ASD: a comprehensive database of allosteric proteins and modulators

PubMed Central

Huang, Zhimin; Zhu, Liang; Cao, Yan; Wu, Geng; Liu, Xinyi; Chen, Yingyi; Wang, Qi; Shi, Ting; Zhao, Yaxue; Wang, Yuefei; Li, Weihua; Li, Yixue; Chen, Haifeng; Chen, Guoqiang; Zhang, Jian

2011-01-01

Allostery is the most direct, rapid and efficient way of regulating protein function, ranging from the control of metabolic mechanisms to signal-transduction pathways. However, an enormous amount of unsystematic allostery information has deterred scientists who could benefit from this field. Here, we present the AlloSteric Database (ASD), the first online database that provides a central resource for the display, search and analysis of structure, function and related annotation for allosteric molecules. Currently, ASD contains 336 allosteric proteins from 101 species and 8095 modulators in three categories (activators, inhibitors and regulators). Proteins are annotated with a detailed description of allostery, biological process and related diseases, and modulators with binding affinity, physicochemical properties and therapeutic area. Integrating the information of allosteric proteins in ASD should allow for the identification of specific allosteric sites of a given subtype among proteins of the same family that can potentially serve as ideal targets for experimental validation. In addition, modulators curated in ASD can be used to investigate potent allosteric targets for the query compound, and also help chemists to implement structure modifications for novel allosteric drug design. Therefore, ASD could be a platform and a starting point for biologists and medicinal chemists for furthering allosteric research. ASD is freely available at http://mdl.shsmu.edu.cn/ASD/. PMID:21051350

Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch.

PubMed

Oakes, Benjamin L; Nadler, Dana C; Flamholz, Avi; Fellmann, Christof; Staahl, Brett T; Doudna, Jennifer A; Savage, David F

2016-06-01

The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disruption of the enzyme's binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the estrogen receptor-α ligand-binding domain. This protein showed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9.

Glutamine 89 is a key residue in the allosteric modulation of human serine racemase activity by ATP.

PubMed

Canosa, Andrea V; Faggiano, Serena; Marchetti, Marialaura; Armao, Stefano; Bettati, Stefano; Bruno, Stefano; Percudani, Riccardo; Campanini, Barbara; Mozzarelli, Andrea

2018-06-13

Serine racemase (SR) catalyses two reactions: the reversible racemisation of L-serine and the irreversible dehydration of L- and D-serine to pyruvate and ammonia. SRs are evolutionarily related to serine dehydratases (SDH) and degradative threonine deaminases (TdcB). Most SRs and TdcBs - but not SDHs - are regulated by nucleotides. SR binds ATP cooperatively and the nucleotide allosterically stimulates the serine dehydratase activity of the enzyme. A H-bond network comprising five residues (T52, N86, Q89, E283 and N316) and water molecules connects the active site with the ATP-binding site. Conservation analysis points to Q89 as a key residue for the allosteric communication, since its mutation to either Met or Ala is linked to the loss of control of activity by nucleotides. We verified this hypothesis by introducing the Q89M and Q89A point mutations in the human SR sequence. The allosteric communication between the active site and the allosteric site in both mutants is almost completely abolished. Indeed, the stimulation of the dehydratase activity by ATP is severely diminished and the binding of the nucleotide is no more cooperative. Ancestral state reconstruction suggests that the allosteric control by nucleotides established early in SR evolution and has been maintained in most eukaryotic lineages.

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

PubMed Central

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

2012-01-01

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

Dynamic Transmission of Protein Allostery without Structural Change: Spatial Pathways or Global Modes?

PubMed Central

McLeish, Tom C.B.; Cann, Martin J.; Rodgers, Thomas L.

2015-01-01

We examine the contrast between mechanisms for allosteric signaling that involve structural change, and those that do not, from the perspective of allosteric pathways. In particular we treat in detail the case of fluctuation-allostery by which amplitude modulation of the thermal fluctuations of the elastic normal modes conveys the allosteric signal, and address the question of what an allosteric pathway means in this case. We find that a perturbation theory of thermal elastic solids and nonperturbative approach (by super-coarse-graining elasticity into internal bending modes) have opposite signatures in their structure of correlated pathways. We illustrate the effect from analysis of previous results from GlxR of Corynebacterium glutamicum, an example of the CRP/FNR transcription family of allosteric homodimers. We find that the visibility of both correlated pathways and disconnected sites of correlated motion in this protein suggests that mechanisms of local elastic stretch and bend are recruited for the purpose of creating and controlling allosteric cooperativity. PMID:26338443

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

PubMed

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

2018-06-07

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

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

NASA Astrophysics Data System (ADS)

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

2014-04-01

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

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

PubMed

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

2014-04-01

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

Engineered Photoactivatable Genetic Switches Based on the Bacterium Phage T7 RNA Polymerase.

PubMed

Han, Tiyun; Chen, Quan; Liu, Haiyan

2017-02-17

Genetic switches in which the activity of T7 RNA polymerase (RNAP) is directly regulated by external signals are obtained with an engineering strategy of splitting the protein into fragments and using regulatory domains to modulate their reconstitutions. Robust switchable systems with excellent dark-off/light-on properties are obtained with the light-activatable VVD domain and its variants as regulatory domains. For the best split position found, working switches exploit either the light-induced interactions between the VVD domains or allosteric effects. The split fragments show high modularity when they are combined with different regulatory domains such as those with chemically inducible interaction, enabling chemically controlled switches. To summarize, the T7 RNA polymerase-based switches are powerful tools to implement light-activated gene expression in different contexts. Moreover, results about the studied split positions and domain organizations may facilitate future engineering studies on this and on related proteins.

Metabolic engineering of Bacillus subtilis fueled by systems biology: Recent advances and future directions.

PubMed

Liu, Yanfeng; Li, Jianghua; Du, Guocheng; Chen, Jian; Liu, Long

By combining advanced omics technology and computational modeling, systems biologists have identified and inferred thousands of regulatory events and system-wide interactions of the bacterium Bacillus subtilis, which is commonly used both in the laboratory and in industry. This dissection of the multiple layers of regulatory networks and their interactions has provided invaluable information for unraveling regulatory mechanisms and guiding metabolic engineering. In this review, we discuss recent advances in the systems biology and metabolic engineering of B. subtilis and highlight current gaps in our understanding of global metabolism and global pathway engineering in this organism. We also propose future perspectives in the systems biology of B. subtilis and suggest ways that this approach can be used to guide metabolic engineering. Specifically, although hundreds of regulatory events have been identified or inferred via systems biology approaches, systematic investigation of the functionality of these events in vivo has lagged, thereby preventing the elucidation of regulatory mechanisms and further rational pathway engineering. In metabolic engineering, ignoring the engineering of multilayer regulation hinders metabolic flux redistribution. Post-translational engineering, allosteric engineering, and dynamic pathway analyses and control will also contribute to the modulation and control of the metabolism of engineered B. subtilis, ultimately producing the desired cellular traits. We hope this review will aid metabolic engineers in making full use of available systems biology datasets and approaches for the design and perfection of microbial cell factories through global metabolism optimization. Copyright © 2016 Elsevier Inc. All rights reserved.

Probing Allosteric Inhibition Mechanisms of the Hsp70 Chaperone Proteins Using Molecular Dynamics Simulations and Analysis of the Residue Interaction Networks.

PubMed

Stetz, Gabrielle; Verkhivker, Gennady M

2016-08-22

Although molecular mechanisms of allosteric regulation in the Hsp70 chaperones have been extensively studied at both structural and functional levels, the current understanding of allosteric inhibition of chaperone activities by small molecules is still lacking. In the current study, using a battery of computational approaches, we probed allosteric inhibition mechanisms of E. coli Hsp70 (DnaK) and human Hsp70 proteins by small molecule inhibitors PET-16 and novolactone. Molecular dynamics simulations and binding free energy analysis were combined with network-based modeling of residue interactions and allosteric communications to systematically characterize and compare molecular signatures of the apo form, substrate-bound, and inhibitor-bound chaperone complexes. The results suggested a mechanism by which the allosteric inhibitors may leverage binding energy hotspots in the interaction networks to stabilize a specific conformational state and impair the interdomain allosteric control. Using the network-based centrality analysis and community detection, we demonstrated that substrate binding may strengthen the connectivity of local interaction communities, leading to a dense interaction network that can promote an efficient allosteric communication. In contrast, binding of PET-16 to DnaK may induce significant dynamic changes and lead to a fractured interaction network and impaired allosteric communications in the DnaK complex. By using a mechanistic-based analysis of distance fluctuation maps and allosteric propensities of protein residues, we determined that the allosteric network in the PET-16 complex may be small and localized due to the reduced communication and low cooperativity of the substrate binding loops, which may promote the higher rates of substrate dissociation and the decreased substrate affinity. In comparison with the significant effect of PET-16, binding of novolactone to HSPA1A may cause only moderate network changes and preserve allosteric coupling between the allosteric pocket and the substrate binding region. The impact of novolactone on the conformational dynamics and allosteric communications in the HSPA1A complex was comparable to the substrate effect, which is consistent with the experimental evidence that PET-16, but not novolactone binding, can significantly decrease substrate affinity. We argue that the unique dynamic and network signatures of PET-16 and novolactone may be linked with the experimentally observed functional effects of these inhibitors on allosteric regulation and substrate binding.

Allosteric Control of Icosahedral Capsid Assembly

PubMed Central

Lazaro, Guillermo R.

2017-01-01

During the lifecycle of a virus, viral proteins and other components self-assemble to form an ordered protein shell called a capsid. This assembly process is subject to multiple competing constraints, including the need to form a thermostable shell while avoiding kinetic traps. It has been proposed that viral assembly satisfies these constraints through allosteric regulation, including the interconversion of capsid proteins among conformations with different propensities for assembly. In this article we use computational and theoretical modeling to explore how such allostery affects the assembly of icosahedral shells. We simulate assembly under a wide range of protein concentrations, protein binding affinities, and two different mechanisms of allosteric control. We find that, above a threshold strength of allosteric control, assembly becomes robust over a broad range of subunit binding affinities and concentrations, allowing the formation of highly thermostable capsids. Our results suggest that allostery can significantly shift the range of protein binding affinities that lead to successful assembly, and thus should be accounted for in models that are used to estimate interaction parameters from experimental data. PMID:27117092

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

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

PubMed

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

2014-11-11

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

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 and direct the chaperone cycle by exerting the precise control over structural stability, global movements and allosteric communications in Hsp90. PMID:23977182

Allosteric activation transitions in enzymes and biomolecular motors: insights from atomistic and coarse-grained simulations.

PubMed

Daily, Michael D; Yu, Haibo; Phillips, George N; Cui, Qiang

2013-01-01

The chemical step in enzymes is usually preceded by a kinetically distinct activation step that involves large-scale conformational transitions. In "simple" enzymes this step corresponds to the closure of the active site; in more complex enzymes, such as biomolecular motors, the activation step is more complex and may involve interactions with other biomolecules. These activation transitions are essential to the function of enzymes and perturbations in the scale and/or rate of these transitions are implicated in various serious human diseases; incorporating key flexibilities into engineered enzymes is also considered a major remaining challenge in rational enzyme design. Therefore it is important to understand the underlying mechanism of these transitions. This is a significant challenge to both experimental and computational studies because of the allosteric and multi-scale nature of such transitions. Using our recent studies of two enzyme systems, myosin and adenylate kinase (AK), we discuss how atomistic and coarse-grained simulations can be used to provide insights into the mechanism of activation transitions in realistic systems. Collectively, the results suggest that although many allosteric transitions can be viewed as domain displacements mediated by flexible hinges, there are additional complexities and various deviations. For example, although our studies do not find any evidence for "cracking" in AK, our results do underline the contribution of intra-domain properties (e.g., dihedral flexibility) to the rate of the transition. The study of mechanochemical coupling in myosin highlights that local changes important to chemistry require stabilization from more extensive structural changes; in this sense, more global structural transitions are needed to activate the chemistry in the active site. These discussions further emphasize the importance of better understanding factors that control the degree of co-operativity for allosteric transitions, again hinting at the intimate connection between protein stability and functional flexibility. Finally, a number of topics of considerable future interest are briefly discussed.

Understanding G Protein-Coupled Receptor Allostery via Molecular Dynamics Simulations: Implications for Drug Discovery.

PubMed

Basith, Shaherin; Lee, Yoonji; Choi, Sun

2018-01-01

Unraveling the mystery of protein allostery has been one of the greatest challenges in both structural and computational biology. However, recent advances in computational methods, particularly molecular dynamics (MD) simulations, have led to its utility as a powerful and popular tool for the study of protein allostery. By capturing the motions of a protein's constituent atoms, simulations can enable the discovery of allosteric hot spots and the determination of the mechanistic basis for allostery. These structural and dynamic studies can provide a foundation for a wide range of applications, including rational drug design and protein engineering. In our laboratory, the use of MD simulations and network analysis assisted in the elucidation of the allosteric hotspots and intracellular signal transduction of G protein-coupled receptors (GPCRs), primarily on one of the adenosine receptor subtypes, A 2A adenosine receptor (A 2A AR). In this chapter, we describe a method for calculating the map of allosteric signal flow in different GPCR conformational states and illustrate how these concepts have been utilized in understanding the mechanism of GPCR allostery. These structural studies will provide valuable insights into the allosteric and orthosteric modulations that would be of great help to design novel drugs targeting GPCRs in pathological states.

The interplay between effector binding and allostery in an engineered protein switch.

PubMed

Choi, Jay H; Xiong, Tina; Ostermeier, Marc

2016-09-01

The protein design rules for engineering allosteric regulation are not well understood. A fundamental understanding of the determinants of ligand binding in an allosteric context could facilitate the design and construction of versatile protein switches and biosensors. Here, we conducted extensive in vitro and in vivo characterization of the effects of 285 unique point mutations at 15 residues in the maltose-binding pocket of the maltose-activated β-lactamase MBP317-347. MBP317-347 is an allosteric enzyme formed by the insertion of TEM-1 β-lactamase into the E. coli maltose binding protein (MBP). We find that the maltose-dependent resistance to ampicillin conferred to the cells by the MBP317-347 switch gene (the switch phenotype) is very robust to mutations, with most mutations slightly improving the switch phenotype. We identified 15 mutations that improved switch performance from twofold to 22-fold, primarily by decreasing the catalytic activity in the absence of maltose, perhaps by disrupting interactions that cause a small fraction of MBP in solution to exist in a partially closed state in the absence of maltose. Other notable mutations include K15D and K15H that increased maltose affinity 30-fold and Y155K and Y155R that compromised switching by diminishing the ability of maltose to increase catalytic activity. The data also provided insights into normal MBP physiology, as select mutations at D14, W62, and F156 retained high maltose affinity but abolished the switch's ability to substitute for MBP in the transport of maltose into the cell. The results reveal the complex relationship between ligand binding and allostery in this engineered switch. © 2016 The Protein Society.

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

PubMed

Hacisuleyman, Aysima; Erman, Burak

2017-06-01

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

Allostery in the Hsp70 chaperone proteins

PubMed Central

Zuiderweg, Erik R.P.; Bertelsen, Eric B.; Rousaki, Aikaterini; Mayer, Matthias P.; Gestwicki, Jason E.; Ahmad, Atta

2013-01-01

Heat shock 70 kDa (Hsp70) chaperones are essential to in-vivo protein folding, protein transport and protein re-folding. They carry out these activities using repeated cycles of binding and release of client proteins. This process is under allosteric control of nucleotide binding and hydrolysis. X-ray crystallography, NMR spectroscopy and other biophysical techniques have contributed much to the understanding of the allosteric mechanism linking these activities and the effect of co-chaperones on this mechanism. In this chapter, these findings are critically reviewed. Studies on the allosteric mechanisms of Hsp70 have gained enhanced urgency, as recent studies have implicated this chaperone as a potential drug target in diseases such as Alzheimer's and cancer. Recent approaches to combat these diseases through interference with the Hsp70 allosteric mechanism are discussed. PMID:22576356

Dancing through Life: Molecular Dynamics Simulations and Network-Centric Modeling of Allosteric Mechanisms in Hsp70 and Hsp110 Chaperone Proteins.

PubMed

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 across diverse chaperone functions. This study reconciles a wide spectrum of structural and functional experiments by demonstrating how integration of molecular simulations and network-centric modeling may explain thermodynamic and mechanistic aspects of allosteric regulation in chaperones.

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 across diverse chaperone functions. This study reconciles a wide spectrum of structural and functional experiments by demonstrating how integration of molecular simulations and network-centric modeling may explain thermodynamic and mechanistic aspects of allosteric regulation in chaperones. PMID:26619280

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

PubMed

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

2016-06-15

One of the most abundant G-protein coupled receptors (GPCRs) in brain, the cannabinoid 1 receptor (CB1R), is a tractable therapeutic target for treating diverse psychobehavioral and somatic disorders. Adverse on-target effects associated with small-molecule CB1R orthosteric agonists and inverse agonists/antagonists have plagued their translational potential. Allosteric CB1R modulators offer a potentially safer modality through which CB1R signaling may be directed for therapeutic benefit. Rational design of candidate, druglike CB1R allosteric modulators requires greater understanding of the architecture of the CB1R allosteric endodomain(s) and the capacity of CB1R allosteric ligands to tune the receptor's information output. We have recently reported the synthesis of a focused library of rationally designed, covalent analogues of Org27569 and PSNCBAM-1, two prototypic CB1R negative allosteric modulators (NAMs). Among the novel, pharmacologically active CB1R NAMs reported, the isothiocyanate GAT100 emerged as the lead by virtue of its exceptional potency in the [(35)S]GTPγS and β-arrestin signaling assays and its ability to label CB1R as a covalent allosteric probe with significantly reduced inverse agonism in the [(35)S]GTPγS assay as compared to Org27569. We report here a comprehensive functional profiling of GAT100 across an array of important downstream cell-signaling pathways and analysis of its potential orthosteric probe-dependence and signaling bias. The results demonstrate that GAT100 is a NAM of the orthosteric CB1R agonist CP55,940 and the endocannabinoids 2-arachidonoylglycerol and anandamide for β-arrestin1 recruitment, PLCβ3 and ERK1/2 phosphorylation, cAMP accumulation, and CB1R internalization in HEK293A cells overexpressing CB1R and in Neuro2a and STHdh(Q7/Q7) cells endogenously expressing CB1R. Distinctively, GAT100 was a more potent and efficacious CB1R NAM than Org27569 and PSNCBAM-1 in all signaling assays and did not exhibit the inverse agonism associated with Org27569 and PSNCBAM-1. Computational docking studies implicate C7.38(382) as a key feature of GAT100 ligand-binding motif. These data help inform the engineering of newer-generation, druggable CB1R allosteric modulators and demonstrate the utility of GAT100 as a covalent probe for mapping structure-function correlates characteristic of the druggable CB1R allosteric space.

Design and Synthesis of Cannabinoid 1 Receptor (CB1R) Allosteric Modulators: Drug Discovery Applications.

PubMed

Kulkarni, Abhijit R; Garai, Sumanta; Janero, David R; Thakur, Ganesh A

2017-01-01

Also expressed in various peripheral tissues, the type-1 cannabinoid receptor (CB1R) is the predominant G protein-coupled receptor (GPCR) in brain, where it is responsible for retrograde control of neurotransmitter release. Cellular signaling mediated by CB1R is involved in numerous physiological processes, and pharmacological CB1R modulation is considered a tenable therapeutic approach for diseases ranging from substance-use disorders and glaucoma to metabolic syndrome. Despite the design and synthesis of a variety of bioactive small molecules targeted to the CB1R orthosteric ligand-binding site, the potential of CB1R as a therapeutic GPCR has been largely unrealized due to adverse events associated with typical orthosteric CB1R agonists and antagonists/inverse agonists. Modulation of CB1R-mediated signal transmission by targeting alternative allosteric ligand-binding site(s) on the receptor has garnered interest as a potentially safer and more effective therapeutic modality. This chapter highlights the design and synthesis of novel, pharmacologically active CB1R allosteric modulators and emphasizes how their molecular properties and the positive and negative allosteric control they exert can lead to improved CB1R-targeted pharmacotherapeutics, as well as designer covalent probes that can be used to map CB1R allosteric binding domains and inform structure-based drug design. © 2017 Elsevier Inc. All rights reserved.

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.

Characteristic Features of Kynurenine Aminotransferase Allosterically Regulated by (Alpha)-Ketoglutarate in Cooperation with Kynurenine

PubMed Central

Okada, Ken; Angkawidjaja, Clement; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

2012-01-01

Kynurenine aminotransferase from Pyrococcus horikoshii OT3 (PhKAT), which is a homodimeric protein, catalyzes the conversion of kynurenine (KYN) to kynurenic acid (KYNA). We analyzed the transaminase reaction mechanisms of this protein with pyridoxal-5′-phosphate (PLP), KYN and α-ketoglutaric acid (2OG) or oxaloacetic acid (OXA). 2OG significantly inhibited KAT activities in kinetic analyses, suggesting that a KYNA biosynthesis is allosterically regulated by 2OG. Its inhibitions evidently were unlocked by KYN. 2OG and KYN functioned as an inhibitor and activator in response to changes in the concentrations of KYN and 2OG, respectively. The affinities of one subunit for PLP or 2OG were different from that of the other subunit, as confirmed by spectrophotometry and isothermal titration calorimetry, suggesting that the difference of affinities between subunits might play a role in regulations of the KAT reaction. Moreover, we identified two active and allosteric sites in the crystal structure of PhKAT-2OG complexes. The crystal structure of PhKAT in complex with four 2OGs demonstrates that two 2OGs in allosteric sites are effector molecules which inhibit the KYNA productions. Thus, the combined data lead to the conclusion that PhKAT probably is regulated by allosteric control machineries, with 2OG as the allosteric inhibitor. PMID:22792273

Engineering integrated digital circuits with allosteric ribozymes for scaling up molecular computation and diagnostics.

PubMed

Penchovsky, Robert

2012-10-19

Here we describe molecular implementations of integrated digital circuits, including a three-input AND logic gate, a two-input multiplexer, and 1-to-2 decoder using allosteric ribozymes. Furthermore, we demonstrate a multiplexer-decoder circuit. The ribozymes are designed to seek-and-destroy specific RNAs with a certain length by a fully computerized procedure. The algorithm can accurately predict one base substitution that alters the ribozyme's logic function. The ability to sense the length of RNA molecules enables single ribozymes to be used as platforms for multiple interactions. These ribozymes can work as integrated circuits with the functionality of up to five logic gates. The ribozyme design is universal since the allosteric and substrate domains can be altered to sense different RNAs. In addition, the ribozymes can specifically cleave RNA molecules with triplet-repeat expansions observed in genetic disorders such as oculopharyngeal muscular dystrophy. Therefore, the designer ribozymes can be employed for scaling up computing and diagnostic networks in the fields of molecular computing and diagnostics and RNA synthetic biology.

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.

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

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

PubMed

Hacisuleyman, Aysima; Erman, Burak

2017-01-01

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

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

PubMed Central

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

2016-01-01

TRPV1 channels in sensory neurons are integrators of painful stimuli and heat, yet how they integrate diverse stimuli and sense temperature remains elusive. Here, we show that external sodium ions stabilize the TRPV1 channel in a closed state, such that removing the external ion leads to channel activation. In studying the underlying mechanism, we find that the temperature sensors in TRPV1 activate in two steps to favor opening, and that the binding of sodium to an extracellular site exerts allosteric control over temperature-sensor activation and opening of the pore. The binding of a tarantula toxin to the external pore also exerts control over temperature-sensor activation, whereas binding of vanilloids influences temperature-sensitivity by largely affecting the open/closed equilibrium. Our results reveal a fundamental role of the external pore in the allosteric control of TRPV1 channel gating and provide essential constraints for understanding how these channels can be tuned by diverse stimuli. DOI: http://dx.doi.org/10.7554/eLife.13356.001 PMID:26882503

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

PubMed Central

Christian, Catherine A.

2013-01-01

Allosteric modulators exert actions on neurotransmitter receptors by positively or negatively altering the effective response of these receptors to their respective neurotransmitter. γ-Aminobutyric acid (GABA) type A ionotropic receptors (GABAARs) are major targets for allosteric modulators such as benzodiazepines, neurosteroids, and barbiturates. Analysis of substances that produce similar effects has been hampered by the lack of techniques to assess the localization and function of such agents in brain slices. Here we describe measurement of the sniffer patch laser uncaging response (SPLURgE), which combines the sniffer patch recording configuration with laser photolysis of caged GABA. This methodology enables the detection of allosteric GABAAR modulators endogenously present in discrete areas of the brain slice and allows for the application of exogenous GABA with spatiotemporal control without altering the release and localization of endogenous modulators within the slice. Here we demonstrate the development and use of this technique for the measurement of allosteric modulation in different areas of the thalamus. Application of this technique will be useful in determining whether a lack of modulatory effect on a particular category of neurons or receptors is due to insensitivity to allosteric modulation or a lack of local release of endogenous ligand. We also demonstrate that this technique can be used to investigate GABA diffusion and uptake. This method thus provides a biosensor assay for rapid detection of endogenous GABAAR modulators and has the potential to aid studies of allosteric modulators that exert effects on other classes of neurotransmitter receptors, such as glutamate, acetylcholine, or glycine receptors. PMID:23843428

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

PubMed

Christian, Catherine A; Huguenard, John R

2013-10-01

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

Sequence Analysis and Molecular Characterization of Clonorchis sinensis Hexokinase, an Unusual Trimeric 50-kDa Glucose-6-Phosphate-Sensitive Allosteric Enzyme

PubMed Central

Chen, Tingjin; Ning, Dan; Sun, Hengchang; Li, Ran; Shang, Mei; Li, Xuerong; Wang, Xiaoyun; Chen, Wenjun; Liang, Chi; Li, Wenfang; Mao, Qiang; Li, Ye; Deng, Chuanhuan; Wang, Lexun; Wu, Zhongdao; Huang, Yan; Xu, Jin; Yu, Xinbing

2014-01-01

Clonorchiasis, which is induced by the infection of Clonorchis sinensis (C. sinensis), is highly associated with cholangiocarcinoma. Because the available examination, treatment and interrupting transmission provide limited opportunities to prevent infection, it is urgent to develop integrated strategies to prevent and control clonorchiasis. Glycolytic enzymes are crucial molecules for trematode survival and have been targeted for drug development. Hexokinase of C. sinensis (CsHK), the first key regulatory enzyme of the glycolytic pathway, was characterized in this study. The calculated molecular mass (Mr) of CsHK was 50.0 kDa. The obtained recombinant CsHK (rCsHK) was a homotrimer with an Mr of approximately 164 kDa, as determined using native PAGE and gel filtration. The highest activity was obtained with 50 mM glycine-NaOH at pH 10 and 100 mM Tris-HCl at pH 8.5 and 10. The kinetics of rCsHK has a moderate thermal stability. Compared to that of the corresponding negative control, the enzymatic activity was significantly inhibited by praziquantel (PZQ) and anti-rCsHK serum. rCsHK was homotropically and allosterically activated by its substrates, including glucose, mannose, fructose, and ATP. ADP exhibited mixed allosteric effect on rCsHK with respect to ATP, while inorganic pyrophosphate (PPi) displayed net allosteric activation with various allosteric systems. Fructose behaved as a dose-dependent V activator with the substrate glucose. Glucose-6-phosphate (G6P) displayed net allosteric inhibition on rCsHK with respect to ATP or glucose with various allosteric systems in a dose-independent manner. There were differences in both mRNA and protein levels of CsHK among the life stages of adult worm, metacercaria, excysted metacercaria and egg of C. sinensis, suggesting different energy requirements during different development stages. Our study furthers the understanding of the biological functions of CsHK and supports the need to screen for small molecule inhibitors of CsHK to interfere with glycolysis in C. sinensis. PMID:25232723

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

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

PubMed

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

2004-01-01

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

Allosteric Tuning of Caspase-7: A Fragment-Based Drug Discovery Approach.

PubMed

Vance, Nicholas R; Gakhar, Lokesh; Spies, M Ashley

2017-11-13

The caspase family of cysteine proteases are highly sought-after drug targets owing to their essential roles in apoptosis, proliferation, and inflammation pathways. High-throughput screening efforts to discover inhibitors have gained little traction. Fragment-based screening has emerged as a powerful approach for the discovery of innovative drug leads. This method has become a central facet of drug discovery campaigns in the pharmaceutical industry and academia. A fragment-based drug discovery campaign against human caspase-7 resulted in the discovery of a novel series of allosteric inhibitors. An X-ray crystal structure of caspase-7 bound to a fragment hit and a thorough kinetic characterization of a zymogenic form of the enzyme were used to investigate the allosteric mechanism of inhibition. This work further advances our understanding of the mechanisms of allosteric control of this class of pharmaceutically relevant enzymes, and provides a new path forward for drug discovery efforts. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Optical control of pain in vivo with a photoactive mGlu5 receptor negative allosteric modulator.

PubMed

Font, Joan; López-Cano, Marc; Notartomaso, Serena; Scarselli, Pamela; Di Pietro, Paola; Bresolí-Obach, Roger; Battaglia, Giuseppe; Malhaire, Fanny; Rovira, Xavier; Catena, Juanlo; Giraldo, Jesús; Pin, Jean-Philippe; Fernández-Dueñas, Víctor; Goudet, Cyril; Nonell, Santi; Nicoletti, Ferdinando; Llebaria, Amadeu; Ciruela, Francisco

2017-04-11

Light-operated drugs constitute a major target in drug discovery, since they may provide spatiotemporal resolution for the treatment of complex diseases (i.e. chronic pain). JF-NP-26 is an inactive photocaged derivative of the metabotropic glutamate type 5 (mGlu 5 ) receptor negative allosteric modulator raseglurant. Violet light illumination of JF-NP-26 induces a photochemical reaction prompting the active-drug's release, which effectively controls mGlu 5 receptor activity both in ectopic expressing systems and in striatal primary neurons. Systemic administration in mice followed by local light-emitting diode (LED)-based illumination, either of the thalamus or the peripheral tissues, induced JF-NP-26-mediated light-dependent analgesia both in neuropathic and in acute/tonic inflammatory pain models. These data offer the first example of optical control of analgesia in vivo using a photocaged mGlu 5 receptor negative allosteric modulator. This approach shows potential for precisely targeting, in time and space, endogenous receptors, which may allow a better management of difficult-to-treat disorders.

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.

Co-Conserved MAPK Features Couple D-Domain Docking Groove to Distal Allosteric Sites via the C-Terminal Flanking Tail

PubMed Central

Nguyen, Tuan; Ruan, Zheng; Oruganty, Krishnadev; Kannan, Natarajan

2015-01-01

Mitogen activated protein kinases (MAPKs) form a closely related family of kinases that control critical pathways associated with cell growth and survival. Although MAPKs have been extensively characterized at the biochemical, cellular, and structural level, an integrated evolutionary understanding of how MAPKs differ from other closely related protein kinases is currently lacking. Here, we perform statistical sequence comparisons of MAPKs and related protein kinases to identify sequence and structural features associated with MAPK functional divergence. We show, for the first time, that virtually all MAPK-distinguishing sequence features, including an unappreciated short insert segment in the β4-β5 loop, physically couple distal functional sites in the kinase domain to the D-domain peptide docking groove via the C-terminal flanking tail (C-tail). The coupling mediated by MAPK-specific residues confers an allosteric regulatory mechanism unique to MAPKs. In particular, the regulatory αC-helix conformation is controlled by a MAPK-conserved salt bridge interaction between an arginine in the αC-helix and an acidic residue in the C-tail. The salt-bridge interaction is modulated in unique ways in individual sub-families to achieve regulatory specificity. Our study is consistent with a model in which the C-tail co-evolved with the D-domain docking site to allosterically control MAPK activity. Our study provides testable mechanistic hypotheses for biochemical characterization of MAPK-conserved residues and new avenues for the design of allosteric MAPK inhibitors. PMID:25799139

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

PubMed

Nickols, Hilary Highfield; Conn, P Jeffrey

2014-01-01

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

The future of type 1 cannabinoid receptor allosteric ligands.

PubMed

Alaverdashvili, Mariam; Laprairie, Robert B

2018-02-01

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

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

NASA Astrophysics Data System (ADS)

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

2013-09-01

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

Development of allosteric modulators of GPCRs for treatment of CNS disorders

PubMed Central

Nickols, Hilary Highfield; Conn, P. Jeffrey

2013-01-01

The discovery of allosteric modulators of G protein-coupled receptors (GPCRs) provides a promising new strategy with potential for developing novel treatments for a variety of central nervous system (CNS) disorders. Traditional drug discovery efforts targeting GPCRs have focused on developing ligands for orthosteric sites which bind endogenous ligands. Allosteric modulators target a site separate from the orthosteric site to modulate receptor function. These allosteric agents can either potentiate (positive allosteric modulator, PAM) or inhibit (negative allosteric modulator, NAM) the receptor response and often provide much greater subtype selectivity than do orthosteric ligands for the same receptors. Experimental evidence has revealed more nuanced pharmacological modes of action of allosteric modulators, with some PAMs showing allosteric agonism in combination with positive allosteric modulation in response to endogenous ligand (ago-potentiators) as well as “bitopic” ligands that interact with both the allosteric and orthosteric sites. Drugs targeting the allosteric site allow for increased drug selectivity and potentially decreased adverse side effects. Promising evidence has demonstrated potential utility of a number of allosteric modulators of GPCRs in multiple CNS disorders, including neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, as well as psychiatric or neurobehavioral diseases such as anxiety, schizophrenia, and addiction. PMID:24076101

Tuning the allosteric regulation of artificial muscarinic and dopaminergic ligand-gated potassium channels by protein engineering of G protein-coupled receptors

PubMed Central

Moreau, Christophe J.; Revilloud, Jean; Caro, Lydia N.; Dupuis, Julien P.; Trouchet, Amandine; Estrada-Mondragón, Argel; Nieścierowicz, Katarzyna; Sapay, Nicolas; Crouzy, Serge; Vivaudou, Michel

2017-01-01

Ligand-gated ion channels enable intercellular transmission of action potential through synapses by transducing biochemical messengers into electrical signal. We designed artificial ligand-gated ion channels by coupling G protein-coupled receptors to the Kir6.2 potassium channel. These artificial channels called ion channel-coupled receptors offer complementary properties to natural channels by extending the repertoire of ligands to those recognized by the fused receptors, by generating more sustained signals and by conferring potassium selectivity. The first artificial channels based on the muscarinic M2 and the dopaminergic D2L receptors were opened and closed by acetylcholine and dopamine, respectively. We find here that this opposite regulation of the gating is linked to the length of the receptor C-termini, and that C-terminus engineering can precisely control the extent and direction of ligand gating. These findings establish the design rules to produce customized ligand-gated channels for synthetic biology applications. PMID:28145461

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.

Allosteric activation via kinetic control: Potassium accelerates a conformational change in IMP dehydrogenase†

PubMed Central

Riera, Thomas V.; Zheng, Lianqing; Josephine, Helen R.; Min, Donghong; Yang, Wei; Hedstrom, Lizbeth

2011-01-01

Allosteric activators are generally believed to shift the equilibrium distribution of enzyme conformations to favor a catalytically productive structure; the kinetics of conformational exchange is seldom addressed. Several observations suggested that the usual allosteric mechanism might not apply to the activation of IMP dehydrogenase (IMPDH) by monovalent cations. Therefore we investigated the mechanism of K+ activation in IMPDH by delineating the kinetic mechanism in the absence of monovalent cations. Surprisingly, the K+-dependence of kcat derives from the rate of flap closure, which increases by ≥65-fold in the presence of K+. We performed both alchemical free energy simulations and potential of mean force calculations using the orthogonal space random walk strategy to computationally analyze how K+ accelerates this conformational change. The simulations recapitulate the preference of IMPDH for K+, validating the computational models. When K+ is replaced with a dummy ion, the residues of the K+ binding site relax into ordered secondary structure, creating a barrier to conformational exchange. K+ mobilizes these residues by providing alternate interactions for the main chain carbonyls. Potential of mean force calculations indicate that K+ changes the shape of the energy well, shrinking the reaction coordinate by shifting the closed conformation toward the open state. This work suggests that allosteric regulation can be under kinetic as well as thermodynamic control. PMID:21870820

Allosteric control of internal electron transfer in cytochrome cd1 nitrite reductase

PubMed Central

Farver, Ole; Kroneck, Peter M. H.; Zumft, Walter G.; Pecht, Israel

2003-01-01

Cytochrome cd1 nitrite reductase is a bifunctional multiheme enzyme catalyzing the one-electron reduction of nitrite to nitric oxide and the four-electron reduction of dioxygen to water. Kinetics and thermodynamics of the internal electron transfer process in the Pseudomonas stutzeri enzyme have been studied and found to be dominated by pronounced interactions between the c and the d1 hemes. The interactions are expressed both in dramatic changes in the internal electron-transfer rates between these sites and in marked cooperativity in their electron affinity. The results constitute a prime example of intraprotein control of the electron-transfer rates by allosteric interactions. PMID:12802018

Role of Protein Dimeric Interface in Allosteric Inhibition of N-Acetyl-Aspartate Hydrolysis by Human Aspartoacylase.

PubMed

Kots, Ekaterina D; Lushchekina, Sofya V; Varfolomeev, Sergey D; Nemukhin, Alexander V

2017-08-28

The results of molecular modeling suggest a mechanism of allosteric inhibition upon hydrolysis of N-acetyl-aspartate (NAA), one of the most abundant amino acid derivatives in brain, by human aspartoacylase (hAsp). Details of this reaction are important to suggest the practical ways to control the enzyme activity. Search for allosteric sites using the Allosite web server and SiteMap analysis allowed us to identify substrate binding pockets located at the interface between the subunits of the hAsp dimer molecule. Molecular docking of NAA to the pointed areas at the dimer interface predicted a specific site, in which the substrate molecule interacts with the Gly237, Arg233, Glu290, and Lys292 residues. Analysis of multiple long-scaled molecular dynamics trajectories (the total simulation time exceeded 1.5 μs) showed that binding of NAA to the identified allosteric site induced significant rigidity to the protein loops with the amino acid side chains forming gates to the enzyme active site. Application of the protein dynamical network algorithms showed that substantial reorganization of the signal propagation pathways of intersubunit communication in the dimer occurred upon allosteric NAA binding to the remote site. The modeling approaches provide an explanation to the observed decrease of the reaction rate of NAA hydrolysis by hAsp at high substrate concentrations.

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-grained methodology is able to capture the effects triggered by allosteric ligands already described in the literature. Conclusions We introduce a simple computational approach to predict the presence and position of allosteric sites in a protein based on the analysis of changes in protein normal modes upon the binding of a coarse-grained ligand at predicted cavities. Its performance has been demonstrated using a newly curated non-redundant set of 91 proteins with reported allosteric properties. The software developed in this work is available upon request from the authors. PMID:23095452

Exploiting protein flexibility to predict the location of allosteric sites.

PubMed

Panjkovich, Alejandro; Daura, Xavier

2012-10-25

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. 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-grained methodology is able to capture the effects triggered by allosteric ligands already described in the literature. We introduce a simple computational approach to predict the presence and position of allosteric sites in a protein based on the analysis of changes in protein normal modes upon the binding of a coarse-grained ligand at predicted cavities. Its performance has been demonstrated using a newly curated non-redundant set of 91 proteins with reported allosteric properties. The software developed in this work is available upon request from the authors.

Automated identification of functional dynamic networks from X-ray crystallography

PubMed Central

van den Bedem, Henry; Bhabha, Gira; Yang, Kun; Wright, Peter E.; Fraser, James S.

2013-01-01

Protein function often depends on the exchange between conformational substates. Allosteric ligand binding or distal mutations can stabilize specific active site conformations and consequently alter protein function. In addition to comparing independently determined X-ray crystal structures, alternative conformations observed at low levels of electron density have the potential to provide mechanistic insights into conformational dynamics. Here, we report a new multi-conformer contact network algorithm (CONTACT) that identifies networks of conformationally heterogeneous residues directly from high-resolution X-ray crystallography data. Contact networks in Escherichia coli dihydrofolate reductase (ecDHFR) predict the long-range pattern of NMR chemical shift perturbations of an allosteric mutation. A comparison of contact networks in wild type and mutant ecDHFR suggests how mutations that alter optimized networks of coordinated motions can impair catalytic function. Thus, CONTACT-guided mutagenesis will allow the structure-dynamics-function relationship to be exploited in protein engineering and design. PMID:23913260

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.

Dual functionality of β-tryptase protomers as both proteases and cofactors in the active tetramer.

PubMed

Maun, Henry R; Liu, Peter S; Franke, Yvonne; Eigenbrot, Charles; Forrest, William F; Schwartz, Lawrence B; Lazarus, Robert A

2018-04-16

Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of the allergic inflammatory responses in asthma. During acute hypersensitivity reactions, mast cells degranulate, releasing active tetramer as a complex with proteoglycans. Extensive efforts have focused on developing therapeutic β-tryptase inhibitors, but its unique activation mechanism is less well explored. Tryptase is active only after proteolytic removal of the pro-domain followed by tetramer formation via two distinct symmetry-related interfaces. We show that the cleaved I16G mutant cannot tetramerize, likely due to impaired insertion of its N-terminus into its 'activation pocket', indicating allosteric linkage at multiple sites on each protomer. We engineered cysteines into each of the two distinct interfaces (Y75C for small or I99C for large) to assess the activity of each tetramer and disulfide-locked dimer. Using size-exclusion chromatography and enzymatic assays, we demonstrate that the two large tetramer interfaces regulate enzymatic activity, elucidating the importance of this protein-protein interaction for allosteric regulation. Notably, the I99C large interface dimer is active, even in the absence of heparin. We show that a monomeric β-tryptase mutant (I99C*:Y75A:Y37bA where C* is cysteinylated Cys99) cannot form a dimer or tetramer, yet is active, but only in the presence of heparin. Thus heparin both stabilizes the tetramer and allosterically conditions the active site. We hypothesize that each β-tryptase protomer in the tetramer has two distinct roles, acting both as a protease and as a cofactor for its neighboring protomer, to allosterically regulate enzymatic activity, providing a rationale for direct correlation of tetramer stability with proteolytic activity. Copyright © 2018, The American Society for Biochemistry and Molecular Biology.

Proof of principle in a de novo designed protein maquette: an allosterically regulated, charge-activated conformational switch in a tetra-alpha-helix bundle.

PubMed

Grosset, A M; Gibney, B R; Rabanal, F; Moser, C C; Dutton, P L

2001-05-08

New understanding of the engineering and allosteric regulation of natural protein conformational switches (such as those that couple chemical and ionic signals, mechanical force, and electro/chemical free energy for biochemical activation, catalysis, and motion) can be derived from simple de novo designed synthetic protein models (maquettes). We demonstrate proof of principle of both reversible switch action and allosteric regulation in a tetra-alpha-helical bundle protein composed of two identical di-helical subunits containing heme coordinated at a specific position close to the disulfide loop region. Individual bundles assume one of two switch states related by large-scale mechanical changes: a syn-topology (helices of the different subunits parallel) or anti-topology (helices antiparallel). Both the spectral properties of a coproporphyrin probe appended to the loop region and the distance-dependent redox interaction between the hemes identify the topologies. Beginning from a syn-topology, introduction of ferric heme in each subunit (either binding or redox change) shifts the topological balance by 25-50-fold (1.9-2.3 kcal/mol) to an anti-dominance. Charge repulsion between the two internal cationic ferric hemes drives the syn- to anti-switch, as demonstrated in two ways. When fixed in the syn-topology, the second ferric heme binding is 25-80-fold (1.9-2.6 kcal/mol) weaker than the first, and adjacent heme redox potentials are split by 80 mV (1.85 kcal/mol), values that energetically match the shift in topological balance. Allosteric and cooperative regulation of the switch by ionic strength exploits the shielded charge interactions between the two hemes and the exposed, cooperative interactions between the coproporphyrin carboxylates.

Allosteric Control of Substrate Specificity of the Escherichia coli ADP-glucose Pyrophosphorylase

NASA Astrophysics Data System (ADS)

Ebrecht, Ana C.; Solamen, Ligin; Hill, Benjamin L.; Iglesias, Alberto A.; Olsen, Kenneth W.; Ballicora, Miguel A.

2017-06-01

The substrate specificity of enzymes is crucial to control the fate of metabolites to different pathways. However, there is growing evidence that many enzymes can catalyze alternative reactions. This promiscuous behavior has important implications in protein evolution and the acquisition of new functions. The question is how the undesirable outcomes of in vivo promiscuity can be prevented. ADP-glucose pyrophosphorylase from Escherichia coli is an example of an enzyme that needs to select the correct substrate from a broad spectrum of alternatives. This selection will guide the flow of carbohydrate metabolism towards the synthesis of reserve polysaccharides. Here, we show that the allosteric activator fructose-1,6-bisphosphate plays a role in such selection by increasing the catalytic efficiency of the enzyme towards the use of ATP rather than other nucleotides. In the presence of fructose-1,6-bisphosphate, the kcat/S0.5 for ATP was near 600-fold higher that other nucleotides, whereas in the absence of activator was only 3-fold higher. We propose that the allosteric regulation of certain enzymes is an evolutionary mechanism of adaptation for the selection of specific substrates.

Enhanced SH3/Linker Interaction Overcomes Abl Kinase Activation by Gatekeeper and Myristic Acid Binding Pocket Mutations and Increases Sensitivity to Small Molecule Inhibitors*

PubMed Central

Panjarian, Shoghag; Iacob, Roxana E.; Chen, Shugui; Wales, Thomas E.; Engen, John R.; Smithgall, Thomas E.

2013-01-01

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control. PMID:23303187

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

PubMed

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.

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.

α2-containing GABAA receptors expressed in hippocampal region CA3 control fast network oscillations

PubMed Central

Heistek, Tim S; Ruiperez-Alonso, Marta; Timmerman, A Jaap; Brussaard, Arjen B; Mansvelder, Huibert D

2013-01-01

GABAA receptors are critically involved in hippocampal oscillations. GABAA receptor α1 and α2 subunits are differentially expressed throughout the hippocampal circuitry and thereby may have distinct contributions to oscillations. It is unknown which GABAA receptor α subunit controls hippocampal oscillations and where these receptors are expressed. To address these questions we used transgenic mice expressing GABAA receptor α1 and/or α2 subunits with point mutations (H101R) that render these receptors insensitive to allosteric modulation at the benzodiazepine binding site, and tested how increased or decreased function of α subunits affects hippocampal oscillations. Positive allosteric modulation by zolpidem prolonged decay kinetics of hippocampal GABAergic synaptic transmission and reduced the frequency of cholinergically induced oscillations. Allosteric modulation of GABAergic receptors in CA3 altered oscillation frequency in CA1, while modulation of GABA receptors in CA1 did not affect oscillations. In mice having a point mutation (H101R) at the GABAA receptor α2 subunit, zolpidem effects on cholinergically induced oscillations were strongly reduced compared to wild-type animals, while zolpidem modulation was still present in mice with the H101R mutation at the α1 subunit. Furthermore, genetic knockout of α2 subunits strongly reduced oscillations, whereas knockout of α1 subunits had no effect. Allosteric modulation of GABAergic receptors was strongly reduced in unitary connections between fast spiking interneurons and pyramidal neurons in CA3 of α2H101R mice, but not of α1H101R mice, suggesting that fast spiking interneuron to pyramidal neuron synapses in CA3 contain α2 subunits. These findings suggest that α2-containing GABAA receptors expressed in the CA3 region provide the inhibition that controls hippocampal rhythm during cholinergically induced oscillations. PMID:23109109

Conformational Control of the Binding of the Transactivation Domain of the MLL Protein and c-Myb to the KIX Domain of CREB

PubMed Central

Korkmaz, Elif Nihal; Nussinov, Ruth; Haliloğlu, Türkan

2012-01-01

The KIX domain of CBP is a transcriptional coactivator. Concomitant binding to the activation domain of proto-oncogene protein c-Myb and the transactivation domain of the trithorax group protein mixed lineage leukemia (MLL) transcription factor lead to the biologically active ternary MLL∶KIX∶c-Myb complex which plays a role in Pol II-mediated transcription. The binding of the activation domain of MLL to KIX enhances c-Myb binding. Here we carried out molecular dynamics (MD) simulations for the MLL∶KIX∶c-Myb ternary complex, its binary components and KIX with the goal of providing a mechanistic explanation for the experimental observations. The dynamic behavior revealed that the MLL binding site is allosterically coupled to the c-Myb binding site. MLL binding redistributes the conformational ensemble of KIX, leading to higher populations of states which favor c-Myb binding. The key element in the allosteric communication pathways is the KIX loop, which acts as a control mechanism to enhance subsequent binding events. We tested this conclusion by in silico mutations of loop residues in the KIX∶MLL complex and by comparing wild type and mutant dynamics through MD simulations. The loop assumed MLL binding conformation similar to that observed in the KIX∶c-Myb state which disfavors the allosteric network. The coupling with c-Myb binding site faded, abolishing the positive cooperativity observed in the presence of MLL. Our major conclusion is that by eliciting a loop-mediated allosteric switch between the different states following the binding events, transcriptional activation can be regulated. The KIX system presents an example how nature makes use of conformational control in higher level regulation of transcriptional activity and thus cellular events. PMID:22438798

Allosteric Modulators of the CB1 Cannabinoid Receptor: A Structural Update Review.

PubMed

Morales, Paula; Goya, Pilar; Jagerovic, Nadine; Hernandez-Folgado, Laura

2016-01-01

In 2005, the first evidence of an allosteric binding site at the CB 1 R was provided by the identification of three indoles of the company Organon that were allosteric enhancers of agonist binding affinity and, functionally, allosteric inhibitors of agonist activity. Since then, structure-activity relationships of indoles as CB 1 R modulators have been reported. Targeting the allosteric site on CB 1 R, new families structurally based on urea and on 3-phenyltropane analogs of cocaine have been discovered as CB 1 R-negative allosteric modulators (NAMs), respectively, by Prosidion and by the Research Triangle Park. Endogenous allosteric ligands of different nature have been identified more recently. Thus, the therapeutic neuroprotection application of lipoxin A4, an arachidonic acid derivative, as an allosteric enhancer of CB 1 R activity has been confirmed in vivo . It was also the case of the steroid hormone, pregnenolone, whose negative allosteric effects on Δ 9 -tetrahydrocannabinol ( Δ 9 -THC) were reproduced in vivo in a behavioral tetrad model and in food intake and memory impairment assays. Curiously, the peroxisome proliferator-activated receptor-γ agonist fenofibrate or polypeptides such as pepcan-12 have been shown to act on the endocannabinoid system through CB 1 R allosteric modulation. The mechanistic bases of the effects of the phytocannabinoid cannabidiol (CBD) are still not fully explained. However, there is evidence that CBD behaves as an NAM of Δ 9 -THC- and 2-AG. Allosteric modulation at CB 1 R offers new opportunities for therapeutic applications. Therefore, further understanding of the chemical features required for allosteric modulation as well as their orthosteric probe dependence may broaden novel approaches for fine-tuning the signaling pathways of the CB 1 R.

Allosteric Modulators of the CB1 Cannabinoid Receptor: A Structural Update Review

PubMed Central

Morales, Paula; Goya, Pilar; Jagerovic, Nadine; Hernandez-Folgado, Laura

2016-01-01

Abstract In 2005, the first evidence of an allosteric binding site at the CB1R was provided by the identification of three indoles of the company Organon that were allosteric enhancers of agonist binding affinity and, functionally, allosteric inhibitors of agonist activity. Since then, structure–activity relationships of indoles as CB1R modulators have been reported. Targeting the allosteric site on CB1R, new families structurally based on urea and on 3-phenyltropane analogs of cocaine have been discovered as CB1R-negative allosteric modulators (NAMs), respectively, by Prosidion and by the Research Triangle Park. Endogenous allosteric ligands of different nature have been identified more recently. Thus, the therapeutic neuroprotection application of lipoxin A4, an arachidonic acid derivative, as an allosteric enhancer of CB1R activity has been confirmed in vivo. It was also the case of the steroid hormone, pregnenolone, whose negative allosteric effects on Δ9-tetrahydrocannabinol (Δ9-THC) were reproduced in vivo in a behavioral tetrad model and in food intake and memory impairment assays. Curiously, the peroxisome proliferator-activated receptor-γ agonist fenofibrate or polypeptides such as pepcan-12 have been shown to act on the endocannabinoid system through CB1R allosteric modulation. The mechanistic bases of the effects of the phytocannabinoid cannabidiol (CBD) are still not fully explained. However, there is evidence that CBD behaves as an NAM of Δ9-THC- and 2-AG. Allosteric modulation at CB1R offers new opportunities for therapeutic applications. Therefore, further understanding of the chemical features required for allosteric modulation as well as their orthosteric probe dependence may broaden novel approaches for fine-tuning the signaling pathways of the CB1R. PMID:28861476

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

PubMed

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

2011-07-01

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

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

PubMed Central

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

2011-01-01

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

Engineering of a membrane-triggered activity switch in coagulation factor VIIa

PubMed Central

Nielsen, Anders L.; Sorensen, Anders B.; Holmberg, Heidi L.; Gandhi, Prafull S.; Karlsson, Johan; Buchardt, Jens; Lamberth, Kasper; Kjelgaard-Hansen, Mads; Ley, Carsten Dan; Sørensen, Brit B.; Ruf, Wolfram; Olsen, Ole H.; Østergaard, Henrik

2017-01-01

Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa. PMID:29109275

The Structural Basis of ATP as an Allosteric Modulator

PubMed Central

Wang, Qi; Shen, Qiancheng; Li, Shuai; Nussinov, Ruth; Zhang, Jian

2014-01-01

Adenosine-5’-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems. PMID:25211773

Identification of regions of rabbit muscle pyruvate kinase important for allosteric regulation by phenylalanine, detected by H/D exchange mass spectrometry†

PubMed Central

Prasannan, Charulata B.; Villar, Maria T.; Artigues, Antonio; Fenton, Aron W.

