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Role of Conformational Entropy in Extremely High Affinity Protein Interactions
Sunday, February 12, 2017 in the human gene for cytochrome c, which result in enhanced mitochondrial apoptotic activity, cause thrombocytopenia 4, an inherited autosomal dominant thrombocytopenia, characterised by a deficiency in the number of platelets in the blood and leading to abnormal bleeding. The first such mutation to be reported was G41S. Here we use stopped-flow kinetic studies of azide binding to human ferricytochrome c, backbone amide H/D exchange and 15N-relaxation dynamics measured by NMR spectroscopy to compare the wild type and G41S forms of human cytochrome c. We show that alternative conformations are kinetically and thermodynamically more readily accessible for the G41S variant than for the wild-type protein. Residue 41 is located in the 40-57 U-loop, and the increased loop dynamics in the G41S variant promote the dissociation from the heme iron of the M80 ligand, revealing a direct conformational link between the loop and the axial ligand to the heme iron. Increased dissociation of M80 increases the population of a peroxidase active state, which is a key non-native conformational state in apoptosis. 166-Plat Global Disordering in Stereo-Specific Protein Association Arun Gupta1, Ines Reinartz2, Alessandro Spilotros3, Venkateswara R. Jonna1, Anders Hofer1, Dmitri I. Svergun3, Alexander Schug2, Magnus Wolf-Watz1. 1 University of Umea˚, Umea˚, Sweden, 2Karlsruhe Institute of Technology, Karlsruhe, Germany, 3European Molecular Biological Laboratory, Hamburg Outstation, Hamburg, Germany. Protein-protein recognition is of fundamental importance for a myriad of biological processes and is ultimately a prerequisite for life as we know it. There exist several established mechanisms that promote formation of stereo-specific protein complexes. Many of these mechanism involve conformational changes of one or both proteins in dimeric assemblies as observed in ‘‘conformational selection’’ and ‘‘coupled folding and binding’’ scenarios. In ‘‘coupled folding and binding’’ events, at least one of the proteins undergoes a global ordering event. By using an integrated computational and experimental approach we have discovered that also global disordering can be a productive route for formation of a stereo-specific protein complex. This mechanism was observed for the chaperone binding domain of the Yersinia effector protein YopH upon binding to its specific chaperone SycH. These two proteins are crucial for type III secretion system mediated infectivity by Yersinia and several other gram negative pathogens. NMR relaxation dispersion experiments demonstrated that the otherwise well folded YopH protein dynamically samples an expanded high-energy state that corresponds to the SycH binding competent conformation. A structure of the protein complex determined from a hybrid SAXS and computational approach revealed that YopH wraps around SycH in a horse shoe like conformation. The binding model was validated by site specific YopH mutations that promoted the disordering event and at the same time displayed improved binding affinity towards SycH. Taken together the data illustrates a tight coupling between a proteins unfolding and functional free energy landscapes and add valuable mechanistic insight into protein-protein recognition. 167-Plat Role of Conformational Entropy in Extremely High Affinity Protein Interactions Jose A. Caro. Biochemistry & Biophysics, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, USA. Interactions of extreme affinity (Kd ~ fM) underlie many biochemical processes necessary to life. The physical determinants of such large binding energies are not well understood. Specific interactions at the interface (DHbinding) and the release of solvating water (TDSsolvation) are usually assumed to dominate the binding energetics. The role of conformational entropy (TDSconf) in determining binding affinity has remained elusive, in part due to the difficulties in measuring such changes in entropy experimentally. Recent developments in the Wand laboratory have bridged this gap by using solution NMR measurements of dynamics to empirically calibrate a ‘‘conformational entropy meter.’’ It has enabled quantitative measurements of the change in conformational entropy in protein-ligand binding. The toxin-antitoxin system studied here, barnase-barstar, forms a complex with fM affinity (DGbinding ~ 19 kcal/mol) without undergoing any major structural changes and retaining a hydrated interface. To explore the role of conformational entropy, the fast (ps-ns timescale) motions of backbone and side chains of the two proteins were measured in both the free (unbound) and the complexed (bound) states using NMR spectroscopy. Furthermore, hydration dynamics were measured in water and in the confined space of a reverse micelle. The dynamic response observed leads to an unfavorable change in TDSconf, with a more rigid, still hydrated interface. This comprehensive study of both protein and ‘‘ligand’’ (in this case, another protein) and the measured site-specific changes in dynamics and hydration sheds
