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• W2021552011 abstract "Functional positive cooperative activation of the extracellular calcium ([Ca2+]o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca2+]o or amino acids elicits intracellular Ca2+ ([Ca2+]i) oscillations. Here, we report the central role of predicted Ca2+-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca2+-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca2+]o. Next, we identify an adjacent l-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca2+]o and l-Phe in eliciting CaSR-mediated [Ca2+]i oscillations. The heterocommunication between Ca2+ and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca2+]o signaling by positively impacting multiple [Ca2+]o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca2+]o and amino acids into intracellular signaling events. Functional positive cooperative activation of the extracellular calcium ([Ca2+]o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca2+]o or amino acids elicits intracellular Ca2+ ([Ca2+]i) oscillations. Here, we report the central role of predicted Ca2+-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca2+-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca2+]o. Next, we identify an adjacent l-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca2+]o and l-Phe in eliciting CaSR-mediated [Ca2+]i oscillations. The heterocommunication between Ca2+ and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca2+]o signaling by positively impacting multiple [Ca2+]o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca2+]o and amino acids into intracellular signaling events. It has long been recognized that Ca2+ acts as a second messenger that is released from intracellular stores and/or taken up from the extracellular environment in response to external stimuli to regulate diverse cellular processes. The discovery of the parathyroid Ca2+-sensing receptor (CaSR) 2The abbreviations used are: CaSRcalcium-sensing receptorGPCRG protein-coupled receptorECDextracellular domainMDmolecular dynamicsPCAprincipal component analysismGluRmetabotropic glutamate receptorAMacetoxymethylester. by Brown et al. (1.Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid.Nature. 1993; 366: 575-580Crossref PubMed Scopus (2358) Google Scholar) has established a new paradigm of Ca2+ signaling. In addition to its known role as a second messenger, extracellular Ca2+ can function as a first messenger by CaSR-mediated triggering of multiple intracellular signaling pathways, including activation of phospholipases C, A2, and D, and various mitogen-activated protein kinases (MAPKs), as well as inhibition of cyclic adenosine monophosphate (cAMP) production (2.Chang W. Shoback D. Extracellular Ca2+-sensing receptors. An overview.Cell Calcium. 2004; 35: 183-196Crossref PubMed Scopus (110) Google Scholar, 3.Breitwieser G.E. Calcium sensing receptors and calcium oscillations. Calcium as a first messenger.Curr. Top. Dev. Biol. 2006; 73: 85-114Crossref PubMed Scopus (44) Google Scholar, 4.Huang C. Miller R.T. The calcium-sensing receptor and its interacting proteins.J. Cell. Mol. Med. 2007; 11: 923-934Crossref PubMed Scopus (62) Google Scholar, 5.Wellendorph P. Bräuner-Osborne H. Molecular basis for amino acid sensing by family C G-protein-coupled receptors.Br. J. Pharmacol. 2009; 156: 869-884Crossref PubMed Scopus (90) Google Scholar, 6.Cheng S.X. Geibel J.P. Hebert S.C. Extracellular polyamines regulate fluid secretion in rat colonic crypts via the extracellular calcium-sensing receptor.Gastroenterology. 2004; 126: 148-158Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 7.Tu C.L. Oda Y. Komuves L. Bikle D.D. The role of the calcium-sensing receptor in epidermal differentiation.Cell Calcium. 2004; 35: 265-273Crossref PubMed Scopus (102) Google Scholar). This receptor is present in the key tissues involved in [Ca2+]o homeostasis (e.g., parathyroid, kidney, and bone) and diverse other nonhomeostatic tissues (e.g., brain, skin, etc.) (8.Mathias R.S. Mathews C.H. Machule C. Gao D. Li W. Denbesten P.K. Identification of the calcium-sensing receptor in the developing tooth organ.J. Bone Miner. Res. 2001; 16: 2238-2244Crossref PubMed Scopus (33) Google Scholar, 9.Buchan A.M. Squires P.E. Ring M. Meloche R.M. Mechanism of action of the calcium-sensing receptor in human antral gastrin cells.Gastroenterology. 