2013-01-01

Mass spectrometry has been used to determine the number of exchangeable backbone amide protons and the associated rate constants that are altered when rabbit muscle pyruvate kinase (rM1-PYK) binds either the allosteric inhibitor (phenylalanine) or a non-allosteric analogue of the inhibitor. Alanine is used as the non-allosteric analogue since it binds competitively with phenylalanine, but elicits a negligible allosteric inhibition, i.e. a negligible reduction of the affinity of rM1-PYK for the substrate, phosphoenolpyruvate (PEP). This experimental design is expected to distinguish changes in the protein caused by effector binding (i.e. those changes common upon the addition of alanine vs. phenylalanine) from changes associated with allosteric regulation (i.e. those elicited by the addition of phenylalanine binding, but not alanine binding). High quality peptic fragments covering 98% of the protein were identified. Changes in both the number of exchangeable protons per peptide and in the rate constant associated with exchange highlight regions of the protein with allosteric roles. The set of allosterically relevant peptides identified by this technique include residues previously identified by mutagenesis to have roles in the allosteric regulation by phenylalanine. PMID:23418858

AlloPred: prediction of allosteric pockets on proteins using normal mode perturbation analysis.

PubMed

Greener, Joe G; Sternberg, Michael J E

2015-10-23

Despite being hugely important in biological processes, allostery is poorly understood and no universal mechanism has been discovered. Allosteric drugs are a largely unexplored prospect with many potential advantages over orthosteric drugs. Computational methods to predict allosteric sites on proteins are needed to aid the discovery of allosteric drugs, as well as to advance our fundamental understanding of allostery. AlloPred, a novel method to predict allosteric pockets on proteins, was developed. AlloPred uses perturbation of normal modes alongside pocket descriptors in a machine learning approach that ranks the pockets on a protein. AlloPred ranked an allosteric pocket top for 23 out of 40 known allosteric proteins, showing comparable and complementary performance to two existing methods. In 28 of 40 cases an allosteric pocket was ranked first or second. The AlloPred web server, freely available at http://www.sbg.bio.ic.ac.uk/allopred/home, allows visualisation and analysis of predictions. The source code and dataset information are also available from this site. Perturbation of normal modes can enhance our ability to predict allosteric sites on proteins. Computational methods such as AlloPred assist drug discovery efforts by suggesting sites on proteins for further experimental study.

Probing Molecular Mechanisms of the Hsp90 Chaperone: Biophysical Modeling Identifies Key Regulators of Functional Dynamics

PubMed Central

Dixit, Anshuman; Verkhivker, Gennady M.

2012-01-01

Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based “conformational selection” of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected residue clusters may be a rather general functional requirement encoded across molecular chaperones. The obtained insights may be useful in guiding discovery of allosteric Hsp90 inhibitors targeting protein interfaces with co-chaperones and protein binding clients. PMID:22624053

An engineered allosteric switch in leucine-zipper oligomerization.

PubMed

Gonzalez, L; Plecs, J J; Alber, T

1996-06-01

Controversy remains about the role of core side-chain packing in specifying protein structure. To investigate the influence of core packing on the oligomeric structure of a coiled coil, we engineered a GCN4 leucine zipper mutant that switches from two to three strands upon binding the hydrophobic ligands cyclohexane and benzene. In solution these ligands increased the apparent thermal stability and the oligomerization order of the mutant leucine zipper. The crystal structure of the peptide-benzene complex shows a single benzene molecule bound at the engineered site in the core of the trimer. These results indicate that coiled coils are well-suited to function as molecular switches and emphasize that core packing is an important determinant of oligomerization specificity.

Allosteric Communication Disrupted by a Small Molecule Binding to the Imidazole Glycerol Phosphate Synthase Protein-Protein Interface.

PubMed

Rivalta, Ivan; Lisi, George P; Snoeberger, Ning-Shiuan; Manley, Gregory; Loria, J Patrick; Batista, Victor S

2016-11-29

Allosteric enzymes regulate a wide range of catalytic transformations, including biosynthetic mechanisms of important human pathogens, upon binding of substrate molecules to an orthosteric (or active) site and effector ligands at distant (allosteric) sites. We find that enzymatic activity can be impaired by small molecules that bind along the allosteric pathway connecting the orthosteric and allosteric sites, without competing with endogenous ligands. Noncompetitive allosteric inhibitors disrupted allostery in the imidazole glycerol phosphate synthase (IGPS) enzyme from Thermotoga maritima as evidenced by nuclear magnetic resonance, microsecond time-scale molecular dynamics simulations, isothermal titration calorimetry, and kinetic assays. The findings are particularly relevant for the development of allosteric antibiotics, herbicides, and antifungal compounds because IGPS is absent in mammals but provides an entry point to fundamental biosynthetic pathways in plants, fungi, and bacteria.

Inducible CRISPR genome-editing tool: classifications and future trends.

PubMed

Dai, Xiaofeng; Chen, Xiao; Fang, Qiuwu; Li, Jia; Bai, Zhonghu

2018-06-01

The discovery of CRISPR-Cas9/dCas9 system has reinforced our ability and revolutionized our history in genome engineering. While Cas9 and dCas9 are programed to modulate gene expression by introducing DNA breaks, blocking transcription factor recruitment or dragging functional groups towards the targeted sites, sgRNAs determine the genomic loci where the modulation occurs. The off-target problem, due to limited sgRNA specificity and genome complexity of many species, has posed concerns for the wide application of this revolutionary technique. To solve this problem and, more importantly, gain power over gene functionality and cell fate control, inducible strategies have been continuously evolved to offer tailored solutions to address specific biological questions. By reviewing recent advances in inducible CRISPR system design and critical elements potentially adding values to such systems, we classify current approaches in this domain into four mechanically distinct categories, namely, "split system", "allosteric system", "combinatorial system", and "transient delivery system", discuss the pros and cons of each system, and point out the under-explored areas and future directions, with the aim of enriching our toolbox of delicate life engineering.

Are there physicochemical differences between allosteric and competitive ligands?

PubMed Central

Lu, Jing; Carlson, Heather A.

2017-01-01

Previous studies have compared the physicochemical properties of allosteric compounds to non-allosteric compounds. Those studies have found that allosteric compounds tend to be smaller, more rigid, more hydrophobic, and more drug-like than non-allosteric compounds. However, previous studies have not properly corrected for the fact that some protein targets have much more data than other systems. This generates concern regarding the possible skew that can be introduced by the inherent bias in the available data. Hence, this study aims to determine how robust the previous findings are to the addition of newer data. This study utilizes the Allosteric Database (ASD v3.0) and ChEMBL v20 to systematically obtain large datasets of both allosteric and competitive ligands. This dataset contains 70,219 and 9,511 unique ligands for the allosteric and competitive sets, respectively. Physically relevant compound descriptors were computed to examine the differences in their chemical properties. Particular attention was given to removing redundancy in the data and normalizing across ligand diversity and varied protein targets. The resulting distributions only show that allosteric ligands tend to be more aromatic and rigid and do not confirm the increase in hydrophobicity or difference in drug-likeness. These results are robust across different normalization schemes. PMID:29125840

Evidence for the Location of the Allosteric Activation Switch in the Multisubunit Phosphorylase Kinase Complex from Mass Spectrometric Identification of Chemically Crosslinked Peptides*

PubMed Central

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

2007-01-01

Phosphorylase kinase (PhK), an (αβγδ)4 complex, regulates glycogenolysis. Its activity, catalyzed by the γ subunit, is tightly controlled by phosphorylation and activators acting through allosteric sites on its regulatory α, β and δ subunits. Activation of the catalytic γ subunit in the PhK complex by phosphorylation is known to be predominantly mediated by the regulatory β subunit, which undergoes a conformational change that is structurally linked with the γ subunit and that is characterized by the ability to form β-β dimers using a short chemical crosslinker. To determine potential regions of interaction of the β and γ subunits, we have used chemical crosslinking and 2-hybrid screening. The β and γ subunits were chemically crosslinked to each other in phosphorylated PhK, and crosslinked peptides were identified in digests of the kinase by Fourier transform mass spectrometry in combination with a search engine developed ‘in house’ that generates a hypothetical list of crosslinked peptides. Such a conjugate between β and γ was identified, verified by MS/MS and shown to correspond to crosslinking between K303 in the C-terminal regulatory domain of γ (γCRD) and R18 in the N-terminal regulatory region of β (β1-31), which contains the phosphorylatable serines 11 and 26. A synthetic peptide corresponding to residues 1-22 of β inhibited the crosslinking between β and γ in the complex, and was itself crosslinked to K303 of γ. Through the use of 2-hybrid screening, the β1-31 region was also shown to control β subunit self-interactions, which 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 β and γ subunits. The sum of our results considered together with previous findings implicates the γCRD as being an allosteric activation switch in PhK that interacts with all three of the enzyme’s regulatory subunits and is proximal to the active site cleft. PMID:17123541

Mechanistic insights into the allosteric regulation of bacterial ADP-glucose pyrophosphorylases

PubMed Central

Comino, Natalia; Cifuente, Javier O.; Marina, Alberto; Orrantia, Ane; Eguskiza, Ander; Guerin, Marcelo E.

2017-01-01

ADP-glucose pyrophosphorylase (AGPase) controls bacterial glycogen and plant starch biosynthetic pathways, the most common carbon storage polysaccharides in nature. AGPase activity is allosterically regulated by a series of metabolites in the energetic flux within the cell. Very recently, we reported the first crystal structures of the paradigmatic AGPase from Escherichia coli (EcAGPase) in complex with its preferred physiological negative and positive allosteric regulators, adenosine 5′-monophosphate (AMP) and fructose 1,6-bisphosphate (FBP), respectively. However, understanding the molecular mechanism by which AMP and FBP allosterically modulates EcAGPase enzymatic activity still remains enigmatic. Here we found that single point mutations of key residues in the AMP-binding site decrease its inhibitory effect but also clearly abolish the overall AMP-mediated stabilization effect in wild-type EcAGPase. Single point mutations of key residues for FBP binding did not revert the AMP-mediated stabilization. Strikingly, an EcAGPase-R130A mutant displayed a dramatic increase in activity when compared with wild-type EcAGPase, and this increase correlated with a significant increment of glycogen content in vivo. The crystal structure of EcAGPase-R130A revealed unprecedented conformational changes in structural elements involved in the allosteric signal transmission. Altogether, we propose a model in which the positive and negative energy reporters regulate AGPase catalytic activity via intra- and interprotomer cross-talk, with a “sensory motif” and two loops, RL1 and RL2, flanking the ATP-binding site playing a significant role. The information reported herein provides exciting possibilities for industrial/biotechnological applications. PMID:28223362

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

The allosteric site regulates the voltage sensitivity of muscarinic receptors.

PubMed

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

2018-01-01

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

Structure and mechanisms of Escherichia coli aspartate transcarbamoylase.

PubMed

Lipscomb, William N; Kantrowitz, Evan R

2012-03-20

Enzymes catalyze a particular reaction in cells, but only a few control the rate of this reaction and the metabolic pathway that follows. One specific mechanism for such enzymatic control of a metabolic pathway involves molecular feedback, whereby a metabolite further down the pathway acts at a unique site on the control enzyme to alter its activity allosterically. This regulation may be positive or negative (or both), depending upon the particular system. Another method of enzymatic control involves the cooperative binding of the substrate, which allows a large change in enzyme activity to emanate from only a small change in substrate concentration. Allosteric regulation and homotropic cooperativity are often known to involve significant conformational changes in the structure of the protein. Escherichia coli aspartate transcarbamoylase (ATCase) is the textbook example of an enzyme that regulates a metabolic pathway, namely, pyrimidine nucleotide biosynthesis, by feedback control and by the cooperative binding of the substrate, L-aspartate. The catalytic and regulatory mechanisms of this enzyme have been extensively studied. A series of X-ray crystal structures of the enzyme in the presence and absence of substrates, products, and analogues have provided details, at the molecular level, of the conformational changes that the enzyme undergoes as it shifts between its low-activity, low-affinity form (T state) to its high-activity, high-affinity form (R state). These structural data provide insights into not only how this enzyme catalyzes the reaction between l-aspartate and carbamoyl phosphate to form N-carbamoyl-L-aspartate and inorganic phosphate, but also how the allosteric effectors modulate this activity. In this Account, we summarize studies on the structure of the enzyme and describe how these structural data provide insights into the catalytic and regulatory mechanisms of the enzyme. The ATCase-catalyzed reaction is regulated by nucleotide binding some 60 Å from the active site, inducing structural alterations that modulate catalytic activity. The delineation of the structure and function in this particular model system will help in understanding the molecular basis of cooperativity and allosteric regulation in other systems as well.

Metal site occupancy and allosteric switching in bacterial metal sensor proteins.

PubMed

Guerra, Alfredo J; Giedroc, David P

2012-03-15

All prokaryotes encode a panel of metal sensor or metalloregulatory proteins that govern the expression of genes that allows an organism to quickly adapt to toxicity or deprivation of both biologically essential transition metal ions, e.g., Zn, Cu, Fe, and heavy metal pollutants. As such, metal sensor proteins can be considered arbiters of intracellular transition metal bioavailability and thus potentially control the metallation state of the metalloproteins in the cell. Metal sensor proteins are specialized allosteric proteins that regulate transcription as a result direct binding of one or two cognate metal ions, to the exclusion of all others. In most cases, the binding of the cognate metal ion induces a structural change in a protein oligomer that either activates or inhibits operator DNA binding. A quantitative measure of the degree to which a particular metal drives metalloregulation of operator DNA-binding is the allosteric coupling free energy, ΔGc. In this review, we summarize recent work directed toward understanding metal occupancy and metal selectivity of these allosteric switches in selected families of metal sensor proteins and examine the structural origins of ΔGc in the functional context a thermodynamic "set-point" model of intracellular metal homeostasis. Copyright © 2011 Elsevier Inc. All rights reserved.

The impact of ions on allosteric functions in human liver pyruvate kinase

PubMed Central

Alontaga, Aileen Y.

2010-01-01

Experimental designs used to monitor the magnitude of an allosteric response can greatly influence observed values. We report here the impact of buffer, monovalent cation, divalent cation and anion on the magnitude of the allosteric regulation of the affinity of human liver pyruvate kinase (hL-PYK) for substrate, phosphoenolpyruvate (PEP). The magnitudes of the allosteric activation by fructose-1,6-bisphosphate (Fru-1,6-BP) and the allosteric inhibition by alanine are independent of most, but not all buffers tested. However, these magnitudes are dependent on whether Mg2+ or Mn2+ is included as the divalent cation. In the presence of Mn2+, any change in Kapp-PEP caused by Fru-1,6-BP is minimal. hL-PYK activity does not appear to require monovalent cation. Monovalent cation binding in the active site impacts PEP affinity with minimum influence on the magnitude of allosteric coupling. However, Na+ and Li+ reduce the magnitude of the allosteric response to Fru-1,6-BP, likely due to mechanisms outside of the active site. Which anion is used to maintain a constant monovalent cation concentration also influences the magnitude of the allosteric response. The value of determining the impact of ions on allosteric function can be appreciated by considering that representative structures used in comparative studies have often been determined using protein crystals grown in diverse buffer and salt conditions. PMID:21609859

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

Emerging Computational Methods for the Rational Discovery of Allosteric Drugs.

PubMed

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

2016-06-08

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

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

PubMed

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

2014-10-01

Allosteric interactions play vital roles in metabolic processes and signal transduction and, more recently, have become the focus of numerous pharmacological studies because of the potential for discovering more target-selective chemical probes and therapeutic agents. In addition to classic early studies on enzymes, there are now examples of small molecule allosteric modulators for all superfamilies of receptors encoded by the genome, including ligand- and voltage-gated ion channels, G protein-coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As a consequence, a vast array of pharmacologic behaviors has been ascribed to allosteric ligands that can vary in a target-, ligand-, and cell-/tissue-dependent manner. The current article presents an overview of allostery as applied to receptor families and approaches for detecting and validating allosteric interactions and gives recommendations for the nomenclature of allosteric ligands and their properties. U.S. Government work not protected by U.S. copyright.

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.

[Compounds modulating parathyroid hormone (PTH) secretion].

PubMed

Nagano, N; Iijima, H

2001-08-01

The control of parathyroid hormone (PTH) secretion is strictly regulated by the parathyroid Ca receptor (CaR). Calcimimetics and calcilytics selectively act on the parathyroid CaR to inhibit and enhance PTH secretion, respectively. According to the recent pharmacological two-state model, calcimimetics act on the CaR as allosteric agonists to stabilize an active conformation of CaR. Conversely, calcilytics act on the CaR as allosteric inverse agonists to stabilize an inactive conformation of CaR. These compounds that can alter circulating levels of PTH and bone turnover might provide novel treatments for adynamic bone disease in patients with chronic renal failure.

Glycogen and its metabolism: some new developments and old themes

PubMed Central

Roach, Peter J.; Depaoli-Roach, Anna A.; Hurley, Thomas D.; Tagliabracci, Vincent S.

2016-01-01

Glycogen is a branched polymer of glucose that acts as a store of energy in times of nutritional sufficiency for utilization in times of need. Its metabolism has been the subject of extensive investigation and much is known about its regulation by hormones such as insulin, glucagon and adrenaline (epinephrine). There has been debate over the relative importance of allosteric compared with covalent control of the key biosynthetic enzyme, glycogen synthase, as well as the relative importance of glucose entry into cells compared with glycogen synthase regulation in determining glycogen accumulation. Significant new developments in eukaryotic glycogen metabolism over the last decade or so include: (i) three-dimensional structures of the biosynthetic enzymes glycogenin and glycogen synthase, with associated implications for mechanism and control; (ii) analyses of several genetically engineered mice with altered glycogen metabolism that shed light on the mechanism of control; (iii) greater appreciation of the spatial aspects of glycogen metabolism, including more focus on the lysosomal degradation of glycogen; and (iv) glycogen phosphorylation and advances in the study of Lafora disease, which is emerging as a glycogen storage disease. PMID:22248338

Metabolic Control of Vesicular Glutamate Transport and Release

PubMed Central

Juge, Narinobu; Gray, John A.; Omote, Hiroshi; Miyaji, Takaaki; Inoue, Tsuyoshi; Hara, Chiaki; Uneyama, Hisayuki; Edwards, Robert H.; Nicoll, Roger A.; Moriyama, Yoshinori

2010-01-01

Fasting has been used to control epilepsy since antiquity, but the mechanism of coupling between metabolic state and excitatory neurotransmission remains unknown. Previous work has shown that the vesicular glutamate transporters (VGLUTs) required for exocytotic release of glutamate undergo an unusual form of regulation by Cl−. Using functional reconstitution of the purified VGLUTs into proteoliposomes, we now show that Cl− acts as an allosteric activator, and the ketone bodies that increase with fasting inhibit glutamate release by competing with Cl− at the site of allosteric regulation. Consistent with these observations, acetoacetate reduced quantal size at hippocampal synapses, and suppresses glutamate release and seizures evoked with 4-aminopyridine in the brain. The results indicate an unsuspected link between metabolic state and excitatory neurotransmission through anion-dependent regulation of VGLUT activity. PMID:20920794

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

PubMed Central

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

2014-01-01

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

Structural and Dynamic Insights into the Mechanism of Allosteric Signal Transmission in ERK2-Mediated MKP3 Activation.

PubMed

Lu, Chang; Liu, Xin; Zhang, Chen-Song; Gong, Haipeng; Wu, Jia-Wei; Wang, Zhi-Xin

2017-11-21

The mitogen-activated protein kinases (MAPKs) are key components of cellular signal transduction pathways, which are down-regulated by the MAPK phosphatases (MKPs). Catalytic activity of the MKPs is controlled both by their ability to recognize selective MAPKs and by allosteric activation upon binding to MAPK substrates. Here, we use a combination of experimental and computational techniques to elucidate the molecular mechanism for the ERK2-induced MKP3 activation. Mutational and kinetic study shows that the 334 FNFM 337 motif in the MKP3 catalytic domain is essential for MKP3-mediated ERK2 inactivation and is responsible for ERK2-mediated MKP3 activation. The long-term molecular dynamics (MD) simulations further reveal a complete dynamic process in which the catalytic domain of MKP3 gradually changes to a conformation that resembles an active MKP catalytic domain over the time scale of the simulation, providing a direct time-dependent observation of allosteric signal transmission in ERK2-induced MKP3 activation.

What Mutagenesis Can and Cannot Reveal About Allostery.

PubMed

Carlson, Gerald M; Fenton, Aron W

2016-05-10

Allosteric regulation of protein function is recognized to be widespread throughout biology; however, knowledge of allosteric mechanisms, the molecular changes within a protein that couple one binding site to another, is limited. Although mutagenesis is often used to probe allosteric mechanisms, we consider herein what the outcome of a mutagenesis study truly reveals about an allosteric mechanism. Arguably, the best way to evaluate the effects of a mutation on allostery is to monitor the allosteric coupling constant (Qax), a ratio of the substrate binding constants in the absence versus presence of an allosteric effector. A range of substitutions at a given residue position in a protein can reveal when a particular substitution causes gain-of-function, which addresses a key challenge in interpreting mutation-dependent changes in the magnitude of Qax. Thus, whole-protein mutagenesis studies offer an acceptable means of identifying residues that contribute to an allosteric mechanism. With this focus on monitoring Qax, and keeping in mind the equilibrium nature of allostery, we consider alternative possibilities for what an allosteric mechanism might be. We conclude that different possible mechanisms (rotation-of-solid-domains, movement of secondary structure, side-chain repacking, changes in dynamics, etc.) will result in different findings in whole-protein mutagenesis studies. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2017-01-01

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

From allosteric drugs to allo-network drugs: state of the art and trends of design, synthesis and computational methods.

PubMed

Csermely, Peter; Nussinov, Ruth; Szilágyi, András

2013-01-01

Allosteric drugs bind to sites which are usually less conserved evolutionarily as compared to orthosteric sites. As such, they can discriminate between closely related proteins, have fewer side effects, and a consequent lower concentration can convey a lesser likelihood of receptor desensitization. However, an allosteric mode of action may also make the results of preclinical and animal experiments less predictive. The sensitivity of the allosteric consequences to the environment further increases the importance of accounting for patient population diversity. Even subtle differences in protein sequence, in cellular metabolic states or in target tissues, can result in different outcomes. This mini-hot-topic issue of CTMC showcases some successes and challenges of allosteric drug development through the examples of seventransmembrane (GPCR), AMPA, NMDA and metabotropic glutamate receptors, as well as the morpheein model of allosterism involved in inherent metabolic errors. Finally, the development of allo-network drugs, which are allosteric drugs acting indirectly on the neighborhood of the pharmacological target in protein-protein interaction or signaling networks, is described.

A Single Amino Acid Residue at Transmembrane Domain 4 of the α Subunit Influences Carisoprodol Direct Gating Efficacy at GABAA Receptors.

PubMed

Kumar, Manoj; Kumar, Manish; Freund, John M; Dillon, Glenn H

2017-09-01

The muscle relaxant carisoprodol has recently been controlled at the federal level as a Schedule IV drug due to its high abuse potential and consequences of misuse, such as withdrawal syndrome, delusions, seizures, and even death. Recent work has shown that carisoprodol can directly gate and allosterically modulate the type A GABA (GABA A ) receptor. These actions are subunit-dependent; compared with other GABA A receptors, carisoprodol has nominal direct gating effects in α 3 β 2 γ 2 receptors. Here, using site-directed mutagenesis and whole-cell patch-clamp electrophysiology in transiently transfected human embryonic kidney 293 cells, we examined the role of GABA A receptor α subunit transmembrane domain 4 (TM4) amino acids in direct gating and allosteric modulatory actions of carisoprodol. Mutation of α 3 valine at position 440 to leucine (present in the equivalent position in the α 1 subunit) significantly increased the direct gating effects of carisoprodol without affecting its allosteric modulatory effects. The corresponding reverse mutation, α 1(L415V), decreased carisoprodol direct gating potency and efficacy. Analysis of a series of amino acid mutations at the 415 position demonstrated that amino acid volume correlated positively with carisoprodol efficacy, whereas polarity inversely correlated with carisoprodol efficacy. We conclude that α 1(415) of TM4 is involved in the direct gating, but not allosteric modulatory, actions of carisoprodol. In addition, the orientation of alkyl or hydroxyl groups at this position influences direct gating effects. These findings support the likelihood that the direct gating and allosteric modulatory effects of carisoprodol are mediated via distinct binding sites. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

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

Molecular Insights into Metabotropic Glutamate Receptor Allosteric Modulation

PubMed Central

Gregory, Karen J.

2015-01-01

The metabotropic glutamate (mGlu) receptors are a group of eight family C G protein–coupled receptors that are expressed throughout the central nervous system (CNS) and periphery. Within the CNS the different subtypes are found in neurons, both pre- and/or postsynaptically, where they mediate modulatory roles and in glial cells. The mGlu receptor family provides attractive targets for numerous psychiatric and neurologic disorders, with the majority of discovery programs focused on targeting allosteric sites, with allosteric ligands now available for all mGlu receptor subtypes. However, the development of allosteric ligands remains challenging. Biased modulation, probe dependence, and molecular switches all contribute to the complex molecular pharmacology exhibited by mGlu receptor allosteric ligands. In recent years we have made significant progress in our understanding of this molecular complexity coupled with an increased understanding of the structural basis of mGlu allosteric modulation. PMID:25808929

Identification of new allosteric sites and modulators of AChE through computational and experimental tools.

PubMed

Roca, Carlos; Requena, Carlos; Sebastián-Pérez, Víctor; Malhotra, Sony; Radoux, Chris; Pérez, Concepción; Martinez, Ana; Antonio Páez, Juan; Blundell, Tom L; Campillo, Nuria E

2018-12-01

Allosteric sites on proteins are targeted for designing more selective inhibitors of enzyme activity and to discover new functions. Acetylcholinesterase (AChE), which is most widely known for the hydrolysis of the neurotransmitter acetylcholine, has a peripheral allosteric subsite responsible for amyloidosis in Alzheimer's disease through interaction with amyloid β-peptide. However, AChE plays other non-hydrolytic functions. Here, we identify and characterise using computational tools two new allosteric sites in AChE, which have allowed us to identify allosteric inhibitors by virtual screening guided by structure-based and fragment hotspot strategies. The identified compounds were also screened for in vitro inhibition of AChE and three were observed to be active. Further experimental (kinetic) and computational (molecular dynamics) studies have been performed to verify the allosteric activity. These new compounds may be valuable pharmacological tools in the study of non-cholinergic functions of AChE.

Understanding cAMP-dependent allostery by NMR spectroscopy: comparative analysis of the EPAC1 cAMP-binding domain in its apo and cAMP-bound states.

PubMed

Mazhab-Jafari, Mohammad T; Das, Rahul; Fotheringham, Steven A; SilDas, Soumita; Chowdhury, Somenath; Melacini, Giuseppe

2007-11-21

cAMP (adenosine 3',5'-cyclic monophosphate) is a ubiquitous second messenger that activates a multitude of essential cellular responses. Two key receptors for cAMP in eukaryotes are protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC), which is a recently discovered guanine nucleotide exchange factor (GEF) for the small GTPases Rap1 and Rap2. Previous attempts to investigate the mechanism of allosteric activation of eukaryotic cAMP-binding domains (CBDs) at atomic or residue resolution have been hampered by the instability of the apo form, which requires the use of mixed apo/holo systems, that have provided only a partial picture of the CBD apo state and of the allosteric networks controlled by cAMP. Here, we show that, unlike other eukaryotic CBDs, both apo and cAMP-bound states of the EPAC1 CBD are stable under our experimental conditions, providing a unique opportunity to define at an unprecedented level of detail the allosteric interactions linking two critical functional sites of this CBD. These are the phosphate binding cassette (PBC), where cAMP binds, and the N-terminal helical bundle (NTHB), which is the site of the inhibitory interactions between the regulatory and catalytic regions of EPAC. Specifically, the combined analysis of the cAMP-dependent changes in chemical shifts, 2 degrees structure probabilities, hydrogen/hydrogen exchange (H/H) and hydrogen/deuterium exchange (H/D) protection factors reveals that the long-range communication between the PBC and the NTHB is implemented by two distinct intramolecular cAMP-signaling pathways, respectively, mediated by the beta2-beta3 loop and the alpha6 helix. Docking of cAMP into the PBC perturbs the NTHB inner core packing and the helical probabilities of selected NTHB residues. The proposed model is consistent with the allosteric role previously hypothesized for L273 and F300 based on site-directed mutagenesis; however, our data show that such a contact is part of a significantly more extended allosteric network that, unlike PKA, involves a tight coupling between the alpha- and beta-subdomains of the EPAC CBD. The proposed mechanism of allosteric activation will serve as a basis to understand agonism and antagonism in the EPAC system and provides also a general paradigm for how small ligands control protein-protein interfaces.

Allostery in disease and in drug discovery.

PubMed

Nussinov, Ruth; Tsai, Chung-Jung

2013-04-11

Allostery is largely associated with conformational and functional transitions in individual proteins. This concept can be extended to consider the impact of conformational perturbations on cellular function and disease states. Here, we clarify the concept of allostery and how it controls physiological activities. We focus on the challenging questions of how allostery can both cause disease and contribute to development of new therapeutics. We aim to increase the awareness of the linkage between disease symptoms on the cellular level and specific aberrant allosteric actions on the molecular level and to emphasize the potential of allosteric drugs in innovative therapies. Copyright © 2013 Elsevier Inc. All rights reserved.

Understanding allosteric interactions in G protein-coupled receptors using Supervised Molecular Dynamics: A prototype study analysing the human A3 adenosine receptor positive allosteric modulator LUF6000.

PubMed

Deganutti, Giuseppe; Cuzzolin, Alberto; Ciancetta, Antonella; Moro, Stefano

2015-07-15

The search for G protein-coupled receptors (GPCRs) allosteric modulators represents an active research field in medicinal chemistry. Allosteric modulators usually exert their activity only in the presence of the orthosteric ligand by binding to protein sites topographically different from the orthosteric cleft. They therefore offer potentially therapeutic advantages by selectively influencing tissue responses only when the endogenous agonist is present. The prediction of putative allosteric site location, however, is a challenging task. In facts, they are usually located in regions showing more structural variation among the family members. In the present work, we applied the recently developed Supervised Molecular Dynamics (SuMD) methodology to interpret at the molecular level the positive allosteric modulation mediated by LUF6000 toward the human adenosine A3 receptor (hA3 AR). Our data suggest at least two possible mechanisms to explain the experimental data available. This study represent, to the best of our knowledge, the first case reported of an allosteric recognition mechanism depicted by means of molecular dynamics simulations. Copyright © 2015 Elsevier Ltd. All rights reserved.

A unified view of "how allostery works".

PubMed

Tsai, Chung-Jung; Nussinov, Ruth

2014-02-01

The question of how allostery works was posed almost 50 years ago. Since then it has been the focus of much effort. This is for two reasons: first, the intellectual curiosity of basic science and the desire to understand fundamental phenomena, and second, its vast practical importance. Allostery is at play in all processes in the living cell, and increasingly in drug discovery. Many models have been successfully formulated, and are able to describe allostery even in the absence of a detailed structural mechanism. However, conceptual schemes designed to qualitatively explain allosteric mechanisms usually lack a quantitative mathematical model, and are unable to link its thermodynamic and structural foundations. This hampers insight into oncogenic mutations in cancer progression and biased agonists' actions. Here, we describe how allostery works from three different standpoints: thermodynamics, free energy landscape of population shift, and structure; all with exactly the same allosteric descriptors. This results in a unified view which not only clarifies the elusive allosteric mechanism but also provides structural grasp of agonist-mediated signaling pathways, and guides allosteric drug discovery. Of note, the unified view reasons that allosteric coupling (or communication) does not determine the allosteric efficacy; however, a communication channel is what makes potential binding sites allosteric.

Druggable orthosteric and allosteric hot spots to target protein-protein interactions.

PubMed

Ma, Buyong; Nussinov, Ruth

2014-01-01

Drug designing targeting protein-protein interactions is challenging. Because structural elucidation and computational analysis have revealed the importance of hot spot residues in stabilizing these interactions, there have been on-going efforts to develop drugs which bind the hot spots and out-compete the native protein partners. The question arises as to what are the key 'druggable' properties of hot spots in protein-protein interactions and whether these mimic the general hot spot definition. Identification of orthosteric (at the protein- protein interaction site) and allosteric (elsewhere) druggable hot spots is expected to help in discovering compounds that can more effectively modulate protein-protein interactions. For example, are there any other significant features beyond their location in pockets in the interface? The interactions of protein-protein hot spots are coupled with conformational dynamics of protein complexes. Currently increasing efforts focus on the allosteric drug discovery. Allosteric drugs bind away from the native binding site and can modulate the native interactions. We propose that identification of allosteric hot spots could similarly help in more effective allosteric drug discovery. While detection of allosteric hot spots is challenging, targeting drugs to these residues has the potential of greatly increasing the hot spot and protein druggability.

Hotspots for allosteric regulation on protein surfaces

PubMed Central

Reynolds, Kimberly A.; McLaughlin, Richard N.; Ranganathan, Rama

2012-01-01

Recent work indicates a general architecture for proteins in which sparse networks of physically contiguous and co-evolving amino acids underlie basic aspects of structure and function. These networks, termed sectors, are spatially organized such that active sites are linked to many surface sites distributed throughout the structure. Using the metabolic enzyme dihydrofolate reductase as a model system, we show that (1) the sector is strongly correlated to a network of residues undergoing millisecond conformational fluctuations associated with enzyme catalysis and (2) sector-connected surface sites are statistically preferred locations for the emergence of allosteric control in vivo. Thus, sectors represent an evolutionarily conserved “wiring” mechanism that can enable perturbations at specific surface positions to rapidly initiate conformational control over protein function. These findings suggest that sectors enable the evolution of intermolecular communication and regulation. PMID:22196731

Metabolic control of vesicular glutamate transport and release.

PubMed

Juge, Narinobu; Gray, John A; Omote, Hiroshi; Miyaji, Takaaki; Inoue, Tsuyoshi; Hara, Chiaki; Uneyama, Hisayuki; Edwards, Robert H; Nicoll, Roger A; Moriyama, Yoshinori

2010-10-06

Fasting has been used to control epilepsy since antiquity, but the mechanism of coupling between metabolic state and excitatory neurotransmission remains unknown. Previous work has shown that the vesicular glutamate transporters (VGLUTs) required for exocytotic release of glutamate undergo an unusual form of regulation by Cl(-). Using functional reconstitution of the purified VGLUTs into proteoliposomes, we now show that Cl(-) acts as an allosteric activator, and the ketone bodies that increase with fasting inhibit glutamate release by competing with Cl(-) at the site of allosteric regulation. Consistent with these observations, acetoacetate reduced quantal size at hippocampal synapses and suppresses glutamate release and seizures evoked with 4-aminopyridine in the brain. The results indicate an unsuspected link between metabolic state and excitatory neurotransmission through anion-dependent regulation of VGLUT activity. Copyright © 2010 Elsevier Inc. All rights reserved.

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.

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

PubMed Central

Takayanagi, Masayoshi; Kurisaki, Ikuo; Nagaoka, Masataka

2014-01-01

Protein allostery is essential for vital activities. Allosteric regulation of human hemoglobin (HbA) with two quaternary states T and R has been a paradigm of allosteric structural regulation of proteins. It is widely accepted that oxygen molecules (O2) act as a “site-specific” homotropic effector, or the successive O2 binding to the heme brings about the quaternary regulation. However, here we show that the site-specific allosteric effect is not necessarily only a unique mechanism of O2 allostery. Our simulation results revealed that the solution environment of high O2 partial pressure enhances the quaternary change from T to R without binding to the heme, suggesting an additional “non-site-specific” allosteric effect of O2. The latter effect should play a complementary role in the quaternary change by affecting the intersubunit contacts. This analysis must become a milestone in comprehensive understanding of the allosteric regulation of HbA from the molecular point of view. PMID:24710521

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.

Effector analogues detect varied allosteric roles for conserved protein-effector interactions in pyruvate kinase isozymes†

PubMed Central

Alontaga, Aileen Y.; Fenton, Aron W.

2011-01-01

The binding site for allosteric inhibitor (amino acid) is highly conserved between human liver pyruvate kinase (hL-PYK) and the rabbit muscle isozyme (rM1-PYK). To detail similarities/differences in the allosteric function of these two homologs, we quantified the binding of 45 amino acid analogues to hL-PYK and their allosteric impact on affinity for the substrate, phosphoenolpyruvate (PEP). This complements a similar study previously completed for rM1-PYK. In hL-PYK, the minimum chemical requirements for effector binding are the same as those identified for rM1-PYK (i.e. the L-2-aminopropanaldehyde substructure of the effector is primarily responsible for binding). However different regions of the effector determine the magnitude of the allosteric response in hL-PYK vs. rM1-PYK. This finding is inconsistent with the idea that allosteric pathways are conserved between homologs of a protein family. PMID:21261284

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

PubMed

Takayanagi, Masayoshi; Kurisaki, Ikuo; Nagaoka, Masataka

2014-04-08

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

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

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

PubMed Central

Mittelstädt, Gerd; Moggré, Gert‐Jan; Panjikar, Santosh; Nazmi, Ali Reza

2016-01-01

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcia Fortanet, Jorge; Chen, Christine Hiu-Tung; Chen, Ying-Nan P.

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

Direct photoaffinity labeling of an allosteric site on subunit protein M1 of mouse ribonucleotide reductase by dTTP.

PubMed Central

Eriksson, S; Caras, I W; Martin, D W

1982-01-01

The protein M1 subunit of ribonucleotide reductase contains at least two allosteric nucleotide binding sites that control the capacity of the enzyme to reduce ribonucleotides to the deoxyribonucleotides required for DNA synthesis. Direct photoaffinity labeling of partially purified protein M1 from mouse T-lymphoma (S49) cells was observed after UV irradiation in the presence of dTTP at 0 degrees C. The relative molar incorporation of nucleotide per subunit was 4-8%. Competition experiments showed that the dTTP was bound to an allosteric domain genetically and kinetically defined as the substrate specificity site of the enzyme. An altered protein M1 isolated from a thymidine-resistant mutant cell line showed significantly decreased photoincorporation of dTTP, consistent with the fact that its CDP reductase activity is resistant to feedback inhibition by dTTP. Specific photolabeling of several other proteins with pyrimidine and purine nucleotides was also found, indicating the general usefulness of direct photoaffinity labeling in the study of enzymes involved in nucleotide and nucleic acid metabolism. Images PMID:7033963

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

PubMed

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

2016-08-01

Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first committed step of the histidine biosynthesis in plants and microorganisms. Here, we present the functional and structural characterization of the ATP-PRT from the pathogenic ε-proteobacteria Campylobacter jejuni (CjeATP-PRT). This enzyme is a member of the long form (HisGL ) ATP-PRT and is allosterically inhibited by histidine, which binds to a remote regulatory domain, and competitively inhibited by AMP. In the crystalline form, CjeATP-PRT was found to adopt two distinctly different hexameric conformations, with an open homohexameric structure observed in the presence of substrate ATP, and a more compact closed form present when inhibitor histidine is bound. CjeATP-PRT was observed to adopt only a hexameric quaternary structure in solution, contradicting previous hypotheses favoring an allosteric mechanism driven by an oligomer equilibrium. Instead, this study supports the conclusion that the ATP-PRT long form hexamer is the active species; the tightening of this structure in response to remote histidine binding results in an inhibited enzyme. © 2016 The Protein Society.

Artificial light-regulation of an allosteric bi-enzyme complex by a photosensitive ligand.

PubMed

Kneuttinger, Andrea C; Winter, Martin; Simeth, Nadja A; Heyn, Kristina; Merkl, Rainer; König, Burkhard; Sterner, Reinhard

2018-05-29

The artificial regulation of proteins by light is an emerging sub-discipline of synthetic biology. Here, we used this concept in order to photo-control both catalysis and allostery within the heterodimeric enzyme complex imidazole glycerol phosphate synthase (ImGP-S). The ImGP-S consists of the cyclase subunit HisF and the glutaminase subunit HisH, which is allosterically stimulated by substrate binding to HisF. We show that a light-sensitive diarylethene (DTE)-based competitive inhibitor in its ring-open state binds with low micromolar affinity to the cyclase subunit and displaces its substrate from the active site. As a consequence, catalysis by HisF and allosteric stimulation of HisH are impaired. Following UV-light irradiation, the DTE-ligand adopts its ring-closed state and loses affinity for HisF, restoring activity and allostery. Our approach allows for the switching of ImGP-S activity and allostery during catalysis and appears to be generally applicable for the light-regulation of other multi-enzyme complexes. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Leakage and slow allostery limit performance of single drug-sensing aptazyme molecules based on the hammerhead ribozyme

PubMed Central

de Silva, Chamaree; Walter, Nils G.

2009-01-01

Engineered “aptazymes” fuse in vitro selected aptamers with ribozymes to create allosteric enzymes as biosensing components and artificial gene regulatory switches through ligand-induced conformational rearrangement and activation. By contrast, activating ligand is employed as an enzymatic cofactor in the only known natural aptazyme, the glmS ribozyme, which is devoid of any detectable conformational rearrangements. To better understand this difference in biosensing strategy, we monitored by single molecule fluorescence resonance energy transfer (FRET) and 2-aminopurine (AP) fluorescence the global conformational dynamics and local base (un)stacking, respectively, of a prototypical drug-sensing aptazyme, built from a theophylline aptamer and the hammerhead ribozyme. Single molecule FRET reveals that a catalytically active state with distal Stems I and III of the hammerhead ribozyme is accessed both in the theophylline-bound and, if less frequently, in the ligand-free state. The resultant residual activity (leakage) in the absence of theophylline contributes to a limited dynamic range of the aptazyme. In addition, site-specific AP labeling shows that rapid local theophylline binding to the aptamer domain leads to only slow allosteric signal transduction into the ribozyme core. Our findings allow us to rationalize the suboptimal biosensing performance of the engineered compared to the natural aptazyme and to suggest improvement strategies. Our single molecule FRET approach also monitors in real time the previously elusive equilibrium docking dynamics of the hammerhead ribozyme between several inactive conformations and the active, long-lived, Y-shaped conformer. PMID:19029309

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

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

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

PubMed

Stetz, Gabrielle; Verkhivker, Gennady M

2017-01-01

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

The selective positive allosteric M1 muscarinic receptor modulator PQCA attenuates learning and memory deficits in the Tg2576 Alzheimer's disease mouse model.

PubMed

Puri, Vanita; Wang, Xiaohai; Vardigan, Joshua D; Kuduk, Scott D; Uslaner, Jason M

2015-01-01

We have recently shown that the M1 muscarinic receptor positive allosteric modulator, PQCA, improves cognitive performance in rodents and non-human primates administered the muscarinic receptor antagonist scopolamine. The purpose of the present experiments was to characterize the effects of PQCA in a model more relevant to the disease pathology of Alzheimer's disease. Tg2576 transgenic mice that have elevated Aβ were tested in the novel object recognition task to characterize recognition memory as a function of age and treatment with the PQCA. The effects of PQCA were compared to the acetylcholinesterase inhibitor donepezil, the standard of care for Alzheimer's disease. In addition, the effect of co-administering PQCA and donepezil was evaluated. Aged Tg2576 mice demonstrated a deficit in recognition memory that was significantly attenuated by PQCA. The positive control donepezil also reversed the deficit. Furthermore, doses of PQCA and donepezil that were inactive on their own were found to improve recognition memory when given together. These studies suggest that M1 muscarinic receptor positive allosteric modulation can ameliorate memory deficits in disease relevant models of Alzheimer's disease. These data, combined with our previous findings demonstrating PQCA improves scopolamine-induced cognitive deficits in both rodents and non-human primates, suggest that M1 positive allosteric modulators have therapeutic potential for the treatment of Alzheimer's disease. Copyright © 2015 Elsevier B.V. All rights reserved.

Structural Evolution of Differential Amino Acid Effector Regulation in Plant Chorismate Mutases*

PubMed Central

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

2014-01-01

Chorismate mutase converts chorismate into prephenate for aromatic amino acid biosynthesis. To understand the molecular basis of allosteric regulation in the plant chorismate mutases, we analyzed the three Arabidopsis thaliana chorismate mutase isoforms (AtCM1–3) and determined the x-ray crystal structures of AtCM1 in complex with phenylalanine and tyrosine. Functional analyses show a wider range of effector control in the Arabidopsis chorismate mutases than previously reported. AtCM1 is activated by tryptophan with phenylalanine and tyrosine acting as negative effectors; however, tryptophan, cysteine, and histidine activate AtCM3. AtCM2 is a nonallosteric form. The crystal structure of AtCM1 in complex with tyrosine and phenylalanine identifies differences in the effector sites of the allosterically regulated yeast enzyme and the other two Arabidopsis isoforms. Site-directed mutagenesis of residues in the effector site reveals key features leading to differential effector regulation in these enzymes. In AtCM1, mutations of Gly-213 abolish allosteric regulation, as observed in AtCM2. A second effector site position, Gly-149 in AtCM1 and Asp-132 in AtCM3, controls amino acid effector specificity in AtCM1 and AtCM3. Comparisons of chorismate mutases from multiple plants suggest that subtle differences in the effector site are conserved in different lineages and may lead to specialized regulation of this branch point enzyme. PMID:25160622

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

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

NASA Astrophysics Data System (ADS)

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

2015-10-01

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

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

PubMed

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

2013-01-01

G protein coupled receptors (GPCRs) are the most historically successful therapeutic targets. Despite this success there are many important aspects of GPCR pharmacology and function that have yet to be exploited to their full therapeutic potential. One in particular that has been gaining attention in recent times is that of GPCR ligands that bind to allosteric sites on the receptor distinct from the orthosteric site of the endogenous ligand. As therapeutics, allosteric ligands possess many theoretical advantages over their orthosteric counterparts, including more complex modes of action, improved safety, more physiologically appropriate responses, better target selectivity, and reduced likelihood of desensitisation and tachyphylaxis. Despite these advantages, the development of allosteric ligands is often difficult from a medicinal chemistry standpoint due to the more complex challenge of identifying allosteric leads and their often flat or confusing SAR. The present review will consider the advantages and challenges associated with allosteric GPCR ligands, and examine how the particular properties of these ligands may be exploited to uncover the therapeutic potential for free fatty acid sensitive GPCRs.

Binding Leverage as a Molecular Basis for Allosteric Regulation

PubMed Central

Mitternacht, Simon; Berezovsky, Igor N.

2011-01-01

Allosteric regulation involves conformational transitions or fluctuations between a few closely related states, caused by the binding of effector molecules. We introduce a quantity called binding leverage that measures the ability of a binding site to couple to the intrinsic motions of a protein. We use Monte Carlo simulations to generate potential binding sites and either normal modes or pairs of crystal structures to describe relevant motions. We analyze single catalytic domains and multimeric allosteric enzymes with complex regulation. For the majority of the analyzed proteins, we find that both catalytic and allosteric sites have high binding leverage. Furthermore, our analysis of the catabolite activator protein, which is allosteric without conformational change, shows that its regulation involves other types of motion than those modulated at sites with high binding leverage. Our results point to the importance of incorporating dynamic information when predicting functional sites. Because it is possible to calculate binding leverage from a single crystal structure it can be used for characterizing proteins of unknown function and predicting latent allosteric sites in any protein, with implications for drug design. PMID:21935347

Hexamethonium-type allosteric modulators of the muscarinic receptors bearing lateral dibenzazepine moieties.

PubMed

Li, R; Tränkle, C; Mohr, K; Holzgrabe, U

2001-04-01

Alkane-bisammonium compounds carrying lateral phthalimido substituents are known to have a high affinity for the allosteric binding site of the acetylcholine M2 receptor. The purpose of this study was to replace the lateral phthalimido moieties with rigid tricyclic skeletons of a large volume in order to learn more about the function of the lateral heterocycles. In addition, methyl groups were introduced into the lateral connecting chains. Allosteric inhibition of the dissociation of [3H]N-methylscopolamine from the M2 receptors in porcine cardiac homogenates served to indicate binding of the test compounds to the allosteric site. The phthalimido groups could be replaced with dibenzazepine moieties without any loss in potency. Interestingly, the additional methyl group in the lateral spacer seems to have a significant influence on the allosteric behaviour.

Cooperative binding mitigates the high-dose hook effect.

PubMed

Roy, Ranjita Dutta; Rosenmund, Christian; Stefan, Melanie I

2017-08-14

The high-dose hook effect (also called prozone effect) refers to the observation that if a multivalent protein acts as a linker between two parts of a protein complex, then increasing the amount of linker protein in the mixture does not always increase the amount of fully formed complex. On the contrary, at a high enough concentration range the amount of fully formed complex actually decreases. It has been observed that allosterically regulated proteins seem less susceptible to this effect. The aim of this study was two-fold: First, to investigate the mathematical basis of how allostery mitigates the prozone effect. And second, to explore the consequences of allostery and the high-dose hook effect using the example of calmodulin, a calcium-sensing protein that regulates the switch between long-term potentiation and long-term depression in neurons. We use a combinatorial model of a "perfect linker protein" (with infinite binding affinity) to mathematically describe the hook effect and its behaviour under allosteric conditions. We show that allosteric regulation does indeed mitigate the high-dose hook effect. We then turn to calmodulin as a real-life example of an allosteric protein. Using kinetic simulations, we show that calmodulin is indeed subject to a hook effect. We also show that this effect is stronger in the presence of the allosteric activator Ca 2+ /calmodulin-dependent kinase II (CaMKII), because it reduces the overall cooperativity of the calcium-calmodulin system. It follows that, surprisingly, there are conditions where increased amounts of allosteric activator actually decrease the activity of a protein. We show that cooperative binding can indeed act as a protective mechanism against the hook effect. This will have implications in vivo where the extent of cooperativity of a protein can be modulated, for instance, by allosteric activators or inhibitors. This can result in counterintuitive effects of decreased activity with increased concentrations of both the allosteric protein itself and its allosteric activators.

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 (Kruse et al., 2013), a structure that provides crucial insights into the mechanisms of orthosteric activation and allosteric modulation of muscarinic receptors. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

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

Allosteric Inhibitors at the Heterodimer Interface of Imidazole Glycerol Phosphate Synthase

NASA Astrophysics Data System (ADS)

Snoeberger, Ning-Shiuan Nicole

Imidazole glycerol phosphate synthase (IGPS) from Thermotoga maritima is a heterodimeric enzyme composed of the HisH and HisF proteins. It is attractive as a pathological target since it is absent in mammals but found in plant and opportunistic human pathogens. IGPS was experimentally determined to be a V-type allosteric enzyme that is involved in an essential biosynthetic pathway of microorganisms. The enzyme catalyzes the hydrolysis of glutamine to form NH3 in the HisH protein, followed by cyclization of NH3 with N'-[(5'-phosphoribulosyl)imino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in the HisF subunit, forming imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) that enter the histidine and purine biosynthetic pathways. Allosteric motions induced upon the binding of the effector PRFAR to HisF propagate through the non-covalent HisH/HisF interface and synchronize catalytic activity at the two distant active sites. However, the nature of the allosteric pathway and the feasibility of manipulating signal transduction by using allosteric drug-like molecules remain to be established. Molecular docking studies of commercial drugs at the HisH/HisF interface were used to identify stable candidates with a potential allosteric effect on the reaction mechanism. Molecular dynamic simulations and calculations of NMR chemical shifts were combined to elucidate the allosteric pathway of IGPS.

