13. 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

  14. Rational Design of Potent, Small, Synthetic Allosteric Inhibitors of Thrombin

    PubMed Central

    Sidhu, Preetpal Singh; Liang, Aiye; Mehta, Akul Y.; Abdel Aziz, May H.; Zhou, Qibing; Desai, Umesh R.

    2011-01-01

    Thrombin is a key enzyme targeted by the majority of current anticoagulants that are direct inhibitors. Allosteric inhibition of thrombin may offer a major advantage of finely tuned regulation. We present here sulfated benzofurans as the first examples of potent, small allosteric inhibitors of thrombin. A sulfated benzofuran library of 15 sulfated monomers and 13 sulfated dimers with different charged, polar and hydrophobic substituents was studied in this work. Synthesis of the sulfated benzofurans was achieved through a multiple step, highly branched strategy, which culminated with microwave-assisted chemical sulfation. Of the 28 potential inhibitors, eleven exhibited reasonable inhibition of human α-thrombin at pH 7.4. Structure activity relationship analysis indicated that sulfation at the 5-position of the benzofuran scaffold was essential for targeting thrombin. A t-butyl 5-sulfated benzofuran derivative was found to be the most potent thrombin inhibitor with an IC50 of 7.3 μM under physiologically relevant conditions. Michaelis-Menten studies showed an allosteric inhibition phenomenon. Plasma clotting assays indicate that the sulfated benzofurans prolong both the activated partial thromboplastin time and prothrombin time. Overall, this work puts forward sulfated benzofurans as the first small, synthetic molecules as powerful lead compounds for the design of a new class of allosteric inhibitors of thrombin. PMID:21714536

  15. Molecular mechanism of allosteric substrate activation in a thiamine diphosphate-dependent decarboxylase.

    PubMed

    Versées, Wim; Spaepen, Stijn; Wood, Martin D H; Leeper, Finian J; Vanderleyden, Jos; Steyaert, Jan

    2007-11-30

    Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.

  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. Allosteric sensitization of nicotinic receptors by galantamine, a new treatment strategy for Alzheimer's disease.

    PubMed

    Maelicke, A; Samochocki, M; Jostock, R; Fehrenbacher, A; Ludwig, J; Albuquerque, E X; Zerlin, M

    2001-02-01

    Cholinesterase inhibitors are the only approved drug treatment for patients with mild to moderately severe Alzheimer's disease. Interestingly, the clinical potency of these drugs does not correlate well with their activity as cholinesterase inhibitors, nor is their action as short lived as would be expected from purely symptomatic treatment. A few cholinesterase inhibitors, including galantamine, produce beneficial effects even after drug treatment has been terminated. These effects assume modes of action other than mere esterase inhibition and are capable of inducing systemic changes. We have recently discovered a mechanism that could account, at least in part, for the above-mentioned unexpected properties of some cholinesterase inhibitors. We have found that a subgroup of cholinesterase inhibitors, including galantamine but excluding tacrine, directly interacts with nicotinic acetylcholine receptors. These compounds, named allosterically potentiating ligands, sensitize nicotinic receptors by increasing the probability of channel opening induced by acetylcholine and nicotinic agonists and by slowing down receptor desensitization. The allosterically potentiating ligand action, which is not necessarily associated with cholinesterase inhibition, has been demonstrated by whole-cell patch-clamp recordings to occur in natural murine and human neurons and in murine and human cell lines expressing various subtypes of neuronal nicotinic acetylcholine receptors.

  18. Thermal activation of 'allosteric-like' large-scale motions in a eukaryotic Lactate Dehydrogenase.

    PubMed

    Katava, Marina; Maccarini, Marco; Villain, Guillaume; Paciaroni, Alessandro; Sztucki, Michael; Ivanova, Oxana; Madern, Dominique; Sterpone, Fabio

    2017-01-23

    Conformational changes occurring during the enzymatic turnover are essential for the regulation of protein functionality. Individuating the protein regions involved in these changes and the associated mechanical modes is still a challenge at both experimental and theoretical levels. We present here a detailed investigation of the thermal activation of the functional modes and conformational changes in a eukaryotic Lactate Dehydrogenase enzyme (LDH). Neutron Spin Echo spectroscopy and Molecular Dynamics simulations were used to uncover the characteristic length- and timescales of the LDH nanoscale motions in the apo state. The modes involving the catalytic loop and the mobile region around the binding site are activated at room temperature, and match the allosteric reorganisation of bacterial LDHs. In a temperature window of about 15 degrees, these modes render the protein flexible enough and capable of reorganising the active site toward reactive configurations. On the other hand an excess of thermal excitation leads to the distortion of the protein matrix with a possible anti-catalytic effect. Thus, the temperature activates eukaryotic LDHs via the same conformational changes observed in the allosteric bacterial LDHs. Our investigation provides an extended molecular picture of eukaryotic LDH's conformational landscape that enriches the static view based on crystallographic studies alone.

  19. Thermal activation of ‘allosteric-like’ large-scale motions in a eukaryotic Lactate Dehydrogenase

    PubMed Central

    Katava, Marina; Maccarini, Marco; Villain, Guillaume; Paciaroni, Alessandro; Sztucki, Michael; Ivanova, Oxana; Madern, Dominique; Sterpone, Fabio

    2017-01-01

    Conformational changes occurring during the enzymatic turnover are essential for the regulation of protein functionality. Individuating the protein regions involved in these changes and the associated mechanical modes is still a challenge at both experimental and theoretical levels. We present here a detailed investigation of the thermal activation of the functional modes and conformational changes in a eukaryotic Lactate Dehydrogenase enzyme (LDH). Neutron Spin Echo spectroscopy and Molecular Dynamics simulations were used to uncover the characteristic length- and timescales of the LDH nanoscale motions in the apo state. The modes involving the catalytic loop and the mobile region around the binding site are activated at room temperature, and match the allosteric reorganisation of bacterial LDHs. In a temperature window of about 15 degrees, these modes render the protein flexible enough and capable of reorganising the active site toward reactive configurations. On the other hand an excess of thermal excitation leads to the distortion of the protein matrix with a possible anti-catalytic effect. Thus, the temperature activates eukaryotic LDHs via the same conformational changes observed in the allosteric bacterial LDHs. Our investigation provides an extended molecular picture of eukaryotic LDH’s conformational landscape that enriches the static view based on crystallographic studies alone. PMID:28112231

  20. 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

  1. Naturally occurring chemical carcinogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Natural products are chemicals found in nature which have unique pharmacological effects. Humans are exposed to many of these bioactive naturally occurring chemicals via the air breathed, the water drunk and the food eaten. Exposure also occurs in clinical settings. Naturally occurring chemicals ...

  2. 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.

  3. 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.

  4. [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.

  5. 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.

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

    PubMed

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

    2016-07-05

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

  7. 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

  8. 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

  9. Novel bivalent positive allosteric modulators of AMPA receptor.

    PubMed

    Lavrov, M I; Grigor'ev, V V; Bachurin, S O; Palyulin, V A; Zefirov, N S

    2015-01-01

    A positive allosteric modulator of AMPA receptors has been designed using computer-aided molecular modeling techniques. It possessed a record high experimentally confirmed potency in the picomolar concentration range and belongs to a new type of bivalent AMPA receptor ligands containing bicyclo[3.3.1]nonane scaffold. The suggested structure could serve as a basis for further optimization and development of drugs for the treatment of neurodegenerative diseases, cognition enhancement, and improvement of memory.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. Dynamics of allosteric transitions in GroEL

    PubMed Central

    Hyeon, Changbong; Lorimer, George H.; Thirumalai, D.

    2006-01-01

    The chaperonin GroEL-GroES, a machine that helps proteins to fold, cycles through a number of allosteric states, the T state, with high affinity for substrate proteins, the ATP-bound R state, and the R″ (GroEL–ADP–GroES) complex. Here, we use a self-organized polymer model for the GroEL allosteric states and a general structure-based technique to simulate the dynamics of allosteric transitions in two subunits of GroEL and the heptamer. The T → R transition, in which the apical domains undergo counterclockwise motion, is mediated by a multiple salt-bridge switch mechanism, in which a series of salt-bridges break and form. The initial event in the R → R″ transition, during which GroEL rotates clockwise, involves a spectacular outside-in movement of helices K and L that results in K80-D359 salt-bridge formation. In both the transitions there is considerable heterogeneity in the transition pathways. The transition state ensembles (TSEs) connecting the T, R, and R″ states are broad with the TSE for the T → R transition being more plastic than the R → R″ TSE. PMID:17135353

  16. 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.

  17. A naturally occurring insertion of a single amino acid rewires transcriptional regulation by glucocorticoid receptor isoforms.

    PubMed

    Thomas-Chollier, Morgane; Watson, Lisa C; Cooper, Samantha B; Pufall, Miles A; Liu, Jennifer S; Borzym, Katja; Vingron, Martin; Yamamoto, Keith R; Meijsing, Sebastiaan H

    2013-10-29

    In addition to guiding proteins to defined genomic loci, DNA can act as an allosteric ligand that influences protein structure and activity. Here we compared genome-wide binding, transcriptional regulation, and, using NMR, the conformation of two glucocorticoid receptor (GR) isoforms that differ by a single amino acid insertion in the lever arm, a domain that adopts DNA sequence-specific conformations. We show that these isoforms differentially regulate gene expression levels through two mechanisms: differential DNA binding and altered communication between GR domains. Our studies suggest a versatile role for DNA in both modulating GR activity and also in directing the use of GR isoforms. We propose that the lever arm is a "fulcrum" for bidirectional allosteric signaling, conferring conformational changes in the DNA reading head that influence DNA sequence selectivity, as well as conferring changes in the dimerization domain that connect functionally with remote regulatory surfaces, thereby influencing which genes are regulated and the magnitude of their regulation.

  18. 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.

  19. 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.

  20. A Mechanistic Understanding of Allosteric Immune Escape Pathways in the HIV-1 Envelope Glycoprotein

    PubMed Central

    Sethi, Anurag; Tian, Jianhui; Derdeyn, Cynthia A.; Korber, Bette; Gnanakaran, S.

    2013-01-01

    The HIV-1 envelope (Env) spike, which consists of a compact, heterodimeric trimer of the glycoproteins gp120 and gp41, is the target of neutralizing antibodies. However, the high mutation rate of HIV-1 and plasticity of Env facilitates viral evasion from neutralizing antibodies through various mechanisms. Mutations that are distant from the antibody binding site can lead to escape, probably by changing the conformation or dynamics of Env; however, these changes are difficult to identify and define mechanistically. Here we describe a network analysis-based approach to identify potential allosteric immune evasion mechanisms using three known HIV-1 Env gp120 protein structures from two different clades, B and C. First, correlation and principal component analyses of molecular dynamics (MD) simulations identified a high degree of long-distance coupled motions that exist between functionally distant regions within the intrinsic dynamics of the gp120 core, supporting the presence of long-distance communication in the protein. Then, by integrating MD simulations with network theory, we identified the optimal and suboptimal communication pathways and modules within the gp120 core. The results unveil both strain-dependent and -independent characteristics of the communication pathways in gp120. We show that within the context of three structurally homologous gp120 cores, the optimal pathway for communication is sequence sensitive, i.e. a suboptimal pathway in one strain becomes the optimal pathway in another strain. Yet the identification of conserved elements within these communication pathways, termed inter-modular hotspots, could present a new opportunity for immunogen design, as this could be an additional mechanism that HIV-1 uses to shield vulnerable antibody targets in Env that induce neutralizing antibody breadth. PMID:23696718

  1. Allosteric Regulation of the Hsp90 Dynamics and Stability by Client Recruiter Cochaperones: Protein Structure Network Modeling

    PubMed Central

    Blacklock, Kristin; Verkhivker, Gennady M.

    2014-01-01

    The fundamental role of the Hsp90 chaperone in supporting functional activity of diverse protein clients is anchored by specific cochaperones. A family of immune sensing client proteins is delivered to the Hsp90 system with the aid of cochaperones Sgt1 and Rar1 that act cooperatively with Hsp90 to form allosterically regulated dynamic complexes. In this work, functional dynamics and protein structure network modeling are combined to dissect molecular mechanisms of Hsp90 regulation by the client recruiter cochaperones. Dynamic signatures of the Hsp90-cochaperone complexes are manifested in differential modulation of the conformational mobility in the Hsp90 lid motif. Consistent with the experiments, we have determined that targeted reorganization of the lid dynamics is a unifying characteristic of the client recruiter cochaperones. Protein network analysis of the essential conformational space of the Hsp90-cochaperone motions has identified structurally stable interaction communities, interfacial hubs and key mediating residues of allosteric communication pathways that act concertedly with the shifts in conformational equilibrium. The results have shown that client recruiter cochaperones can orchestrate global changes in the dynamics and stability of the interaction networks that could enhance the ATPase activity and assist in the client recruitment. The network analysis has recapitulated a broad range of structural and mutagenesis experiments, particularly clarifying the elusive role of Rar1 as a regulator of the Hsp90 interactions and a stability enhancer of the Hsp90-cochaperone complexes. Small-world organization of the interaction networks in the Hsp90 regulatory complexes gives rise to a strong correspondence between highly connected local interfacial hubs, global mediator residues of allosteric interactions and key functional hot spots of the Hsp90 activity. We have found that cochaperone-induced conformational changes in Hsp90 may be determined by specific

  2. "Naturally occurring asbestos

    NASA Astrophysics Data System (ADS)

    Cagnard, F.; Lahondère, D.; Blein, O.; Lahfid, A.; Wille, G.

    2012-04-01

    The term asbestos refers to six silicate minerals from amphibole and serpentine groups. By definition, it consists in bundles of thin and flexible long fibers, with high-tensile strength, and chemical and heat resistance. In contrast to asbestos found within commercial products and mining, the specific term ''naturally occurring asbestos'' (NOA) refers to asbestiform minerals occurring within rocks or soils that can be released by human activities or weathering processes. The fact that the exposure to asbestos is related to lung pathologies is now widely demonstrated (e.g. asbestosis, mesothelioma and lung cancer). However, if health risks associated with exposure to NOA exist, they are not yet well documented. The crystallization of natural asbestos occurs in specific Mg-rich lithologies associated with peculiar structural and metamorphic conditions. By recognizing and combining such specific geologic criteria, the presence or the absence of asbestos in bedrock terrains can be reasonably predicted and maps of NOA hazard can be drawn. We present here new results of geological mapping and petrological study concerning the evaluation of the NOA hazard in the Alps and Corsica, in France. The three folds approach consists in (1) a determination of lithologies with potential NOA from a bibliographic compilation and extraction of target zones from a geological geodatabase (2) a geological mapping of the target zones followed by a petrological characterization of sampled asbestiform minerals in the laboratory (optical microscopy, TEM, SEM, and Raman spectroscopy technics), and (3) the drawing of the final map of NOA hazard, at regional-scale. Occurrence criteria can be retained as follows: 1. NOA are abundant in the internal zones of the Alps and Corsica, especially within ophiolitic complexes. Natural asbestos are mostly concentrated within ultramafic rocks but can also occur within basic lithologies such as Mg-metagabbros, metabasalts and meta-pillow-lavas, 2. Asbestos

  3. 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

  4. Selective GABAA α5 Positive Allosteric Modulators Improve Cognitive Function in Aged Rats with Memory Impairment

    PubMed Central

    Koh, Ming Teng; Rosenzweig-Lipson, Sharon; Gallagher, Michela

    2012-01-01

    A condition of excess activity in the hippocampal formation is observed in the aging brain and in conditions that confer additional risk during aging for Alzheimer’s disease. Compounds that act as positive allosteric modulators at GABAA α5 receptors might be useful in targeting this condition because GABAA α5 receptors mediate tonic inhibition of principal neurons in the affected network. While agents to improve cognitive function in the past focused on inverse agonists, which are negative allosteric modulators at GABAA α5 receptors, research supporting that approach used only young animals and predated current evidence for excessive hippocampal activity in age-related conditions of cognitive impairment. Here, we used two compounds, Compound 44 [6,6-dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one] and Compound 6 [methyl 3,5-diphenylpyridazine-4-carboxylate], with functional activity as potentiators of γ-aminobutyric acid at GABAA α5 receptors, to test their ability to improve hippocampal-dependent memory in aged rats with identified cognitive impairment. Improvement was obtained in aged rats across protocols differing in motivational and performance demands and across varying retention intervals. Significant memory improvement occurred after either intracereboventricular infusion with Compound 44 (100 μg) in a water maze task or systemic administration with Compound 6 (3 mg/kg) in a radial arm maze task. Furthermore, systemic administration improved behavioral performance at dosing shown to provide drug exposure in the brain and in vivo receptor occupancy in the hippocampus. These data suggest a novel approach to improve neural network function in clinical conditions of excess hippocampal activity. PMID:22732440

  5. Molecular Dynamics Investigation of a Mechanism of Allosteric Signal Transmission in Ribosomes.

    PubMed

    Makarov, G I; Golovin, A V; Sumbatyan, N V; Bogdanov, A A

    2015-08-01

    The ribosome is a molecular machine that synthesizes all cellular proteins via translation of genetic information encoded in polynucleotide chain of messenger RNA. Transition between different stages of the ribosome working cycle is strictly coordinated by changes in structure and mutual position both of subunits of the ribosome and its ligands. Therein, information regarding structural transformations is transmitted between functional centers of the ribosome through specific signals. Usually, functional centers of ribosomes are located at a distance reaching up to several tens of angstroms, and it is believed that such signals are transduced allosterically. In our study, we attempted to answer the question of how allosteric signal can be transmitted from one of the so-called sensory elements of ribosomal tunnel (RT) to the peptidyl transferase center (PTC). A segment of RT wall from the E. coli ribosome composed of nucleotide residues A2058, A2059, m(2)A2503, G2061, A2062, and C2063 of its 23S rRNA was examined by molecular dynamics simulations. It was found that a potential signal transduction pathway A2058-C2063 acted as a dynamic ensemble of interdependent conformational states, wherein cascade-like changes can occur. It was assumed that structural rearrangement in the A2058-C2063 RT segment results in reversible inactivation of PTC due to a strong stacking contact between functionally important U2585 residue of the PTC and nucleotide residue C2063. A potential role for the observed conformational transition in the A2058-C2063 segment for regulating ribosome activity is discussed.

  6. Kinetic response of a photoperturbed allosteric protein.

    PubMed

    Buchli, Brigitte; Waldauer, Steven A; Walser, Reto; Donten, Mateusz L; Pfister, Rolf; Blöchliger, Nicolas; Steiner, Sandra; Caflisch, Amedeo; Zerbe, Oliver; Hamm, Peter

    2013-07-16

    By covalently linking an azobenzene photoswitch across the binding groove of a PDZ domain, a conformational transition, similar to the one occurring upon ligand binding to the unmodified domain, can be initiated on a picosecond timescale by a laser pulse. The protein structures have been characterized in the two photoswitch states through NMR spectroscopy and the transition between them through ultrafast IR spectroscopy and molecular dynamics simulations. The binding groove opens on a 100-ns timescale in a highly nonexponential manner, and the molecular dynamics simulations suggest that the process is governed by the rearrangement of the water network on the protein surface. We propose this rearrangement of the water network to be another possible mechanism of allostery.

  7. 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

  8. 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.

  9. 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.

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

    SciTech Connect

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

    2012-08-07

    Ligand-dependent activity has been engineered into enzymes for purposes ranging from controlling cell morphology to reprogramming cellular signaling pathways. Where these successes have typically fused a naturally allosteric domain to the enzyme of interest, here we instead demonstrate an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. We present two examples, W33G in a {beta}-glycosidase enzyme ({beta}-gly) and W492G in a {beta}-glucuronidase enzyme ({beta}-gluc), in which we engineer indole-dependent activity into enzymes by removing a buried tryptophan side chain that serves as a buttress for the active site architecture. In both cases, we observe a loss of function, and in both cases we find that the subsequent addition of indole can be used to restore activity. Through a detailed analysis of {beta}-gly W33G kinetics, we demonstrate that this rescued enzyme is fully functionally equivalent to the corresponding wild-type enzyme. We then present the apo and indole-bound crystal structures of {beta}-gly W33G, which together establish the structural basis for enzyme inactivation and rescue. Finally, we use this designed switch to modulate {beta}-glycosidase activity in living cells using indole. Disruption and recovery of protein structure may represent a general technique for introducing allosteric control into enzymes, and thus may serve as a starting point for building a variety of bioswitches and sensors.

  11. Allosteric regulation of glycogen synthase in liver. A physiological dilemma.

    PubMed

    Nuttall, F Q; Gannon, M C

    1993-06-25

    Glycogen synthase catalyzes the transfer of the glucosyl moiety from UDP-glucose to the terminal branch of the glycogen molecule and is considered to be the rate-limiting enzyme for glycogen synthesis. However, under ideal assay conditions, i.e. 37 degrees C with saturating concentrations of UDP-glucose and the activator, glucose-6-P, the maximal catalytic activity of glycogen synthase was only 78% of the in vivo glycogen synthetic rate. Using concentrations of UDP-glucose and glucose-6-P likely to be present in vivo, the rate was only approximately 30%. This prompted us to reassess a possible role of allosteric effectors on synthase activity. Glycogen synthase was assayed at 37 degrees C using dilute, pH 7.0, buffered extracts, initial rate conditions, and UDP-glucose and glucose-6-P concentrations, which approximate those calculated to be present in total liver cell water. Several allosteric effectors were tested. Magnesium and AMP had little effect on activity. Pi, ADP, ATP, and UTP inhibited activity. When a combination of effectors were added at concentrations approximating those present in cell water, synthase activity could account for only 2% of the glycogen synthetic rate. Thus, although allosteric effectors are likely to be playing a major role in regulating synthase enzymic activity in liver cells, to date, a metabolite that can stimulate activity and/or overcome nucleotide inhibition has yet to be identified. If such a metabolite cannot be identified, an additional or alternative pathway for glycogen synthesis must be considered.

  12. 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.

  13. Experimental evidence for allosteric modifier saturation as predicted by the bi-substrate Hill equation.

    PubMed

    Hanekom, A J; Hofmeyr, J H S; Snoep, J L; Rohwer, J M

    2006-09-01

    The cooperative enzyme reaction rates predicted by the bi-substrate Hill equation and the bi-substrate Monod-Wyman-Changeux (MWC) equation when allosterically inhibited are compared in silico. Theoretically, the Hill equation predicts that when the maximum inhibitory effect at a certain substrate condition has been reached, an increase in allosteric inhibitor concentration will have no effect on reaction rate, that is the Hill equation shows allosteric inhibitor saturation. This saturating inhibitory effect is not present in the MWC equation. Experimental in vitro data for pyruvate kinase, a bi-substrate cooperative enzyme that is allosterically inhibited, are presented. This enzyme also shows inhibitor saturation, and therefore serves as experimental evidence that the bi-substrate Hill equation predicts more realistic allosteric inhibitor behaviour than the bi-substrate MWC equation.

  14. Bioinformatic scaling of allosteric interactions in biomedical isozymes

    NASA Astrophysics Data System (ADS)

    Phillips, J. C.

    2016-09-01

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

  15. Naturally occurring insecticides.

    PubMed Central

    Soloway, S B

    1976-01-01

    Naturally occurring insecticides are abundant and varied in their effects, though but a few are articles of commerce. Even for these, pyrethrum, nicotine, rotenone, hellebore, ryania, and sabadilla, there is a paucity of information on mammalian toxicology and environmental effects. In general, these materials are characterized favorably by low acute toxicity and ready dissipation in nature. Unfavorable aspects of natural insecticides are the contained mixture of active and inactive components and the low active ingredient content on a crop yield basis pointing to a high unit cost. Natural insecticides can serve additionally as leads to unnatural mimics, of which the commercially successful synthetic pyrethroids are prime examples. The chemical nature, relationship of insecticidal activity to chemical structure, occurrence, production, and utilization, registered uses, metabolism, and insect and mammalian toxicity are reviewed. PMID:789058

  16. 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.

  17. 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

  18. Engineering alternate cooperative-communications in the lactose repressor protein scaffold.

    PubMed

    Meyer, Sarai; Ramot, Roee; Kishore Inampudi, Krishna; Luo, Beibei; Lin, Chenyu; Amere, Swathi; Wilson, Corey J

    2013-06-01

    To expand our understanding of the hallmarks of allosteric control we used directed-evolution to engineer alternate cooperative communication in the lactose repressor protein (LacI) scaffold. Starting with an I(s) type LacI mutant D88A (i.e. a LacI variant that is insensitive to the exogenous ligand isopropyl-β-d-thiogalactoside (IPTG) and remains bound to operator DNA, + or -IPTG) we used error-prone polymerase chain reaction to introduce compensatory mutations to restore modulated DNA binding function to the allosterically 'dead' I(s)(D88A) background. Five variants were generated, three variants (C4, C32 and C80) with wild-type like function and two co-repressor variants (C101 and C140) that are functionally inverted. To better resolve the residues that define new allosteric networks in the LacI variants, we conducted mutational tolerance analysis via saturation mutagenesis at each of the evolved positions to assess sensitivity to mutation--a hallmark of allosteric residues. To better understand the physicochemical bases of alternate allosteric function, variant LacI(C80) was characterized to assess IPTG ligand binding at equilibrium, kinetically using stopped-flow, and via isothermal titration calorimetry. These data suggest that the conferred modulated DNA binding function observed for LacI(C80), while thermodynamically similar to wild-type LacI, is mechanistically different from the wild-type repressor, suggesting a new allosteric network and communication route.

  19. Naturally Occurring Food Toxins

    PubMed Central

    Dolan, Laurie C.; Matulka, Ray A.; Burdock, George A.

    2010-01-01

    Although many foods contain toxins as a naturally-occurring constituent or, are formed as the result of handling or processing, the incidence of adverse reactions to food is relatively low. The low incidence of adverse effects is the result of some pragmatic solutions by the US Food and Drug Administration (FDA) and other regulatory agencies through the creative use of specifications, action levels, tolerances, warning labels and prohibitions. Manufacturers have also played a role by setting limits on certain substances and developing mitigation procedures for process-induced toxins. Regardless of measures taken by regulators and food producers to protect consumers from natural food toxins, consumption of small levels of these materials is unavoidable. Although the risk for toxicity due to consumption of food toxins is fairly low, there is always the possibility of toxicity due to contamination, overconsumption, allergy or an unpredictable idiosyncratic response. The purpose of this review is to provide a toxicological and regulatory overview of some of the toxins present in some commonly consumed foods, and where possible, discuss the steps that have been taken to reduce consumer exposure, many of which are possible because of the unique process of food regulation in the United States. PMID:22069686

  20. Communicating up

    ERIC Educational Resources Information Center

    Lum, Lydia

    2013-01-01

    Chief communicators at many U.S. institutions are interested in forging closer ties with governing boards. Proponents say such relationships can increase board trust and confidence in communicators before a crisis occurs, making it easier to manage the institution's reputation and limit negative publicity when one does. At some institutions, such…

  1. 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.

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

    PubMed

    Grimm, Sasha S; Isacoff, Ehud Y

    2016-04-01

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

  3. Allosteric substrate switching in a voltage sensing lipid phosphatase

    PubMed Central

    Grimm, Sasha S.; Isacoff, Ehud Y.

    2016-01-01

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

  4. 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.

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

    PubMed Central

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

    2014-01-01

    PARP-1 is a nuclear protein that has important roles in maintenance of genomic integrity. During genotoxic stress, PARP-1 recruits to sites of DNA damage where PARP-1 domain architecture initiates catalytic activation and subsequent poly(ADP-ribose)-dependent DNA repair. PARP-1 inhibition is a promising new way to selectively target cancers harboring DNA repair deficiencies. However, current inhibitors target other PARPs raising important questions concerning long-term off-target effects. Here we propose a new strategy that targets PARP-1 allosteric regulation as a selective way of inhibiting PARP-1. We found that disruption of PARP-1 domain-domain contacts through mutagenesis held no cellular consequences on recruitment to DNA damage or a model system of transcriptional regulation, but prevented DNA-damage dependent catalytic activation. Further, PARP-1 mutant overexpression in a pancreatic cancer cell line (MIA PaCa-2) increased sensitivity to platinum-based anti-cancer agents. These results not only highlight the potential of a synergistic drug combination of allosteric PARP inhibitors with DNA damaging agents in genomically unstable cancer cells (regardless of homologous recombination status), but also signify important applications of selective PARP-1 inhibition. Lastly, the development of a high-throughput (HT) PARP-1 assay is described as a tool to promote discovery of novel PARP-1 selective inhibitors. PMID:24189460

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

    PubMed

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

    2014-01-01

    PARP-1 is a nuclear protein that has important roles in maintenance of genomic integrity. During genotoxic stress, PARP-1 recruits to sites of DNA damage where PARP-1 domain architecture initiates catalytic activation and subsequent poly(ADP-ribose)-dependent DNA repair. PARP-1 inhibition is a promising new way to selectively target cancers harboring DNA repair deficiencies. However, current inhibitors target other PARPs, raising important questions about long-term off-target effects. Here, we propose a new strategy that targets PARP-1 allosteric regulation as a selective way of inhibiting PARP-1. We found that disruption of PARP-1 domain-domain contacts through mutagenesis held no cellular consequences on recruitment to DNA damage or a model system of transcriptional regulation, but prevented DNA-damage-dependent catalytic activation. Furthermore, PARP-1 mutant overexpression in a pancreatic cancer cell line (MIA PaCa-2) increased sensitivity to platinum-based anticancer agents. These results not only highlight the potential of a synergistic drug combination of allosteric PARP inhibitors with DNA-damaging agents in genomically unstable cancer cells (regardless of homologous recombination status), but also signify important applications of selective PARP-1 inhibition. Finally, the development of a high-throughput PARP-1 assay is described as a tool to promote discovery of novel PARP-1 selective inhibitors.

  7. 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.

  8. 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

  9. Regulation of DNA Strand Displacement Using Allosteric DNA Toehold.

    PubMed

    Yang, Xiaolong; Tang, Yanan; Traynor, Sarah M; Li, Feng

    2016-10-05

    Toehold-mediated DNA strand displacement is the fundamental basis for the construction and operation of diverse DNA devices, including circuits, machines, sensors, and reconfigurable structures. Controllable activation and regulation of toeholds are critical to construct devices with multistep, autonomous, and complex behaviors. A handful of unique toehold activation mechanisms, including toehold-exchange, associative toehold, and remote toehold, have been developed and are often combined to achieve desired strand displacement behaviors and functions. Here we report an allosteric DNA toehold (A-toehold) design that allows the flexible regulation of DNA strand displacement by splitting an input strand into an A-toehold and branch migration domain. Because of its simplicity, the A-toehold mechanism can be a useful addition to the current toolbox of DNA strand displacement techniques. We demonstrated that A-toehold enabled a number of interesting functions that were previously shown using more sophisticated DNA strand displacement systems, including 1) continuously tuning the rate of strand displacement, 2) dynamic control of strand displacement reactions, and 3) selective activation of multiple strand displacement reactions. Moreover, by combining A-toehold and toehold-exchange mechanisms, we have successfully constructed a non-covalent DNA catalysis network that resembles an allosteric enzyme.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. Allosteric Modulation of Alpha7 Nicotinic Receptors: Mechanistic Insight through Metadynamics and Essential Dynamics.

    PubMed

    Grazioso, Giovanni; Sgrignani, Jacopo; Capelli, Romina; Matera, Carlo; Dallanoce, Clelia; De Amici, Marco; Cavalli, Andrea

    2015-12-28

    Increasing attention has recently been devoted to allosteric modulators, as they can provide inherent advantages over classic receptor agonists. In the field of nicotinic receptors (nAChRs), the main advantage is that allosteric modulators can trigger pharmacological responses, limiting receptor desensitization. Most of the known allosteric ligands are "positive allosteric modulators" (PAMs), which increase both sensitivity to receptor agonists and current amplitude. Intriguingly, some allosteric modulators are also able to activate the α7 receptor (α7-nAChR) even in the absence of orthosteric agonists. These compounds have been named "ago-allosteric modulators" and GAT107 has been studied in depth because of its unique mechanism of action. We here investigate by molecular dynamics simulations, metadynamics, and essential dynamics the activation mechanism of α7-nAChR, in the presence of different nicotinic modulators. We determine the free energy profiles associated with the closed-to-open motion of the loop C, and we highlight mechanistic differences observed in the presence of different modulators. In particular, we demonstrate that GAT107 triggers conformational motions and cross-talk similar to those observed when the α7-nACh receptor is in complex with both an agonist and an allosteric modulator.