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light on the thermodynamic contributions that enable fM affinities. Supported by grants from the NIH, The Mathers Foundation and NSF. 168-Plat Pressure Effects on Dissociation of CheY-FliM Complex Studied by Molecular Dynamics Simulations Hiroaki Hata1, Yasutaka Nishihara1, Masayoshi Nishiyama2, Ikuro Kawagishi3, Akio Kitao1. 1 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan, 2The Hakubi Center for Advanced Research, Kyoto University, Kyoto, Japan, 3Department of Frontier Bioscience, Hosei University, Tokyo, Japan. The rotational switching of the bacteria flagella motor is controlled by binding of the signaling molecule CheY onto FliM which is a part of motor basal body. The rotational switching plays a central role in the bacterial chemotaxis. Recently, it was reported that high hydrostatic pressures of >120 MPa can induce the rotational switching even in the absence of CheY [1]. It was also suggested that hydration of the switch complex at high pressure induces structural changes similar to those caused by the binding of CheY. To gain further insights into the high pressure effect on the motor switching, we investigated differences in conformation of monomeric CheY and also CheY-FliM complex at different pressure conditions using molecular dynamics (MD) simulations. Then, pressure effects on the binding stability of the CheY-FliM complex was studied by dissociating the complex. The dissociation of the protein complex was observed using an efficient sampling method, PaCS-MD (Parallel Cascade Selection Molecular Dynamics) [2]. In PaCS-MD, the cycle of short MD simulations and selection of the structures close to the product structure for the next cycle are repeated, which enhances the conformational transitions without any additional external biases. From the obtained MD trajectories, the dissociation behavior was characterized using coordinates such as the center of mass (COM) distance and the number of native contacts between CheY and FliM. Moreover, potentials of mean force along the COM distance were calculated from probability distributions in steady state obtained by Markov state models. Those potentials of mean force provided binding free energies of the protein complex. Based on the results, we will also discuss mechanisms underlying influences of high hydrostatic pressures on the binding. Such insights would provide a further understanding towards an accurate regulation of protein-protein interactions. [1] Nishiyama, M. et al. 2013. J. Bacteriol. 195:1809-1814. doi: 10.1128/ JB.02139-12. [2] Harada, R. and Kitao, A. 2013. J. Chem. Phys. 139:035103. doi: 10.1063/ 1.4813023.
Platform: General Protein-Lipid Interactions I 169-Plat Predicting Cholesterol Interaction Sites on GPCRs by Molecular Simulation Edward R. Lyman1, Clement Arnarez2, Eric Rouviere2. 1 Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, USA, 2Department of Physics and Astronomy, University of Delaware, Newark, DE, USA. G-protein coupled receptor function depends on the lipid environment, in particular on cholesterol. Given that brute force mutagenesis of the entire membrane-facing surface is not practical, an approach is presented to identify putative cholesterol interaction sites on the surface of GPCRs. In unbiased simulations in the presence of cholesterol, specific residues are identified as loci of cholesterol interaction, identified on the basis of long-lived, reproducible cholesterol binding. Results will be presented for several GPCRs, with a special focus on the A2A adenosine receptor. 170-Plat Membrane Cholesterol and the Adenosine A2a Receptor Claire McGraw, Anne S. Robinson. Tulane University, New Orleans, LA, USA. G-protein coupled receptors (GPCRs) represent the largest family of receptor proteins in the living world, having approximately 800 human genes predicted; however the high-resolution crystal structures of only 26 GPCRs have been reported (Ghosh et al., 2015). The first human GPCR to be crystallized was the b2-adrenergic receptor (b2AR) in 2007 (Cherezov et al., 2007; Rasmussen et al., 2007; Rosenbaum et al., 2007). Shortly thereafter an alternate crystal form of the b2AR revealed a specific cholesterol binding site between helices I, II, III and IV. From this work a cholesterol consensus motif (CCM) was established, which defined specific interactions between cholesterol and the receptor. Utilization of this CCM predicted that as many as 25% of all class A GPCRs could have a specific interaction with cholesterol (Hanson et al., 2008).