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

PubMed

Farb, Joshua N; Morrical, Scott W

2009-01-16

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

The rice endosperm ADP-glucose pyrophosphorylase large subunit is essential for optimal catalysis and allosteric regulation of the heterotetrameric enzyme.

PubMed

Tuncel, Aytug; Kawaguchi, Joe; Ihara, Yasuharu; Matsusaka, Hiroaki; Nishi, Aiko; Nakamura, Tetsuhiro; Kuhara, Satoru; Hirakawa, Hideki; Nakamura, Yasunori; Cakir, Bilal; Nagamine, Ai; Okita, Thomas W; Hwang, Seon-Kap; Satoh, Hikaru

2014-06-01

Although an alternative pathway has been suggested, the prevailing view is that starch synthesis in cereal endosperm is controlled by the activity of the cytosolic isoform of ADPglucose pyrophosphorylase (AGPase). In rice, the cytosolic AGPase isoform is encoded by the OsAGPS2b and OsAGPL2 genes, which code for the small (S2b) and large (L2) subunits of the heterotetrameric enzyme, respectively. In this study, we isolated several allelic missense and nonsense OsAGPL2 mutants by N-methyl-N-nitrosourea (MNU) treatment of fertilized egg cells and by TILLING (Targeting Induced Local Lesions in Genomes). Interestingly, seeds from three of the missense mutants (two containing T139I and A171V) were severely shriveled and had seed weight and starch content comparable with the shriveled seeds from OsAGPL2 null mutants. Results from kinetic analysis of the purified recombinant enzymes revealed that the catalytic and allosteric regulatory properties of these mutant enzymes were significantly impaired. The missense heterotetramer enzymes and the S2b homotetramer had lower specific (catalytic) activities and affinities for the activator 3-phosphoglycerate (3-PGA). The missense heterotetramer enzymes showed more sensitivity to inhibition by the inhibitor inorganic phosphate (Pi) than the wild-type AGPase, while the S2b homotetramer was profoundly tolerant to Pi inhibition. Thus, our results provide definitive evidence that starch biosynthesis during rice endosperm development is controlled predominantly by the catalytic activity of the cytoplasmic AGPase and its allosteric regulation by the effectors. Moreover, our results show that the L2 subunit is essential for both catalysis and allosteric regulatory properties of the heterotetramer enzyme. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Orthosteric and allosteric potentiation of heteromeric neuronal nicotinic acetylcholine receptors.

PubMed

Wang, Jingyi; Lindstrom, Jon

2018-06-01

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

Synergistic Allosteric Mechanism of Fructose-1,6-bisphosphate and Serine for Pyruvate Kinase M2 via Dynamics Fluctuation Network Analysis.

PubMed

Yang, Jingxu; Liu, Hao; Liu, Xiaorui; Gu, Chengbo; Luo, Ray; Chen, Hai-Feng

2016-06-27

Pyruvate kinase M2 (PKM2) plays a key role in tumor metabolism and regulates the rate-limiting final step of glycolysis. In tumor cells, there are two allosteric effectors for PKM2: fructose-1,6-bisphosphate (FBP) and serine. However, the relationship between FBP and serine for allosteric regulation of PKM2 is unknown. Here we constructed residue/residue fluctuation correlation network based on all-atom molecular dynamics simulations to reveal the regulation mechanism. The results suggest that the correlation network in bound PKM2 is distinctly different from that in the free state, FBP/PKM2, or Ser/PKM2. The community network analysis indicates that the information can freely transfer from the allosteric sites of FBP and serine to the substrate site in bound PKM2, while there exists a bottleneck for information transfer in the network of the free state. Furthermore, the binding free energy between the substrate and PKM2 for bound PKM2 is significantly lower than either of FBP/PKM2 or Ser/PKM2. Thus, a hypothesis of "synergistic allosteric mechanism" is proposed for the allosteric regulation of FBP and serine. This hypothesis was further confirmed by the perturbational and mutational analyses of community networks and binding free energies. Finally, two possible synergistic allosteric pathways of FBP-K433-T459-R461-A109-V71-R73-MG2-OXL and Ser-I47-C49-R73-MG2-OXL were identified based on the shortest path algorithm and were confirmed by the network perturbation analysis. Interestingly, no similar pathways could be found in the free state. The process targeting on the allosteric pathways can better regulate the glycolysis of PKM2 and significantly inhibit the progression of tumor.

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

PubMed

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

2009-10-25

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

Implementation of the NMR CHEmical Shift Covariance Analysis (CHESCA): A Chemical Biologist's Approach to Allostery.

PubMed

Boulton, Stephen; Selvaratnam, Rajeevan; Ahmed, Rashik; Melacini, Giuseppe

2018-01-01

Mapping allosteric sites is emerging as one of the central challenges in physiology, pathology, and pharmacology. Nuclear Magnetic Resonance (NMR) spectroscopy is ideally suited to map allosteric sites, given its ability to sense at atomic resolution the dynamics underlying allostery. Here, we focus specifically on the NMR CHEmical Shift Covariance Analysis (CHESCA), in which allosteric systems are interrogated through a targeted library of perturbations (e.g., mutations and/or analogs of the allosteric effector ligand). The atomic resolution readout for the response to such perturbation library is provided by NMR chemical shifts. These are then subject to statistical correlation and covariance analyses resulting in clusters of allosterically coupled residues that exhibit concerted responses to the common set of perturbations. This chapter provides a description of how each step in the CHESCA is implemented, starting from the selection of the perturbation library and ending with an overview of different clustering options.

Scaffold-based novel SHP2 allosteric inhibitors design using Receptor-Ligand pharmacophore model, virtual screening and molecular dynamics.

PubMed

Jin, Wen-Yan; Ma, Ying; Li, Wei-Ya; Li, Hong-Lian; Wang, Run-Ling

2018-04-01

SHP2 is a potential target for the development of novel therapies for SHP2-dependent cancers. In our research, with the aid of the 'Receptor-Ligand Pharmacophore' technique, a 3D-QSAR method was carried out to explore structure activity relationship of SHP2 allosteric inhibitors. Structure-based drug design was employed to optimize SHP099, an efficacious, potent, and selective SHP2 allosteric inhibitor. A novel class of selective SHP2 allosteric inhibitors was discovered by using the powerful 'SBP', 'ADMET' and 'CDOCKER' techniques. By means of molecular dynamics simulations, it was observed that these novel inhibitors not only had the same function as SHP099 did in inhibiting SHP2, but also had more favorable conformation for binding to the receptor. Thus, this report may provide a new method in discovering novel and selective SHP2 allosteric inhibitors. Copyright © 2018 Elsevier Ltd. All rights reserved.

The pH dependence of the allosteric response of human liver pyruvate kinase to fructose-1,6-bisphosphate, ATP, and alanine

PubMed Central

Fenton, Aron W.; Hutchinson, Myra

2009-01-01

The allosteric regulation of human liver pyruvate kinase (hL-PYK) by fructose-1,6-bisphosphate (Fru-1,6-BP; activator), ATP (inhibitor) and alanine (Ala; inhibitor) was monitored over a pH range from 6.5 to 8.0 at 37°C. As a function of increasing pH, hL-PYK's affinity for the substrate phosphoenolpyruvate (PEP), and for Fru-1,6-BP decreases, while affinities for ATP and Ala slightly increases. At pH 6.5, Fru-1,6-BP and ATP elicit only small allosteric impacts on PEP affinity. As pH increases, Fru-1,6-BP and ATP elicit greater allosteric responses, but the response to Ala is relatively constant. Since the magnitudes of the allosteric coupling for ATP and for Ala inhibition are different and the pH dependences of these magnitudes are not similar, these inhibitors likely elicit their responses using different molecular mechanisms. In addition, our results fail to support a general correlation between pH dependent changes in effector affinity and pH dependent changes in the corresponding allosteric response. PMID:19467627

OptoGluNAM4.1, a Photoswitchable Allosteric Antagonist for Real-Time Control of mGlu4 Receptor Activity.

PubMed

Rovira, Xavier; Trapero, Ana; Pittolo, Silvia; Zussy, Charleine; Faucherre, Adèle; Jopling, Chris; Giraldo, Jesús; Pin, Jean-Philippe; Gorostiza, Pau; Goudet, Cyril; Llebaria, Amadeu

2016-08-18

OptoGluNAM4.1, a negative allosteric modulator (NAM) of metabotropic glutamate receptor 4 (mGlu4) contains a reactive group that covalently binds to the receptor and a blue-light-activated, fast-relaxing azobenzene group that allows reversible receptor activity photocontrol in vitro and in vivo. OptoGluNAM4.1 induces light-dependent behavior in zebrafish and reverses the activity of the mGlu4 agonist LSP4-2022 in a mice model of chronic pain, defining a photopharmacological tool to better elucidate the physiological roles of the mGlu4 receptor in the nervous system. Copyright © 2016 Elsevier Ltd. All rights reserved.

Insulin analogs for the treatment of diabetes mellitus: therapeutic applications of protein engineering.

PubMed

Berenson, Daniel F; Weiss, Allison R; Wan, Zhu-Li; Weiss, Michael A

2011-12-01

The engineering of insulin analogs represents a triumph of structure-based protein design. A framework has been provided by structures of insulin hexamers. Containing a zinc-coordinated trimer of dimers, such structures represent a storage form of the active insulin monomer. Initial studies focused on destabilization of subunit interfaces. Because disassembly facilitates capillary absorption, such targeted destabilization enabled development of rapid-acting insulin analogs. Converse efforts were undertaken to stabilize the insulin hexamer and promote higher-order self-assembly within the subcutaneous depot toward the goal of enhanced basal glycemic control with reduced risk of hypoglycemia. Current products either operate through isoelectric precipitation (insulin glargine, the active component of Lantus(®); Sanofi-Aventis) or employ an albumin-binding acyl tether (insulin detemir, the active component of Levemir(®); Novo-Nordisk). To further improve pharmacokinetic properties, modified approaches are presently under investigation. Novel strategies have recently been proposed based on subcutaneous supramolecular assembly coupled to (a) large-scale allosteric reorganization of the insulin hexamer (the TR transition), (b) pH-dependent binding of zinc ions to engineered His-X(3)-His sites at hexamer surfaces, or (c) the long-range vision of glucose-responsive polymers for regulated hormone release. Such designs share with wild-type insulin and current insulin products a susceptibility to degradation above room temperature, and so their delivery, storage, and use require the infrastructure of an affluent society. Given the global dimensions of the therapeutic supply chain, we envisage that concurrent engineering of ultra-stable protein analog formulations would benefit underprivileged patients in the developing world.

Often Ignored Facts about the Control of the 2-Oxoglutarate Dehydrogenase Complex

ERIC Educational Resources Information Center

Strumilo, Slawomir

2005-01-01

Information about the control of the activity of the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme in the citric acid cycle, is not well covered in the biochemical education literature, especially as it concerns the allosteric regulation of OGDHC by adenine nucleotide and ortophosphate. From experimental work published during the last…

Microsecond molecular dynamics simulations provide insight into the ATP-competitive inhibitor-induced allosteric protection of Akt kinase phosphorylation.

PubMed

Mou, Linkai; Cui, Tongwei; Liu, Weiguang; Zhang, Hong; Cai, Zhanxiu; Lu, Shaoyong; Gao, Guojun

2017-05-01

Akt is a serine/threonine protein kinase, a critical mediator of growth factor-induced survival in key cellular pathways. Allosteric signaling between protein intramolecular domains requires long-range communication mediated by hotspot residues, often triggered by ligand binding. Here, based on extensive 3 μs explicit solvent molecular dynamics (MD) simulations of Akt1 kinase domain in the unbound (apo) and ATP-competitive inhibitor, GDC-0068-bound states, we propose a molecular mechanism for allosteric regulation of Akt1 kinase phosphorylation by GDC-0068 binding to the ATP-binding site. MD simulations revealed that the apo Akt1 is flexible with two disengaged N- and C-lobes, equilibrated between the open and closed conformations. GDC-0068 occupancy of the ATP-binding site shifts the conformational equilibrium of Akt1 from the open conformation toward the closed conformation and stabilizes the closed state. This effect enables allosteric signal propagation from the GDC-0068 to the phosphorylated T308 (pT308) in the activation loop and restrains phosphatase access to pT308, thereby protecting the pT308 in the GDC-0068-bound Akt1. Importantly, functional hotspots involved in the allosteric communication from the GDC-0068 to the pT308 are identified. Our analysis of GDC-0068-induced allosteric protection of Akt kinase phosphorylation yields important new insights into the molecular mechanism of allosteric regulation of Akt kinase activity. © 2016 John Wiley & Sons A/S.

The Energy Landscape Analysis of Cancer Mutations in Protein Kinases

PubMed Central

Dixit, Anshuman; Verkhivker, Gennady M.

2011-01-01

The growing interest in quantifying the molecular basis of protein kinase activation and allosteric regulation by cancer mutations has fueled computational studies of allosteric signaling in protein kinases. In the present study, we combined computer simulations and the energy landscape analysis of protein kinases to characterize the interplay between oncogenic mutations and locally frustrated sites as important catalysts of allostetric kinase activation. While structurally rigid kinase core constitutes a minimally frustrated hub of the catalytic domain, locally frustrated residue clusters, whose interaction networks are not energetically optimized, are prone to dynamic modulation and could enable allosteric conformational transitions. The results of this study have shown that the energy landscape effect of oncogenic mutations may be allosteric eliciting global changes in the spatial distribution of highly frustrated residues. We have found that mutation-induced allosteric signaling may involve a dynamic coupling between structurally rigid (minimally frustrated) and plastic (locally frustrated) clusters of residues. The presented study has demonstrated that activation cancer mutations may affect the thermodynamic equilibrium between kinase states by allosterically altering the distribution of locally frustrated sites and increasing the local frustration in the inactive form, while eliminating locally frustrated sites and restoring structural rigidity of the active form. The energy landsape analysis of protein kinases and the proposed role of locally frustrated sites in activation mechanisms may have useful implications for bioinformatics-based screening and detection of functional sites critical for allosteric regulation in complex biomolecular systems. PMID:21998754

Hotspot Mutations in KIT Receptor Differentially Modulate Its Allosterically Coupled Conformational Dynamics: Impact on Activation and Drug Sensitivity

PubMed Central

Chauvot de Beauchêne, Isaure; Allain, Ariane; Panel, Nicolas; Laine, Elodie; Trouvé, Alain; Dubreuil, Patrice; Tchertanov, Luba

2014-01-01

Receptor tyrosine kinase KIT controls many signal transduction pathways and represents a typical allosterically regulated protein. The mutation-induced deregulation of KIT activity impairs cellular physiological functions and causes serious human diseases. The impact of hotspots mutations (D816H/Y/N/V and V560G/D) localized in crucial regulatory segments, the juxtamembrane region (JMR) and the activation (A-) loop, on KIT internal dynamics was systematically studied by molecular dynamics simulations. The mutational outcomes predicted in silico were correlated with in vitro and in vivo activation rates and drug sensitivities of KIT mutants. The allosteric regulation of KIT in the native and mutated forms is described in terms of communication between the two remote segments, JMR and A-loop. A strong correlation between the communication profile and the structural and dynamical features of KIT in the native and mutated forms was established. Our results provide new insight on the determinants of receptor KIT constitutive activation by mutations and resistance of KIT mutants to inhibitors. Depiction of an intra-molecular component of the communication network constitutes a first step towards an integrated description of vast communication pathways established by KIT in physiopathological contexts. PMID:25079768

Dual allosteric activation mechanisms in monomeric human glucokinase

PubMed Central

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

2015-01-01

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 1H-13C heteronuclear multiple quantum coherence (HMQC) spectrum of 13C-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 1H-13C 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. PMID:26283387

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.

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.

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

PubMed

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

2017-11-20

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

Structural and functional characterization of the Mycobacterium tuberculosis uridine monophosphate kinase: insights into the allosteric regulation.

PubMed

Labesse, Gilles; Benkali, Khaled; Salard-Arnaud, Isabelle; Gilles, Anne-Marie; Munier-Lehmann, Hélène

2011-04-01

Nucleoside Monophosphate Kinases (NMPKs) family are key enzymes in nucleotide metabolism. Bacterial UMPKs depart from the main superfamily of NMPKs. Having no eukaryotic counterparts they represent attractive therapeutic targets. They are regulated by GTP and UTP, while showing different mechanisms in Gram(+), Gram(-) and archaeal bacteria. In this work, we have characterized the mycobacterial UMPK (UMPKmt) combining enzymatic and structural investigations with site-directed mutagenesis. UMPKmt exhibits cooperativity toward ATP and an allosteric regulation by GTP and UTP. The crystal structure of the complex of UMPKmt with GTP solved at 2.5 Å, was merely identical to the modelled apo-form, in agreement with SAXS experiments. Only a small stretch of residues was affected upon nucleotide binding, pointing out the role of macromolecular dynamics rather than major structural changes in the allosteric regulation of bacterial UMPKs. We further probe allosteric regulation by site-directed mutagenesis. In particular, a key residue involved in the allosteric regulation of this enzyme was identified.

Nootropic α7 nicotinic receptor allosteric modulator derived from GABAA receptor modulators

PubMed Central

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

2007-01-01

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

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

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.

Molecular mechanism of Aurora A kinase autophosphorylation and its allosteric activation by TPX2

DOE PAGES

Zorba, Adelajda; Buosi, Vanessa; Kutter, Steffen; ...

2014-05-27

We elucidate the molecular mechanisms of two distinct activation strategies (autophosphorylation and TPX2-mediated activation) in human Aurora A kinase. Classic allosteric activation is in play where either activation loop phosphorylation or TPX2 binding to a conserved hydrophobic groove shifts the equilibrium far towards the active conformation. We resolve the controversy about the mechanism of autophosphorylation by demonstrating intermolecular autophosphorylation in a long-lived dimer by combining X-ray crystallography with functional assays. We then address the allosteric activation by TPX2 through activity assays and the crystal structure of a domain-swapped dimer of dephosphorylated Aurora A and TPX21−25. While autophosphorylation is the keymore » regulatory mechanism in the centrosomes in the early stages of mitosis, allosteric activation by TPX2 of dephosphorylated Aurora A could be at play in the spindle microtubules. The mechanistic insights into autophosphorylation and allosteric activation by TPX2 binding proposed here, may have implications for understanding regulation of other protein kinases.« less

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

Structural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition.

PubMed

Atkinson, Sarah C; Dogovski, Con; Downton, Matthew T; Czabotar, Peter E; Dobson, Renwick C J; Gerrard, Juliet A; Wagner, John; Perugini, Matthew A

2013-03-01

Lysine is one of the most limiting amino acids in plants and its biosynthesis is carefully regulated through inhibition of the first committed step in the pathway catalyzed by dihydrodipicolinate synthase (DHDPS). This is mediated via a feedback mechanism involving the binding of lysine to the allosteric cleft of DHDPS. However, the precise allosteric mechanism is yet to be defined. We present a thorough enzyme kinetic and thermodynamic analysis of lysine inhibition of DHDPS from the common grapevine, Vitis vinifera (Vv). Our studies demonstrate that lysine binding is both tight (relative to bacterial DHDPS orthologs) and cooperative. The crystal structure of the enzyme bound to lysine (2.4 Å) identifies the allosteric binding site and clearly shows a conformational change of several residues within the allosteric and active sites. Molecular dynamics simulations comparing the lysine-bound (PDB ID 4HNN) and lysine free (PDB ID 3TUU) structures show that Tyr132, a key catalytic site residue, undergoes significant rotational motion upon lysine binding. This suggests proton relay through the catalytic triad is attenuated in the presence of lysine. Our study reveals for the first time the structural mechanism for allosteric inhibition of DHDPS from the common grapevine.

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.

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

Allostery and the dynamic oligomerization of porphobilinogen synthase

PubMed Central

Jaffe, Eileen K.; Lawrence, Sarah H.

2011-01-01

The structural basis for allosteric regulation of porphobilinogen synthase (PBGS) is modulation of a quaternary structure equilibrium between octamer and hexamer (via dimers), which is represented schematically as 8mer ⇔ 2mer ⇔ 2mer* ⇔ 6mer*. The “*” represents a reorientation between two domains of each subunit that occurs in the dissociated state because it is sterically forbidden in the larger multimers. Allosteric effectors of PBGS are both intrinsic and extrinsic and are phylogenetically variable. In some species this equilibrium is modulated intrinsically by magnesium which binds at a site specific to the 8mer. In other species this equilibrium is modulated intrinsically by pH; the guanidinium group of an arginine being spatially equivalent to the allosteric magnesium ion. In humans, disease associated variants all shift the equilibrium toward the 6mer* relative to wild type. The 6mer* has a surface cavity that is not present in the 8mer and is proposed as a small molecule allosteric binding site. In silico and in vitro approaches have revealed species-specific allosteric PBGS inhibitors that stabilize the 6mer*. Some of these inhibitors are drugs in clinical use leading to the hypothesis that extrinsic allosteric inhibition of human PBGS could be a mechanism for drug side effects. PMID:22037356

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruvinsky, Anatoly M., E-mail: anatoly.ruvinsky@astrazeneca.com; Center for Bioinformatics, The University of Kansas, Lawrence, Kansas 66047; Vakser, Ilya A.

Ferritin-like molecules show a remarkable combination of the evolutionary conserved activity of iron uptake and release that engage different pores in the conserved ferritin shell. It was hypothesized that pore selection and iron traffic depend on dynamic allostery with no conformational changes in the backbone. In this study, we detect the allosteric networks in Pseudomonas aeruginosa bacterioferritin (BfrB), bacterial ferritin (FtnA), and bullfrog M and L ferritins (Ftns) by a network-weaving algorithm (NWA) that passes threads of an allosteric network through highly correlated residues using hierarchical clustering. The residue-residue correlations are calculated in the packing-on elastic network model that introducesmore » atom packing into the common packing-off model. Applying NWA revealed that each of the molecules has an extended allosteric network mostly buried inside the ferritin shell. The structure of the networks is consistent with experimental observations of iron transport: The allosteric networks in BfrB and FtnA connect the ferroxidase center with the 4-fold pores and B-pores, leaving the 3-fold pores unengaged. In contrast, the allosteric network directly links the 3-fold pores with the 4-fold pores in M and L Ftns. The majority of the network residues are either on the inner surface or buried inside the subunit fold or at the subunit interfaces. We hypothesize that the ferritin structures evolved in a way to limit the influence of functionally unrelated events in the cytoplasm on the allosteric network to maintain stability of the translocation mechanisms. We showed that the residue-residue correlations and the resultant long-range cooperativity depend on the ferritin shell packing, which, in turn, depends on protein sequence composition. Switching from the packing-on to the packing-off model reduces correlations by 35%–38% so that no allosteric network can be found. The influence of the side-chain packing on the allosteric networks explains the diversity in mechanisms of iron traffic suggested by experimental approaches.« less

Creation of a Ligand-Dependent Enzyme by Fusing Circularly Permuted Antibody Variable Region Domains.

PubMed

Iwai, Hiroto; Kojima-Misaizu, Miki; Dong, Jinhua; Ueda, Hiroshi

2016-04-20

Allosteric control of enzyme activity with exogenous substances has been hard to achieve, especially using antibody domains that potentially allow control by any antigens of choice. Here, in order to attain this goal, we developed a novel antibody variable region format introduced with circular permutations, called Clampbody. The two variable-region domains of the antibone Gla protein (BGP) antibody were each circularly permutated to have novel termini at the loops near their domain interface. Through their attachment to the N- and C-termini of a circularly permutated TEM-1 β-lactamase (cpBLA), we created a molecular switch that responds to the antigen peptide. The fusion protein specifically recognized the antigen, and in the presence of some detergent or denaturant, its catalytic activity was enhanced up to 4.7-fold in an antigen-dependent manner, due to increased resistance to these reagents. Hence, Clampbody will be a powerful tool for the allosteric regulation of enzyme and other protein activities and especially useful to design robust biosensors.

The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility.

PubMed

Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I; Hantschel, Oliver

2014-11-17

The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility

NASA Astrophysics Data System (ADS)

Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I.; Hantschel, Oliver

2014-11-01

The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

Caffeine inhibition of glycogen phosphorylase from Mytilus galloprovincialis mantle tissue.

PubMed

San Juan Serrano, F; Sánchez López, J L; García Martín, L O

1995-09-01

A different caffeine inhibition of both phosphorylated and unphosphorylated forms of glycogen phosphorylase from Mytilus mantle has been demonstrated. Caffeine increases the allosteric constant of phosphorylase b 30-fold, acting as an allosteric inhibitor (nH = 2) of mixed type with respect to inorganic phosphate (Pi) and AMP, and of single competitive type with respect to glycogen. The Mytilus phosphorylated form is also caffeine inhibited through competitive inhibition in relation to Pi and glycogen. In this case, the inhibitor does not modify the allosteric constant (near 2), neither does it display allosteric effects (nH = 1). The results demonstrate the notable modification of the nucleotide site promoted by the phosphorylation process and the existence of a functional inhibitory nucleoside site in Mytilus phosphorylase.

SPACER: server for predicting allosteric communication and effects of regulation

PubMed Central

Goncearenco, Alexander; Mitternacht, Simon; Yong, Taipang; Eisenhaber, Birgit; Eisenhaber, Frank; Berezovsky, Igor N.

2013-01-01

The SPACER server provides an interactive framework for exploring allosteric communication in proteins with different sizes, degrees of oligomerization and function. SPACER uses recently developed theoretical concepts based on the thermodynamic view of allostery. It proposes easily tractable and meaningful measures that allow users to analyze the effect of ligand binding on the intrinsic protein dynamics. The server shows potential allosteric sites and allows users to explore communication between the regulatory and functional sites. It is possible to explore, for instance, potential effector binding sites in a given structure as targets for allosteric drugs. As input, the server only requires a single structure. The server is freely available at http://allostery.bii.a-star.edu.sg/. PMID:23737445

Combinatorial Enzyme Design Probes Allostery and Cooperativity in the Trypsin Fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Page, Michael J.; Di Cera, Enrico; St. Louis-MED)

2010-06-14

Converting one enzyme into another is challenging due to the uneven distribution of important amino acids for function in both protein sequence and structure. We report a strategy for protein engineering allowing an organized mixing and matching of genetic material that leverages lower throughput with increased quality of screens. Our approach successfully tested the contribution of each surface-exposed loop in the trypsin fold alone and the cooperativity of their combinations towards building the substrate selectivity and Na{sup +}-dependent allosteric activation of the protease domain of human coagulation factor Xa into a bacterial trypsin. As the created proteases lack additional proteinmore » domains and protein co-factor activation mechanism requisite for the complexity of blood coagulation, they are stepping-stones towards further understanding and engineering of artificial clotting factors.« less

Allosteric Regulation of Lactobacillus plantarum Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase (Xfp)

PubMed Central

Glenn, Katie

2015-01-01

ABSTRACT Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which catalyzes the conversion of xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P) to acetyl phosphate, plays a key role in carbohydrate metabolism in a number of bacteria. Recently, we demonstrated that the fungal Cryptococcus neoformans Xfp2 exhibits both substrate cooperativity for all substrates (X5P, F6P, and Pi) and allosteric regulation in the forms of inhibition by phosphoenolpyruvate (PEP), oxaloacetic acid (OAA), and ATP and activation by AMP (K. Glenn, C. Ingram-Smith, and K. S. Smith. Eukaryot Cell 13:657–663, 2014). Allosteric regulation has not been reported previously for the characterized bacterial Xfps. Here, we report the discovery of substrate cooperativity and allosteric regulation among bacterial Xfps, specifically the Lactobacillus plantarum Xfp. L. plantarum Xfp is an allosteric enzyme inhibited by PEP, OAA, and glyoxylate but unaffected by the presence of ATP or AMP. Glyoxylate is an additional inhibitor to those previously reported for C. neoformans Xfp2. As with C. neoformans Xfp2, PEP and OAA share the same or possess overlapping sites on L. plantarum Xfp. Glyoxylate, which had the lowest half-maximal inhibitory concentration of the three inhibitors, binds at a separate site. This study demonstrates that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfp enzymes, yet important differences exist between the enzymes in these two domains. IMPORTANCE Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) plays a key role in carbohydrate metabolism in a number of bacteria. Although we recently demonstrated that the fungal Cryptococcus Xfp is subject to substrate cooperativity and allosteric regulation, neither phenomenon has been reported for a bacterial Xfp. Here, we report that the Lactobacillus plantarum Xfp displays substrate cooperativity and is allosterically inhibited by phosphoenolpyruvate and oxaloacetate, as is the case for Cryptococcus Xfp. The bacterial enzyme is unaffected by the presence of AMP or ATP, which act as a potent activator and inhibitor of the fungal Xfp, respectively. Our results demonstrate that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfps, yet important differences exist between the enzymes in these two domains. PMID:25605308

Allosteric regulation of Lactobacillus plantarum xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp).

PubMed

Glenn, Katie; Smith, Kerry S

2015-04-01

Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which catalyzes the conversion of xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P) to acetyl phosphate, plays a key role in carbohydrate metabolism in a number of bacteria. Recently, we demonstrated that the fungal Cryptococcus neoformans Xfp2 exhibits both substrate cooperativity for all substrates (X5P, F6P, and Pi) and allosteric regulation in the forms of inhibition by phosphoenolpyruvate (PEP), oxaloacetic acid (OAA), and ATP and activation by AMP (K. Glenn, C. Ingram-Smith, and K. S. Smith. Eukaryot Cell 13: 657-663, 2014). Allosteric regulation has not been reported previously for the characterized bacterial Xfps. Here, we report the discovery of substrate cooperativity and allosteric regulation among bacterial Xfps, specifically the Lactobacillus plantarum Xfp. L. plantarum Xfp is an allosteric enzyme inhibited by PEP, OAA, and glyoxylate but unaffected by the presence of ATP or AMP. Glyoxylate is an additional inhibitor to those previously reported for C. neoformans Xfp2. As with C. neoformans Xfp2, PEP and OAA share the same or possess overlapping sites on L. plantarum Xfp. Glyoxylate, which had the lowest half-maximal inhibitory concentration of the three inhibitors, binds at a separate site. This study demonstrates that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfp enzymes, yet important differences exist between the enzymes in these two domains. Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) plays a key role in carbohydrate metabolism in a number of bacteria. Although we recently demonstrated that the fungal Cryptococcus Xfp is subject to substrate cooperativity and allosteric regulation, neither phenomenon has been reported for a bacterial Xfp. Here, we report that the Lactobacillus plantarum Xfp displays substrate cooperativity and is allosterically inhibited by phosphoenolpyruvate and oxaloacetate, as is the case for Cryptococcus Xfp. The bacterial enzyme is unaffected by the presence of AMP or ATP, which act as a potent activator and inhibitor of the fungal Xfp, respectively. Our results demonstrate that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfps, yet important differences exist between the enzymes in these two domains. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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 ATP site, and may enhance allosteric cooperativity with the substrate binding region by increasing communication capabilities of mediating residues. PMID:25427151

Shift in the equilibrium between on and off states of the allosteric switch in Ras-GppNHp affected by small molecules and bulk solvent composition.

PubMed

Holzapfel, Genevieve; Buhrman, Greg; Mattos, Carla

2012-08-07

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-β. 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.

Modular architecture of protein structures and allosteric communications: potential implications for signaling proteins and regulatory linkages

PubMed Central

del Sol, Antonio; Araúzo-Bravo, Marcos J; Amoros, Dolors; Nussinov, Ruth

2007-01-01

Background Allosteric communications are vital for cellular signaling. Here we explore a relationship between protein architectural organization and shortcuts in signaling pathways. Results We show that protein domains consist of modules interconnected by residues that mediate signaling through the shortest pathways. These mediating residues tend to be located at the inter-modular boundaries, which are more rigid and display a larger number of long-range interactions than intra-modular regions. The inter-modular boundaries contain most of the residues centrally conserved in the protein fold, which may be crucial for information transfer between amino acids. Our approach to modular decomposition relies on a representation of protein structures as residue-interacting networks, and removal of the most central residue contacts, which are assumed to be crucial for allosteric communications. The modular decomposition of 100 multi-domain protein structures indicates that modules constitute the building blocks of domains. The analysis of 13 allosteric proteins revealed that modules characterize experimentally identified functional regions. Based on the study of an additional functionally annotated dataset of 115 proteins, we propose that high-modularity modules include functional sites and are the basic functional units. We provide examples (the Gαs subunit and P450 cytochromes) to illustrate that the modular architecture of active sites is linked to their functional specialization. Conclusion Our method decomposes protein structures into modules, allowing the study of signal transmission between functional sites. A modular configuration might be advantageous: it allows signaling proteins to expand their regulatory linkages and may elicit a broader range of control mechanisms either via modular combinations or through modulation of inter-modular linkages. PMID:17531094

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

PubMed

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

2014-11-25

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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) canmore » 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.« less

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

2017-06-01

D 2 and D 3 dopamine receptors belong to the largest family of cell surface proteins in eukaryotes, the G protein-coupled receptors (GPCRs). Considering their crucial physiologic 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 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 N -[(1r,4r)-4-[2-(7-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-1H-indole-2-carboxamide (SB269652), a compound supposed to interact with the orthosteric binding site of dopamine receptors, was actually a negative allosteric modulator of D 2 - and D 3 -receptor dimers, thus identifying the first allosteric small molecule acting on these important therapeutic targets. This review addresses the progress in understanding the molecular mechanisms of interaction between the negative modulator SB269652 and D 2 and D 3 dopamine receptor monomers and dimers, and surveys the prospects for developing new dopamine receptor allosteric drugs with SB269652 as the leading compound. U.S. Government work not protected by U.S. copyright.

Glutamine Hydrolysis by Imidazole Glycerol Phosphate Synthase Displays Temperature Dependent Allosteric Activation

PubMed Central

Lisi, George P.; Currier, Allen A.; Loria, J. Patrick

2018-01-01

The enzyme imidazole glycerol phosphate synthase (IGPS) is a model for studies of long-range allosteric regulation in enzymes. Binding of the allosteric effector ligand N'-[5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) stimulates millisecond (ms) timescale motions in IGPS that enhance its catalytic function. We studied the effect of temperature on these critical conformational motions and the catalytic mechanism of IGPS from the hyperthermophile Thermatoga maritima in an effort to understand temperature-dependent allostery. Enzyme kinetic and NMR dynamics measurements show that apo and PRFAR-activated IGPS respond differently to changes in temperature. Multiple-quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments performed at 303, 323, and 343 K (30, 50, and 70°C) reveal that millisecond flexibility is enhanced to a higher degree in apo IGPS than in the PRFAR-bound enzyme as the sample temperature is raised. We find that the flexibility of the apo enzyme is nearly identical to that of its PRFAR activated state at 343 K, whereas conformational motions are considerably different between these two forms of the enzyme at room temperature. Arrhenius analyses of these flexible sites show a varied range of activation energies that loosely correlate to allosteric communities identified by computational methods and reflect local changes in dynamics that may facilitate conformational sampling of the active conformation. In addition, kinetic assays indicate that allosteric activation by PRFAR decreases to 65-fold at 343 K, compared to 4,200-fold at 303 K, which mirrors the decreased effect of PRFAR on ms motions relative to the unactivated enzyme. These studies indicate that at the growth temperature of T. maritima, PFRAR is a weaker allosteric activator than it is at room temperature and illustrate that the allosteric mechanism of IGPS is temperature dependent. PMID:29468164

‘Partial’ competition of heterobivalent ligand binding may be mistaken for allosteric interactions: a comparison of different target interaction models

PubMed Central

Vauquelin, Georges; Hall, David; Charlton, Steven J

2015-01-01

Background and Purpose Non-competitive drugs that confer allosteric modulation of orthosteric ligand binding are of increasing interest as therapeutic agents. Sought-after advantages include a ceiling level to drug effect and greater receptor-subtype selectivity. It is thus important to determine the mode of interaction of newly identified receptor ligands early in the drug discovery process and binding studies with labelled orthosteric ligands constitute a traditional approach for this. According to the general allosteric ternary complex model, allosteric ligands that exhibit negative cooperativity may generate distinctive ‘competition’ curves: they will not reach baseline levels and their nadir will increase in par with the orthosteric ligand concentration. This behaviour is often considered a key hallmark of allosteric interactions. Experimental Approach The present study is based on differential equation-based simulations. Key Results The differential equation-based simulations revealed that the same ‘competition binding’ pattern was also obtained when a monovalent ligand binds to one of the target sites of a heterobivalent ligand, even if this process is exempt of allosteric interactions. This pattern was not strictly reciprocal when the binding of each of the ligands was recorded. The prominence of this phenomenon may vary from one heterobivalent ligand to another and we suggest that this phenomenon may take place with ligands that have been proposed to bind according to ‘two-domain’ and ‘charnière’ models. Conclusions and Implications The present findings indicate a familiar experimental situation where bivalency may give rise to observations that could inadvertently be interpreted as allosteric binding. Yet, both mechanisms could be differentiated based on alternative experiments and structural considerations. PMID:25537684

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

PubMed Central

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

2012-01-01

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

The allosteric citalopram binding site differentially interferes with neuronal firing rate and SERT trafficking in serotonergic neurons.

PubMed

Matthäus, Friederike; Haddjeri, Nasser; Sánchez, Connie; Martí, Yasmina; Bahri, Senda; Rovera, Renaud; Schloss, Patrick; Lau, Thorsten

2016-11-01

Citalopram is a clinically applied selective serotonin re-uptake inhibitor for antidepressant pharmacotherapy. It consists of two enantiomers, S-citalopram (escitalopram) and R-citalopram, of which escitalopram exerts the antidepressant therapeutic effect and has been shown to be one of the most efficient antidepressants, while R-citalopram antagonizes escitalopram via an unknown molecular mechanism that may depend on binding to a low-affinity allosteric binding site of the serotonin transporter. However, the precise mechanism of antidepressant regulation of the serotonin transporter by citalopram enantiomers still remains elusive. Here we investigate escitalopram׳s acute effect on (1) serotonergic neuronal firing in transgenic mice that express the human serotonin transporter without and with a mutation that disables the allosteric binding site, and (2) regulation of the serotonin transporter׳s cell surface localization in stem cell-derived serotonergic neurons. Our results demonstrate that escitalopram inhibited neuronal firing less potently in the mouse line featuring a mutation that abolishes the function of the allosteric binding site and induced serotonin transporter internalization independently of the allosteric binding site mechanism. Furthermore, citalopram enantiomers dose-dependently induced serotonin transporter internalization. In conclusion, this study provides new insight into antidepressant effects exerted by citalopram enantiomers in presence and absence of a functional allosteric binding site. Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.

Medicinal chemistry of P2X receptors: allosteric modulators.

PubMed

Müller, Christa E

2015-01-01

P2X receptors are trimeric ligand-gated ion channels whose potential as novel drug targets for a number of diseases has been recognized. They are mainly involved in inflammatory processes, including neuroinflammation, and pain sensation. The orthosteric binding site is lined by basic amino acid residues that bind the negatively charged agonist ATP. Therefore it is not easy to develop orthosteric ligands that possess drug-like properties for such a highly polar binding site. However, ligand-gated ion channels offer multiple additional binding sites for allosteric ligands, positive or negative allosteric modulators enhancing or blocking receptor function. So far, the P2X3 (and P2X2/3), as well as the P2X7 receptor subtype have been the main focus of drug development efforts. A number of potent and selective allosteric antagonists have been developed to block these receptors. We start to see the development of novel allosteric ligands also for the other P2X receptor subtypes, P2X1, P2X2 and especially P2X4. The times when only poor, non-selective, non-drug-like tools for studying P2X receptor function were available have been overcome. The first clinical studies with allosteric P2X3 and P2X7 antagonists suggest that P2X therapeutics may soon become a reality.

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.

Kuwanon-L as a New Allosteric HIV-1 Integrase Inhibitor: Molecular Modeling and Biological Evaluation.

PubMed

Esposito, Francesca; Tintori, Cristina; Martini, Riccardo; Christ, Frauke; Debyser, Zeger; Ferrarese, Roberto; Cabiddu, Gianluigi; Corona, Angela; Ceresola, Elisa Rita; Calcaterra, Andrea; Iovine, Valentina; Botta, Bruno; Clementi, Massimo; Canducci, Filippo; Botta, Maurizio; Tramontano, Enzo

2015-11-01

HIV-1 integrase (IN) active site inhibitors are the latest class of drugs approved for HIV treatment. The selection of IN strand-transfer drug-resistant HIV strains in patients supports the development of new agents that are active as allosteric IN inhibitors. Here, a docking-based virtual screening has been applied to a small library of natural ligands to identify new allosteric IN inhibitors that target the sucrose binding pocket. From theoretical studies, kuwanon-L emerged as the most promising binder and was thus selected for biological studies. Biochemical studies showed that kuwanon-L is able to inhibit the HIV-1 IN catalytic activity in the absence and in the presence of LEDGF/p75 protein, the IN dimerization, and the IN/LEDGF binding. Kuwanon-L also inhibited HIV-1 replication in cell cultures. Overall, docking and biochemical results suggest that kuwanon-L binds to an allosteric binding pocket and can be considered an attractive lead for the development of new allosteric IN antiviral agents. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wellington, Samantha; Nag, Partha P.; Michalska, Karolina

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

A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wellington, Samantha; Nag, Partha P.; Michalska, Karolina

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

Allosteric Inhibition via R-state Destabilization in ATP Sulfurylase from Penicillium chrysogenum

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 bymore » 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.« less

Discovery and SAR of muscarinic receptor subtype 1 (M1) allosteric activators from a molecular libraries high throughput screen. Part 1: 2,5-dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones as positive allosteric modulators.

PubMed

Han, Changho; Chatterjee, Arindam; Noetzel, Meredith J; Panarese, Joseph D; Smith, Emery; Chase, Peter; Hodder, Peter; Niswender, Colleen; Conn, P Jeffrey; Lindsley, Craig W; Stauffer, Shaun R

2015-01-15

Results from a 2012 high-throughput screen of the NIH Molecular Libraries Small Molecule Repository (MLSMR) against the human muscarinic receptor subtype 1 (M1) for positive allosteric modulators is reported. A content-rich screen utilizing an intracellular calcium mobilization triple-addition protocol allowed for assessment of all three modes of pharmacology at M1, including agonist, positive allosteric modulator, and antagonist activities in a single screening platform. We disclose a dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-one hit (DBPQ, CID 915409) and examine N-benzyl pharmacophore/SAR relationships versus previously reported quinolin-3(5H)-ones and isatins, including ML137. SAR and consideration of recently reported crystal structures, homology modeling, and structure-function relationships using point mutations suggests a shared binding mode orientation at the putative common allosteric binding site directed by the pendant N-benzyl substructure. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

Role of Terahertz (THz) Fluctuations in the Allosteric Properties of the PDZ Domains.

PubMed

Conti Nibali, Valeria; Morra, Giulia; Havenith, Martina; Colombo, Giorgio

2017-11-09

With the aim of investigating the relationship between the fast fluctuations of proteins and their allosteric behavior, we perform molecular dynamics simulations of two model PDZ domains with differential allosteric responses. We focus on protein dynamics in the THz regime (0.1-3 THz) as opposed to lower frequencies. By characterizing the dynamic modulation of the protein backbone induced by ligand binding in terms of single residue and pairwise distance fluctuations, we identify a response nucleus modulated by the ligand that is visible only at THz frequencies. The residues of this nucleus undergo a significant stiffening and an increase in mutual coordination upon binding. Additionally, we find that the dynamic modulation is significantly more intense for the side chains, where it is also redistributed to distal regions not immediately in contact with the ligand allowing us to better define the response nucleus at THz frequencies. The overlap between the known allosterically responding residues of the investigated PDZ domains and the modulated region highlighted here suggests that fast THz dynamics could play a role in allosteric mechanisms.

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

PubMed

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

2014-09-25

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

Design, synthesis, and action of oxotremorine-related hybrid-type allosteric modulators of muscarinic acetylcholine receptors.

PubMed

Disingrini, Teresa; Muth, Mathias; Dallanoce, Clelia; Barocelli, Elisabetta; Bertoni, Simona; Kellershohn, Kerstin; Mohr, Klaus; De Amici, Marco; Holzgrabe, Ulrike

2006-01-12

A novel series of muscarinic receptor ligands of the hexamethonio-type was prepared which contained, on one side, the phthalimidopropane or 1,8-naphthalimido-2,2-dimethylpropane moiety typical for subtype selective allosteric antagonists and, on the other, the acetylenic fragment typical for the nonselective orthosteric muscarinic agonists oxotremorine, oxotremorine-M, and related muscarinic agonists. Binding experiments in M(2) receptors using [(3)H]N-methylscopolamine as an orthosteric probe proved an allosteric action of both groups of hybrids, 7a-10a and 8b-10b. The difference in activity between a-group and b-group hybrids corresponded with the activity difference between the allosteric parent compounds. In M(1)-M(3) muscarinic isolated organ preparations, most of the hybrids behaved as subtype selective antagonists. [(35)S]GTPgammaS binding assays using human M(2) receptors overexpressed in CHO cells revealed that a weak intrinsic efficacy was preserved in 8b-10b. Thus, attaching muscarinic allosteric antagonist moieties to orthosteric muscarinic agonists may lead to hybrid compounds in which functions of both components are mixed.

Dynamic hysteresis in a one-dimensional Ising model: application to allosteric proteins.

PubMed

Graham, I; Duke, T A J

2005-06-01

We solve exactly the problem of dynamic hysteresis for a finite one-dimensional Ising model at low temperature. We find that the area of the hysteresis loop, as the field is varied periodically, scales as the square root of the field frequency for a large range of frequencies. Below a critical frequency there is a correction to the scaling law, resulting in a linear relationship between hysteresis area and frequency. The one-dimensional Ising model provides a simplified description of switchlike behavior in allosteric proteins, such as hemoglobin. Thus our analysis predicts the switching dynamics of allosteric proteins when they are exposed to a ligand concentration which changes with time. Many allosteric proteins bind a regulator that is maintained at a nonequilibrium concentration by active signal transduction processes. In the light of our analysis, we discuss to what extent allosteric proteins can respond to changes in regulator concentration caused by an upstream signaling event, while remaining insensitive to the intrinsic nonequilibrium fluctuations in regulator level which occur in the absence of a signal.

Coherent Conformational Degrees of Freedom as a Structural Basis for Allosteric Communication

PubMed Central

Mitternacht, Simon; Berezovsky, Igor N.

2011-01-01

Conformational changes in allosteric regulation can to a large extent be described as motion along one or a few coherent degrees of freedom. The states involved are inherent to the protein, in the sense that they are visited by the protein also in the absence of effector ligands. Previously, we developed the measure binding leverage to find sites where ligand binding can shift the conformational equilibrium of a protein. Binding leverage is calculated for a set of motion vectors representing independent conformational degrees of freedom. In this paper, to analyze allosteric communication between binding sites, we introduce the concept of leverage coupling, based on the assumption that only pairs of sites that couple to the same conformational degrees of freedom can be allosterically connected. We demonstrate how leverage coupling can be used to analyze allosteric communication in a range of enzymes (regulated by both ligand binding and post-translational modifications) and huge molecular machines such as chaperones. Leverage coupling can be calculated for any protein structure to analyze both biological and latent catalytic and regulatory sites. PMID:22174669

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

PubMed Central

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

2017-01-01

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

Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to "Biased Opioids"?

PubMed

Root-Bernstein, Robert; Turke, Miah; Subhramanyam, Udaya K Tiruttani; Churchill, Beth; Labahn, Joerg

2018-01-17

Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists.

Intrinsic K-Ras dynamics: A novel molecular dynamics data analysis method shows causality between residue pair motions

NASA Astrophysics Data System (ADS)

Vatansever, Sezen; Gümüş, Zeynep H.; Erman, Burak

2016-11-01

K-Ras is the most frequently mutated oncogene in human cancers, but there are still no drugs that directly target it in the clinic. Recent studies utilizing dynamics information show promising results for selectively targeting mutant K-Ras. However, despite extensive characterization, the mechanisms by which K-Ras residue fluctuations transfer allosteric regulatory information remain unknown. Understanding the direction of information flow can provide new mechanistic insights for K-Ras targeting. Here, we present a novel approach -conditional time-delayed correlations (CTC) - using the motions of all residue pairs of a protein to predict directionality in the allosteric regulation of the protein fluctuations. Analyzing nucleotide-dependent intrinsic K-Ras motions with the new approach yields predictions that agree with the literature, showing that GTP-binding stabilizes K-Ras motions and leads to residue correlations with relatively long characteristic decay times. Furthermore, our study is the first to identify driver-follower relationships in correlated motions of K-Ras residue pairs, revealing the direction of information flow during allosteric modulation of its nucleotide-dependent intrinsic activity: active K-Ras Switch-II region motions drive Switch-I region motions, while α-helix-3L7 motions control both. Our results provide novel insights for strategies that directly target mutant K-Ras.

Levamisole: A Positive Allosteric Modulator for the α3β4 Nicotinic Acetylcholine Receptors Prevents Weight Gain in the CD-1 Mice on a High Fat Diet.

PubMed

Lewis, Jeanne A; Yakel, Jerrel L; Pandya, Anshul A

2017-01-01

Neuronal nicotinic acetylcholine receptors (nAChRs) regulate the function of multiple neurotransmitter pathways throughout the central nervous system. This includes nAChRs found on the proopiomelanocortin neurons in the hypothalamus. Activation of these nAChRs by nicotine causes a decrease in the consumption of food in rodents. This study tested the effect of subtype selective allosteric modulators for nAChRs on the body weight of CD-1 mice. Levamisole, an allosteric modulator for the α3β4 subtype of nAChRs, prevented weight gain in mice that were fed a high fat diet. PNU-120596 and desformylflustrabromine were observed to be selective PAMs for the α7 and α4β2 nAChR, respectively. Both of these compounds failed to prevent weight gain in the CD-1 mice. These results suggest that the modulation of hypothalamic α3β4 nAChRs is an important factor in regulating food intake, and the PAMs for these receptors need further investigation as potential therapeutic agents for controlling weight gain. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Regulation of Son of sevenless by the membrane-actin linker protein ezrin

PubMed Central

Geißler, Katja J.; Jung, M. Juliane; Riecken, Lars Björn; Sperka, Tobias; Cui, Yan; Schacke, Stephan; Merkel, Ulrike; Markwart, Robby; Rubio, Ignacio; Than, Manuel E.; Breithaupt, Constanze; Peuker, Sebastian; Seifert, Reinhard; Kaupp, Ulrich Benjamin; Herrlich, Peter; Morrison, Helen

2013-01-01

Receptor tyrosine kinases participate in several signaling pathways through small G proteins such as Ras (rat sarcoma). An important component in the activation of these G proteins is Son of sevenless (SOS), which catalyzes the nucleotide exchange on Ras. For optimal activity, a second Ras molecule acts as an allosteric activator by binding to a second Ras-binding site within SOS. This allosteric Ras-binding site is blocked by autoinhibitory domains of SOS. We have reported recently that Ras activation also requires the actin-binding proteins ezrin, radixin, and moesin. Here we report the mechanism by which ezrin modulates SOS activity and thereby Ras activation. Active ezrin enhances Ras/MAPK signaling and interacts with both SOS and Ras in vivo and in vitro. Moreover, in vitro kinetic assays with recombinant proteins show that ezrin also is important for the activity of SOS itself. Ezrin interacts with GDP-Ras and with the Dbl homology (DH)/pleckstrin homology (PH) domains of SOS, bringing GDP-Ras to the proximity of the allosteric site of SOS. These actions of ezrin are antagonized by the neurofibromatosis type 2 tumor-suppressor protein merlin. We propose an additional essential step in SOS/Ras control that is relevant for human cancer as well as all physiological processes involving Ras. PMID:24297905

Supermolecular drug challenge to overcome drug resistance in cancer cells.