  15. Allosteric modulation in monomers and oligomers of a G protein-coupled receptor

    PubMed Central

    Shivnaraine, Rabindra V; Kelly, Brendan; Sankar, Krishana S; Redka, Dar'ya S; Han, Yi Rang; Huang, Fei; Elmslie, Gwendolynne; Pinto, Daniel; Li, Yuchong; Rocheleau, Jonathan V; Gradinaru, Claudiu C; Ellis, John; Wells, James W

    2016-01-01

    The M2 muscarinic receptor is the prototypic model of allostery in GPCRs, yet the molecular and the supramolecular determinants of such effects are unknown. Monomers and oligomers of the M2 muscarinic receptor therefore have been compared to identify those allosteric properties that are gained in oligomers. Allosteric interactions were monitored by means of a FRET-based sensor of conformation at the allosteric site and in pharmacological assays involving mutants engineered to preclude intramolecular effects. Electrostatic, steric, and conformational determinants of allostery at the atomic level were examined in molecular dynamics simulations. Allosteric effects in monomers were exclusively negative and derived primarily from intramolecular electrostatic repulsion between the allosteric and orthosteric ligands. Allosteric effects in oligomers could be positive or negative, depending upon the allosteric-orthosteric pair, and they arose from interactions within and between the constituent protomers. The complex behavior of oligomers is characteristic of muscarinic receptors in myocardial preparations. DOI: http://dx.doi.org/10.7554/eLife.11685.001 PMID:27151542

  16. 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.

  17. 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

  18. The Mechanism of Allosteric Inhibition of Protein Tyrosine Phosphatase 1B

    PubMed Central

    Lu, Shaoyong; Huang, Wenkang; Geng, Lv; Shen, Qiancheng; Zhang, Jian

    2014-01-01

    As the prototypical member of the PTP family, protein tyrosine phosphatase 1B (PTP1B) is an attractive target for therapeutic interventions in type 2 diabetes. The extremely conserved catalytic site of PTP1B renders the design of selective PTP1B inhibitors intractable. Although discovered allosteric inhibitors containing a benzofuran sulfonamide scaffold offer fascinating opportunities to overcome selectivity issues, the allosteric inhibitory mechanism of PTP1B has remained elusive. Here, molecular dynamics (MD) simulations, coupled with a dynamic weighted community analysis, were performed to unveil the potential allosteric signal propagation pathway from the allosteric site to the catalytic site in PTP1B. This result revealed that the allosteric inhibitor compound-3 induces a conformational rearrangement in helix α7, disrupting the triangular interaction among helix α7, helix α3, and loop11. Helix α7 then produces a force, pulling helix α3 outward, and promotes Ser190 to interact with Tyr176. As a result, the deviation of Tyr176 abrogates the hydrophobic interactions with Trp179 and leads to the downward movement of the WPD loop, which forms an H-bond between Asp181 and Glu115. The formation of this H-bond constrains the WPD loop to its open conformation and thus inactivates PTP1B. The discovery of this allosteric mechanism provides an overall view of the regulation of PTP1B, which is an important insight for the design of potent allosteric PTP1B inhibitors. PMID:24831294

  19. 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

  20. Allosteric signalling in the outer membrane translocation domain of PapC usher

    PubMed Central

    Farabella, Irene; Pham, Thieng; Henderson, Nadine S; Geibel, Sebastian; Phan, Gilles; Thanassi, David G; Delcour, Anne H; Waksman, Gabriel; Topf, Maya

    2014-01-01

    PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili in uropathogenic E. coli. Their translocation domain is a large β-barrel occluded by a plug domain, which is displaced to allow the translocation of pilus subunits across the membrane. Previous studies suggested that this gating mechanism is controlled by a β-hairpin and an α-helix. To investigate the role of these elements in allosteric signal communication, we developed a method combining evolutionary and molecular dynamics studies of the native translocation domain and mutants lacking the β-hairpin and/or the α-helix. Analysis of a hybrid residue interaction network suggests distinct regions (residue ‘communities’) within the translocation domain (especially around β12–β14) linking these elements, thereby modulating PapC gating. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four of these communities. This study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability. DOI: http://dx.doi.org/10.7554/eLife.03532.001 PMID:25271373

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

    PubMed

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

    2013-05-13

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

  2. Virus Assembly and Maturation: Auto-regulation Through Allosteric Molecular Switches

    PubMed Central

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

    2013-01-01

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

  3. Interrogation of the intersubunit interface of the open Hv1 proton channel with a probe of allosteric coupling

    PubMed Central

    Hong, Liang; Singh, Vikrant; Wulff, Heike; Tombola, Francesco

    2015-01-01

    The Hv1 voltage-gated proton channel is a dimeric complex consisting of two voltage-sensing domains (VSDs), each containing a gated proton permeation pathway. Dimerization is controlled by a cytoplasmic coiled-coil domain. The transitions from the closed to the open state in the two VSDs are known to occur cooperatively; however, the underlying mechanism is poorly understood. Intersubunit interfaces play a critical role in allosteric processes; but, such interfaces have not been determined in the open Hv1 channel. Here we show that 2-guanidinothiazole derivatives block the two Hv1 VSDs in a cooperative way, and use one of the compounds as a probe of allosteric coupling between open subunits. We find that the extracellular ends of the first transmembrane segments of the VSDs form the intersubunit interface that mediates coupling between binding sites, while the coiled-coil domain does not directly participate in the process. We also find strong evidence that the channel’s proton selectivity filter controls blocker binding cooperativity. PMID:26365828

  4. Structural insights into Ca2+-activated long-range allosteric channel gating of RyR1

    PubMed Central

    Wei, Risheng; Wang, Xue; Zhang, Yan; Mukherjee, Saptarshi; Zhang, Lei; Chen, Qiang; Huang, Xinrui; Jing, Shan; Liu, Congcong; Li, Shuang; Wang, Guangyu; Xu, Yaofang; Zhu, Sujie; Williams, Alan J; Sun, Fei; Yin, Chang-Cheng

    2016-01-01

    Ryanodine receptors (RyRs) are a class of giant ion channels with molecular mass over 2.2 mega-Daltons. These channels mediate calcium signaling in a variety of cells. Since more than 80% of the RyR protein is folded into the cytoplasmic assembly and the remaining residues form the transmembrane domain, it has been hypothesized that the activation and regulation of RyR channels occur through an as yet uncharacterized long-range allosteric mechanism. Here we report the characterization of a Ca2+-activated open-state RyR1 structure by cryo-electron microscopy. The structure has an overall resolution of 4.9 Å and a resolution of 4.2 Å for the core region. In comparison with the previously determined apo/closed-state structure, we observed long-range allosteric gating of the channel upon Ca2+ activation. In-depth structural analyses elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism of RyR1. Our work not only provides structural insights into the molecular mechanisms of channel gating and regulation of RyRs, but also sheds light on structural basis for channel-gating and ion selectivity mechanisms for the six-transmembrane-helix cation channel family. PMID:27573175

  5. Allosteric MEK1/2 inhibitors including cobimetanib and trametinib in the treatment of cutaneous melanomas.

    PubMed

    Roskoski, Robert

    2017-03-01

    The Ras-Raf-MEK-ERK (Map kinase) cellular pathway is a highly conserved eukaryotic signaling module that transduces extracellular signals from growth factors and cytokines into intracellular regulatory events that are involved in cell growth and proliferation or the contrary pathway of cell differentiation. Dysregulation of this pathway occurs in more than one-third of all malignancies, a process that has fostered the development of targeted Map kinase pathway inhibitors. Cutaneous melanomas, which arise from skin melanocytes, are the most aggressive form of skin cancer. Mutations that activate the Map kinase pathway occur in more than 90% of these melanomas. This has led to the development of the combination of dabrafenib and trametinib or vemurafenib and cobimetanib for the treatment of BRAF V600E mutant melanomas. Dabrafenib and vemurafenib target V600E/K BRAF mutants while trametinib and cobimetanib target MEK1/2. The latter two agents bind to MEK1/2 at a site that is adjacent to, but separate from, the ATP-binding site and are therefore classified as type III allosteric protein kinase inhibitors. These agents form a hydrogen bond with a conserved β3-lysine and they make numerous hydrophobic contacts with residues within the αC-helix, the β5 strand, and within the activation segment, regions of the protein kinase domain that exhibit greater diversity than those found within the ATP-binding site. One advantage of such allosteric inhibitors is that they do not have to compete with millimolar concentrations of cellular ATP, which most FDA-approved small molecule competitive inhibitors such as imatinib must do. Owing to the wide spread activation of this pathway in numerous neoplasms, trametinib and cobimetinib are being studied in combination with other targeted and cytotoxic drugs in a variety of clinical situations. Except for BRAF and NRAS mutations, there are no other biomarkers correlated with treatment responses following MEK1/2 inhibition and the

  6. 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.

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

    PubMed Central

    2017-01-01

    The N-methyl-D-aspartate receptors (NMDARs) are subtype glutamate receptors that play important roles in excitatory neurotransmission and synaptic plasticity. Their hypo- or hyperactivation are proposed to contribute to the genesis or progression of various brain diseases, including stroke, schizophrenia, depression, and Alzheimer's disease. Past efforts in targeting NMDARs for therapeutic intervention have largely been on inhibitors of NMDARs. In light of the discovery of NMDAR hypofunction in psychiatric disorders and perhaps Alzheimer's disease, efforts in boosting NMDAR activity/functions have surged in recent years. In this review, we will focus on enhancing NMDAR functions, especially on the recent progress in the generation of subunit-selective, allosteric positive modulators (PAMs) of NMDARs. We shall also discuss the usefulness of these newly developed NMDAR-PAMs. PMID:28163934

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

    PubMed

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

    2016-02-01

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

  9. 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.

  10. 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)

  11. Structure-Based Network Analysis of Activation Mechanisms in the ErbB Family of Receptor Tyrosine Kinases: The Regulatory Spine Residues Are Global Mediators of Structural Stability and Allosteric Interactions

    PubMed Central

    James, Kevin A.; Verkhivker, Gennady M.

    2014-01-01

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

  12. 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).

  13. 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.

  14. Allosteric Optical Control of a Class B G‐Protein‐Coupled Receptor

    PubMed Central

    Broichhagen, Johannes; Johnston, Natalie R.; von Ohlen, Yorrick; Meyer‐Berg, Helena; Jones, Ben J.; Bloom, Stephen R.; Rutter, Guy A.

    2016-01-01

    Abstract Allosteric regulation promises to open up new therapeutic avenues by increasing drug specificity at G‐protein‐coupled receptors (GPCRs). However, drug discovery efforts are at present hampered by an inability to precisely control the allosteric site. Herein, we describe the design, synthesis, and testing of PhotoETP, a light‐activated positive allosteric modulator of the glucagon‐like peptide‐1 receptor (GLP‐1R), a class B GPCR involved in the maintenance of glucose homeostasis in humans. PhotoETP potentiates Ca2+, cAMP, and insulin responses to glucagon‐like peptide‐1 and its metabolites following illumination of cells with blue light. PhotoETP thus provides a blueprint for the production of small‐molecule class B GPCR allosteric photoswitches, and may represent a useful tool for understanding positive cooperativity at the GLP‐1R. PMID:27059784

  15. Discovery and Characterization of Biased Allosteric Agonists of the Chemokine Receptor CXCR3.

    PubMed

    Milanos, Lampros; Brox, Regine; Frank, Theresa; Poklukar, Gašper; Palmisano, Ralf; Waibel, Reiner; Einsiedel, Jürgen; Dürr, Maximilian; Ivanović-Burmazović, Ivana; Larsen, Olav; Hjortø, Gertrud Malene; Rosenkilde, Mette Marie; Tschammer, Nuska

    2016-03-10

    In this work we report a design, synthesis, and detailed functional characterization of unique strongly biased allosteric agonists of CXCR3 that contain tetrahydroisoquinoline carboxamide cores. Compound 11 (FAUC1036) is the first strongly biased allosteric agonist of CXCR3 that selectively induces weak chemotaxis and leads to receptor internalization and the β-arrestin 2 recruitment with potency comparable to that of the chemokine CXCL11 without any activation of G proteins. A subtle structural change (addition of a methoxy group, 14 (FAUC1104)) led to a contrasting biased allosteric partial agonist that activated solely G proteins, induced chemotaxis, but failed to induce receptor internalization or β-arrestin 2 recruitment. Concomitant structure-activity relationship studies indicated very steep structure-activity relationships, which steer the ligand bias between the β-arrestin 2 and G protein pathway. Overall, the information presented provides a powerful platform for further development and rational design of strongly biased allosteric agonists of CXCR3.

  16. 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.

  17. 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.

  18. Ultrahigh Enzyme Activity Assembled in Layered Double Hydroxides via Mg(2+)-Allosteric Effector.

    PubMed

    Wang, Min; Huang, Shu-Wan; Xu, Dan; Bao, Wen-Jing; Xia, Xing-Hua

    2015-06-02

    It is well-known that some metal ions could be allosteric effectors of allosteric enzymes to activate/inhibit the catalytic activities of enzymes. In nanobiocatalytic systems constructed based on the positive metal ion-induced allosteric effect, the incorporated enzymes will be activated and thus exhibit excellent catalytic performance. Herein, we present an environmentally friendly strategy to construct a novel allosteric effect-based β-galactosidase/Mg-Al layered double hydroxide (β-gal/Mg-Al-LDH) nanobiocatalytic system via the delamination-reconstruction method. The intercalated β-gal in the LDH galleries changes its conformation significantly due to the Mg(2+)-induced allosteric interactions and other weak interactions, which causes the activation of enzymatic activity. The β-gal/Mg-Al-LDH nanobiocatalytic system shows much higher catalytic activity and affinity toward its substrate and about 30 times higher catalytic reaction velocity than the free β-gal, which suggests that Mg(2+)-induced allosteric effect plays a vital role in the improvement of enzymatic performance.

  19. Dissecting Enzyme Regulation by Multiple Allosteric Effectors: Nucleotide Regulation of Aspartate Transcarbamoylase†

    PubMed Central

    Rabinowitz, Joshua D.; Hsiao, Jennifer J.; Gryncel, Kimberly R.; Kantrowitz, Evan R.; Feng, Xiao-Jiang; Li, Genyuan; Rabitz, Herschel

    2009-01-01

    The enzyme aspartate transcarbamoylase (ATCase, EC 2.1.3.2 of Escherichia coli), which catalyzes the committed step of pyrimidine biosynthesis, is allosterically regulated by all four ribonucleoside triphosphates (NTPs) in a nonlinear manner. Here, we dissect this regulation using the recently developed approach of random sampling–high-dimensional model representation (RS–HDMR). ATCase activity was measured in vitro at 300 random NTP concentration combinations, each involving (consistent with in vivo conditions) all four NTPs being present. These data were then used to derive a RS–HDMR model of ATCase activity over the full four-dimensional NTP space. The model accounted for 90% of the variance in the experimental data. Its main elements were positive ATCase regulation by ATP and negative by CTP, with the negative effects of CTP dominating the positive ones of ATP when both regulators were abundant (i.e., a negative cooperative effect of ATP × CTP). Strong sensitivity to both ATP and CTP concentrations occurred in their physiological concentration ranges. UTP had only a slight effect, and GTP had almost none. These findings support a predominant role of CTP and ATP in ATCase regulation. The general approach provides a new paradigm for dissecting multifactorial regulation of biological molecules and processes. PMID:18454556

  20. FRET-Based Sensors Unravel Activation and Allosteric Modulation of the GABAB Receptor.

    PubMed

    Lecat-Guillet, Nathalie; Monnier, Carine; Rovira, Xavier; Kniazeff, Julie; Lamarque, Laurent; Zwier, Jurriaan M; Trinquet, Eric; Pin, Jean-Philippe; Rondard, Philippe

    2017-03-06

    The main inhibitory neurotransmitter, γ-aminobutyric acid (GABA), modulates many synapses by activating the G protein-coupled receptor GABAB, which is a target for various therapeutic applications. It is an obligatory heterodimer made of GB1 and GB2 that can be regulated by positive allosteric modulators (PAMs). The molecular mechanism of activation of the GABAB receptor remains poorly understood. Here, we have developed FRET-based conformational GABAB sensors compatible with high-throughput screening. We identified conformational changes occurring within the extracellular and transmembrane domains upon receptor activation, which are smaller than those observed in the related metabotropic glutamate receptors. These sensors also allow discrimination between agonists of different efficacies and between PAMs that have different modes of action, which has not always been possible using conventional functional assays. Our study brings important new information on the activation mechanism of the GABAB receptor and should facilitate the screening and identification of new chemicals targeting this receptor.

  1. Allosteric inhibition of aminopeptidase N functions related to tumor growth and virus infection

    PubMed Central

    Santiago, César; Mudgal, Gaurav; Reguera, Juan; Recacha, Rosario; Albrecht, Sébastien; Enjuanes, Luis; Casasnovas, José M.

    2017-01-01

    Cell surface aminopeptidase N (APN) is a membrane-bound ectoenzyme that hydrolyzes proteins and peptides and regulates numerous cell functions. APN participates in tumor cell expansion and motility, and is a target for cancer therapies. Small drugs that bind to the APN active site inhibit catalysis and suppress tumor growth. APN is also a major cell entry receptor for coronavirus, which binds to a region distant from the active site. Three crystal structures that we determined of human and pig APN ectodomains defined the dynamic conformation of the protein. These structures offered snapshots of closed, intermediate and open APN, which represent distinct functional states. Coronavirus envelope proteins specifically recognized the open APN form, prevented ectodomain progression to the closed form and substrate hydrolysis. In addition, drugs that bind the active site inhibited both coronavirus binding to cell surface APN and infection; the drugs probably hindered APN transition to the virus-specific open form. We conclude that allosteric inhibition of APN functions occurs by ligand suppression of ectodomain motions necessary for catalysis and virus cell entry, as validated by locking APN with disulfides. Blocking APN dynamics can thus be a valuable approach to development of drugs that target this ectoenzyme. PMID:28393915

  2. Allosteric action in real time: Time-resolved crystallographic studies of a cooperative dimeric hemoglobin

    PubMed Central

    Knapp, James E.; Pahl, Reinhard; Šrajer, Vukica; Royer, William E.

    2006-01-01

    Protein allostery provides mechanisms for regulation of biological function at the molecular level. We present here an investigation of global, ligand-induced allosteric transition in a protein by time-resolved x-ray diffraction. The study provides a view of structural changes in single crystals of Scapharca dimeric hemoglobin as they proceed in real time, from 5 ns to 80 μs after ligand photodissociation. A tertiary intermediate structure forms rapidly (<5 ns) as the protein responds to the presence of an unliganded heme within each R-state protein subunit, with key structural changes observed in the heme groups, neighboring residues, and interface water molecules. This intermediate lays a foundation for the concerted tertiary and quaternary structural changes that occur on a microsecond time scale and are associated with the transition to a low-affinity T-state structure. Reversal of these changes shows a considerable lag as a T-like structure persists well after ligand rebinding, suggesting a slow T-to-R transition. PMID:16684887

  3. Allosteric inhibition of aminopeptidase N functions related to tumor growth and virus infection.

    PubMed

    Santiago, César; Mudgal, Gaurav; Reguera, Juan; Recacha, Rosario; Albrecht, Sébastien; Enjuanes, Luis; Casasnovas, José M

    2017-04-10

    Cell surface aminopeptidase N (APN) is a membrane-bound ectoenzyme that hydrolyzes proteins and peptides and regulates numerous cell functions. APN participates in tumor cell expansion and motility, and is a target for cancer therapies. Small drugs that bind to the APN active site inhibit catalysis and suppress tumor growth. APN is also a major cell entry receptor for coronavirus, which binds to a region distant from the active site. Three crystal structures that we determined of human and pig APN ectodomains defined the dynamic conformation of the protein. These structures offered snapshots of closed, intermediate and open APN, which represent distinct functional states. Coronavirus envelope proteins specifically recognized the open APN form, prevented ectodomain progression to the closed form and substrate hydrolysis. In addition, drugs that bind the active site inhibited both coronavirus binding to cell surface APN and infection; the drugs probably hindered APN transition to the virus-specific open form. We conclude that allosteric inhibition of APN functions occurs by ligand suppression of ectodomain motions necessary for catalysis and virus cell entry, as validated by locking APN with disulfides. Blocking APN dynamics can thus be a valuable approach to development of drugs that target this ectoenzyme.

  4. Bupivacaine Blocks N-Type Inactivating Kv Channels in the Open State: No Allosteric Effect on Inactivation Kinetics

    PubMed Central

    Nilsson, Johanna; Madeja, Michael; Elinder, Fredrik; Århem, Peter

    2008-01-01

    Local anesthetics bind to ion channels in a state-dependent manner. For noninactivating voltage-gated K channels the binding mainly occurs in the open state, while for voltage-gated inactivating Na channels it is assumed to occur mainly in inactivated states, leading to an allosterically caused increase in the inactivation probability, reflected in a negative shift of the steady-state inactivation curve, prolonged recovery from inactivation, and a frequency-dependent block. How local anesthetics bind to N-type inactivating K channels is less explored. In this study, we have compared bupivacaine effects on inactivating (Shaker and Kv3.4) and noninactivating (Shaker-IR and Kv3.2) channels, expressed in Xenopus oocytes. Bupivacaine was found to block these channels time-dependently without shifting the steady-state inactivation curve markedly, without a prolonged recovery from inactivation, and without a frequency-dependent block. An analysis, including computational testing of kinetic models, suggests binding to the channel mainly in the open state, with affinities close to those estimated for corresponding noninactivating channels (300 and 280 μM for Shaker and Shaker-IR, and 60 and 90 μM for Kv3.4 and Kv3.2). The similar magnitudes of Kd, as well as of blocking and unblocking rate constants for inactivating and noninactivating Shaker channels, most likely exclude allosteric interactions between the inactivation mechanism and the binding site. The relevance of these results for understanding the action of local anesthetics on Na channels is discussed. PMID:18790854

  5. 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.

  6. Allosteric reversion of Haemophilus influenzae β-carbonic anhydrase via a proline shift.

    PubMed

    Hoffmann, Katherine M; Million-Perez, H Rachael; Merkhofer, Richard; Nicholson, Hilary; Rowlett, Roger S

    2015-01-20

    Haemophilus influenzae β-carbonic anhydrase (HICA) has been reverse-engineered in the allosteric site region to resemble the nonallosteric Pisum sativum enzyme in order to identify critical features of allostery and intersusbunit communication. Three variants (W39V/G41A, P48S/A49P, and W39V/G41A/P48S/A49P) were identified, through a comparison with a crystal structure of nonallosteric P. sativum β-carbonic anhydrase (PSCA, PDB 1EKJ ), to potentially revert HICA to a nonallosteric enzyme. The W39V/G41A and P48S/A49P mutations decreased the apparent kcat/Km proton dependence from 4 to 2 and 1, respectively, increasing the overall maximal kcat/Km to 16 ± 2 μM(-1) s(-1) (380% of wild type) and 17 ± 3 μM(-1) s(-1) (405% of wild type). The pKa values of the metal-bound water molecule based on the pH-rate profile kinetics (8.32 ± 0.04 for W39V/G41A and 8.3 ± 0.1 for P48S/A49P) were also slightly higher than that for the wild-type enzyme (7.74 ± 0.04). The P48S/A49P variant has lost all pH-rate cooperativity. The W39V/G41A/P48S/A49P variant's kinetics were unusual and were fit with a log-linear function with a slope 0.9 ± 0.2. The crystal structure of the W39V/G41A variant revealed an active site very similar to the T-state wild-type oligomer with bicarbonate trapped in the escort site. By contrast, the X-ray crystal structure of a proline shift variant (P48S/A49P) reveals that it has adopted an active site conformation nearly identical to that of nonallosteric β-carbonic anhydrase (R-state) for one chain, including a tight association with the dimer-exchanged N-terminal helices; the second chain in the asymmetric unit is associated in a biologically relevant oligomer, but it adopts a T-state conformation that is not capped by dimer-exchanged N-terminal helices. The hybrid R/T nature of HICA P48S/A49P structurally recapitulates the interruption of pH-rate cooperativity observed for this variant. Comparison of the conformations of the R and T chains of P48S/A49P

  7. 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.

  8. ASSESSMENT OF SUBUNIT-DEPENDENT DIRECT GATING AND ALLOSTERIC MODULATORY EFFECTS OF CARISOPRODOL AT GABAA RECEPTORS

    PubMed Central

    Kumar, Manoj; González, Lorie A.; Dillon, Glenn H.

    2016-01-01

    Carisoprodol is a widely prescribed muscle relaxant, abuse of which has grown considerably in recent years. It directly activates and allosterically modulates α1β2γ2 GABAARs, although the site(s) of action are unknown. To gain insight into the actions of carisoprodol, subunit-dependent effects of this drug were assessed. Whole-cell patch clamp recordings were obtained from HEK293 cells expressing α1β2, α1β3 or αxβzγ2 (where x = 1–6 and z = 1–3) GABAARs, and in receptors incorporating the δ subunit (modeling extrasynaptic receptors). The ability to directly gate and allosterically potentiate GABA-gated currents was observed for all configurations. Presence or absence of the γ2 subunit did not affect the ability of carisoprodol to directly gate or allosterically modulate the receptor. Presence of the β1 subunit conferred highest efficacy for direct activation relative to maximum GABA currents, while presence of the β2 subunit conferred highest efficacy for allosteric modulation of the GABA response. With regard to α subunits, carisoprodol was most efficacious at enhancing the actions of GABA in receptors incorporating the α1 subunit. The ability to directly gate the receptor was generally comparable regardless of the α subunit isoform, although receptors incorporating the α3 subunit showed significantly reduced direct gating efficacy and affinity. In extrasynaptic (α1β3δ and α4β3δ) receptors, carisoprodol had greater efficacy than GABA as a direct gating agonist. In addition, carisoprodol allosterically potentiated both EC20 and saturating GABA concentrations in these receptors. In assessing voltage-dependence, we found direct gating and inhibitory effects were insensitive to membrane voltage, whereas allosteric modulatory effects were affected by membrane voltage. Our findings demonstrate direct and allosteric effects of carisoprodol at synaptic and extrasynpatic GABAARs and that subunit isoform influences these effects. PMID:25896767

  9. Chemical shift imprint of intersubunit communication in a symmetric homodimer

    PubMed Central

    Falk, Bradley T.; Sapienza, Paul J.; Lee, Andrew L.

    2016-01-01

    Allosteric communication is critical for protein function and cellular homeostasis, and it can be exploited as a strategy for drug design. However, unlike many protein–ligand interactions, the structural basis for the long-range communication that underlies allostery is not well understood. This lack of understanding is most evident in the case of classical allostery, in which a binding event in one protomer is sensed by a second symmetric protomer. A primary reason why study of interdomain signaling is challenging in oligomeric proteins is the difficulty in characterizing intermediate, singly bound species. Here, we use an NMR approach to isolate and characterize a singly ligated state (“lig1”) of a homodimeric enzyme that is otherwise obscured by rapid exchange with apo and saturated forms. Mixed labeled dimers were prepared that simultaneously permit full population of the lig1 state and isotopic labeling of either protomer. Direct visualization of peaks from lig1 yielded site-specific ligand-state multiplets that provide a convenient format for assessing mechanisms of intersubunit communication from a variety of NMR measurements. We demonstrate this approach on thymidylate synthase from Escherichia coli, a homodimeric enzyme known to be half-the-sites reactive. Resolving the dUMP1 state shows that active site communication occurs not upon the first dUMP binding, but upon the second. Surprisingly, for many sites, dUMP1 peaks are found beyond the limits set by apo and dUMP2 peaks, indicating that binding the first dUMP pushes the enzyme ensemble to further conformational extremes than the apo or saturated forms. The approach used here should be generally applicable to homodimers. PMID:27466406

  10. Lipid-Mediated Regulation of Embedded Receptor Kinases via Parallel Allosteric Relays.

    PubMed

    Ghosh, Madhubrata; Wang, Loo Chien; Ramesh, Ranita; Morgan, Leslie K; Kenney, Linda J; Anand, Ganesh S

    2017-02-28

    Membrane-anchored receptors are essential cellular signaling elements for stimulus sensing, propagation, and transmission inside cells. However, the contributions of lipid interactions to the function and dynamics of embedded receptor kinases have not been described in detail. In this study, we used amide hydrogen/deuterium exchange mass spectrometry, a sensitive biophysical approach, to probe the dynamics of a membrane-embedded receptor kinase, EnvZ, together with functional assays to describe the role of lipids in receptor kinase function. Our results reveal that lipids play an important role in regulating receptor function through interactions with transmembrane segments, as well as through peripheral interactions with nonembedded domains. Specifically, the lipid membrane allosterically modulates the activity of the embedded kinase by altering the dynamics of a glycine-rich motif that is critical for phosphotransfer from ATP. This allostery in EnvZ is independent of membrane composition and involves direct interactions with transmembrane and periplasmic segments, as well as peripheral interactions with nonembedded domains of the protein. In the absence of the membrane-spanning regions, lipid allostery is propagated entirely through peripheral interactions. Whereas lipid allostery impacts the phosphotransferase function of the kinase, extracellular stimulus recognition is mediated via a four-helix bundle subdomain located in the cytoplasm, which functions as the osmosensing core through osmolality-dependent helical stabilization. Our findings emphasize the functional modularity in a membrane-embedded kinase, separated into membrane association, phosphotransferase function, and stimulus recognition. These components are integrated through long-range communication relays, with lipids playing an essential role in regulation.

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

    PubMed

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

    2015-09-10

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

  12. Allosteric Small-Molecule Inhibitors of the AKT Kinase

    NASA Astrophysics Data System (ADS)

    Dalafave, D. S.

    This research addresses computational design of small druglike molecules for possible anticancer applications. AKT and SGK are kinases that control important cellular functions. They are highly homologous, having similar activators and targets. Cancers with increased SGK activity may develop resistance to AKT-specific inhibitors. Our goal was to design new molecules that would bind both AKT and SGK, thus preventing the development of drug resistance. Most kinase inhibitors target the kinase ATP-binding site. However, the high similarity in this site among kinases makes it difficult to target specifically. Furthermore, mutations in this site can cause resistance to ATP-competitive kinase inhibitors. We used existing AKT inhibitors as initial templates to design molecules that could potentially bind the allosteric sites of both AKT and SGK. Molecules with no implicit toxicities and optimal drug-like properties were used for docking studies. Binding energies of the stable complexes that the designed molecules formed with AKT and SGK were calculated. Possible applications of the designed putative inhibitors against cancers with overexpressed AKT/SGK is discussed.

  13. Allosteric activation of apicomplexan calcium-dependent protein kinases

    PubMed Central

    Ingram, Jessica R.; Knockenhauer, Kevin E.; Markus, Benedikt M.; Mandelbaum, Joseph; Ramek, Alexander; Shan, Yibing; Shaw, David E.; Schwartz, Thomas U.; Ploegh, Hidde L.; Lourido, Sebastian

    2015-01-01

    Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics, the effects of 1B7–kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes. PMID:26305940

  14. Ignavine: a novel allosteric modulator of the μ opioid receptor

    PubMed Central

    Ohbuchi, Katsuya; Miyagi, Chika; Suzuki, Yasuyuki; Mizuhara, Yasuharu; Mizuno, Keita; Omiya, Yuji; Yamamoto, Masahiro; Warabi, Eiji; Sudo, Yuka; Yokoyama, Akinobu; Miyano, Kanako; Hirokawa, Takatsugu; Uezono, Yasuhito

    2016-01-01

    Processed Aconiti tuber (PAT) is used to treat pain associated with various disorders. Although it has been demonstrated that the κ opioid receptor (KOR) signaling pathway is a mediator of the analgesic effect of PAT, active components affecting opioid signaling have not yet been identified. In this study, we explored candidate components of PAT by pharmacokinetic analysis and identified ignavine, which is a different structure from aconitine alkaloids. A receptor binding assay of opioid receptors showed that ignavine specifically binds the μ opioid receptor (MOR), not the KOR. Receptor internalization assay in MOR-expressing cell lines revealed that ignavine augmented the responses produced by D-Ala(2)-N-Me-Phe(4)-Gly-ol(5)-enkephalin (DAMGO), a representative MOR agonist, at a low concentration and inhibited it at a higher concentration. Ignavine also exerted positive modulatory activity for DAMGO, endomorphin-1 and morphine in cAMP assay. Additionally, ignavine alone showed an analgesic effect in vivo. In silico simulation analysis suggested that ignavine would induce a unique structural change distinguished from those induced by a representative MOR agonist and antagonist. These data collectively suggest the possibility that ignavine could be a novel allosteric modulator of the MOR. The present results may open the way for the development of a novel pain management strategy. PMID:27530869

  15. Allosteric Inhibition of Anti-Apoptotic MCL-1

    PubMed Central

    Lee, Susan; Wales, Thomas E.; Escudero, Silvia; Cohen, Daniel T.; Luccarelli, James; Gallagher, Catherine; Cohen, Nicole A.; Huhn, Annissa J.; Bird, Gregory H.; Engen, John R.; Walensky, Loren D.

