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Positive Allosteric Modulators Induced Conformational Changes in the Metabotropic Glutamate Receptor 2 - In Silico Predictions and Experimental Tests
90a
Sunday, February 28, 2016
add support to the growing evidence for use of selective b1-blockers to reverse detrimental remodeling of the failing RV in PAH patients. 461-Pos Board B241 Positive Allosteric Modulators Induced Conformational Changes in the Metabotropic Glutamate Receptor 2 - In Silico Predictions and Experimental Tests Yu Xu, Amr Ellaithy, Guoqing Xiang, Manolakou Danai, Balatsoukas Agisilaos, Takeharu Kawano, Meng Cui, Diomedes E. Logothetis. Physiology & Biophysics, Virginia Commonwealth University, Richmond, VA, USA. Background: Metabotropic glutamate type 2 receptor (mGlu2R) is a Class C Gprotein coupled receptor (GPCR), widely distributed in the brain where it mainly functions to limit excess glutamate release. mGlu2R positive allosteric modulators (PAMs) provide a novel way to regulate glutamatergic function since they bind to the transmembrane domain (TMD) and enhance responses of mGlu2R to extracellular orthosteric ligands via modulation of affinity and/ or efficacy. Compared to orthosteric agonists, PAMs have the unique ability to modulate glutamate release in a ‘state-dependent’ manner that helps finetune physiological responses. Methods: We docked different PAMs to a mGlu2R homology model based on the mGlu1R X-ray structure and performed 300 ns molecular dynamics (MD) simulations on receptor-ligand complexes. Mutagenesis and voltage-clamp experiments are being used to test the model’s predictions on the effects of PAMs on wild-type and mutant mGlu2R expressed in the Xenopus laevis oocyte heterologous expression system. Results and Conclusions: Preliminary computational results indicate that PAMs can induce allosteric conformational changes in mGlu2R TMD helices and intracellular loop regions that accompany G-protein binding and/or activation, and thus enhance formation of an active mGlu2R conformation. In addition, our simulation results also show that PAM binding can affect the interaction between the pre-TMH1 and the extracellular loop 1, which may influence the orthosteric ligand binding in the extracellular Venus flytrap domain. These predictions are being validated experimentally. Our simulations suggest that mGlu2R PAM binding alone produces a unique PAM-bound conformation that may resemble an intermediate conformation formed on the pathway to full receptor activation. These results provide insight into the mechanisms of allosteric modulation of mGlu2R, a potential therapeutic target for a variety of neuropsychiatric disorders. 462-Pos Board B242 In Vivo Studies of VEGFR2 Interactions in the Presence and Absence of VEGF Christopher King, Kalina Hristova. Johns Hopkins University, Baltimore, MD, USA. Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) is a receptor tyrosine kinase (RTK) that is critical for vasculogenesis and angiogenesis. Enhanced VEGFR2 signaling is often correlated with malignancy. It has been shown that full-length VEGFR2 exists in a monomer-dimer equilibrium in the absence of bound VEGF. Thus, the canonical model of RTK activation does not seem to adequately describe the behavior of VEGFR2 in the cell membrane. In order to understand the role that VEGFR2 extracellular domain plays in unliganded dimerization in live cells, we utilize Fully Quantified Spectral Imaging (FSI) to probe the interactions of VEGFR2 mutant constructs with rationally truncated EC domains. In addition, we investigate the stoichiometry of ligand binding to VEGFR2 EC domain as a function of VEGF concentration and total receptor expression. 463-Pos Board B243 The WNT Proteins Induce Ca2D Signaling through the Activation of the Polycystin Complex Vasyl Nesin, Seokho Kim, Hongguang Nie, Leonidas Tsiokas. Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. WNT ligands can induce Ca2þ signaling on target cells. PKD1 (Polycystin 1) is an atypical G protein coupled receptor complexed with TRPP2 (Polycystin 2 or PKD2), a Ca2þ-permeable ion channel. Inactivating mutations in the PKD1 or PKD2 genes cause autosomal dominant polycystic kidney disease (ADPKD), one of the most common genetic diseases of unknown mechanism. Here, we show that secreted WNTs specifically bind to the extracellular domain of PKD1 and induce large whole cell currents and Ca2þ influx. Pathogenic PKD1 or PKD2 mutations that abrogate complex formation, compromise cell surface expression of PKD1, or diminish TRPP2 channel activity suppress activation by a WNT protein. Cells derived from Pkd2-/- mice show complete loss of WNT-induced Ca2þ currents and are unable to polarize in response to WNT-