PubMed

Onishi, Yasuhiko; Eshita, Yuki; Ji, Rui-Cheng; Kobayashi, Takashi; Onishi, Masayasu; Mizuno, Masaaki; Yoshida, Jun; Kubota, Naoji

2018-06-04

Overcoming multidrug resistance (MDR) of cancer cells can be accomplished using drug delivery systems in large-molecular-weight ATP-binding cassette transporters before entry into phagolysosomes and by particle-cell-surface interactions. However, these hypotheses do not address the intratumoral heterogeneity in cancer. Anti-MDR must be related to alterations of drug targets, expression of detoxification, as well as altered proliferation. In this study, it is shown that the excellent efficacy and sustainability of anti-MDR is due to a stable ES complex because of the allosteric facilities of artificial enzymes when they are used as supramolecular complexes. The allosteric effect of supermolecular drugs can be explained by the induced-fit model and can provide stable feedback control systems through the loop transfer function of the Hill equation. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

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

Changes in small angle X-ray scattering parameters observed upon ligand binding to rabbit muscle pyruvate kinase are not correlated with allosteric transitions†

PubMed Central

Fenton, Aron W.; Williams, Rachel; Trewhella, Jill

2010-01-01

Protein fluorescence and small-angle X-ray scattering (SAXS) have been used to monitor effector affinity and conformational changes previously associated with allosteric regulation in rabbit muscle pyruvate kinase (M1-PYK). In the absence of substrate (phosphoenolpyruvate; PEP), SAXS-monitored conformational changes in M1-PYK elicited by the binding of phenylalanine (an allosteric inhibitor that reduces the affinity of M1-PYK for PEP) are similar to those observed upon binding of alanine or 2-aminobutyric acid. Under the current assay conditions, these small amino acids bind to the protein, but elicit a minimal change in the affinity of the protein for PEP. Therefore, if changes in scattering signatures represent cleft closure via domain rotation as previously interpreted, it can be concluded that these motions are not sufficient to elicit allosteric inhibition. Additionally, although PEP has similar affinities for the free enzyme and the M1-PYK/small-amino-acid complexes (i.e. the small amino acids have minimal allosteric effects), PEP binding elicits different changes in the SAXS signature of the free enzyme vs. the M1-PYK/small-amino-acid complexes. PMID:20712377

Allosteric binding sites in Rab11 for potential drug candidates

PubMed Central

2018-01-01

Rab11 is an important protein subfamily in the RabGTPase family. These proteins physiologically function as key regulators of intracellular membrane trafficking processes. Pathologically, Rab11 proteins are implicated in many diseases including cancers, neurodegenerative diseases and type 2 diabetes. Although they are medically important, no previous study has found Rab11 allosteric binding sites where potential drug candidates can bind to. In this study, by employing multiple clustering approaches integrating principal component analysis, independent component analysis and locally linear embedding, we performed structural analyses of Rab11 and identified eight representative structures. Using these representatives to perform binding site mapping and virtual screening, we identified two novel binding sites in Rab11 and small molecules that can preferentially bind to different conformations of these sites with high affinities. After identifying the binding sites and the residue interaction networks in the representatives, we computationally showed that these binding sites may allosterically regulate Rab11, as these sites communicate with switch 2 region that binds to GTP/GDP. These two allosteric binding sites in Rab11 are also similar to two allosteric pockets in Ras that we discovered previously. PMID:29874286

The allosteric communication pathways in KIX domain of CBP.

PubMed

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

2013-08-27

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

Recent Advances in the Realm of Allosteric Modulators for Opioid Receptors for Future Therapeutics.

PubMed

Remesic, Michael; Hruby, Victor J; Porreca, Frank; Lee, Yeon Sun

2017-06-21

Opioids, and more specifically μ-opioid receptor (MOR) agonists such as morphine, have long been clinically used as therapeutics for severe pain states but often come with serious side effects such as addiction and tolerance. Many studies have focused on bringing about analgesia from the MOR with attenuated side effects, but its underlying mechanism is not fully understood. Recently, focus has been geared toward the design and elucidation of the orthosteric site with ligands of various biological profiles and mixed subtype opioid activities and selectivities, but targeting the allosteric site is an area of increasing interest. It has been shown that allosteric modulators play key roles in influencing receptor function such as its tolerance to a ligand and affect downstream pathways. There has been a high variance of chemical structures that provide allosteric modulation at a given receptor, but recent studies and reviews tend to focus on the altered cellular mechanisms instead of providing a more rigorous description of the allosteric ligand's structure-function relationship. In this review, we aim to explore recent developments in the structural motifs that potentiate orthosteric binding and their influences on cellular pathways in an effort to present novel approaches to opioid therapeutic design.

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

PubMed

Ahmed, Ahmed H; Oswald, Robert E

2010-03-11

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

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

PubMed Central

Ahmed, Ahmed H.; Oswald, Robert E.

2010-01-01

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

Nonequilibrium dissipation-free transport in F₁-ATPase and the thermodynamic role of asymmetric allosterism.

PubMed

Kawaguchi, Kyogo; Sasa, Shin-Ichi; Sagawa, Takahiro

2014-06-03

F1-ATPase (or F1), the highly efficient and reversible biochemical engine, has motivated physicists as well as biologists to imagine the design principles governing machines in the fluctuating world. Recent experiments have clarified yet another interesting property of F1; the dissipative heat inside the motor is very small, irrespective of the velocity of rotation and energy transport. Conceptual interest is devoted to the fact that the amount of internal dissipation is not simply determined by the sequence of equilibrium pictures, but also relies on the rotational-angular dependence of nucleotide affinity, which is a truly nonequilibrium aspect. We propose that the totally asymmetric allosteric model (TASAM), where adenosine triphosphate (ATP) binding to F1 is assumed to have low dependence on the angle of the rotating shaft, produces results that are most consistent with the experiments. Theoretical analysis proves the crucial role of two time scales in the model, which explains the universal mechanism to produce the internal dissipation-free feature. The model reproduces the characteristic torque dependence of the rotational velocity of F1 and predicts that the internal dissipation upon the ATP synthesis direction rotation becomes large at the low nucleotide condition. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Computational Study on the Inhibitor Binding Mode and Allosteric Regulation Mechanism in Hepatitis C Virus NS3/4A Protein

PubMed Central

Xue, Weiwei; Yang, Ying; Wang, Xiaoting; Liu, Huanxiang; Yao, Xiaojun

2014-01-01

HCV NS3/4A protein is an attractive therapeutic target responsible for harboring serine protease and RNA helicase activities during the viral replication. Small molecules binding at the interface between the protease and helicase domains can stabilize the closed conformation of the protein and thus block the catalytic function of HCV NS3/4A protein via an allosteric regulation mechanism. But the detailed mechanism remains elusive. Here, we aimed to provide some insight into the inhibitor binding mode and allosteric regulation mechanism of HCV NS3/4A protein by using computational methods. Four simulation systems were investigated. They include: apo state of HCV NS3/4A protein, HCV NS3/4A protein in complex with an allosteric inhibitor and the truncated form of the above two systems. The molecular dynamics simulation results indicate HCV NS3/4A protein in complex with the allosteric inhibitor 4VA adopts a closed conformation (inactive state), while the truncated apo protein adopts an open conformation (active state). Further residue interaction network analysis suggests the communication of the domain-domain interface play an important role in the transition from closed to open conformation of HCV NS3/4A protein. However, the inhibitor stabilizes the closed conformation through interaction with several key residues from both the protease and helicase domains, including His57, Asp79, Asp81, Asp168, Met485, Cys525 and Asp527, which blocks the information communication between the functional domains interface. Finally, a dynamic model about the allosteric regulation and conformational changes of HCV NS3/4A protein was proposed and could provide fundamental insights into the allosteric mechanism of HCV NS3/4A protein function regulation and design of new potent inhibitors. PMID:24586263

Real-time characterization of cannabinoid receptor 1 (CB1 ) allosteric modulators reveals novel mechanism of action.

PubMed

Cawston, Erin E; Redmond, William J; Breen, Courtney M; Grimsey, Natasha L; Connor, Mark; Glass, Michelle

2013-10-01

The cannabinoid receptor type 1 (CB1 ) has an allosteric binding site. The drugs ORG27569 {5-chloro-3-ethyl-N-[2-[4-(1-piperidinyl)phenyl]ethyl]-1H-indole-2-carboxamide} and PSNCBAM-1 {1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea} have been extensively characterized with regard to their effects on signalling of the orthosteric ligand CP55,940 {(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol}, and studies have suggested that these allosteric modulators increase binding affinity but act as non-competitive antagonists in functional assays. To gain a deeper understanding of allosteric modulation of CB1 , we examined real-time signalling and trafficking responses of the receptor in the presence of allosteric modulators. Studies of CB1 signalling were carried out in HEK 293 and AtT20 cells expressing haemagglutinin-tagged human and rat CB1 . We measured real-time accumulation of cAMP, activation and desensitization of potassium channel-mediated cellular hyperpolarization and CB1 internalization. ORG27569 and PSNCBAM-1 produce a complex, concentration and time-dependent modulation of agonist-mediated regulation of cAMP levels, as well as an increased rate of desensitization of CB1 -mediated cellular hyperpolarization and a decrease in agonist-induced receptor internalization. Contrary to previous studies characterizing allosteric modulators at CB1, this study suggests that the mechanism of action is not non-competitive antagonism of signalling, but rather that enhanced binding results in an increased rate of receptor desensitization and reduced internalization, which results in time-dependent modulation of cAMP signalling. The observed effect of the allosteric modulators is therefore dependent on the time frame over which the signalling response occurs. This finding may have important consequences for the potential therapeutic application of these compounds. © 2013 The British Pharmacological Society.

CYP2E1 Metabolism of Styrene Involves Allostery

PubMed Central

Hartman, Jessica H.; Boysen, Gunnar

2012-01-01

We are the first to report allosterism during styrene oxidation by recombinant CYP2E1 and human liver microsomes. At low styrene concentrations, oxidation is inefficient because of weak binding to CYP2E1 (Ks = 830 μM). A second styrene molecule then binds CYP2E1 with higher affinity (Kss = 110 μM) and significantly improves oxidation to achieve a kcat of 6.3 nmol · min−1 · nmol CYP2E1−1. The transition between these metabolic cycles coincides with reported styrene concentrations in blood from exposed workers; thus, this CYP2E1 mechanism may be relevant in vivo. Scaled modeling of the in vitro-positive allosteric mechanism for styrene metabolism to its in vivo clearance led to significant deviations from the traditional model based on Michaelis-Menten kinetics. Low styrene levels were notably much less toxic than generally assumed. We interrogated the allosteric mechanism using the CYP2E1-specific inhibitor and drug 4-methylpyrazole, which we have shown binds two CYP2E1 sites. From the current studies, styrene was a positive allosteric effector on 4-methylpyrazole binding, based on a 10-fold increase in 4-methylpyrazole binding affinity from Ki 0.51 to Ksi 0.043 μM. The inhibitor was a negative allosteric effector on styrene oxidation, because kcat decreased 6-fold to 0.98 nmol · min−1 · nmol CYP2E1−1. Consequently, mixtures of styrene and other molecules can induce allosteric effects on binding and metabolism by CYP2E1 and thus mitigate the efficiency of their metabolism and corresponding effects on human health. Taken together, our elucidation of mechanisms for these allosteric reactions provides a powerful tool for further investigating the complexities of CYP2E1 metabolism of drugs and pollutants. PMID:22807108

Deregulation of Feedback Inhibition of Phosphoenolpyruvate Carboxylase for Improved Lysine Production in Corynebacterium glutamicum

PubMed Central

Chen, Zhen; Bommareddy, Rajesh Reddy; Frank, Doinita; Rappert, Sugima

2014-01-01

Allosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. In this study, the feedback inhibition of Corynebacterium glutamicum PEPC was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. Based on rational protein design, six PEPC mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. Introducing one of the point mutations (N917G) into the ppc gene, encoding PEPC of the lysine-producing strain C. glutamicum LC298, resulted in ∼37% improved lysine production. In vitro enzyme assays and 13C-based metabolic flux analysis showed ca. 20 and 30% increases in the PEPC activity and corresponding flux, respectively, in the mutant strain. Higher demand for NADPH in the mutant strain increased the flux toward pentose phosphate pathway, which increased the supply of NADPH for enhanced lysine production. The present study highlights the importance of allosteric regulation on the flux control of central metabolism. The strategy described here can also be implemented to improve other oxaloacetate-derived products. PMID:24334667

Deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in Corynebacterium glutamicum.

PubMed

Chen, Zhen; Bommareddy, Rajesh Reddy; Frank, Doinita; Rappert, Sugima; Zeng, An-Ping

2014-02-01

Allosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. In this study, the feedback inhibition of Corynebacterium glutamicum PEPC was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. Based on rational protein design, six PEPC mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. Introducing one of the point mutations (N917G) into the ppc gene, encoding PEPC of the lysine-producing strain C. glutamicum LC298, resulted in ∼37% improved lysine production. In vitro enzyme assays and (13)C-based metabolic flux analysis showed ca. 20 and 30% increases in the PEPC activity and corresponding flux, respectively, in the mutant strain. Higher demand for NADPH in the mutant strain increased the flux toward pentose phosphate pathway, which increased the supply of NADPH for enhanced lysine production. The present study highlights the importance of allosteric regulation on the flux control of central metabolism. The strategy described here can also be implemented to improve other oxaloacetate-derived products.

Allosteric control of transcription in GntR family of transcription regulators: A structural overview.

PubMed

Jain, Deepti

2015-07-01

The GntR family of transcription regulators constitutes one of the most abundant family of transcription factors. These modulators are involved in a variety of mechanisms controlling various metabolic processes. GntR family members are typically two domain proteins with a smaller N-terminus domain (NTD) with conserved architecture of winged-helix-turn-helix (wHTH) for DNA binding and a larger C-terminus domain (CTD) or the effector binding domain which is also involved in oligomerization. Interestingly, the CTD shows structural heterogeneity depending upon the type of effector molecule that it binds and displays structural homology to various classes of proteins. Binding of the effector molecule to the CTD brings about a conformational change in the transcription factor such that its affinity for its cognate DNA sequence is altered. This review summarizes the structural information available on the members of GntR family and discusses the common features of the DNA binding and operator recognition within the family. The variation in the allosteric mechanism employed by the members of this family is also discussed. © 2015 International Union of Biochemistry and Molecular Biology.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malaby, Andrew W.; Das, Sanchaita; Chakravarthy, Srinivas

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

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.

Anisotropic energy flow and allosteric ligand binding in albumin

NASA Astrophysics Data System (ADS)

Li, Guifeng; Magana, Donny; Dyer, R. Brian

2014-01-01

Allosteric interactions in proteins generally involve propagation of local structural changes through the protein to a remote site. Anisotropic energy transport is thought to couple the remote sites, but the nature of this process is poorly understood. Here, we report the relationship between energy flow through the structure of bovine serum albumin and allosteric interactions between remote ligand binding sites of the protein. Ultrafast infrared spectroscopy is used to probe the flow of energy through the protein backbone following excitation of a heater dye, a metalloporphyrin or malachite green, bound to different binding sites in the protein. We observe ballistic and anisotropic energy flow through the protein structure following input of thermal energy into the flexible ligand binding sites, without local heating of the rigid helix bundles that connect these sites. This efficient energy transport mechanism enables the allosteric propagation of binding energy through the connecting helix structures.

Anisotropic energy flow and allosteric ligand binding in albumin.

PubMed

Li, Guifeng; Magana, Donny; Dyer, R Brian

2014-01-01

Allosteric interactions in proteins generally involve propagation of local structural changes through the protein to a remote site. Anisotropic energy transport is thought to couple the remote sites, but the nature of this process is poorly understood. Here, we report the relationship between energy flow through the structure of bovine serum albumin and allosteric interactions between remote ligand binding sites of the protein. Ultrafast infrared spectroscopy is used to probe the flow of energy through the protein backbone following excitation of a heater dye, a metalloporphyrin or malachite green, bound to different binding sites in the protein. We observe ballistic and anisotropic energy flow through the protein structure following input of thermal energy into the flexible ligand binding sites, without local heating of the rigid helix bundles that connect these sites. This efficient energy transport mechanism enables the allosteric propagation of binding energy through the connecting helix structures.

Anisotropic energy flow and allosteric ligand binding in albumin

PubMed Central

Li, Guifeng; Magana, Donny; Dyer, R. Brian

2014-01-01

Allosteric interactions in proteins generally involve propagation of local structural changes through the protein to a remote site. Anisotropic energy transport is thought to couple the remote sites, but the nature of this process is poorly understood. Here, we report the relationship between energy flow through the structure of bovine serum albumin and allosteric interactions between remote ligand binding sites of the protein. Ultrafast infrared spectroscopy is used to probe the flow of energy through the protein backbone following excitation of a heater dye, a metalloporphyrin or malachite green, bound to different binding sites in the protein. We observe ballistic and anisotropic energy flow through the protein structure following input of thermal energy into the flexible ligand binding sites, without local heating of the rigid helix bundles that connect these sites. This efficient energy transport mechanism enables the allosteric propagation of binding energy through the connecting helix structures. PMID:24445265

Allostery: an illustrated definition for the ‘second secret of life’

PubMed Central

Fenton, Aron W.

2008-01-01

Although allosteric regulation is the ‘second secret of life’, the molecular mechanisms that give rise to allostery currently elude understanding. In my opinion, experimental progress is hampered by a commonly used but misleading definition of allostery as protein structural changes that are elicited by the binding of a single ligand. Allostery is more strictly defined in functional terms as a comparison of how one ligand binds in the absence, versus the presence, of a second ligand. Therefore, as each of the two binding events involves two protein complexes, a study of allostery must consider four complexes and not just two. Such a comparison can distinguish allosteric from non-allosteric protein changes, the importance of which is frequently overlooked. When a study of all four complexes is not feasible, an alternative, albeit limited, strategy can identify subsets of allosteric-specific changes. PMID:18706817

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

NASA Astrophysics Data System (ADS)

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

2017-03-01

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

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

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

PubMed

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

2016-02-26

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

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

Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor

PubMed Central

Spurny, Radovan; Debaveye, Sarah; Farinha, Ana; Veys, Ken; Vos, Ann M.; Gossas, Thomas; Atack, John; Bertrand, Sonia; Bertrand, Daniel; Danielson, U. Helena; Tresadern, Gary; Ulens, Chris

2015-01-01

The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential. PMID:25918415

Harnessing Proteasome Dynamics and Allostery in Drug Design

PubMed Central

Osmulski, Pawel A.

2014-01-01

Abstract Significance: The proteasome is the essential protease that is responsible for regulated cleavage of the bulk of intracellular proteins. Its central role in cellular physiology has been exploited in therapies against aggressive cancers where proteasome-specific competitive inhibitors that block proteasome active centers are very effectively used. However, drugs regulating this essential protease are likely to have broader clinical usefulness. The non-catalytic sites of the proteasome emerge as an attractive alternative target in search of highly specific and diverse proteasome regulators. Recent Advances: Crystallographic models of the proteasome leave the false impression of fixed structures with minimal molecular dynamics lacking long-distance allosteric signaling. However, accumulating biochemical and structural observations strongly support the notion that the proteasome is regulated by precise allosteric interactions arising from protein dynamics, encouraging the active search for allosteric regulators. Here, we discuss properties of several promising compounds that affect substrate gating and processing in antechambers, and interactions of the catalytic core with regulatory proteins. Critical Issues: Given the structural complexity of proteasome assemblies, it is a painstaking process to better understand their allosteric regulation and molecular dynamics. Here, we discuss the challenges and achievements in this field. We place special emphasis on the role of atomic force microscopy imaging in probing the allostery and dynamics of the proteasome, and in dissecting the mechanisms involving small-molecule allosteric regulators. Future Directions: New small-molecule allosteric regulators may become a next generation of drugs targeting the proteasome, which is critical to the development of new therapies in cancers and other diseases. Antioxid. Redox Signal. 21, 2286–2301. PMID:24410482

Behavioral Effects of the Benzodiazepine-Positive Allosteric Modulator SH-053-2’F-S-CH3 in an Immune-Mediated Neurodevelopmental Disruption Model

PubMed Central

Richetto, Juliet; Labouesse, Marie A.; Poe, Michael M.; Cook, James M.; Grace, Anthony A.; Riva, Marco A.

2015-01-01

Background: Impaired γ-aminobutyric acid (GABA) signaling may contribute to the emergence of cognitive deficits and subcortical dopaminergic hyperactivity in patients with schizophrenia and related psychotic disorders. Against this background, it has been proposed that pharmacological interventions targeting GABAergic dysfunctions may prove useful in correcting such cognitive impairments and dopaminergic imbalances. Methods: Here, we explored possible beneficial effects of the benzodiazepine-positive allosteric modulator SH-053-2’F-S-CH3, with partial selectivity at the α2, α3, and α5 subunits of the GABAA receptor in an immune-mediated neurodevelopmental disruption model. The model is based on prenatal administration of the viral mimetic polyriboinosinic-polyribocytidilic acid [poly(I:C)] in mice, which is known to capture various GABAergic, dopamine-related, and cognitive abnormalities implicated in schizophrenia and related disorders. Results: Real-time polymerase chain reaction analyses confirmed the expected alterations in GABAA receptor α subunit gene expression in the medial prefrontal cortices and ventral hippocampi of adult poly(I:C) offspring relative to control offspring. Systemic administration of SH-053-2’F-S-CH3 failed to normalize the poly(I:C)-induced deficits in working memory and social interaction, but instead impaired performance in these cognitive and behavioral domains both in control and poly(I:C) offspring. In contrast, SH-053-2’F-S-CH3 was highly effective in mitigating the poly(I:C)-induced amphetamine hypersensitivity phenotype without causing side effects in control offspring. Conclusions: Our preclinical data suggest that benzodiazepine-like positive allosteric modulators with activity at the α2, α3, and α5 subunits of the GABAA receptor may be particularly useful in correcting pathological overactivity of the dopaminergic system, but they may be ineffective in targeting multiple pathological domains that involve the co-existence of psychotic, social, and cognitive dysfunctions. PMID:25636893

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

PubMed Central

Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J.; Smithgall, Thomas E.

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system. PMID:26222440

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

PubMed

Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J; Smithgall, Thomas E

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system.

Enhanced heterotetrameric assembly of potato ADP-glucose pyrophosphorylase using reverse genetics.

PubMed

Seferoglu, A Bengisu; Koper, Kaan; Can, F Betul; Cevahir, Gul; Kavakli, I Halil

2014-08-01

ADP-glucose pyrophosphorylase (AGPase) is a key allosteric enzyme in plant starch biosynthesis. Plant AGPase is a heterotetrameric enzyme that consists of large (LS) and small subunits (SS), which are encoded by two different genes. Computational and experimental studies have revealed that the heterotetrameric assembly of AGPase is thermodynamically weak. Modeling studies followed by the mutagenesis of the LS of the potato AGPase identified a heterotetramer-deficient mutant, LS(R88A). To enhance heterotetrameric assembly, LS(R88A) cDNA was subjected to error-prone PCR, and second-site revertants were identified according to their ability to restore glycogen accumulation, as assessed with iodine staining. Selected mutations were introduced into the wild-type (WT) LS and co-expressed with the WT SS in Escherichia coli glgC(-). The biochemical characterization of revertants revealed that LS(I90V)SS(WT), LS(Y378C)SS(WT) and LS(D410G)SS(WT) mutants displayed enhanced heterotetrameric assembly with the WT SS. Among these mutants, LS(Y378C)SS(WT) AGPase displayed increased heat stability compared with the WT enzyme. Kinetic characterization of the mutants indicated that the LS(I90V)SS(WT) and LS(Y378C)SS(WT) AGPases have comparable allosteric and kinetic properties. However, the LS(D410G)SS(WT) mutant exhibited altered allosteric properties of being less responsive and more sensitive to 3-phosphoglyceric acid activation and inorganic phosphate inhibition. This study not only enhances our understanding of the interaction between the SS and the LS of AGPase but also enables protein engineering to obtain enhanced assembled heat-stable variants of AGPase, which can be used for the improvement of plant yields. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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)

Allosteric mechanism of water channel gating by Ca2+–calmodulin

PubMed Central

Reichow, Steve L.; Clemens, Daniel M.; Freites, J. Alfredo; Németh-Cahalan, Karin L.; Heyden, Matthias; Tobias, Douglas J.; Hall, James E.; Gonen, Tamir

2013-01-01

Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, our understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudo-atomic structure of full-length mammalian aquaporin-0 (AQP0, Bos Taurus) in complex with CaM using electron microscopy to understand how this signaling protein modulates water channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits. PMID:23893133

Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to “Biased Opioids”?

PubMed Central

Turke, Miah; Subhramanyam, Udaya K. Tiruttani; Churchill, Beth; Labahn, Joerg

2018-01-01

Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists. PMID:29342106

K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions

PubMed Central

Ostrem, Jonathan M.; Peters, Ulf; Sos, Martin L.; Wells, James A.; Shokat, Kevan M.

2014-01-01

Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies1–3. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP4 and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis5,6. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP7 and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner. PMID:24256730

Remote Control by Inter-Enzyme Allostery: A Novel Paradigm for Regulation of the Shikimate Pathway.

PubMed

Munack, Steffi; Roderer, Kathrin; Ökvist, Mats; Kamarauskaite, Jurate; Sasso, Severin; van Eerde, André; Kast, Peter; Krengel, Ute

2016-03-27

DAHP synthase and chorismate mutase catalyze key steps in the shikimate biosynthetic pathway en route to aromatic amino acids. In Mycobacterium tuberculosis, chorismate mutase (MtCM; Rv0948c), located at the branch point toward phenylalanine and tyrosine, has poor activity on its own. However, it is efficiently activated by the first enzyme of the pathway, DAHP synthase (MtDS; Rv2178c), through formation of a non-covalent MtCM-MtDS complex. Here, we show how MtDS serves as an allosteric platform for feedback regulation of both enzymes, using X-ray crystallography, small-angle X-ray scattering, size-exclusion chromatography, and multi-angle light scattering. Crystal structures of the fully inhibited MtDS and the allosterically down-regulated MtCM-MtDS complex, solved at 2.8 and 2.7Å, respectively, reveal how effector binding at the internal MtDS subunit interfaces regulates the activity of MtDS and MtCM. While binding of all three metabolic end products to MtDS shuts down the entire pathway, the binding of phenylalanine jointly with tyrosine releases MtCM from the MtCM-MtDS complex, hence suppressing MtCM activation by 'inter-enzyme allostery'. This elegant regulatory principle, invoking a transient allosteric enzyme interaction, seems to be driven by dynamics and is likely a general strategy used by nature. Copyright © 2016 Elsevier Ltd. All rights reserved.

mGluR4 positive allosteric modulators with potential for the treatment of Parkinson's disease: WO09010455.

PubMed

East, Stephen P; Gerlach, Kai

2010-03-01

Stimulation of the metabotropic glutamate receptor 4 (mGluR4) represents a promising new approach to the symptomatic treatment of the neurodegenerative disorder Parkinson's disease (PD). Preclinical models using both agonists and positive allosteric modulators of mGluR4 have demonstrated the potential for this receptor for the treatment of PD. The present article evaluates a recent patent filed by Addex Pharma S.A. claiming a novel series of mGluR4 positive allosteric modulators. Many of the examples disclosed are active at EC(50)'s < 500 nM.

Sequential allosteric mechanism of ATP hydrolysis by the CCT/TRiC chaperone is revealed through Arrhenius analysis

PubMed Central

Gruber, Ranit; Levitt, Michael; Horovitz, Amnon

2017-01-01

Knowing the mechanism of allosteric switching is important for understanding how molecular machines work. The CCT/TRiC chaperonin nanomachine undergoes ATP-driven conformational changes that are crucial for its folding function. Here, we demonstrate that insight into its allosteric mechanism of ATP hydrolysis can be achieved by Arrhenius analysis. Our results show that ATP hydrolysis triggers sequential ‟conformational waves.” They also suggest that these waves start from subunits CCT6 and CCT8 (or CCT3 and CCT6) and proceed clockwise and counterclockwise, respectively. PMID:28461478

Sequential allosteric mechanism of ATP hydrolysis by the CCT/TRiC chaperone is revealed through Arrhenius analysis.

PubMed

Gruber, Ranit; Levitt, Michael; Horovitz, Amnon

2017-05-16

Knowing the mechanism of allosteric switching is important for understanding how molecular machines work. The CCT/TRiC chaperonin nanomachine undergoes ATP-driven conformational changes that are crucial for its folding function. Here, we demonstrate that insight into its allosteric mechanism of ATP hydrolysis can be achieved by Arrhenius analysis. Our results show that ATP hydrolysis triggers sequential ‟conformational waves." They also suggest that these waves start from subunits CCT6 and CCT8 (or CCT3 and CCT6) and proceed clockwise and counterclockwise, respectively.

Allosteric regulation of epigenetic modifying enzymes.

PubMed

Zucconi, Beth E; Cole, Philip A

2017-08-01

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

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

PubMed Central

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

2014-01-01

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

Structures of pyruvate kinases display evolutionarily divergent allosteric strategies

PubMed Central

Morgan, Hugh P.; Zhong, Wenhe; McNae, Iain W.; Michels, Paul A. M.; Fothergill-Gilmore, Linda A.; Walkinshaw, Malcolm D.

2014-01-01

The transition between the inactive T-state (apoenzyme) and active R-state (effector bound enzyme) of Trypanosoma cruzi pyruvate kinase (PYK) is accompanied by a symmetrical 8° rigid body rocking motion of the A- and C-domain cores in each of the four subunits, coupled with the formation of additional salt bridges across two of the four subunit interfaces. These salt bridges provide increased tetramer stability correlated with an enhanced specificity constant (kcat/S0.5). A detailed kinetic and structural comparison between the potential drug target PYKs from the pathogenic protists T. cruzi, T. brucei and Leishmania mexicana shows that their allosteric mechanism is conserved. By contrast, a structural comparison of trypanosomatid PYKs with the evolutionarily divergent PYKs of humans and of bacteria shows that they have adopted different allosteric strategies. The underlying principle in each case is to maximize (kcat/S0.5) by stabilizing and rigidifying the tetramer in an active R-state conformation. However, bacterial and mammalian PYKs have evolved alternative ways of locking the tetramers together. In contrast to the divergent allosteric mechanisms, the PYK active sites are highly conserved across species. Selective disruption of the varied allosteric mechanisms may therefore provide a useful approach for the design of species-specific inhibitors. PMID:26064527

Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase*

PubMed Central

Zhang, Shengnan; Fitzpatrick, Paul F.

2016-01-01

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. PMID:26823465

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

Molecular Dynamics Simulations and Dynamic Network Analysis Reveal the Allosteric Unbinding of Monobody to H-Ras Triggered by R135K Mutation.

PubMed

Ni, Duan; Song, Kun; Zhang, Jian; Lu, Shaoyong

2017-10-26

Ras proteins, as small GTPases, mediate cell proliferation, survival and differentiation. Ras mutations have been associated with a broad spectrum of human cancers and thus targeting Ras represents a potential way forward for cancer therapy. A recently reported monobody NS1 allosterically disrupts the Ras-mediated signaling pathway, but its efficacy is reduced by R135K mutation in H-Ras. However, the detailed mechanism is unresolved. Here, using molecular dynamics (MD) simulations and dynamic network analysis, we explored the molecular mechanism for the unbinding of NS1 to H-Ras and shed light on the underlying allosteric network in H-Ras. MD simulations revealed that the overall structures of the two complexes did not change significantly, but the H-Ras-NS1 interface underwent significant conformational alteration in the mutant Binding free energy analysis showed that NS1 binding was unfavored after R135K mutation, which resulted in the unfavorable binding of NS1. Furthermore, the critical residues on H-Ras responsible for the loss of binding of NS1 were identified. Importantly, the allosteric networks for these important residues were revealed, which yielded a novel insight into the allosteric regulatory mechanism of H-Ras.

Molecular Dynamics Simulations and Dynamic Network Analysis Reveal the Allosteric Unbinding of Monobody to H-Ras Triggered by R135K Mutation

PubMed Central

Song, Kun; Zhang, Jian; Lu, Shaoyong

2017-01-01

Ras proteins, as small GTPases, mediate cell proliferation, survival and differentiation. Ras mutations have been associated with a broad spectrum of human cancers and thus targeting Ras represents a potential way forward for cancer therapy. A recently reported monobody NS1 allosterically disrupts the Ras-mediated signaling pathway, but its efficacy is reduced by R135K mutation in H-Ras. However, the detailed mechanism is unresolved. Here, using molecular dynamics (MD) simulations and dynamic network analysis, we explored the molecular mechanism for the unbinding of NS1 to H-Ras and shed light on the underlying allosteric network in H-Ras. MD simulations revealed that the overall structures of the two complexes did not change significantly, but the H-Ras–NS1 interface underwent significant conformational alteration in the mutant Binding free energy analysis showed that NS1 binding was unfavored after R135K mutation, which resulted in the unfavorable binding of NS1. Furthermore, the critical residues on H-Ras responsible for the loss of binding of NS1 were identified. Importantly, the allosteric networks for these important residues were revealed, which yielded a novel insight into the allosteric regulatory mechanism of H-Ras. PMID:29072601

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

PubMed

Joseph, Thomas T; Osman, Roman

2012-01-01

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

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 effects of a large number of internal allosteric pathways. PMID:23028290

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 interaction networks that can inhibit or promote progression of the ATPase cycle and thus control the recruitment of client proteins. PMID:24466147

Allosteric regulation of the Hsp90 dynamics and stability by client recruiter cochaperones: protein structure network modeling.

PubMed

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 interaction networks that can inhibit or promote progression of the ATPase cycle and thus control the recruitment of client proteins.

Rationalizing context-dependent performance of dynamic RNA regulatory devices.

PubMed

Kent, Ross; Halliwell, Samantha; Young, Kate; Swainston, Neil; Dixon, Neil

2018-06-21

The ability of RNA to sense, regulate and store information is an attractive attribute for a variety of functional applications including the development of regulatory control devices for synthetic biology. RNA folding and function is known to be highly context sensitive, which limits the modularity and reuse of RNA regulatory devices to control different heterologous sequences and genes. We explored the cause and effect of sequence context sensitivity for translational ON riboswitches located in the 5' UTR, by constructing and screening a library of N-terminal synonymous codon variants. By altering the N-terminal codon usage we were able to obtain RNA devices with a broad range of functional performance properties (ON, OFF, fold-change). Linear regression and calculated metrics were used to rationalize the major determining features leading to optimal riboswitch performance, and to identify multiple interactions between the explanatory metrics. Finally, partial least squared (PLS) analysis was employed in order to understand the metrics and their respective effect on performance. This PLS model was shown to provide good explanation of our library. This study provides a novel multi-variant analysis framework by which to rationalize the codon context performance of allosteric RNA-devices. The framework will also serve as a platform for future riboswitch context engineering endeavors.

New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase

NASA Astrophysics Data System (ADS)

Han, Xinya; Huang, Yunyuan; Zhang, Rui; Xiao, San; Zhu, Shuaihuan; Qin, Nian; Hong, Zongqin; Wei, Lin; Feng, Jiangtao; Ren, Yanliang; Feng, Lingling; Wan, Jian

2016-08-01

Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2‧,3‧-O-(2,4,6-trinitrophenyl)adenosine 5‧-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase.

New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase.

PubMed

Han, Xinya; Huang, Yunyuan; Zhang, Rui; Xiao, San; Zhu, Shuaihuan; Qin, Nian; Hong, Zongqin; Wei, Lin; Feng, Jiangtao; Ren, Yanliang; Feng, Lingling; Wan, Jian

2016-08-05

Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

Cressman, William J; Beckett, Dorothy

2016-01-19

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

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.

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2013-01-18

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

The molecular mechanism of flop-selectivity and subsite recognition for an AMPA receptor allosteric modulator: Structures of GluA2 and GluA3 complexed with PEPA

PubMed Central

Ahmed, Ahmed H.; Ptak, Christopher P.; Oswald, Robert E.

2011-01-01

Glutamate receptors are important potential drug targets for cognitive enhancement and the treatment of schizophrenia in part because they are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system. One approach to the application of therapeutic agents to the AMPA subtype of glutamate receptors is the use of allosteric modulators, which promote dimerization by binding to a dimer interface thereby reducing desensitization and deactivation. AMPA receptors exist in two alternatively spliced variants (flip and flop) that differ in desensitization and receptor activation profiles. Most of the structural information on modulators of the AMPA receptor target the flip subtype. We report here the crystal structure of the flop-selective allosteric modulator, PEPA, bound to the binding domains of the GluA2 and GluA3 flop isoforms of AMPA receptors. Specific hydrogen bonding patterns can explain the preference for the flop isoform. This includes a bidentate hydrogen bonding pattern between PEPA and N754 of the flop isoforms of GluA2 and GluA3 (the corresponding position in the flip isoform is S754). Comparison with other allosteric modulators provides a framework for the development of new allosteric modulators with preferences for either the flip or flop isoforms. In addition to interactions with N/S754, specific interactions of the sulfonamide with conserved residues in the binding site are characteristics of a number of allosteric modulators. These, in combination, with variable interactions with five subsites on the binding surface lead to different stoichiometries, orientations within the binding pockets, and functional outcomes. PMID:20199107

Molecular Dynamics Simulation of the Allosteric Regulation of eIF4A Protein from the Open to Closed State, Induced by ATP and RNA Substrates

PubMed Central

Meng, Hongqing; Li, Chaoqun; Wang, Yan; Chen, Guangju

2014-01-01

Background Eukaryotic initiation factor 4A (eIF4A) plays a key role in the process of protein translation initiation by facilitating the melting of the 5′ proximal secondary structure of eukaryotic mRNA for ribosomal subunit attachment. It was experimentally postulated that the closed conformation of the eIF4A protein bound by the ATP and RNA substrates is coupled to RNA duplex unwinding to promote protein translation initiation, rather than an open conformation in the absence of ATP and RNA substrates. However, the allosteric process of eIF4A from the open to closed state induced by the ATP and RNA substrates are not yet fully understood. Methodology In the present work, we constructed a series of diplex and ternary models of the eIF4A protein bound by the ATP and RNA substrates to carry out molecular dynamics simulations, free energy calculations and conformation analysis and explore the allosteric properties of eIF4A. Results The results showed that the eIF4A protein completes the conformational transition from the open to closed state via two allosteric processes of ATP binding followed by RNA and vice versa. Based on cooperative allosteric network analysis, the ATP binding to the eIF4A protein mainly caused the relative rotation of two domains, while the RNA binding caused the proximity of two domains via the migration of RNA bases in the presence of ATP. The cooperative binding of ATP and RNA for the eIF4A protein plays a key role in the allosteric transition. PMID:24465900

Allosterism in human complement component 5a ((h)C5a): a damper of C5a receptor (C5aR) signaling.

PubMed

Rana, Soumendra; Sahoo, Amita Rani; Majhi, Bharat Kumar

2016-06-01

The phenomena of allosterism continues to advance the field of drug discovery, by illuminating gainful insights for many key processes, related to the structure-function relationships in proteins and enzymes, including the transmembrane G-protein coupled receptors (GPCRs), both in normal as well as in the disease states. However, allosterism is completely unexplored in the native protein ligands, especially when a small covalent change significantly modulates the pharmacology of the protein ligands toward the signaling axes of the GPCRs. One such example is the human C5a ((h)C5a), the potent cationic anaphylatoxin that engages C5aR and C5L2 to elicit numerous immunological and non-immunological responses in humans. From the recently available structure-function data, it is clear that unlike the mouse C5a ((m)C5a), the (h)C5a displays conformational heterogeneity. However, the molecular basis of such conformational heterogeneity, otherwise allosterism in (h)C5a and its precise contribution toward the overall C5aR signaling is not known. This study attempts to decipher the functional role of allosterism in (h)C5a, by exploring the inherent conformational dynamics in (m)C5a, (h)C5a and in its point mutants, including the proteolytic mutant des-Arg(74)-(h)C5a. Prima facie, the comparative molecular dynamics study, over total 500 ns, identifies Arg(74)-Tyr(23) and Arg(37)-Phe(51) "cation-π" pairs as the molecular "allosteric switches" on (h)C5a that potentially functions as a damper of C5aR signaling.

Consequences of Energetic Frustration on the Ligand-Coupled Folding/Dimerization Dynamics of Allosteric Protein S100A12.

PubMed

Ren, Weitong; Li, Wenfei; Wang, Jun; Zhang, Jian; Wang, Wei

2017-10-26

Allosteric proteins are featured by energetic degeneracy of two (or more) functionally relevant conformations, therefore their energy landscapes are often locally frustrated. How such frustration affects the protein folding/binding dynamics is not well understood. Here, by using molecular simulations we study the consequences of local frustration in the dimerization dynamics of allosteric proteins based on a homodimer protein S100A12. Despite of the structural symmetry of the two EF-hand motifs in the three-dimensional structures, the S100A12 homodimer shows allosteric behaviors and local frustration only in half of its structural elements, i.e., the C-terminal EF-hand. We showed that such spatially asymmetric location of frustration leads to asymmetric dimerization pathways, in which the dimerization is dominantly initiated by the interchain binding of the minimally frustrated N-terminal EF-hands, achieving optimal balance between the requirements of rapid conformational switching and interchain assembling to the energy landscapes. We also showed that the local frustration, as represented by the double-basin topography of the energy landscape, gives rise to multiple cross-linked dimerization pathways, in which the dimerization is coupled with the allosteric motions of the C-terminal EF-hands. Binding of metal ions tends to reshape the energy landscape and modulate the dimerization pathways. In addition, by employing the frustratometer method, we showed that the highly frustrated residue-pairs in the C-terminal EF-hand are partially unfolded during the conformational transitions of the native homodimer, leading to lowing of free energy barrier. Our results revealed tight interplay between the local frustration of the energy landscape and the dimerization dynamics for allosteric proteins.

PF-06827443 Displays Robust Allosteric Agonist and Positive Allosteric Modulator Activity in High Receptor Reserve and Native Systems.

PubMed

Moran, Sean P; Cho, Hyekyung P; Maksymetz, James; Remke, Daniel H; Hanson, Ryan M; Niswender, Colleen M; Lindsley, Craig W; Rook, Jerri M; Conn, P Jeffrey

2018-04-25

Positive allosteric modulators (PAMs) of the M 1 subtype of muscarinic acetylcholine receptor have attracted intense interest as an exciting new approach for improving the cognitive deficits in schizophrenia and Alzheimer's disease. Recent evidence suggests that the presence of intrinsic agonist activity of some M 1 PAMs may reduce efficacy and contribute to adverse effect liability. However, the M 1 PAM PF-06827443 was reported to have only weak agonist activity at human M 1 receptors but produced M 1 -dependent adverse effects. We now report that PF-06827443 is an allosteric agonist in cell lines expressing rat, dog, and human M 1 and use of inducible cell lines shows that agonist activity of PF-06827443 is dependent on receptor reserve. Furthermore, PF-06827443 is an agonist in native tissue preparations and induces behavioral convulsions in mice similar to other ago-PAMs. These findings suggest that PF-06827443 is a robust ago-PAM, independent of species, in cell lines and native systems.

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

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

Identifiability, reducibility, and adaptability in allosteric macromolecules.

PubMed

Bohner, Gergő; Venkataraman, Gaurav

2017-05-01

The ability of macromolecules to transduce stimulus information at one site into conformational changes at a distant site, termed "allostery," is vital for cellular signaling. Here, we propose a link between the sensitivity of allosteric macromolecules to their underlying biophysical parameters, the interrelationships between these parameters, and macromolecular adaptability. We demonstrate that the parameters of a canonical model of the mSlo large-conductance Ca 2+ -activated K + (BK) ion channel are non-identifiable with respect to the equilibrium open probability-voltage relationship, a common functional assay. We construct a reduced model with emergent parameters that are identifiable and expressed as combinations of the original mechanistic parameters. These emergent parameters indicate which coordinated changes in mechanistic parameters can leave assay output unchanged. We predict that these coordinated changes are used by allosteric macromolecules to adapt, and we demonstrate how this prediction can be tested experimentally. We show that these predicted parameter compensations are used in the first reported allosteric phenomena: the Bohr effect, by which hemoglobin adapts to varying pH. © 2017 Bohner and Venkataraman.

Identifiability, reducibility, and adaptability in allosteric macromolecules

PubMed Central

Bohner, Gergő

2017-01-01

The ability of macromolecules to transduce stimulus information at one site into conformational changes at a distant site, termed “allostery,” is vital for cellular signaling. Here, we propose a link between the sensitivity of allosteric macromolecules to their underlying biophysical parameters, the interrelationships between these parameters, and macromolecular adaptability. We demonstrate that the parameters of a canonical model of the mSlo large-conductance Ca2+-activated K+ (BK) ion channel are non-identifiable with respect to the equilibrium open probability-voltage relationship, a common functional assay. We construct a reduced model with emergent parameters that are identifiable and expressed as combinations of the original mechanistic parameters. These emergent parameters indicate which coordinated changes in mechanistic parameters can leave assay output unchanged. We predict that these coordinated changes are used by allosteric macromolecules to adapt, and we demonstrate how this prediction can be tested experimentally. We show that these predicted parameter compensations are used in the first reported allosteric phenomena: the Bohr effect, by which hemoglobin adapts to varying pH. PMID:28416647

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

PubMed

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

2018-02-05

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

Time-resolved observation of protein allosteric communication

PubMed Central

Buchenberg, Sebastian; Sittel, Florian; Stock, Gerhard

2017-01-01

Allostery represents a fundamental mechanism of biological regulation that is mediated via long-range communication between distant protein sites. Although little is known about the underlying dynamical process, recent time-resolved infrared spectroscopy experiments on a photoswitchable PDZ domain (PDZ2S) have indicated that the allosteric transition occurs on multiple timescales. Here, using extensive nonequilibrium molecular dynamics simulations, a time-dependent picture of the allosteric communication in PDZ2S is developed. The simulations reveal that allostery amounts to the propagation of structural and dynamical changes that are genuinely nonlinear and can occur in a nonlocal fashion. A dynamic network model is constructed that illustrates the hierarchy and exceeding structural heterogeneity of the process. In compelling agreement with experiment, three physically distinct phases of the time evolution are identified, describing elastic response (≲0.1 ns), inelastic reorganization (∼100 ns), and structural relaxation (≳1μs). Issues such as the similarity to downhill folding as well as the interpretation of allosteric pathways are discussed. PMID:28760989

Characterization of inhibitory anti-insulin-like growth factor receptor antibodies with different epitope specificity and ligand-blocking properties: implications for mechanism of action in vivo.

PubMed

Doern, Adam; Cao, Xianjun; Sereno, Arlene; Reyes, Christopher L; Altshuler, Angelina; Huang, Flora; Hession, Cathy; Flavier, Albert; Favis, Michael; Tran, Hon; Ailor, Eric; Levesque, Melissa; Murphy, Tracey; Berquist, Lisa; Tamraz, Susan; Snipas, Tracey; Garber, Ellen; Shestowsky, William S; Rennard, Rachel; Graff, Christilyn P; Wu, Xiufeng; Snyder, William; Cole, Lindsay; Gregson, David; Shields, Michael; Ho, Steffan N; Reff, Mitchell E; Glaser, Scott M; Dong, Jianying; Demarest, Stephen J; Hariharan, Kandasamy

2009-04-10

Therapeutic antibodies directed against the type 1 insulin-like growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting down-stream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 microm) to investigate allosteric versus competitive blocking mechanisms. Four distinct inhibitory classes were found as follows: 1) allosteric IGF-1 blockers, 2) allosteric IGF-2 blockers, 3) allosteric IGF-1 and IGF-2 blockers, and 4) competitive IGF-1 and IGF-2 blockers. The epitopes of representative antibodies from each of these classes were mapped using a purified IGF-1R library containing 64 mutations. Most of these antibodies bound overlapping surfaces on the cysteine-rich repeat and L2 domains. One class of allosteric IGF-1 and IGF-2 blocker was identified that bound a separate epitope on the outer surface of the FnIII-1 domain. Using various biophysical techniques, we show that the dual IGF blockers inhibit ligand binding using a spectrum of mechanisms ranging from highly allosteric to purely competitive. Binding of IGF-1 or the inhibitory antibodies was associated with conformational changes in IGF-1R, linked to the ordering of dynamic or unstructured regions of the receptor. These results suggest IGF-1R uses disorder/order within its polypeptide sequence to regulate its activity. Interestingly, the activity of representative allosteric and competitive inhibitors on H322M tumor cell growth in vitro was reflective of their individual ligand-blocking properties. Many of the antibodies in the clinic likely adopt one of the inhibitory mechanisms described here, and the outcome of future clinical studies may reveal whether a particular inhibitory mechanism leads to optimal clinical efficacy.

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-activated STAT5 proteins. Our theoretical prediction based on results obtained with MONETA was validated for KIT by in vitro experiments. MONETA is a versatile analytical and visualization tool entirely devoted to the understanding of the functioning/malfunctioning of allosteric regulation in proteins - a crucial basis to guide the discovery of next-generation allosteric drugs.

Bisquaternary dimers of strychnine and brucine. A new class of potent enhancers of antagonist binding to muscarinic M2 receptors.

PubMed

Zlotos, D P; Buller, S; Holzgrabe, U; Mohr, K

2003-06-12

Bisquaternary dimers of strychnine and brucine were synthesized and their allosteric effect on muscarinic acetylcholine M(2) receptors was examined. The compounds retarded the dissociation of the antagonist [(3)H]N-methylscopolamine ([(3)H]NMS) from porcine cardiac cholinoceptors. This action indicated ternary complex formation. All compounds exhibited higher affinity to the allosteric site of [(3)H]NMS-occupied M(2) receptors than the monomeric strychnine and brucine, while the positive cooperativity with NMS was fully maintained. SAR studies revealed the unchanged strychnine ring as an important structural feature for high allosteric potency.

Insights into the Impact of Linker Flexibility and Fragment Ionization on the Design of CK2 Allosteric Inhibitors: Comparative Molecular Dynamics Simulation Studies.

PubMed

Zhou, Yue; Zhang, Na; Qi, Xiaoqian; Tang, Shan; Sun, Guohui; Zhao, Lijiao; Zhong, Rugang; Peng, Yongzhen

2018-01-01

Protein kinase is a novel therapeutic target for human diseases. The off-target and side effects of ATP-competitive inhibitors preclude them from the clinically relevant drugs. The compounds targeting the druggable allosteric sites outside the highly conversed ATP binding pocket have been identified as promising alternatives to overcome current barriers of ATP-competitive inhibitors. By simultaneously interacting with the αD region (new allosteric site) and sub-ATP binding pocket, the attractive compound CAM4066 was named as allosteric inhibitor of CK2α. It has been demonstrated that the rigid linker and non-ionizable substituted fragment resulted in significant decreased inhibitory activities of compounds. The molecular dynamics simulations and energy analysis revealed that the appropriate coupling between the linker and pharmacophore fragments were essential for binding of CAM4066 with CK2α. The lower flexible linker of compound 21 lost the capability of coupling fragments A and B to αD region and positive area, respectively, whereas the methyl benzoate of fragment B induced the re-orientated Pre-CAM4066 with the inappropriate polar interactions. Most importantly, the match between the optimized linker and pharmacophore fragments is the challenging work of fragment-linking based drug design. These results provide rational clues to further structural modification and development of highly potent allosteric inhibitors of CK2.

Metabotropic glutamate receptor 4 (mGlu4)-positive allosteric modulators for the treatment of Parkinson's disease: historical perspective and review of the patent literature.

PubMed

Lindsley, Craig W; Hopkins, Corey R

2012-05-01

Metabotropic glutamate receptor 4 (mGlu(4)) is a group III GPCR and has been demonstrated to play a major role in a number of therapeutic areas within the CNS. As the orthosteric site of all glutamate receptors is highly conserved, modulating mGlu(4) via allosteric modulation has emerged as a very attractive mode-of-action and has been validated preclinically in a number of animal models for Parkinson's disease, anxiety, pain, and neuroinflammation. In this review, the patent literature for mGlu(4)-positive allosteric modulators over the past 4 years will be provided. Patents from all companies are discussed and an overview of the chemical matter and relevant biological properties will be given. Although there has yet to be an mGlu(4)-positive allosteric modulator progressed into clinical trials, there is a wealth of preclinical data from the primary literature that shows the promise of this emerging target. A number of academic and industry laboratories have recently published exciting patent data covering a multitude of chemical matter. Positive allosteric modulation of mGlu(4) remains one of the more attractive non-dopaminergic therapies for Parkinson's disease, as well as emerging data for other indications such as pain, neuroinflammation, schizophrenia and diabetes, which could potentially make mGlu(4) a significant therapeutic target going forward.

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

Insights into the Impact of Linker Flexibility and Fragment Ionization on the Design of CK2 Allosteric Inhibitors: Comparative Molecular Dynamics Simulation Studies

PubMed Central

Zhou, Yue; Zhang, Na; Qi, Xiaoqian; Tang, Shan; Zhao, Lijiao; Zhong, Rugang; Peng, Yongzhen

2018-01-01

Protein kinase is a novel therapeutic target for human diseases. The off-target and side effects of ATP-competitive inhibitors preclude them from the clinically relevant drugs. The compounds targeting the druggable allosteric sites outside the highly conversed ATP binding pocket have been identified as promising alternatives to overcome current barriers of ATP-competitive inhibitors. By simultaneously interacting with the αD region (new allosteric site) and sub-ATP binding pocket, the attractive compound CAM4066 was named as allosteric inhibitor of CK2α. It has been demonstrated that the rigid linker and non-ionizable substituted fragment resulted in significant decreased inhibitory activities of compounds. The molecular dynamics simulations and energy analysis revealed that the appropriate coupling between the linker and pharmacophore fragments were essential for binding of CAM4066 with CK2α. The lower flexible linker of compound 21 lost the capability of coupling fragments A and B to αD region and positive area, respectively, whereas the methyl benzoate of fragment B induced the re-orientated Pre-CAM4066 with the inappropriate polar interactions. Most importantly, the match between the optimized linker and pharmacophore fragments is the challenging work of fragment-linking based drug design. These results provide rational clues to further structural modification and development of highly potent allosteric inhibitors of CK2. PMID:29301250

Allosteric Effect of Adenosine Triphosphate on Peptide Recognition by 3'5'-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits.