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light on the thermodynamic contributions that enable fM affinities. Supported by grants from the NIH, The Mathers Foundation and NSF. 168-Plat Pressure Effects on Dissociation of CheY-FliM Complex Studied by Molecular Dynamics Simulations Hiroaki Hata1, Yasutaka Nishihara1, Masayoshi Nishiyama2, Ikuro Kawagishi3, Akio Kitao1. 1 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan, 2The Hakubi Center for Advanced Research, Kyoto University, Kyoto, Japan, 3Department of Frontier Bioscience, Hosei University, Tokyo, Japan. The rotational switching of the bacteria flagella motor is controlled by binding of the signaling molecule CheY onto FliM which is a part of motor basal body. The rotational switching plays a central role in the bacterial chemotaxis. Recently, it was reported that high hydrostatic pressures of >120 MPa can induce the rotational switching even in the absence of CheY [1]. It was also suggested that hydration of the switch complex at high pressure induces structural changes similar to those caused by the binding of CheY. To gain further insights into the high pressure effect on the motor switching, we investigated differences in conformation of monomeric CheY and also CheY-FliM complex at different pressure conditions using molecular dynamics (MD) simulations. Then, pressure effects on the binding stability of the CheY-FliM complex was studied by dissociating the complex. The dissociation of the protein complex was observed using an efficient sampling method, PaCS-MD (Parallel Cascade Selection Molecular Dynamics) [2]. In PaCS-MD, the cycle of short MD simulations and selection of the structures close to the product structure for the next cycle are repeated, which enhances the conformational transitions without any additional external biases. From the obtained MD trajectories, the dissociation behavior was characterized using coordinates such as the center of mass (COM) distance and the number of native contacts between CheY and FliM. Moreover, potentials of mean force along the COM distance were calculated from probability distributions in steady state obtained by Markov state models. Those potentials of mean force provided binding free energies of the protein complex. Based on the results, we will also discuss mechanisms underlying influences of high hydrostatic pressures on the binding. Such insights would provide a further understanding towards an accurate regulation of protein-protein interactions. [1] Nishiyama, M. et al. 2013. J. Bacteriol. 195:1809-1814. doi: 10.1128/ JB.02139-12. [2] Harada, R. and Kitao, A. 2013. J. Chem. Phys. 139:035103. doi: 10.1063/ 1.4813023.
Platform: General Protein-Lipid Interactions I 169-Plat Predicting Cholesterol Interaction Sites on GPCRs by Molecular Simulation Edward R. Lyman1, Clement Arnarez2, Eric Rouviere2. 1 Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, USA, 2Department of Physics and Astronomy, University of Delaware, Newark, DE, USA. G-protein coupled receptor function depends on the lipid environment, in particular on cholesterol. Given that brute force mutagenesis of the entire membrane-facing surface is not practical, an approach is presented to identify putative cholesterol interaction sites on the surface of GPCRs. In unbiased simulations in the presence of cholesterol, specific residues are identified as loci of cholesterol interaction, identified on the basis of long-lived, reproducible cholesterol binding. Results will be presented for several GPCRs, with a special focus on the A2A adenosine receptor. 170-Plat Membrane Cholesterol and the Adenosine A2a Receptor Claire McGraw, Anne S. Robinson. Tulane University, New Orleans, LA, USA. G-protein coupled receptors (GPCRs) represent the largest family of receptor proteins in the living world, having approximately 800 human genes predicted; however the high-resolution crystal structures of only 26 GPCRs have been reported (Ghosh et al., 2015). The first human GPCR to be crystallized was the b2-adrenergic receptor (b2AR) in 2007 (Cherezov et al., 2007; Rasmussen et al., 2007; Rosenbaum et al., 2007). Shortly thereafter an alternate crystal form of the b2AR revealed a specific cholesterol binding site between helices I, II, III and IV. From this work a cholesterol consensus motif (CCM) was established, which defined specific interactions between cholesterol and the receptor. Utilization of this CCM predicted that as many as 25% of all class A GPCRs could have a specific interaction with cholesterol (Hanson et al., 2008).