    2016-01-01

    MCL-1 is an anti-apoptotic BCL-2 family protein that has emerged as a major pathogenic factor in human cancer. Like BCL-2, MCL-1 bears a surface groove whose function is to sequester the BH3 killer domains of pro-apoptotic BCL-2 family members, a mechanism harnessed by cancer cells to establish formidable apoptotic blockades. Whereas drugging the BH3-binding groove has been achieved for BCL-2, translating this approach to MCL-1 has been challenging. Here, we report an alternative mechanism for MCL-1 inhibition by small molecule covalent modification of C286 at a novel interaction site distant from the BH3-binding groove. Our structure-function analyses revealed that the BH3-binding capacity of MCL-1 and its suppression of BAX are impaired by molecular engagement, a phenomenon recapitulated by C286W mutagenic mimicry in vitro and in cells. Thus, we characterize an allosteric mechanism for disrupting the anti-apoptotic, BH3-binding activity of MCL-1, informing a new strategy for disarming MCL-1 in cancer. PMID:27159560

  16. 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.

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

    PubMed Central

    Uteshev, Victor V.

    2014-01-01

    In the central nervous system, deficits in cholinergic neurotransmission correlate with decreased attention and cognitive impairment, while stimulation of neuronal nicotinic acetylcholine receptors improves attention, cognitive performance and neuronal resistance to injury as well as produces robust analgesic and anti-inflammatory effects. The rational basis for the therapeutic use of orthosteric agonists and positive allosteric modulators (PAMs) of nicotinic receptors arises from the finding that functional nicotinic receptors are ubiquitously expressed in neuronal and non-neuronal tissues including brain regions highly vulnerable to traumatic and ischemic types of injury (e.g., cortex and hippocampus). Moreover, functional nicotinic receptors do not vanish in age-, disease- and trauma-related neuropathologies, but their expression and/or activation levels decline in a subunit- and brain region-specific manner. Therefore, augmenting the endogenous cholinergic tone by nicotinic agents is possible and may offset neurological impairments associated with cholinergic hypofunction. Importantly, because neuronal damage elevates extracellular levels of choline (a selective agonist of α7 nicotinic acetylcholine receptors) near the site of injury, α7-PAM-based treatments may augment pathology-activated α7-dependent auto-therapies where and when they are most needed (i.e., in the penumbra, post-injury). Thus, the nicotinic-PAM-based treatments are expected to be highly efficacious with fewer side effects as compared to a more indiscriminate action of exogenous orthosteric agonists. In this review, I will summarize the existing trends in therapeutic applications of nicotinic PAMs. PMID:24530419

  18. Allosteric activation of apicomplexan calcium-dependent protein kinases

    SciTech Connect

    Ingram, Jessica R.; Knockenhauer, Kevin E.; Markus, Benedikt M.; Mandelbaum, Joseph; Ramek, Alexander; Shan, Yibing; Shaw, David E.; Schwartz, Thomas U.; Ploegh, Hidde L.; Lourido, Sebastian

    2015-08-24

    Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics, the effects of 1B7–kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.

  19. Allosteric activation of apicomplexan calcium-dependent protein kinases

    DOE PAGES

    Ingram, Jessica R.; Knockenhauer, Kevin E.; Markus, Benedikt M.; ...

    2015-08-24

    Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics,more » the effects of 1B7–kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.« less

  20. Functional impact of allosteric agonist activity of selective positive allosteric modulators of metabotropic glutamate receptor subtype 5 in regulating central nervous system function.

    PubMed

    Noetzel, Meredith J; Rook, Jerri M; Vinson, Paige N; Cho, Hyekyung P; Days, Emily; Zhou, Y; Rodriguez, Alice L; Lavreysen, Hilde; Stauffer, Shaun R; Niswender, Colleen M; Xiang, Zixiu; Daniels, J Scott; Jones, Carrie K; Lindsley, Craig W; Weaver, C David; Conn, P Jeffrey

    2012-02-01

    Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu(5)) have emerged as an exciting new approach for the treatment of schizophrenia and other central nervous system (CNS) disorders. Of interest, some mGlu(5) PAMs act as pure PAMs, only potentiating mGlu(5) responses to glutamate whereas others [allosteric agonists coupled with PAM activity (ago-PAMs)] potentiate responses to glutamate and have intrinsic allosteric agonist activity in mGlu(5)-expressing cell lines. All mGlu(5) PAMs previously shown to have efficacy in animal models act as ago-PAMs in cell lines, raising the possibility that allosteric agonist activity is critical for in vivo efficacy. We have now optimized novel mGlu(5) pure PAMs that are devoid of detectable agonist activity and structurally related mGlu(5) ago-PAMs that activate mGlu(5) alone in cell lines. Studies of mGlu(5) PAMs in cell lines revealed that ago-PAM activity is dependent on levels of mGlu(5) receptor expression in human embryonic kidney 293 cells, whereas PAM potency is relatively unaffected by levels of receptor expression. Furthermore, ago-PAMs have no agonist activity in the native systems tested, including cortical astrocytes and subthalamic nucleus neurons and in measures of long-term depression at the hippocampal Schaffer collateral-CA1 synapse. Finally, studies with pure PAMs and ago-PAMs chemically optimized to provide comparable CNS exposure revealed that both classes of mGlu(5) PAMs have similar efficacy in a rodent model predictive of antipsychotic activity. These data suggest that the level of receptor expression influences the ability of mGlu(5) PAMs to act as allosteric agonists in vitro and that ago-PAM activity observed in cell-based assays may not be important for in vivo efficacy.

  1. Inversion of allosteric effect of arginine on N-acetylglutamate synthase, a molecular marker for evolution of tetrapods

    PubMed Central

    Haskins, Nantaporn; Panglao, Maria; Qu, Qiuhao; Majumdar, Himani; Cabrera-Luque, Juan; Morizono, Hiroki; Tuchman, Mendel; Caldovic, Ljubica

    2008-01-01

    Background The efficient conversion of ammonia, a potent neurotoxin, into non-toxic metabolites was an essential adaptation that allowed animals to move from the aquatic to terrestrial biosphere. The urea cycle converts ammonia into urea in mammals, amphibians, turtles, snails, worms and many aquatic animals and requires N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI) in mammals and amphibians, and carbamylphosphate synthetase III (CPSIII) in fish and invertebrates. NAG-dependent CPSI and CPSIII catalyze the formation of carbamylphosphate in the first and rate limiting step of ureagenesis. NAG is produced enzymatically by N-acetylglutamate synthase (NAGS), which is also found in bacteria and plants as the first enzyme of arginine biosynthesis. Arginine is an allosteric inhibitor of microbial and plant NAGS, and allosteric activator of mammalian NAGS. Results Information from mutagenesis studies of E. coli and P. aeruginosa NAGS was combined with structural information from the related bacterial N-acetylglutamate kinases to identify four residues in mammalian NAGS that interact with arginine. Substitutions of these four residues were engineered in mouse NAGS and into the vertebrate-like N-acetylglutamate synthase-kinase (NAGS-K) of Xanthomonas campestris, which is inhibited by arginine. All mutations resulted in arginine losing the ability to activate mouse NAGS, and inhibit X. campestris NAGS-K. To examine at what point in evolution inversion of arginine effect on NAGS occur, we cloned NAGS from fish and frogs and examined the arginine response of their corresponding proteins. Fish NAGS were partially inhibited by arginine and frog NAGS were activated by arginine. Conclusion Difference in arginine effect on bacterial and mammalian NAGS most likely stems from the difference in the type of conformational change triggered by arginine binding to these proteins. The change from arginine inhibition of NAGS to activation

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

    PubMed

    Mansari, Mostafa El; Wiborg, Ove; Mnie-Filali, Ouissame; Benturquia, Nadia; Sánchez, Connie; Haddjeri, Nasser

    2007-02-01

    Clinical and preclinical studies have shown that the effect of citalopram on serotonin (5-HT) reuptake inhibition and its antidepressant activity resides in the S-enantiomer. In addition, using a variety of in-vivo and in-vitro paradigms, it was shown that R-citalopram counteracts the effect of escitalopram. This effect was suggested to occur via an allosteric modulation at the level of the 5-HT transporter. Using in-vitro binding assays at membranes from COS-1 cells expressing the human 5-HT transporter (hSERT) and in-vivo electrophysiological and microdialysis techniques in rats, the present study was directed at determining whether R-citalopram modifies the action of selective serotonin reuptake inhibitors (SSRIs) known to act on allosteric sites namely escitalopram, and to a lesser extent paroxetine, compared to fluoxetine, which has no affinity for these sites. In-vitro binding studies showed that R-citalopram attenuated the association rates of escitalopram and paroxetine to the 5-HT transporter, but had no effect on the association rates of fluoxetine, venlafaxine or sertraline. In the rat dorsal raphe nucleus, R-citalopram (250 microg/kg i.v.) blocked the suppressant effect on neuronal firing activity of both escitalopram (100 microg/kg i.v.) and paroxetine (500 microg/kg i.v.), but not fluoxetine (10 mg/kg i.v.). Interestingly, administration of R-citalopram (8 mg/kg i.p.) attenuated the increase of extracellular levels of 5-HT ([5-HT]ext) in the ventral hippocampus induced by both escitalopram (0.28 microM) and paroxetine (0.75 microM), but not fluoxetine (10 microM). In conclusion, the present in-vitro and in-vivo studies show that R-citalopram counteracts the activity of escitalopram and paroxetine, but not fluoxetine, by acting at the allosteric binding site of the 5-HT transporter, either located in the dorsal raphe nucleus or post-synaptically in the ventral hippocampus. This conclusion is strengthened by the observation that the inhibitory effect of

  3. 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.

  4. Conformational flexibility and the mechanisms of allosteric transitions in topologically similar proteins

    NASA Astrophysics Data System (ADS)

    Tripathi, Swarnendu; Portman, John J.

    2011-08-01

    Conformational flexibility plays a central role in allosteric transition of proteins. In this paper, we extend the analysis of our previous study [S. Tripathi and J. J. Portman, Proc. Natl. Acad. Sci. U.S.A. 106, 2104 (2009)] to investigate how relatively minor structural changes of the meta-stable states can significantly influence the conformational flexibility and allosteric transition mechanism. We use the allosteric transitions of the domains of calmodulin as an example system to highlight the relationship between the transition mechanism and the inter-residue contacts present in the meta-stable states. In particular, we focus on the origin of transient local unfolding (cracking), a mechanism that can lower free energy barriers of allosteric transitions, in terms of the inter-residue contacts of the meta-stable states and the pattern of local strain that develops during the transition. We find that the magnitude of the local strain in the protein is not the sole factor determining whether a region will ultimately crack during the transition. These results emphasize that the residue interactions found exclusively in one of the two meta-stable states is the key in understanding the mechanism of allosteric conformational change.

  5. Conformational flexibility and the mechanisms of allosteric transitions in topologically similar proteins.

    PubMed

    Tripathi, Swarnendu; Portman, John J

    2011-08-21

    Conformational flexibility plays a central role in allosteric transition of proteins. In this paper, we extend the analysis of our previous study [S. Tripathi and J. J. Portman, Proc. Natl. Acad. Sci. U.S.A. 106, 2104 (2009)] to investigate how relatively minor structural changes of the meta-stable states can significantly influence the conformational flexibility and allosteric transition mechanism. We use the allosteric transitions of the domains of calmodulin as an example system to highlight the relationship between the transition mechanism and the inter-residue contacts present in the meta-stable states. In particular, we focus on the origin of transient local unfolding (cracking), a mechanism that can lower free energy barriers of allosteric transitions, in terms of the inter-residue contacts of the meta-stable states and the pattern of local strain that develops during the transition. We find that the magnitude of the local strain in the protein is not the sole factor determining whether a region will ultimately crack during the transition. These results emphasize that the residue interactions found exclusively in one of the two meta-stable states is the key in understanding the mechanism of allosteric conformational change.

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

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

    PubMed Central

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

    2016-01-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators. PMID:27032695

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal chorismate mutase.

    PubMed

    Helmstaedt, Kerstin; Heinrich, Gabriele; Lipscomb, William N; Braus, Gerhard H

    2002-05-14

    The yeast chorismate mutase is regulated by tyrosine as feedback inhibitor and tryptophan as crosspathway activator. The monomer consists of a catalytic and a regulatory domain covalently linked by the loop L220s (212-226), which functions as a molecular hinge. Two monomers form the active dimeric enzyme stabilized by hydrophobic interactions in the vicinity of loop L220s. The role of loop L220s and its environment for enzyme regulation, dimerization, and stability was analyzed. Substitution of yeast loop L220s in place of the homologous loop from the corresponding and similarly regulated Aspergillus enzyme (and the reverse substitution) changed tyrosine inhibition to activation. Yeast loop L220s substituted into the Aspergillus enzyme resulted in a tryptophan-inhibitable enzyme. Monomeric yeast chorismate mutases could be generated by substituting two hydrophobic residues in and near the hinge region. The resulting Thr-212-->Asp-Phe-28-->Asp enzyme was as stable as wild type, but lost allosteric regulation and showed reduced catalytic activity. These results underline the crucial role of this molecular hinge for inhibition, activation, quaternary structure, and stability of yeast chorismate mutase.

  17. 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.

  18. Allosteric coupling between proximal C-terminus and selectivity filter is facilitated by the movement of transmembrane segment 4 in TREK-2 channel

    PubMed Central

    Zhuo, Ren-Gong; Peng, Peng; Liu, Xiao-Yan; Yan, Hai-Tao; Xu, Jiang-Ping; Zheng, Jian-Quan; Wei, Xiao-Li; Ma, Xiao-Yun

    2016-01-01

    TREK-2, a member of two-pore-domain potassium channel family, regulates cellular excitability in response to diverse stimuli. However, how such stimuli control channel function remains unclear. Here, by characterizing the responses of cytosolic proximal C-terminus deletant (ΔpCt) and transmembrane segment 4 (M4)-glycine hinge mutant (G312A) to 2-Aminoethoxydiphenyl borate (2-APB), an activator of TREK-2, we show that the transduction initiated from pCt domain is allosterically coupled with the conformation of selectivity filter (SF) via the movements of M4, without depending on the original status of SF. Moreover, ΔpCt and G312A also exhibited blunted responses to extracellular alkalization, a model to induce SF conformational transition. These results suggest that the coupling between pCt domain and SF is bidirectional, and M4 movements are involved in both processes. Further mechanistic exploration reveals that the function of Phe316, a residue close to the C-terminus of M4, is associated with such communications. However, unlike TREK-2, M4-hinge of TREK-1 only controls the transmission from pCt to SF, rather than SF conformational changes triggered by pHo changes. Together, our findings uncover the unique gating properties of TREK-2, and elucidate the mechanisms for how the extracellular and intracellular stimuli harness the pore gating allosterically. PMID:26879043

  19. 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.

  20. 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

  1. 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.

  2. The concept of allosteric interaction and its consequences for the chemistry of the brain.

    PubMed

    Changeux, Jean-Pierre

    2013-09-20

    Throughout this Reflections article, I have tried to follow up on the genesis in the 1960s and subsequent evolution of the concept of allosteric interaction and to examine its consequences within the past decades, essentially in the field of the neuroscience. The main conclusion is that allosteric mechanisms built on similar structural principles operate in bacterial regulatory enzymes, gene repressors (and the related nuclear receptors), rhodopsin, G-protein-coupled receptors, neurotransmitter receptors, ion channels, and so on from prokaryotes up to the human brain yet with important features of their own. Thus, future research on these basic cybernetic sensors is expected to develop in two major directions: at the elementary level, toward the atomic structure and molecular dynamics of the conformational changes involved in signal recognition and transduction, but also at a higher level of organization, the contribution of allosteric mechanisms to the modulation of brain functions.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. The Concept of Allosteric Interaction and Its Consequences for the Chemistry of the Brain

    PubMed Central

    Changeux, Jean-Pierre

    2013-01-01

    Throughout this Reflections article, I have tried to follow up on the genesis in the 1960s and subsequent evolution of the concept of allosteric interaction and to examine its consequences within the past decades, essentially in the field of the neuroscience. The main conclusion is that allosteric mechanisms built on similar structural principles operate in bacterial regulatory enzymes, gene repressors (and the related nuclear receptors), rhodopsin, G-protein-coupled receptors, neurotransmitter receptors, ion channels, and so on from prokaryotes up to the human brain yet with important features of their own. Thus, future research on these basic cybernetic sensors is expected to develop in two major directions: at the elementary level, toward the atomic structure and molecular dynamics of the conformational changes involved in signal recognition and transduction, but also at a higher level of organization, the contribution of allosteric mechanisms to the modulation of brain functions. PMID:23878193

  8. 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.

  9. 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.

  10. Dual allosteric activation mechanisms in monomeric human glucokinase.

    PubMed

    Whittington, A Carl; Larion, Mioara; Bowler, Joseph M; Ramsey, Kristen M; Brüschweiler, Rafael; Miller, Brian G

    2015-09-15

    Cooperativity in human glucokinase (GCK), the body's primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme's small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the (1)H-(13)C heteronuclear multiple quantum coherence (HMQC) spectrum of (13)C-Ile-labeled enzyme resembles the glucose-bound state. In β-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the (1)H-(13)C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems.

  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. 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

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

    PubMed Central

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

    2016-01-01

    Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A — on the opposite face of the molecule — more liable to adopt an ‘open’ state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin–enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the ‘open’ state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation. PMID:27407165

  14. 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.

  15. 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

  16. 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.

  17. 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.

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

    SciTech Connect

    Holzapfel, Genevieve; Buhrman, Greg; Mattos, Carla

    2012-08-31

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

  19. 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.

  20. 2013 Philip S. Portoghese Medicinal Chemistry Lectureship: Drug Discovery Targeting Allosteric Sites†

    PubMed Central

    2015-01-01

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

  1. 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.

  2. 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

  3. 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).

  4. 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

  5. Elucidation of a four-site allosteric network in fibroblast growth factor receptor tyrosine kinases

    PubMed Central

    Chen, Huaibin; Marsiglia, William M; Cho, Min-Kyu; Huang, Zhifeng; Deng, Jingjing; Blais, Steven P; Gai, Weiming; Bhattacharya, Shibani; Neubert, Thomas A; Traaseth, Nathaniel J; Mohammadi, Moosa

    2017-01-01

    Receptor tyrosine kinase (RTK) signaling is tightly regulated by protein allostery within the intracellular tyrosine kinase domains. Yet the molecular determinants of allosteric connectivity in tyrosine kinase domain are incompletely understood. By means of structural (X-ray and NMR) and functional characterization of pathogenic gain-of-function mutations affecting the FGF receptor (FGFR) tyrosine kinase domain, we elucidated a long-distance allosteric network composed of four interconnected sites termed the ‘molecular brake’, ‘DFG latch’, ‘A-loop plug’, and ‘αC tether’. The first three sites repress the kinase from adopting an active conformation, whereas the αC tether promotes the active conformation. The skewed design of this four-site allosteric network imposes tight autoinhibition and accounts for the incomplete mimicry of the activated conformation by pathogenic mutations targeting a single site. Based on the structural similarity shared among RTKs, we propose that this allosteric model for FGFR kinases is applicable to other RTKs. DOI: http://dx.doi.org/10.7554/eLife.21137.001 PMID:28166054

  6. 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.

  7. 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

  8. 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.

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

    PubMed Central

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

    2016-01-01

    At least 120 non-olfactory G protein-coupled receptors in the human genome are ”orphans” for which endogenous ligands are unknown, and many have no selective ligands, hindering elucidation of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Yeast-based screens against GPR68 identified the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. Over 3000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators many of which were confirmed in functional assays. One potent GPR68 modulator—ogerin– suppressed recall in fear conditioning in wild-type, but not in GPR68 knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs. PMID:26550826

  10. Nootropic alpha7 nicotinic receptor allosteric modulator derived from GABAA receptor modulators.

    PubMed

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

    2007-05-08

    Activation of brain alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) has broad therapeutic potential in CNS diseases related to cognitive dysfunction, including Alzheimer's disease and schizophrenia. In contrast to direct agonist activation, positive allosteric modulation of alpha7 nAChRs would deliver the clinically validated benefits of allosterism to these indications. We have generated a selective alpha7 nAChR-positive allosteric modulator (PAM) from a library of GABAA receptor PAMs. Compound 6 (N-(4-chlorophenyl)-alpha-[[(4-chloro-phenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide) evokes robust positive modulation of agonist-induced currents at alpha7 nAChRs, while preserving the rapid native characteristics of desensitization, and has little to no efficacy at other ligand-gated ion channels. In rodent models, it corrects sensory-gating deficits and improves working memory, effects consistent with cognitive enhancement. Compound 6 represents a chemotype for allosteric activation of alpha7 nAChRs, with therapeutic potential in CNS diseases with cognitive dysfunction.

  11. 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.

  12. Glucosamine-6-phosphate deaminase from beef kidney is an allosteric system of the V-type.

    PubMed

    Lara-Lemus, R; Calcagno, M L

    1998-10-14

    The enzyme glucosamine-6-phosphate deaminase from beef kidney has been purified to homogeneity by allosteric-site affinity chromatography. Its amino acid composition and the N-terminal sequence (1-42), were obtained. The amino acid sequence of this segment is essentially identical to the corresponding regions of the human and hamster glucosamine-6-phosphate deaminases. The beef enzyme is a hexamer of 32.5 kDa subunits; this is nearly 2.5 kDa higher than the molecular mass of the homologous enzyme from Escherichia coli. Beef kidney deaminase exhibits a notable difference from the bacterial enzyme in its allosteric activation by N-acetylglucosamine 6-phosphate This metabolite, which is also is the allosteric activator of the bacterial glucosamine-6-phosphate deaminase, activates the enzyme by increasing its kcat without any change in the Km values for glucosamine 6-phosphate, over a wide range of activator concentration. This observation places beef kidney deaminase in the class of V-type allosteric systems.

  13. 2013 Philip S. Portoghese Medicinal Chemistry Lectureship: drug discovery targeting allosteric sites.

    PubMed

    Lindsley, Craig W

    2014-09-25

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

  14. 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

  15. 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 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.

  16. Repetition Blindness Occurs in Nonwords

    ERIC Educational Resources Information Center

    Harris, Catherine L.; Morris, Alison L.

    2004-01-01

    Theorists have predicted that repetition blindness (RB) should be absent for nonwords because they do not activate preexisting mental types. The authors hypothesized that RB would be observed for nonwords because RB can occur at a sublexical level. Four experiments showed that RB is observed for word-nonword pairs (noon noof), orthographically…

  17. Allosteric and hyperekplexic mutant phenotypes investigated on an α1 glycine receptor transmembrane structure.

    PubMed

    Moraga-Cid, Gustavo; Sauguet, Ludovic; Huon, Christèle; Malherbe, Laurie; Girard-Blanc, Christine; Petres, Stéphane; Murail, Samuel; Taly, Antoine; Baaden, Marc; Delarue, Marc; Corringer, Pierre-Jean

    2015-03-03

    The glycine receptor (GlyR) is a pentameric ligand-gated ion channel (pLGIC) mediating inhibitory transmission in the nervous system. Its transmembrane domain (TMD) is the target of allosteric modulators such as general anesthetics and ethanol and is a major locus for hyperekplexic congenital mutations altering the allosteric transitions of activation or desensitization. We previously showed that the TMD of the human α1GlyR could be fused to the extracellular domain of GLIC, a bacterial pLGIC, to form a functional chimera called Lily. Here, we overexpress Lily in Schneider 2 insect cells and solve its structure by X-ray crystallography at 3.5 Å resolution. The TMD of the α1GlyR adopts a closed-channel conformation involving a single ring of hydrophobic residues at the center of the pore. Electrophysiological recordings show that the phenotypes of key allosteric mutations of the α1GlyR, scattered all along the pore, are qualitatively preserved in this chimera, including those that confer decreased sensitivity to agonists, constitutive activity, decreased activation kinetics, or increased desensitization kinetics. Combined structural and functional data indicate a pore-opening mechanism for the α1GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the α1GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor.

  18. 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.

  19. 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.

  20. Allosteric and hyperekplexic mutant phenotypes investigated on an α1 glycine receptor transmembrane structure

    PubMed Central

    Moraga-Cid, Gustavo; Sauguet, Ludovic; Huon, Christèle; Malherbe, Laurie; Girard-Blanc, Christine; Petres, Stéphane; Murail, Samuel; Taly, Antoine; Baaden, Marc; Delarue, Marc; Corringer, Pierre-Jean

    2015-01-01

    The glycine receptor (GlyR) is a pentameric ligand-gated ion channel (pLGIC) mediating inhibitory transmission in the nervous system. Its transmembrane domain (TMD) is the target of allosteric modulators such as general anesthetics and ethanol and is a major locus for hyperekplexic congenital mutations altering the allosteric transitions of activation or desensitization. We previously showed that the TMD of the human α1GlyR could be fused to the extracellular domain of GLIC, a bacterial pLGIC, to form a functional chimera called Lily. Here, we overexpress Lily in Schneider 2 insect cells and solve its structure by X-ray crystallography at 3.5 Å resolution. The TMD of the α1GlyR adopts a closed-channel conformation involving a single ring of hydrophobic residues at the center of the pore. Electrophysiological recordings show that the phenotypes of key allosteric mutations of the α1GlyR, scattered all along the pore, are qualitatively preserved in this chimera, including those that confer decreased sensitivity to agonists, constitutive activity, decreased activation kinetics, or increased desensitization kinetics. Combined structural and functional data indicate a pore-opening mechanism for the α1GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the α1GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor. PMID:25730860

  1. 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

  2. Naturally Occurring Radioactive Materials (NORM)

    SciTech Connect

    Gray, P.

    1997-02-01

    This paper discusses the broad problems presented by Naturally Occuring Radioactive Materials (NORM). Technologically Enhanced naturally occuring radioactive material includes any radionuclides whose physical, chemical, radiological properties or radionuclide concentration have been altered from their natural state. With regard to NORM in particular, radioactive contamination is radioactive material in an undesired location. This is a concern in a range of industries: petroleum; uranium mining; phosphorus and phosphates; fertilizers; fossil fuels; forestry products; water treatment; metal mining and processing; geothermal energy. The author discusses in more detail the problem in the petroleum industry, including the isotopes of concern, the hazards they present, the contamination which they cause, ways to dispose of contaminated materials, and regulatory issues. He points out there are three key programs to reduce legal exposure and problems due to these contaminants: waste minimization; NORM assesment (surveys); NORM compliance (training).

  3. 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

  4. 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

  5. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2008-12-01

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

  7. 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. 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.

  9. 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.

  10. Allosteric regulation of catalytic activity: Escherichia coli aspartate transcarbamoylase versus yeast chorismate mutase.

    PubMed

    Helmstaedt, K; Krappmann, S; Braus, G H

    2001-09-01

    Allosteric regulation of key metabolic enzymes is a fascinating field to study the structure-function relationship of induced conformational changes of proteins. In this review we compare the principles of allosteric transitions of the complex classical model aspartate transcarbamoylase (ATCase) from Escherichia coli, consisting of 12 polypeptides, and the less complicated chorismate mutase derived from baker's yeast, which functions as a homodimer. Chorismate mutase presumably represents the minimal oligomerization state of a cooperative enzyme which still can be either activated or inhibited by different heterotropic effectors. Detailed knowledge of the number of possible quaternary states and a description of molecular triggers for conformational changes of model enzymes such as ATCase and chorismate mutase shed more and more light on allostery as an important regulatory mechanism of any living cell. The comparison of wild-type and engineered mutant enzymes reveals that current textbook models for regulation do not cover the entire picture needed to describe the function of these enzymes in detail.

  11. 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.

  12. 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

  13. 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

  14. Allosteric ACTion: the varied ACT domains regulating enzymes of amino-acid metabolism.

    PubMed

    Lang, Eric J M; Cross, Penelope J; Mittelstädt, Gerd; Jameson, Geoffrey B; Parker, Emily J

    2014-12-01

    Allosteric regulation of enzyme activity plays important metabolic roles. Here we review the allostery of enzymes of amino-acid metabolism conferred by a discrete domain known as the ACT domain. This domain of 60-70 residues has a βαββαβ topology leading to a four-stranded β4β1β3β2 antiparallel sheet with two antiparallel helices on one face. Extensive sequence variation requires a combined sequence/structure/function analysis for identification of the ACT domain. Common features include highly varied modes of self-association of ACT domains, ligand binding at domain interfaces, and transmittal of allosteric signals through conformational changes and/or the manipulation of quaternary equilibria. A recent example illustrates the relatively facile adoption of this versatile module of allostery by gene fusion.

  15. 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.

  16. Allosteric modulation by benzodiazepines of GABA-gated chloride channels of an identified insect motor neurone.

    PubMed

    Buckingham, Steven D; Higashino, Yoshiaki; Sattelle, David B

    2009-11-01

    The actions of benzodiazepines were studied on the responses to GABA of the fast coxal depressor (D(f)) motor neurone of the cockroach, Periplaneta americana. Ro5-4864, diazepam and clonazepam were investigated. Responses to GABA receptors were enhanced by both Ro5-4864 and diazepam, whereas clonazepam, a potent-positive allosteric modulator of human GABA(A) receptors, was ineffective on the native insect GABA receptors of the D(f) motor neurone. Thus, clear pharmacological differences exist between insect and mammalian native GABA-gated chloride channels with respect to the actions of benzodiazepines. The results enhance our understanding of invertebrate GABA-gated chloride channels which have recently proved important in (a) comparative studies aimed at identifying human allosteric drug-binding sites and (b) understanding the actions of compounds used to control ectoparasites and insect crop pests.

  17. Evidence for an active T-state pig kidney fructose 1,6-bisphosphatase: interface residue Lys-42 is important for allosteric inhibition and AMP cooperativity.

    PubMed Central

    Lu, G.; Stec, B.; Giroux, E. L.; Kantrowitz, E. R.