PubMed

Kivi, Rait; Solovjova, Karina; Haljasorg, Tõiv; Arukuusk, Piret; Järv, Jaak

2016-12-01

The allosteric influence of adenosine triphosphate (ATP) on the binding effectiveness of a series of peptide inhibitors with the catalytic subunit of 3'5'-cyclic adenosine monophosphate dependent protein kinase was investigated, and the dependence of this effect on peptide structure was analyzed. The allosteric effect was calculated as ratio of peptide binding effectiveness with the enzyme-ATP complex and with the free enzyme, quantified by the competitive inhibition of the enzyme in the presence of ATP excess, and by the enzyme-peptide complex denaturation assay, respectively It was found that the principle "better binding-stronger allostery" holds for interactions of the studied peptides with the enzyme, indicating that allostery and peptide binding with the free enzyme are governed by the same specificity pattern. This means that the allosteric regulation does not include new ligand-protein interactions, but changes the intensity (strength) of the interatomic forces that govern the complex formation in the case of each individual ligand. We propose that the allosteric regulation can be explained by the alteration of the intrinsic dynamics of the protein by ligand binding, and that this phenomenon, in turn, modulates the ligand off-rate from its binding site as well as the binding affinity. The positive allostery could therefore be induced by a reduction in the enzyme's overall intrinsic dynamics.

Biophysical Optimization of a Therapeutic Protein by Nonstandard Mutagenesis

PubMed Central

Pandyarajan, Vijay; Phillips, Nelson B.; Cox, Gabriela P.; Yang, Yanwu; Whittaker, Jonathan; Ismail-Beigi, Faramarz; Weiss, Michael A.

2014-01-01

Insulin provides a model for the therapeutic application of protein engineering. A paradigm in molecular pharmacology was defined by design of rapid-acting insulin analogs for the prandial control of glycemia. Such analogs, a cornerstone of current diabetes regimens, exhibit accelerated subcutaneous absorption due to more rapid disassembly of oligomeric species relative to wild-type insulin. This strategy is limited by a molecular trade-off between accelerated disassembly and enhanced susceptibility to degradation. Here, we demonstrate that this trade-off may be circumvented by nonstandard mutagenesis. Our studies employed LysB28, ProB29-insulin (“lispro”) as a model prandial analog that is less thermodynamically stable and more susceptible to fibrillation than is wild-type insulin. We have discovered that substitution of an invariant tyrosine adjoining the engineered sites in lispro (TyrB26) by 3-iodo-Tyr (i) augments its thermodynamic stability (ΔΔGu 0.5 ±0.2 kcal/mol), (ii) delays onset of fibrillation (lag time on gentle agitation at 37 °C was prolonged by 4-fold), (iii) enhances affinity for the insulin receptor (1.5 ± 0.1-fold), and (iv) preserves biological activity in a rat model of diabetes mellitus. 1H NMR studies suggest that the bulky iodo-substituent packs within a nonpolar interchain crevice. Remarkably, the 3-iodo-TyrB26 modification stabilizes an oligomeric form of insulin pertinent to pharmaceutical formulation (the R6 zinc hexamer) but preserves rapid disassembly of the oligomeric form pertinent to subcutaneous absorption (T6 hexamer). By exploiting this allosteric switch, 3-iodo-TyrB26-lispro thus illustrates how a nonstandard amino acid substitution can mitigate the unfavorable biophysical properties of an engineered protein while retaining its advantages. PMID:24993826

Structural basis of detection and signaling of DNA single-strand breaks by human PARP-1

DOE PAGES

Eustermann, Sebastian; Wu, Wing -Fung; Langelier, Marie -France; ...

2015-11-25

Poly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1’s function remained obscure; inherent dynamics of SSBs and PARP-1’s multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1’s signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformabilitymore » of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodifcation in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Finally, our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins.« less

Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

PubMed Central

Heintz, Udo; Schlichting, Ilme

2016-01-01

The design of synthetic optogenetic tools that allow precise spatiotemporal control of biological processes previously inaccessible to optogenetic control has developed rapidly over the last years. Rational design of such tools requires detailed knowledge of allosteric light signaling in natural photoreceptors. To understand allosteric communication between sensor and effector domains, characterization of all relevant signaling states is required. Here, we describe the mechanism of light-dependent DNA binding of the light-oxygen-voltage (LOV) transcription factor Aureochrome 1a from Phaeodactylum tricornutum (PtAu1a) and present crystal structures of a dark state LOV monomer and a fully light-adapted LOV dimer. In combination with hydrogen/deuterium-exchange, solution scattering data and DNA-binding experiments, our studies reveal a light-sensitive interaction between the LOV and basic region leucine zipper DNA-binding domain that together with LOV dimerization results in modulation of the DNA affinity of PtAu1a. We discuss the implications of these results for the design of synthetic LOV-based photosensors with application in optogenetics. DOI: http://dx.doi.org/10.7554/eLife.11860.001 PMID:26754770

Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu

2012-03-15

The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structuremore » of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.« less

Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence*

PubMed Central

Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L.; Embrey, Kevin J.; Golovanov, Alexander P.

2016-01-01

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly 15N-labeled Ras as well as [13C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions. PMID:26565026

Structural Characterization of the Interaction of the Fibroblast Growth Factor Receptor with a Small Molecule Allosteric Inhibitor.

PubMed

Kappert, Franziska; Sreeramulu, Sridhar; Jonker, Hendrik R A; Richter, Christian; Rogov, Vladimir V; Proschak, Ewgenij; Hargittay, Bruno; Saxena, Krishna; Schwalbe, Harald

2018-06-04

The interaction of fibroblast growth factors (FGFs) with their fibroblast growth factor receptors (FGFRs) are important in the signaling network of cell growth and development. SSR128129E (SSR), a ligand of small molecular weight with potential anti-cancer properties, acts allosterically on the extracellular domains of FGFRs. Up to now, the structural basis of SSR binding to the D3 domain of FGFR remained elusive. This work reports the structural characterization of the interaction of SSR with one specific receptor, FGFR3, by NMR spectroscopy. This information provides a basis for rational drug design for allosteric FGFR inhibitors. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Native and engineered sensors for Ca2+ and Zn2+: lessons from calmodulin and MTF1.

PubMed

Carpenter, Margaret C; Palmer, Amy E

2017-05-09

Ca 2+ and Zn 2+ dynamics have been identified as important drivers of physiological processes. In order for these dynamics to encode function, the cell must have sensors that transduce changes in metal concentration to specific downstream actions. Here we compare and contrast the native metal sensors: calmodulin (CaM), the quintessential Ca 2+ sensor and metal-responsive transcription factor 1 (MTF1), a candidate Zn 2+ sensor. While CaM recognizes and modulates the activity of hundreds of proteins through allosteric interactions, MTF1 recognizes a single DNA motif that is distributed throughout the genome regulating the transcription of many target genes. We examine how the different inorganic chemistries of these two metal ions may shape these different mechanisms transducing metal ion concentration into changing physiologic activity. In addition to native metal sensors, scientists have engineered sensors to spy on the dynamic changes of metals in cells. The inorganic chemistry of the metals shapes the possibilities in the design strategies of engineered sensors. We examine how different strategies to tune the affinities of engineered sensors mirror the strategies nature developed to sense both Ca 2+ and Zn 2+ in cells. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Engineered Context-Sensitive Agonism: Tissue-Selective Drug Signaling through a G Protein-Coupled Receptor.

PubMed

Seemann, Wiebke K; Wenzel, Daniela; Schrage, Ramona; Etscheid, Justine; Bödefeld, Theresa; Bartol, Anna; Warnken, Mareille; Sasse, Philipp; Klöckner, Jessica; Holzgrabe, Ulrike; DeAmici, Marco; Schlicker, Eberhard; Racké, Kurt; Kostenis, Evi; Meyer, Rainer; Fleischmann, Bernd K; Mohr, Klaus

2017-02-01

Drug discovery strives for selective ligands to achieve targeted modulation of tissue function. Here we introduce engineered context-sensitive agonism as a postreceptor mechanism for tissue-selective drug action through a G protein-coupled receptor. Acetylcholine M 2 -receptor activation is known to mediate, among other actions, potentially dangerous slowing of the heart rate. This unwanted side effect is one of the main reasons that limit clinical application of muscarinic agonists. Herein we show that dualsteric (orthosteric/allosteric) agonists induce less cardiac depression ex vivo and in vivo than conventional full agonists. Exploration of the underlying mechanism in living cells employing cellular dynamic mass redistribution identified context-sensitive agonism of these dualsteric agonists. They translate elevation of intracellular cAMP into a switch from full to partial agonism. Designed context-sensitive agonism opens an avenue toward postreceptor pharmacologic selectivity, which even works in target tissues operated by the same subtype of pharmacologic receptor. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Engineering vanilloid-sensitivity into the rat TRPV2 channel

PubMed Central

Zhang, Feng; Hanson, Sonya M; Jara-Oseguera, Andres; Krepkiy, Dmitriy; Bae, Chanhyung; Pearce, Larry V; Blumberg, Peter M; Newstead, Simon; Swartz, Kenton J

2016-01-01

The TRPV1 channel is a detector of noxious stimuli, including heat, acidosis, vanilloid compounds and lipids. The gating mechanisms of the related TRPV2 channel are poorly understood because selective high affinity ligands are not available, and the threshold for heat activation is extremely high (>50°C). Cryo-EM structures of TRPV1 and TRPV2 reveal that they adopt similar structures, and identify a putative vanilloid binding pocket near the internal side of TRPV1. Here we use biochemical and electrophysiological approaches to investigate the resiniferatoxin(RTx) binding site in TRPV1 and to explore the functional relationships between TRPV1 and TRPV2. Collectively, our results support the interaction of vanilloids with the proposed RTx binding pocket, and demonstrate an allosteric influence of a tarantula toxin on vanilloid binding. Moreover, we show that sensitivity to RTx can be engineered into TRPV2, demonstrating that the gating and permeation properties of this channel are similar to TRPV1. DOI: http://dx.doi.org/10.7554/eLife.16409.001 PMID:27177419

Engineering vanilloid-sensitivity into the rat TRPV2 channel.

PubMed

Zhang, Feng; Hanson, Sonya M; Jara-Oseguera, Andres; Krepkiy, Dmitriy; Bae, Chanhyung; Pearce, Larry V; Blumberg, Peter M; Newstead, Simon; Swartz, Kenton J

2016-05-13

The TRPV1 channel is a detector of noxious stimuli, including heat, acidosis, vanilloid compounds and lipids. The gating mechanisms of the related TRPV2 channel are poorly understood because selective high affinity ligands are not available, and the threshold for heat activation is extremely high (>50°C). Cryo-EM structures of TRPV1 and TRPV2 reveal that they adopt similar structures, and identify a putative vanilloid binding pocket near the internal side of TRPV1. Here we use biochemical and electrophysiological approaches to investigate the resiniferatoxin(RTx) binding site in TRPV1 and to explore the functional relationships between TRPV1 and TRPV2. Collectively, our results support the interaction of vanilloids with the proposed RTx binding pocket, and demonstrate an allosteric influence of a tarantula toxin on vanilloid binding. Moreover, we show that sensitivity to RTx can be engineered into TRPV2, demonstrating that the gating and permeation properties of this channel are similar to TRPV1.

Mechanistic insights into allosteric regulation of the A 2A adenosine G protein-coupled receptor by physiological cations

DOE PAGES

Ye, Libin; Neale, Chris Andrew; Sljoka, Adnan; ...

2018-04-10

Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19F NMR is used to delineate the effects of cations on functional states of the adenosine A 2A GPCR. While Na + reinforces an inactive ensemble and a partial-agonist stabilized state, Ca 2+ and Mg 2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridgemore » specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. Lastly, an understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.« less

[Pharmacological characteristics of drugs targeted on calcium-sensing receptor.-properties of cinacalcet hydrochloride as allosteric modulator].

PubMed

Nagano, Nobuo; Tsutsui, Takaaki

2016-06-01

Calcimimetics act as positive allosteric modulators of the calcium-sensing receptor (CaSR), thereby decreasing parathyroid hormone (PTH) secretion from the parathyroid glands. On the other hand, negative allosteric modulators of the CaSR with stimulatory effect on PTH secretion are termed calcilytics. The calcimimetic cinacalcet hydrochloride (cinacalcet) is the world's first allosteric modulator of G protein-coupled receptor to enter the clinical market. Cinacalcet just tunes the physiological effects of Ca(2+), an endogenous ligand, therefore, shows high selectivity and low side effects. Calcimimetics also increase cell surface CaSR expression by acting as pharmacological chaperones (pharmacoperones). It is considered that the cinacalcet-induced upper gastrointestinal problems are resulted from enhanced physiological responses to Ca(2+) and amino acids via increased sensitivity of digestive tract CaSR by cinacalcet. While clinical developments of calcilytics for osteoporosis were unfortunately halted or terminated due to paucity of efficacy, it is expected that calcilytics may be useful for the treatment of patients with activating CaSR mutations, asthma, and idiopathic pulmonary artery hypertension.

Crystal structures of the M 1 and M 4 muscarinic acetylcholine receptors

DOE PAGES

Thal, David M.; Sun, Bingfa; Feng, Dan; ...

2016-03-09

Muscarinic M1–M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer’s disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. In this paper, we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 andmore » M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. Finally, we also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.« less

Crystal structures of the M 1 and M 4 muscarinic acetylcholine receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thal, David M.; Sun, Bingfa; Feng, Dan

Muscarinic M1–M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer’s disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. In this paper, we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 andmore » M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. Finally, we also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.« less

Mechanistic insights into allosteric regulation of the A 2A adenosine G protein-coupled receptor by physiological cations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ye, Libin; Neale, Chris Andrew; Sljoka, Adnan

Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19F NMR is used to delineate the effects of cations on functional states of the adenosine A 2A GPCR. While Na + reinforces an inactive ensemble and a partial-agonist stabilized state, Ca 2+ and Mg 2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridgemore » specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. Lastly, an understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.« less

Effects of neuronal nicotinic acetylcholine receptor allosteric modulators in animal behavior studies

PubMed Central

Pandya, Anshul. A.; Yakel, Jerrel L.

2013-01-01

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation-conducting transmembrane channels from the cys-loop receptor superfamily. The neuronal subtypes of these receptors (e.g. the α7 and α4β2 subtypes) are involved in neurobehavioral processes such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and a number of cognitive functions like learning and memory. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders, and behavioral studies in animals are useful models to assess the effects of compounds that act on these receptors. Allosteric modulators are ligands that bind to the receptors at sites other than the orthosteric site where acetylcholine, the endogenous agonist for the nAChRs, binds. While conventional ligands for the neuronal nAChRs have been studied for their behavioral effects in animals, allosteric modulators for these receptors have only recently gained attention, and research on their behavioral effects is growing rapidly. Here we will discuss the behavioral effects of allosteric modulators of the neuronal nAChRs. PMID:23732296

Protein Allostery and Conformational Dynamics.

PubMed

Guo, Jingjing; Zhou, Huan-Xiang

2016-06-08

The functions of many proteins are regulated through allostery, whereby effector binding at a distal site changes the functional activity (e.g., substrate binding affinity or catalytic efficiency) at the active site. Most allosteric studies have focused on thermodynamic properties, in particular, substrate binding affinity. Changes in substrate binding affinity by allosteric effectors have generally been thought to be mediated by conformational transitions of the proteins or, alternatively, by changes in the broadness of the free energy basin of the protein conformational state without shifting the basin minimum position. When effector binding changes the free energy landscape of a protein in conformational space, the change affects not only thermodynamic properties but also dynamic properties, including the amplitudes of motions on different time scales and rates of conformational transitions. Here we assess the roles of conformational dynamics in allosteric regulation. Two cases are highlighted where NMR spectroscopy and molecular dynamics simulation have been used as complementary approaches to identify residues possibly involved in allosteric communication. Perspectives on contentious issues, for example, the relationship between picosecond-nanosecond local and microsecond-millisecond conformational exchange dynamics, are presented.

Dynamics and allostery of the ionotropic glutamate receptors and the ligand binding domain.

PubMed

Tobi, Dror

2016-02-01

The dynamics of the ligand-binding domain (LBD) and the intact ionotropic glutamate receptor (iGluR) were studied using Gaussian Network Model (GNM) analysis. The dynamics of LBDs with various allosteric modulators is compared using a novel method of multiple alignment of GNM modes of motion. The analysis reveals that allosteric effectors change the dynamics of amino acids at the upper lobe interface of the LBD dimer as well as at the hinge region between the upper- and lower- lobes. For the intact glutamate receptor the analysis show that the clamshell-like movement of the LBD upper and lower lobes is coupled to the bending of the trans-membrane domain (TMD) helices which may open the channel pore. The results offer a new insight on the mechanism of action of allosteric modulators on the iGluR and support the notion of TMD helices bending as a possible mechanism for channel opening. In addition, the study validates the methodology of multiple GNM modes alignment as a useful tool to study allosteric effect and its relation to proteins dynamics. © 2015 Wiley Periodicals, Inc.

A structural comparison of 'real' and 'model' calmodulin clarified allosteric interactions regulating domain motion.

PubMed

Shimoyama, Hiromitsu

2018-05-07

Calmodulin (CaM) is a multifunctional calcium-binding protein, which regulates various biochemical processes. CaM acts via structural changes and complex forming with its target enzymes. CaM has two globular domains (N-lobe and C-lobe) connected by a long linker region. Upon calcium binding, the N-lobe and C-lobe undergo local conformational changes, after that, entire CaM wraps the target enzyme through a large conformational change. However, the regulation mechanism, such as allosteric interactions regulating the conformational changes, is still unclear. In order to clarify the allosteric interactions, in this study, experimentally obtained 'real' structures are compared to 'model' structures lacking the allosteric interactions. As the allosteric interactions would be absent in calcium-free CaM (apo-CaM), allostery-eliminated calcium-bound CaM (holo-CaM) models were constructed by combining the apo-CaM's linker and the holo-CaM's N- and C-lobe. Before the comparison, the 'real' and 'model' structures were clustered and cluster-cluster relationship was determined by a principal component analysis. The structures were compared based on the relationship, then, a distance map and a contact probability analysis clarified that the inter-domain motion is regulated by several groups of inter-domain contacting residue pairs. The analyses suggested that these residues cause inter-domain translation and rotation, and as a consequence, the motion encourage structural diversity. The resultant diversity would contribute to the functional versatility of CaM.

Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase.

PubMed

Abdurakhmanov, Eldar; Øie Solbak, Sara; Danielson, U Helena

2017-06-16

Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a.

Defining NADH-Driven Allostery Regulating Apoptosis-Inducing Factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brosey, Chris A.; Ho, Chris; Long, Winnie Z.

Apoptosis-inducing factor (AIF) is critical for mitochondrial respiratory complex biogenesis and for mediating necroptotic parthanatos; these functions are seemingly regulated by enigmatic allosteric switching driven by NADH charge-transfer complex (CTC) formation. In this paper, we define molecular pathways linking AIF's active site to allosteric switching regions by characterizing dimer-permissive mutants using small-angle X-ray scattering (SAXS) and crystallography and by probing AIF-CTC communication networks using molecular dynamics simulations. Collective results identify two pathways propagating allostery from the CTC active site: (1) active-site H454 links to S480 of AIF's central β-strand to modulate a hydrophobic border at the dimerization interface, and (2)more » an interaction network links AIF's FAD cofactor, central β-strand, and Cβ-clasp whereby R529 reorientation initiates C-loop release during CTC formation. Finally, this knowledge of AIF allostery and its flavoswitch mechanism provides a foundation for biologically understanding and biomedically controlling its participation in mitochondrial homeostasis and cell death.« less

Structure-based design and mechanisms of allosteric inhibitors for mitochondrial branched-chain α-ketoacid dehydrogenase kinase

PubMed Central

Qi, Xiangbing; Gui, Wen-Jun; Morlock, Lorraine K.; Wallace, Amy L.; Ahmed, Kamran; Laxman, Sunil; Campeau, Philippe M.; Lee, Brendan H.; Hutson, Susan M.; Tu, Benjamin P.; Williams, Noelle S.; Tambar, Uttam K.; Wynn, R. Max; Chuang, David T.

2013-01-01

The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are elevated in maple syrup urine disease, heart failure, obesity, and type 2 diabetes. BCAA homeostasis is controlled by the mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC), which is negatively regulated by the specific BCKD kinase (BDK). Here, we used structure-based design to develop a BDK inhibitor, (S)-α-chloro-phenylpropionic acid [(S)-CPP]. Crystal structures of the BDK-(S)-CPP complex show that (S)-CPP binds to a unique allosteric site in the N-terminal domain, triggering helix movements in BDK. These conformational changes are communicated to the lipoyl-binding pocket, which nullifies BDK activity by blocking its binding to the BCKDC core. Administration of (S)-CPP to mice leads to the full activation and dephosphorylation of BCKDC with significant reduction in plasma BCAA concentrations. The results buttress the concept of targeting mitochondrial BDK as a pharmacological approach to mitigate BCAA accumulation in metabolic diseases and heart failure. PMID:23716694

Defining NADH-Driven Allostery Regulating Apoptosis-Inducing Factor

DOE PAGES

Brosey, Chris A.; Ho, Chris; Long, Winnie Z.; ...

2016-11-03

Apoptosis-inducing factor (AIF) is critical for mitochondrial respiratory complex biogenesis and for mediating necroptotic parthanatos; these functions are seemingly regulated by enigmatic allosteric switching driven by NADH charge-transfer complex (CTC) formation. In this paper, we define molecular pathways linking AIF's active site to allosteric switching regions by characterizing dimer-permissive mutants using small-angle X-ray scattering (SAXS) and crystallography and by probing AIF-CTC communication networks using molecular dynamics simulations. Collective results identify two pathways propagating allostery from the CTC active site: (1) active-site H454 links to S480 of AIF's central β-strand to modulate a hydrophobic border at the dimerization interface, and (2)more » an interaction network links AIF's FAD cofactor, central β-strand, and Cβ-clasp whereby R529 reorientation initiates C-loop release during CTC formation. Finally, this knowledge of AIF allostery and its flavoswitch mechanism provides a foundation for biologically understanding and biomedically controlling its participation in mitochondrial homeostasis and cell death.« less

Allosteric auto-inhibition and activation of the Nedd4 family E3 ligase Itch.

PubMed

Zhu, Kang; Shan, Zelin; Chen, Xing; Cai, Yuqun; Cui, Lei; Yao, Weiyi; Wang, Zhen; Shi, Pan; Tian, Changlin; Lou, Jizhong; Xie, Yunli; Wen, Wenyu

2017-09-01

The Nedd4 family E3 ligases are key regulators of cell growth and proliferation and are often misregulated in human cancers and other diseases. The ligase activities of Nedd4 E3s are tightly controlled via auto-inhibition. However, the molecular mechanism underlying Nedd4 E3 auto-inhibition and activation is poorly understood. Here, we show that the WW domains proceeding the catalytic HECT domain play an inhibitory role by binding directly to HECT in the Nedd4 E3 family member Itch. Our structural and biochemical analyses of Itch reveal that the WW2 domain and a following linker allosterically lock HECT in an inactive state inhibiting E2-E3 transthiolation. Binding of the Ndfip1 adaptor or JNK1-mediated phosphorylation relieves the auto-inhibition of Itch in a WW2-dependent manner. Aberrant activation of Itch leads to migration defects of cortical neurons during development. Our study provides a new mechanism governing the regulation of Itch. © 2017 The Authors.

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

PubMed

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

2011-05-01

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

Intracellular calcium levels determine differential modulation of allosteric interactions within G protein-coupled receptor heteromers.

PubMed

Navarro, Gemma; Aguinaga, David; Moreno, Estefania; Hradsky, Johannes; Reddy, Pasham P; Cortés, Antoni; Mallol, Josefa; Casadó, Vicent; Mikhaylova, Marina; Kreutz, Michael R; Lluís, Carme; Canela, Enric I; McCormick, Peter J; Ferré, Sergi

2014-11-20

The pharmacological significance of the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer is well established and it is being considered as an important target for the treatment of Parkinson’s disease and other neuropsychiatric disorders. However, the physiological factors that control its distinctive biochemical properties are still unknown. We demonstrate that different intracellular Ca2+ levels exert a differential modulation of A2AR-D2R heteromer-mediated adenylyl-cyclase and MAPK signaling in striatal cells. This depends on the ability of low and high Ca2+ levels to promote a selective interaction of the heteromer with the neuronal Ca2+-binding proteins NCS-1 and calneuron-1, respectively. These Ca2+-binding proteins differentially modulate allosteric interactions within the A2AR-D2R heteromer, which constitutes a unique cellular device that integrates extracellular (adenosine and dopamine) and intracellular (Ca+2) signals to produce a specific functional response.

Allosteric conformational barcodes direct signaling in the cell.

PubMed

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

2013-09-03

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

A mutation uncouples the tubulin conformational and GTPase cycles, revealing allosteric control of microtubule dynamics

PubMed Central

Geyer, Elisabeth A; Burns, Alexander; Lalonde, Beth A; Ye, Xuecheng; Piedra, Felipe-Andres; Huffaker, Tim C; Rice, Luke M

2015-01-01

Microtubule dynamic instability depends on the GTPase activity of the polymerizing αβ-tubulin subunits, which cycle through at least three distinct conformations as they move into and out of microtubules. How this conformational cycle contributes to microtubule growing, shrinking, and switching remains unknown. Here, we report that a buried mutation in αβ-tubulin yields microtubules with dramatically reduced shrinking rate and catastrophe frequency. The mutation causes these effects by suppressing a conformational change that normally occurs in response to GTP hydrolysis in the lattice, without detectably changing the conformation of unpolymerized αβ-tubulin. Thus, the mutation weakens the coupling between the conformational and GTPase cycles of αβ-tubulin. By showing that the mutation predominantly affects post-GTPase conformational and dynamic properties of microtubules, our data reveal that the strength of the allosteric response to GDP in the lattice dictates the frequency of catastrophe and the severity of rapid shrinking. DOI: http://dx.doi.org/10.7554/eLife.10113.001 PMID:26439009

The Core of Allosteric Motion in Thermus caldophilus l-Lactate Dehydrogenase*

PubMed Central

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

2014-01-01

For Thermus caldophilus l-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S0.5 value 103-fold and increased the Vmax 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 His179 of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased Vmax 4-fold but reduced pyruvate S0.5 only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase Vmax, but 102-reduced pyruvate S0.5, 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. PMID:25258319

Carboxypeptidase M Is a Positive Allosteric Modulator of the Kinin B1 Receptor*

PubMed Central

Zhang, Xianming; Tan, Fulong; Skidgel, Randal A.

2013-01-01

Ligand binding to extracellular domains of G protein-coupled receptors can result in novel and nuanced allosteric effects on receptor signaling. We previously showed that the protein-protein interaction of carboxypeptidase M (CPM) and kinin B1 receptor (B1R) enhances B1R signaling in two ways; 1) kinin binding to CPM causes a conformational activation of the B1R, and 2) CPM-generated des-Arg-kinin agonist is efficiently delivered to the B1R. Here, we show CPM is also a positive allosteric modulator of B1R signaling to its agonist, des-Arg10-kallidin (DAKD). In HEK cells stably transfected with B1R, co-expression of CPM enhanced DAKD-stimulated increases in intracellular Ca2+ or phosphoinositide turnover by a leftward shift of the dose-response curve without changing the maximum. CPM increased B1R affinity for DAKD by ∼5-fold but had no effect on basal B1R-dependent phosphoinositide turnover. Soluble, recombinant CPM bound to HEK cells expressing B1Rs without stimulating receptor signaling. CPM positive allosteric action was independent of enzyme activity but depended on interaction of its C-terminal domain with the B1R extracellular loop 2. Disruption of the CPM/B1R interaction or knockdown of CPM in cytokine-treated primary human endothelial cells inhibited the allosteric enhancement of CPM on B1R DAKD binding or ERK1/2 activation. CPM also enhanced the DAKD-induced B1R conformational change as detected by increased intramolecular fluorescence or bioluminescence resonance energy transfer. Thus, CPM binding to extracellular loop 2 of the B1R results in positive allosteric modulation of B1R signaling, and disruption of this interaction could provide a novel therapeutic approach to reduce pathological B1R signaling. PMID:24108126

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 may be a universal requirement encoded in protein structures to balance the inherent tension between resilience and efficiency of the residue interaction networks. PMID:24922508

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

PubMed

Blacklock, Kristin; Verkhivker, Gennady M

2014-06-01

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 may be a universal requirement encoded in protein structures to balance the inherent tension between resilience and efficiency of the residue interaction networks.

The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation

PubMed Central

Guerrero-Mendiola, Carlos; García-Trejo, José J.; Encalada, Rusely; Saavedra, Emma

2017-01-01

In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K+-dependent, whereas those with Lys117 were K+-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in Vibrio cholerae. The main findings of this work showed that the isozyme with Glu117 is an active K+-dependent enzyme. At the same substrate concentration, its Vmax in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K+-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K+-independent enzyme, Mn2+ may “mimic” the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in Vibrio cholerae have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that VcIPK and VcIIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium. PMID:28686591

Sensing new chemicals with bacterial transcription factors.

PubMed

Libis, Vincent; Delépine, Baudoin; Faulon, Jean-Loup

2016-10-01

Bacteria rely on allosteric transcription factors (aTFs) to sense a wide range of chemicals. The variety of effectors has contributed in making aTFs the most used input system in synthetic biological circuits. Considering their enabling role in biotechnology, an important question concerns the size of the chemical space that can potentially be detected by these biosensors. From digging into the ever changing repertoire of natural regulatory circuits, to advances in aTF engineering, we review here different strategies that are pushing the boundaries of this chemical space. We also review natural and synthetic cases of indirect sensing, where aTFs work in combination with metabolism to enable detection of new molecules. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Repellent DEET Potentiates Carbamate Effects via Insect Muscarinic Receptor Interactions: An Alternative Strategy to Control Insect Vector-Borne Diseases.

PubMed

Abd-Ella, Aly; Stankiewicz, Maria; Mikulska, Karolina; Nowak, Wieslaw; Pennetier, Cédric; Goulu, Mathilde; Fruchart-Gaillard, Carole; Licznar, Patricia; Apaire-Marchais, Véronique; List, Olivier; Corbel, Vincent; Servent, Denis; Lapied, Bruno

2015-01-01

Insect vector-borne diseases remain one of the principal causes of human mortality. In addition to conventional measures of insect control, repellents continue to be the mainstay for personal protection. Because of the increasing pyrethroid-resistant mosquito populations, alternative strategies to reconstitute pyrethroid repellency and knock-down effects have been proposed by mixing the repellent DEET (N,N-Diethyl-3-methylbenzamide) with non-pyrethroid insecticide to better control resistant insect vector-borne diseases. By using electrophysiological, biochemichal, in vivo toxicological techniques together with calcium imaging, binding studies and in silico docking, we have shown that DEET, at low concentrations, interacts with high affinity with insect M1/M3 mAChR allosteric site potentiating agonist effects on mAChRs coupled to phospholipase C second messenger pathway. This increases the anticholinesterase activity of the carbamate propoxur through calcium-dependent regulation of acetylcholinesterase. At high concentrations, DEET interacts with low affinity on distinct M1/M3 mAChR site, counteracting the potentiation. Similar dose-dependent dual effects of DEET have also been observed at synaptic mAChR level. Additionally, binding and in silico docking studies performed on human M1 and M3 mAChR subtypes indicate that DEET only displays a low affinity antagonist profile on these M1/M3 mAChRs. These results reveal a selective high affinity positive allosteric site for DEET in insect mAChRs. Finally, bioassays conducted on Aedes aegypti confirm the synergistic interaction between DEET and propoxur observed in vitro, resulting in a higher mortality of mosquitoes. Our findings reveal an unusual allosterically potentiating action of the repellent DEET, which involves a selective site in insect. These results open exciting research areas in public health particularly in the control of the pyrethroid-resistant insect-vector borne diseases. Mixing low doses of DEET and a non-pyrethroid insecticide will lead to improvement in the efficiency treatments thus reducing both the concentration of active ingredients and side effects for non-target organisms. The discovery of this insect specific site may pave the way for the development of new strategies essential in the management of chemical use against resistant mosquitoes.

The Repellent DEET Potentiates Carbamate Effects via Insect Muscarinic Receptor Interactions: An Alternative Strategy to Control Insect Vector-Borne Diseases

PubMed Central

Abd-Ella, Aly; Stankiewicz, Maria; Mikulska, Karolina; Nowak, Wieslaw; Pennetier, Cédric; Goulu, Mathilde; Fruchart-Gaillard, Carole; Licznar, Patricia; Apaire-Marchais, Véronique; List, Olivier; Corbel, Vincent; Servent, Denis; Lapied, Bruno

2015-01-01

Insect vector-borne diseases remain one of the principal causes of human mortality. In addition to conventional measures of insect control, repellents continue to be the mainstay for personal protection. Because of the increasing pyrethroid-resistant mosquito populations, alternative strategies to reconstitute pyrethroid repellency and knock-down effects have been proposed by mixing the repellent DEET (N,N-Diethyl-3-methylbenzamide) with non-pyrethroid insecticide to better control resistant insect vector-borne diseases. By using electrophysiological, biochemichal, in vivo toxicological techniques together with calcium imaging, binding studies and in silico docking, we have shown that DEET, at low concentrations, interacts with high affinity with insect M1/M3 mAChR allosteric site potentiating agonist effects on mAChRs coupled to phospholipase C second messenger pathway. This increases the anticholinesterase activity of the carbamate propoxur through calcium-dependent regulation of acetylcholinesterase. At high concentrations, DEET interacts with low affinity on distinct M1/M3 mAChR site, counteracting the potentiation. Similar dose-dependent dual effects of DEET have also been observed at synaptic mAChR level. Additionally, binding and in silico docking studies performed on human M1 and M3 mAChR subtypes indicate that DEET only displays a low affinity antagonist profile on these M1/M3 mAChRs. These results reveal a selective high affinity positive allosteric site for DEET in insect mAChRs. Finally, bioassays conducted on Aedes aegypti confirm the synergistic interaction between DEET and propoxur observed in vitro, resulting in a higher mortality of mosquitoes. Our findings reveal an unusual allosterically potentiating action of the repellent DEET, which involves a selective site in insect. These results open exciting research areas in public health particularly in the control of the pyrethroid-resistant insect-vector borne diseases. Mixing low doses of DEET and a non-pyrethroid insecticide will lead to improvement in the efficiency treatments thus reducing both the concentration of active ingredients and side effects for non-target organisms. The discovery of this insect specific site may pave the way for the development of new strategies essential in the management of chemical use against resistant mosquitoes. PMID:25961834

Genetically encoding a light switch in an ionotropic glutamate receptor reveals subunit-specific interfaces.

PubMed

Zhu, Shujia; Riou, Morgane; Yao, C Andrea; Carvalho, Stéphanie; Rodriguez, Pamela C; Bensaude, Olivier; Paoletti, Pierre; Ye, Shixin

2014-04-22

Reprogramming receptors to artificially respond to light has strong potential for molecular studies and interrogation of biological functions. Here, we design a light-controlled ionotropic glutamate receptor by genetically encoding a photoreactive unnatural amino acid (UAA). The photo-cross-linker p-azido-L-phenylalanine (AzF) was encoded in NMDA receptors (NMDARs), a class of glutamate-gated ion channels that play key roles in neuronal development and plasticity. AzF incorporation in the obligatory GluN1 subunit at the GluN1/GluN2B N-terminal domain (NTD) upper lobe dimer interface leads to an irreversible allosteric inhibition of channel activity upon UV illumination. In contrast, when pairing the UAA-containing GluN1 subunit with the GluN2A subunit, light-dependent inactivation is completely absent. By combining electrophysiological and biochemical analyses, we identify subunit-specific structural determinants at the GluN1/GluN2 NTD dimer interfaces that critically dictate UV-controlled inactivation. Our work reveals that the two major NMDAR subtypes differ in their ectodomain-subunit interactions, in particular their electrostatic contacts, resulting in GluN1 NTD coupling more tightly to the GluN2B NTD than to the GluN2A NTD. It also paves the way for engineering light-sensitive ligand-gated ion channels with subtype specificity through the genetic code expansion.

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

PubMed Central

Thie, Holger

2017-01-01

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

Discovery and Characterization of Novel Subtype-Selective Allosteric Agonists for the Investigation of M1 Receptor Function in the Central Nervous System

PubMed Central

2009-01-01

Cholinergic transmission in the forebrain is mediated primarily by five subtypes of muscarinic acetylcholine receptors (mAChRs), termed M1−M5. Of the mAChR subtypes, M1 is among the most heavily expressed in regions that are critical for learning and memory and has been viewed as the most critical mAChR subtype for memory and attention mechanisms. Unfortunately, it has been difficult to develop selective activators of M1 and other individual mAChR subtypes, which has prevented detailed studies of the functional roles of selective activation of M1. Using a functional high-throughput screening and subsequent diversity-oriented synthesis approach, we have discovered a novel series of highly selective M1 allosteric agonists. These compounds activate M1 with EC50 values in the 150−500 nM range and have unprecedented, clean ancillary pharmacology (no substantial activity at 10 μM across a large panel of targets). Targeted mutagenesis revealed a potentially novel allosteric binding site in the third extracellular loop of the M1 receptor for these allosteric agonists. Optimized compounds, such as VU0357017, provide excellent brain exposure after systemic dosing and have robust in vivo efficacy in reversing scopolamine-induced deficits in a rodent model of contextual fear conditioning. This series of selective M1 allosteric agonists provides critical research tools to allow dissection of M1-mediated effects in the CNS and potential leads for novel treatments for Alzheimer’s disease and schizophrenia. PMID:21961051

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

PubMed

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

2015-03-01

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

Internalization of the chemokine receptor CCR4 can be evoked by orthosteric and allosteric receptor antagonists

PubMed Central

Ajram, Laura; Begg, Malcolm; Slack, Robert; Cryan, Jenni; Hall, David; Hodgson, Simon; Ford, Alison; Barnes, Ashley; Swieboda, Dawid; Mousnier, Aurelie; Solari, Roberto

2014-01-01

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. PMID:24534492

B-973, a novel piperazine positive allosteric modulator of the α7 nicotinic acetylcholine receptor.

PubMed

Post-Munson, Debra J; Pieschl, Rick L; Molski, Thaddeus F; Graef, John D; Hendricson, Adam W; Knox, Ronald J; McDonald, Ivar M; Olson, Richard E; Macor, John E; Weed, Michael R; Bristow, Linda J; Kiss, Laszlo; Ahlijanian, Michael K; Herrington, James

2017-03-15

The alpha7 (α7) nicotinic acetylcholine receptor is a therapeutic target for cognitive disorders. Here we describe 3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propanamide (B-973), a novel piperazine-containing molecule that acts as a positive allosteric modulator of the α7 receptor. We characterize the action of B-973 on the α7 receptor using electrophysiology and radioligand binding. At 0.1mM acetylcholine, 1μM B-973 potentiated peak acetylcholine-induced currents 6-fold relative to maximal acetylcholine (3mM) and slowed channel desensitization, resulting in a 6900-fold increase in charge transfer. The EC 50 of B-973 was approximately 0.3μM at acetylcholine concentrations ranging from 0.03 to 3mM. At a concentration of 1μM, B-973 shifted the acetylcholine EC 50 of peak currents from 0.30mM in control to 0.007mM. B-973 slowed channel deactivation upon acetylcholine removal (τ=50s) and increased the affinity of the α7 agonist [ 3 H]A-585539. In the absence of exogenously added acetylcholine, application of B-973 at concentrations >1μM induced large methyllycaconitine-sensitive currents, suggesting B-973 can function as an Ago-PAM at high concentrations. B-973 will be a useful probe for investigating the biological consequences of increasing α7 receptor activity through allosteric modulation. Copyright © 2017 Elsevier B.V. All rights reserved.

Evidence for an allosteric mechanism of substrate release from membrane-transporter accessory binding proteins.

PubMed

Marinelli, Fabrizio; Kuhlmann, Sonja I; Grell, Ernst; Kunte, Hans-Jörg; Ziegler, Christine; Faraldo-Gómez, José D

2011-12-06

Numerous membrane importers rely on accessory water-soluble proteins to capture their substrates. These substrate-binding proteins (SBP) have a strong affinity for their ligands; yet, substrate release onto the low-affinity membrane transporter must occur for uptake to proceed. It is generally accepted that release is facilitated by the association of SBP and transporter, upon which the SBP adopts a conformation similar to the unliganded state, whose affinity is sufficiently reduced. Despite the appeal of this mechanism, however, direct supporting evidence is lacking. Here, we use experimental and theoretical methods to demonstrate that an allosteric mechanism of enhanced substrate release is indeed plausible. First, we report the atomic-resolution structure of apo TeaA, the SBP of the Na(+)-coupled ectoine TRAP transporter TeaBC from Halomonas elongata DSM2581(T), and compare it with the substrate-bound structure previously reported. Conformational free-energy landscape calculations based upon molecular dynamics simulations are then used to dissect the mechanism that couples ectoine binding to structural change in TeaA. These insights allow us to design a triple mutation that biases TeaA toward apo-like conformations without directly perturbing the binding cleft, thus mimicking the influence of the membrane transporter. Calorimetric measurements demonstrate that the ectoine affinity of the conformationally biased triple mutant is 100-fold weaker than that of the wild type. By contrast, a control mutant predicted to be conformationally unbiased displays wild-type affinity. This work thus demonstrates that substrate release from SBPs onto their membrane transporters can be facilitated by the latter through a mechanism of allosteric modulation of the former.

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

The Activation of c-Src Tyrosine Kinase: Conformational Transition Pathway and Free Energy Landscape.

PubMed

Fajer, Mikolai; Meng, Yilin; Roux, Benoît

2017-04-20

Tyrosine kinases are important cellular signaling allosteric enzymes that regulate cell growth, proliferation, metabolism, differentiation, and migration. Their activity must be tightly controlled, and malfunction can lead to a variety of diseases, particularly cancer. The nonreceptor tyrosine kinase c-Src, a prototypical model system and a representative member of the Src-family, functions as complex multidomain allosteric molecular switches comprising SH2 and SH3 domains modulating the activity of the catalytic domain. The broad picture of self-inhibition of c-Src via the SH2 and SH3 regulatory domains is well characterized from a structural point of view, but a detailed molecular mechanism understanding is nonetheless still lacking. Here, we use advanced computational methods based on all-atom molecular dynamics simulations with explicit solvent to advance our understanding of kinase activation. To elucidate the mechanism of regulation and self-inhibition, we have computed the pathway and the free energy landscapes for the "inactive-to-active" conformational transition of c-Src for different configurations of the SH2 and SH3 domains. Using the isolated c-Src catalytic domain as a baseline for comparison, it is observed that the SH2 and SH3 domains, depending upon their bound orientation, promote either the inactive or active state of the catalytic domain. The regulatory structural information from the SH2-SH3 tandem is allosterically transmitted via the N-terminal linker of the catalytic domain. Analysis of the conformational transition pathways also illustrates the importance of the conserved tryptophan 260 in activating c-Src, and reveals a series of concerted events during the activation process.

Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

PubMed

Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L; Embrey, Kevin J; Golovanov, Alexander P

2016-01-22

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed Central

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

2011-01-01

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. PMID:21575610

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. Copyright © 2011 Elsevier Inc. All rights reserved.

Fast non-genomic effects of progesterone-derived neurosteroids on nociceptive thresholds and pain symptoms.

PubMed

Charlet, Alexandre; Lasbennes, François; Darbon, Pascal; Poisbeau, Pierrick

2008-10-31

Fast Inhibitory controls mediated by glycine (GlyRs) and GABAA receptors (GABAARs) play an important role to prevent the apparition of pathological pain symptoms of allodynia and hyperalgesia. The use of positive allosteric modulators of these receptors, specifically expressed in the spinal cord, may represent an interesting strategy to limit or block pain expression. In this study, we have used stereoisomers of progesterone metabolites, acting only via non-genomic effects, in order to evaluate the contribution of GlyRs and GABAARs for the reduction of mechanical and thermal heat hypernociception. We show that 3alpha neurosteroids were particularly efficient to elevate nociceptive thresholds in naive animal. It also reduced mechanical allodynia and thermal heat hyperalgesia in the carrageenan model of inflammatory pain. This effect is likely to be mediated by GABAA receptors since 3beta isomer was inefficient. More interestingly, 3alpha5beta neurosteroid was only efficient on mechanical allodynia while having no effect on thermal heat hyperalgesia. We characterized these paradoxical effects of 3alpha5beta neurosteroid using the strychnine and bicuculline models of allodynia. We clearly show that 3alpha5beta neurosteroid exerts an antinociceptive effect via a positive allosteric modulation of GABAARs but, at the same time, is pronociceptive by reducing GlyR function. This illustrates the importance of the inhibitory amino acid receptor channels and their allosteric modulators in spinal pain processing. Moreover, our results indicate that neurosteroids, which are synthesized in the dorsal horn of the spinal cord and have limited side effects, may be of significant interest in order to treat pathological pain symptoms.

Deeper Insights into the Allosteric Modulation of Ionotropic Glutamate Receptors.

PubMed

Regan, Michael C; Furukawa, Hiro

2016-09-21

Two articles in this issue of Neuron (Yelshanskaya et al., 2016; Yi et al., 2016) explore the structural basis of allosteric inhibition in ionotropic glutamate receptors, providing key insights into how iGluRs function in the brain as well as how they might be pharmacologically modulated in neurological disorders and disease. Copyright © 2016. Published by Elsevier Inc.

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

USDA-ARS?s Scientific Manuscript database

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...

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

USDA-ARS?s Scientific Manuscript database

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...

Flavonoids as potent allosteric inhibitors of protein tyrosine phosphatase 1B: molecular dynamics simulation and free energy calculation.

PubMed

Zargari, Farshid; Lotfi, Maryam; Shahraki, Omolbanin; Nikfarjam, Zahra; Shahraki, Jafar

2017-12-11

Protein tyrosine phosphatase 1B (PTP1B) is a member of the PTP superfamily which is considered to be a negative regulator of insulin receptor (IR) signaling pathway. PTP1B is a promising drug target for the treatment of type 2 diabetes, obesity, and cancer. The existence of allosteric site in PTP1B has turned the researcher's attention to an alternate strategy for inhibition of this enzyme. Herein, the molecular interactions between the allosteric site of PTP1B with three non-competitive flavonoids, (MOR), (MOK), and (DPO) have been investigated. Three ligands were docked into allosteric site of the enzyme. The resulting protein-ligand complexes were used for molecular dynamics studies. Principal component and free-energy landscape (FEL) as well as cluster analyses were used to investigate the conformational and dynamical properties of the protein and identify representative enzyme substrates bounded to the inhibitors. Per residue energy decomposition analysis attributed dissimilar affinities of three inhibitors to the several hydrogen bonds and non-bonded interactions. In conclusion, our results exhibited an inhibitory pattern of the ligands against PTP1B.

Two-track virtual screening approach to identify both competitive and allosteric inhibitors of human small C-terminal domain phosphatase 1

NASA Astrophysics Data System (ADS)

Park, Hwangseo; Lee, Hye Seon; Ku, Bonsu; Lee, Sang-Rae; Kim, Seung Jun

2017-08-01

Despite a wealth of persuasive evidence for the involvement of human small C-terminal domain phosphatase 1 (Scp1) in the impairment of neuronal differentiation and in Huntington's disease, small-molecule inhibitors of Scp1 have been rarely reported so far. This study aims to the discovery of both competitive and allosteric Scp1 inhibitors through the two-track virtual screening procedure. By virtue of the improvement of the scoring function by implementing a new molecular solvation energy term and by reoptimizing the atomic charges for the active-site Mg2+ ion cluster, we have been able to identify three allosteric and five competitive Scp1 inhibitors with low-micromolar inhibitory activity. Consistent with the results of kinetic studies on the inhibitory mechanisms, the allosteric inhibitors appear to be accommodated in the peripheral binding pocket through the hydrophobic interactions with the nonpolar residues whereas the competitive ones bind tightly in the active site with a direct coordination to the central Mg2+ ion. Some structural modifications to improve the biochemical potency of the newly identified inhibitors are proposed based on the binding modes estimated with docking simulations.

Seven Transmembrane Receptors as Shapeshifting Proteins: The Impact of Allosteric Modulation and Functional Selectivity on New Drug Discovery

PubMed Central

Miller, Laurence J.

2010-01-01

It is useful to consider seven transmembrane receptors (7TMRs) as disordered proteins able to allosterically respond to a number of binding partners. Considering 7TMRs as allosteric systems, affinity and efficacy can be thought of in terms of energy flow between a modulator, conduit (the receptor protein), and a number of guests. These guests can be other molecules, receptors, membrane-bound proteins, or signaling proteins in the cytosol. These vectorial flows of energy can yield standard canonical guest allostery (allosteric modification of drug effect), effects along the plane of the cell membrane (receptor oligomerization), or effects directed into the cytosol (differential signaling as functional selectivity). This review discusses these apparently diverse pharmacological effects in terms of molecular dynamics and protein ensemble theory, which tends to unify 7TMR behavior toward cells. Special consideration will be given to functional selectivity (biased agonism and biased antagonism) in terms of mechanism of action and potential therapeutic application. The explosion of technology that has enabled observation of diverse 7TMR behavior has also shown how drugs can have multiple (pluridimensional) efficacies and how this can cause paradoxical drug classification and nomenclatures. PMID:20392808

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Quinlan, Casey L.; Kaiser, Stephen E.; Bolaños, Ben

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

Signalling networks and dynamics of allosteric transitions in bacterial chaperonin GroEL: implications for iterative annealing of misfolded proteins.

PubMed

Thirumalai, D; Hyeon, Changbong

2018-06-19

Signal transmission at the molecular level in many biological complexes occurs through allosteric transitions. Allostery describes the responses of a complex to binding of ligands at sites that are spatially well separated from the binding region. We describe the structural perturbation method, based on phonon propagation in solids, which can be used to determine the signal-transmitting allostery wiring diagram (AWD) in large but finite-sized biological complexes. Application to the bacterial chaperonin GroEL-GroES complex shows that the AWD determined from structures also drives the allosteric transitions dynamically. From both a structural and dynamical perspective these transitions are largely determined by formation and rupture of salt-bridges. The molecular description of allostery in GroEL provides insights into its function, which is quantitatively described by the iterative annealing mechanism. Remarkably, in this complex molecular machine, a deep connection is established between the structures, reaction cycle during which GroEL undergoes a sequence of allosteric transitions, and function, in a self-consistent manner.This article is part of a discussion meeting issue 'Allostery and molecular machines'. © 2018 The Author(s).

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

PubMed

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

2018-05-10

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

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

PubMed Central

Das, Debasis; Krantz, Bryan A.

2016-01-01

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

Revealing the favorable dissociation pathway of type II kinase inhibitors via enhanced sampling simulations and two-end-state calculations.

PubMed

Sun, Huiyong; Tian, Sheng; Zhou, Shunye; Li, Youyong; Li, Dan; Xu, Lei; Shen, Mingyun; Pan, Peichen; Hou, Tingjun

2015-02-13

How does a type II inhibitor bind to/unbind from a kinase target is still a confusing question because the small molecule occupies both the ATP pocket and the allosteric pocket of the kinase binding site. Here, by using enhanced sampling simulations (umbrella sampling, US) and two-end-state free energy calculations (MM/GSBA), we systemically studied the dissociation processes of two distinct small molecules escaping from the binding pocket of p38 MAP kinase through the allosteric channel and the ATP channel. The results show that the unbinding pathways along the allosteric channel have much lower PMF depths than those along the ATP channel, suggesting that the allosteric channel is more favorable for the dissociations of the two inhibitors and thereby supporting the general understanding that the largest channel of a target is usually the entry/exit pathway for the binding/dissociation of small molecules. Interestingly, the MM/GBSA approach yielded similar PMF profiles compared with those based on US, a much time consuming approach, indicating that for a general study, such as detecting the important transition state of a ligand binding/unbinding process, MM/GBSA may be a feasible choice.

The Effects of Protein-Ligand Associations on the Subunit Interactions of Phosphofructokinase from B. stearothermophilus†

PubMed Central

Quinlan, R. Jason; Reinhart, Gregory D.