induced cell migration. These data define PKD1 as a new class of WNT (co)receptors coupled to Ca2þ signaling and directly implicate defective WNT/Ca2þ pathway as the primary cause of ADPKD. 464-Pos Board B244 Predicted mode of Binding of Non-Nitrogenous m-Opioid Receptor Ligands by Metadynamics Sebastian Schneider1, Davide Provasi1, Rachel Saylor2, Thomas Prisinzano2, Marta Filizola1. 1 Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 2Department of Medicinal Chemistry, University of Kansas School of Pharmacy, Lawrence, KS, USA. Classical opioid receptor drugs, like morphine and its derivatives, have been used for centuries to manage chronic pain despite frequent occurrence of adverse effects, including addiction, respiratory depression, vomiting, constipation, and severe sedation. A recent campaign to identify m-opioid receptor agonists that are effective as analgesics but devoid of side effects led to the discovery of a selective and potent analogue of the non-nitrogenous ligand herkinorin, a semi-synthetic derivative of the naturally occurring k-opioid receptor agonist salvinorin A. Several different crystal structures of opioid receptors have offered insight into the binding of opioid ligands with positively charged amine groups to their receptors. These ligands all bind at a well-characterized orthosteric binding pocket through a salt-bridge interaction between the amino group and a conserved aspartic acid in transmembrane helix 3 of the receptor. The obvious absence of this interaction in the binding of non-nitrogenous ligands, and the uncertainty surrounding both their binding pocket and energetically preferred poses, prompted us to carry out all-atom multiple-walker metadynamics simulations to study the binding of herkinorin analogues to a fully flexible m-opioid receptor in an explicit lipid-water environment. Clustering of all the bound conformations sampled reveals different, but energetically indistinguishable, binding modes for the studied ligands. Analysis of the ligand-receptor interactions formed by each conformation in the various clusters suggests testable hypotheses of molecular determinants responsible for the affinity and/or efficacy of herkinorin analogues. 465-Pos Board B245 Deterministic and Stochastic Mathematical Modeling of Melanopsin’s Light Response in ipRGCs and HEK Cells Phyllis R. Robinson1, Kathleen Hoffman2, Hye-Won Kang2. 1 Biological Sciences, UMBC, Baltimore, MD, USA, 2Mathematics and Statistics, UMBC, Baltimore, MD, USA. Melanopsin, a visual pigment expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs), primarily affects non-image forming vision with a hypothesized G-protein phototransduction pathway similar to the phototransduction pathway found in Drosophila photoreceptors. The biochemical cascade underlying the light response in ipRGCs has not yet been fully elucidated. A deterministic and stochastic mathematical model of the hypothesized pathway is developed to probe key elements of the pathway. The rate constants, initial conditions and parameters are fit to voltage clamp data from ipRGCs and also calcium imaging data from HEK cells transfected with mouse melanopsin. Sensitivity analysis reveals key steps in the phototransduction pathway, independent of the experimental conditions, as well as key differences attributable to the different experimental conditions. 466-Pos Board B246 Covalent Ras Dimerization on Membrane Surfaces through Photosensitized Oxidation Jean K. Chung. Chemistry, University of California Berkeley, Berkeley, CA, USA. Ras, a small GTPase found primarily on the inner leaflet of the plasma membrane, is an important signaling node and an attractive target for anticancer therapies. Lateral organization of Ras on cellular membranes has long been a subject to intense research; in particular, whether it forms dimers on membranes as part of its regulatory function has been a point of great interest. Here we report Ras dimers readily formed on membranes by Type II photosensitization reactions, in which molecular oxygen in ambient solution mediates the radicalization of proteins under typical fluorescence experimental conditions. The presence of Ras dimers on membranes was detected by diffusion-based fluorescence techniques including fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT), and molecular weights of the stable covalently coupled species were confirmed by gel electrophoresis. Fluorescence spectroscopy implicates interprotein dityrosine as one of the dimerization motifs. The specific surface tyrosine distribution on Ras renders the protein especially sensitive to this reaction and point mutations affecting surface tyrosines are observed to alter dimerization