    1996-01-01

    During the R-->T transition in the tetrameric pig kidney fructose-1,6-bisphosphatase (Fru-1,6-P2ase, EC 3.1.3.11) a major change in the quaternary structure of the enzyme occurs that is induced by the binding of the allosteric inhibitor AMP (Ke HM, Liang JY, Zhang Y, Lipscomb WN, 1991, Biochemistry 30:4412-4420). The change in quaternary structure involving the rotation of the upper dimer by 17 degrees relative to the lower dimer is coupled to a series of structural changes on the secondary and tertiary levels. The structural data indicate that Lys-42 is involved in a complex set of intersubunit interactions across the dimer-dimer interface with residues of the 190's loop, a loop located at the pivot of the allosteric rotation. In order to test the function of Lys-42, we have replaced it with alanine using site-specific mutagenesis. The kcat and K(m) values for Lys-42-->Ala Fru-1,6-P2ase were 11 s-1 and 3.3 microM, respectively, resulting in a mutant enzyme that was slightly less efficient catalytically than the normal pig kidney enzyme. Although the Lys-42-->Ala Fru-1,6-P2ase was similar kinetically in terms of K(m) and kcat, the response to inhibition by AMP was significantly different than that of the normal pig kidney enzyme. Not only was AMP inhibition no longer cooperative, but also it occurred in two stages, corresponding to high- and low-affinity binding sites. Saturation of the high-affinity sites only reduced the activity by 30%, compared to 100% for the wild-type enzyme. In order to determine in what structural state the enzyme was after saturation of the high-affinity sites, the Lys-42-->Ala enzyme was crystallized in the presence of Mn2+, fructose-6-phosphate (Fru-6-P), and 100 microM AMP and the data collected to 2.3 A resolution. The X-ray structure showed the T state with AMP binding with full occupancy to the four regulatory sites and the inhibitor Fru-6-P bound at the active sites. The results reported here suggest that, in the normal pig kidney

  18. Mutant Analysis Reveals Allosteric Regulation of ClpB Disaggregase

    PubMed Central

    Franke, Kamila B.; Bukau, Bernd; Mogk, Axel

    2017-01-01

    The members of the hexameric AAA+ disaggregase of E. coli and S. cerevisiae, ClpB, and Hsp104, cooperate with the Hsp70 chaperone system in the solubilization of aggregated proteins. Aggregate solubilization relies on a substrate threading activity of ClpB/Hsp104 fueled by ATP hydrolysis in both ATPase rings (AAA-1, AAA-2). ClpB/Hsp104 ATPase activity is controlled by the M-domains, which associate to the AAA-1 ring to downregulate ATP hydrolysis. Keeping M-domains displaced from the AAA-1 ring by association with Hsp70 increases ATPase activity due to enhanced communication between protomers. This communication involves conserved arginine fingers. The control of ClpB/Hsp104 activity is crucial, as hyperactive mutants with permanently dissociated M-domains exhibit cellular toxicity. Here, we analyzed AAA-1 inter-ring communication in relation to the M-domain mediated ATPase regulation, by subjecting a conserved residue of the AAA-1 domain subunit interface of ClpB (A328) to mutational analysis. While all A328X mutants have reduced disaggregation activities, their ATPase activities strongly differed. ClpB-A328I/L mutants have reduced ATPase activity and when combined with the hyperactive ClpB-K476C M-domain mutation, suppress cellular toxicity. This underlines that ClpB ATPase activation by M-domain dissociation relies on increased subunit communication. The ClpB-A328V mutant in contrast has very high ATPase activity and exhibits cellular toxicity on its own, qualifying it as novel hyperactive ClpB mutant. ClpB-A328V hyperactivity is however, different from that of M-domain mutants as M-domains stay associated with the AAA-1 ring. The high ATPase activity of ClpB-A328V primarily relies on the AAA-2 ring and correlates with distinct conformational changes in the AAA-2 catalytic site. These findings characterize the subunit interface residue A328 as crucial regulatory element to control ATP hydrolysis in both AAA rings. PMID:28275610

  19. Mutant Analysis Reveals Allosteric Regulation of ClpB Disaggregase.

    PubMed

    Franke, Kamila B; Bukau, Bernd; Mogk, Axel

    2017-01-01

    The members of the hexameric AAA+ disaggregase of E. coli and S. cerevisiae, ClpB, and Hsp104, cooperate with the Hsp70 chaperone system in the solubilization of aggregated proteins. Aggregate solubilization relies on a substrate threading activity of ClpB/Hsp104 fueled by ATP hydrolysis in both ATPase rings (AAA-1, AAA-2). ClpB/Hsp104 ATPase activity is controlled by the M-domains, which associate to the AAA-1 ring to downregulate ATP hydrolysis. Keeping M-domains displaced from the AAA-1 ring by association with Hsp70 increases ATPase activity due to enhanced communication between protomers. This communication involves conserved arginine fingers. The control of ClpB/Hsp104 activity is crucial, as hyperactive mutants with permanently dissociated M-domains exhibit cellular toxicity. Here, we analyzed AAA-1 inter-ring communication in relation to the M-domain mediated ATPase regulation, by subjecting a conserved residue of the AAA-1 domain subunit interface of ClpB (A328) to mutational analysis. While all A328X mutants have reduced disaggregation activities, their ATPase activities strongly differed. ClpB-A328I/L mutants have reduced ATPase activity and when combined with the hyperactive ClpB-K476C M-domain mutation, suppress cellular toxicity. This underlines that ClpB ATPase activation by M-domain dissociation relies on increased subunit communication. The ClpB-A328V mutant in contrast has very high ATPase activity and exhibits cellular toxicity on its own, qualifying it as novel hyperactive ClpB mutant. ClpB-A328V hyperactivity is however, different from that of M-domain mutants as M-domains stay associated with the AAA-1 ring. The high ATPase activity of ClpB-A328V primarily relies on the AAA-2 ring and correlates with distinct conformational changes in the AAA-2 catalytic site. These findings characterize the subunit interface residue A328 as crucial regulatory element to control ATP hydrolysis in both AAA rings.

  20. 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.

  1. 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

  2. Potentiation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site.

    PubMed

    Young, Gareth T; Zwart, Ruud; Walker, Alison S; Sher, Emanuele; Millar, Neil S

    2008-09-23

    Positive allosteric modulators of alpha7 nicotinic acetylcholine receptors (nAChRs) have attracted considerable interest as potential tools for the treatment of neurological and psychiatric disorders such as Alzheimer's disease and schizophrenia. However, despite the potential therapeutic usefulness of these compounds, little is known about their mechanism of action. Here, we have examined two allosteric potentiators of alpha7 nAChRs (PNU-120596 and LY-2087101). From studies with a series of subunit chimeras, we have identified the transmembrane regions of alpha7 as being critical in facilitating potentiation of agonist-evoked responses. Furthermore, we have identified five transmembrane amino acids that, when mutated, significantly reduce potentiation of alpha7 nAChRs. The amino acids we have identified are located within the alpha-helical transmembrane domains TM1 (S222 and A225), TM2 (M253), and TM4 (F455 and C459). Mutation of either A225 or M253 individually have particularly profound effects, reducing potentiation of EC(20) concentrations of acetylcholine to a tenth of the level seen with wild-type alpha7. Reference to homology models of the alpha7 nAChR, based on the 4A structure of the Torpedo nAChR, indicates that the side chains of all five amino acids point toward an intrasubunit cavity located between the four alpha-helical transmembrane domains. Computer docking simulations predict that the allosteric compounds such as PNU-120596 and LY-2087101 may bind within this intrasubunit cavity, much as neurosteroids and volatile anesthetics are thought to interact with GABA(A) and glycine receptors. Our findings suggest that this is a conserved modulatory allosteric site within neurotransmitter-gated ion channels.

  3. Allosteric inhibitors of hepatitis C polymerase: discovery of potent and orally bioavailable carbon-linked dihydropyrones.

    PubMed

    Li, Hui; Linton, Angelica; Tatlock, John; Gonzalez, Javier; Borchardt, Allen; Abreo, Mel; Jewell, Tanya; Patel, Leena; Drowns, Matthew; Ludlum, Sarah; Goble, Mike; Yang, Michele; Blazel, Julie; Rahavendran, Ravi; Skor, Heather; Shi, Stephanie; Lewis, Cristina; Fuhrman, Shella

    2007-08-23

    The discovery and optimization of a novel class of carbon-linked dihydropyrones as allosteric HCV NS5B polymerase inhibitors are presented. Replacement of the sulfur linker atom with carbon reduced compound acidity and greatly increased cell permeation. Further structure-activity relationship (SAR) studies led to the identification of compounds, exemplified by 23 and 24, with significantly improved antiviral activities in the cell-based replicon assay and favorable pharmacokinetic profiles.

  4. 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.

  5. Identifying Cytochrome P450 Functional Networks and Their Allosteric Regulatory Elements

    DTIC Science & Technology

    2013-12-03

    Adx is a small iron - sulfur protein that transfers electrons from NADPH to terminal CYP as part of the overall metabolic processing of substrates. We...needed to create a systematic approach that allows us to identify putative allosteric sites. Here, we present combined co-evolutionary analysis and...isoform. The cysteine residue, which connects to iron , is conserved among the isoforms, implying that the catalytic function is co- evolved and similar for

  6. Overcoming EGFR T790M and C797S resistance with mutant-selective allosteric inhibitors

    PubMed Central

    Jia, Yong; Yun, Cai-Hong; Park, Eunyoung; Ercan, Dalia; Manuia, Mari; Juarez, Jose; Xu, Chunxiao; Rhee, Kevin; Chen, Ting; Zhang, Haikuo; Palakurthi, Sangeetha; Jang, Jaebong; Lelais, Gerald; DiDonato, Michael; Bursulaya, Badry; Michellys, Pierre-Yves; Epple, Robert; Marsilje, Thomas H.; McNeill, Matthew; Lu, Wenshuo; Harris, Jennifer; Bender, Steven; Wong, Kwok-Kin; Jänne, Pasi A.; Eck, Michael J.

    2016-01-01

    EGFR tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harboring activating mutations in the EGFR kinase1,2, but resistance arises rapidly, most frequently due to the secondary T790M mutation within the ATP-site of the receptor.3,4 Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant5,6, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond7. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternate mechanisms of action. Here we describe rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild type receptor. A crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays, but as a single agent is not effective in blocking EGFR-driven proliferation in cells due to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state8. We observe dramatic synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization9,10, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by L858R/T790M EGFR and by L858R/T790M/C797S EGFR, a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors. PMID:27251290

  7. When Yawning Occurs in Elephants

    PubMed Central

    Rossman, Zoë T.; Hart, Benjamin L.; Greco, Brian J.; Young, Debbie; Padfield, Clare; Weidner, Lisa; Gates, Jennifer; Hart, Lynette A.

    2017-01-01

    Yawning is a widely recognized behavior in mammalian species. One would expect that elephants yawn, although to our knowledge, no one has reported observations of yawning in any species of elephant. After confirming a behavioral pattern matching the criteria of yawning in two Asian elephants (Elephas maximus) in a zoological setting, this study was pursued with nine captive African elephants (Loxodonta africana) at a private reserve in the Western Cape, South Africa, the Knysna Elephant Park. Observations were made in June–September and in December. In the daytime, handlers managed seven of the elephants for guided interactions with visitors. At night, all elephants were maintained in a large enclosure with six having limited outdoor access. With infrared illumination, the elephants were continuously recorded by video cameras. During the nights, the elephants typically had 1–3 recumbent sleeping/resting bouts, each lasting 1–2 h. Yawning was a regular occurrence upon arousal from a recumbency, especially in the final recumbency of the night. Yawning was significantly more frequent in some elephants. Yawning was rare during the daytime and during periods of standing around in the enclosure at night. In six occurrences of likely contagious yawning, one elephant yawned upon seeing another elephant yawning upon arousal from a final recumbency; we recorded the sex and age category of the participants. The generality of yawning in both African and Asian elephants in other environments was documented in video recordings from 39 zoological facilities. In summary, the study provides evidence that yawning does occur in both African and Asian elephants, and in African elephants, yawning was particularly associated with arousal from nighttime recumbencies. PMID:28293560

  8. When Yawning Occurs in Elephants.

    PubMed

    Rossman, Zoë T; Hart, Benjamin L; Greco, Brian J; Young, Debbie; Padfield, Clare; Weidner, Lisa; Gates, Jennifer; Hart, Lynette A

    2017-01-01

    Yawning is a widely recognized behavior in mammalian species. One would expect that elephants yawn, although to our knowledge, no one has reported observations of yawning in any species of elephant. After confirming a behavioral pattern matching the criteria of yawning in two Asian elephants (Elephas maximus) in a zoological setting, this study was pursued with nine captive African elephants (Loxodonta africana) at a private reserve in the Western Cape, South Africa, the Knysna Elephant Park. Observations were made in June-September and in December. In the daytime, handlers managed seven of the elephants for guided interactions with visitors. At night, all elephants were maintained in a large enclosure with six having limited outdoor access. With infrared illumination, the elephants were continuously recorded by video cameras. During the nights, the elephants typically had 1-3 recumbent sleeping/resting bouts, each lasting 1-2 h. Yawning was a regular occurrence upon arousal from a recumbency, especially in the final recumbency of the night. Yawning was significantly more frequent in some elephants. Yawning was rare during the daytime and during periods of standing around in the enclosure at night. In six occurrences of likely contagious yawning, one elephant yawned upon seeing another elephant yawning upon arousal from a final recumbency; we recorded the sex and age category of the participants. The generality of yawning in both African and Asian elephants in other environments was documented in video recordings from 39 zoological facilities. In summary, the study provides evidence that yawning does occur in both African and Asian elephants, and in African elephants, yawning was particularly associated with arousal from nighttime recumbencies.

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

    PubMed Central

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

    2014-01-01

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

  10. 2-Aminothienopyridazines as Novel Adenosine A1 Receptor Allosteric Modulators and Antagonists

    PubMed Central

    Ferguson, Gemma N.; Valant, Celine; Horne, James; Figler, Heidi; Flynn, Bernard L.; Linden, Joel; Chalmers, David K.; Sexton, Patrick M.; Christopoulos, Arthur; Scammells, Peter J.

    2008-01-01

    A pharmacophore-based screen identified 32 compounds including ethyl 5-amino-3-(4-tert-butylphenyl)-4-oxo-3,4-dihydrothieno[3,4-d]pyridazine-1-carboxylate (8) as a new allosteric modulator of the adenosine A1 receptor (A1AR). On the basis of this lead, various derivatives were prepared and evaluated for activity at the human A1AR. A number of the test compounds allosterically stabilized agonist-receptor-G protein ternary complexes in dissociation kinetic assays, but were found to be more potent as antagonists in subsequent functional assays of ERK1/2 phosphorylation. Additional experiments on the most potent antagonist, 13b, investigating A1AR-mediated [35S]GTPγS binding and [3H]CCPA equilibrium binding confirmed its antagonistic mode of action and also identified inverse agonism. This study has thus identified a new class of A1AR antagonists that can also recognize the receptor’s allosteric site with lower potency. PMID:18771255

  11. Allosteric Site Variants of Haemophilus influenzae β-Carbonic Anhdyrase†, ‡

    PubMed Central

    Rowlett, Roger S.; Tu, Chingkuang; Lee, Joseph; Herman, Ariel G.; Chapnick, Douglas A.; Shah, Shalini H.; Gareiss, Peter C.

    2009-01-01

    Haemophilus influenzae β-carbonic anhydrase (HICA) is hypothesized to be an allosteric protein that is regulated by the binding of bicarbonate ion to a non-catalytic (inhibitory) site that controls the ligation of Asp44 to the catalytically essential zinc ion. We report here the X-ray crystallographic structures of two variants (W39F and Y181F) involved in the binding of bicarbonate ion in the non-catalytic site and an active site variant (D44N) that is incapable of forming a strong zinc ligand. The alteration of Trp39 to Phe increases the apparent Ki for bicarbonate inhibition by 4.8-fold. While the structures of W39F and Y181F are very similar to the wild-type enzyme, the X-ray crystal structure of the D44N variant reveals that it has adopted an active site conformation nearly identical to that of non-allosteric β-carbonic anhydrases. We propose that the structure of the D44N variant is likely to be representative of the active conformation of the enzyme. These results lend additional support to the hypothesis that HICA is an allosteric enzyme that can adopt active and inactive conformations, the latter of which is stabilized by bicarbonate ion binding to a non-catalytic site. PMID:19459702

  12. 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.

  13. Identification of natural allosteric inhibitor for Akt1 protein through computational approaches and in vitro evaluation.

    PubMed

    Pragna Lakshmi, T; Kumar, Amit; Vijaykumar, Veena; Natarajan, Sakthivel; Krishna, Ramadas

    2017-03-01

    Akt, a serine/threonine protein kinase, is often hyper activated in breast and prostate cancers, but with poor prognosis. Allosteric inhibitors regulate aberrant kinase activity by stabilizing the protein in inactive conformation. Several natural compounds have been reported as inhibitors for kinases. In this study, to identify potential natural allosteric inhibitor for Akt1, we generated a seven-point pharmacophore model and screened it through natural compound library. Quercetin-7-O-β-d-glucopyranoside or Q7G was found to be the best among selected molecules based on its hydrogen bond occupancy with key allosteric residues, persistent polar contacts and salt bridges that stabilize Akt1 in inactive conformation and minimum binding free energy during molecular dynamics simulation. Q7G induced dose-dependent inhibition of breast cancer cells (MDA MB-231) and arrested them in G1 and sub-G phase. This was associated with down-regulation of anti-apoptotic protein Bcl-2, up-regulation of cleaved caspase-3 and PARP. Expression of p-Akt (Ser473) was also down-regulated which might be due to Akt1 inhibition in inactive conformation. We further confirmed the Akt1 and Q7G interaction which was observed to have a dissociation constant (Kd) of 0.246μM. With these computational, biological and thermodynamic studies, we suggest Q7G as a lead molecule and propose for its further optimization.

  14. Selective small molecule inhibitor of the Mycobacterium tuberculosis fumarate hydratase reveals an allosteric regulatory site

    PubMed Central

    Kasbekar, Monica; Fischer, Gerhard; Mott, Bryan T.; Yasgar, Adam; Hyvönen, Marko; Boshoff, Helena I. M.; Abell, Chris; Barry, Clifton E.; Thomas, Craig J.

    2016-01-01

    Enzymes in essential metabolic pathways are attractive targets for the treatment of bacterial diseases, but in many cases, the presence of homologous human enzymes makes them impractical candidates for drug development. Fumarate hydratase, an essential enzyme in the tricarboxylic acid (TCA) cycle, has been identified as one such potential therapeutic target in tuberculosis. We report the discovery of the first small molecule inhibitor, to our knowledge, of the Mycobacterium tuberculosis fumarate hydratase. A crystal structure at 2.0-Å resolution of the compound in complex with the protein establishes the existence of a previously unidentified allosteric regulatory site. This allosteric site allows for selective inhibition with respect to the homologous human enzyme. We observe a unique binding mode in which two inhibitor molecules interact within the allosteric site, driving significant conformational changes that preclude simultaneous substrate and inhibitor binding. Our results demonstrate the selective inhibition of a highly conserved metabolic enzyme that contains identical active site residues in both the host and the pathogen. PMID:27325754

  15. Discovery, synthesis, and molecular pharmacology of selective positive allosteric modulators of the δ-opioid receptor.

    PubMed

    Burford, Neil T; Livingston, Kathryn E; Canals, Meritxell; Ryan, Molly R; Budenholzer, Lauren M L; Han, Ying; Shang, Yi; Herbst, John J; O'Connell, Jonathan; Banks, Martyn; Zhang, Litao; Filizola, Marta; Bassoni, Daniel L; Wehrman, Tom S; Christopoulos, Arthur; Traynor, John R; Gerritz, Samuel W; Alt, Andrew

    2015-05-28

    Allosteric modulators of G protein-coupled receptors (GPCRs) have a number of potential advantages compared to agonists or antagonists that bind to the orthosteric site of the receptor. These include the potential for receptor selectivity, maintenance of the temporal and spatial fidelity of signaling in vivo, the ceiling effect of the allosteric cooperativity which may prevent overdose issues, and engendering bias by differentially modulating distinct signaling pathways. Here we describe the discovery, synthesis, and molecular pharmacology of δ-opioid receptor-selective positive allosteric modulators (δ PAMs). These δ PAMs increase the affinity and/or efficacy of the orthosteric agonists leu-enkephalin, SNC80 and TAN67, as measured by receptor binding, G protein activation, β-arrestin recruitment, adenylyl cyclase inhibition, and extracellular signal-regulated kinases (ERK) activation. As such, these compounds are useful pharmacological tools to probe the molecular pharmacology of the δ receptor and to explore the therapeutic potential of δ PAMs in diseases such as chronic pain and depression.

  16. Negative allosteric modulation of nicotinic acetylcholine receptors blocks nicotine self-administration in rats.

    PubMed

    Yoshimura, Ryan F; Hogenkamp, Derk J; Li, Wen Y; Tran, Minhtam B; Belluzzi, James D; Whittemore, Edward R; Leslie, Frances M; Gee, Kelvin W

    2007-12-01

    Drugs that antagonize nicotinic acetylcholine receptors (nAChRs) can be used to inhibit nicotine-induced behavior in both humans and animals. The aim of our experiments is to establish a proof-of-principle that antagonism of nAChRs by negative allosteric modulation can alter behavior in a relevant animal model of addiction, nicotine self-administration. We have identified a novel, negative allosteric modulator of nAChRs, UCI-30002 [N-(1,2,3,4-tetrahydro-1-naphthyl)-4-nitroaniline], with selectivity for the major neuronal nAChR subtypes over muscle-type nAChRs. After systemic administration, UCI-30002 significantly reduces nicotine self-administration in rats on both fixed ratio and progressive ratio schedules of reinforcement. The minimum effective dose that significantly alters nicotine self-administration corresponds to brain concentrations of UCI-30002 that produce at least 30% inhibition of the major neuronal nAChR subtypes measured in vitro. UCI-30002 has no effect on responding for food reinforcement in rats on either type of schedule, indicating that there is no effect on general responding or natural reward. UCI-30002 represents validation of the concept that negative allosteric modulators may have significant benefits as a strategy for treating nicotine addiction and encourages the development of subtype-selective modulators.

  17. 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.

  18. Allosteric Inhibition of Phosphoenolpyruvate Carboxylases is Determined by a Single Amino Acid Residue in Cyanobacteria

    PubMed Central

    Takeya, Masahiro; Hirai, Masami Yokota; Osanai, Takashi

    2017-01-01

    Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme for CO2 fixation and primary metabolism in photosynthetic organisms including cyanobacteria. The kinetics and allosteric regulation of PEPCs have been studied in many organisms, but the biochemical properties of PEPC in the unicellular, non-nitrogen-fixing cyanobacterium Synechocystis sp. PCC 6803 have not been clarified. In this study, biochemical analysis revealed that the optimum pH and temperature of Synechocystis 6803 PEPC proteins were 7.3 and 30 °C, respectively. Synechocystis 6803 PEPC was found to be tolerant to allosteric inhibition by several metabolic effectors such as malate, aspartate, and fumarate compared with other cyanobacterial PEPCs. Comparative sequence and biochemical analysis showed that substitution of the glutamate residue at position 954 with lysine altered the enzyme so that it was inhibited by malate, aspartate, and fumarate. PEPC of the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 was purified, and its activity was inhibited in the presence of malate. Substitution of the lysine at position 946 (equivalent to position 954 in Synechocystis 6803) with glutamate made Anabaena 7120 PEPC tolerant to malate. These results demonstrate that the allosteric regulation of PEPC in cyanobacteria is determined by a single amino acid residue, a characteristic that is conserved in different orders. PMID:28117365

  19. 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

  20. Transracial Communication.

    ERIC Educational Resources Information Center

    Smith, Arthur L.

    This book explores and explains communication among different racial groups within the scope of existing communication theory. Following a brief introduction, chapters cover "Directions in Transracial Communication" (definitions, process, structurization, and purpose); "Culture and Transracial Communication" (a viewpoint on…

  1. Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations.

    PubMed Central

    Fang, Jie; Hsu, Betty Y L; MacMullen, Courtney M; Poncz, Mortimer; Smith, Thomas J; Stanley, Charles A

    2002-01-01

    Glutamate dehydrogenase (GDH) catalyses the reversible oxidative deamination of l-glutamate to 2-oxoglutarate in the mitochondrial matrix. In mammals, this enzyme is highly regulated by allosteric effectors. The major allosteric activator and inhibitor are ADP and GTP, respectively; allosteric activation by leucine may play an important role in amino acid-stimulated insulin secretion. The physiological significance of this regulation has been highlighted by the identification of children with an unusual hyperinsulinism/hyperammonaemia syndrome associated with dominant mutations in GDH that cause a loss in GTP inhibition. In order to determine the effects of these mutations on the function of the human GDH homohexamer, we studied the expression, purification and characterization of two of these regulatory mutations (H454Y, which affects the putative GTP-binding site, and S448P, which affects the antenna region) and a mutation designed to alter the putative binding site for ADP (R463A). The sensitivity to GTP inhibition was impaired markedly in the purified H454Y (ED(50), 210 microM) and S448P (ED(50), 3.1 microM) human GDH mutants compared with the wild-type human GDH (ED(50), 42 nM) or GDH isolated from heterozygous patient cells (ED(50), 290 and 280 nM, respectively). Sensitivity to ADP or leucine stimulation was unaffected by these mutations, confirming that they interfere specifically with the inhibitory GTP-binding site. Conversely, the R463A mutation completely eliminated ADP activation of human GDH, but had little effect on either GTP inhibition or leucine activation. The effects of these three mutations on ATP regulation indicated that this nucleotide inhibits human GDH through binding of its triphosphate tail to the GTP site and, at higher concentrations, activates the enzyme through binding of the nucleotide to the ADP site. These data confirm the assignment of the GTP and ADP allosteric regulatory sites on GDH based on X-ray crystallography and provide

  2. The core of allosteric motion in Thermus caldophilus L-lactate dehydrogenase.

    PubMed

    Ikehara, Yoko; Arai, Kazuhito; Furukawa, Nayuta; Ohno, Tadashi; Miyake, Tatsuya; Fushinobu, Shinya; Nakajima, Masahiro; Miyanaga, Akimasa; Taguchi, Hayao

    2014-11-07

    For Thermus caldophilus L-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S(0.5) value 10(3)-fold and increased the V(max) value 4-fold at 30 °C and pH 7.0, indicating that TcLDH has a much more T state-sided allosteric equilibrium than Thermus thermophilus L-lactate dehydrogenase, which has only two amino acid replacements, A154G and H179Y. The inactive (T) and active (R) state structures of TcLDH were determined at 1.8 and 2.0 Å resolution, respectively. The structures indicated that two mobile regions, MR1 (positions 172-185) and MR2 (positions 211-221), form a compact core for allosteric motion, and His(179) of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased V(max) 4-fold but reduced pyruvate S(0.5) only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase V(max), but 10(2)-reduced pyruvate S(0.5), and additively increased the FBP-independent activity of the Q4(R) enzyme. The two types of mutation consistently increased the thermal stability of the enzyme. The MR1-MR2 area is a positively charged cluster, and its center approaches another positively charged cluster (N domain cluster) across the Q-axis subunit interface by 5 Å, when the enzyme undergoes the T to R transition. Structural and kinetic analyses thus revealed the simple and unique allosteric machinery of TcLDH, where the MR1-MR2 area pivotally moves during the allosteric motion and mediates the allosteric equilibrium through electrostatic repulsion within the protein molecule.

  3. 5-Chloroindole: a potent allosteric modulator of the 5-HT3 receptor

    PubMed Central

    Newman, Amy S; Batis, Nikolaos; Grafton, Gillian; Caputo, Francesca; Brady, Catherine A; Lambert, Jeremy J; Peters, John A; Gordon, John; Brain, Keith L; Powell, Andrew D; Barnes, Nicholas M

    2013-01-01

    Background and Purpose The 5-HT3 receptor is a ligand-gated ion channel that is modulated allosterically by various compounds including colchicine, alcohols and volatile anaesthetics. However the positive allosteric modulators (PAMs) identified to date have low affinity, which hinders investigation because of non-selective effects at pharmacologically active concentrations. The present study identifies 5-chloroindole (Cl-indole) as a potent PAM of the 5-HT3 receptor. Experimental Approach 5-HT3 receptor function was assessed by the increase in intracellular calcium and single-cell electrophysiological recordings in HEK293 cells stably expressing the h5-HT3A receptor and also the mouse native 5-HT3 receptor that increases neuronal contraction of bladder smooth muscle. Key Results Cl-indole (1–100 μM) potentiated agonist (5-HT) and particularly partial agonist [(S)-zacopride, DDP733, RR210, quipazine, dopamine, 2-methyl-5-HT, SR57227A, meta chlorophenyl biguanide] induced h5-HT3A receptor-mediated responses. This effect of Cl-indole was also apparent at the mouse native 5-HT3 receptor. Radioligand-binding studies identified that Cl-indole induced a small (∼twofold) increase in the apparent affinity of 5-HT for the h5-HT3A receptor, whereas there was no effect upon the affinity of the antagonist, tropisetron. Cl-indole was able to reactivate desensitized 5-HT3 receptors. In contrast to its effect on the 5-HT3 receptor, Cl-indole did not alter human nicotinic α7 receptor responses. Conclusions and Implications The present study identifies Cl-indole as a relatively potent and selective PAM of the 5-HT3 receptor; such compounds will aid investigation of the molecular basis for allosteric modulation of the 5-HT3 receptor and may assist the discovery of novel therapeutic drugs targeting this receptor. Linked Articles Recent reviews on allosteric modulation can be found at: Kenakin, T (2013). New concepts in pharmacological efficacy at 7TM receptors: IUPHAR Review 2

  4. Mechanistic analysis of the function of agonists and allosteric modulators: reconciling two-state and operational models

    PubMed Central

    Roche, David; Gil, Debora; Giraldo, Jesús

    2013-01-01

    Two-state and operational models of both agonism and allosterism are compared to identify and characterize common pharmacological parameters. To account for the receptor-dependent basal response, constitutive receptor activity is considered in the operational models. By arranging two-state models as the fraction of active receptors and operational models as the fractional response relative to the maximum effect of the system, a one-by-one correspondence between parameters is found. The comparative analysis allows a better understanding of complex allosteric interactions. In particular, the inclusion of constitutive receptor activity in the operational model of allosterism allows the characterization of modulators able to lower the basal response of the system; that is, allosteric modulators with negative intrinsic efficacy. Theoretical simulations and overall goodness of fit of the models to simulated data suggest that it is feasible to apply the models to experimental data and constitute one step forward in receptor theory formalism. Linked Articles Another recent review on allosteric modulation can be found at: Kenakin, T (2013). New concepts in pharmacological efficacy at 7TM receptors: IUPHAR Review 2. British Journal of Pharmacology 168: 554–575. doi: 10.1111/j.1476-5381.2012.02223.x And in this issue of BJP there is an article on a new allosteric modulator: Newman AS, Batis N, Grafton G, Caputo F, Brady CA, Lambert J, Peters JA, Gordon J, Brain KL, Powell AD and Barnes NM (2013). 5-Chloroindole: a potent allosteric modulator of the 5-HT3 receptor. British Journal of Pharmacology 169: 1228–1238. doi: 10.1111/bph.12213 PMID:23647200

  5. Communication Graph Generator for Parallel Programs

    SciTech Connect

    2014-04-08

    Graphator is a collection of relatively simple sequential programs that generate communication graphs/matrices for commonly occurring patterns in parallel programs. Currently, there is support for five communication patterns: two-dimensional 4-point stencil, four-dimensional 8-point stencil, all-to-alls over sub-communicators, random near-neighbor communication, and near-neighbor communication.

  6. 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

  7. AIM for Allostery: Using the Ising Model to Understand Information Processing and Transmission in Allosteric Biomolecular Systems.

    PubMed

    LeVine, Michael V; Weinstein, Harel

    2015-05-01

    In performing their biological functions, molecular machines must process and transmit information with high fidelity. Information transmission requires dynamic coupling between the conformations of discrete structural components within the protein positioned far from one another on the molecular scale. This type of biomolecular "action at a distance" is termed allostery. Although allostery is ubiquitous in biological regulation and signal transduction, its treatment in theoretical models has mostly eschewed quantitative descriptions involving the system's underlying structural components and their interactions. Here, we show how Ising models can be used to formulate an approach to allostery in a structural context of interactions between the constitutive components by building simple allosteric constructs we termed Allosteric Ising Models (AIMs). We introduce the use of AIMs in analytical and numerical calculations that relate thermodynamic descriptions of allostery to the structural context, and then show that many fundamental properties of allostery, such as the multiplicative property of parallel allosteric channels, are revealed from the analysis of such models. The power of exploring mechanistic structural models of allosteric function in more complex systems by using AIMs is demonstrated by building a model of allosteric signaling for an experimentally well-characterized asymmetric homodimer of the dopamine D2 receptor.

  8. 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.