2008-01-01

Differences between the crystal structures of inhibitor-bound and uninihibited forms of phosphofructokinase (PFK) from B. stearothermophilus have led to a structural model for allosteric inhibition by phosphenolpyruvate (PEP) wherein a dimer-dimer interface within the tetrameric enzyme undergoes a quaternary shift. We have developed a labeling and hybridization technique to generate a tetramer with subunits containing two different extrinsic fluorophores simultaneously in known subunit orientations. This construct has been utilized in the examination of the effects of allosteric ligand and substrate binding on the subunit affinities of tetrameric PFK using several biophysical and spectroscopic techniques including 2-photon, dual-channel Fluorescence Correlation Spectroscopy (FCS). We demonstrate that PEP-binding at the allosteric site is sufficient to reduce the affinity of the active site interface from beyond the limits of experimental detection to nanomolar affinity, while conversely strengthening the interface at which it is bound. The reduced interface affinity is specific to inhibitor-binding, as binding the activator ADP at the same allosteric site causes no reduction in subunit affinity. With inhibitor bound, the weakened subunit affinity has allowed the kinetics of dimer association to be elucidated. PMID:16981693

Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer

PubMed Central

Bonaventura, Jordi; Navarro, Gemma; Casadó-Anguera, Verònica; Azdad, Karima; Rea, William; Moreno, Estefanía; Brugarolas, Marc; Mallol, Josefa; Canela, Enric I.; Lluís, Carme; Cortés, Antoni; Volkow, Nora D.; Schiffmann, Serge N.; Ferré, Sergi; Casadó, Vicent

2015-01-01

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain. PMID:26100888

Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer.

PubMed

Bonaventura, Jordi; Navarro, Gemma; Casadó-Anguera, Verònica; Azdad, Karima; Rea, William; Moreno, Estefanía; Brugarolas, Marc; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Cortés, Antoni; Volkow, Nora D; Schiffmann, Serge N; Ferré, Sergi; Casadó, Vicent

2015-07-07

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain.

The structure of brain glycogen phosphorylase-from allosteric regulation mechanisms to clinical perspectives.

PubMed

Mathieu, Cécile; Dupret, Jean-Marie; Rodrigues Lima, Fernando

2017-02-01

Glycogen phosphorylase (GP) is the key enzyme that regulates glycogen mobilization in cells. GP is a complex allosteric enzyme that comprises a family of three isozymes: muscle GP (mGP), liver GP (lGP), and brain GP (bGP). Although the three isozymes display high similarity and catalyze the same reaction, they differ in their sensitivity to the allosteric activator adenosine monophosphate (AMP). Moreover, inactivating mutations in mGP and lGP have been known to be associated with glycogen storage diseases (McArdle and Hers disease, respectively). The determination, decades ago, of the structure of mGP and lGP have allowed to better understand the allosteric regulation of these two isoforms and the development of specific inhibitors. Despite its important role in brain glycogen metabolism, the structure of the brain GP had remained elusive. Here, we provide an overview of the human brain GP structure and its relationship with the two other members of this key family of the metabolic enzymes. We also summarize how this structure provides valuable information to understand the regulation of bGP and to design specific ligands of potential pharmacological interest. © 2016 Federation of European Biochemical Societies.

Allosteric regulation of phosphofructokinase controls the emergence of glycolytic oscillations in isolated yeast cells.

PubMed

Gustavsson, Anna-Karin; van Niekerk, David D; Adiels, Caroline B; Kooi, Bob; Goksör, Mattias; Snoep, Jacky L

2014-06-01

Oscillations are widely distributed in nature and synchronization of oscillators has been described at the cellular level (e.g. heart cells) and at the population level (e.g. fireflies). Yeast glycolysis is the best known oscillatory system, although it has been studied almost exclusively at the population level (i.e. limited to observations of average behaviour in synchronized cultures). We studied individual yeast cells that were positioned with optical tweezers in a microfluidic chamber to determine the precise conditions for autonomous glycolytic oscillations. Hopf bifurcation points were determined experimentally in individual cells as a function of glucose and cyanide concentrations. The experiments were analyzed in a detailed mathematical model and could be interpreted in terms of an oscillatory manifold in a three-dimensional state-space; crossing the boundaries of the manifold coincides with the onset of oscillations and positioning along the longitudinal axis of the volume sets the period. The oscillatory manifold could be approximated by allosteric control values of phosphofructokinase for ATP and AMP. The mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.mib.ac.uk/webMathematica/UItester.jsp?modelName=gustavsson5. [Database section added 14 May 2014 after original online publication]. © 2014 FEBS.

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

PubMed Central

Ferguson, Alicia; Boomer, Ryan M.; Kurz, Markus; Keene, Sara C.; Diener, John L.; Keefe, Anthony D.; Wilson, Charles; Cload, Sharon T.

2004-01-01

We have utilized in vitro selection technology to develop allosteric ribozyme sensors that are specific for the small molecule analytes caffeine or aspartame. Caffeine- or aspartame-responsive ribozymes were converted into fluorescence-based RiboReporter™ sensor systems that were able to detect caffeine or aspartame in solution over a concentration range from 0.5 to 5 mM. With read-times as short as 5 min, these caffeine- or aspartame-dependent ribozymes function as highly specific and facile molecular sensors. Interestingly, successful isolation of allosteric ribozymes for the analytes described here was enabled by a novel selection strategy that incorporated elements of both modular design and activity-based selection methods typically used for generation of catalytic nucleic acids. PMID:15026535

Identifying Cytochrome P450 Functional Networks and Their Allosteric Regulatory Elements

DTIC Science & Technology

2013-12-03

drug alters the metabolism of another drug, potentially altering the pharmacological properties of either drug and leading to adverse toxic effects ...Despite extensive efforts in past decades to understand the mechanism behind these effects , drug metabolism and drug-drug interactions remain...important isoforms that is responsible for >50% of all drug metabolism, exhibits atypical kinetics due to allosteric effects [6]. Recently, Woods et al

Engineering naturally occurring trans-acting non-coding RNAs to sense molecular signals

PubMed Central

Qi, Lei; Lucks, Julius B.; Liu, Chang C.; Mutalik, Vivek K.; Arkin, Adam P.

2012-01-01

Non-coding RNAs (ncRNAs) are versatile regulators in cellular networks. While most trans-acting ncRNAs possess well-defined mechanisms that can regulate transcription or translation, they generally lack the ability to directly sense cellular signals. In this work, we describe a set of design principles for fusing ncRNAs to RNA aptamers to engineer allosteric RNA fusion molecules that modulate the activity of ncRNAs in a ligand-inducible way in Escherichia coli. We apply these principles to ncRNA regulators that can regulate translation (IS10 ncRNA) and transcription (pT181 ncRNA), and demonstrate that our design strategy exhibits high modularity between the aptamer ligand-sensing motif and the ncRNA target-recognition motif, which allows us to reconfigure these two motifs to engineer orthogonally acting fusion molecules that respond to different ligands and regulate different targets in the same cell. Finally, we show that the same ncRNA fused with different sensing domains results in a sensory-level NOR gate that integrates multiple input signals to perform genetic logic. These ligand-sensing ncRNA regulators provide useful tools to modulate the activity of structurally related families of ncRNAs, and building upon the growing body of RNA synthetic biology, our ability to design aptamer–ncRNA fusion molecules offers new ways to engineer ligand-sensing regulatory circuits. PMID:22383579

An allosteric model of the molecular interactions of excitation- contraction coupling in skeletal muscle

PubMed Central

1993-01-01

A contact interaction is proposed to exist between the voltage sensor of the transverse tubular membrane of skeletal muscle and the calcium release channel of the sarcoplasmic reticulum. This interaction is given a quantitative formulation inspired in the Monod, Wyman, and Changeux model of allosteric transitions in hemoglobin (Monod, J., J. Wyman, and J.-P. Changeux. 1965. Journal of Molecular Biology. 12:88- 118), and analogous to one proposed by Marks and Jones for voltage- dependent Ca channels (Marks, T. N., and S. W. Jones. 1992. Journal of General Physiology. 99:367-390). The allosteric protein is the calcium release channel, a homotetramer, with two accessible states, closed and open. The kinetics and equilibrium of this transition are modulated by voltage sensors (dihydropyridine receptors) pictured as four units per release channel, each undergoing independent voltage-driven transitions between two states (resting and activating). For each voltage sensor that moves to the activating state, the tendency of the channel to open increases by an equal (large) factor. The equilibrium and kinetic equations of the model are solved and shown to reproduce well a number of experimentally measured relationships including: charge movement (Q) vs. voltage, open probability of the release channel (Po) vs. voltage, the transfer function relationship Po vs. Q, and the kinetics of charge movement, release activation, and deactivation. The main consequence of the assumption of allosteric coupling is that primary effects on the release channel are transmitted backward to the voltage sensor and give secondary effects. Thus, the model reproduces well the effects of perchlorate, described in the two previous articles, under the assumption that the primary effect is to increase the intrinsic tendency of the release channel to open, with no direct effects on the voltage sensor. This modification of the open-closed equilibrium of the release channel causes a shift in the equilibrium dependency of charge movement with voltage. The paradoxical slowing of charge movement by perchlorate also results from reciprocal effects of the channel on the allosterically coupled voltage sensors. The observations of the previous articles plus the simulations in this article constitute functional evidence of allosteric transmission. PMID:8245819

Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor from the Active Site of O-Acetyl Serine Sulfhydrylase Reveals a Competitive-Allostery Mechanism.

PubMed

Singh, Appu Kumar; Ekka, Mary Krishna; Kaushik, Abhishek; Pandya, Vaibhav; Singh, Ravi P; Banerjee, Shrijita; Mittal, Monica; Singh, Vijay; Kumaran, S

2017-09-19

By classical competitive antagonism, a substrate and competitive inhibitor must bind mutually exclusively to the active site. The competitive inhibition of O-acetyl serine sulfhydrylase (OASS) by the C-terminus of serine acetyltransferase (SAT) presents a paradox, because the C-terminus of SAT binds to the active site of OASS with an affinity that is 4-6 log-fold (10 4 -10 6 ) greater than that of the substrate. Therefore, we employed multiple approaches to understand how the substrate gains access to the OASS active site under physiological conditions. Single-molecule and ensemble approaches showed that the active site-bound high-affinity competitive inhibitor is actively dissociated by the substrate, which is not consistent with classical views of competitive antagonism. We employed fast-flow kinetic approaches to demonstrate that substrate-mediated dissociation of full length SAT-OASS (cysteine regulatory complex) follows a noncanonical "facilitated dissociation" mechanism. To understand the mechanism by which the substrate induces inhibitor dissociation, we resolved the crystal structures of enzyme·inhibitor·substrate ternary complexes. Crystal structures reveal a competitive allosteric binding mechanism in which the substrate intrudes into the inhibitor-bound active site and disengages the inhibitor before occupying the site vacated by the inhibitor. In summary, here we reveal a new type of competitive allosteric binding mechanism by which one of the competitive antagonists facilitates the dissociation of the other. Together, our results indicate that "competitive allostery" is the general feature of noncanonical "facilitated/accelerated dissociation" mechanisms. Further understanding of the mechanistic framework of "competitive allosteric" mechanism may allow us to design a new family of "competitive allosteric drugs/small molecules" that will have improved selectivity and specificity as compared to their competitive and allosteric counterparts.

Allosteric modulation of sigma-1 receptors elicits anti-seizure activities.

PubMed

Guo, Lin; Chen, Yanke; Zhao, Rui; Wang, Guanghui; Friedman, Eitan; Zhang, Ao; Zhen, Xuechu

2015-08-01

Application of orthosteric sigma-1 receptor agonists as anti-seizure drugs has been hindered by questionable efficacy and potential adverse effects. Here, we have investigated the anti-seizure effects of the novel and potent allosteric modulator of sigma-1 receptors, SKF83959 and its derivative SOMCL-668 (3-methyl-phenyl-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-ol). The anti-seizure effects of SKF83959 were investigated in three mouse models, maximal electroshock seizures, pentylenetetrazole-induced convulsions and kainic acid-induced 'status epilepticus'. Also, in rats, the cortical epileptiform activity induced by topical application of picrotoxin was recorded in electrocorticograms. In rat hippocampal brain slices, effects of the drugs on the high potassium-evoked epileptiform local field potentials were studied. Anti-seizure activities of SOMCL-668, a newly developed sigma-1 receptor selective allosteric modulator, were also investigated. SKF83959 (20, 40 mg·kg(-1) ) exhibited anti -seizure actitity in the three mouse models and reduced the cortical epileptiform activity without alteration of spontaneous motor activity and motor coordination. These effects were blocked by the sigma-1 receptor antagonist BD1047, but not the dopamine D1 receptor antagonist SCH23390. SKF83959 alone did not directly inhibit the epileptiform firing of CA3 neurons induced by high potassium in hippocampal slices, but did potentiate inhibition by the orthosteric sigma-1 receptor agonist SKF10047. Lastly, a selective sigma-1 receptor allosteric modulator SOMCL-668, which does not bind to dopamine receptors, exerted similar anti-seizure activities. SKF83959 and SOMCL-668 displayed anti-seizure activities, indicating that allosteric modulation of sigma-1 receptors may provide a novel approach for discovering new anti-seizure drugs. © 2015 The British Pharmacological Society.

The interaction of escitalopram and R-citalopram at the human serotonin transporter investigated in the mouse.

PubMed

Jacobsen, Jacob P R; Plenge, Per; Sachs, Benjamin D; Pehrson, Alan L; Cajina, Manuel; Du, Yunzhi; Roberts, Wendy; Rudder, Meghan L; Dalvi, Prachiti; Robinson, Taylor J; O'Neill, Sharon P; Khoo, King S; Morillo, Connie Sanchez; Zhang, Xiaodong; Caron, Marc G

2014-12-01

Escitalopram appears to be a superior antidepressant to racemic citalopram. It has been hypothesized that binding of R-citalopram to the serotonin transporter (SERT) antagonizes escitalopram binding to and inhibition of the SERT, there by curtailing the elevation of extracellular 5-hydroxytryptamine (5-HTExt), and hence anti-depressant efficacy. Further, it has been suggested that a putative allosteric binding site is important for binding of escitalopram to the primary, orthosteric, site, and for R-citalopram's inhibition here of. Primary: Investigate at the human (h)SERT, at clinical relevant doses, whether R-citalopram antagonizes escitalopram-induced 5-HTExt elevation. Secondary: Investigate whether abolishing the putative allosteric site affects escitalopram-induced 5-HTExt elevation and/or modulates the effect of R-citalopram. Recombinant generation of hSERT transgenic mice; in vivo microdialysis; SERT binding; pharmacokinetics; 5-HT sensitive behaviors (tail suspension, marble burying). We generated mice expressing either the wild-type human SERT (hSERT(WT)) or hSERT carrying amino acid substitutions (A505V, L506F, I507L, S574T and I575T) collectively abolishing the putative allosteric site (hSERT(ALI/VFL+SI/TT)). One mg/kg escitalopram yielded clinical relevant plasma levels and brain levels consistent with therapeutic SERT occupancy. The hSERT mice showed normal basal 5-HTExt levels. Escitalopram-induced 5-HTExt elevation was not decreased by R-citalopram co-treatment and was unaffected by loss of the allosteric site. The behavioral effects of the clinically relevant escitalopram dose were small and tended to be enhanced by R-citalopram co-administration. We find no evidence that R-citalopram directly antagonizes escitalopram or that the putative allosteric site is important for hSERT inhibition by escitalopram.

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

PubMed

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

2017-10-01

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

Agonist activation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

PubMed Central

Gill, JasKiran K.; Savolainen, Mari; Young, Gareth T.; Zwart, Ruud; Sher, Emanuele; Millar, Neil S.

2011-01-01

Conventional nicotinic acetylcholine receptor (nAChR) agonists, such as acetylcholine, act at an extracellular “orthosteric” binding site located at the interface between two adjacent subunits. Here, we present evidence of potent activation of α7 nAChRs via an allosteric transmembrane site. Previous studies have identified a series of nAChR-positive allosteric modulators (PAMs) that lack agonist activity but are able to potentiate responses to orthosteric agonists, such as acetylcholine. It has been shown, for example, that TQS acts as a conventional α7 nAChR PAM. In contrast, we have found that a compound with close chemical similarity to TQS (4BP-TQS) is a potent allosteric agonist of α7 nAChRs. Whereas the α7 nAChR antagonist metyllycaconitine acts competitively with conventional nicotinic agonists, metyllycaconitine is a noncompetitive antagonist of 4BP-TQS. Mutation of an amino acid (M253L), located in a transmembrane cavity that has been proposed as being the binding site for PAMs, completely blocks agonist activation by 4BP-TQS. In contrast, this mutation had no significant effect on agonist activation by acetylcholine. Conversely, mutation of an amino acid located within the known orthosteric binding site (W148F) has a profound effect on agonist potency of acetylcholine (resulting in a shift of ∼200-fold in the acetylcholine dose-response curve), but had little effect on the agonist dose-response curve for 4BP-TQS. Computer docking studies with an α7 homology model provides evidence that both TQS and 4BP-TQS bind within an intrasubunit transmembrane cavity. Taken together, these findings provide evidence that agonist activation of nAChRs can occur via an allosteric transmembrane site. PMID:21436053

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

PubMed

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

2018-01-01

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

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.

The apelin receptor inhibits the angiotensin II type 1 receptor via allosteric trans-inhibition

PubMed Central

Siddiquee, K; Hampton, J; McAnally, D; May, LT; Smith, LH

2013-01-01

Background and Purpose The apelin receptor (APJ) is often co-expressed with the angiotensin II type-1 receptor (AT1) and acts as an endogenous counter-regulator. Apelin antagonizes Ang II signalling, but the precise molecular mechanism has not been elucidated. Understanding this interaction may lead to new therapies for the treatment of cardiovascular disease. Experimental Approach The physical interaction of APJ and AT1 receptors was detected by co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). Functional and pharmacological interactions were measured by G-protein-dependent signalling and recruitment of β-arrestin. Allosterism and cooperativity between APJ and AT1 were measured by radioligand binding assays. Key Results Apelin, but not Ang II, induced APJ : AT1 heterodimerization forced AT1 into a low-affinity state, reducing Ang II binding. Likewise, apelin mediated a concentration-dependent depression in the maximal production of inositol phosphate (IP1) and β-arrestin recruitment to AT1 in response to Ang II. The signal depression approached a limit, the magnitude of which was governed by the cooperativity indicative of a negative allosteric interaction. Fitting the data to an operational model of allosterism revealed that apelin-mediated heterodimerization significantly reduces Ang II signalling efficacy. These effects were not observed in the absence of apelin. Conclusions and Implications Apelin-dependent heterodimerization between APJ and AT1 causes negative allosteric regulation of AT1 function. As AT1 is significant in the pathogenesis of cardiovascular disease, these findings suggest that impaired apelin and APJ function may be a common underlying aetiology. Linked Article This article is commented on by Goupil et al., pp. 1101–1103 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12040 PMID:22935142

Evolution of allosteric regulation in chorismate mutases from early plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

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

PubMed

Jiang, Ludi; Zhang, Xianbao; Chen, Xi; He, Yusu; Qiao, Liansheng; Zhang, Yanling; Li, Gongyu; Xiang, Yuhong

2015-07-15

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

The Hsp70 interdomain linker is a dynamic switch that enables allosteric communication between two structured domains.

PubMed

English, Charles A; Sherman, Woody; Meng, Wenli; Gierasch, Lila M

2017-09-08

Hsp70 molecular chaperones play key roles in cellular protein homeostasis by binding to exposed hydrophobic regions of incompletely folded or aggregated proteins. This crucial Hsp70 function relies on allosteric communication between two well-structured domains: an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD), which are tethered by an interdomain linker. ATP or ADP binding to the NBD alters the substrate-binding affinity of the SBD, triggering functionally essential cycles of substrate binding and release. The interdomain linker is a well-structured participant in the interdomain interface in ATP-bound Hsp70s. By contrast, in the ADP-bound state, exemplified by the Escherichia coli Hsp70 DnaK, the interdomain linker is flexible. Hsp70 interdomain linker sequences are highly conserved; moreover, mutations in this region compromise interdomain allostery. To better understand the role of this region in Hsp70 allostery, we used molecular dynamics simulations to explore the conformational landscape of the interdomain linker in ADP-bound DnaK and supported our simulations by strategic experimental data. We found that while the interdomain linker samples many conformations, it behaves as three relatively ordered segments connected by hinges. As a consequence, the distances and orientations between the NBD and SBD are limited. Additionally, the C-terminal region of the linker forms previously unreported, transient interactions with the SBD, and the predominant linker-docking site is available in only one allosteric state, that with high affinity for substrate. This preferential binding implicates the interdomain linker as a dynamic allosteric switch. The linker-binding site on the SBD is a potential target for small molecule modulators of the Hsp70 allosteric cycle. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Targeting the Allosteric Site of Oncoprotein BCR-ABL as an Alternative Strategy for Effective Target Protein Degradation.

PubMed

Shimokawa, Kenichiro; Shibata, Norihito; Sameshima, Tomoya; Miyamoto, Naoki; Ujikawa, Osamu; Nara, Hiroshi; Ohoka, Nobumichi; Hattori, Takayuki; Cho, Nobuo; Naito, Mikihiko

2017-10-12

Protein degradation technology based on hybrid small molecules is an emerging drug modality that has significant potential in drug discovery and as a unique method of post-translational protein knockdown in the field of chemical biology. Here, we report the first example of a novel and potent protein degradation inducer that binds to an allosteric site of the oncogenic BCR-ABL protein. BCR-ABL allosteric ligands were incorporated into the SNIPER (Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers) platform, and a series of in vitro biological assays of binding affinity, target protein modulation, signal transduction, and growth inhibition were carried out. One of the designed compounds, 6 (SNIPER(ABL)-062), showed desirable binding affinities against ABL1, cIAP1/2, and XIAP and consequently caused potent BCR-ABL degradation.

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

PubMed

Fay, Jonathan F; Farrens, David L

2012-09-28

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

Regulatory properties of 6-phosphofructokinase and control of glycolysis in boar spermatozoa.

PubMed

Kamp, G; Schmidt, H; Stypa, H; Feiden, S; Mahling, C; Wegener, G

2007-01-01

Glycolysis is crucial for sperm functions (motility and fertilization), but how this pathway is regulated in spermatozoa is not clear. This prompted to study the location and the regulatory properties of 6-phosphofructokinase (PFK, EC 2.7.1.11), the most important element for control of glycolytic flux. Unlike some other glycolytic enzymes, PFK showed no tight binding to sperm structures. It could readily be extracted from ejaculated boar spermatozoa by sonication and was then chromatographically purified. At physiological pH, the enzyme was allosterically inhibited by near-physiological concentrations of its co-substrate ATP, which induced co-operativity, i.e. reduced the affinity for the substrate fructose 6-phosphate. Inhibition by ATP was reinforced by citrate and H+. Above pH 8, PFK lost all its regulatory properties and showed maximum activity. However, in the physiological pH range, PFK activity was very sensitive to small changes in effectors. At near-physiological substrate concentrations, PFK activity requires activators (de-inhibitors) of which the combination of AMP and fructose 2,6-bisphosphate (F2,6P2) was most efficient as a result of synergistic effects. The kinetics of PFK suggest AMP, F2,6P2, H+, and citrate as allosteric effectors controlling PFK activity in boar spermatozoa. Using immunogold labeling, PFK was localized in the mid-piece and principal piece of the flagellum as well as in the acrosomal area at the top of the head and in the cytoplasmic droplets released from the mid-piece after ejaculation.

IP3-mediated gating mechanism of the IP3 receptor revealed by mutagenesis and X-ray crystallography.

PubMed

Hamada, Kozo; Miyatake, Hideyuki; Terauchi, Akiko; Mikoshiba, Katsuhiko

2017-05-02

The inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R) is an IP 3 -gated ion channel that releases calcium ions (Ca 2+ ) from the endoplasmic reticulum. The IP 3 -binding sites in the large cytosolic domain are distant from the Ca 2+ conducting pore, and the allosteric mechanism of how IP 3 opens the Ca 2+ channel remains elusive. Here, we identify a long-range gating mechanism uncovered by channel mutagenesis and X-ray crystallography of the large cytosolic domain of mouse type 1 IP 3 R in the absence and presence of IP 3 Analyses of two distinct space group crystals uncovered an IP 3 -dependent global translocation of the curvature α-helical domain interfacing with the cytosolic and channel domains. Mutagenesis of the IP 3 R channel revealed an essential role of a leaflet structure in the α-helical domain. These results suggest that the curvature α-helical domain relays IP 3 -controlled global conformational dynamics to the channel through the leaflet, conferring long-range allosteric coupling from IP 3 binding to the Ca 2+ channel.

GABAB receptor ligands for the treatment of alcohol use disorder: preclinical and clinical evidence

PubMed Central

Agabio, Roberta; Colombo, Giancarlo

2014-01-01

The present paper summarizes the preclinical and clinical studies conducted to define the “anti-alcohol” pharmacological profile of the prototypic GABAB receptor agonist, baclofen, and its therapeutic potential for treatment of alcohol use disorder (AUD). Numerous studies have reported baclofen-induced suppression of alcohol drinking (including relapse- and binge-like drinking) and alcohol reinforcing, motivational, stimulating, and rewarding properties in rodents and monkeys. The majority of clinical surveys conducted to date—including case reports, retrospective chart reviews, and randomized placebo-controlled studies—suggest the ability of baclofen to suppress alcohol consumption, craving for alcohol, and alcohol withdrawal symptomatology in alcohol-dependent patients. The recent identification of a positive allosteric modulatory binding site, together with the synthesis of in vivo effective ligands, represents a novel, and likely more favorable, option for pharmacological manipulations of the GABAB receptor. Accordingly, data collected to date suggest that positive allosteric modulators of the GABAB receptor reproduce several “anti-alcohol” effects of baclofen and display a higher therapeutic index (with larger separation—in terms of doses—between “anti-alcohol” effects and sedation). PMID:24936171

Allosteric mechanism controls traffic in the chaperone/usher pathway.

PubMed

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

2012-11-07

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leonardi, Roberta; Zhang, Yong-Mei; Yun, Mi-Kyung

2010-09-27

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

Mechanism of UCH-L5 Activation and Inhibition by DEUBAD Domains in RPN13 and INO80G

PubMed Central

Sahtoe, Danny D.; van Dijk, Willem J.; El Oualid, Farid; Ekkebus, Reggy; Ovaa, Huib; Sixma, Titia K.

2015-01-01

Summary Deubiquitinating enzymes (DUBs) control vital processes in eukaryotes by hydrolyzing ubiquitin adducts. Their activities are tightly regulated, but the mechanisms remain elusive. In particular, the DUB UCH-L5 can be either activated or inhibited by conserved regulatory proteins RPN13 and INO80G, respectively. Here we show how the DEUBAD domain in RPN13 activates UCH-L5 by positioning its C-terminal ULD domain and crossover loop to promote substrate binding and catalysis. The related DEUBAD domain in INO80G inhibits UCH-L5 by exploiting similar structural elements in UCH-L5 to promote a radically different conformation, and employs molecular mimicry to block ubiquitin docking. In this process, large conformational changes create small but highly specific interfaces that mediate activity modulation of UCH-L5 by altering the affinity for substrates. Our results establish how related domains can exploit enzyme conformational plasticity to allosterically regulate DUB activity. These allosteric sites may present novel insights for pharmaceutical intervention in DUB activity. PMID:25702870

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…

Vibrational resonance, allostery, and activation in rhodopsin-like G protein-coupled receptors

PubMed Central

Woods, Kristina N.; Pfeffer, Jürgen; Dutta, Arpana; Klein-Seetharaman, Judith

2016-01-01

G protein-coupled receptors are a large family of membrane proteins activated by a variety of structurally diverse ligands making them highly adaptable signaling molecules. Despite recent advances in the structural biology of this protein family, the mechanism by which ligands induce allosteric changes in protein structure and dynamics for its signaling function remains a mystery. Here, we propose the use of terahertz spectroscopy combined with molecular dynamics simulation and protein evolutionary network modeling to address the mechanism of activation by directly probing the concerted fluctuations of retinal ligand and transmembrane helices in rhodopsin. This approach allows us to examine the role of conformational heterogeneity in the selection and stabilization of specific signaling pathways in the photo-activation of the receptor. We demonstrate that ligand-induced shifts in the conformational equilibrium prompt vibrational resonances in the protein structure that link the dynamics of conserved interactions with fluctuations of the active-state ligand. The connection of vibrational modes creates an allosteric association of coupled fluctuations that forms a coherent signaling pathway from the receptor ligand-binding pocket to the G-protein activation region. Our evolutionary analysis of rhodopsin-like GPCRs suggest that specific allosteric sites play a pivotal role in activating structural fluctuations that allosterically modulate functional signals. PMID:27849063

Discovery of Allosteric and Selective Inhibitors of Inorganic Pyrophosphatase from Mycobacterium tuberculosis.

PubMed

Pang, Allan H; Garzan, Atefeh; Larsen, Martha J; McQuade, Thomas J; Garneau-Tsodikova, Sylvie; Tsodikov, Oleg V

2016-11-18

Inorganic pyrophosphatase (PPiase) is an essential enzyme that hydrolyzes inorganic pyrophosphate (PP i ), driving numerous metabolic processes. We report a discovery of an allosteric inhibitor (2,4-bis(aziridin-1-yl)-6-(1-phenylpyrrol-2-yl)-s-triazine) of bacterial PPiases. Analogues of this lead compound were synthesized to target specifically Mycobacterium tuberculosis (Mtb) PPiase (MtPPiase). The best analogue (compound 16) with a K i of 11 μM for MtPPiase is a species-specific inhibitor. Crystal structures of MtPPiase in complex with the lead compound and one of its analogues (compound 6) demonstrate that the inhibitors bind in a nonconserved interface between monomers of the hexameric MtPPiase in a yet unprecedented pairwise manner, while the remote conserved active site of the enzyme is occupied by a bound PP i substrate. Consistent with the structural studies, the kinetic analysis of the most potent inhibitor has indicated that it functions uncompetitively, by binding to the enzyme-substrate complex. The inhibitors appear to allosterically lock the active site in a closed state causing its dysfunctionalization and blocking the hydrolysis. These inhibitors are the first examples of allosteric, species-selective inhibitors of PPiases, serving as a proof-of-principle that PPiases can be selectively targeted.

A comparative review of escitalopram, paroxetine, and sertraline: are they all alike?

PubMed Central

Reines, Elin H.; Montgomery, Stuart A.

2014-01-01

It is known that newer antidepressants, such as the selective serotonin reuptake inhibitors (SSRIs), provide advantages in tolerability over antidepressants such as the tricyclics. However, even within the SSRI class, differences in efficacy or tolerability exist between the individual drugs. Among the three most widely prescribed SSRIs are paroxetine, sertraline, and escitalopram. Escitalopram is commonly referred to as an SSRI, but also has well-documented allosteric properties, and thus can be further classed as an allosteric serotonin reuptake inhibitor. All three antidepressants are efficacious compared with placebo, but there is evidence that escitalopram is more effective than a range of other antidepressants. There are no direct data to regard either paroxetine or sertraline as a superior antidepressant. Escitalopram is superior compared with paroxetine, which has a less favorable tolerability profile. Paroxetine is associated with cholinergic muscarinic antagonism and potent inhibition of CYP2D6, and sertraline has moderate drug interaction issues in comparison with escitalopram. Overall, as an allosteric serotonin reuptake inhibitor that is somewhat different from classical SSRIs, escitalopram is the first choice judged by combined efficacy and tolerability, and nonclinical data have offered possible mechanisms through which escitalopram could be more efficacious, based on its interaction with orthosteric and allosteric binding sites at the serotonin transporter. PMID:24424469

A comparative review of escitalopram, paroxetine, and sertraline: Are they all alike?

PubMed

Sanchez, Connie; Reines, Elin H; Montgomery, Stuart A

2014-07-01

It is known that newer antidepressants, such as the selective serotonin reuptake inhibitors (SSRIs), provide advantages in tolerability over antidepressants such as the tricyclics. However, even within the SSRI class, differences in efficacy or tolerability exist between the individual drugs. Among the three most widely prescribed SSRIs are paroxetine, sertraline, and escitalopram. Escitalopram is commonly referred to as an SSRI, but also has well-documented allosteric properties, and thus can be further classed as an allosteric serotonin reuptake inhibitor. All three antidepressants are efficacious compared with placebo, but there is evidence that escitalopram is more effective than a range of other antidepressants. There are no direct data to regard either paroxetine or sertraline as a superior antidepressant. Escitalopram is superior compared with paroxetine, which has a less favorable tolerability profile. Paroxetine is associated with cholinergic muscarinic antagonism and potent inhibition of CYP2D6, and sertraline has moderate drug interaction issues in comparison with escitalopram. Overall, as an allosteric serotonin reuptake inhibitor that is somewhat different from classical SSRIs, escitalopram is the first choice judged by combined efficacy and tolerability, and nonclinical data have offered possible mechanisms through which escitalopram could be more efficacious, based on its interaction with orthosteric and allosteric binding sites at the serotonin transporter.

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

PubMed

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

2017-08-16

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

An atomistic view of Hsp70 allosteric crosstalk: from the nucleotide to the substrate binding domain and back

PubMed Central

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

2016-01-01

The Hsp70 is an allosterically regulated family of molecular chaperones. They consist of two structural domains, NBD and SBD, connected by a flexible linker. ATP hydrolysis at the NBD modulates substrate recognition at the SBD, while peptide binding at the SBD enhances ATP hydrolysis. In this study we apply Molecular Dynamics (MD) to elucidate the molecular determinants underlying the allosteric communication from the NBD to the SBD and back. We observe that local structural and dynamical modulation can be coupled to large-scale rearrangements, and that different combinations of ligands at NBD and SBD differently affect the SBD domain mobility. Substituting ADP with ATP in the NBD induces specific structural changes involving the linker and the two NBD lobes. Also, a SBD-bound peptide drives the linker docking by increasing the local dynamical coordination of its C-terminal end: a partially docked DnaK structure is achieved by combining ATP in the NBD and peptide in the SBD. We propose that the MD-based analysis of the inter domain dynamics and structure modulation could be used as a tool to computationally predict the allosteric behaviour and functional response of Hsp70 upon introducing mutations or binding small molecules, with potential applications for drug discovery. PMID:27025773

Electrostatic Interactions as Mediators in the Allosteric Activation of Protein Kinase A RIα.

PubMed

P Barros, Emília; Malmstrom, Robert D; Nourbakhsh, Kimya; Del Rio, Jason C; Kornev, Alexandr P; Taylor, Susan S; Amaro, Rommie E

2017-03-14

Close-range electrostatic interactions that form salt bridges are key components of protein stability. Here we investigate the role of these charged interactions in modulating the allosteric activation of protein kinase A (PKA) via computational and experimental mutational studies of a conserved basic patch located in the regulatory subunit's B/C helix. Molecular dynamics simulations evidenced the presence of an extended network of fluctuating salt bridges spanning the helix and connecting the two cAMP binding domains in its extremities. Distinct changes in the flexibility and conformational free energy landscape induced by the separate mutations of Arg239 and Arg241 suggested alteration of cAMP-induced allosteric activation and were verified through in vitro fluorescence polarization assays. These observations suggest a mechanical aspect to the allosteric transition of PKA, with Arg239 and Arg241 acting in competition to promote the transition between the two protein functional states. The simulations also provide a molecular explanation for the essential role of Arg241 in allowing cooperative activation, by evidencing the existence of a stable interdomain salt bridge with Asp267. Our integrated approach points to the role of salt bridges not only in protein stability but also in promoting conformational transition and function.

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

An allosteric Akt inhibitor effectively blocks Akt signaling and tumor growth with only transient effects on glucose and insulin levels in vivo

PubMed Central

Cherrin, Craig; Haskell, Kathleen; Howell, Bonnie; Jones, Raymond; Leander, Karen; Robinson, Ronald; Watkins, Aubrey; Bilodeau, Mark; Hoffman, Jacob; Sanderson, Philip; Hartman, George; Mahan, Elizabeth; Prueksaritanont, Thomayant; Jiang, Guoqiang; She, Qing-Bai; Rosen, Neal; Sepp-Lorenzino, Laura; Defeo-Jones, Deborah; Huber, Hans E.

2010-01-01

The PI3K-Akt pathway is dysregulated in the majority of solid tumors. Pharmacological inhibition of Akt is a promising strategy for treating tumors resistant to growth factor receptor antagonists due to mutations in PI3K or PTEN. We have developed allosteric, isozyme-specific inhibitors of Akt activity and activation, as well as ex vivo kinase assays to measure inhibition of individual Akt isozymes in tissues. Here we describe the relationship between PK, Akt inhibition, hyperglycemia and tumor efficacy for a selective inhibitor of Akt1 and Akt2 (AKTi). In nude mice, AKTi treatment caused transient insulin resistance and reversible, dose-dependent hyperglycemia and hyperinsulinemia. Akt1 and Akt2 phosphorylation was inhibited in mouse lung with EC50 values of 1.6 and 7 μM, respectively, and with similar potency in other tissues and xenograft tumors. Weekly subcutaneous dosing of AKTi resulted in dose-dependent inhibition of LNCaP prostate cancer xenografts, an AR-dependent tumor with PTEN deletion and constitutively activated Akt. Complete tumor growth inhibition was achieved at 200 mpk, a dose that maintained inhibition of Akt1 and Akt2 of greater than 80% and 50%, respectively, for at least 12 hours in xenograft tumor and mouse lung. Hyperglycemia could be controlled by reducing Cmax, while maintaining efficacy in the LNCaP model, but not by insulin administration. AKTi treatment was well tolerated, without weight loss or gross toxicities. These studies supported the rationale for clinical development of allosteric Akt inhibitors and provide the basis for further refining of pharmacokinetic properties and dosing regimens of this class of inhibitors. PMID:20139722

Dependence of prevalence of contiguous pathways in proteins on structural complexity.

PubMed

Thayer, Kelly M; Galganov, Jesse C; Stein, Avram J

2017-01-01

Allostery is a regulatory mechanism in proteins where an effector molecule binds distal from an active site to modulate its activity. Allosteric signaling may occur via a continuous path of residues linking the active and allosteric sites, which has been suggested by large conformational changes evident in crystal structures. An alternate possibility is that the signal occurs in the realm of ensemble dynamics via an energy landscape change. While the latter was first proposed on theoretical grounds, increasing evidence suggests that such a control mechanism is plausible. A major difficulty for testing the two methods is the ability to definitively determine that a residue is directly involved in allosteric signal transduction. Statistical Coupling Analysis (SCA) is a method that has been successful at predicting pathways, and experimental tests involving mutagenesis or domain substitution provide the best available evidence of signaling pathways. However, ascertaining energetic pathways which need not be contiguous is far more difficult. To date, simple estimates of the statistical significance of a pathway in a protein remain to be established. The focus of this work is to estimate such benchmarks for the statistical significance of contiguous pathways for the null model of selecting residues at random. We found that when 20% of residues in proteins are randomly selected, contiguous pathways at the 6 Å cutoff level were found with success rates of 51% in PDZ, 30% in p53, and 3% in MutS. The results suggest that the significance of pathways may have system specific factors involved. Furthermore, the possible existence of false positives for contiguous pathways implies that signaling could be occurring via alternate routes including those consistent with the energetic landscape model.

Identical linkage and cooperativity of oxygen and carbon monoxide binding to Octopus dofleini hemocyanin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Connelly, P.R.; Gill, S.J.; Miller, K.I.

1989-02-21

Employment of high-precision thin-layer methods has enabled detailed functional characterization of oxygen and carbon monoxide binding for (1) the fully assembled form with 70 binding sites and (2) the isolated chains with 7 binding sites of octopus dofleini hemocyanin. The striking difference in the cooperativities of the two ligands for the assembled decamer is revealed through an examination of the binding capacities and the partition coefficient, determined as functions of the activities of both ligands. A global analysis of the data sets supported by a two-state allosteric model assuming an allosteric unit of 7. Higher level allosteric interactions were notmore » indicated. This contrasts to results obtained for arthropod hemocyanins. Oxygen and carbon monoxide experiments performed on the isolated subunit chain confirmed the presence of functional heterogeneity reported previously. The analysis shows two types of binding sites in the ratio of 4:3.« less

Allosteric Regulation of E-Cadherin Adhesion*

PubMed Central

Shashikanth, Nitesh; Petrova, Yuliya I.; Park, Seongjin; Chekan, Jillian; Maiden, Stephanie; Spano, Martha; Ha, Taekjip; Gumbiner, Barry M.; Leckband, Deborah E.

2015-01-01

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120ctn, increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120ctn dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120ctn dephosphorylation. PMID:26175155

Site-Mutation of Hydrophobic Core Residues Synchronically Poise Super Interleukin 2 for Signaling: Identifying Distant Structural Effects through Affordable Computations.

PubMed

Mei, Longcan; Zhou, Yanping; Zhu, Lizhe; Liu, Changlin; Wu, Zhuo; Wang, Fangkui; Hao, Gefei; Yu, Di; Yuan, Hong; Cui, Yanfang

2018-03-20

A superkine variant of interleukin-2 with six site mutations away from the binding interface developed from the yeast display technique has been previously characterized as undergoing a distal structure alteration which is responsible for its super-potency and provides an elegant case study with which to get insight about how to utilize allosteric effect to achieve desirable protein functions. By examining the dynamic network and the allosteric pathways related to those mutated residues using various computational approaches, we found that nanosecond time scale all-atom molecular dynamics simulations can identify the dynamic network as efficient as an ensemble algorithm. The differentiated pathways for the six core residues form a dynamic network that outlines the area of structure alteration. The results offer potentials of using affordable computing power to predict allosteric structure of mutants in knowledge-based mutagenesis.

Universal fieldable assay with unassisted visual detection

NASA Technical Reports Server (NTRS)

Chelyapov, Nicolas (Inventor)

2012-01-01

A universal detection system based on allosteric aptamers, signal amplification cascade, and eye-detectable phrase transition. A broadly applicable homogeneous detection system is provided. It utilizes components of the blood coagulation cascade in the presence of polystyrene microspheres (MS) as a signal amplifier. Russell's viper venom factor X activator (RVV-X) triggers the cascade, which results in an eye-visible phase transition--precipitation of MS bound to clotted fibrin. An allosteric RNA aptamer, RNA132, with affinity for RVV-X and human vascular endothelial growth factor (VEGF.sub.165) was created. RNA132 inhibits enzymatic activity of RVV-X. The effector molecule, VEGF.sub.165, reverses the inhibitory activity of RNA132 on RVV-X and restores its enzymatic activity, thus triggering the cascade and enabling the phase transition. Similar results were obtained for another allosteric aptamer modulated by a protein tyrosine phosphatase. The assay is instrumentation-free for both processing and readout.

Solution NMR Spectroscopy for the Study of Enzyme Allostery

PubMed Central

Lisi, George P.; Loria, J. Patrick

2016-01-01

Allostery is a ubiquitous biological regulatory process in which distant binding sites within a protein or enzyme are functionally and thermodynamically coupled. Allosteric interactions play essential roles in many enzymological mechanisms, often facilitating formation of enzyme-substrate complexes and/or product release. Thus, elucidating the forces that drive allostery is critical to understanding the complex transformations of biomolecules. Currently, a number of models exist to describe allosteric behavior, taking into account energetics as well as conformational rearrangements and fluctuations. In the following review, we discuss the use of solution NMR techniques designed to probe allosteric mechanisms in enzymes. NMR spectroscopy is unequaled in its ability to detect structural and dynamical changes in biomolecules, and the case studies presented herein demonstrate the range of insights to be gained from this valuable method. We also provide a detailed technical discussion of several specialized NMR experiments that are ideally suited for the study of enzymatic allostery. PMID:26734986

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.

Crystal structures of C4 form maize and quaternary complex of E. coli phosphoenolpyruvate carboxylases.

PubMed

Matsumura, Hiroyoshi; Xie, Yong; Shirakata, Shunsuke; Inoue, Tsuyoshi; Yoshinaga, Takeo; Ueno, Yoshihisa; Izui, Katsura; Kai, Yasushi

2002-12-01

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the first step in the fixation of atmospheric CO(2) during C(4) photosynthesis. The crystal structure of C(4) form maize PEPC (ZmPEPC), the first structure of the plant PEPCs, has been determined at 3.0 A resolution. The structure includes a sulfate ion at the plausible binding site of an allosteric activator, glucose 6-phosphate. The crystal structure of E. coli PEPC (EcPEPC) complexed with Mn(2+), phosphoenolpyruvate analog (3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate), and an allosteric inhibitor, aspartate, has also been determined at 2.35 A resolution. Dynamic movements were found in the ZmPEPC structure, compared with the EcPEPC structure, around two loops near the active site. On the basis of these molecular structures, the mechanisms for the carboxylation reaction and for the allosteric regulation of PEPC are proposed.

An allosteric photoredox catalyst inspired by photosynthetic machinery

PubMed Central

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

2015-01-01

Biological photosynthetic machinery allosterically regulate light harvesting via conformational and electronic changes at the antenna protein complexes as a response to specific chemical inputs. Fundamental limitations in current approaches to regulating inorganic light-harvesting mimics prevent their use in catalysis. Here we show that a light-harvesting antenna/reaction centre mimic can be regulated by utilizing a coordination framework incorporating antenna hemilabile ligands and assembled via a high-yielding, modular approach. As in nature, allosteric regulation is afforded by coupling the conformational changes to the disruptions in the electrochemical landscape of the framework upon recognition of specific coordinating analytes. The hemilabile ligands enable switching using remarkably mild and redox-inactive inputs, allowing one to regulate the photoredox catalytic activity of the photosynthetic mimic reversibly and in situ. Thus, we demonstrate that bioinspired regulatory mechanisms can be applied to inorganic light-harvesting arrays displaying switchable catalytic properties and with potential uses in solar energy conversion and photonic devices. PMID:25817586

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

NASA Astrophysics Data System (ADS)

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

2016-09-01

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

A Chimeric Kinesin-1 Head/Kinesin-5 Tail Motor Switches between Diffusive and Processive Motility

PubMed Central

Thiede, Christina; Lakämper, Stefan; Wessel, Alok D.; Kramer, Stefanie; Schmidt, Christoph F.

2013-01-01

Homotetrameric kinesin-5 motors are essential for chromosome separation and assembly of the mitotic spindle. These kinesins bind between two microtubules (MTs) and slide them apart, toward the spindle poles. This process must be tightly regulated in mitosis. In in vitro assays, Eg5 moves diffusively on single MTs and switches to a directed mode between MTs. How allosteric communication between opposing motor domains works remains unclear, but kinesin-5 tail domains may be involved. Here we present a single-molecule fluorescence study of a tetrameric kinesin-1 head/kinesin-5 tail chimera, DK4mer. This motor exhibited fast processive motility on single MTs interrupted by pauses. Like Eg5, DK4mer diffused along MTs with ADP, and slid antiparallel MTs apart with ATP. In contrast to Eg5, diffusive and processive periods were clearly distinguishable. This allowed us to measure transition rates among states and for unbinding as a function of buffer ionic strength. These data, together with results from controls using tail-less dimers, indicate that there are two modes of interaction with MTs, separated by an energy barrier. This result suggests a scheme of motor regulation that involves switching between two bound states, possibly allosterically controlled by the opposing tetramer end. Such a scheme is likely to be relevant for the regulation of native kinesin-5 motors. PMID:23442865

Accelerated Disassembly of IgE:Receptor Complexes by a Disruptive Macromolecular Inhibitor

PubMed Central

Kim, Beomkyu; Eggel, Alexander; Tarchevskaya, Svetlana S.; Vogel, Monique; Prinz, Heino; Jardetzky, Theodore S.

2012-01-01

IgE antibodies bind the high affinity IgE Fc receptor (FcεRI), found primarily on mast cells and basophils, and trigger inflammatory cascades of the allergic response1,2. Inhibitors of IgE:FcεRI binding have been identified and an anti-IgE therapeutic antibody (omalizumab) is used to treat severe allergic asthma3,4. However, preformed IgE:FcεRI complexes that prime cells prior to allergen exposure dissociate extremely slowly5 and cannot be disrupted by strictly competitive inhibitors. IgE-Fc conformational flexibility indicated that inhibition could be mediated by allosteric or other non-classical mechanisms6–8. Here we demonstrate that an engineered protein inhibitor, DARPin E2_799–11, acts through a non-classical inhibition mechanism, not only blocking IgE:FcεRI interactions, but actively stimulating the dissociation of preformed ligand-receptor complexes. The structure of the E2_79:IgE-Fc3-4 complex predicts the presence of two non-equivalent E2_79 sites in the asymmetric IgE:FcεRI complex, with Site 1 distant from the receptor and Site 2 exhibiting partial steric overlap. While the structure is suggestive of an allosteric inhibition mechanism, mutational studies and quantitative kinetic modeling indicate that E2_79 acts through a facilitated dissociation mechanism at Site 2 alone. These results demonstrate that high affinity IgE:FcεRI complexes can be actively dissociated to block the allergic response and suggest that protein:protein complexes may be more generally amenable to active disruption by macromolecular inhibitors. PMID:23103871

Inherent flexibility determines the transition mechanisms of the EF-hands of calmodulin.

PubMed

Tripathi, Swarnendu; Portman, John J

2009-02-17

We explore how inherent flexibility of a protein molecule influences the mechanism controlling allosteric transitions by using a variational model inspired from work in protein folding. The striking differences in the predicted transition mechanism for the opening of the two domains of calmodulin (CaM) emphasize that inherent flexibility is key to understanding the complex conformational changes that occur in proteins. In particular, the C-terminal domain of CaM (cCaM), which is inherently less flexible than its N-terminal domain (nCaM), reveals "cracking" or local partial unfolding during the open/closed transition. This result is in harmony with the picture that cracking relieves local stresses caused by conformational deformations of a sufficiently rigid protein. We also compare the conformational transition in a recently studied even-odd paired fragment of CaM. Our results rationalize the different relative binding affinities of the EF-hands in the engineered fragment compared with the intact odd-even paired EF-hands (nCaM and cCaM) in terms of changes in flexibility along the transition route. Aside from elucidating general theoretical ideas about the cracking mechanism, these studies also emphasize how the remarkable intrinsic plasticity of CaM underlies conformational dynamics essential for its diverse functions.

Discovery of 1,5-Disubstituted Pyridones: A New Class of Positive Allosteric Modulators of the Metabotropic Glutamate 2 Receptor

PubMed Central

2010-01-01

A series of 1,5-disubstituted pyridones was identified as positive allosteric modulators (PAMs) of the metabotropic glutamate receptor 2 (mGluR2) via high throughput screening (HTS). Subsequent SAR exploration led to the identification of several compounds with improved in vitro activity. Lead compound 8 was further profiled and found to attenuate the increase in PCP induced locomotor activity in mice. PMID:22778815

Discovery of Intramolecular Signal Transduction Network Based on a New Protein Dynamics Model of Energy Dissipation

PubMed Central

Ma, Cheng-Wei; Xiu, Zhi-Long; Zeng, An-Ping

2012-01-01

A novel approach to reveal intramolecular signal transduction network is proposed in this work. To this end, a new algorithm of network construction is developed, which is based on a new protein dynamics model of energy dissipation. A key feature of this approach is that direction information is specified after inferring protein residue-residue interaction network involved in the process of signal transduction. This enables fundamental analysis of the regulation hierarchy and identification of regulation hubs of the signaling network. A well-studied allosteric enzyme, E. coli aspartokinase III, is used as a model system to demonstrate the new method. Comparison with experimental results shows that the new approach is able to predict all the sites that have been experimentally proved to desensitize allosteric regulation of the enzyme. In addition, the signal transduction network shows a clear preference for specific structural regions, secondary structural types and residue conservation. Occurrence of super-hubs in the network indicates that allosteric regulation tends to gather residues with high connection ability to collectively facilitate the signaling process. Furthermore, a new parameter of propagation coefficient is defined to determine the propagation capability of residues within a signal transduction network. In conclusion, the new approach is useful for fundamental understanding of the process of intramolecular signal transduction and thus has significant impact on rational design of novel allosteric proteins. PMID:22363664

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.

Mn²⁺/Mg ²⁺-dependent pyruvate kinase from a D-lactic acid-producing bacterium Sporolactobacillus inulinus: characterization of a novel Mn²⁺-mediated allosterically regulated enzyme.