Sunday, February 28, 2016
add support to the growing evidence for use of selective b1-blockers to reverse detrimental remodeling of the failing RV in PAH patients. 461-Pos Board B241 Positive Allosteric Modulators Induced Conformational Changes in the Metabotropic Glutamate Receptor 2 - In Silico Predictions and Experimental Tests Yu Xu, Amr Ellaithy, Guoqing Xiang, Manolakou Danai, Balatsoukas Agisilaos, Takeharu Kawano, Meng Cui, Diomedes E. Logothetis. Physiology & Biophysics, Virginia Commonwealth University, Richmond, VA, USA. Background: Metabotropic glutamate type 2 receptor (mGlu2R) is a Class C Gprotein coupled receptor (GPCR), widely distributed in the brain where it mainly functions to limit excess glutamate release. mGlu2R positive allosteric modulators (PAMs) provide a novel way to regulate glutamatergic function since they bind to the transmembrane domain (TMD) and enhance responses of mGlu2R to extracellular orthosteric ligands via modulation of affinity and/ or efficacy. Compared to orthosteric agonists, PAMs have the unique ability to modulate glutamate release in a ‘state-dependent’ manner that helps finetune physiological responses. Methods: We docked different PAMs to a mGlu2R homology model based on the mGlu1R X-ray structure and performed 300 ns molecular dynamics (MD) simulations on receptor-ligand complexes. Mutagenesis and voltage-clamp experiments are being used to test the model’s predictions on the effects of PAMs on wild-type and mutant mGlu2R expressed in the Xenopus laevis oocyte heterologous expression system. Results and Conclusions: Preliminary computational results indicate that PAMs can induce allosteric conformational changes in mGlu2R TMD helices and intracellular loop regions that accompany G-protein binding and/or activation, and thus enhance formation of an active mGlu2R conformation. In addition, our simulation results also show that PAM binding can affect the interaction between the pre-TMH1 and the extracellular loop 1, which may influence the orthosteric ligand binding in the extracellular Venus flytrap domain. These predictions are being validated experimentally. Our simulations suggest that mGlu2R PAM binding alone produces a unique PAM-bound conformation that may resemble an intermediate conformation formed on the pathway to full receptor activation. These results provide insight into the mechanisms of allosteric modulation of mGlu2R, a potential therapeutic target for a variety of neuropsychiatric disorders. 462-Pos Board B242 In Vivo Studies of VEGFR2 Interactions in the Presence and Absence of VEGF Christopher King, Kalina Hristova. Johns Hopkins University, Baltimore, MD, USA. Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) is a receptor tyrosine kinase (RTK) that is critical for vasculogenesis and angiogenesis. Enhanced VEGFR2 signaling is often correlated with malignancy. It has been shown that full-length VEGFR2 exists in a monomer-dimer equilibrium in the absence of bound VEGF. Thus, the canonical model of RTK activation does not seem to adequately describe the behavior of VEGFR2 in the cell membrane. In order to understand the role that VEGFR2 extracellular domain plays in unliganded dimerization in live cells, we utilize Fully Quantified Spectral Imaging (FSI) to probe the interactions of VEGFR2 mutant constructs with rationally truncated EC domains. In addition, we investigate the stoichiometry of ligand binding to VEGFR2 EC domain as a function of VEGF concentration and total receptor expression. 463-Pos Board B243 The WNT Proteins Induce Ca2D Signaling through the Activation of the Polycystin Complex Vasyl Nesin, Seokho Kim, Hongguang Nie, Leonidas Tsiokas. Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. WNT ligands can induce Ca2þ signaling on target cells. PKD1 (Polycystin 1) is an atypical G protein coupled receptor complexed with TRPP2 (Polycystin 2 or PKD2), a Ca2þ-permeable ion channel. Inactivating mutations in the PKD1 or PKD2 genes cause autosomal dominant polycystic kidney disease (ADPKD), one of the most common genetic diseases of unknown mechanism. Here, we show that secreted WNTs specifically bind to the extracellular domain of PKD1 and induce large whole cell currents and Ca2þ influx. Pathogenic PKD1 or PKD2 mutations that abrogate complex formation, compromise cell surface expression of PKD1, or diminish TRPP2 channel activity suppress activation by a WNT protein. Cells derived from Pkd2-/- mice show complete loss of WNT-induced Ca2þ currents and are unable to polarize in response to WNT-