  9. The Five-To-Six-Coordination Transition of Ferric Human Serum Heme-Albumin Is Allosterically-Modulated by Ibuprofen and Warfarin: A Combined XAS and MD Study

    PubMed Central

    Bionducci, Monica; Fanali, Gabriella; Meli, Massimiliano; Colombo, Giorgio; Fasano, Mauro; Ascenzi, Paolo; Mobilio, Settimio

    2014-01-01

    Human serum albumin (HSA) is involved physiologically in heme scavenging; in turn, heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, the allosteric effect of ibuprofen and warfarin on the local atomic structure around the ferric heme-Fe (heme-Fe(III)) atom of HSA-heme-Fe (HSA-heme-Fe(III)) has been probed by Fe-K edge X-ray absorption spectroscopy (XAS). The quantitative analysis of the Fe-K edge extended X-ray absorption fine structure (EXAFS) signals and modeling of the near edge (XANES) spectral features demonstrated that warfarin and ibuprofen binding modify the local structure of the heme-Fe(III). Combined XAS data analysis and targeted molecular dynamics (MD) simulations provided atomic resolution insights of protein structural rearrangements required to accommodate the heme-Fe(III) upon ibuprofen and warfarin binding. In the absence of drugs, the heme-Fe(III) atom is penta-coordinated having distorted 4+1 configuration made by the nitrogen atoms of the porphyrin ring and the oxygen phenoxy atom of the Tyr161 residue. MD simulations show that ibuprofen and warfarin association to the secondary fatty acid (FA) binding site 2 (FA2) induces a reorientation of domain I of HSA-heme-Fe(III), this leads to the redirection of the His146 residue providing an additional bond to the heme-Fe(III) atom, providing the 5+1 configuration. The comparison of Fe-K edge XANES spectra calculated using MD structures with those obtained experimentally confirms the reliability of the proposed structural model. As a whole, combining XAS and MD simulations it has been possible to provide a reliable model of the heme-Fe(III) atom coordination state and to understand the complex allosteric transition occurring in HSA-heme-Fe(III) upon ibuprofen and warfarin binding. PMID:25153171

  10. Group II metabotropic glutamate receptor type 2 allosteric potentiators prevent sodium lactate-induced panic-like response in panic-vulnerable rats

    PubMed Central

    Johnson, Philip L; Fitz, Stephanie D; Engleman, Eric A; Svensson, Kjell A; Schkeryantz, Jeffrey M; Shekhar, Anantha

    2015-01-01

    Rats with chronic inhibition of GABA synthesis by infusion of l-allyglycine, a glutamic acid decarboxylase inhibitor, into their dorsomedial/perifornical hypothalamus are anxious and exhibit panic-like cardio-respiratory responses to treatment with intravenous (i.v.) sodium lactate (NaLac) infusions, in a manner similar to what occurs in patients with panic disorder. We previously showed that either NMDA receptor antagonists or metabotropic glutamate receptor type 2/3 receptor agonists can block such a NaLac response, suggesting that a glutamate mechanism is contributing to this panic-like state. Using this animal model of panic, we tested the efficacy of CBiPES and THIIC, which are selective group II metabotropic glutamate type 2 receptor allosteric potentiators (at 10–30mg/kg i.p.), in preventing NaLac-induced panic-like behavioral and cardiovascular responses. The positive control was alprazolam (3mg/kg i.p.), a clinically effective anti-panic benzodiazepine. As predicted, panic-prone rats given a NaLac challenge displayed NaLac-induced panic-like cardiovascular (i.e. tachycardia and hypertensive) responses and “anxiety” (i.e. decreased social interaction time) and “flight” (i.e. increased locomotion) -associated behaviors; however, systemic injection of the panic-prone rats with CBiPES, THIIC or alprazolam prior to the NaLac dose blocked all NaLac-induced panic-like behaviors and cardiovascular responses. These data suggested that in a rat animal model, selective group II metabotropic glutamate type 2 receptor allosteric potentiators show an anti-panic efficacy similar to alprazolam. PMID:22914798

  11. Regulation by membrane fluidity of the allosteric behavior of the (Ca2)-adenosine triphosphatase from Escherichia coli.

    PubMed

    Siñeriz, F; Bloj, B; Farías, R N; Trucco, R E

    1973-09-01

    The allosteric properties of the membrane-bound (Ca(2+))-adenosine triphosphatase of an unsaturated fatty acid auxotroph of Escherichia coli were studied in membranes with different fatty acid compositions. The Hill coefficient of the inhibition by Na(+) ranged from 1.4, in the case where the auxotroph was grown with cis-vaccenic acid as supplement, to 2.8 when grown on linolenic acid. The results indicate that no fatty acid is particularly involved in the allosteric phenomena. A correlation between the values of the Hill coefficient and the double bond index or the ratio of the double bond index saturated to the fatty acids of the membrane was found. These facts are interpreted as a modulation by the membrane fluidity of the allosteric behavior of the membrane-bound enzyme. The general biological character of this phenomenon is discussed in this paper.

  12. Satellite Communication.

    ERIC Educational Resources Information Center

    Technology Teacher, 1985

    1985-01-01

    Presents a discussion of communication satellites: explains the principles of satellite communication, describes examples of how governments and industries are currently applying communication satellites, analyzes issues confronting satellite communication, links mathematics and science to the study of satellite communication, and applies…

  13. Subunit Interfaces Contribute Differently to Activation and Allosteric Modulation of Neuronal Nicotinic Acetylcholine Receptors

    PubMed Central

    Short, Caitlin A.; Cao, Angela T.; Wingfield, Molly A.; Doers, Matthew E.; Jobe, Emily M.; Wang, Nan; Levandoski, Mark M.

    2015-01-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in the nervous system and are implicated in many normal and pathological processes. The structural determinants of allostery in nAChRs are not well understood. One class of nAChR allosteric modulators, including the small molecule morantel (Mor), acts from a site that is structurally homologous to the canonical agonist site but exists in the β(+)/α(–) subunit interface. We hypothesized that all nAChR subunits move with respect to each other during channel activation and allosteric modulation. We therefore studied five pairs of residues predicted to span the interfaces of α3β2 receptors, one at the agonist interface and four at the modulator interface. Substituting cysteines in these positions, we used disulfide trapping to perturb receptor function. The pair α3Y168-β2D190, involving the C loop region of the β2 subunit, mediates modulation and agonist activation, because evoked currents were reduced up to 50% following oxidation (H2O2) treatment. The pair α3S125-β2Q39, below the canonical site, is also involved in channel activation, in accord with previous studies of the muscle-type receptor; however, the pair is differentially sensitive to ACh activation and Mor modulation (currents decreased 60% and 80%, respectively). The pairs α3Q37-β2A127 and α3E173-β2R46, both in the non-canonical interface, showed increased currents following oxidation, suggesting that subunit movements are not symmetrical. Together, our results from disulfide trapping and further mutation analysis indicate that subunit interface movement is important for allosteric modulation of nAChRs, but that the two types of interfaces contribute unequally to receptor activation. PMID:25486620

  14. An allosteric role for receptor activity-modifying proteins in defining GPCR pharmacology

    PubMed Central

    J Gingell, Joseph; Simms, John; Barwell, James; Poyner, David R; Watkins, Harriet A; Pioszak, Augen A; Sexton, Patrick M; Hay, Debbie L

    2016-01-01

    G protein-coupled receptors are allosteric proteins that control transmission of external signals to regulate cellular response. Although agonist binding promotes canonical G protein signalling transmitted through conformational changes, G protein-coupled receptors also interact with other proteins. These include other G protein-coupled receptors, other receptors and channels, regulatory proteins and receptor-modifying proteins, notably receptor activity-modifying proteins (RAMPs). RAMPs have at least 11 G protein-coupled receptor partners, including many class B G protein-coupled receptors. Prototypic is the calcitonin receptor, with altered ligand specificity when co-expressed with RAMPs. To gain molecular insight into the consequences of this protein–protein interaction, we combined molecular modelling with mutagenesis of the calcitonin receptor extracellular domain, assessed in ligand binding and functional assays. Although some calcitonin receptor residues are universally important for peptide interactions (calcitonin, amylin and calcitonin gene-related peptide) in calcitonin receptor alone or with receptor activity-modifying protein, others have RAMP-dependent effects, whereby mutations decreased amylin/calcitonin gene-related peptide potency substantially only when RAMP was present. Remarkably, the key residues were completely conserved between calcitonin receptor and AMY receptors, and between subtypes of AMY receptor that have different ligand preferences. Mutations at the interface between calcitonin receptor and RAMP affected ligand pharmacology in a RAMP-dependent manner, suggesting that RAMP may allosterically influence the calcitonin receptor conformation. Supporting this, molecular dynamics simulations suggested that the calcitonin receptor extracellular N-terminal domain is more flexible in the presence of receptor activity-modifying protein 1. Thus, RAMPs may act in an allosteric manner to generate a spectrum of unique calcitonin receptor

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

    PubMed

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

    2015-09-01

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

  16. 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.

  17. 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

  18. 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.

  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

    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.

  20. 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.

  1. Positive allosteric modulators of the μ-opioid receptor: a novel approach for future pain medications

    PubMed Central

    Burford, N T; Traynor, J R; Alt, A

    2015-01-01

    Morphine and other agonists of the μ-opioid receptor are used clinically for acute and chronic pain relief and are considered to be the gold standard for pain medication. However, these opioids also have significant side effects, which are also mediated via activation of the μ-opioid receptor. Since the latter half of the twentieth century, researchers have sought to tease apart the mechanisms underlying analgesia, tolerance and dependence, with the hope of designing drugs with fewer side effects. These efforts have revolved around the design of orthosteric agonists with differing pharmacokinetic properties and/or selectivity profiles for the different opioid receptor types. Recently, μ-opioid receptor-positive allosteric modulators (μ-PAMs) were identified, which bind to a (allosteric) site on the μ-opioid receptor separate from the orthosteric site that binds an endogenous agonist. These allosteric modulators have little or no detectable functional activity when bound to the receptor in the absence of orthosteric agonist, but can potentiate the activity of bound orthosteric agonist, seen as an increase in apparent potency and/or efficacy of the orthosteric agonist. In this review, we describe the potential advantages that a μ-PAM approach might bring to the design of novel therapeutics for pain that may lack the side effects currently associated with opioid therapy. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24460691

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

    PubMed Central

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

    2016-01-01

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

  3. 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.

  4. Octahydropyrrolo[3,4-c]pyrrole negative allosteric modulators of mGlu1

    PubMed Central

    Manka, Jason T.; Rodriguez, Alice L.; Morrison, Ryan D.; Venable, Daryl F.; Cho, Hyekyung P.; Blobaum, Anna L.; Daniels, J. Scott; Niswender, Colleen M.; Conn, P. Jeffrey; Lindsley, Craig W.; Emmitte, Kyle A.

    2014-01-01

    Development of SAR in an octahydropyrrolo[3,4-c]pyrrole series of negative allosteric modulators of mGlu1 using a functional cell-based assay is described in this Letter. The octahydropyrrolo[3,4-c]pyrrole scaffold was chosen as an isosteric replacement for the piperazine ring found in the initial hit compound. Characterization of selected compounds in protein binding assays was used to identify the most promising analogs, which were then profiled in P450 inhibition assays in order to further assess the potential for drug-likeness within this series of compounds. PMID:23932792

  5. Allosteric mechanisms of G protein coupled receptor signaling: a structural perspective

    PubMed Central

    Thaker, Tarjani M.; Kaya, Ali I.; Preininger, Anita M.; Hamm, Heidi E.; Iverson, T.M.

    2012-01-01

    G protein-Coupled Receptors (GPCRs) use a complex series of intramolecular conformational changes to couple agonist binding to the binding and activation of cognate heterotrimeric G protein (Gαβγ). The mechanisms underlying this long-range activation have been identified using a variety of biochemical and structural approaches and have primarily used visual signal transduction via the GPCR rhodopsin and cognate heterotrimeric G protein transducin (Gt) as a model system. In this chapter, we will review the methods that have revealed allosteric signaling through rhodopsin and transducin. These methods can be applied to a variety of GPCR-mediated signaling pathways. PMID:22052489

  6. Positive Allosteric Modulators of Metabotropic Glutamate 2 Receptors in Schizophrenia Treatment

    PubMed Central

    Ellaithy, Amr; Younkin, Jason; Gonzalez-Maeso, Javier; Logothetis, Diomedes E.

    2015-01-01

    The last two decades have witnessed a rise in the “NMDA receptor hypofunction” hypothesis for schizophrenia, a devastating disorder that affects around 1% of the population worldwide. A variety of presynaptic, postsynaptic and regulatory proteins involved in glutamatergic signaling have thus been proposed as potential therapeutic targets. This Review focuses on positive allosteric modulation of metabotropic glutamate 2 receptors (mGlu2Rs) and discusses how recent preclinical epigenetic data may provide a molecular explanation for the discrepant results of clinical studies, further stimulating the field to exploit the promise of mGlu2R as a target for schizophrenia treatment. PMID:26148747

  7. 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

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

    PubMed Central

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

    2012-01-01

    During the stringent response, Escherichia coli enzyme RelA produces the ppGpp alarmone, which in turn regulates transcription, translation and replication. We show that ppGpp dramatically increases the turnover rate of its own ribosome-dependent synthesis by RelA, resulting in direct positive regulation of an enzyme by its product. Positive allosteric regulation therefore constitutes a new mechanism of enzyme activation. By integrating the output of individual RelA molecules and ppGpp degradation pathways, this regulatory circuit contributes to a fast and coordinated transition to stringency. PMID:22814757

  9. Energetics of allosteric negative coupling in the zinc sensor S. aureus CzrA.

    PubMed

    Grossoehme, Nicholas E; Giedroc, David P

    2009-12-16

    The linked equilibria of an allosterically regulated protein are defined by the structures, residue-specific dynamics and global energetics of interconversion among all relevant allosteric states. Here, we use isothermal titration calorimetry (ITC) to probe the global thermodynamics of allosteric negative regulation of the binding of the paradigm ArsR-family zinc sensing repressor Staphylococcus aureus CzrA to the czr DNA operator (CzrO) by Zn(2+). Zn(2+) binds to the two identical binding sites on the free CzrA homodimer in two discernible steps. A larger entropic driving force Delta(-TDeltaS) of -4.7 kcal mol(-1) and a more negative DeltaC(p) characterize the binding of the first Zn(2+) relative to the second. These features suggest a modest structural transition in forming the Zn(1) state followed by a quenching of the internal dynamics on filling the second zinc site, which collectively drive homotropic negative cooperativity of Zn(2+) binding (Delta(DeltaG) = 1.8 kcal mol(-1)). Negative homotropic cooperativity also characterizes Zn(2+) binding to the CzrA*CzrO complex (Delta(DeltaG) = 1.3 kcal mol(-1)), although the underlying energetics are vastly different, with homotropic Delta(DeltaH) and Delta(-TDeltaS) values both small and slightly positive. In short, Zn(2+) binding to the complex fails to induce a large structural or dynamical change in the CzrA bound to the operator. The strong heterotropic negative linkage in this system (DeltaG(c)(t) = 6.3 kcal mol(-1)) therefore derives from the vastly different structures of the apo-CzrA and CzrA*CzrO reference states (DeltaH(c)(t) = 9.4 kcal mol(-1)) in a way that is reinforced by a global rigidification of the allosterically inhibited Zn(2) state off the DNA (TDeltaS(c)(t) = -3.1 kcal mol(-1), i.e., DeltaS(c)(t) > 0). The implications of these findings for other metalloregulatory proteins are discussed.

  10. 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

  11. 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.

  12. Discovery of Peptidomimetic Ligands of EED as Allosteric Inhibitors of PRC2.

    PubMed

    Barnash, Kimberly D; The, Juliana; Norris-Drouin, Jacqueline L; Cholensky, Stephanie H; Worley, Beau M; Li, Fengling; Stuckey, Jacob I; Brown, Peter J; Vedadi, Masoud; Arrowsmith, Cheryl H; Frye, Stephen V; James, Lindsey I

    2017-03-13

    The function of EED within polycomb repressive complex 2 (PRC2) is mediated by a complex network of protein-protein interactions. Allosteric activation of PRC2 by binding of methylated proteins to the embryonic ectoderm development (EED) aromatic cage is essential for full catalytic activity, but details of this regulation are not fully understood. EED's recognition of the product of PRC2 activity, histone H3 lysine 27 trimethylation (H3K27me3), stimulates PRC2 methyltransferase activity at adjacent nucleosomes leading to H3K27me3 propagation and, ultimately, gene repression. By coupling combinatorial chemistry and structure-based design, we optimized a low-affinity methylated jumonji, AT-rich interactive domain 2 (Jarid2) peptide to a smaller, more potent peptidomimetic ligand (Kd = 1.14 ± 0.14 μM) of the aromatic cage of EED. Our strategy illustrates the effectiveness of applying combinatorial chemistry to achieve both ligand potency and property optimization. Furthermore, the resulting ligands, UNC5114 and UNC5115, demonstrate that targeted disruption of EED's reader function can lead to allosteric inhibition of PRC2 catalytic activity.

  13. Allosteric drug discrimination is coupled to mechanochemical changes in the kinesin-5 motor core.

    PubMed

    Kim, Elizabeth D; Buckley, Rebecca; Learman, Sarah; Richard, Jessica; Parke, Courtney; Worthylake, David K; Wojcik, Edward J; Walker, Richard A; Kim, Sunyoung

    2010-06-11

    Essential in mitosis, the human Kinesin-5 protein is a target for >80 classes of allosteric compounds that bind to a surface-exposed site formed by the L5 loop. Not established is why there are differing efficacies in drug inhibition. Here we compare the ligand-bound states of two L5-directed inhibitors against 15 Kinesin-5 mutants by ATPase assays and IR spectroscopy. Biochemical kinetics uncovers functional differences between individual residues at the N or C termini of the L5 loop. Infrared evaluation of solution structures and multivariate analysis of the vibrational spectra reveal that mutation and/or ligand binding not only can remodel the allosteric binding surface but also can transmit long range effects. Changes in L5-localized 3(10) helix and disordered content, regardless of substitution or drug potency, are experimentally detected. Principal component analysis couples these local structural events to two types of rearrangements in beta-sheet hydrogen bonding. These transformations in beta-sheet contacts are correlated with inhibitory drug response and are corroborated by wild type Kinesin-5 crystal structures. Despite considerable evolutionary divergence, our data directly support a theorized conserved element for long distance mechanochemical coupling in kinesin, myosin, and F(1)-ATPase. These findings also suggest that these relatively rapid IR approaches can provide structural biomarkers for clinical determination of drug sensitivity and drug efficacy in nucleotide triphosphatases.

  14. Herbacetin is a novel allosteric inhibitor of ornithine decarboxylase with antitumor activity

    PubMed Central

    Lee, Mee-Hyun; Oi, Naomi; Lim, Do Young; Kim, Myoung Ok; Cho, Young-Yeon; Pugliese, Angelo; Shim, Jung-Hyun; Chen, Hanyong; Cho, Eun Jin; Kim, Jong-Eun; Kang, Sun Chul; Paul, Souren; Kang, Hee Eun; Jung, Ji Won; Lee, Sung-Young; Kim, Sung-Hyun; Reddy, Kanamata; Yeom, Young Il; Bode, Ann M; Dong, Zigang

    2015-01-01

    Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the first step of polyamine biosynthesis that is associated with cell growth and tumor formation. Existing catalytic inhibitors of ODC have lacked efficacy in clinical testing or displayed unacceptable toxicity. In this study, we report the identification of an effective and nontoxic allosteric inhibitor of ODC. Using computer docking simulation and an in vitro ODC enzyme assay, we identified herbacetin, a natural compound found in flax and other plants, as a novel ODC inhibitor. Mechanistic investigations defined aspartate 44 in ODC as critical for binding. Herbacetin exhibited potent anticancer activity in colon cancer cell lines expressing high levels of ODC. Intraperitoneal or oral administration of herbacetin effectively suppressed HCT116 xenograft tumor growth and also reduced the number and size of polyps in a mouse model of APC-driven colon cancer (ApcMin/+). Unlike the well established ODC inhibitor DFMO, herbacetin treatment was not associated with hearing loss. Taken together, our findings defined the natural product herbacetin as an allosteric inhibitor of ODC with chemopreventive and antitumor activity in preclinical models of colon cancer, prompting its further investigation in clinical trials. PMID:26676750

  15. Cholesterol-mediated allosteric regulation of the mitochondrial translocator protein structure

    PubMed Central

    Jaipuria, Garima; Leonov, Andrei; Giller, Karin; Vasa, Suresh Kumar; Jaremko, Łukasz; Jaremko, Mariusz; Linser, Rasmus; Becker, Stefan; Zweckstetter, Markus

    2017-01-01

    Cholesterol is an important regulator of membrane protein function. However, the exact mechanisms involved in this process are still not fully understood. Here we study how the tertiary and quaternary structure of the mitochondrial translocator protein TSPO, which binds cholesterol with nanomolar affinity, is affected by this sterol. Residue-specific analysis of TSPO by solid-state NMR spectroscopy reveals a dynamic monomer–dimer equilibrium of TSPO in the membrane. Binding of cholesterol to TSPO's cholesterol-recognition motif leads to structural changes across the protein that shifts the dynamic equilibrium towards the translocator monomer. Consistent with an allosteric mechanism, a mutation within the oligomerization interface perturbs transmembrane regions located up to 35 Å away from the interface, reaching TSPO's cholesterol-binding motif. The lower structural stability of the intervening transmembrane regions provides a mechanistic basis for signal transmission. Our study thus reveals an allosteric signal pathway that connects membrane protein tertiary and quaternary structure with cholesterol binding. PMID:28358007

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

    PubMed Central

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

    2015-01-01

    Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named “(p)ppGpp”] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) from Bacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (SN2) reaction. We show that pppGpp—but not ppGpp—positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles. PMID:26460002

  17. A conserved motif mediates both multimer formation and allosteric activation of phosphoglycerate mutase 5.

    PubMed

    Wilkins, Jordan M; McConnell, Cyrus; Tipton, Peter A; Hannink, Mark

    2014-09-05

    Phosphoglycerate mutase 5 (PGAM5) is an atypical mitochondrial Ser/Thr phosphatase that modulates mitochondrial dynamics and participates in both apoptotic and necrotic cell death. The mechanisms that regulate the phosphatase activity of PGAM5 are poorly understood. The C-terminal phosphoglycerate mutase domain of PGAM5 shares homology with the catalytic domains found in other members of the phosphoglycerate mutase family, including a conserved histidine that is absolutely required for catalytic activity. However, this conserved domain is not sufficient for maximal phosphatase activity. We have identified a highly conserved amino acid motif, WDXNWD, located within the unique N-terminal region, which is required for assembly of PGAM5 into large multimeric complexes. Alanine substitutions within the WDXNWD motif abolish the formation of multimeric complexes and markedly reduce phosphatase activity of PGAM5. A peptide containing the WDXNWD motif dissociates the multimeric complex and reduces but does not fully abolish phosphatase activity. Addition of the WDXNWD-containing peptide in trans to a mutant PGAM5 protein lacking the WDXNWD motif markedly increases phosphatase activity of the mutant protein. Our results are consistent with an intermolecular allosteric regulation mechanism for the phosphatase activity of PGAM5, in which the assembly of PGAM5 into multimeric complexes, mediated by the WDXNWD motif, results in maximal activation of phosphatase activity. Our results suggest the possibility of identifying small molecules that function as allosteric regulators of the phosphatase activity of PGAM5.

  18. Activation of nanoscale allosteric protein domain motion revealed by neutron spin echo spectroscopy

    NASA Astrophysics Data System (ADS)

    Bu, Zimei; Farago, Bela; Callaway, David

    2012-02-01

    NHERF1 is a multi-domain scaffolding protein that assembles the signaling complexes, and regulates the cell surface expression and endocytic recycling of a variety of membrane proteins. The ability of the two PDZ domains in NHERF1 to assemble protein complexes is allosterically modulated by a membrane-cytoskeleton linker protein ezrin, whose binding site is located as far as 110 angstroms away from the PDZ domains. Here, using neutron spin echo (NSE) spectroscopy, selective deuterium labeling, and theoretical analyses, we reveal the activation of interdomain motion in NHERF1 on nanometer length scales and on sub-microsecond time scales upon forming a complex with ezrin. We show that a much simplified coarse-grained model is sufficient to describe inter-domain motion of a multi-domain protein or protein complex. We expect that future NSE experiments will benefit by exploiting our approach of selective deuteration to resolve the specific domain motions of interest from a plethora of global translational and rotational motions. The results demonstrate that propagation of allosteric signals to distal sites involves the activation of long-range coupled domain motions on submicrosecond time scales, and that these coupled motions can be distinguished and characterized by NSE.

  19. Positive Allosteric Modulation of Insect Olfactory Receptor Function by ORco Agonists

    PubMed Central

    Tsitoura, Panagiota; Iatrou, Kostas

    2016-01-01

    Insect olfactory receptors (ORs) are heteromeric ligand-gated cation channels composed of a common olfactory receptor subunit (ORco) and a variable subunit (ORx) of as yet unknown structures and undetermined stoichiometries. In this study, we examined the allosteric modulation exerted on Anopheles gambiae heteromeric ORx/ORco olfactory receptors in vitro by a specific class of ORco agonists (OAs) comprising ORcoRAM2 and VUAA1. High OA concentrations produced stronger functional responses in cells expressing heteromeric receptor channels relative to cells expressing ORco alone. These OA-induced responses of ORx/ORco channels were also notably much stronger than those obtained upon administration of ORx-specific ligands to the same receptors. Most importantly, small concentrations of OAs were found to act as strong potentiators of ORx/ORco function, increasing dramatically both the efficacy and potency of ORx-specific odorants. These results suggest that insect heteromeric ORs are highly dynamic complexes adopting different conformations that change in a concerted fashion as a result of the interplay between the subunits of the oligomeric assemblies, and that allosteric modulation may constitute an important element in the modulation and fining tuning of olfactory reception function. PMID:28018173

  20. 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

  1. 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.

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

    PubMed

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

    2014-10-27

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

  3. Structure, Dynamics, and Allosteric Potential of Ionotropic Glutamate Receptor N-Terminal Domains

    PubMed Central

    Krieger, James; Bahar, Ivet; Greger, Ingo H.

    2015-01-01

    Ionotropic glutamate receptors (iGluRs) are tetrameric cation channels that mediate synaptic transmission and plasticity. They have a unique modular architecture with four domains: the intracellular C-terminal domain (CTD) that is involved in synaptic targeting, the transmembrane domain (TMD) that forms the ion channel, the membrane-proximal ligand-binding domain (LBD) that binds agonists such as L-glutamate, and the distal N-terminal domain (NTD), whose function is the least clear. The extracellular portion, comprised of the LBD and NTD, is loosely arranged, mediating complex allosteric regulation and providing a rich target for drug development. Here, we briefly review recent work on iGluR NTD structure and dynamics, and further explore the allosteric potential for the NTD in AMPA-type iGluRs using coarse-grained simulations. We also investigate mechanisms underlying the established NTD allostery in NMDA-type iGluRs, as well as the fold-related metabotropic glutamate and GABAB receptors. We show that the clamshell motions intrinsically favored by the NTD bilobate fold are coupled to dimeric and higher-order rearrangements that impact the iGluR LBD and ultimately the TMD. Finally, we explore the dynamics of intact iGluRs and describe how it might affect receptor operation in a synaptic environment. PMID:26255587

  4. Designing Allosteric Inhibitors of Factor XIa. Lessons from the Interactions of Sulfated Pentagalloylglucopyranosides

    PubMed Central

    2015-01-01

    We recently introduced sulfated pentagalloylglucopyranoside (SPGG) as an allosteric inhibitor of factor XIa (FXIa) (Al-Horani et al., J. Med Chem.2013, 56, 867–87823316863). To better understand the SPGG–FXIa interaction, we utilized eight SPGG variants and a range of biochemical techniques. The results reveal that SPGG’s sulfation level moderately affected FXIa inhibition potency and selectivity over thrombin and factor Xa. Variation in the anomeric configuration did not affect potency. Interestingly, zymogen factor XI bound SPGG with high affinity, suggesting its possible use as an antidote. Acrylamide quenching experiments suggested that SPGG induced significant conformational changes in the active site of FXIa. Inhibition studies in the presence of heparin showed marginal competition with highly sulfated SPGG variants but robust competition with less sulfated variants. Resolution of energetic contributions revealed that nonionic forces contribute nearly 87% of binding energy suggesting a strong possibility of specific interaction. Overall, the results indicate that SPGG may recognize more than one anion-binding, allosteric site on FXIa. An SPGG molecule containing approximately 10 sulfate groups on positions 2 through 6 of the pentagalloylglucopyranosyl scaffold may be the optimal FXIa inhibitor for further preclinical studies. PMID:24844380

  5. Allosteric substrate inhibition of Arabidopsis NAD-dependent malic enzyme 1 is released by fumarate.

    PubMed

    Tronconi, Marcos Ariel; Wheeler, Mariel Claudia Gerrard; Martinatto, Andrea; Zubimendi, Juan Pablo; Andreo, Carlos Santiago; Drincovich, María Fabiana

    2015-03-01

    Plant mitochondria can use L-malate and fumarate, which accumulate in large levels, as respiratory substrates. In part, this property is due to the presence of NAD-dependent malic enzymes (NAD-ME) with particular biochemical characteristics. Arabidopsis NAD-ME1 exhibits a non-hyperbolic behavior for the substrate L-malate, and its activity is strongly stimulated by fumarate. Here, the possible structural connection between these properties was explored through mutagenesis, kinetics, and fluorescence studies. The results indicated that NAD-ME1 has a regulatory site for L-malate that can also bind fumarate. L-Malate binding to this site elicits a sigmoidal and low substrate-affinity response, whereas fumarate binding turns NAD-ME1 into a hyperbolic and high substrate affinity enzyme. This effect was also observed when the allosteric site was either removed or altered. Hence, fumarate is not really an activator, but suppresses the inhibitory effect of l-malate. In addition, residues Arg50, Arg80 and Arg84 showed different roles in organic acid binding. These residues form a triad, which is the basis of the homo and heterotrophic effects that characterize NAD-ME1. The binding of L-malate and fumarate at the same allosteric site is herein reported for a malic enzyme and clearly indicates an important role of NAD-ME1 in processes that control flow of C4 organic acids in Arabidopsis mitochondrial metabolism.

  6. A Potent, Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 3 (PRMT3)**

    PubMed Central

    Kaniskan, H. Ümit; Szewczyk, Magdalena M.; Yu, Zhengtian; Eram, Mohammad S.; Yang, Xiaobao; Schmidt, Keith; Luo, Xiao; Dai, Miao; He, Feng; Zang, Irene; Lin, Ying; Kennedy, Steven; Li, Fengling; Dobrovetsky, Elena; Dong, Aiping; Smil, David; Min, Sun-Joon; Landon, Melissa; Lin-Jones, Jennifer; Huang, Xi-Ping; Roth, Bryan L.; Schapira, Matthieu; Atadja, Peter; Barsyte-Lovejoy, Dalia; Arrowsmith, Cheryl H.; Brown, Peter J.; Zhao, Kehao; Jin, Jian; Vedadi, Masoud

    2015-01-01

    PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is essential for maturation of ribosomes, may have a role in lipogenesis, and is implicated in several diseases. A potent, selective, and cell- active PRMT3 inhibitor would be a valuable tool for further investigating PRMT3 biology. Here we report the discovery of the first PRMT3 chemical probe, SGC707, by structure-based optimization of the allosteric PRMT3 inhibitors we reported previously, and thorough characterization of this probe in biochemical, biophysical, and cellular assays. SGC707 is a potent PRMT3 inhibitor (IC50 = 31 ± 2 nm, KD = 53 ± 2 nm) with outstanding selectivity (selective against 31 other methyltransferases and more than 250 non-epigenetic targets). The mechanism of action studies and crystal structure of the PRMT3-SGC707 complex confirm the allosteric inhibition mode. Importantly, SGC707 engages PRMT3 and potently inhibits its methyltransferase activity in cells. It is also bioavailable and suitable for animal studies. This well- characterized chemical probe is an excellent tool to further study the role of PRMT3 in health and disease. PMID:25728001

  7. Allosteric Features of KCNQ1 Gating Revealed by Alanine Scanning Mutagenesis

    PubMed Central

    Ma, Li-Juan; Ohmert, Iris; Vardanyan, Vitya

    2011-01-01

    Controlled opening and closing of an ion-selective pathway in response to changes of membrane potential is a fundamental feature of voltage-gated ion channels. In recent decades, various details of this process have been revealed with unprecedented precision based on studies of prototypic potassium channels. Though current scientific efforts are focused more on a thorough description of voltage-sensor movement, much less is known about the similarities and differences of the gating mechanisms among potassium channels. Here, we describe the peculiarities of the KCNQ1 gating process in parallel comparison to Shaker. We applied alanine scanning mutagenesis to the S4-S5 linker and pore region and followed the regularities of gating perturbations in KCNQ1. We found a fractional constitutive conductance for wild-type KCNQ1. This component increased significantly in mutants with considerably leftward-shifted steady-state activation curves. In contrast to Shaker, no correlation between V1/2 and Z parameters was observed for the voltage-dependent fraction of KCNQ1. Our experimental findings are explained by a simple allosteric gating scheme with voltage-driven and voltage-independent transitions. Allosteric features are discussed in the context of extreme gating adaptability of KCNQ1 upon interaction with KCNE β-subunits. PMID:21320432

  8. Long-range allosteric signaling in red light–regulated diguanylyl cyclases

    PubMed Central

    Gourinchas, Geoffrey; Etzl, Stefan; Göbl, Christoph; Vide, Uršula; Madl, Tobias; Winkler, Andreas

    2017-01-01

    Nature has evolved an astonishingly modular architecture of covalently linked protein domains with diverse functionalities to enable complex cellular networks that are critical for cell survival. The coupling of sensory modules with enzymatic effectors allows direct allosteric regulation of cellular signaling molecules in response to diverse stimuli. We present molecular details of red light–sensing bacteriophytochromes linked to cyclic dimeric guanosine monophosphate–producing diguanylyl cyclases. Elucidation of the first crystal structure of a full-length phytochrome with its enzymatic effector, in combination with the characterization of light-induced changes in conformational dynamics, reveals how allosteric light regulation is fine-tuned by the architecture and composition of the coiled-coil sensor-effector linker and also the central helical spine. We anticipate that consideration of molecular principles of sensor-effector coupling, going beyond the length of the characteristic linker, and the appreciation of dynamically driven allostery will open up new directions for the design of novel red light–regulated optogenetic tools. PMID:28275738

  9. Dynamics Correlation Network for Allosteric Switching of PreQ1 Riboswitch

    PubMed Central

    Wang, Wei; Jiang, Cheng; Zhang, Jinmai; Ye, Wei; Luo, Ray; Chen, Hai-Feng

    2016-01-01

    Riboswitches are a class of metabolism control elements mostly found in bacteria. Due to their fundamental importance in bacteria gene regulation, riboswitches have been proposed as antibacterial drug targets. Prequeuosine (preQ1) is the last free precursor in the biosynthetic pathway of queuosine that is crucial for translation efficiency and fidelity. However, the regulation mechanism for the preQ1 riboswitch remains unclear. Here we constructed fluctuation correlation network based on all-atom molecular dynamics simulations to reveal the regulation mechanism. The results suggest that the correlation network in the bound riboswitch is distinctly different from that in the apo riboswitch. The community network indicates that the information freely transfers from the binding site of preQ1 to the expression platform of the P3 helix in the bound riboswitch and the P3 helix is a bottleneck in the apo riboswitch. Thus, a hypothesis of “preQ1-binding induced allosteric switching” is proposed to link riboswitch and translation regulation. The community networks of mutants support this hypothesis. Finally, a possible allosteric pathway of A50-A51-A52-U10-A11-G12-G56 was also identified based on the shortest path algorithm and confirmed by mutations and network perturbation. The novel fluctuation network analysis method can be used as a general strategy in studies of riboswitch structure-function relationship. PMID:27484311

  10. Structural basis for the allosteric regulation and substrate recognition of human cytosolic 5'-nucleotidase II.

    PubMed

    Walldén, Karin; Nordlund, Pär

    2011-05-13

    Cytosolic 5'-nucleotidase II (cN-II) catalyzes the dephosphorylation of 6-hydroxypurine nucleoside 5'-monophosphates and participates in the regulation of purine nucleotide pools within the cell. It interferes with the phosphorylation-dependent activation of nucleoside analogues used in the treatment of cancer and viral diseases. It is allosterically activated by a number of phosphate-containing cellular metabolites such as ATP, diadenosine polyphosphates, and 2,3-bisphosphoglycerate, which couple its activity with the metabolic state of the cell. We present seven high-resolution structures of human cN-II, including a ligand-free form and complexes with various substrates and effectors. These structures reveal the structural basis for the allosteric activation of cN-II, uncovering a mechanism where an effector-induced disorder-to-order transition generates rearrangements within the catalytic site and the subsequent coordination of the catalytically essential magnesium. Central to the activation is the large transition of the catalytically essential Asp356. This study also provides the structural basis for the substrate specificity of cN-II, where Arg202, Asp206, and Phe157 seem to be important residues for purine/pyrimidine selectivity. These structures provide a comprehensive structural basis for the design of cN-II inhibitors. They also contribute to the understanding of how the nucleotide salvage pathway is regulated at a molecular level.