PubMed

Zheng, Lu; Xu, Tingting; Bai, Zhongzhong; He, Bingfang

2014-02-01

Sporolactobacillus inulinus has attracted scientific and commercial interest due to its high efficiency in D-lactic acid production. Pyruvate kinase (PYK) is one of the key regulatory points in glycolysis, and well-activated PYK can improve D-lactic acid production. A novel Mn(2+)/Mg(2+)-dependent PYK from S. inulinus was expressed in Escherichia coli and purified to homogeneity. Kinetic characterization demonstrated that the S. inulinus PYK had drastically higher activity and affinity toward substrates in the presence of Mn(2+) compared to those of the common PYK cofactor Mg(2+), and the circular dichroism spectra of the S. inulinus PYK suggested a Mn(2+)-mediated allosteric activation. The S. inulinus PYK was also allosterically regulated by ribose-5-phosphate or AMP activation and inorganic phosphate or ATP inhibition. The inhibition could be marked reduced or fully eliminated in the presence of activators. The result of fermentations by S. inulinus Y2-8 showed that the extracellular-added MnSO₄ and KH₂PO₄ significantly affected glycolysis flux and D-lactic acid production, which is consistent with the allosteric regulation of Mn(2+) and inorganic phosphate on PYK. The sophisticated regulatory role of PYK would establish the foundation of substantial disturbance or restructuring of cellular metabolism for improving the S. inulinus D-lactic acid production.

An allosteric antibody to the leptin receptor reduces body weight and reverses the diabetic phenotype in the Lep(ob) /Lep(ob) mouse.

PubMed

Bhaskar, Vinay; Goldfine, Ira D; Gerstner, Resi; Michelson, Kristen; Tran, Catarina; Nonet, Genevieve; Bohmann, David; Pongo, Elizabeth; Zhao, Jingsong; Horwitz, Arnold H; Takeuchi, Toshihiko; White, Mark; Corbin, John A

2016-08-01

Leptin (LEP) deficiency results in major metabolic perturbations, including obesity, dyslipidemia, and diabetes. Although LEP deficiency can be treated with daily injections of a recombinant LEP, generation of an antibody activating the LEP receptor (LEPR) that has both an intrinsically long half-life and low immunogenicity could be useful in the treatment of this condition. Phage display technology coupled with flow cytometry and cell-based in vitro assays were employed to identify an allosteric agonist of the mouse LEPR. LEP-deficient Lep(ob) /Lep(ob) mice were used to compare in vivo effects of LEP to antibody administration. To evaluate hypothalamic effects of treatment, changes in mRNA levels of neuropeptide Y and proopiomelanocortin were measured. XPA.80.037 is a monoclonal antibody that demonstrates allosteric agonism of the mouse LEPR. Treatment of Lep(ob) /Lep(ob) mice with XPA.80.037 markedly reduced hyperphagia and body weight, normalized blood glucose and plasma insulin levels, and corrected dyslipidemia. These metabolic alterations correlated with changes in mRNA levels of neuropeptide Y and proopiomelanocortin, suggesting that XPA.80.037 had hypothalamic effects. Agonist allosteric monoclonal antibodies to the LEPR can correct metabolic effects associated with LEP deficiency in vivo and thereby have the potential to treat conditions of LEP deficiency. © 2016 The Obesity Society.

Impact of mutations on the allosteric conformational equilibrium

PubMed Central

Weinkam, Patrick; Chen, Yao Chi; Pons, Jaume; Sali, Andrej

2012-01-01

Allostery in a protein involves effector binding at an allosteric site that changes the structure and/or dynamics at a distant, functional site. In addition to the chemical equilibrium of ligand binding, allostery involves a conformational equilibrium between one protein substate that binds the effector and a second substate that less strongly binds the effector. We run molecular dynamics simulations using simple, smooth energy landscapes to sample specific ligand-induced conformational transitions, as defined by the effector-bound and unbound protein structures. These simulations can be performed using our web server: http://salilab.org/allosmod/. We then develop a set of features to analyze the simulations and capture the relevant thermodynamic properties of the allosteric conformational equilibrium. These features are based on molecular mechanics energy functions, stereochemical effects, and structural/dynamic coupling between sites. Using a machine-learning algorithm on a dataset of 10 proteins and 179 mutations, we predict both the magnitude and sign of the allosteric conformational equilibrium shift by the mutation; the impact of a large identifiable fraction of the mutations can be predicted with an average unsigned error of 1 kBT. With similar accuracy, we predict the mutation effects for an 11th protein that was omitted from the initial training and testing of the machine-learning algorithm. We also assess which calculated thermodynamic properties contribute most to the accuracy of the prediction. PMID:23228330

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

Deficits in the extinction of ethanol-seeking behavior following chronic intermittent ethanol exposure are attenuated with positive allosteric modulation of mGlu5.

PubMed

Gass, J T; McGonigal, J T; Chandler, L J

2017-02-01

Alcoholism is a chronic relapsing disorder characterized by periods of heavy alcohol consumption and unsuccessful attempts at abstinence. Relapse is one of the most problematic aspects in the treatment of alcoholism and is triggered by ethanol-associated cues. Extinction-based cue exposure therapies have proven ineffective in the treatment of alcoholism. However, positive allosteric modulation of mGlu5 with CDPPB enhances the extinction learning of alcohol-seeking behavior. The current study investigated the impact of chronic alcohol exposure on the extinction of ethanol-seeking behavior. Adult Wistar rats were trained to self-administer alcohol with a light/tone stimulus serving as the alcohol cue. After training, one group of rats was exposed to chronic intermittent ethanol (CIE) daily for a period of 2 weeks to induce ethanol dependence. Control rats were exposed to air for the same period of time. Both groups were then retrained to self-administer ethanol and subsequently tested for changes in extinction learning. CIE exposed rats consumed more ethanol compared to their pre-CIE levels and to control rats. During extinction training, CIE rats responded significantly more on the previously active lever and required more sessions to reach extinction criteria compared to control rats. Treatment with CDPPB facilitated extinction in control rats and attenuated the increased resistance to extinction in CIE-exposed rats. These results demonstrate that chronic ethanol exposure not only alters ethanol intake, but also the extinction of ethanol-seeking behaviors. The ability to attenuate deficits through modulation of mGlu5 provides a potential target for pharmacological manipulation that could ultimately reduce relapse in alcoholics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Allosteric modulation of semicarbazide-sensitive amine oxidase activities in vitro by imidazoline receptor ligands

PubMed Central

Holt, Andrew; Wieland, Barbara; Baker, Glen B

2004-01-01

Evidence indicates that imidazoline I2 binding sites (I2BSs) are present on monoamine oxidase (MAO) and on soluble (plasma) semicarbazide-sensitive amine oxidase enzymes. The binding site on MAO has been described as a modulatory site, although no effects on activity are thought to have been observed as a result of ligands binding to these sites. We examined the effects in vitro of several imidazoline binding site ligands on activities of bovine plasma amine oxidase (BPAO) and porcine kidney diamine oxidase (PKDAO) in a spectrophotometric protocol. While both enzymes were inhibited at high concentrations of all ligands, clonidine, cirazoline and oxymetazoline were seen, at lower concentrations, to increase activity of BPAO versus benzylamine, but not of PKDAO versus putrescine. This effect was substrate dependent, with mixed or biphasic inhibition of spermidine, methylamine, p-tyramine and β-phenylethylamine oxidation observed at cirazoline concentrations that increased benzylamine oxidation. With benzylamine as substrate, clonidine decreased KM (EC50 8.82 μM, Emax 75.1% of control) and increased Vmax (EC50 164.6 μM, Emax 154.1% of control). Cirazoline decreased Vmax (EC50 2.15 μM, Emax 91.4% of control), then decreased KM (EC50 5.63 μM, Emax 42.6% of control) and increased Vmax (EC50 49.0 μM, Emax 114.4% of decreased Vmax value). Data for clonidine fitted a mathematical model for two-site nonessential activation plus linear intersecting noncompetitive inhibition. Data for cirazoline were consistent with involvement of a fourth site. These results reveal an ability of imidazoline ligands to modulate BPAO kinetics allosterically. The derived mechanism may have functional significance with respect to modulation of MAO by I2BS ligands. PMID:15451775

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

PubMed

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

1999-12-01

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

Allosteric regulation of tryptophan synthase channeling: the internal aldimine probed by trans-3-indole-3'-acrylate binding.

PubMed

Casino, Patricia; Niks, Dimitri; Ngo, Huu; Pan, Peng; Brzovic, Peter; Blumenstein, Lars; Barends, Thomas Reinier; Schlichting, Ilme; Dunn, Michael F

2007-07-03

Substrate channeling in the tryptophan synthase bienzyme complex from Salmonella typhimurium is regulated by allosteric interactions triggered by binding of ligand to the alpha-site and covalent reaction at the beta-site. These interactions switch the enzyme between low-activity forms with open conformations and high-activity forms with closed conformations. Previously, allosteric interactions have been demonstrated between the alpha-site and the external aldimine, alpha-aminoacrylate, and quinonoid forms of the beta-site. Here we employ the chromophoric l-Trp analogue, trans-3-indole-3'-acrylate (IA), and noncleavable alpha-site ligands (ASLs) to probe the allosteric properties of the internal aldimine, E(Ain). The ASLs studied are alpha-d,l-glycerol phosphate (GP) and d-glyceraldehyde 3-phosphate (G3P), and examples of two new classes of high-affinity alpha-site ligands, N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), that were previously shown to bind to the alpha-site by optical spectroscopy and X-ray crystal structures [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Synthesis and characterization of allosteric probes of substrate channeling in the tryptophan synthase bienzyme complex, Biochemistry 46, 7713-7727]. The binding of IA to the beta-site is stimulated by the binding of GP, G3P, F6, or F9 to the alpha-site. The binding of ASLs was found to increase the affinity of the beta-site of E(Ain) for IA by 4-5-fold, demonstrating for the first time that the beta-subunit of the E(Ain) species undergoes a switching between low- and high-affinity states in response to the binding of ASLs.

The interaction of escitalopram and R-citalopram at the human serotonin transporter investigated in the mouse

PubMed Central

Jacobsen, Jacob P.R.; Plenge, Per; Sachs, Benjamin D.; Pehrson, Alan L.; Cajina, Manuel; Du, Yunzhi; Roberts, Wendy; Rudder, Meghan L.; Dalvi, Prachiti; Robinson, Taylor J.; O’Neill, Sharon P.; Khoo, King S.; Morillo, Connie Sanchez; Zhang, Xiaodong; Caron, Marc G.

2015-01-01

Rationale Escitalopram is a superior antidepressant to racemic citalopram. It has been hypothesized that binding of R-citalopram to the serotonin transporter (SERT) antagonizes escitalopram binding to and inhibition of the SERT, curtailing the elevation of extracellular 5-hydroxytryptamine (5-HTExt), and antidepressant efficacy. Further, it has been suggested that a putative allosteric binding site is important for binding of escitalopram to the primary, orthosteric, site, and for R-citalopram’s inhibition hereof. Objectives Primary: Investigate at the human (h)SERT, at clinical relevant doses, whether R-citalopram antagonizes escitalopram-induced 5-HTExt elevation. Secondary: Investigate whether abolishing the putative allosteric site affects escitalopram-induced 5-HTExt elevation and/or modulates the effect of R-citalopram. Methods Recombinant technology; in vivo microdialysis; receptor binding; pharmacokinetics; 5-HT sensitive behaviors (tail suspension, marble burying). Results We generated mice expressing either the wild-type human SERT (hSERTWT) or hSERT carrying amino acid substitutions (A505V, L506F, I507L, S574T and I575T) collectively abolishing the putative allosteric site (hSERTALI/VFL+SI/TT). One mg/kg escitalopram yielded clinical relevant plasma levels and brain levels consistent with therapeutic SERT occupancy. Importantly, escitalopram-induced 5-HTExt elevation was not decreased by R-citalopram co-treatment. Further, escitalopram-induced 5-HTExt elevation was not affected by loss of the allosteric site. The behavioral effects of the clinically relevant escitalopram dose were small, tending to be enhanced by R-citalopram co-administration. Conclusions We find no evidence that R-citalopram directly antagonizes escitalopram or that the putative allosteric site is important for hSERT inhibition by escitalopram. Our findings points to mechanisms for R-citalopram antagonism of escitalopram’s antidepressant action other than direct antagonistic binding interactions at the hSERT. PMID:24810106

Effect of novel negative allosteric modulators of neuronal nicotinic receptors on cells expressing native and recombinant nicotinic receptors: implications for drug discovery.

PubMed

González-Cestari, Tatiana F; Henderson, Brandon J; Pavlovicz, Ryan E; McKay, Susan B; El-Hajj, Raed A; Pulipaka, Aravinda B; Orac, Crina M; Reed, Damon D; Boyd, R Thomas; Zhu, Michael X; Li, Chenglong; Bergmeier, Stephen C; McKay, Dennis B

2009-02-01

Allosteric modulation of nAChRs is considered to be one of the most promising approaches for drug design targeting nicotinic acetylcholine receptors (nAChRs). We have reported previously on the pharmacological activity of several compounds that seem to act noncompetitively to inhibit the activation of alpha3beta4(*) nAChRs. In this study, the effects of 51 structurally similar molecules on native and recombinant alpha3beta4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native alpha3beta4(*) nAChR, with IC(50) values ranging from 0.4 to 13.0 microM. Using cells expressing recombinant alpha3beta4 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity), with IC(50) values ranging from 0.7 to 38.2 microM. Radiolabeled nAChR binding studies to orthosteric sites showed no inhibitory activity on either native or recombinant nAChRs. Correlation analyses of the data from both functional assays suggested additional, non-nAChR activity of the molecules. To test this hypothesis, the effects of the drugs on neurosecretion stimulated through non-nAChR mechanisms were investigated; inhibitory effects ranged from no inhibition to 95% inhibition at concentrations of 10 microM. Correlation analyses of the functional data confirmed this hypothesis. Several of the molecules (24/51) increased agonist binding to native nAChRs, supporting allosteric interactions with nAChRs. Computational modeling and blind docking identified a binding site for our negative allosteric modulators near the orthosteric binding site of the receptor. In summary, this study identified several molecules for potential development as negative allosteric modulators and documented the importance of multiple screening assays for nAChR drug discovery.

Effect of Novel Negative Allosteric Modulators of Neuronal Nicotinic Receptors on Cells Expressing Native and Recombinant Nicotinic Receptors: Implications for Drug Discovery

PubMed Central

González-Cestari, Tatiana F.; Henderson, Brandon J.; Pavlovicz, Ryan E.; McKay, Susan B.; El-Hajj, Raed A.; Pulipaka, Aravinda B.; Orac, Crina M.; Reed, Damon D.; Boyd, R. Thomas; Zhu, Michael X.; Li, Chenglong; Bergmeier, Stephen C.; McKay, Dennis B.

2009-01-01

Allosteric modulation of nAChRs is considered to be one of the most promising approaches for drug design targeting nicotinic acetylcholine receptors (nAChRs). We have reported previously on the pharmacological activity of several compounds that seem to act noncompetitively to inhibit the activation of α3β4* nAChRs. In this study, the effects of 51 structurally similar molecules on native and recombinant α3β4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native α3β4* nAChR, with IC50 values ranging from 0.4 to 13.0 μM. Using cells expressing recombinant α3β4 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity), with IC50 values ranging from 0.7 to 38.2 μM. Radiolabeled nAChR binding studies to orthosteric sites showed no inhibitory activity on either native or recombinant nAChRs. Correlation analyses of the data from both functional assays suggested additional, non-nAChR activity of the molecules. To test this hypothesis, the effects of the drugs on neurosecretion stimulated through non-nAChR mechanisms were investigated; inhibitory effects ranged from no inhibition to 95% inhibition at concentrations of 10 μM. Correlation analyses of the functional data confirmed this hypothesis. Several of the molecules (24/51) increased agonist binding to native nAChRs, supporting allosteric interactions with nAChRs. Computational modeling and blind docking identified a binding site for our negative allosteric modulators near the orthosteric binding site of the receptor. In summary, this study identified several molecules for potential development as negative allosteric modulators and documented the importance of multiple screening assays for nAChR drug discovery. PMID:18984653

P2X4 Receptor in Silico and Electrophysiological Approaches Reveal Insights of Ivermectin and Zinc Allosteric Modulation

PubMed Central

Latapiat, Verónica; Rodríguez, Felipe E.; Godoy, Francisca; Montenegro, Felipe A.; Barrera, Nelson P.; Huidobro-Toro, Juan P.

2017-01-01

Protein allosteric modulation is a pillar of metabolic regulatory mechanisms; this concept has been extended to include ion channel regulation. P2XRs are ligand-gated channels activated by extracellular ATP, sensitive to trace metals and other chemicals. By combining in silico calculations with electrophysiological recordings, we investigated the molecular basis of P2X4R modulation by Zn(II) and ivermectin, an antiparasite drug currently used in veterinary medicine. To this aim, docking studies, molecular dynamics simulations and non-bonded energy calculations for the P2X4R in the apo and holo states or in the presence of ivermectin and/or Zn(II) were accomplished. Based on the crystallized Danio rerio P2X4R, the rat P2X4R, P2X2R, and P2X7R structures were modeled, to determine ivermectin binding localization. Calculations revealed that its allosteric site is restricted to transmembrane domains of the P2X4R; the role of Y42 and W46 plus S341 and non-polar residues were revealed as essential, and are not present in the homologous P2X2R or P2X7R transmembrane domains. This finding was confirmed by preferential binding conformations and electrophysiological data, revealing P2X4R modulator specificity. Zn(II) acts in the P2X4R extracellular domain neighboring the SS3 bridge. Molecular dynamics in the different P2X4R states revealed allosterism-induced stability. Pore and lateral fenestration measurements of the P2X4R showed conformational changes in the presence of both modulators compatible with a larger opening of the extracellular vestibule. Electrophysiological studies demonstrated additive effects in the ATP-gated currents by joint applications of ivermectin plus Zn(II). The C132A P2X4R mutant was insensitive to Zn(II); but IVM caused a 4.9 ± 0.7-fold increase in the ATP-evoked currents. Likewise, the simultaneous application of both modulators elicited a 7.1 ± 1.7-fold increase in the ATP-gated current. Moreover, the C126A P2X4R mutant evoked similar ATP-gated currents comparable to those of wild-type P2X4R. Finally, a P2X4/2R chimera did not respond to IVM but Zn(II) elicited a 2.7 ± 0.6-fold increase in the ATP-gated current. The application of IVM plus Zn(II) evoked a 2.7 ± 0.9-fold increase in the ATP-gated currents. In summary, allosteric modulators caused additive ATP-gated currents; consistent with lateral fenestration enlargement. Energy calculations demonstrated a favorable transition of the holo receptor state following both allosteric modulators binding, as expected for allosteric interactions. PMID:29326590

Membrane-induced Allosteric Control of Phospholipase C-β Isozymes*

PubMed Central

Charpentier, Thomas H.; Waldo, Gary L.; Barrett, Matthew O.; Huang, Weigang; Zhang, Qisheng; Harden, T. Kendall; Sondek, John

2014-01-01

All peripheral membrane proteins must negotiate unique constraints intrinsic to the biological interface of lipid bilayers and the cytosol. Phospholipase C-β (PLC-β) isozymes hydrolyze the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to propagate diverse intracellular responses that underlie the physiological action of many hormones, neurotransmitters, and growth factors. PLC-β isozymes are autoinhibited, and several proteins, including Gαq, Gβγ, and Rac1, directly engage distinct regions of these phospholipases to release autoinhibition. To understand this process, we used a novel, soluble analog of PIP2 that increases in fluorescence upon cleavage to monitor phospholipase activity in real time in the absence of membranes or detergents. High concentrations of Gαq or Gβ1γ2 did not activate purified PLC-β3 under these conditions despite their robust capacity to activate PLC-β3 at membranes. In addition, mutants of PLC-β3 with crippled autoinhibition dramatically accelerated the hydrolysis of PIP2 in membranes without an equivalent acceleration in the hydrolysis of the soluble analog. Our results illustrate that membranes are integral for the activation of PLC-β isozymes by diverse modulators, and we propose a model describing membrane-mediated allosterism within PLC-β isozymes. PMID:25193662

Molecular basis for zinc potentiation at strychnine-sensitive glycine receptors.

PubMed

Miller, Paul S; Da Silva, Helena M A; Smart, Trevor G

2005-11-11

The divalent cation Zn(2+) is a potent potentiator at the strychnine-sensitive glycine receptor (GlyR). This occurs at nanomolar concentrations, which are the predicted endogenous levels of extracellular neuronal Zn(2+). Using structural modeling and functional mutagenesis, we have identified the molecular basis for the elusive Zn(2+) potentiation site on GlyRs and account for the differential sensitivity of GlyR alpha(1) and GlyR alpha(2) to Zn(2+) potentiation. In addition, juxtaposed to this Zn(2+) site, which is located externally on the N-terminal domain of the alpha subunit, another residue was identified in the nearby Cys loop, a region that is critical for receptor gating in all Cys loop ligand-gated ion channels. This residue acted as a key control element in the allosteric transduction pathway for Zn(2+) potentiation, enabling either potentiation or overt inhibition of receptor activation depending upon the moiety resident at this location. Overall, we propose that Zn(2+) binds to a site on the extracellular outer face of the GlyR alpha subunit and exerts its positive allosteric effect via an interaction with the Cys loop to increase the efficacy of glycine receptor gating.

Metabolic products of linalool and modulation of GABAA receptors

NASA Astrophysics Data System (ADS)

Milanos, Sinem; Elsharif, Shaimaa A.; Janzen, Dieter; Buettner, Andrea; Villmann, Carmen

2017-06-01

Terpenoids are major subcomponents in aroma substances which harbor sedative physiological potential. We have demonstrated that various monoterpenoids such as the acyclic linalool enhance GABAergic currents in an allosteric manner in vitro upon overexpression of inhibitory a1b2 GABAA receptors in various expression systems. However, in plants or humans, i.e. following intake via inhalation or ingestion, linalool undergoes metabolic modifications including oxygenation and acetylation, which may affect the modulatory efficacy of the generated linalool derivatives. Here, we analyzed the modulatory potential of linalool derivatives at a1b2g2 GABAA receptors upon transient overexpression. Following receptor expression control, electrophysiological recordings in a whole cell configuration were used to determine the chloride influx upon co-application of GABA EC5-10 together with the modulatory substance. Our results show that only oxygenated linalool metabolites at carbon 8 positively affect GABAergic currents whereas derivatives hydroxylated or carboxylated at carbon 8 were rather ineffective. Acetylated linalool derivatives resulted in non-significant changes of GABAergic currents. We can conclude that metabolism of linalool reduces its positive allosteric potential at GABAA receptors compared to the significant potentiation effects of the parent molecule linalool itself.

Protein–Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP)*

PubMed Central

Narayan, Vikram; Landré, Vivien; Ning, Jia; Hernychova, Lenka; Muller, Petr; Verma, Chandra; Walkinshaw, Malcolm D.; Blackburn, Elizabeth A.; Ball, Kathryn L.

2015-01-01

CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control. PMID:26330542

IP3-mediated gating mechanism of the IP3 receptor revealed by mutagenesis and X-ray crystallography

PubMed Central

Hamada, Kozo; Miyatake, Hideyuki; Terauchi, Akiko; Mikoshiba, Katsuhiko

2017-01-01

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is an IP3-gated ion channel that releases calcium ions (Ca2+) from the endoplasmic reticulum. The IP3-binding sites in the large cytosolic domain are distant from the Ca2+ conducting pore, and the allosteric mechanism of how IP3 opens the Ca2+ channel remains elusive. Here, we identify a long-range gating mechanism uncovered by channel mutagenesis and X-ray crystallography of the large cytosolic domain of mouse type 1 IP3R in the absence and presence of IP3. Analyses of two distinct space group crystals uncovered an IP3-dependent global translocation of the curvature α-helical domain interfacing with the cytosolic and channel domains. Mutagenesis of the IP3R channel revealed an essential role of a leaflet structure in the α-helical domain. These results suggest that the curvature α-helical domain relays IP3-controlled global conformational dynamics to the channel through the leaflet, conferring long-range allosteric coupling from IP3 binding to the Ca2+ channel. PMID:28416699

Allosteric Ligand Binding and Anisotropic Energy Flow in Albumin

NASA Astrophysics Data System (ADS)

Dyer, Brian

2014-03-01

Protein allostery usually involves propagation of local structural changes through the protein to a remote site. Coupling of structural changes at remote sites is thought to occur through anisotropic energy transport, but the nature of this process is poorly understood. We have studied the relationship between allosteric interactions of remote ligand binding sites of the protein and energy flow through the structure of bovine serum albumin (BSA). We applied ultrafast infrared spectroscopy to probe the flow of energy through the protein backbone following excitation of a heater dye, a metalloporphyrin or malachite green, bound to different binding sites in the protein. We observe ballistic flow through the protein structure following input of thermal energy into the flexible ligand binding sites. We also observe anisotropic heat flow through the structure, without local heating of the rigid helix bundles that connect these sites. We will discuss the implications of this efficient energy transport mechanism with regard to the allosteric propagation of binding energy through the connecting helix structures.

Allosteric Regulation of E-Cadherin Adhesion.

PubMed

Shashikanth, Nitesh; Petrova, Yuliya I; Park, Seongjin; Chekan, Jillian; Maiden, Stephanie; Spano, Martha; Ha, Taekjip; Gumbiner, Barry M; Leckband, Deborah E

2015-08-28

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120(ctn), increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120(ctn) dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120(ctn) dephosphorylation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Discovery of 3-(3-(4-(1-Aminocyclobutyl)phenyl)-5-phenyl-3 H -imidazo[4,5- b ]pyridin-2-yl)pyridin-2-amine (ARQ 092): An Orally Bioavailable, Selective, and Potent Allosteric AKT Inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lapierre, Jean-Marc; Eathiraj, Sudharshan; Vensel, David

The work in this paper describes the optimization of the 3-(3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine chemical series as potent, selective allosteric inhibitors of AKT kinases, leading to the discovery of ARQ 092 (21a). The cocrystal structure of compound 21a bound to full-length AKT1 confirmed the allosteric mode of inhibition of this chemical class and the role of the cyclobutylamine moiety. Compound 21a demonstrated high enzymatic potency against AKT1, AKT2, and AKT3, as well as potent cellular inhibition of AKT activation and the phosphorylation of the downstream target PRAS40. Compound 21a also served as a potent inhibitor of the AKT1-E17K mutant protein and inhibited tumormore » growth in a human xenograft mouse model of endometrial adenocarcinoma.« less

X-ray structures and mechanism of the human serotonin transporter.

PubMed

Coleman, Jonathan A; Green, Evan M; Gouaux, Eric

2016-04-21

The serotonin transporter (SERT) terminates serotonergic signalling through the sodium- and chloride-dependent reuptake of neurotransmitter into presynaptic neurons. SERT is a target for antidepressant and psychostimulant drugs, which block reuptake and prolong neurotransmitter signalling. Here we report X-ray crystallographic structures of human SERT at 3.15 Å resolution bound to the antidepressants (S)-citalopram or paroxetine. Antidepressants lock SERT in an outward-open conformation by lodging in the central binding site, located between transmembrane helices 1, 3, 6, 8 and 10, directly blocking serotonin binding. We further identify the location of an allosteric site in the complex as residing at the periphery of the extracellular vestibule, interposed between extracellular loops 4 and 6 and transmembrane helices 1, 6, 10 and 11. Occupancy of the allosteric site sterically hinders ligand unbinding from the central site, providing an explanation for the action of (S)-citalopram as an allosteric ligand. These structures define the mechanism of antidepressant action in SERT, and provide blueprints for future drug design.

Insight into structural requirements for selective and/or dual CXCR3 and CXCR4 allosteric modulators.

PubMed

Kolarič, Anja; Švajger, Urban; Tomašič, Tihomir; Brox, Regine; Frank, Theresa; Minovski, Nikola; Tschammer, Nuska; Anderluh, Marko

2018-05-11

Based on the previously published pyrazolopyridine-based hit compound for which negative allosteric modulation of both CXCR3 and CXCR4 receptors was disclosed, we designed, synthesized and biologically evaluated a set of novel, not only negative, but also positive allosteric modulators with preserved pyrazolopyridine core. Compound 9e is a dual negative modulator, inhibiting G protein activity of both receptors. For CXCR4 receptor para-substituted aromatic group of compounds distinguishes between negative and positive modulation. Para-methoxy substitution leads to functional antagonism, while para-chloro triggers agonism. Additionally, we discovered that chemotaxis is not completely correlated with G protein pathways. This is the first work in which we have on a series of compounds successfully demonstrated that it is possible to produce selective as well as dual-acting modulators of chemokine receptors, which is very promising for future research in the field of discovery of selective or dual modulators of chemokine receptors. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Functional anatomy of an allosteric protein

NASA Astrophysics Data System (ADS)

Purohit, Prasad; Gupta, Shaweta; Jadey, Snehal; Auerbach, Anthony

2013-12-01

Synaptic receptors are allosteric proteins that switch on and off to regulate cell signalling. Here, we use single-channel electrophysiology to measure and map energy changes in the gating conformational change of a nicotinic acetylcholine receptor. Two separated regions in the α-subunits—the transmitter-binding sites and αM2-αM3 linkers in the membrane domain—have the highest ϕ-values (change conformation the earliest), followed by the extracellular domain, most of the membrane domain and the gate. Large gating-energy changes occur at the transmitter-binding sites, α-subunit interfaces, the αM1 helix and the gate. We hypothesize that rearrangements of the linkers trigger the global allosteric transition, and that the hydrophobic gate unlocks in three steps. The mostly local character of side-chain energy changes and the similarly high ϕ-values of separated domains, both with and without ligands, suggest that gating is not strictly a mechanical process initiated by the affinity change for the agonist.

An allosteric photoredox catalyst inspired by photosynthetic machinery

DOE PAGES

Lifschitz, Alejo M.; Young, Ryan M.; Mendez-Arroyo, Jose; ...

2015-03-30

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

The insect repellent N,N-diethyl-m-toluamide (DEET) induces angiogenesis via allosteric modulation of the M3 muscarinic receptor in endothelial cells.

PubMed

Legeay, Samuel; Clere, Nicolas; Hilairet, Grégory; Do, Quoc-Tuan; Bernard, Philippe; Quignard, Jean-François; Apaire-Marchais, Véronique; Lapied, Bruno; Faure, Sébastien

2016-06-27

The insect repellent N,N-diethyl-m-toluamide (DEET) has been reported to inhibit AChE (acetylcholinesterase) and to possess potential carcinogenic properties with excessive vascularization. In the present paper, we demonstrate that DEET specifically stimulates endothelial cells that promote angiogenesis which increases tumor growth. DEET activates cellular processes that lead to angiogenesis including proliferation, migration and adhesion. This is associated with an enhancement of NO production and VEGF expression in endothelial cells. M3 silencing or the use of a pharmacological M3 inhibitor abrogates all of these effects which reveals that DEET-induced angiogenesis is M3 sensitive. The experiments involving calcium signals in both endothelial and HEK cells overexpressing M3 receptors, as well as binding and docking studies demonstrate that DEET acts as an allosteric modulator of the M3 receptor. In addition, DEET inhibited AChE which increased acetylcholine bioavailability and binding to M3 receptors and also strengthened proangiogenic effects by an allosteric modulation.

Dynamic allostery of protein alpha helical coiled-coils

PubMed Central

Hawkins, Rhoda J; McLeish, Tom C.B

2005-01-01

Alpha helical coiled-coils appear in many important allosteric proteins such as the dynein molecular motor and bacteria chemotaxis transmembrane receptors. As a mechanism for transmitting the information of ligand binding to a distant site across an allosteric protein, an alternative to conformational change in the mean static structure is an induced change in the pattern of the internal dynamics of the protein. We explore how ligand binding may change the intramolecular vibrational free energy of a coiled-coil, using parameterized coarse-grained models, treating the case of dynein in detail. The models predict that coupling of slide, bend and twist modes of the coiled-coil transmits an allosteric free energy of ∼2kBT, consistent with experimental results. A further prediction is a quantitative increase in the effective stiffness of the coiled-coil without any change in inherent flexibility of the individual helices. The model provides a possible and experimentally testable mechanism for transmission of information through the alpha helical coiled-coil of dynein. PMID:16849225

Calcium/calmodulin-dependent kinase II phosphorylation of the GABAA receptor alpha1 subunit modulates benzodiazepine binding.

PubMed

Churn, Severn B; Rana, Aniruddha; Lee, Kangmin; Parsons, J Travis; De Blas, Angel; Delorenzo, Robert J

2002-09-01

gamma-Aminobutyric acid (GABA) is the primary neurotransmitter that is responsible for the fast inhibitory synaptic transmission in the central nervous system. A major post-translational mechanism that can rapidly regulate GABAAR function is receptor phosphorylation. This study was designed to test the effect of endogenous calcium and calmodulin-dependent kinase II (CaM kinase II) activation on both allosteric modulator binding and GABAA receptor subunit phosphorylation. Endogenous CaM kinase II activity was stimulated, and GABAA receptors were subsequently analyzed for bothallosteric modulator binding properties and immunoprecipitated and analyzed for subunit phosphorylation levels. A significant increase in allosteric-modulator binding of the GABAAR was observed under conditions maximal for CaM kinase II activation. In addition, CaM kinase II activation resulted in a direct increase in phosphorylation of the GABAA receptor alpha1 subunit. The data suggest that the CaM kinase II-dependent phosphorylation of the GABAA receptor alpha1 subunit modulated allosteric modulator binding to the GABAA receptor.

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

PubMed

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

2016-05-03

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

Beyond radio-displacement techniques for Identification of CB1 Ligands: The First Application of a Fluorescence-quenching Assay

PubMed Central

Bruno, Agostino; Lembo, Francesca; Novellino, Ettore; Stornaiuolo, Mariano; Marinelli, Luciana

2014-01-01

Cannabinoid type 1 Receptor (CB1) belongs to the GPCR family and it has been targeted, so far, for the discovery of drugs aimed at the treatment of neuropathic pain, nausea, vomit, and food intake disorders. Here, we present the development of the first fluorescent assay enabling the measurement of kinetic binding constants for CB1orthosteric ligands. The assay is based on the use of T1117, a fluorescent analogue of AM251. We prove that T1117 binds endogenous and recombinant CB1 receptors with nanomolar affinity. Moreover, T1117 binding to CB1 is sensitive to the allosteric ligand ORG27569 and thus it is applicable to the discovery of new allosteric drugs. The herein presented assay constitutes a sustainable valid alternative to the expensive and environmental impacting radiodisplacement techniques and paves the way for an easy, fast and cheap high-throughput drug screening toward CB1 for identification of new orthosteric and allosteric modulators. PMID:24441508

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

Metabotropic glutamate receptor subtype 5: molecular pharmacology, allosteric modulation and stimulus bias

PubMed Central

Sengmany, K

2015-01-01

The metabotropic glutamate receptor subtype 5 (mGlu5) is a family C GPCR that has been implicated in various neuronal processes and, consequently, in several CNS disorders. Over the past few decades, GPCR‐based drug discovery, including that for mGlu5 receptors, has turned considerable attention to targeting allosteric binding sites. Modulation of endogenous agonists by allosteric ligands offers the advantages of spatial and temporal fine‐tuning of receptor activity, increased selectivity and reduced adverse effects with the potential to elicit improved clinical outcomes. Further, with greater appreciation of the multifaceted nature of the transduction of mGlu5 receptor signalling, it is increasingly apparent that drug discovery must take into consideration unique receptor conformations and the potential for stimulus‐bias. This novel paradigm proposes that different ligands may differentially modulate distinct signalling pathways arising from the same receptor. We review our current understanding of the complexities of mGlu5 receptor signalling and regulation, and how these relate to allosteric ligands. Ultimately, a deeper appreciation of these relationships will provide the foundation for targeted drug design of compounds with increased selectivity, not only for the desired receptor but also for the desired signalling outcome from the receptor. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein‐Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc PMID:26276909

Trace metals in the brain: allosteric modulators of ligand-gated receptor channels, the case of ATP-gated P2X receptors.

PubMed

Huidobro-Toro, J Pablo; Lorca, Ramón A; Coddou, Claudio

2008-03-01

Zinc and copper are indispensable trace metals for life with a recognized role as catalysts in enzyme actions. We now review evidence supporting the role of trace metals as novel allosteric modulators of ionotropic receptors: a new and fundamental physiological role for zinc and copper in neuronal and brain excitability. The review is focussed on ionotropic receptor channels including nucleotide receptors, in particular the P2X receptor family. Since zinc and copper are stored within synaptic vesicles in selected brain regions, and released to the synaptic cleft upon electrical nerve ending depolarization, it is plausible that zinc and copper reach concentrations in the synapse that profoundly affect ligand-gated ionic channels, including the ATP-gated currents of P2X receptors. The identification of key P2X receptor amino acids that act as ligands for trace metal coordination, carves the structural determinants underlying the allosteric nature of the trace metal modulation. The recognition that the identified key residues such as histidines, aspartic and glutamic acids or cysteines in the extracellular domain are different for each P2X receptor subtype and may be different for each metal, highlights the notion that each P2X receptor subtype evolved independent strategies for metal coordination, which form upon the proper three-dimensional folding of the receptor channels. The understanding of the molecular mechanism of allosteric modulation of ligand-operated ionic channels by trace metals is a new contribution to metallo-neurobiology.

Insights into Brain Glycogen Metabolism

PubMed Central

Mathieu, Cécile; de la Sierra-Gallay, Ines Li; Duval, Romain; Xu, Ximing; Cocaign, Angélique; Léger, Thibaut; Woffendin, Gary; Camadro, Jean-Michel; Etchebest, Catherine; Haouz, Ahmed; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

2016-01-01

Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation. However, alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease. Glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, catalyzes the rate-limiting step of glycogen mobilization. Moreover, the allosteric regulation of the three GP isozymes (muscle, liver, and brain) by metabolites and phosphorylation, in response to hormonal signaling, fine-tunes glycogenolysis to fulfill energetic and metabolic requirements. Whereas the structures of muscle and liver GPs have been known for decades, the structure of brain GP (bGP) has remained elusive despite its critical role in brain glycogen metabolism. Here, we report the crystal structure of human bGP in complex with PEG 400 (2.5 Å) and in complex with its allosteric activator AMP (3.4 Å). These structures demonstrate that bGP has a closer structural relationship with muscle GP, which is also activated by AMP, contrary to liver GP, which is not. Importantly, despite the structural similarities between human bGP and the two other mammalian isozymes, the bGP structures reveal molecular features unique to the brain isozyme that provide a deeper understanding of the differences in the activation properties of these allosteric enzymes by the allosteric effector AMP. Overall, our study further supports that the distinct structural and regulatory properties of GP isozymes contribute to the different functions of muscle, liver, and brain glycogen. PMID:27402852

Molecular dynamics simulations reveal the allosteric effect of F1174C resistance mutation to ceritinib in ALK-associated lung cancer.

PubMed

Ni, Zhong; Wang, Xiting; Zhang, Tianchen; Jin, Rong Zhong

2016-12-01

Anaplastic lymphoma kinase (ALK) has become as an important target for the treatment of various human cancers, especially non-small-cell lung cancer. A mutation, F1174C, suited in the C-terminal helix αC of ALK and distal from the small-molecule inhibitor ceritinib bound to the ATP-binding site, causes the emergence of drug resistance to ceritinib. However, the detailed mechanism for the allosteric effect of F1174C resistance mutation to ceritinib remains unclear. Here, molecular dynamics (MD) simulations and binding free energy calculations [Molecular Mechanics/Generalized Born Surface Area (MM/GBSA)] were carried out to explore the advent of drug resistance mutation in ALK. MD simulations observed that the exquisite aromatic-aromatic network formed by residues F1098, F1174, F1245, and F1271 in the wild-type ALK-ceritinib complex was disrupted by the F1174C mutation. The resulting mutation allosterically affected the conformational dynamic of P-loop and caused the upward movement of the P-loop from the ATP-binding site, thereby weakening the interaction between ceritinib and the P-loop. The subsequent MM/GBSA binding free energy calculations and decomposition analysis of binding free energy validated this prediction. This study provides mechanistic insight into the allosteric effect of F1174C resistance mutation to ceritinib in ALK and is expected to contribute to design the next-generation of ALK inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buhrman, Greg; O; #8242

2012-09-17

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

Structural Determinants of Sleeping Beauty Transposase Activity

PubMed Central

Abrusán, György; Yant, Stephen R; Szilágyi, András; Marsh, Joseph A; Mátés, Lajos; Izsvák, Zsuzsanna; Barabás, Orsolya; Ivics, Zoltán

2016-01-01

Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here, we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein coevolutionary information can be used to classify groups of physically connected, coevolving residues into elements called “sectors”, which have proven useful for understanding the folding, allosteric interactions, and enzymatic activity of proteins. Using extensive mutagenesis data, protein modeling and analysis of folding energies, we show that (i) The Sleeping Beauty transposase contains two sectors, which span across conserved domains, and are enriched in DNA-binding residues, indicating that the DNA binding and endonuclease functions of the transposase coevolve; (ii) Sector residues are highly sensitive to mutations, and most mutations of these residues strongly reduce transposition rate; (iii) Mutations with a strong effect on free energy of folding in the DDE domain of the transposase significantly reduce transposition rate. (iv) Mutations that influence DNA and protein-protein interactions generally reduce transposition rate, although most hyperactive mutants are also located on the protein surface, including residues with protein-protein interactions. This suggests that hyperactivity results from the modification of protein interactions, rather than the stabilization of protein fold. PMID:27401040

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

PubMed

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

2016-02-15

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

Development of small molecule biosensors by coupling the recognition of the bacterial allosteric transcription factor with isothermal strand displacement amplification.

PubMed

Yao, Yongpeng; Li, Shanshan; Cao, Jiaqian; Liu, Weiwei; Fan, Keqiang; Xiang, Wensheng; Yang, Keqian; Kong, Deming; Wang, Weishan

2018-05-08

Here, we demonstrate an easy-to-implement and general biosensing strategy by coupling the small-molecule recognition of the bacterial allosteric transcription factor (aTF) with isothermal strand displacement amplification (SDA) in vitro. Based on this strategy, we developed two biosensors for the detection of an antiseptic, p-hydroxybenzoic acid, and a disease marker, uric acid, using bacterial aTF HosA and HucR, respectively, highlighting the great potential of this strategy for the development of small-molecule biosensors.

Unconventional plasticity of HIV-1 reverse transcriptase: how inhibitors could open a connection "gate" between allosteric and catalytic sites.

PubMed

Bellucci, Luca; Angeli, Lucilla; Tafi, Andrea; Radi, Marco; Botta, Maurizio

2013-12-23

Targeted molecular dynamics (TMD) simulations allowed for identifying the chemical/structural features of the nucleotide-competitive HIV-1 inhibitor DAVP-1, which is responsible for the disruption of the T-shape motif between Try183 and Trp229 of the reverse transcriptase (RT). DAVP-1 promoted the opening of a connection "gate" between allosteric and catalytic sites of HIV-1 RT, thus explaining its peculiar mechanism of action and providing useful insights to develop novel nucleotide-competitive RT inhibitors.

Positive allosteric modulators of the metabotropic glutamate receptor subtype 4 (mGluR4). Part II: Challenges in hit-to-lead

PubMed Central

Williams, Richard; Niswender, Colleen M.; Luo, Qingwei; Le, Uyen; Conn, P. Jeffrey; Lindsley, Craig W.

2013-01-01

This Letter describes the synthesis and SAR of two mGluR4 positive allosteric modulator leads, 6 and 7. VU001171 (6) represents the most potent (EC50 = 650 nM), efficacious (141% Glu Max) and largest fold shift (36-fold) of any mGluR4 PAM reported to date. However, this work highlights the challenges in hit-to-lead for mGluR4 PAMs, with multiple confirmed HTS hits displaying little or no tractable SAR. PMID:19097893

A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination.

PubMed

Ribeiro, Lucas Ferreira; Tullman, Jennifer; Nicholes, Nathan; Silva, Sérgio Ruschi Bergamachi; Vieira, Davi Serradella; Ostermeier, Marc; Ward, Richard John

2016-01-01

Saccharification of lignocellulosic material by xylanases and other glycoside hydrolases is generally conducted at high concentrations of the final reaction products, which frequently inhibit the enzymes used in the saccharification process. Using a random nonhomologous recombination strategy, we have fused the GH11 xylanase from Bacillus subtilis (XynA) with the xylose binding protein from Escherichia coli (XBP) to produce an enzyme that is allosterically stimulated by xylose. The pT7T3GFP_XBP plasmid containing the XBP coding sequence was randomly linearized with DNase I, and ligated with the XynA coding sequence to create a random XynA-XBP insertion library, which was used to transform E. coli strain JW3538-1 lacking the XBP gene. Screening for active XBP was based on the expression of GFP from the pT7T3GFP_XBP plasmid under the control of a xylose inducible promoter. In the presence of xylose, cells harboring a functional XBP domain in the fusion protein (XBP+) showed increased GFP fluorescence and were selected using FACS. The XBP+ cells were further screened for xylanase activity by halo formation around xylanase producing colonies (XynA+) on LB-agar-xylan media after staining with Congo red. The xylanase activity ratio with xylose/without xylose in supernatants from the XBP+/XynA+ clones was measured against remazol brilliant blue xylan. A clone showing an activity ratio higher than 1.3 was selected where the XynA was inserted after the asparagine 271 in the XBP, and this chimera was denominated as XynA-XBP271. The XynA-XBP271 was more stable than XynA at 55 °C, and in the presence of xylose the catalytic efficiency was ~3-fold greater than the parental xylanase. Molecular dynamics simulations predicted the formation of an extended protein-protein interface with coupled movements between the XynA and XBP domains. In the XynA-XBP271 with xylose bound to the XBP domain, the mobility of a β-loop in the XynA domain results in an increased access to the active site, and may explain the observed allosteric activation. The approach presented here provides an important advance for the engineering enzymes that are stimulated by the final product.

Temperature-dependence of isometric tension and cross-bridge kinetics of cardiac muscle fibers reconstituted with a tropomyosin internal deletion mutant.

PubMed

Lu, Xiaoying; Tobacman, Larry S; Kawai, Masataka

2006-12-01

The effect of temperature on isometric tension and cross-bridge kinetics was studied with a tropomyosin (Tm) internal deletion mutant AS-Delta23Tm (Ala-Ser-Tm Delta(47-123)) in bovine cardiac muscle fibers by using the thin filament extraction and reconstitution technique. The results are compared with those from actin reconstituted alone, cardiac muscle-derived control acetyl-Tm, and recombinant control AS-Tm. In all four reconstituted muscle groups, isometric tension and stiffness increased linearly with temperature in the range 5-40 degrees C for fibers activated in the presence of saturating ATP and Ca(2+). The slopes of the temperature-tension plots of the two controls were very similar, whereas the slope derived from fibers with actin alone had approximately 40% the control value, and the slope from mutant Tm had approximately 36% the control value. Sinusoidal analysis was performed to study the temperature dependence of cross-bridge kinetics. All three exponential processes A, B, and C were identified in the high temperature range (30-40 degrees C); only processes B and C were identified in the mid-temperature range (15-25 degrees C), and only process C was identified in the low temperature range (5-10 degrees C). At a given temperature, similar apparent rate constants (2pia, 2pib, 2pic) were observed in all four muscle groups, whereas their magnitudes were markedly less in the order of AS-Delta23Tm < Actin < AS-Tm approximately Acetyl-Tm groups. Our observations are consistent with the hypothesis that Tm enhances hydrophobic and stereospecific interactions (positive allosteric effect) between actin and myosin, but Delta23Tm decreases these interactions (negative allosteric effect). Our observations further indicate that tension/cross-bridge is increased by Tm, but is diminished by Delta23Tm. We conclude that Tm affects the conformation of actin so as to increase the area of hydrophobic interaction between actin and myosin molecules.

Acid-base-controlled stereoselective metalation of overhanging carboxylic acid porphyrins: consequences for the formation of heterobimetallic complexes.

PubMed

Le Gac, Stéphane; Najjari, Btissam; Dorcet, Vincent; Roisnel, Thierry; Fusaro, Luca; Luhmer, Michel; Furet, Eric; Halet, Jean-François; Boitrel, Bernard

2013-08-12

Overhanging carboxylic acid porphyrins have revealed promising ditopic ligands offering a new entry in the field of supramolecular coordination chemistry of porphyrinoids. Notably, the adjunction of a so-called hanging-atop (HAT) Pb(II) cation to regular Pb(II) porphyrin complexes allowed a stereoselective incorporation of the N-core bound cation, and an allosterically controlled Newton's cradle-like motion of the two Pb(II) ions also emerged from such bimetallic complexes. In this contribution, we have extended this work to other ligands and metal ions, aiming at understanding the parameters that control the HAT Pb(II) coordination. The nature of the N-core bound metal ion (Zn(II), Cd(II)), the influence of the deprotonation state of the overhanging COOH group and the presence of a neutral ligand on the opposite side (exogenous or intramolecular), have been examined through (1)H NMR spectroscopic experiments with the help of radiocrystallographic structures and DFT calculations. Single and bis-strap ligands have been considered. They all incorporate a COOH group hung over the N-core on one side. For the bis-strap ligands, either an ester or an amide group has been introduced on the other side. In the presence of a base, the mononuclear Zn(II) or Cd(II) complexes incorporate the carbonyl of the overhanging carboxylate as apical ligand, decreasing its availability for the binding of a HAT Pb(II). An allosteric effector (e.g., 4-dimethylaminopyridine (DMAP), in the case of a single-strap ligand) or an intramolecular ligand (e.g., an amide group), strong enough to compete with the carbonyl of the hung COO(-), is required to switch the N-core bound cation to the opposite side with concomitant release of the COO(-), thereby allowing HAT Pb(II) complexation. In the absence of a base, Zn(II) or Cd(II) binds preferentially the carbonyl of the intramolecular ester or amide groups in apical position rather than that of the COOH. This better preorganization, with the overhanging COOH fully available, is responsible for a stronger binding of the HAT Pb(II). Thus, either allosteric or acid-base control is achieved through stereoselective metalation of Zn(II) or Cd(II). In the latter case, according to the deprotonation state of the COOH group, the best electron-donating ligand is located on one or the other side of the porphyrin (COO(-)>CONHR>COOR>COOH): the lower affinity of COOH for Zn(II) and Cd(II), the higher for a HAT Pb(II). These insights provide new opportunities for the elaboration of innovative bimetallic molecular switches. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Gating by Cyclic Gmp and Voltage in the α Subunit of the Cyclic Gmp–Gated Channel from Rod Photoreceptors

PubMed Central

Benndorf, Klaus; Koopmann, Rolf; Eismann, Elisabeth; Kaupp, U. Benjamin

1999-01-01

Gating by cGMP and voltage of the α subunit of the cGMP-gated channel from rod photoreceptor was examined with a patch-clamp technique. The channels were expressed in Xenopus oocytes. At low [cGMP] (<20 μM), the current displayed strong outward rectification. At low and high (700 μM) [cGMP], the channel activity was dominated by only one conductance level. Therefore, the outward rectification at low [cGMP] results solely from an increase in the open probability, P o. Kinetic analysis of single-channel openings revealed two exponential distributions. At low [cGMP], the larger P o at positive voltages with respect to negative voltages is caused by an increased frequency of openings in both components of the open-time distribution. In macroscopic currents, depolarizing voltage steps, starting from −100 mV, generated a time-dependent current that increased with the step size (activation). At low [cGMP] (20 μM), the degree of activation was large and the time course was slow, whereas at saturating [cGMP] (7 mM) the respective changes were small and fast. The dose–response relation at −100 mV was shifted to the right and saturated at significantly lower P o values with respect to that at +100 mV (0.77 vs. 0.96). P o was determined as function of the [cGMP] (at +100 and −100 mV) and voltage (at 20, 70, and 700 μM, and 7 mM cGMP). Both relations could be fitted with an allosteric state model consisting of four independent cGMP-binding reactions and one voltage-dependent allosteric opening reaction. At saturating [cGMP] (7 mM), the activation time course was monoexponential, which allowed us to determine the individual rate constants for the allosteric reaction. For the rapid rate constants of cGMP binding and unbinding, lower limits are determined. It is concluded that an allosteric model consisting of four independent cGMP-binding reactions and one voltage-dependent allosteric reaction, describes the cGMP- and voltage-dependent gating of cGMP-gated channels adequately. PMID:10498668

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

PubMed

Zhen, Juan; Reith, Maarten E A

2016-09-01

The structures of the leucine transporter, drosophila dopamine transporter, and human serotonin transporter show a secondary binding site (designated S2 ) for drugs and substrate in the extracellular vestibule toward the membrane exterior in relation to the primary substrate recognition site (S1 ). The present experiments are aimed at disrupting S2 by mutating Asp476 and Ile159 to Ala. Both mutants displayed a profound decrease in [(3) H]DA uptake compared with wild-type associated with a reduced turnover rate kcat . This was not caused by a conformational bias as the mutants responded to Zn(2+) (10 μM) similarly as WT. The dopamine transporters with either the D476A or I159A mutation both displayed a higher Ki for dopamine for the inhibition of [3H](-)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane binding than did the WT transporter, in accordance with an allosteric interaction between the S1 and S2 sites. The results provide evidence in favor of a general applicability of the two-site allosteric model of the Javitch/Weinstein group from LeuT to dopamine transporter and possibly other monoamine transporters. X-ray structures of transporters closely related to the dopamine (DA) transporter show a secondary binding site S2 in the extracellular vestibule proximal to the primary binding site S1 which is closely linked to one of the Na(+) binding sites. This work examines the relationship between S2 and S1 sites. We found that S2 site impairment severely reduced DA transport and allosterically reduced S1 site affinity for the cocaine analog [(3) H]CFT. Our results are the first to lend direct support for the application of the two-site allosteric model, advanced for bacterial LeuT, to the human DA transporter. The model states that, after binding of the first DA molecule (DA1 ) to the primary S1 site (along with Na(+) ), binding of a second DA (DA2 ) to the S2 site triggers, through an allosteric interaction, the release of DA1 and Na(+) into the cytoplasm. © 2016 International Society for Neurochemistry.