induced cell migration. These data define PKD1 as a new class of WNT (co)receptors coupled to Ca2þ signaling and directly implicate defective WNT/Ca2þ pathway as the primary cause of ADPKD. 464-Pos Board B244 Predicted mode of Binding of Non-Nitrogenous m-Opioid Receptor Ligands by Metadynamics Sebastian Schneider1, Davide Provasi1, Rachel Saylor2, Thomas Prisinzano2, Marta Filizola1. 1 Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 2Department of Medicinal Chemistry, University of Kansas School of Pharmacy, Lawrence, KS, USA. Classical opioid receptor drugs, like morphine and its derivatives, have been used for centuries to manage chronic pain despite frequent occurrence of adverse effects, including addiction, respiratory depression, vomiting, constipation, and severe sedation. A recent campaign to identify m-opioid receptor agonists that are effective as analgesics but devoid of side effects led to the discovery of a selective and potent analogue of the non-nitrogenous ligand herkinorin, a semi-synthetic derivative of the naturally occurring k-opioid receptor agonist salvinorin A. Several different crystal structures of opioid receptors have offered insight into the binding of opioid ligands with positively charged amine groups to their receptors. These ligands all bind at a well-characterized orthosteric binding pocket through a salt-bridge interaction between the amino group and a conserved aspartic acid in transmembrane helix 3 of the receptor. The obvious absence of this interaction in the binding of non-nitrogenous ligands, and the uncertainty surrounding both their binding pocket and energetically preferred poses, prompted us to carry out all-atom multiple-walker metadynamics simulations to study the binding of herkinorin analogues to a fully flexible m-opioid receptor in an explicit lipid-water environment. Clustering of all the bound conformations sampled reveals different, but energetically indistinguishable, binding modes for the studied ligands. Analysis of the ligand-receptor interactions formed by each conformation in the various clusters suggests testable hypotheses of molecular determinants responsible for the affinity and/or efficacy of herkinorin analogues. 465-Pos Board B245 Deterministic and Stochastic Mathematical Modeling of Melanopsin’s Light Response in ipRGCs and HEK Cells Phyllis R. Robinson1, Kathleen Hoffman2, Hye-Won Kang2. 1 Biological Sciences, UMBC, Baltimore, MD, USA, 2Mathematics and Statistics, UMBC, Baltimore, MD, USA. Melanopsin, a visual pigment expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs), primarily affects non-image forming vision with a hypothesized G-protein phototransduction pathway similar to the phototransduction pathway found in Drosophila photoreceptors. The biochemical cascade underlying the light response in ipRGCs has not yet been fully elucidated. A deterministic and stochastic mathematical model of the hypothesized pathway is developed to probe key elements of the pathway. The rate constants, initial conditions and parameters are fit to voltage clamp data from ipRGCs and also calcium imaging data from HEK cells transfected with mouse melanopsin. Sensitivity analysis reveals key steps in the phototransduction pathway, independent of the experimental conditions, as well as key differences attributable to the different experimental conditions. 466-Pos Board B246 Covalent Ras Dimerization on Membrane Surfaces through Photosensitized Oxidation Jean K. Chung. Chemistry, University of California Berkeley, Berkeley, CA, USA. Ras, a small GTPase found primarily on the inner leaflet of the plasma membrane, is an important signaling node and an attractive target for anticancer therapies. Lateral organization of Ras on cellular membranes has long been a subject to intense research; in particular, whether it forms dimers on membranes as part of its regulatory function has been a point of great interest. Here we report Ras dimers readily formed on membranes by Type II photosensitization reactions, in which molecular oxygen in ambient solution mediates the radicalization of proteins under typical fluorescence experimental conditions. The presence of Ras dimers on membranes was detected by diffusion-based fluorescence techniques including fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT), and molecular weights of the stable covalently coupled species were confirmed by gel electrophoresis. Fluorescence spectroscopy implicates interprotein dityrosine as one of the dimerization motifs. The specific surface tyrosine distribution on Ras renders the protein especially sensitive to this reaction and point mutations affecting surface tyrosines are observed to alter dimerization