  11. Crystal structure of human cytosolic 5'-nucleotidase II: insights into allosteric regulation and substrate recognition.

    PubMed

    Walldén, Karin; Stenmark, Pål; Nyman, Tomas; Flodin, Susanne; Gräslund, Susanne; Loppnau, Peter; Bianchi, Vera; Nordlund, Pär

    2007-06-15

    Cytosolic 5'-nucleotidase II catalyzes the dephosphorylation of 6-hydroxypurine nucleoside 5'-monophosphates and regulates the IMP and GMP pools within the cell. It possesses phosphotransferase activity and thereby also catalyzes the reverse reaction. Both reactions are allosterically activated by adenine-based nucleotides and 2,3-bisphosphoglycerate. We have solved structures of cytosolic 5'-nucleotidase II as native protein (2.2 Angstrom) and in complex with adenosine (1.5 Angstrom) and beryllium trifluoride (2.15 Angstrom) The tetrameric enzyme is structurally similar to enzymes of the haloacid dehalogenase (HAD) superfamily, including mitochondrial 5'(3')-deoxyribonucleotidase and cytosolic 5'-nucleotidase III but possesses additional regulatory regions that contain two allosteric effector sites. At effector site 1 located near a subunit interface we modeled diadenosine tetraphosphate with one adenosine moiety in each subunit. This efficiently glues the tetramer subunits together in pairs. The model shows why diadenosine tetraphosphate but not diadenosine triphosphate activates the enzyme and supports a role for cN-II during apoptosis when the level of diadenosine tetraphosphate increases. We have also modeled 2,3-bisphosphoglycerate in effector site 1 using one phosphate site from each subunit. By comparing the structure of cytosolic 5'-nucleotidase II with that of mitochondrial 5'(3')-deoxyribonucleotidase in complex with dGMP, we identified residues involved in substrate recognition.

  12. Molecular mechanism of the allosteric enhancement of the umami taste sensation.

    PubMed

    Mouritsen, Ole G; Khandelia, Himanshu

    2012-09-01

    The fifth taste quality, umami, arises from binding of glutamate to the umami receptor T1R1/T1R3. The umami taste is enhanced several-fold upon addition of free nucleotides such as guanosine-5'-monophosphate (GMP) to glutamate-containing food. GMP may operate via binding to the ligand-binding domain of the T1R1 part of the umami receptor at an allosteric site. Using molecular dynamics simulations, we show that GMP can stabilize the closed (active) state of T1R1 by binding to the outer vestibule of the so-called Venus flytrap domain of the receptor. The transition between the closed and open conformations was accessed in the simulations. Using principal component analysis, we show that the dynamics of the Venus flytrap domain along the hinge-bending motion that activates signaling is dampened significantly upon binding of glutamate, and further slows down upon binding of GMP at an allosteric site, thus suggesting a molecular mechanism of cooperativity between GMP and glutamate.

  13. 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

  14. Allosteric Mechanisms of Molecular Machines at the Membrane: Transport by Sodium-Coupled Symporters.

    PubMed

    LeVine, Michael V; Cuendet, Michel A; Khelashvili, George; Weinstein, Harel

    2016-06-08

    Solute transport across cell membranes is ubiquitous in biology as an essential physiological process. Secondary active transporters couple the unfavorable process of solute transport against its concentration gradient to the energetically favorable transport of one or several ions. The study of such transporters over several decades indicates that their function involves complex allosteric mechanisms that are progressively being revealed in atomistic detail. We focus on two well-characterized sodium-coupled symporters: the bacterial amino acid transporter LeuT, which is the prototype for the "gated pore" mechanism in the mammalian synaptic monoamine transporters, and the archaeal GltPh, which is the prototype for the "elevator" mechanism in the mammalian excitatory amino acid transporters. We present the evidence for the role of allostery in the context of a quantitative formalism that can reconcile biochemical and biophysical data and thereby connects directly to recent insights into the molecular structure and dynamics of these proteins. We demonstrate that, while the structures and mechanisms of these transporters are very different, the available data suggest a common role of specific models of allostery in their functions. We argue that such allosteric mechanisms appear essential not only for sodium-coupled symport in general but also for the function of other types of molecular machines in the membrane.

  15. Structure and Energetics of Allosteric Regulation of HCN2 Ion Channels by Cyclic Nucleotides*

    PubMed Central

    DeBerg, Hannah A.; Brzovic, Peter S.; Flynn, Galen E.; Zagotta, William N.; Stoll, Stefan

    2016-01-01

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels play an important role in regulating electrical activity in the heart and brain. They are gated by the binding of cyclic nucleotides to a conserved, intracellular cyclic nucleotide-binding domain (CNBD), which is connected to the channel pore by a C-linker region. Binding of cyclic nucleotides increases the rate and extent of channel activation and shifts it to less hyperpolarized voltages. We probed the allosteric mechanism of different cyclic nucleotides on the CNBD and on channel gating. Electrophysiology experiments showed that cAMP, cGMP, and cCMP were effective agonists of the channel and produced similar increases in the extent of channel activation. In contrast, electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) on the isolated CNBD indicated that the induced conformational changes and the degrees of stabilization of the active conformation differed for the three cyclic nucleotides. We explain these results with a model where different allosteric mechanisms in the CNBD all converge to have the same effect on the C-linker and render all three cyclic nucleotides similarly potent activators of the channel. PMID:26559974

  16. Agonistic aptamer to the insulin receptor leads to biased signaling and functional selectivity through allosteric modulation

    PubMed Central

    Yunn, Na-Oh; Koh, Ara; Han, Seungmin; Lim, Jong Hun; Park, Sehoon; Lee, Jiyoun; Kim, Eui; Jang, Sung Key; Berggren, Per-Olof; Ryu, Sung Ho

    2015-01-01

    Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo. Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors. PMID:26245346

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

    PubMed Central

    Manna, Moutusi; Niemelä, Miia; Tynkkynen, Joona; Javanainen, Matti; Kulig, Waldemar; Müller, Daniel J; Rog, Tomasz; Vattulainen, Ilpo

    2016-01-01

    There is evidence that lipids can be allosteric regulators of membrane protein structure and activation. However, there are no data showing how exactly the regulation emerges from specific lipid-protein interactions. Here we show in atomistic detail how the human β2-adrenergic receptor (β2AR) – a prototypical G protein-coupled receptor – is modulated by cholesterol in an allosteric fashion. Extensive atomistic simulations show that cholesterol regulates β2AR by limiting its conformational variability. The mechanism of action is based on the binding of cholesterol at specific high-affinity sites located near the transmembrane helices 5–7 of the receptor. The alternative mechanism, where the β2AR conformation would be modulated by membrane-mediated interactions, plays only a minor role. Cholesterol analogues also bind to cholesterol binding sites and impede the structural flexibility of β2AR, however cholesterol generates the strongest effect. The results highlight the capacity of lipids to regulate the conformation of membrane receptors through specific interactions. DOI: http://dx.doi.org/10.7554/eLife.18432.001 PMID:27897972

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

    PubMed

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

    2016-02-17

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

  19. Modulation of γ-secretase specificity using small molecule allosteric inhibitors

    PubMed Central

    Shelton, Christopher C.; Zhu, Lei; Chau, Deming; Yang, Li; Wang, Rong; Djaballah, Hakim; Zheng, Hui; Li, Yue-Ming

    2009-01-01

    γ-Secretase cleaves multiple substrates within the transmembrane domain that include the amyloid precursor protein as well as the Notch family of receptors. These substrates are associated with Alzheimer disease and cancer. Despite extensive investigation of this protease, little is known regarding the regulation of γ-secretase specificity. To discover selective inhibitors for drug development and for probing the mechanisms of γ-secretase specificity, we screened chemical libraries and consequently developed a di-coumarin family of inhibitors that preferentially inhibit γ-secretase-mediated production of Aβ42 over other cleavage activities. These coumarin dimer-based compounds interact with γ-secretase by binding to an allosteric site. By developing a multiple photo-affinity probe approach, we demonstrate that this allosteric binding causes a conformational change within the active site of γ-secretase at the S2 and S1 sub-sites that leads to selective inhibition of Aβ42. In conclusion, by using these di-coumarin compounds, we reveal a mechanism by which γ-secretase specificity is regulated and provide insights into the molecular basis by which familial presenilin mutations may affect the active site and specificity of γ-secretase. Furthermore, this class of selective inhibitors provides the basis for development of Alzheimer disease therapeutic agents. PMID:19906985

  20. 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.

  1. Notes on stochastic (bio)-logic gates: computing with allosteric cooperativity

    NASA Astrophysics Data System (ADS)

    Agliari, Elena; Altavilla, Matteo; Barra, Adriano; Dello Schiavo, Lorenzo; Katz, Evgeny

    2015-05-01

    Recent experimental breakthroughs have finally allowed to implement in-vitro reaction kinetics (the so called enzyme based logic) which code for two-inputs logic gates and mimic the stochastic AND (and NAND) as well as the stochastic OR (and NOR). This accomplishment, together with the already-known single-input gates (performing as YES and NOT), provides a logic base and paves the way to the development of powerful biotechnological devices. However, as biochemical systems are always affected by the presence of noise (e.g. thermal), standard logic is not the correct theoretical reference framework, rather we show that statistical mechanics can work for this scope: here we formulate a complete statistical mechanical description of the Monod-Wyman-Changeaux allosteric model for both single and double ligand systems, with the purpose of exploring their practical capabilities to express noisy logical operators and/or perform stochastic logical operations. Mixing statistical mechanics with logics, and testing quantitatively the resulting findings on the available biochemical data, we successfully revise the concept of cooperativity (and anti-cooperativity) for allosteric systems, with particular emphasis on its computational capabilities, the related ranges and scaling of the involved parameters and its differences with classical cooperativity (and anti-cooperativity).

  2. A water-mediated allosteric network governs activation of Aurora kinase A.

    PubMed

    Cyphers, Soreen; Ruff, Emily F; Behr, Julie M; Chodera, John D; Levinson, Nicholas M

    2017-04-01

    The catalytic activity of many protein kinases is controlled by conformational changes of a conserved Asp-Phe-Gly (DFG) motif. We used an infrared probe to track the DFG motif of the mitotic kinase Aurora A (AurA) and found that allosteric activation by the spindle-associated protein Tpx2 involves an equilibrium shift toward the active DFG-in state. Förster resonance energy transfer experiments show that the activation loop undergoes a nanometer-scale movement that is tightly coupled to the DFG equilibrium. Tpx2 further activates AurA by stabilizing a water-mediated allosteric network that links the C-helix to the active site through an unusual polar residue in the regulatory spine. The polar spine residue and water network of AurA are essential for phosphorylation-driven activation, but an alternative form of the water network found in related kinases can support Tpx2-driven activation, suggesting that variations in the water-mediated hydrogen bond network mediate regulatory diversification in protein kinases.

  3. Neuroprotection by selective allosteric potentiators of the EP2 prostaglandin receptor

    PubMed Central

    Jiang, Jianxiong; Ganesh, Thota; Du, Yuhong; Thepchatri, Pahk; Rojas, Asheebo; Lewis, Iestyn; Kurtkaya, Serdar; Li, Lian; Qui, Min; Serrano, Geidy; Shaw, Renee; Sun, Aiming; Dingledine, Ray

    2010-01-01

    Activation of the Gαs-coupled EP2 receptor for prostaglandin E2 (PGE2) promotes cell survival in several models of tissue damage. To advance understanding of EP2 functions, we designed experiments to develop allosteric potentiators of this key prostaglandin receptor. Screens of 292,000 compounds identified 93 that at 20 μM (i) potentiated the cAMP response to a low concentration of PGE2 by > 50%; (ii) had no effect on EP4 or β2 adrenergic receptors, the cAMP assay itself, or the parent cell line; and (iii) increased the potency of PGE2 on EP2 receptors at least 3-fold. In aqueous solution, the active compounds are largely present as nanoparticles that appear to serve as active reservoirs for bioactive monomer. From 94 compounds synthesized or purchased, based on the modification of one hit compound, the most active increased the potency of PGE2 on EP2 receptors 4- to 5-fold at 10 to 20 μM and showed substantial neuroprotection in an excitotoxicity model. These small molecules represent previously undescribed allosteric modulators of a PGE2 receptor. Our results strongly reinforce the notion that activation of EP2 receptors by endogenous PGE2 released in a cell-injury setting is neuroprotective. PMID:20080612

  4. Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics*

    PubMed Central

    Zhang, Ping; Leger, Andrew J.; Baleja, James D.; Rana, Rajashree; Corlin, Tiffany; Nguyen, Nga; Koukos, Georgios; Bohm, Andrew; Covic, Lidija; Kuliopulos, Athan

    2015-01-01

    G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class. PMID:25934391

  5. [Escitalopram: a selective inhibitor and allosteric modulator of the serotonin transporter].

    PubMed

    Mnie-Filali, O; El Mansari, M; Scarna, H; Zimmer, L; Sánchez, C; Haddjeri, N

    2007-12-01

    , while chronic treatment with R-citalopram did not modify the basal proliferation rate in the dentate gyrus, it blocked the increase induced by escitalopram when coadministered. This suggests that neuronal adaptive changes, which are essential for antidepressant response, are rapidly induced by escitalopram but prevented by R-citalopram coadministration. The attenuating effect of R-citalopram was suggested to underlie the delayed recovery of 5-HT neuronal activity following long-term treatment with citalopram versus escitalopram. This is confirmed since a treatment with R-citalopram antagonized the recovery of firing observed in escitalopram-treated rats. The exact mechanism by which R-citalopram exerts its action is not yet fully defined; however, an allosteric interaction between the enantiomers and the 5-HT transporter (SERT) has been proposed. In this context, in vitro studies have revealed the existence of at least two binding sites on SERT: (1) a primary high-affinity binding site or orthosteric site that mediates the inhibition of 5-HT reuptake and (2) an allosteric low-affinity binding site that modulates the binding of ligands at the primary site. In presence of escitalopram alone, both the primary and the allosteric sites are occupied. Thus, escitalopram exerts a stabilizing effect on this association to SERT, resulting in an effective inhibition of 5-HT reuptake activity. On the other hand, in the presence of the two enantiomers, R-citalopram binds to the allosteric site and decreases the escitalopram action on SERT. Such an innovative mechanism of action can constitute a basis for development of new allosteric antidepressants that demonstrate higher efficacy and earlier onset of therapeutic effect.

  6. Information Needs While A Disaster Is Occurring

    NASA Astrophysics Data System (ADS)

    Perry, S. C.

    2010-12-01

    Evidence from recent earthquakes, wildfires, and debris flows in southern California indicates that many people - local officials as well as residents and visitors - lack important understanding during the time that a disaster is unfolding, a time of uncertainty and confusion. While some of the uncertainty is inherent, some could be alleviated. Physical scientists and engineers know what to expect as the event unfolds. Social scientists know how humans will react during a disaster, and how to effectively communicate the warnings or evacuation orders that may precede it. Such knowledge can improve public safety. As just a few of many examples: - Based on questions posed at numerous public talks, many individuals who practice "Drop Cover and Hold" during earthquake drills do not understand what they are protecting themselves against, and thus cannot determine what to do when an earthquake strikes and they have no cover available. Similarly, they do not know how to act during the aftershocks that follow. - The 2009 Station Fire in the San Gabriel Mountains put foothills communities at risk, first from the wildfire and then from debris flows. Some neighborhoods received multiple evacuation notices during a few days or months. Local officials have expressed frustration and concern about an evacuation compliance rate that is steadily dropping and is now below 50%. The debris flow danger will persist over the next 2-4 winters yet evacuation compliance may drop lower still. - On February 6, 2010, a significant rainstorm brought the threat of imminent debris flows to areas burned by the Station Fire. In one neighborhood, residents loaded their cars with important belongings then waited for indications that they should evacuate. Powerful debris flows suddenly appeared, sweeping the cars downhill and destroying both cars and belongings. Some residents did understand that rainfall intensity would control the generation of debris flows in that storm. But they didn't understand

  7. Speech Communication.

    ERIC Educational Resources Information Center

    Brooks, William D.

    Presented in this book is a view of speech communication which enables an individual to become fully aware of his or her role as both initiator and recipient of messages. Communication is treated broadly with emphasis on the understanding and skills relating to various types of speech communication across the broad spectrum of human communication.…

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2016-05-27

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

  10. 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.

  11. 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

  12. Effects of structural analogues of the substrate and allosteric regulator of the human mitochondrial NAD(P)+-dependent malic enzyme.

    PubMed

    Su, Kuo-Liang; Chang, Kuan-Yu; Hung, Hui-Chih

    2009-08-01

    Fumarate, a four-carbon trans dicarboxylic acid, is the allosteric activator of the human mitochondrial NAD(P)(+)-dependent malic enzyme (m-NAD(P)-ME). In this paper, we discuss the effects of the structural analogues of fumarate on human m-NAD(P)-ME. Succinate, a dicarboxylic acid with a carbon-carbon single bond, can also activate the enzyme, but the activating effect of succinate is less than that of fumarate. Succinamide, a diamide of succinate, cannot activate the enzyme and is a poor active-site inhibitor. The cis isomer of fumarate, maleic acid, significantly inhibits the ME activity, suggesting that the trans configuration of fumarate is crucial for operating the allosteric regulation of the enzyme. Other dicarboxylic acids, including glutaconic acid, malonic acid and alpha-ketoglutarate, cannot activate the enzyme and inversely inhibit enzyme activity. Our data suggest that these structural analogues are mainly active-site inhibitors, although they may enter the allosteric site to inhibit the enzyme. Furthermore, these data also suggest that the dicarboxylic acid must be in a trans conformation for allosteric activation of the enzyme.

  13. A Structural Basis for the Allosteric Regulatin of Non-Hydrolysing UDP-G1cNAc 2-Epimerases

    SciTech Connect

    Velloso,L.; Bhaskaran, S.; Schuch, R.; Fischetti, V.; Stebbins, C.

    2008-01-01

    The non-hydrolysing bacterial UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) catalyses the conversion of UDP-GlcNAc into UDP-N-acetylmannosamine, an intermediate in the biosynthesis of several cell-surface polysaccharides. This enzyme is allosterically regulated by its substrate UDP-GlcNAc. The structure of the ternary complex between the Bacillus anthracis UDP-GlcNAc 2-epimerase, its substrate UDP-GlcNAc and the reaction intermediate UDP, showed direct interactions between UDP and its substrate, and between the complex and highly conserved enzyme residues, identifying the allosteric site of the enzyme. The binding of UDP-GlcNAc is associated with conformational changes in the active site of the enzyme. Kinetic data and mutagenesis of the highly conserved UDP-GlcNAc-interacting residues confirm their importance in the substrate binding and catalysis of the enzyme. This constitutes the first example to our knowledge, of an enzymatic allosteric activation by direct interaction between the substrate and the allosteric activator.

  14. Discovery of Potential Orthosteric and Allosteric Antagonists of P2Y1R from Chinese Herbs by Molecular Simulation Methods

    PubMed Central

    Lu, Fang; Jiang, Lu-di; Qiao, Lian-sheng; Xiang, Yu-hong

    2016-01-01

    P2Y1 receptor (P2Y1R), which belongs to G protein-coupled receptors (GPCRs), is an important target in ADP-induced platelet aggregation. The crystal structure of P2Y1R has been solved recently, which revealed orthosteric and allosteric ligand-binding sites with the details of ligand-protein binding modes. And it suggests that P2Y1R antagonists, which recognize two distinct sites, could potentially provide an efficacious and safe antithrombotic profile. In present paper, 2D similarity search, pharmacophore based screening, and molecular docking were used to explore the potential natural P2Y1R antagonists. 2D similarity search was used to classify orthosteric and allosteric antagonists of P2Y1R. Based on the result, pharmacophore models were constructed and validated by the test set. Optimal models were selected to discover potential P2Y1R antagonists of orthosteric and allosteric sites from Traditional Chinese Medicine (TCM). And the hits were filtered by Lipinski's rule. Then molecular docking was used to refine the results of pharmacophore based screening and analyze the binding mode of the hits and P2Y1R. Finally, two orthosteric and one allosteric potential compounds were obtained, which might be used in future P2Y1R antagonists design. This work provides a reliable guide for discovering natural P2Y1R antagonists acting on two distinct sites from TCM. PMID:27635149

  15. 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.

  16. 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.

  17. 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

  18. Identification of Regions Critically Affecting Kinetics and Allosteric Regulation of the Escherichia coli ADP-Glucose Pyrophosphorylase by Modeling and Pentapeptide-Scanning Mutagenesis▿

    PubMed Central

    Ballicora, Miguel A.; Erben, Esteban D.; Yazaki, Terutaka; Bertolo, Ana L.; Demonte, Ana M.; Schmidt, Jennifer R.; Aleanzi, Mabel; Bejar, Clarisa M.; Figueroa, Carlos M.; Fusari, Corina M.; Iglesias, Alberto A.; Preiss, Jack

    2007-01-01

    ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the enzyme responsible for the regulation of bacterial glycogen synthesis. To perform a structure-function relationship study of the Escherichia coli ADP-Glc PPase enzyme, we studied the effects of pentapeptide insertions at different positions in the enzyme and analyzed the results with a homology model. We randomly inserted 15 bp in a plasmid with the ADP-Glc PPase gene. We obtained 140 modified plasmids with single insertions of which 21 were in the coding region of the enzyme. Fourteen of them generated insertions of five amino acids, whereas the other seven created a stop codon and produced truncations. Correlation of ADP-Glc PPase activity to these modifications validated the enzyme model. Six of the insertions and one truncation produced enzymes with sufficient activity for the E. coli cells to synthesize glycogen and stain in the presence of iodine vapor. These were in regions away from the substrate site, whereas the mutants that did not stain had alterations in critical areas of the protein. The enzyme with a pentapeptide insertion between Leu102 and Pro103 was catalytically competent but insensitive to activation. We postulate this region as critical for the allosteric regulation of the enzyme, participating in the communication between the catalytic and regulatory domains. PMID:17496097

  19. 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.

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

    PubMed Central

    Al-Horani, Rami A.; Karuturi, Rajesh; Lee, Michael; Afosah, Daniel K.

    2016-01-01

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

  1. Allosteric site-mediated active site inhibition of PBP2a using Quercetin 3-O-rutinoside and its combination.

    PubMed

    Rani, Nidhi; Vijayakumar, Saravanan; P T V, Lakshmi; Arunachalam, Annamalai

    2016-08-01

    Recent crystallographic study revealed the involvement of allosteric site in active site inhibition of penicillin binding protein (PBP2a), where one molecule of Ceftaroline (Cef) binds to the allosteric site of PBP2a and paved way for the other molecule (Cef) to bind at the active site. Though Cef has the potency to inhibit the PBP2a, its adverse side effects are of major concern. Previous studies have reported the antibacterial property of Quercetin derivatives, a group of natural compounds. Hence, the present study aims to evaluate the effect of Quercetin 3-o-rutinoside (Rut) in allosteric site-mediated active site inhibition of PBP2a. The molecular docking studies between allosteric site and ligands (Rut, Que, and Cef) revealed a better binding efficiency (G-score) of Rut (-7.790318) and Cef (-6.194946) with respect to Que (-5.079284). Molecular dynamic (MD) simulation studies showed significant changes at the active site in the presence of ligands (Rut and Cef) at allosteric site. Four different combinations of Rut and Cef were docked and their G-scores ranged between -6.320 and -8.623. MD studies revealed the stability of the key residue (Ser403) with Rut being at both sites, compared to other complexes. Morphological analysis through electron microscopy confirmed that combination of Rut and Cefixime was able to disturb the bacterial cell membrane in a similar fashion to that of Rut and Cefixime alone. The results of this study indicate that the affinity of Rut at both sites were equally good, with further validations Rut could be considered as an alternative for inhibiting MRSA growth.

  2. Drug-induced Skin Lesions in Cynomolgus Macaques Treated with Metabotropic Glutamate Receptor 5 (mGluR5) Negative Allosteric Modulators.

    PubMed

    Palanisamy, Gopinath S; Marcek, John M; Cappon, Gregg D; Whritenour, Jessica; Shaffer, Christopher L; Brady, Joseph T; Houle, Christopher

    2015-10-01

    Three orally administered metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulators caused skin lesions consistent with delayed type-IV hypersensitivity in cynomolgus macaques in 2- and 12-week toxicity studies. Several monkeys developed macroscopic skin lesions in multiple locations after 8 to 9 days of dosing; the most prominent effects involved the genital region of males and generalized erythema occurred in both sexes. Microscopic lesions occurred in both clinically affected and unaffected areas and were characterized by lymphocytic interface inflammation, subepidermal bullae, and individual keratinocyte vacuolation/necrosis. In the 12-week study, clinical effects in 2 animals resolved with continued dosing, whereas in others the inflammatory process progressed with 1 female exhibiting systemic lymphocytic inflammation in multiple tissues. The inflammatory infiltrate consisted of CD3 and CD4 positive T lymphocytes with minimal CD68 positive macrophages and only rare CD8 positive T lymphocytes. A subset of animals given a dosing holiday was subsequently rechallenged with similar lesions developing but with a more rapid clinical onset. These skin lesions were consistent with type-IV delayed hypersensitivity with some features comparable to bullous drug eruptions in humans. A relationship between these findings and the intended mode of action for these compounds could not be ruled out, given the occurrence across different chemotypes.

  3. Evidence for the location of the allosteric activation switch in the multisubunit phosphorylase kinase complex from mass spectrometric identification of chemically crosslinked peptides.

    PubMed

    Nadeau, Owen W; Anderson, David W; Yang, Qing; Artigues, Antonio; Paschall, Justin E; Wyckoff, Gerald J; McClintock, Jennifer L; Carlson, Gerald M

    2007-02-02

    Phosphorylase kinase (PhK), an (alphabetagammadelta)(4) complex, regulates glycogenolysis. Its activity, catalyzed by the gamma subunit, is tightly controlled by phosphorylation and activators acting through allosteric sites on its regulatory alpha, beta and delta subunits. Activation by phosphorylation is predominantly mediated by the regulatory beta subunit, which undergoes a conformational change that is structurally linked with the gamma subunit and that is characterized by the ability of a short chemical crosslinker to form beta-beta dimers. To determine potential regions of interaction of the beta and gamma subunits, we have used chemical crosslinking and two-hybrid screening. The beta and gamma subunits were crosslinked to each other in phosphorylated PhK, and crosslinked peptides from digests were identified by Fourier transform mass spectrometry, beginning with a search engine developed "in house" that generates a hypothetical list of crosslinked peptides. A conjugate between beta and gamma that was verified by MS/MS corresponded to crosslinking between K303 in the C-terminal regulatory domain of gamma (gammaCRD) and R18 in the N-terminal regulatory region of beta (beta1-31), which contains the phosphorylatable serines 11 and 26. A synthetic peptide corresponding to residues 1-22 of beta inhibited the crosslinking between beta and gamma, and was itself crosslinked to K303 of gamma. In two-hybrid screening, the beta1-31 region controlled beta subunit self-interactions, in that they were favored by truncation of this region or by mutation of the phosphorylatable serines 11 and 26, thus providing structural evidence for a phosphorylation-dependent subunit communication network in the PhK complex involving at least these two regulatory regions of the beta and gamma subunits. The sum of our results considered together with previous findings implicates the gammaCRD as being an allosteric activation switch in PhK that interacts with all three of the enzyme

  4. Extracellular Calcium Modulates Actions of Orthosteric and Allosteric Ligands on Metabotropic Glutamate Receptor 1α*

    PubMed Central

    Jiang, Jason Y.; Nagaraju, Mulpuri; Meyer, Rebecca C.; Zhang, Li; Hamelberg, Donald; Hall, Randy A.; Brown, Edward M.; Conn, P. Jeffrey; Yang, Jenny J.