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

PubMed

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

2016-12-23

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

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

PubMed Central

Liu, Binbin; Zhang, Jing; Koetzner, Cheri A.; Jones, Susan A.; Lin, Qishan

2017-01-01

The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 1.8 μM, 11.4 μM, and 4.8 μM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 μM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis experiments unambiguously demonstrated an allosteric mechanism for inhibition of the viral protease by NSC135618. PMID:28542603

New screening strategy and analysis for identification of allosteric modulators for glucagon-like peptide-1 receptor using GLP-1 (9-36) amide.

PubMed

Nakane, Atsushi; Gotoh, Yusuke; Ichihara, Junji; Nagata, Hidetaka

2015-12-15

The glucagon-like peptide-1 receptor (GLP-1R) is an important physiologic regulator of insulin secretion and a major therapeutic target for diabetes mellitus. GLP-1 (7-36) amide (active form of GLP-1) is truncated to GLP-1 (9-36) amide, which has been described as a weak agonist of GLP-1R and the major form of GLP-1 in the circulation. New classes of positive allosteric modulators (PAMs) for GLP-1R may offer improved therapeutic profiles. To identify these new classes, we developed novel and robust primary and secondary high-throughput screening (HTS) systems in which PAMs were identified to enhance the GLP-1R signaling induced by GLP-1 (9-36) amide. Screening enabled identification of two compounds, HIT-465 and HIT-736, which possessed new patterns of modulation of GLP-1R. We investigated the ability of these compounds to modify GLP-1R signaling enhanced GLP-1 (9-36) amide- and/or GLP-1 (7-36) amide-mediated cyclic adenosine monophosphate (cAMP) accumulation. These compounds also had unique profiles with regard to allosteric modulation of multiple downstream signaling (PathHunter β-arrestin signaling, PathHunter internalization signaling, microscopy-based internalization assay). We found allosteric modulation patterns to be obviously different among HIT-465, HIT-736, and Novo Nordisk compound 2. This work may enable the design of new classes of drug candidates by targeting modulation of GLP-1 (7-36) amide and GLP-1 (9-36) amide. Copyright © 2015 Elsevier Inc. All rights reserved.

Tunable allosteric library of caspase-3 identifies coupling between conserved water molecules and conformational selection

PubMed Central

Maciag, Joseph J.; Mackenzie, Sarah H.; Tucker, Matthew B.; Schipper, Joshua L.; Swartz, Paul; Clark, A. Clay

2016-01-01

The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 high-resolution (<2.0 Å) structures of wild-type caspase-3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of posttranslational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of 19 caspase-3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies by more than four orders of magnitude. Altogether, our database consists of 37 high-resolution structures of caspase-3 variants, and we demonstrate that the decrease in activity correlates with a loss of conserved water molecules. The data show that the activity of caspase-3 can be fine-tuned through globally desolvating the active conformation within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine-tune activity through conformational selection. PMID:27681633

Heme Regulates Allosteric Activation of the Slo1 BK Channel

PubMed Central

Horrigan, Frank T.; Heinemann, Stefan H.; Hoshi, Toshinori

2005-01-01

Large conductance calcium-dependent (Slo1 BK) channels are allosterically activated by membrane depolarization and divalent cations, and possess a rich modulatory repertoire. Recently, intracellular heme has been identified as a potent regulator of Slo1 BK channels (Tang, X.D., R. Xu, M.F. Reynolds, M.L. Garcia, S.H. Heinemann, and T. Hoshi. 2003. Nature. 425:531–535). Here we investigated the mechanism of the regulatory action of heme on heterologously expressed Slo1 BK channels by separating the influences of voltage and divalent cations. In the absence of divalent cations, heme generally decreased ionic currents by shifting the channel's G–V curve toward more depolarized voltages and by rendering the curve less steep. In contrast, gating currents remained largely unaffected by heme. Simulations suggest that a decrease in the strength of allosteric coupling between the voltage sensor and the activation gate and a concomitant stabilization of the open state account for the essential features of the heme action in the absence of divalent ions. At saturating levels of divalent cations, heme remained similarly effective with its influence on the G–V simulated by weakening the coupling of both Ca2+ binding and voltage sensor activation to channel opening. The results thus show that heme dampens the influence of allosteric activators on the activation gate of the Slo1 BK channel. To account for these effects, we consider the possibility that heme binding alters the structure of the RCK gating ring and thereby disrupts both Ca2+- and voltage-dependent gating as well as intrinsic stability of the open state. PMID:15955873

Allosteric effects of gold nanoparticles on human serum albumin.

PubMed

Shao, Qing; Hall, Carol K

2017-01-07

The ability of nanoparticles to alter protein structure and dynamics plays an important role in their medical and biological applications. We investigate allosteric effects of gold nanoparticles on human serum albumin protein using molecular simulations. The extent to which bound nanoparticles influence the structure and dynamics of residues distant from the binding site is analyzed. The root mean square deviation, root mean square fluctuation and variation in the secondary structure of individual residues on a human serum albumin protein are calculated for four protein-gold nanoparticle binding complexes. The complexes are identified in a brute-force search process using an implicit-solvent coarse-grained model for proteins and nanoparticles. They are then converted to atomic resolution and their structural and dynamic properties are investigated using explicit-solvent atomistic molecular dynamics simulations. The results show that even though the albumin protein remains in a folded structure, the presence of a gold nanoparticle can cause more than 50% of the residues to decrease their flexibility significantly, and approximately 10% of the residues to change their secondary structure. These affected residues are distributed on the whole protein, even on regions that are distant from the nanoparticle. We analyze the changes in structure and flexibility of amino acid residues on a variety of binding sites on albumin and confirm that nanoparticles could allosterically affect the ability of albumin to bind fatty acids, thyroxin and metals. Our simulations suggest that allosteric effects must be considered when designing and deploying nanoparticles in medical and biological applications that depend on protein-nanoparticle interactions.

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

PubMed

Zhong, Huailing; Haddjeri, Nasser; Sánchez, Connie

2012-01-01

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

Tunable allosteric library of caspase-3 identifies coupling between conserved water molecules and conformational selection.

PubMed

Maciag, Joseph J; Mackenzie, Sarah H; Tucker, Matthew B; Schipper, Joshua L; Swartz, Paul; Clark, A Clay

2016-10-11

The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 high-resolution (<2.0 Å) structures of wild-type caspase-3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of posttranslational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of 19 caspase-3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies by more than four orders of magnitude. Altogether, our database consists of 37 high-resolution structures of caspase-3 variants, and we demonstrate that the decrease in activity correlates with a loss of conserved water molecules. The data show that the activity of caspase-3 can be fine-tuned through globally desolvating the active conformation within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine-tune activity through conformational selection.

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

Bitopic Binding Mode of an M1 Muscarinic Acetylcholine Receptor Agonist Associated with Adverse Clinical Trial Outcomes

PubMed Central

Bradley, Sophie J.; Molloy, Colin; Bundgaard, Christoffer; Mogg, Adrian J.; Thompson, Karen J.; Dwomoh, Louis; Sanger, Helen E.; Crabtree, Michael D.; Brooke, Simon M.; Sexton, Patrick M.; Felder, Christian C.; Christopoulos, Arthur; Broad, Lisa M.

2018-01-01

The realization of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer’s disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1H-benzimidazol-2-one), described previously as a potent M1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that “pure” positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses. PMID:29695609

Insights into Brain Glycogen Metabolism: THE STRUCTURE OF HUMAN BRAIN GLYCOGEN PHOSPHORYLASE.

PubMed

Mathieu, Cécile; Li de la Sierra-Gallay, Ines; Duval, Romain; Xu, Ximing; Cocaign, Angélique; Léger, Thibaut; Woffendin, Gary; Camadro, Jean-Michel; Etchebest, Catherine; Haouz, Ahmed; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

2016-08-26

Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation. However, alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease. Glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, catalyzes the rate-limiting step of glycogen mobilization. Moreover, the allosteric regulation of the three GP isozymes (muscle, liver, and brain) by metabolites and phosphorylation, in response to hormonal signaling, fine-tunes glycogenolysis to fulfill energetic and metabolic requirements. Whereas the structures of muscle and liver GPs have been known for decades, the structure of brain GP (bGP) has remained elusive despite its critical role in brain glycogen metabolism. Here, we report the crystal structure of human bGP in complex with PEG 400 (2.5 Å) and in complex with its allosteric activator AMP (3.4 Å). These structures demonstrate that bGP has a closer structural relationship with muscle GP, which is also activated by AMP, contrary to liver GP, which is not. Importantly, despite the structural similarities between human bGP and the two other mammalian isozymes, the bGP structures reveal molecular features unique to the brain isozyme that provide a deeper understanding of the differences in the activation properties of these allosteric enzymes by the allosteric effector AMP. Overall, our study further supports that the distinct structural and regulatory properties of GP isozymes contribute to the different functions of muscle, liver, and brain glycogen. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

NbIT - A New Information Theory-Based Analysis of Allosteric Mechanisms Reveals Residues that Underlie Function in the Leucine Transporter LeuT

PubMed Central

LeVine, Michael V.; Weinstein, Harel

2014-01-01

Complex networks of interacting residues and microdomains in the structures of biomolecular systems underlie the reliable propagation of information from an input signal, such as the concentration of a ligand, to sites that generate the appropriate output signal, such as enzymatic activity. This information transduction often carries the signal across relatively large distances at the molecular scale in a form of allostery that is essential for the physiological functions performed by biomolecules. While allosteric behaviors have been documented from experiments and computation, the mechanism of this form of allostery proved difficult to identify at the molecular level. Here, we introduce a novel analysis framework, called N-body Information Theory (NbIT) analysis, which is based on information theory and uses measures of configurational entropy in a biomolecular system to identify microdomains and individual residues that act as (i)-channels for long-distance information sharing between functional sites, and (ii)-coordinators that organize dynamics within functional sites. Application of the new method to molecular dynamics (MD) trajectories of the occluded state of the bacterial leucine transporter LeuT identifies a channel of allosteric coupling between the functionally important intracellular gate and the substrate binding sites known to modulate it. NbIT analysis is shown also to differentiate residues involved primarily in stabilizing the functional sites, from those that contribute to allosteric couplings between sites. NbIT analysis of MD data thus reveals rigorous mechanistic elements of allostery underlying the dynamics of biomolecular systems. PMID:24785005

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

Bitopic Binding Mode of an M1 Muscarinic Acetylcholine Receptor Agonist Associated with Adverse Clinical Trial Outcomes.

PubMed

Bradley, Sophie J; Molloy, Colin; Bundgaard, Christoffer; Mogg, Adrian J; Thompson, Karen J; Dwomoh, Louis; Sanger, Helen E; Crabtree, Michael D; Brooke, Simon M; Sexton, Patrick M; Felder, Christian C; Christopoulos, Arthur; Broad, Lisa M; Tobin, Andrew B; Langmead, Christopher J

2018-06-01

The realization of the therapeutic potential of targeting the M 1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer's disease has prompted the discovery of M 1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1 H -benzimidazol-2-one), described previously as a potent M 1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M 1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that "pure" positive allosteric modulators showing selectivity for the M 1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses. Copyright © 2018 by The Author(s).

Ionotropic and Metabotropic Mechanisms of Allosteric Modulation of α7 Nicotinic Receptor Intracellular Calcium.

PubMed

King, Justin R; Ullah, Aman; Bak, Ellen; Jafri, M Saleet; Kabbani, Nadine

2018-06-01

The pharmacological targeting of the α 7 nicotinic acetylcholine receptor ( α 7) is a promising strategy in the development of new drugs for neurologic diseases. Because α 7 receptors regulate cellular calcium, we investigated how the prototypical type II-positive allosteric modulator PNU120596 affects α 7-mediated calcium signaling. Live imaging experiments show that PNU120596 augments ryanodine receptor-driven calcium-induced calcium release (CICR), inositol-induced calcium release (IICR), and phospholipase C activation by the α 7 receptor. Both influx of calcium through the α 7 nicotinic acetylcholine receptor (nAChR) channel as well as the binding of intracellular G proteins were involved in the effect of PNU120596 on intracellular calcium. This is evidenced by the findings that chelation of extracellular calcium, expression of α 7 D44A or α 7 345-348A mutant subunits, or blockade of calcium store release compromised the ability of PNU120596 to increase intracellular calcium transients generated by α 7 ligand activation. Spatiotemporal stochastic modeling of calcium transient responses corroborates these results and indicates that α 7 receptor activation enables calcium microdomains locally and to lesser extent in the distant cytosol. From the model, allosteric modulation of the receptor activates CICR locally via ryanodine receptors and augments IICR through enhanced calcium influx due to prolonged α 7 nAChR opening. These findings provide a new mechanistic framework for understanding the effect of α 7 receptor allosteric modulation on both local and global calcium dynamics. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS

PubMed Central

Jun, Jesse E.; Yang, Ming; Chen, Hang; Chakraborty, Arup K.

2013-01-01

Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation. PMID:23589333

Kinetics of Interaction between ADP-ribosylation Factor-1 (Arf1) and the Sec7 Domain of Arno Guanine Nucleotide Exchange Factor, Modulation by Allosteric Factors, and the Uncompetitive Inhibitor Brefeldin A

PubMed Central

Rouhana, Jad; Padilla, André; Estaran, Sébastien; Bakari, Sana; Delbecq, Stephan; Boublik, Yvan; Chopineau, Joel; Pugnière, Martine; Chavanieu, Alain

2013-01-01

The GDP/GTP nucleotide exchange of Arf1 is catalyzed by nucleotide exchange factors (GEF), such as Arno, which act through their catalytic Sec7 domain. This exchange is a complex mechanism that undergoes conformational changes and intermediate complex species involving several allosteric partners such as nucleotides, Mg2+, and Sec7 domains. Using a surface plasmon resonance approach, we characterized the kinetic binding parameters for various intermediate complexes. We first confirmed that both GDP and GTP counteract equivalently to the free-nucleotide binary Arf1-Arno complex stability and revealed that Mg2+ potentiates by a factor of 2 the allosteric effect of GDP. Then we explored the uncompetitive inhibitory mechanism of brefeldin A (BFA) that conducts to an abortive pentameric Arf1-Mg2+-GDP-BFA-Sec7 complex. With BFA, the association rate of the abortive complex is drastically reduced by a factor of 42, and by contrast, the 15-fold decrease of the dissociation rate concurs to stabilize the pentameric complex. These specific kinetic signatures have allowed distinguishing the level and nature as well as the fate in real time of formed complexes according to experimental conditions. Thus, we showed that in the presence of GDP, the BFA-resistant Sec7 domain of Arno can also associate to form a pentameric complex, which suggests that the uncompetitive inhibition by BFA and the nucleotide allosteric effect combine to stabilize such abortive complex. PMID:23255605

Enzymological Basis for Growth Inhibition by l-Phenylalanine in the Cyanobacterium Synechocystis sp. 29108

PubMed Central

Hall, Geraldine C.; Jensen, Roy A.

1980-01-01

The pattern of allosteric control in the biosynthetic pathway for aromatic amino acids provides a basis to explain vulnerability to growth inhibition by l-phenylalanine (0.2 mM or greater) in the unicellular cyanobacterium Synechocystis sp. 29108. We attribute growth inhibition to the hypersensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase to feedback inhibition by l-phenylalanine. Hyperregulation of this initial enzyme of aromatic biosynthesis depletes the supply of precursors needed for biosynthesis of l-tyrosine and l-tryptophan. Consistent with this mechanism is the total reversal of phenylalanine inhibition by a combination of tyrosine and tryptophan. Inhibited cultures also contained decreased levels of phycocyanin pigments, a characteristic previously correlated with amino acid starvation in cyanobacteria. l-Phenylalanine is a potent noncompetitive inhibitor (with both substrates) of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase, whereas l-tyrosine is a very weak inhibitor. Prephenate dehydratase also displays allosteric sensitivity to phenylalanine (inhibition) and to tyrosine (activation). Both 2-fluoro and 4-fluoro derivatives of phenylalanine were potent analog antimetabolites, and these were used in addition to l-phenylalanine as selective agents for resistant mutants. Mutants were isolated which excreted both phenylalanine and tyrosine, the consequence of an altered 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase no longer sensitive to feedback inhibition. Simultaneous insensitivity to l-tyrosine suggests that l-tyrosine acts as a weak analog mimic of l-phenylalanine at a common binding site. Prephenate dehydratase in the regulatory mutants was unaltered. Surprisingly, in view of the lack of regulation in the tyrosine branchlet of the pathway, such mutants excrete more phenylalanine than tyrosine, indicating that l-tyrosine activation dominates l-phenylalanine inhibition of prephenate dehydratase in vivo. In mutant Phe r19 the loss in allosteric sensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase was accompanied by a threefold increase in specific activity. This could suggest that existence of a modest degree of repression control (autogenous) over 3-deoxy-d-arabinoheptulosonate synthase, although other explanations are possible. Specific activities of chorismate mutase, prephenate dehydratase, shikimate/nicotinamide adenine dinucleotide phosphate dehydrogenase, and arogenate/nicotinamide adenine dinucleotide phosphate dehydrogenase in mutant Phe r19 were identical with those of the wild type. PMID:6108316

Efficacy of Selective PDE4D Negative Allosteric Modulators in the Object Retrieval Task in Female Cynomolgus Monkeys (Macaca fascicularis)

PubMed Central

Sutcliffe, Jane S.; Beaumont, Vahri; Watson, James M.; Chew, Chang Sing; Beconi, Maria; Hutcheson, Daniel M.; Dominguez, Celia; Munoz-Sanjuan, Ignacio

2014-01-01

Cyclic adenosine monophosphate (cAMP) signalling plays an important role in synaptic plasticity and information processing in the hippocampal and basal ganglia systems. The augmentation of cAMP signalling through the selective inhibition of phosphodiesterases represents a viable strategy to treat disorders associated with dysfunction of these circuits. The phosphodiesterase (PDE) type 4 inhibitor rolipram has shown significant pro-cognitive effects in neurological disease models, both in rodents and primates. However, competitive non-isoform selective PDE4 inhibitors have a low therapeutic index which has stalled their clinical development. Here, we demonstrate the pro-cognitive effects of selective negative allosteric modulators (NAMs) of PDE4D, D159687 and D159797 in female Cynomolgous macaques, in the object retrieval detour task. The efficacy displayed by these NAMs in a primate cognitive task which engages the corticostriatal circuitry, together with their suitable pharmacokinetic properties and safety profiles, suggests that clinical development of these allosteric modulators should be considered for the treatment of a variety of brain disorders associated with cognitive decline. PMID:25050979

Small-molecule agonists for the glucagon-like peptide 1 receptor

PubMed Central

Knudsen, Lotte Bjerre; Kiel, Dan; Teng, Min; Behrens, Carsten; Bhumralkar, Dilip; Kodra, János T.; Holst, Jens J.; Jeppesen, Claus B.; Johnson, Michael D.; de Jong, Johannes Cornelis; Jorgensen, Anker Steen; Kercher, Tim; Kostrowicki, Jarek; Madsen, Peter; Olesen, Preben H.; Petersen, Jacob S.; Poulsen, Fritz; Sidelmann, Ulla G.; Sturis, Jeppe; Truesdale, Larry; May, John; Lau, Jesper

2007-01-01

The peptide hormone glucagon-like peptide (GLP)-1 has important actions resulting in glucose lowering along with weight loss in patients with type 2 diabetes. As a peptide hormone, GLP-1 has to be administered by injection. Only a few small-molecule agonists to peptide hormone receptors have been described and none in the B family of the G protein coupled receptors to which the GLP-1 receptor belongs. We have discovered a series of small molecules known as ago-allosteric modulators selective for the human GLP-1 receptor. These compounds act as both allosteric activators of the receptor and independent agonists. Potency of GLP-1 was not changed by the allosteric agonists, but affinity of GLP-1 for the receptor was increased. The most potent compound identified stimulates glucose-dependent insulin release from normal mouse islets but, importantly, not from GLP-1 receptor knockout mice. Also, the compound stimulates insulin release from perfused rat pancreas in a manner additive with GLP-1 itself. These compounds may lead to the identification or design of orally active GLP-1 agonists. PMID:17213325

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

2008-10-08

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

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

Design, synthesis, and activity of 2,3-diphosphoglycerate analogs as allosteric modulators of hemoglobin O2 affinity.

PubMed

Kassa, Tigist W; Zhang, Ning; Palmer, Andre F; Matthews, Jason Shastri

2013-04-01

Four phosphonate derivates of 2,3-diphosphoglycerate (2,3-DPG), in which the phosphate group is replaced by a methylene or difluoromethylene, were successfully synthesized for use as allosteric modulators of hemoglobin (Hb) O2 affinity. The syntheses were accomplished in four steps and the reagents were converted to their potassium salts to allow for effective binding with Hb in aqueous media. O2 equilibrium measurements of the chemically modified Hbs exhibited P50 values in the range 8.9-12.8 with Hill coefficients in the range of 1.5-2.4.

Discovery and characterization of a novel series of N-phenylsulfonyl-1H-pyrrole picolinamides as positive allosteric modulators of the metabotropic glutamate receptor 4 (mGlu4).

PubMed

Gogliotti, Rocco D; Blobaum, Anna L; Morrison, Ryan M; Daniels, J Scott; Salovich, James M; Cheung, Yiu-Yin; Rodriguez, Alice L; Loch, Matthew T; Conn, P Jeffrey; Lindsley, Craig W; Niswender, Colleen M; Hopkins, Corey R

2016-07-01

Herein we report the synthesis and characterization of a novel series of N-phenylsulfonyl-1H-pyrrole picolinamides as novel positive allosteric modulators of mGlu4. We detail our work towards finding phenyl replacements for the core scaffold of previously reported phenyl sulfonamides and phenyl sulfone compounds. Our efforts culminated in the identification of N-(1-((3,4-dimethylphenyl)sulfonyl)-1H-pyrrol-3-yl)picolinamide as a potent PAM of mGlu4. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structural analysis of a 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase with an N-terminal chorismate mutase-like regulatory domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Light, Samuel H.; Halavaty, Andrei S.; Minasov, George

2012-06-27

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes the first step in the biosynthesis of a number of aromatic metabolites. Likely because this reaction is situated at a pivotal biosynthetic gateway, several DAHPS classes distinguished by distinct mechanisms of allosteric regulation have independently evolved. One class of DAHPSs contains a regulatory domain with sequence homology to chorismate mutase - an enzyme further downstream of DAHPS that catalyzes the first committed step in tyrosine/phenylalanine biosynthesis - and is inhibited by chorismate mutase substrate (chorismate) and product (prephenate). Described in this work, structures of the Listeria monocytogenes chorismate/prephenate regulated DAHPS in complex with Mn{sup 2+}more » and Mn{sup 2+} + phosphoenolpyruvate reveal an unusual quaternary architecture: DAHPS domains assemble as a tetramer, from either side of which chorismate mutase-like (CML) regulatory domains asymmetrically emerge to form a pair of dimers. This domain organization suggests that chorismate/prephenate binding promotes a stable interaction between the discrete regulatory and catalytic domains and supports a mechanism of allosteric inhibition similar to tyrosine/phenylalanine control of a related DAHPS class. We argue that the structural similarity of chorismate mutase enzyme and CML regulatory domain provides a unique opportunity for the design of a multitarget antibacterial.« less

Ube2V2 Is a Rosetta Stone Bridging Redox and Ubiquitin Codes, Coordinating DNA Damage Responses.

PubMed

Zhao, Yi; Long, Marcus J C; Wang, Yiran; Zhang, Sheng; Aye, Yimon

2018-02-28

Posttranslational modifications (PTMs) are the lingua franca of cellular communication. Most PTMs are enzyme-orchestrated. However, the reemergence of electrophilic drugs has ushered mining of unconventional/non-enzyme-catalyzed electrophile-signaling pathways. Despite the latest impetus toward harnessing kinetically and functionally privileged cysteines for electrophilic drug design, identifying these sensors remains challenging. Herein, we designed "G-REX"-a technique that allows controlled release of reactive electrophiles in vivo. Mitigating toxicity/off-target effects associated with uncontrolled bolus exposure, G-REX tagged first-responding innate cysteines that bind electrophiles under true k cat / K m conditions. G-REX identified two allosteric ubiquitin-conjugating proteins-Ube2V1/Ube2V2-sharing a novel privileged-sensor-cysteine. This non-enzyme-catalyzed-PTM triggered responses specific to each protein. Thus, G-REX is an unbiased method to identify novel functional cysteines. Contrasting conventional active-site/off-active-site cysteine-modifications that regulate target activity, modification of Ube2V2 allosterically hyperactivated its enzymatically active binding-partner Ube2N, promoting K63-linked client ubiquitination and stimulating H2AX-dependent DNA damage response. This work establishes Ube2V2 as a Rosetta-stone bridging redox and ubiquitin codes to guard genome integrity.

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

PubMed

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

2012-02-01

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

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

NASA Astrophysics Data System (ADS)

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

2012-02-01

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

Allosteric activation of exopolysaccharide synthesis through cyclic di-GMP-stimulated protein–protein interaction

PubMed Central

Steiner, Samuel; Lori, Christian; Boehm, Alex; Jenal, Urs

2013-01-01

In many bacterial pathogens, the second messenger c-di-GMP stimulates the production of an exopolysaccharide (EPS) matrix to shield bacteria from assaults of the immune system. How c-di-GMP induces EPS biogenesis is largely unknown. Here, we show that c-di-GMP allosterically activates the synthesis of poly-β-1,6-N-acetylglucosamine (poly-GlcNAc), a major extracellular matrix component of Escherichia coli biofilms. C-di-GMP binds directly to both PgaC and PgaD, the two inner membrane components of the poly-GlcNAc synthesis machinery to stimulate their glycosyltransferase activity. We demonstrate that the PgaCD machinery is a novel type c-di-GMP receptor, where ligand binding to two proteins stabilizes their interaction and promotes enzyme activity. This is the first example of a c-di-GMP-mediated process that relies on protein–protein interaction. At low c-di-GMP concentrations, PgaD fails to interact with PgaC and is rapidly degraded. Thus, when cells experience a c-di-GMP trough, PgaD turnover facilitates the irreversible inactivation of the Pga machinery, thereby temporarily uncoupling it from c-di-GMP signalling. These data uncover a mechanism of c-di-GMP-mediated EPS control and provide a frame for c-di-GMP signalling specificity in pathogenic bacteria. PMID:23202856

Cocaine Effects on Dopaminergic Transmission Depend on a Balance between Sigma-1 and Sigma-2 Receptor Expression.

PubMed

Aguinaga, David; Medrano, Mireia; Vega-Quiroga, Ignacio; Gysling, Katia; Canela, Enric I; Navarro, Gemma; Franco, Rafael

2018-01-01

Sigma σ 1 and σ 2 receptors are targets of cocaine. Despite sharing a similar name, the two receptors are structurally unrelated and their physiological role is unknown. Cocaine increases the level of dopamine, a key neurotransmitter in CNS motor control and reward areas. While the drug also affects dopaminergic signaling by allosteric modulations exerted by σ 1 R interacting with dopamine D 1 and D 2 receptors, the potential regulation of dopaminergic transmission by σ 2 R is also unknown. We here demonstrate that σ 2 R may form heteroreceptor complexes with D 1 but not with D 2 receptors. Remarkably σ 1 , σ 2 , and D 1 receptors may form heterotrimers with particular signaling properties. Determination of cAMP levels, MAP kinase activation and label-free assays demonstrate allosteric interactions within the trimer. Importantly, the presence of σ 2 R induces bias in signal transduction as σ 2 R ligands increase cAMP signaling whereas reduce MAP kinase activation. These effects, which are opposite to those exerted via σ 1 R, suggest that the D 1 receptor-mediated signaling depends on the degree of trimer formation and the differential balance of sigma receptor and heteroreceptor expression in acute versus chronic cocaine consumption. Although the physiological role is unknown, the heteroreceptor complex formed by σ 1 , σ 2 , and D 1 receptors arise as relevant to convey the cocaine actions on motor control and reward circuits and as a key factor in acquisition of the addictive habit.

Cocaine Effects on Dopaminergic Transmission Depend on a Balance between Sigma-1 and Sigma-2 Receptor Expression

PubMed Central

Aguinaga, David; Medrano, Mireia; Vega-Quiroga, Ignacio; Gysling, Katia; Canela, Enric I.; Navarro, Gemma; Franco, Rafael

2018-01-01

Sigma σ1 and σ2 receptors are targets of cocaine. Despite sharing a similar name, the two receptors are structurally unrelated and their physiological role is unknown. Cocaine increases the level of dopamine, a key neurotransmitter in CNS motor control and reward areas. While the drug also affects dopaminergic signaling by allosteric modulations exerted by σ1R interacting with dopamine D1 and D2 receptors, the potential regulation of dopaminergic transmission by σ2R is also unknown. We here demonstrate that σ2R may form heteroreceptor complexes with D1 but not with D2 receptors. Remarkably σ1, σ2, and D1 receptors may form heterotrimers with particular signaling properties. Determination of cAMP levels, MAP kinase activation and label-free assays demonstrate allosteric interactions within the trimer. Importantly, the presence of σ2R induces bias in signal transduction as σ2R ligands increase cAMP signaling whereas reduce MAP kinase activation. These effects, which are opposite to those exerted via σ1R, suggest that the D1 receptor-mediated signaling depends on the degree of trimer formation and the differential balance of sigma receptor and heteroreceptor expression in acute versus chronic cocaine consumption. Although the physiological role is unknown, the heteroreceptor complex formed by σ1, σ2, and D1 receptors arise as relevant to convey the cocaine actions on motor control and reward circuits and as a key factor in acquisition of the addictive habit. PMID:29483862

Dual regulatory switch confers tighter control on HtrA2 proteolytic activity.

PubMed

Singh, Nitu; D'Souza, Areetha; Cholleti, Anuradha; Sastry, G Madhavi; Bose, Kakoli

2014-05-01

High-temperature requirement protease A2 (HtrA2), a multitasking serine protease that is involved in critical biological functions and pathogenicity, such as apoptosis and cancer, is a potent therapeutic target. It is established that the C-terminal post-synaptic density protein, Drosophila disc large tumor suppressor, zonula occludens-1 protein (PDZ) domain of HtrA2 plays pivotal role in allosteric modulation, substrate binding and activation, as commonly reported in other members of this family. Interestingly, HtrA2 exhibits an additional level of functional modulation through its unique N-terminus, as is evident from 'inhibitor of apoptosis proteins' binding and cleavage. This phenomenon emphasizes multiple activation mechanisms, which so far remain elusive. Using conformational dynamics, binding kinetics and enzymology studies, we addressed this complex behavior with respect to defining its global mode of regulation and activity. Our findings distinctly demonstrate a novel N-terminal ligand-mediated triggering of an allosteric switch essential for transforming HtrA2 to a proteolytically competent state in a PDZ-independent yet synergistic activation process. Dynamic analyses suggested that it occurs through a series of coordinated structural reorganizations at distal regulatory loops (L3, LD, L1), leading to a population shift towards the relaxed conformer. This precise synergistic coordination among different domains might be physiologically relevant to enable tighter control upon HtrA2 activation for fostering its diverse cellular functions. Understanding this complex rheostatic dual switch mechanism offers an opportunity for targeting various disease conditions with tailored site-specific effector molecules. © 2014 FEBS.

Rational Design and Tuning of Functional RNA Switch to Control an Allosteric Intermolecular Interaction.

PubMed

Endoh, Tamaki; Sugimoto, Naoki

2015-08-04

Conformational transitions of biomolecules in response to specific stimuli control many biological processes. In natural functional RNA switches, often called riboswitches, a particular RNA structure that has a suppressive or facilitative effect on gene expression transitions to an alternative structure with the opposite effect upon binding of a specific metabolite to the aptamer region. Stability of RNA secondary structure (-ΔG°) can be predicted based on thermodynamic parameters and is easily tuned by changes in nucleobases. We envisioned that tuning of a functional RNA switch that causes an allosteric interaction between an RNA and a peptide would be possible based on a predicted switching energy (ΔΔG°) that corresponds to the energy difference between the RNA secondary structure before (-ΔG°before) and after (-ΔG°after) the RNA conformational transition. We first selected functional RNA switches responsive to neomycin with predicted ΔΔG° values ranging from 5.6 to 12.2 kcal mol(-1). We then demonstrated a simple strategy to rationally convert the functional RNA switch to switches responsive to natural metabolites thiamine pyrophosphate, S-adenosyl methionine, and adenine based on the predicted ΔΔG° values. The ΔΔG° values of the designed RNA switches proportionally correlated with interaction energy (ΔG°interaction) between the RNA and peptide, and we were able to tune the sensitivity of the RNA switches for the trigger molecule. The strategy demonstrated here will be generally applicable for construction of functional RNA switches and biosensors in which mechanisms are based on conformational transition of nucleic acids.

Using Evolution to Guide Protein Engineering: The Devil IS in the Details.

PubMed

Swint-Kruse, Liskin

2016-07-12

For decades, protein engineers have endeavored to reengineer existing proteins for novel applications. Overall, protein folds and gross functions can be readily transferred from one protein to another by transplanting large blocks of sequence (i.e., domain recombination). However, predictably fine-tuning function (e.g., by adjusting ligand affinity, specificity, catalysis, and/or allosteric regulation) remains a challenge. One approach has been to use the sequences of protein families to identify amino acid positions that change during the evolution of functional variation. The rationale is that these nonconserved positions could be mutated to predictably fine-tune function. Evolutionary approaches to protein design have had some success, but the engineered proteins seldom replicate the functional performances of natural proteins. This Biophysical Perspective reviews several complexities that have been revealed by evolutionary and experimental studies of protein function. These include 1) challenges in defining computational and biological thresholds that define important amino acids; 2) the co-occurrence of many different patterns of amino acid changes in evolutionary data; 3) difficulties in mapping the patterns of amino acid changes to discrete functional parameters; 4) the nonconventional mutational outcomes that occur for a particular group of functionally important, nonconserved positions; 5) epistasis (nonadditivity) among multiple mutations; and 6) the fact that a large fraction of a protein's amino acids contribute to its overall function. To overcome these challenges, new goals are identified for future studies. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

PSNCBAM-1, a novel allosteric antagonist at cannabinoid CB1 receptors with hypophagic effects in rats.

PubMed

Horswill, J G; Bali, U; Shaaban, S; Keily, J F; Jeevaratnam, P; Babbs, A J; Reynet, C; Wong Kai In, P

2007-11-01

Rimonabant (Acomplia, SR141716A), a cannabinoid CB1 receptor inverse agonist, has recently been approved for the treatment of obesity. There are, however, concerns regarding its side effect profile. Developing a CB1 antagonist with a different pharmacological mechanism may lead to a safer alternative. To this end we have screened a proprietary small molecule library and have discovered a novel class of allosteric antagonist at CB1 receptors. Herein, we have characterized an optimized prototypical molecule, PSNCBAM-1, and its hypophagic effects in vivo. A CB1 yeast reporter assay was used as a primary screen. PSNCBAM-1 was additionally characterized in [35S]-GTPgammaS, cAMP and radioligand binding assays. An acute rat feeding model was used to evaluate its effects on food intake and body weight in vivo. In CB1 receptor yeast reporter assays, PSNCBAM-1 blocked the effects induced by agonists such as CP55,940, WIN55212-2, anandamide (AEA) or 2-arachidonoyl glycerol (2-AG). The antagonist characteristics of PSNCBAM-1 were confirmed in [35S]-GTPgammaS binding and cAMP assays and was shown to be non-competitive by Schild analyses. PSNCBAM-1 did not affect CB2 receptors. In radioligand binding assays, PSNCBAM-1 increased the binding of [3H]CP55,940 despite its antagonist effects. In an acute rat feeding model, PSNCBAM-1 decreased food intake and body weight. PSNCBAM-1 exerted its effects through selective allosteric modulation of the CB1 receptor. The acute effects on food intake and body weight induced in rats provide a first report of in vivo activity for an allosteric CB1 receptor antagonist.

PSNCBAM-1, a novel allosteric antagonist at cannabinoid CB1 receptors with hypophagic effects in rats

PubMed Central

Horswill, J G; Bali, U; Shaaban, S; Keily, J F; Jeevaratnam, P; Babbs, A J; Reynet, C; Wong Kai In, P

2007-01-01

Background and purpose: Rimonabant (AcompliaTM, SR141716A), a cannabinoid CB1 receptor inverse agonist, has recently been approved for the treatment of obesity. There are, however, concerns regarding its side effect profile. Developing a CB1 antagonist with a different pharmacological mechanism may lead to a safer alternative. To this end we have screened a proprietary small molecule library and have discovered a novel class of allosteric antagonist at CB1 receptors. Herein, we have characterized an optimized prototypical molecule, PSNCBAM-1, and its hypophagic effects in vivo. Experimental approach: A CB1 yeast reporter assay was used as a primary screen. PSNCBAM-1 was additionally characterized in [35S]-GTPγS, cAMP and radioligand binding assays. An acute rat feeding model was used to evaluate its effects on food intake and body weight in vivo. Key results: In CB1 receptor yeast reporter assays, PSNCBAM-1 blocked the effects induced by agonists such as CP55,940, WIN55212-2, anandamide (AEA) or 2-arachidonoyl glycerol (2-AG). The antagonist characteristics of PSNCBAM-1 were confirmed in [35S]-GTPγS binding and cAMP assays and was shown to be non-competitive by Schild analyses. PSNCBAM-1 did not affect CB2 receptors. In radioligand binding assays, PSNCBAM-1 increased the binding of [3H]CP55,940 despite its antagonist effects. In an acute rat feeding model, PSNCBAM-1 decreased food intake and body weight. Conclusions and implications: PSNCBAM-1 exerted its effects through selective allosteric modulation of the CB1 receptor. The acute effects on food intake and body weight induced in rats provide a first report of in vivo activity for an allosteric CB1 receptor antagonist. PMID:17592509

Conformational Transition Pathway in the Activation Process of Allosteric Glucokinase

PubMed Central

Shi, Ting; Zhao, Yaxue; Chen, Yingyi; Li, Xiaobai; Liu, Xinyi; Huang, Zhimin; Zhang, Jian

2013-01-01

Glucokinase (GK) is a glycolytic enzyme that plays an important role in regulating blood glucose level, thus acting as a potentially attractive target for drug discovery in the treatment of diabetes of the young type 2 and persistent hyperinsulinemic hypoglycemia of infancy. To characterize the activation mechanism of GK from the super-open state (inactive state) to the closed state (active state), a series of conventional molecular dynamics (MD) and targeted MD (TMD) simulations were performed on this enzyme. Conventional MD simulation showed a specific conformational ensemble of GK when the enzyme is inactive. Seven TMD simulations depicted a reliably conformational transition pathway of GK from the inactive state to the active state, and the components important to the conformational change of GK were identified by analyzing the detailed structures of the TMD trajectories. In combination with the inactivation process, our findings showed that the whole conformational pathway for the activation-inactivation-activation of GK is a one-direction circulation, and the active state is less stable than the inactive state in the circulation. Additionally, glucose was demonstrated to gradually modulate its binding pose with the help of residues in the large domain and connecting region of GK during the activation process. Furthermore, the obtained energy barriers were used to explain the preexisting equilibrium and the slow binding kinetic process of the substrate by GK. The simulated results are in accordance with the recent findings from the mutagenesis experiments and kinetic analyses. Our observations reveal a complicated conformational process in the allosteric protein, resulting in new knowledge about the delicate mechanisms for allosteric biological macromolecules that will be useful in drug design for targeting allosteric proteins. PMID:23409066

Toward a detailed description of the pathways of allosteric communication in the GroEL chaperonin through atomistic simulation.

PubMed

Piggot, Thomas J; Sessions, Richard B; Burston, Steven G

2012-02-28

GroEL, along with its coprotein GroES, is essential for ensuring the correct folding of unfolded or newly synthesized proteins in bacteria. GroEL is a complex, allosteric molecule, composed of two heptameric rings stacked back to back, that undergoes large structural changes during its reaction cycle. These structural changes are driven by the cooperative binding and subsequent hydrolysis of ATP, by GroEL. Despite numerous previous studies, the precise mechanisms of allosteric communication and the associated structural changes remain elusive. In this paper, we describe a series of all-atom, unbiased, molecular dynamics simulations over relatively long (50-100 ns) time scales of a single, isolated GroEL subunit and also a heptameric GroEL ring, in the presence and absence of ATP. Combined with results from a distance restraint-biased simulation of the single ring, the atomistic details of the earliest stages of ATP-driven structural changes within this complex molecule are illuminated. Our results are in broad agreement with previous modeling studies of isolated subunits and with a coarse-grained, forcing simulation of the single ring. These are the first reported all-atom simulations of the GroEL single-ring complex and provide a unique insight into the role of charged residues K80, K277, R284, R285, and E388 at the subunit interface in transmission of the allosteric signal. These simulations also demonstrate the feasibility of performing all-atom simulations of very large systems on sufficiently long time scales on typical high performance computing facilities to show the origins of the earliest events in biologically relevant processes.

Conserved Allosteric Hot Spots in the Transmembrane Domains of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels and Multidrug Resistance Protein (MRP) Pumps*

PubMed Central

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

2014-01-01

ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs. PMID:24876383

Structural modulation of factor VIIa by full-length tissue factor (TF1-263): implication of novel interactions between EGF2 domain and TF.

PubMed

Prasad, Ramesh; Sen, Prosenjit

2018-02-01

Tissue factor (TF)-mediated factor VII (FVII) activation and a subsequent proteolytic TF-FVIIa binary complex formation is the key step initiating the coagulation cascade, with implications in various homeostatic and pathologic scenarios. TF binding allosterically modifies zymogen-like free FVIIa to its highly catalytically active form. As a result of unresolved crystal structure of the full-length TF 1-263 -FVIIa binary complex and free FVIIa, allosteric alterations in FVIIa following its binding to full-length TF and the consequences of these on function are not entirely clear. The present study aims to map and identify structural alterations in FVIIa and TF resulting from full-length TF binding to FVIIa and the key events responsible for enhanced FVIIa activity in coagulation. We constructed the full-length TF 1-263 -FVIIa membrane bound complex using computational modeling and subjected it to molecular dynamics (MD) simulations. MD simulations showed that TF alters the structure of each domain of FVIIa and these combined alterations contribute to enhanced TF-FVIIa activity. Detailed, domain-wise investigation revealed several new non-covalent interactions between TF and FVIIa that were not found in the truncated soluble TF-FVIIa crystal structure. The structural modulation of each FVIIa domain imparted by TF indicated that both inter and intra-domain communication is crucial for allosteric modulation of FVIIa. Our results suggest that these newly formed interactions can provide additional stability to the protease domain and regulate its activity profile by governing catalytic triad (CT) orientation and localization. The unexplored newly formed interactions between EGF2 and TF provides a possible explanation for TF-induced allosteric activation of FVIIa.

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

PubMed

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

2014-07-18

ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Switch I-dependent allosteric signaling in a G-protein chaperone-B12 enzyme complex.

PubMed

Campanello, Gregory C; Lofgren, Michael; Yokom, Adam L; Southworth, Daniel R; Banerjee, Ruma

2017-10-27

G-proteins regulate various processes ranging from DNA replication and protein synthesis to cytoskeletal dynamics and cofactor assimilation and serve as models for uncovering strategies deployed for allosteric signal transduction. MeaB is a multifunctional G-protein chaperone, which gates loading of the active 5'-deoxyadenosylcobalamin cofactor onto methylmalonyl-CoA mutase (MCM) and precludes loading of inactive cofactor forms. MeaB also safeguards MCM, which uses radical chemistry, against inactivation and rescues MCM inactivated during catalytic turnover by using the GTP-binding energy to offload inactive cofactor. The conserved switch I and II signaling motifs used by G-proteins are predicted to mediate allosteric regulation in response to nucleotide binding and hydrolysis in MeaB. Herein, we targeted conserved residues in the MeaB switch I motif to interrogate the function of this loop. Unexpectedly, the switch I mutations had only modest effects on GTP binding and on GTPase activity and did not perturb stability of the MCM-MeaB complex. However, these mutations disrupted multiple MeaB chaperone functions, including cofactor editing, loading, and offloading. Hence, although residues in the switch I motif are not essential for catalysis, they are important for allosteric regulation. Furthermore, single-particle EM analysis revealed, for the first time, the overall architecture of the MCM-MeaB complex, which exhibits a 2:1 stoichiometry. These EM studies also demonstrate that the complex exhibits considerable conformational flexibility. In conclusion, the switch I element does not significantly stabilize the MCM-MeaB complex or influence the affinity of MeaB for GTP but is required for transducing signals between MeaB and MCM. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed Central

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

2009-01-01

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

Designing small molecules to target cryptic pockets yields both positive and negative allosteric modulators

PubMed Central

Moeder, Katelyn E.; Ho, Chris M. W.; Zimmerman, Maxwell I.; Frederick, Thomas E.; Bowman, Gregory R.

2017-01-01

Allosteric drugs, which bind to proteins in regions other than their main ligand-binding or active sites, make it possible to target proteins considered “undruggable” and to develop new therapies that circumvent existing resistance. Despite growing interest in allosteric drug discovery, rational design is limited by a lack of sufficient structural information about alternative binding sites in proteins. Previously, we used Markov State Models (MSMs) to identify such “cryptic pockets,” and here we describe a method for identifying compounds that bind in these cryptic pockets and modulate enzyme activity. Experimental tests validate our approach by revealing both an inhibitor and two activators of TEM β-lactamase (TEM). To identify hits, a library of compounds is first virtually screened against either the crystal structure of a known cryptic pocket or an ensemble of structures containing the same cryptic pocket that is extracted from an MSM. Hit compounds are then screened experimentally and characterized kinetically in individual assays. We identify three hits, one inhibitor and two activators, demonstrating that screening for binding to allosteric sites can result in both positive and negative modulation. The hit compounds have modest effects on TEM activity, but all have higher affinities than previously identified inhibitors, which bind the same cryptic pocket but were found, by chance, via a computational screen targeting the active site. Site-directed mutagenesis of key contact residues predicted by the docking models is used to confirm that the compounds bind in the cryptic pocket as intended. Because hit compounds are identified from docking against both the crystal structure and structures from the MSM, this platform should prove suitable for many proteins, particularly targets whose crystal structures lack obvious druggable pockets, and for identifying both inhibitory and activating small-molecule modulators. PMID:28570708

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

PubMed Central

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

2013-01-01

The modulatory role of allosteric receptor-receptor interactions in the pain pathways of the Central Nervous System and the peripheral nociceptors has become of increasing interest. As integrators of nociceptive and antinociceptive wiring and volume transmission signals, with a major role for the opioid receptor heteromers, they likely have an important role in the pain circuits and may be involved in acupuncture. The delta opioid receptor (DOR) exerts an antagonistic allosteric influence on the mu opioid receptor (MOR) function in a MOR-DOR heteromer. This heteromer contributes to morphine-induced tolerance and dependence, since it becomes abundant and develops a reduced G-protein-coupling with reduced signaling mainly operating via β-arrestin2 upon chronic morphine treatment. A DOR antagonist causes a return of the Gi/o binding and coupling to the heteromer and the biological actions of morphine. The gender- and ovarian steroid-dependent recruitment of spinal cord MOR/kappa opioid receptor (KOR) heterodimers enhances antinociceptive functions and if impaired could contribute to chronic pain states in women. MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, mediating morphine induced itch. Other mechanism for the antinociceptive actions of acupuncture along meridians may be that it enhances the cross-desensitization of the TRPA1 (chemical nociceptor)-TRPV1 (capsaicin receptor) heteromeric channel complexes within the nociceptor terminals located along these meridians. Selective ionotropic cannabinoids may also produce cross-desensitization of the TRPA1-TRPV1 heteromeric nociceptor channels by being negative allosteric modulators of these channels leading to antinociception and antihyperalgesia. PMID:23956775

The α7 nicotinic receptor dual allosteric agonist and positive allosteric modulator GAT107 reverses nociception in mouse models of inflammatory and neuropathic pain

PubMed Central

Wilkerson, Jenny L; Kulkarni, Abhijit; Toma, Wisam; AlSharari, Shakir; Gul, Zulfiye; Lichtman, Aron H; Papke, Roger L; Damaj, M Imad

2016-01-01

Background and Purpose Orthosteric agonists and positive allosteric modulators (PAMs) of the α7 nicotinic ACh receptor (nAChR) represent novel therapeutic approaches for pain modulation. Moreover, compounds with dual function as allosteric agonists and PAMs, known as ago‐PAMs, add further regulation of receptor function. Experimental Approach Initial studies examined the α7 ago‐PAM, GAT107, in the formalin, complete Freund's adjuvant (CFA), LPS inflammatory pain models, the chronic constriction injury neuropathic pain model and the tail flick and hot plate acute thermal nociceptive assays. Additional studies examined the locus of action of GAT107 and immunohistochemical markers in the dorsal horn of the spinal cord in the CFA model. Key Results Complementary pharmacological and genetic approaches confirmed that the dose‐dependent antinociceptive effects of GAT107 were mediated through α7 nAChR. However, GAT107 was inactive in the tail flick and hot plate assays. In addition, GAT107 blocked conditioned place aversion elicited by acetic acid injection. Furthermore, intrathecal, but not intraplantar, injections of GAT107 reversed nociception in the CFA model, suggesting a spinal component of action. Immunohistochemical evaluation revealed an increase in the expression of astrocyte‐specific glial fibrillary acidic protein and phosphorylated p38MAPK within the spinal cords of mice treated with CFA, which was attenuated by intrathecal GAT107 treatment. Importantly, GAT107 did not elicit motor impairment and continued to produce antinociceptive effects after subchronic administration in both phases of the formalin test. Conclusions and Implications Collectively, these results provide the first proof of principle that α7 ago‐PAMs represent an effective pharmacological strategy for treating inflammatory and neuropathic pain. PMID:27243753

Characterization of the allosteric binding pocket of human liver fructose-1,6-bisphosphatase by protein crystallography and inhibitor activity studies.

PubMed

Iversen, L F; Brzozowski, M; Hastrup, S; Hubbard, R; Kastrup, J S; Larsen, I K; Naerum, L; Nørskov-Lauridsen, L; Rasmussen, P B; Thim, L; Wiberg, F C; Lundgren, K

1997-05-01

The structures of three complexes of human fructose-1,6-bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5-Amino-4-carboxamido-1 beta-D-5-phosphate-ribofuranosyl-1H-imidazole (AICAR-P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR-P is rotated 180 degrees compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR-P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR-P were determined to be 1.7, 1.4, and 20.9 microM, respectively. Thus, the approximately 10 times lower potency of AICAR-P is difficult to explain solely from the variations observed in the binding pocket. Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR-P complexes. This implies an important role of the conserved water molecule in binding of the ligand.

Characterization of the allosteric binding pocket of human liver fructose-1,6-bisphosphatase by protein crystallography and inhibitor activity studies.

PubMed Central

Iversen, L. F.; Brzozowski, M.; Hastrup, S.; Hubbard, R.; Kastrup, J. S.; Larsen, I. K.; Naerum, L.; Nørskov-Lauridsen, L.; Rasmussen, P. B.; Thim, L.; Wiberg, F. C.; Lundgren, K.

1997-01-01

The structures of three complexes of human fructose-1,6-bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5-Amino-4-carboxamido-1 beta-D-5-phosphate-ribofuranosyl-1H-imidazole (AICAR-P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR-P is rotated 180 degrees compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR-P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR-P were determined to be 1.7, 1.4, and 20.9 microM, respectively. Thus, the approximately 10 times lower potency of AICAR-P is difficult to explain solely from the variations observed in the binding pocket. Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR-P complexes. This implies an important role of the conserved water molecule in binding of the ligand. PMID:9144768