    2014-01-01

    Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca2+. However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca2+-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca2+-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca2+ enhanced mGluR1α-mediated intracellular Ca2+ responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca2+ diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca2+, respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca2+ binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca2+. These studies reveal that binding of extracellular Ca2+ to the predicted Ca2+-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α. PMID:24280223

  5. 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

  6. A linkage analysis toolkit for studying allosteric networks in ion channels

    PubMed Central

    2013-01-01

    A thermodynamic approach to studying allosterically regulated ion channels such as the large-conductance voltage- and Ca2+-dependent (BK) channel is presented, drawing from principles originally introduced to describe linkage phenomena in hemoglobin. In this paper, linkage between a principal channel component and secondary elements is derived from a four-state thermodynamic cycle. One set of parallel legs in the cycle describes the “work function,” or the free energy required to activate the principal component. The second are “lever operations” activating linked elements. The experimental embodiment of this linkage cycle is a plot of work function versus secondary force, whose asymptotes are a function of the parameters (displacements and interaction energies) of an allosteric network. Two essential work functions play a role in evaluating data from voltage-clamp experiments. The first is the conductance Hill energy WH[g], which is a “local” work function for pore activation, and is defined as kT times the Hill transform of the conductance (G-V) curve. The second is the electrical capacitance energy WC[q], representing “global” gating charge displacement, and is equal to the product of total gating charge per channel times the first moment (VM) of normalized capacitance (slope of Q-V curve). Plots of WH[g] and WC[q] versus voltage and Ca2+ potential can be used to measure thermodynamic parameters in a model-independent fashion of the core gating constituents (pore, voltage-sensor, and Ca2+-binding domain) of BK channel. The method is easily generalized for use in studying other allosterically regulated ion channels. The feasibility of performing linkage analysis from patch-clamp data were explored by simulating gating and ionic currents of a 17-particle model BK channel in response to a slow voltage ramp, which yielded interaction energies deviating from their given values in the range of 1.3 to 7.2%. PMID:23250867

  7. Ibuprofen Impairs Allosterically Peroxynitrite Isomerization by Ferric Human Serum Heme-Albumin*

    PubMed Central

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

    2009-01-01

    Human serum albumin (HSA) participates in heme scavenging; in turn, heme endows HSA with myoglobin-like reactivity and spectroscopic properties. Here, the allosteric effect of ibuprofen on peroxynitrite isomerization to NO3− catalyzed by ferric human serum heme-albumin (HSA-heme-Fe(III)) is reported. Data were obtained at 22.0 °C. HSA-heme-Fe(III) catalyzes peroxynitrite isomerization in the absence and presence of CO2; the values of the second order catalytic rate constant (kon) are 4.1 × 105 and 4.5 × 105 m−1 s−1, respectively. Moreover, HSA-heme-Fe(III) prevents peroxynitrite-mediated nitration of free added l-tyrosine. The pH dependence of kon (pKa = 6.9) suggests that peroxynitrous acid reacts preferentially with the heme-Fe(III) atom, in the absence and presence of CO2. The HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite has been ascribed to the reactive pentacoordinated heme-Fe(III) atom. In the absence and presence of CO2, ibuprofen impairs dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) and facilitates the nitration of free added l-tyrosine; the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(III) (L) ranges between 7.7 × 10−4 and 9.7 × 10−4 m. Under conditions where [ibuprofen] is ≫L, the kinetics of HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite is superimposable to that obtained in the absence of HSA-heme-Fe(III) or in the presence of non-catalytic HSA-heme-Fe(III)-cyanide complex and HSA. Ibuprofen binding impairs allosterically peroxynitrite isomerization by HSA-heme-Fe(III), inducing the hexacoordination of the heme-Fe(III) atom. These results represent the first evidence for peroxynitrite isomerization by HSA-heme-Fe(III), highlighting the allosteric modulation of HSA-heme-Fe(III) reactivity by heterotropic interaction(s), and outlining the role of drugs in modulating HSA functions. The present results could be relevant for the drug-dependent protective role

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

    SciTech Connect

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

    2009-05-19

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

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

    PubMed

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

    2011-11-01

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

  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. Discovery of substituted benzamides as follicle stimulating hormone receptor allosteric modulators.

    PubMed

    Yu, Henry N; Richardson, Thomas E; Nataraja, Selva; Fischer, David J; Sriraman, Venkataraman; Jiang, Xuliang; Bharathi, Pandi; Foglesong, Robert J; Haxell, Thomas F N; Heasley, Brian H; Jenks, Mathew; Li, Jane; Dugas, Melanie S; Collis, Regina; Tian, Hui; Palmer, Stephen; Goutopoulos, Andreas

    2014-05-01

    Follicle-stimulating hormone (FSH), acting on its receptor (FSHR), plays a pivotal role in the stimulation of follicular development and maturation. Multiple injections of protein formulations are used during clinical protocols for ovulation induction and for in vitro fertilization that are followed by a selection of assisted reproductive technologies. In order to increase patient convenience and compliance several research groups have searched for orally bioavailable FSH mimetics for innovative fertility medicines. We report here the discovery of a series of substituted benzamides as positive allosteric modulators (PAM) targeting FSHR. Optimization of this series has led to enhanced activity in primary rat granulosa cells, as well as remarkable selectivity against the closely related luteinizing hormone receptor (LHR) and thyroid stimulating hormone receptor (TSHR). Two modulators, 9j and 9k, showed promising in vitro and pharmacokinetic profiles.

  12. Allosteric transcriptional regulation via changes in the overall topology of the core promoter

    DOE PAGES

    Philips, Steven J.; Canalizo-Hernandez, Monica; Yildirim, Ilyas; ...

    2015-08-21

    Many transcriptional activators act at a distance from core promoter elements and work by recruiting RNA polymerase through protein-protein interactions. We show here how the prokaryotic regulatory protein CueR both represses and activates transcription by differentially modulating local DNA structure within the promoter. Structural studies reveal that the repressor state slightly bends the promoter DNA, precluding optimal RNA polymerase-promoter recognition. Upon binding a metal ion in the allosteric site, CueR switches into an activator conformation. It maintains all protein-DNA contacts but introduces torsional stresses that kink and undertwist the promoter, stabilizing an A-DNA-like conformation. Finally, these factors switch on andmore » off transcription by exerting dynamic control of DNA stereochemistry, reshaping the core promoter and making it a better or worse substrate for polymerase.« less

  13. DNA-PKcs structure suggests an allosteric mechanism modulating DNA double-strand break repair.

    PubMed

    Sibanda, Bancinyane L; Chirgadze, Dimitri Y; Ascher, David B; Blundell, Tom L

    2017-02-03

    DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a central component of nonhomologous end joining (NHEJ), repairing DNA double-strand breaks that would otherwise lead to apoptosis or cancer. We have solved its structure in complex with the C-terminal peptide of Ku80 at 4.3 angstrom resolution using x-ray crystallography. We show that the 4128-amino acid structure comprises three large structural units: the N-terminal unit, the Circular Cradle, and the Head. Conformational differences between the two molecules in the asymmetric unit are correlated with changes in accessibility of the kinase active site, which are consistent with an allosteric mechanism to bring about kinase activation. The location of KU80ct194 in the vicinity of the breast cancer 1 (BRCA1) binding site suggests competition with BRCA1, leading to pathway selection between NHEJ and homologous recombination.

  14. Difluorocyclobutylacetylenes as positive allosteric modulators of mGluR5 with reduced bioactivation potential.

    PubMed

    Degnan, Andrew P; Maxwell, Darrell; Balakrishnan, Anand; Brown, Jeffrey M; Easton, Amy; Gulianello, Michael; Hanumegowda, Umesh; Hill-Drzewi, Melissa; Miller, Regina; Santone, Kenneth S; Senapati, Arun; Shields, Eric E; Sivarao, Digavalli V; Westphal, Ryan; Whiterock, Valerie J; Zhuo, Xiaoliang; Bronson, Joanne J; Macor, John E

    2016-12-15

    Schizophrenia is a serious illness that affects millions of patients and has been associated with N-methyl-d-aspartate receptor (NMDAR) hypofunction. It has been demonstrated that activation of metabotropic glutamate receptor 5 (mGluR5) enhances NMDA receptor function, suggesting the potential utility of mGluR5 positive allosteric modulators (PAMs) in the treatment of schizophrenia. Herein we describe the optimization of an mGluR5 PAM by replacement of a phenyl with aliphatic heterocycles and carbocycles as a strategy to reduce bioactivation in a biaryl acetylene chemotype. Replacement with a difluorocyclobutane followed by further optimization culminated in the identification of compound 32, a low fold shift PAM with reduced bioactivation potential. Compound 32 demonstrated favorable brain uptake and robust efficacy in mouse novel object recognition (NOR) at low doses.

  15. Triazolopyridine ethers as potent, orally active mGlu2 positive allosteric modulators for treating schizophrenia.

    PubMed

    Higgins, Mendi A; Marcin, Lawrence R; Christopher Zusi, F; Gentles, Robert; Ding, Min; Pearce, Bradley C; Easton, Amy; Kostich, Walter A; Seager, Matthew A; Bourin, Clotilde; Bristow, Linda J; Johnson, Kim A; Miller, Regina; Hogan, John; Whiterock, Valerie; Gulianello, Michael; Ferrante, Meredith; Huang, Yanling; Hendricson, Adam; Alt, Andrew; Macor, John E; Bronson, Joanne J

    2017-01-15

    Triazolopyridine ethers with mGlu2 positive allosteric modulator (PAM) activity are disclosed. The synthesis, in vitro activity, and metabolic stability data for a series of analogs is provided. The effort resulted in the discovery of a potent, selective, and brain penetrant lead molecule BMT-133218 ((+)-7m). After oral administration at 10mg/kg, BMT-133218 demonstrated full reversal of PCP-stimulated locomotor activity and prevented MK-801-induced working memory deficits in separate mouse models. Also, reversal of impairments in executive function were observed in rat set-shifting studies at 3 and 10mg/kg (p.o.). Extensive plasma protein binding as the result of high lipophilicity likely limited activity at lower doses. Optimized triazolopyridine ethers offer utility as mGlu2 PAMs for the treatment of schizophrenia and merit further preclinical investigation.

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

    PubMed Central

    2016-01-01

    The rotational speed of an overcrowded alkene-based molecular rotary motor, having an integrated 4,5-diazafluorenyl coordination motif, can be regulated allosterically via the binding of metal ions. DFT calculations have been used to predict the relative speed of rotation of three different (i.e., zinc, palladium, and platinum) metal dichloride complexes. The photochemical and thermal isomerization behavior of these complexes has been studied in detail using UV–vis and 1H NMR spectroscopy. Our results confirm that metal coordination induces a contraction of the diazafluorenyl lower half, resulting in a reduction of the steric hindrance in the “fjord” region of the molecule, which causes an increase of the rotational speed. Importantly, metal complexation can be accomplished in situ and is found to be reversible upon the addition of a competing ligand. Consequently, the rotational behavior of these molecular motors can be dynamically controlled with chemical additives. PMID:27669358

  17. Experiments on Hemoglobin in Single Crystals and Silica Gels Distinguish among Allosteric Models

    PubMed Central

    Henry, Eric R.; Mozzarelli, Andrea; Viappiani, Cristiano; Abbruzzetti, Stefania; Bettati, Stefano; Ronda, Luca; Bruno, Stefano; Eaton, William A.

    2015-01-01

    Trapping quaternary structures of hemoglobin in single crystals or by encapsulation in silica gels has provided a demanding set of data to test statistical mechanical models of allostery. In this work, we compare the results of those experiments with predictions of the four major allosteric models for hemoglobin: the quaternary two-state model of Monod, Wyman, and Changeux; the tertiary two-state model of Henry et al., which is the simplest extension of the Monod-Wyman-Changeux model to include pre-equilibria of tertiary as well as quaternary conformations; the structure-based model of Szabo and Karplus; and the modification of the latter model by Lee and Karplus. We show that only the tertiary two-state model can provide a near quantitative explanation of the single-crystal and gel experimental results. PMID:26038112

  18. Allosteric regulation of SecA: magnesium-mediated control of conformation and activity.

    PubMed

    Gold, Vicki A M; Robson, Alice; Clarke, Anthony R; Collinson, Ian

    2007-06-15

    In bacteria, the SecA protein associates with a ubiquitous protein channel SecYEG where it drives the post-translational secretion of pre-proteins across the plasma membrane. The high-resolution structures of both proteins have been determined in their resting states; however, the mechanism that couples ATP hydrolysis to active transport of substrate proteins through the membrane is not well understood. An analysis of the steady-state ATPase activity of the enzyme reveals that there is an allosteric binding site for magnesium distinct from that associated with hydrolysis of ATP. We have demonstrated that this regulation involves a large conformational change to the SecA dimer, which exerts a strong influence on the turnover and affinity for ATP, as well as the affinity for ADP. The strong inhibitory influence of magnesium on the ATPase activity can be countered by cardiolipin and conditions that promote protein translocation.

  19. An allosteric self-splicing ribozyme triggered by a bacterial second messenger.

    PubMed

    Lee, Elaine R; Baker, Jenny L; Weinberg, Zasha; Sudarsan, Narasimhan; Breaker, Ronald R

    2010-08-13

    Group I self-splicing ribozymes commonly function as components of selfish mobile genetic elements. We identified an allosteric group I ribozyme, wherein self-splicing is regulated by a distinct riboswitch class that senses the bacterial second messenger c-di-GMP. The tandem RNA sensory system resides in the 5' untranslated region of the messenger RNA for a putative virulence gene in the pathogenic bacterium Clostridium difficile. c-di-GMP binding by the riboswitch induces folding changes at atypical splice site junctions to modulate alternative RNA processing. Our findings indicate that some self-splicing ribozymes are not selfish elements but are harnessed by cells as metabolite sensors and genetic regulators.

  20. TRANSCRIPTION. Allosteric transcriptional regulation via changes in the overall topology of the core promoter.

    PubMed

    Philips, Steven J; Canalizo-Hernandez, Monica; Yildirim, Ilyas; Schatz, George C; Mondragón, Alfonso; O'Halloran, Thomas V

    2015-08-21

    Many transcriptional activators act at a distance from core promoter elements and work by recruiting RNA polymerase through protein-protein interactions. We show here how the prokaryotic regulatory protein CueR both represses and activates transcription by differentially modulating local DNA structure within the promoter. Structural studies reveal that the repressor state slightly bends the promoter DNA, precluding optimal RNA polymerase-promoter recognition. Upon binding a metal ion in the allosteric site, CueR switches into an activator conformation. It maintains all protein-DNA contacts but introduces torsional stresses that kink and undertwist the promoter, stabilizing an A-form DNA-like conformation. These factors switch on and off transcription by exerting dynamic control of DNA stereochemistry, reshaping the core promoter and making it a better or worse substrate for polymerase.

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

    PubMed

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

    2016-06-07

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

  2. 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.

  3. Allosteric transcriptional regulation via changes in the overall topology of the core promoter

    SciTech Connect

    Philips, Steven J.; Canalizo-Hernandez, Monica; Yildirim, Ilyas; Schatz, George C.; Mondragon, Alfonso; O'Halloran, Thomas V.

    2015-08-21

    Many transcriptional activators act at a distance from core promoter elements and work by recruiting RNA polymerase through protein-protein interactions. We show here how the prokaryotic regulatory protein CueR both represses and activates transcription by differentially modulating local DNA structure within the promoter. Structural studies reveal that the repressor state slightly bends the promoter DNA, precluding optimal RNA polymerase-promoter recognition. Upon binding a metal ion in the allosteric site, CueR switches into an activator conformation. It maintains all protein-DNA contacts but introduces torsional stresses that kink and undertwist the promoter, stabilizing an A-DNA-like conformation. Finally, these factors switch on and off transcription by exerting dynamic control of DNA stereochemistry, reshaping the core promoter and making it a better or worse substrate for polymerase.

  4. 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.

  5. Positive allosteric modulators as an approach to nicotinic acetylcholine receptor-targeted therapeutics: advantages and limitations.

    PubMed

    Williams, Dustin K; Wang, Jingyi; Papke, Roger L

    2011-10-15

    Neuronal nicotinic acetylcholine receptors (nAChR), recognized targets for drug development in cognitive and neuro-degenerative disorders, are allosteric proteins with dynamic interconversions between multiple functional states. Activation of the nAChR ion channel is primarily controlled by the binding of ligands (agonists, partial agonists, competitive antagonists) at conventional agonist binding sites, but is also regulated in either negative or positive ways by the binding of ligands to other modulatory sites. In this review, we discuss models for the activation and desensitization of nAChR, and the discovery of multiple types of ligands that influence those processes in both heteromeric nAChR, such as the high-affinity nicotine receptors of the brain, and homomeric α7-type receptors. In recent years, α7 nAChRs have been identified as a potential target for therapeutic indications leading to the development of α7-selective agonists and partial agonists. However, unique properties of α7 nAChR, including low probability of channel opening and rapid desensitization, may limit the therapeutic usefulness of ligands binding exclusively to conventional agonist binding sites. New enthusiasm for the therapeutic targeting of α7 has come from the identification of α7-selective positive allosteric modulators (PAMs) that work effectively on the intrinsic factors that limit α7 ion channel activation. While these new drugs appear promising for therapeutic development, we also consider potential caveats and possible limitations for their use, including PAM-insensitive forms of desensitization and cytotoxicity issues.

  6. Assessment of direct gating and allosteric modulatory effects of meprobamate in recombinant GABAA receptors

    PubMed Central

    Kumar, Manish; Dillon, Glenn H.

    2016-01-01

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

  7. A Novel Allosteric Activator of Free Fatty Acid 2 Receptor Displays Unique Gi-functional Bias*

    PubMed Central

    Bolognini, Daniele; Moss, Catherine E.; Nilsson, Karolina; Petersson, Annika U.; Donnelly, Iona; Sergeev, Eugenia; König, Gabriele M.; Kostenis, Evi; Kurowska-Stolarska, Mariola; Miller, Ashley; Dekker, Niek; Tobin, Andrew B.

    2016-01-01

    The short chain fatty acid receptor FFA2 is able to stimulate signaling via both Gi- and Gq/G11-promoted pathways. These pathways are believed to control distinct physiological end points but FFA2 receptor ligands appropriate to test this hypothesis have been lacking. Herein, we characterize AZ1729, a novel FFA2 regulator that acts as a direct allosteric agonist and as a positive allosteric modulator, increasing the activity of the endogenously produced short chain fatty acid propionate in Gi-mediated pathways, but not at those transduced by Gq/G11. Using AZ1729 in combination with direct inhibitors of Gi and Gq/G11 family G proteins demonstrated that although both arms contribute to propionate-mediated regulation of phospho-ERK1/2 MAP kinase signaling in FFA2-expressing 293 cells, the Gq/G11-mediated pathway is predominant. We extend these studies by employing AZ1729 to dissect physiological FFA2 signaling pathways. The capacity of AZ1729 to act at FFA2 receptors to inhibit β-adrenoreceptor agonist-promoted lipolysis in primary mouse adipocytes and to promote chemotaxis of isolated human neutrophils confirmed these as FFA2 processes mediated by Gi signaling, whereas, in concert with blockade by the Gq/G11 inhibitor FR900359, the inability of AZ1729 to mimic or regulate propionate-mediated release of GLP-1 from mouse colonic preparations defined this physiological response as an end point transduced via activation of Gq/G11. PMID:27385588

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

    PubMed

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

    2015-08-01

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

  9. Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators

    PubMed Central

    Wang, Wei; Hong, Jeong S.; Rab, Andras; Sorscher, Eric J.; Kirk, Kevin L.

    2016-01-01

    W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3–5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein. PMID:27007499

  10. pH-dependent interconvertible allosteric forms of murine melanoma tyrosinase. Physiological implications.

    PubMed

    Devi, C C; Tripathi, R K; Ramaiah, A

    1987-08-03

    Murine melanoma melanosomal tyrosinase, solubilised at pH 6.8 and 1% Igepal, exhibits a lag in cresolase activity which increases with increasing concentration of tyrosine. The enzyme, solubilised at pH 5.0 and assayed at pH 5.0, does not exhibit lag even at inhibitory concentrations of tyrosine while the same enzyme when assayed at pH 6.8 exhibits characteristic lag. When the enzyme was solubilised from a melanosomal fraction with detergent/water without any buffer, significant linear activity for 2 h was seen at an inhibitory concentration of tyrosine, indicating for the first time the presence of a form of tyrosinase without lag and inhibition by excess tyrosine. Exposure of the enzyme solubilised in buffer/detergent at pH 6.8 to rapid decrease in pH to 5.0 or 4.7 makes the enzyme remain irreversibly in the form without characteristic lag, even at an inhibitory concentration of tyrosine and at pH 6.8. These results may be interpreted as follows. The enzyme at pH 6.8 exists in the E form with an allosteric site for tyrosine. Decrease of the pH of the enzyme solution from 6.8 to 5.0 or 4.7 by dialysis results in the reversible protonation of the enzyme, which no longer binds tyrosine at its allosteric site and consequently inhibition by excess tyrosine and lag were not observed at acidic pH. However, if the enzyme was rapidly brought to pH 5.0 from 6.8 it remains irreversibly in the protonated form even at pH 6.8. Ascorbic acid acts as an effective reductant for the hydroxylation of tyrosine by tyrosinase, while 3,4-dihydroxyphenylalanine is both an effective reductant and counteracts the inhibition by tyrosine at pH 6.8.

  11. 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

  12. Allosteric Gating of a Large Conductance Ca-activated K+ Channel

    PubMed Central

    Cox, D.H.; Cui, J.; Aldrich, R.W.

    1997-01-01

    Large-conductance Ca-activated potassium channels (BK channels) are uniquely sensitive to both membrane potential and intracellular Ca2+. Recent work has demonstrated that in the gating of these channels there are voltage-sensitive steps that are separate from Ca2+ binding steps. Based on this result and the macroscopic steady state and kinetic properties of the cloned BK channel mslo, we have recently proposed a general kinetic scheme to describe the interaction between voltage and Ca2+ in the gating of the mslo channel (Cui, J., D.H. Cox, and R.W. Aldrich. 1997. J. Gen. Physiol. In press.). This scheme supposes that the channel exists in two main conformations, closed and open. The conformational change between closed and open is voltage dependent. Ca2+ binds to both the closed and open conformations, but on average binds more tightly to the open conformation and thereby promotes channel opening. Here we describe the basic properties of models of this form and test their ability to mimic mslo macroscopic steady state and kinetic behavior. The simplest form of this scheme corresponds to a voltage-dependent version of the Monod-Wyman-Changeux (MWC) model of allosteric proteins. The success of voltage-dependent MWC models in describing many aspects of mslo gating suggests that these channels may share a common molecular mechanism with other allosteric proteins whose behaviors have been modeled using the MWC formalism. We also demonstrate how this scheme can arise as a simplification of a more complex scheme that is based on the premise that the channel is a homotetramer with a single Ca2+ binding site and a single voltage sensor in each subunit. Aspects of the mslo data not well fitted by the simplified scheme will likely be better accounted for by this more general scheme. The kinetic schemes discussed in this paper may be useful in interpreting the effects of BK channel modifications or mutations. PMID:9276753

  13. 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.

  14. Mechanism of Kir6.2 channel inhibition by sulfhydryl modification: pore block or allosteric gating?

    PubMed

    Cui, Yijun; Fan, Zheng

    2002-05-01

    Chemical modification can inhibit ion channels either by reacting with pore-lining residues and directly occluding the channel or by closing the channel allosterically. A general method to distinguish between these two mechanisms does not exist. Previously, sulfhydryl (SH) modification has been shown to inhibit ATP-sensitive K(+) (K(ATP)) channels. The crucial modification has been localized to C42 near the N-terminus of Kir6.2, a pore-forming subunit of K(ATP) channels, but little is known about how SH modification of C42 causes channel inhibition. To investigate this mechanism, we used the membrane-impermeable methanethiosulfonates, MTSET and MTS-TEAH, to modify Kir6.2 channels. While intracellular application of MTSET irreversibly inhibited channels, MTS-TEAH failed to do so. Instead, MTS-TEAH treatment prolonged channel openings and prevented the effect of subsequent MTSET treatment. Similar observations were made in mutants in which cysteines other than C42 had been mutated. Neither MTSET nor MTS-TEAH, however, affected mutant channels in which valines were substituted for C42 residues in all subunits. The reagents were effective when two of four C42 residues in the tetramer were replaced by valines. These results can be interpreted as indicating that both reagents modify C42. We then employed spermine, a known inner pore blocker, as a probe to examine whether MTS-TEAH modification alters pore accessibility. We found that spermine block was not changed by MTS-TEAH modification. Based on these data, we postulate that C42 faces either the cytoplasm or a vestibule section wide enough to allow spermine to pass freely after modification by MTS-TEAH. Our study suggests that channel inhibition caused by SH modification of Kir6.2 is an allosteric effect, and is not caused by direct pore blockage.

  15. Insights into the allosteric regulation of Syk association with receptor ITAM, a multi-state equilibrium.

    PubMed

    Feng, Chao; Post, Carol Beth

    2016-02-17

    The phosphorylation of interdomain A (IA), a linker region between tandem SH2 domains of Syk tyrosine kinase, regulates the binding affinity for association of Syk with doubly-phosphorylated ITAM regions of the B cell receptor. The mechanism of this allosteric regulation has been suggested to be a switch from the high-affinity bifunctional binding, mediated through both SH2 domains binding two phosphotyrosine residues of ITAM, to a substantially lower-affinity binding of only one SH2 domain. IA phosphorylation triggers the switch by inducing disorder in IA and weakening the SH2-SH2 interaction. The postulated switch to a single-SH2-domain binding mode is examined using NMR to monitor site-specific binding to each SH2 domain of Syk variants engineered to have IA regions that differ in conformational flexibility. The combined analysis of titration curves and NMR line-shapes provides sufficient information to determine the energetics of inter-molecular binding at each SH2 site along with an intra-molecular binding or isomerization step. A less favorable isomerization equilibrium associated with the changes in the SH2-SH2 conformational ensemble and IA flexibility accounts for the inhibition of Syk association with membrane ITAM regions when IA is phosphorylated, and refutes the proposed switch to single-SH2-domain binding. Syk localizes in the cell through its SH2 interactions, and this basis for allosteric regulation of ITAM association proposes for the first time a phosphorylation-dependent model to regulate Syk binding to alternate receptors and other signaling proteins that differ either in the number of residues separating ITAM phosphotyrosines or by having only one phosphotyrosine, a half ITAM.

  16. Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1

    PubMed Central

    Dowal, Louisa; Sim, Derek S.; Dilks, James R.; Blair, Price; Beaudry, Sarah; Denker, Bradley M.; Koukos, Georgios; Kuliopulos, Athan; Flaumenhaft, Robert

    2011-01-01

    G protein-coupled receptors (GPCRs) can assume multiple conformations and possess multiple binding sites. Whereas endogenous agonists acting at the orthosteric binding site stabilize the active receptor conformation, small molecules that act at nonorthosteric sites can stabilize alternative conformations. The large majority of these allosteric modulators associate with extracellular loops of GPCRs. The role of intracellular domains in mediating allosteric modulation is largely unknown. In screening a small-molecule library for inhibitors of platelet activation, we identified a family of compounds that modified PAR1-mediated granule secretion. The most potent inhibitory compound, termed JF5, also demonstrated noncompetitive inhibition of the α2A-adrenergic receptor. Aggregation studies using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to GPCRs that possessed a constrained eighth helix, as defined by a C-terminal palmitoylation site and interactions with TM7 and the i1 loop. Inhibition by JF5 was overcome in a PAR1 mutant in which the eighth helix was deleted, confirming a role for helix 8 in JF5 activity. Evaluation of downstream signaling showed that JF5 was selective with regard to G protein coupling, blocking signaling mediated by Gαq but not Gα12. The compound inhibited thrombus formation in vivo following vascular injury with an IC50 of ∼1 mg/kg. These results indicate a role for helix 8 in conferring sensitivity to small molecules, and show that this sensitivity can be exploited to control platelet activation during thrombus formation. PMID:21282664

  17. Small Molecule Positive Allosteric Modulation of TRPV1 Activation by Vanilloids and Acidic pHS⃞

    PubMed Central

    Kaszas, Krisztian; Keller, Jason M.; Coddou, Claudio; Mishra, Santosh K.; Hoon, Mark A.; Stojilkovic, Stanko; Jacobson, Kenneth A.

    2012-01-01

    Transient receptor potential cation channel subfamily V member 1 (TRPV1) is a high-conductance, nonselective cation channel strongly expressed in nociceptive primary afferent neurons of the peripheral nervous system and functions as a multimodal nociceptor gated by temperatures greater than 43°C, protons, and small-molecule vanilloid ligands such as capsaicin. The ability to respond to heat, low pH, vanilloids, and endovanilloids and altered sensitivity and expression in experimental inflammatory and neuropathic pain models made TRPV1 a major target for the development of novel, nonopioid analgesics and resulted in the discovery of potent antagonists. In human clinical trials, observations of hyperthermia and the potential for thermal damage by suppressing the ability to sense noxious heat suggested that full-scale blockade of TRPV1 function can be counterproductive and subtler pharmacological approaches are necessary. Here we show that the dihydropyridine derivative 4,5-diethyl-3-(2-methoxyethylthio)-2-methyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS1477) behaves as a positive allosteric modulator of both proton and vanilloid activation of TRPV1. Under inflammatory-mimetic conditions of low pH (6.0) and protein kinase C phosphorylation, addition of MRS1477 further increased sensitivity of already sensitized TPRV1 toward capsaicin. MRS1477 does not affect inhibition by capsazepine or ruthenium red and remains effective in potentiating activation by pH in the presence of an orthosteric vanilloid antagonist. These results indicate a distinct site on TRPV1 for positive allosteric modulation that may bind endogenous compounds or novel pharmacological agents. Positive modulation of TRPV1 sensitivity suggests that it may be possible to produce a selective analgesia through calcium overload restricted to highly active nociceptive nerve endings at sites of tissue damage and inflammation. PMID:22005042

  18. Nucleoside Analogue Triphosphates Allosterically Regulate Human Ribonucleotide Reductase and Identify Chemical Determinants That Drive Substrate Specificity.

    PubMed

    Knappenberger, Andrew J; Ahmad, Md Faiz; Viswanathan, Rajesh; Dealwis, Chris G; Harris, Michael E

    2016-10-18

    Class I ribonucleotide reductase (RR) maintains balanced pools of deoxyribonucleotide substrates for DNA replication by converting ribonucleoside diphosphates (NDPs) to 2'-deoxyribonucleoside diphosphates (dNDPs). Binding of deoxynucleoside triphosphate (dNTP) effectors (ATP/dATP, dGTP, and dTTP) modulates the specificity of class I RR for CDP, UDP, ADP, and GDP substrates. Crystal structures of bacterial and eukaryotic RRs show that dNTP effectors and NDP substrates bind on either side of a flexible nine-amino acid loop (loop 2). Interactions with the effector nucleobase alter loop 2 geometry, resulting in changes in specificity among the four NDP substrates of RR. However, the functional groups proposed to drive specificity remain untested. Here, we use deoxynucleoside analogue triphosphates to determine the nucleobase functional groups that drive human RR (hRR) specificity. The results demonstrate that the 5-methyl, O4, and N3 groups of dTTP contribute to specificity for GDP. The O6 and protonated N1 of dGTP direct specificity for ADP. In contrast, the unprotonated N1 of adenosine is the primary determinant of ATP/dATP-directed specificity for CDP. Structural models from X-ray crystallography of eukaryotic RR suggest that the side chain of D287 in loop 2 is involved in binding of dGTP and dTTP, but not dATP/ATP. This feature is consistent with experimental results showing that a D287A mutant of hRR is deficient in allosteric regulation by dGTP and dTTP, but not ATP/dATP. Together, these data define the effector functional groups that are the drivers of human RR specificity and provide constraints for evaluating models of allosteric regulation.

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

    PubMed

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

    2015-09-30

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

  20. Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators.

    PubMed

    Wang, Wei; Hong, Jeong S; Rab, Andras; Sorscher, Eric J; Kirk, Kevin L

    2016-01-01

    W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3-5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein.

  1. Identification of a Novel Inhibitory Allosteric Site in p38α

    PubMed Central

    Gomez-Gutierrez, Patricia; Campos, Pedro M.; Vega, Miguel; Perez, Juan J.

    2016-01-01

    In the present study, we report the discovery of a novel allosteric inhibitory site for p38α, a subclass of the mitogen-activated protein kinases (MAPK) family. The putative site was discovered after inspection of the crystallographic structure of the p38α-MK2 complex. MK2 (MAPK-activated protein kinase 2) is an interesting protein playing a dual role as modulator and substrate of p38α. This intriguing behavior is due to the ability of the two proteins to form distinctive heterodimers when p38α is phosphorylated or not. We hypothesized that the regulatory action of MK2 is due to its capability to keep p38α in an inactive conformation and consequently, we investigated the atomic structure of the p38α-MK2 complex to understand such regulatory behavior at the molecular level. After inspection of the complex structure, two peptides designed from the MK2 regulatory loop in contact with p38α with sequences Tyr1-Ser2-Asn3-His4-Gly5-Leu6 (peptide-1) and [Phe0]-peptide-1 (peptide-2) in their zwitterionic form were investigated for their phosphorylation inhibitory capability in vitro. Since both peptides exhibited inhibitory capability of the p38α kinase mediated phosphorylation of MEF2A, in a subsequent step we pursued the discovery of small molecule peptidomimetics. For this purpose we characterized in detail the peptide-p38α interaction using molecular dynamics simulations, leading to the definition of a pharmacophore for the peptide-protein interaction. This hypothesis was used as query for a in silico screening, leading to the discovery of a fused ring compound with micromolar inhibitory activity. Site-directed mutagenesis studies support that the compound binds to the putative novel allosteric site in p38α. PMID:27898710

  2. Lightwave Communications.

    ERIC Educational Resources Information Center

    Rheam, Harry

    1993-01-01

    Describes simple and inexpensive labs for introducing students to fiber optic communications. Students investigate light as a carrier wave; look into the difficulties associated with "light" communication; and learn about modulation, optical fibers, and critical angles. (PR)

  3. Data communications

    SciTech Connect

    Preckshot, G.G.

    1993-08-01

    The purpose of this paper is to recommend regulatory guidance for reviewers examining computer communication systems used in nuclear power plants. The recommendations cover three areas important to these communications systems: system design, communication protocols, and communication media. The first area, system design, considers three aspects of system design--questions about architecture, specific risky design elements or omissions to look for in designs being reviewed, and recommendations for multiplexed data communication systems used in safety systems. The second area reviews pertinent aspects of communication protocol design and makes recommendations for newly designed protocols or the selection of existing protocols for safety system, information display, and non-safety control system use. The third area covers communication media selection, which differs significantly from traditional wire and cable. The recommendations for communication media extend or enhance the concerns of published IEEE standards about three subjects: data rate, imported hazards and maintainability.

  4. Communicating science

    USGS Publications Warehouse

    Farris, Gaye S.

    2005-01-01

    For science to have an impact, it must be communicated and easily accessible. The USGS National Wetlands Research Center communicates its research findings through several ways: publishing, the Web, the library, and education and outreach.

  5. TENORM (Technologically Enhanced Naturally Occurring Radioactive Materials)

    MedlinePlus

    Jump to main content US EPA United States Environmental Protection Agency Search Search Radiation Protection Share Facebook Twitter Google+ Pinterest Contact Us Technologically Enhanced Naturally Occurring Radioactive ...

  6. Communication (action with communicative content).

    PubMed

    Russo, M T

    2010-01-01

    The term Communication generally designate the transmission of a message of concepts, feelings or needs from a speaker to a receiver by means of verbal or no verbal language. The pragmatic approach to human communication has put in evidence a further implication of this concept: every behaviour therefore has a value even when it is not intentional. Recently, a more dynamic concept of communication has been elaborated where communication means communicative action. This interpretation is the starting point for the theory of the "communicative acting" and subsequently of the so called discourse ethic elaborated by J. Habermas.

  7. Communication, Communication, Communication! Growth through Laboratory Instructing

    ERIC Educational Resources Information Center

    Peterson, Jamie J.; DeAngelo, Samantha; Mack, Nancy; Thompson, Claudia; Cooper, Jennifer; Sesma, Arturo, Jr.

    2014-01-01

    This study examined gains undergraduate students made in their communication and collaboration skills when they served as peer teachers, i.e., laboratory instructors (LIs), for a General Psychology laboratory. Self-ratings of communication and collaboration skills were completed before and after teaching the laboratory. When compared to before the…

  8. Ripple Communication.

    ERIC Educational Resources Information Center

    Wilcox, R. Stimson

    1980-01-01

    Discusses how surface-dwelling animals use the water surface as a mode of communication by making ripple signals while they swim about. Provides information about surfaces and surface waves, ripple communication in water striders, ripple signal characteristics, sensing and orienting, other modes of communication, and evolution of ripple…

  9. Communicating Risk.

    ERIC Educational Resources Information Center

    Feldman, Joyce

    1993-01-01

    Communicating the environmental risk involved in projects like public incinerators is part of the education of the community. Presents an outline for communicating with the community that includes communication within the project office; solicitation of public input; development of small group informational activities; shared responsibilities;…

  10. Existential Communication.

    ERIC Educational Resources Information Center

    Self, Charles C.

    Focusing on the seminal work "Being and Nothingness," this paper explores the implications of the ideas of Jean-Paul Sartre for the study of communication in society. The paper redefines communication from an existential point of view, explores some implications of this redefinition for the study of communication within the social…

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

    SciTech Connect

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

    2012-11-01

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

  12. Empirical Analysis of Systematic Communication Errors.

    DTIC Science & Technology

    1981-09-01

    human o~ . .... 8 components in communication systems. (Systematic errors were defined to be those that occur regularly in human communication links...phase of the human communication process and focuses on the linkage between a specific piece of information (and the receiver) and the transmission...communication flow. (2) Exchange. Exchange is the next phase in human communication and entails a concerted effort on the part of the sender and receiver to share

  13. Wilderness communications.

    PubMed

    Worley, Gordon H

    2011-09-01

    When an emergency situation arises in a remote location, the ability to communicate with outside sources of assistance can prove very valuable. This article reviews the different types of communications technologies available to individuals in remote locations, including satellite telephones, personal locator beacons, satellite messengers, cellular telephones, and the different licensed and non-licensed 2-way radio services available for personal use. It also discusses basic radio communications techniques, emergency communication, requesting ground or air casualty evacuation, and selecting communications devices for different applications.

  14. Evaluation of alpha7 nicotinic acetylcholine receptor agonists and positive allosteric modulators using the parallel oocyte electrophysiology test station.

    PubMed

    Malysz, John; Grønlien, Jens H; Timmermann, Daniel B; Håkerud, Monika; Thorin-Hagene, Kirsten; Ween, Hilde; Trumbull, Jonathan D; Xiong, Yongli; Briggs, Clark A; Ahring, Philip K; Dyhring, Tino; Gopalakrishnan, Murali

    2009-08-01

    Neuronal acetylcholine receptors (nAChRs) of the alpha7 subtype are ligand-gated ion channels that are widely distributed throughout the central nervous system and considered as attractive targets for the treatment of various neuropsychiatric and neurodegenerative diseases. Both agonists and positive allosteric modulators (PAMs) are being developed as means to enhance the function of alpha7 nAChRs. The in vitro characterization of alpha7 ligands, including agonists and PAMs, relies on multiple technologies, but only electrophysiological measurements assess the channel activity directly. Traditional electrophysiological approaches utilizing two-electrode voltage clamp or patch clamp in isolated cells have very low throughput to significantly impact drug discovery. Abbott (Abbott Park, IL) has developed a two-electrode voltage clamp-based system, the Parallel Oocyte Electrophysiology Test Station (POETs()), that allows for the investigation of ligand-gated ion channels such as alpha7 nAChRs in a higher-throughput manner. We describe the utility of this technology in the discovery of selective alpha7 agonists and PAMs. With alpha7 agonists, POETs experiments involved both single- and multiple-point concentration-response testing revealing diverse activation profiles (zero efficacy desensitizing, partial, and full agonists). In the characterization of alpha7 PAMs, POETs testing has served as a reliable primary or secondary screen identifying compounds that fall into distinct functional types depending on the manner in which current potentiation occurred. Type I PAMs (eg, genistein, NS1738, and 5-hydroxyindole) increase predominantly the peak amplitude response, type II PAMs affect the peak current and current decay (eg, PNU-120,596 and 4-(naphthalen-1-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide), and anothertype slowing the current decay kinetics in the absence of increases in the peak current. In summary, POETs technology allows for significant

  15. The First Negative Allosteric Modulator for Dopamine D2 and D3 Receptors SB269652 May Lead to a New Generation of Antipsychotic Drugs.

    PubMed

    Rossi, Mario; Fasciani, Irene; Marampon, Francesco; Maggio, Roberto; Scarselli, Marco

    2017-03-06

    D2 and D3 dopamine receptors belong to the largest family of cell surface proteins in eukaryotes, the G protein-coupled receptors (GPCRs). Considering their crucial physiological functions and their relatively accessible cellular locations, GPCRs represent one of the most important classes of therapeutic targets. Until recently, the only strategy to develop drugs regulating GPCR activity was through the identification of compounds that directly acted on the orthosteric sites for endogenous ligands. However, many efforts have recently been made in order to identify small molecules that are able to interact with allosteric sites. These sites are less well-conserved; therefore, allosteric ligands have greater selectivity on the specific receptor. Strikingly, the use of allosteric modulators can provide specific advantages, such as an increased selectivity for GPCR subunits and the ability to introduce specific beneficial therapeutic effects without disrupting the integrity of complex physiologically-regulated networks. In 2010, our group unexpectedly found that SB269652, a compound supposed to interact with the orthosteric binding site of dopamine receptors, was actually a negative allosteric modulator of D2 and D3 receptor dimers, thus identifying the first allosteric small molecule acting on these important therapeutic targets. This review addresses the progresses in the understanding of the molecular mechanisms of interaction between the negative modulator SB269652 and D2 and D3 dopamine receptor monomers and dimers, and also the perspectives of developing new dopamine receptor allosteric drugs based on SB269652 as the leading compound.

  16. Inversion of the allosteric response of Escherichia coli glucosamine-6-P deaminase to N-acetylglucosamine 6-P, by single amino acid replacements.

    PubMed

    Cisneros, David A; Montero-Morán, Gabriela M; Lara-González, Samuel; Calcagno, Mario L

    2004-01-01

    Amino acid replacements in the active site of glucosamine-6-P deaminase from Escherichia coli (GlcN6P deaminase, EC 3.5.99.6) involving the residues D141 and E148 produce atypical allosteric kinetics. These residues are located in the chain segment 139-156 which is part of the active site and which also forms several intersubunit contacts close to the allosteric site. In the D141N and E148Q mutant forms of this deaminase, there is an inversion of the effect of its physiological allosteric effector, N-acetylglucosamine 6-P, which becomes an inhibitor at substrate concentrations above a critical value. For both mutants, this particular point appears at low substrate concentration and the inhibition by the allosteric activator is the dominant effect in velocity versus substrate curves. These effects are analyzed as a particular case of the concerted allosteric model, assuming that the R state, the conformer displaying the higher affinity for the substrate, is the less catalytic state, thus producing an inverted allosteric response.

  17. Coaching Communication Partners: A Preliminary Investigation of Communication Intervention during Mealtime in Rett Syndrome

    ERIC Educational Resources Information Center

    Bartolotta, Theresa E.; Remshifski, Patricia A.

    2013-01-01

    Rett syndrome (RTT) occurs primarily in females and is characterized by deficits in cognition, communication, hand use and ambulation. This quasi-experimental study explored the use of a coaching program to increase communicative interactions between girls with RTT and their communication partners. Communication coaching strategies were provided…

  18. Exploration of Allosteric Agonism Structure-Activity Relationships within an Acetylene Series of Metabotropic Glutamate Receptor 5 (mGlu5) Positive Allosteric Modulators (PAMs): discovery of 5-((3-fluorophenyl)ethynyl)-N-(3-methyloxetan-3-yl)picolinamide (ML254)

    PubMed Central

    Turlington, Mark; Noetzel, Meredith J.; Chun, Aspen; Zhou, Ya; Gogliotti, Rocco D.; Nguyen, Elizabeth D.; Gregory, Karen J.; Vinson, Paige N.; Rook, Jerri M.; Gogi, Kiran K.; Xiang, Zixiu; Bridges, Thomas M.; Daniels, J. Scott; Jones, Carrie; Niswender, Colleen M.; Meiler, Jens; Conn, P. Jeffrey; Lindsley, Craig W.; Stauffer, Shaun R.

    2014-01-01

    Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGlu5) represent a promising therapeutic strategy for the treatment of schizophrenia. Both allosteric agonism and high glutamate fold-shift have been implicated in the neurotoxic profile of some mGlu5 PAMs; however, these hypotheses remain to be adequately addressed. To develop tool compounds to probe these hypotheses, the structure-activity relationship of allosteric agonism was examined within an acetylenic series of mGlu5 PAMs exhibiting allosteric agonism in addition to positive allosteric modulation (ago-PAMs). PAM 38t, a low glutamate fold-shift allosteric ligand (maximum fold-shift ~3.0), was selected as a potent PAM with no agonism in the in vitro system used for compound characterization and in two native electrophysiological systems using rat hippocampal slices. PAM 38t (ML254) will be useful to probe the relative contribution of cooperativity and allosteric agonism to the adverse effect liability and neurotoxicity associated with this class of mGlu5 PAMs. PMID:24050755

  19. Exploration of allosteric agonism structure-activity relationships within an acetylene series of metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs): discovery of 5-((3-fluorophenyl)ethynyl)-N-(3-methyloxetan-3-yl)picolinamide (ML254).

    PubMed

    Turlington, Mark; Noetzel, Meredith J; Chun, Aspen; Zhou, Ya; Gogliotti, Rocco D; Nguyen, Elizabeth D; Gregory, Karen J; Vinson, Paige N; Rook, Jerri M; Gogi, Kiran K; Xiang, Zixiu; Bridges, Thomas M; Daniels, J Scott; Jones, Carrie; Niswender, Colleen M; Meiler, Jens; Conn, P Jeffrey; Lindsley, Craig W; Stauffer, Shaun R

    2013-10-24

    Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGlu5) represent a promising therapeutic strategy for the treatment of schizophrenia. Both allosteric agonism and high glutamate fold-shift have been implicated in the neurotoxic profile of some mGlu5 PAMs; however, these hypotheses remain to be adequately addressed. To develop tool compounds to probe these hypotheses, the structure-activity relationship of allosteric agonism was examined within an acetylenic series of mGlu5 PAMs exhibiting allosteric agonism in addition to positive allosteric modulation (ago-PAMs). PAM 38t, a low glutamate fold-shift allosteric ligand (maximum fold-shift ~ 3.0), was selected as a potent PAM with no agonism in the in vitro system used for compound characterization and in two native electrophysiological systems using rat hippocampal slices. PAM 38t (ML254) will be useful to probe the relative contribution of cooperativity and allosteric agonism to the adverse effect liability and neurotoxicity associated with this class of mGlu5 PAMs.

  20. Inhibition of tumor angiogenesis and growth by a small-molecule multi-FGF receptor blocker with allosteric properties.

    PubMed

    Bono, Françoise; De Smet, Frederik; Herbert, Corentin; De Bock, Katrien; Georgiadou, Maria; Fons, Pierre; Tjwa, Marc; Alcouffe, Chantal; Ny, Annelii; Bianciotto, Marc; Jonckx, Bart; Murakami, Masahiro; Lanahan, Anthony A; Michielsen, Christof; Sibrac, David; Dol-Gleizes, Frédérique; Mazzone, Massimiliano; Zacchigna, Serena; Herault, Jean-Pascal; Fischer, Christian; Rigon, Patrice; Ruiz de Almodovar, Carmen; Claes, Filip; Blanc, Isabelle; Poesen, Koen; Zhang, Jie; Segura, Inmaculada; Gueguen, Geneviève; Bordes, Marie-Françoise; Lambrechts, Diether; Broussy, Roselyne; van de Wouwer, Marlies; Michaux, Corinne; Shimada, Toru; Jean, Isabelle; Blacher, Silvia; Noel, Agnès; Motte, Patrick; Rom, Eran; Rakic, Jean-Marie; Katsuma, Susumu; Schaeffer, Paul; Yayon, Avner; Van Schepdael, Ann; Schwalbe, Harald; Gervasio, Francesco Luigi; Carmeliet, Geert; Rozensky, Jef; Dewerchin, Mieke; Simons, Michael; Christopoulos, Arthur; Herbert, Jean-Marc; Carmeliet, Peter

    2013-04-15

    Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally-active extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment.

  1. Ligand-Based Discovery of a New Scaffold for Allosteric Modulation of the μ-Opioid Receptor.

    PubMed

    Bisignano, Paola; Burford, Neil T; Shang, Yi; Marlow, Brennica; Livingston, Kathryn E; Fenton, Abigail M; Rockwell, Kristin; Budenholzer, Lauren; Traynor, John R; Gerritz, Samuel W; Alt, Andrew; Filizola, Marta

    2015-09-28

    With the hope of discovering effective analgesics with fewer side effects, attention has recently shifted to allosteric modulators of the opioid receptors. In the past two years, the first chemotypes of positive or silent allosteric modulators (PAMs or SAMs, respectively) of μ- and δ-opioid receptor types have been reported in the literature. During a structure-guided lead optimization campaign with μ-PAMs BMS-986121 and BMS-986122 as starting compounds, we discovered a new chemotype that was confirmed to display μ-PAM or μ-SAM activity depending on the specific substitutions as assessed by endomorphin-1-stimulated β-arrestin2 recruitment assays in Chinese Hamster Ovary (CHO)-μ PathHunter cells. The most active μ-PAM of this series was analyzed further in competition binding and G-protein activation assays to understand its effects on ligand binding and to investigate the nature of its probe dependence.

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

    PubMed Central

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

    2015-01-01

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

  3. Stereoselective reactivity of the SH groups of yeast glyceraldehydephosphate dehydrogenase in the allosteric T and R states.

    PubMed

    Eisele, B; Wallenfels, K

    1968-07-01

    Yeast glyceraldehyde-3-phosphate dehydrogenase as a typical SH enzyme is inactivated by the antipodes of a-iodopropionic acid and its amide at different rates. The apoenzyme reacts faster with the D(+) antipode of the free a-iodopropionic acid (k(D)/k(L) = 6.8) and the L(-) antipode of the amide (k(L)/k(D) = 3). On addition of NAD(+) the stereoselectivity of the SH group towards a-iodopropionic acid is inverted, that towards the amide is enlarged, the rate relationships depending on the NAD(+) concentration.The results were interpreted by the assumption, that the allosteric T state of the enzyme reacts most rapidly with the D(+) antipodes, whereas the R state favours the L(-) antipodes of the alkylation reagents. The dependence of the reaction rates on the NAD(+) concentration could be fitted to the allosteric function of state R.

  4. Chalcones as positive allosteric modulators of α7 nicotinic acetylcholine receptors: a new target for a privileged structure.

    PubMed

    Balsera, Beatriz; Mulet, José; Fernández-Carvajal, Asia; de la Torre-Martínez, Roberto; Ferrer-Montiel, Antonio; Hernández-Jiménez, José G; Estévez-Herrera, Judith; Borges, Ricardo; Freitas, Andiara E; López, Manuela G; García-López, M Teresa; González-Muñiz, Rosario; Pérez de Vega, María Jesús; Valor, Luis M; Svobodová, Lucie; Sala, Salvador; Sala, Francisco; Criado, Manuel

    2014-10-30

    The α7 acetylcholine nicotine receptor is a ligand-gated ion channel that is involved in cognition disorders, schizophrenia, pain and inflammation among other diseases. Therefore, the development of new agents that target this receptor has great significance. Positive allosteric modulators might be advantageous, since they facilitate receptor responses without directly interacting with the agonist binding site. Here we report the search for and further design of new positive allosteric modulators having the relatively simple chalcone structure. From the natural product isoliquiritigenin as starting point, chalcones substituted with hydroxyl groups at defined locations were identified as optimal and specific promoters of α7 nicotinic function. The most potent compound (2,4,2',5'-tetrahydroxychalcone, 111) was further characterized showing its potential as neuroprotective, analgesic and cognitive enhancer, opening the way for future developments around the chalcone structure.

  5. 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.

  6. Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGluR5) attenuate microglial activation.

    PubMed

    Xue, Fengtian; Stoica, Bogdan A; Hanscom, Marie; Kabadi, Shruti V; Faden, Alan I

    2014-01-01

    Traumatic brain injury causes progressive neurodegeneration associated with chronic microglial activation. Recent studies show that neurodegeneration and neuroinflammation after traumatic brain injury can be inhibited as late as one month in animals by the activation of the metabotropic glutamate receptor 5 in microglia using (RS)-2-chloro-5- hydroxy-phenylglycine. However, the therapeutic potential of this agonist is limited due to its relatively weak potency and brain permeability. To address such concerns, we evaluated the anti-inflammatory activities of several positive allosteric modulators using various in vitro assays, and found that 3,3'-difluorobenzaldazine, 3-cyano-N-(1,3-diphenyl-1H-pyrazol- 5-yl)benzamide and 4-nitro-N-(1-(2-fluorophenyl)-3-phenyl-1H-pyrazol-5-yl)benzamide showed significantly improved potency which makes them potential lead compounds for further development of positive allosteric modulators for the treatment of traumatic brain injury.

  7. Role of the Fourth Transmembrane α Helix in the Allosteric Modulation of Pentameric Ligand-Gated Ion Channels.

    PubMed

    Carswell, Casey L; Hénault, Camille M; Murlidaran, Sruthi; Therien, J P Daniel; Juranka, Peter F; Surujballi, Julian A; Brannigan, Grace; Baenziger, John E

    2015-09-01

    The gating of pentameric ligand-gated ion channels is sensitive to a variety of allosteric modulators that act on structures peripheral to those involved in the allosteric pathway leading from the agonist site to the channel gate. One such structure, the lipid-exposed transmembrane α helix, M4, is the target of lipids, neurosteroids, and disease-causing mutations. Here we show that M4 interactions with the adjacent transmembrane α helices, M1 and M3, modulate pLGIC function. Enhanced M4 interactions promote channel function while ineffective interactions reduce channel function. The interface chemistry governs the intrinsic strength of M4-M1/M3 inter-helical interactions, both influencing channel gating and imparting distinct susceptibilities to the potentiating effects of a lipid-facing M4 congenital myasthenic syndrome mutation. Through aromatic substitutions, functional studies, and molecular dynamics simulations, we elucidate a mechanism by which M4 modulates channel function.

  8. Allergies and Asthma: They Often Occur Together

    MedlinePlus

    ... occur together. The same substances that trigger your hay fever symptoms, such as pollen, dust mites and pet ... a major risk factor for allergic asthma. Having hay fever or other allergies yourself also increases your risk ...

  9. Naturally Occurring Radon and 120(h) transfers

    EPA Pesticide Factsheets

    This page contains a discussion regarding how the presence of naturally occurring radon on closing military bases affects the United States' ability to transfer parcels under §120(h) (3) and §120(h) (4).

  10. Molten Metal Explosions are Still Occurring

    DTIC Science & Technology

    2009-02-01

    recycling plant. Another recent 665 Light Metals 2009 Edited by: Geoff Bearne TMS (The Minerals, Metals & Materials Society), 2009 catastrophic...occurred recently in a recycling plant casting small ingots over a water tank. An explosion occurred that extensively damaged the machine and...have been held in Europe as a joint activity with the European Aluminium Association (EAA) and the International Aluminium Institute (IAI). These

  11. Trichotillomania and Co-occurring Anxiety

    PubMed Central

    Grant, Jon E.; Redden, Sarah A.; Leppink, Eric W.; Chamberlain, Samuel R.

    2017-01-01

    Background Trichotillomania appears to be a fairly common disorder, with high rates of co-occurring anxiety disorders. Many individuals with trichotillomania also report that pulling worsens during periods of increased anxiety. Even with these clinical links to anxiety, little research has explored whether trichotillomania with co-occurring anxiety is a meaningful subtype. Methods 165 adults with trichotillomania were examined on a variety of clinical measures including symptom severity, functioning, and comorbidity. Participants also underwent cognitive testing assessing motor inhibition and cognitive flexibility. Clinical features and cognitive functioning were compared between those with current co-occurring anxiety disorders (i.e. social anxiety, generalized anxiety disorder, panic disorder, and anxiety disorder NOS) (n=38) and those with no anxiety disorder (n=127). Results Participants with trichotillomania and co-occurring anxiety reported significantly worse hair pulling symptoms, were more likely to have co-occurring depression, and were more likely to have a first-degree relative with obsessive compulsive disorder. Those with anxiety disorders also exhibited significantly worse motor inhibitory performance on a task of motor inhibition (stop-signal task). Conclusions This study suggests that anxiety disorders affect the clinical presentation of hair pulling behavior. Further research is needed to validate our findings and to consider whether treatments should be specially tailored differently for adults with trichotillomania who have co-occurring anxiety disorders, or more pronounced cognitive impairment. PMID:27668531

  12. Consequences of Co-Occurring Attention-Deficit/Hyperactivity Disorder on Children's Language Impairments

    ERIC Educational Resources Information Center

    Redmond, Sean M.; Ash, Andrea C.; Hogan, Tiffany P.

    2015-01-01

    Purpose: Co-occurring attention-deficit/hyperactivity disorder (ADHD) and communication disorders represent a frequently encountered challenge for school-based practitioners. The purpose of the present study was to examine in more detail the clinical phenomenology of co-occurring ADHD and language impairments (LIs). Method: Measures of nonword…

  13. 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.

  14. 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

  15. Influence of macrocyclization on allosteric, juxtamembrane-derived, stapled peptide inhibitors of the epidermal growth factor receptor (EGFR).

    PubMed

    Sinclair, Julie K-L; Schepartz, Alanna

    2014-09-19

    The hydrocarbon-stapled peptide E1(S) allosterically inhibits the kinase activity of the epidermal growth factor receptor (EGFR) by blocking a distant but essential protein-protein interaction: a coiled coil formed from the juxtamembrane segment (JM) of each member of the dimeric partnership.1 Macrocyclization is not required for activity: the analogous unstapled (but alkene-bearing) peptide is equipotent in cell viability, immunoblot, and bipartite display experiments to detect coiled coil formation on the cell surface.

  16. Development of M1 mAChR Allosteric and Bitopic Ligands: Prospective Therapeutics for the Treatment of Cognitive Deficits

    PubMed Central

    2013-01-01

    Since the cholinergic hypothesis of memory dysfunction was first reported, extensive research efforts have focused on elucidating the mechanisms by which this intricate system contributes to the regulation of processes such as learning, memory, and higher executive function. Several cholinergic therapeutic targets for the treatment of cognitive deficits, psychotic symptoms, and the underlying pathophysiology of neurodegenerative disorders, such as Alzheimer’s disease and schizophrenia, have since emerged. Clinically approved drugs now exist for some of these targets; however, they all may be considered suboptimal therapeutics in that they produce undesirable off-target activity leading to side effects, fail to address the wide variety of symptoms and underlying pathophysiology that characterize these disorders, and/or afford little to no therapeutic effect in subsets of patient populations. A promising target for which there are presently no approved therapies is the M1 muscarinic acetylcholine receptor (M1 mAChR). Despite avid investigation, development of agents that selectively activate this receptor via the orthosteric site has been hampered by the high sequence homology of the binding site between the five muscarinic receptor subtypes and the wide distribution of this receptor family in both the central nervous system (CNS) and the periphery. Hence, a plethora of ligands targeting less structurally conserved allosteric sites of the M1 mAChR have been investigated. This Review aims to explain the rationale behind allosterically targeting the M1 mAChR, comprehensively summarize and critically evaluate the M1 mAChR allosteric ligand literature to date, highlight the challenges inherent in allosteric ligand investigation that are impeding their clinical advancement, and discuss potential methods for resolving these issues. PMID:23659787

  17. Development of M1 mAChR allosteric and bitopic ligands: prospective therapeutics for the treatment of cognitive deficits.

    PubMed

    Davie, Briana J; Christopoulos, Arthur; Scammells, Peter J

    2013-07-17

    Since the cholinergic hypothesis of memory dysfunction was first reported, extensive research efforts have focused on elucidating the mechanisms by which this intricate system contributes to the regulation of processes such as learning, memory, and higher executive function. Several cholinergic therapeutic targets for the treatment of cognitive deficits, psychotic symptoms, and the underlying pathophysiology of neurodegenerative disorders, such as Alzheimer's disease and schizophrenia, have since emerged. Clinically approved drugs now exist for some of these targets; however, they all may be considered suboptimal therapeutics in that they produce undesirable off-target activity leading to side effects, fail to address the wide variety of symptoms and underlying pathophysiology that characterize these disorders, and/or afford little to no therapeutic effect in subsets of patient populations. A promising target for which there are presently no approved therapies is the M1 muscarinic acetylcholine receptor (M1 mAChR). Despite avid investigation, development of agents that selectively activate this receptor via the orthosteric site has been hampered by the high sequence homology of the binding site between the five muscarinic receptor subtypes and the wide distribution of this receptor family in both the central nervous system (CNS) and the periphery. Hence, a plethora of ligands targeting less structurally conserved allosteric sites of the M1 mAChR have been investigated. This Review aims to explain the rationale behind allosterically targeting the M1 mAChR, comprehensively summarize and critically evaluate the M1 mAChR allosteric ligand literature to date, highlight the challenges inherent in allosteric ligand investigation that are impeding their clinical advancement, and discuss potential methods for resolving these issues.

  18. 5-(N, N-Hexamethylene) amiloride is a GABA-A ρ1 receptor positive allosteric modulator.

    PubMed

    Snell, Heather D; Gonzales, Eric B

    2016-11-01

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

  19. Overcoming EGFR(T790M) and EGFR(C797S) resistance with mutant-selective allosteric inhibitors.

    PubMed

    Jia, Yong; Yun, Cai-Hong; Park, Eunyoung; Ercan, Dalia; Manuia, Mari; Juarez, Jose; Xu, Chunxiao; Rhee, Kevin; Chen, Ting; Zhang, Haikuo; Palakurthi, Sangeetha; Jang, Jaebong; Lelais, Gerald; DiDonato, Michael; Bursulaya, Badry; Michellys, Pierre-Yves; Epple, Robert; Marsilje, Thomas H; McNeill, Matthew; Lu, Wenshuo; Harris, Jennifer; Bender, Steven; Wong, Kwok-Kin; Jänne, Pasi A; Eck, Michael J

    2016-06-02

    The epidermal growth factor receptor (EGFR)-directed tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harbouring activating mutations in the EGFR kinase, but resistance arises rapidly, most frequently owing to the secondary T790M mutation within the ATP site of the receptor. Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternative mechanisms of action. Here we describe the rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild-type receptor. The crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays. However, as a single agent it is not effective in blocking EGFR-driven proliferation in cells owing to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state. We observe marked synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR(L858R/T790M) and by EGFR(L858R/T790M/C797S), a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors.

  20. Identification and Quantification of a New Family of Peptide Endocannabinoids (Pepcans) Showing Negative Allosteric Modulation at CB1 Receptors*

    PubMed Central

    Bauer, Mark; Chicca, Andrea; Tamborrini, Marco; Eisen, David; Lerner, Raissa; Lutz, Beat; Poetz, Oliver; Pluschke, Gerd; Gertsch, Jürg

    2012-01-01

    The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB1). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB1 receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [3H]CP55,940 and [3H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [3H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB1 receptors. Competition binding studies revealed Ki values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [35S]GTPγS binding, and CB1 receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB1 receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling. PMID:22952224

  1. 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.

  2. Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site

    PubMed Central

    Snell, Heather D.

    2015-01-01

    Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cys-loop family of ligand-gated ion channels, such as the heteromeric GABA-A αβγ receptors. The actions of these guanidine compounds on the homomeric GABA-A ρ1 receptor remains unclear, especially in light of how many GABA-A αβγ receptor modulators have different effects in the GABA-A ρ1 receptors. We sought to characterize the influence of amiloride and GMQ on the human GABA-A ρ1 receptors using whole-cell patch-clamp electrophysiology. The diuretic amiloride potentiated the human GABA-A ρ1 GABA-mediated current, whereas GMQ antagonized the receptor. Furthermore, a GABA-A second transmembrane domain site, the intersubunit site, responsible for allosteric modulation in the heteromeric GABA-A receptors mediated amiloride’s positive allosteric actions. In contrast, the mutation did not remove GMQ antagonism but only changed the guanidine compound’s potency within the human GABA-A ρ1 receptor. Through modeling and introduction of point mutations, we propose that the GABA-A ρ1 intersubunit site plays a role in mediating the allosteric effects of amiloride and GMQ. PMID:25829529

  3. Continuous Allosteric Regulation of a Viral Packaging Motor by a Sensor that Detects the Density and Conformation of Packaged DNA

    PubMed Central

    Berndsen, Zachary T.; Keller, Nicholas; Smith, Douglas E.

    2015-01-01

    We report evidence for an unconventional type of allosteric regulation of a biomotor. We show that the genome-packaging motor of phage ϕ29 is regulated by a sensor that detects the density and conformation of the DNA packaged inside the viral capsid, and slows the motor by a mechanism distinct from the effect of a direct load force on the motor. Specifically, we show that motor-ATP interactions are regulated by a signal that is propagated allosterically from inside the viral shell to the motor mounted on the outside. This signal continuously regulates the motor speed and pausing in response to changes in either density or conformation of the packaged DNA, and slows the motor before the buildup of large forces resisting DNA confinement. Analysis of motor slipping reveals that the force resisting packaging remains low (<1 pN) until ∼70% and then rises sharply to ∼23 pN at high filling, which is a several-fold lower value than was previously estimated under the assumption that force alone slows the motor. These findings are consistent with recent studies of the stepping kinetics of the motor. The allosteric regulatory mechanism we report allows double-stranded DNA viruses to achieve rapid, high-density packing of their genomes by limiting the buildup of nonequilibrium load forces on the motor. PMID:25606680

  4. Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site.

    PubMed

    Snell, Heather D; Gonzales, Eric B

    2015-06-01

    Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cys-loop family of ligand-gated ion channels, such as the heteromeric GABA-A αβγ receptors. The actions of these guanidine compounds on the homomeric GABA-A ρ1 receptor remains unclear, especially in