2001; 120: 1128-1139Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 10.Hofer A.M. Brown E.M. Extracellular calcium sensing and signalling.Nat. Rev. Mol. Cell Biol. 2003; 4: 530-538Crossref PubMed Scopus (520) Google Scholar, 11.Brown E.M. MacLeod R.J. Extracellular calcium sensing and extracellular calcium signaling.Physiol. Rev. 2001; 81: 239-297Crossref PubMed Scopus (1224) Google Scholar). CaSR consists of a large N-terminal extracellular domain (ECD) (∼600 residues) folded into a Venus flytrap motif, followed by a seven-pass transmembrane region and a cytosolic C terminus. The ECD has been shown to play an important role in the cooperative response of the CaSR to [Ca2+]o. Elevations in [Ca2+]o activate the CaSR, evoking increases in the intracellular Ca2+ concentration ([Ca2+]i), producing [Ca2+]i oscillations, modulating the rate of parathyroid hormone secretion, and regulating gene expression (3.Breitwieser G.E. Calcium sensing receptors and calcium oscillations. Calcium as a first messenger.Curr. Top. Dev. Biol. 2006; 73: 85-114Crossref PubMed Scopus (44) Google Scholar, 12.Dolmetsch R.E. Xu K. Lewis R.S. Calcium oscillations increase the efficiency and specificity of gene expression.Nature. 1998; 392: 933-936Crossref PubMed Scopus (1673) Google Scholar, 13.Miedlich S. Gama L. Breitwieser G.E. Calcium sensing receptor activation by a calcimimetic suggests a link between cooperativity and intracellular calcium oscillations.J. Biol. Chem. 2002; 277: 49691-49699Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 14.Young S.H. Rozengurt E. Amino acids and Ca2+ stimulate different patterns of Ca2+ oscillations through the Ca2+-sensing receptor.Am. J. Physiol. Cell Physiol. 2002; 282: C1414-C1422Crossref PubMed Scopus (71) Google Scholar). The pattern of [Ca2+]i oscillations is one of the most important signatures reflecting the state of CaSR activity. calcium-sensing receptor G protein-coupled receptor extracellular domain molecular dynamics principal component analysis metabotropic glutamate receptor acetoxymethylester. More than 200 naturally occurring mutations have been identified in the CaSR that either inactivate the receptor (reducing sensitivity to [Ca2+]o), leading to familial hypocalciuric hypercalcemia or neonatal severe hyperparathyroidism, or activate it (increasing sensitivity to [Ca2+]o), thereby causing autosomal dominant hypoparathyroidism (15.Hannan F.M. Nesbit M.A. Zhang C. Cranston T. Curley A.J. Harding B. Fratter C. Rust N. Christie P.T. Turner J.J. Lemos M.C. Bowl M.R. Bouillon R. Brain C. Bridges N. Burren C. Connell J.M. Jung H. Marks E. McCredie D. Mughal Z. Rodda C. Tollefsen S. Brown E.M. Yang J.J. Thakker R.V. Identification of 70 calcium-sensing receptor mutations in hyper- and hypo-calcaemic patients. Evidence for clustering of extracellular domain mutations at calcium-binding sites.Hum. Mol. Genet. 2012; 21: 2768-2778Crossref PubMed Scopus (131) Google Scholar, 16.Hu J. Spiegel A.M. Naturally occurring mutations of the extracellular Ca2+-sensing receptor. Implications for its structure and function.Trends Endocrinol. Metab. 2003; 14: 282-288Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 17.Hendy G.N. Guarnieri V. Canaff L. Calcium-sensing receptor and associated diseases.Prog. Mol. Biol. Transl. Sci. 2009; 89: 31-95Crossref PubMed Scopus (89) Google Scholar). Several of these naturally occurring mutations of CaSR exhibit altered functional cooperativity (15.Hannan F.M. Nesbit M.A. Zhang C. Cranston T. Curley A.J. Harding B. Fratter C. Rust N. Christie P.T. Turner J.J. Lemos M.C. Bowl M.R. Bouillon R. Brain C. Bridges N. Burren C. Connell J.M. Jung H. Marks E. McCredie D. Mughal Z. Rodda C. Tollefsen S. Brown E.M. Yang J.J. Thakker R.V. Identification of 70 calcium-sensing receptor mutations in hyper- and hypo-calcaemic patients. Evidence for clustering of extracellular domain mutations at calcium-binding sites.Hum. Mol. Genet. 2012; 21: 2768-2778Crossref PubMed Scopus (131) Google Scholar). Functional cooperativity of CaSR (i.e., based on biological activity determined using functional assays rather than a direct binding assay), particularly the functional positive homotropic cooperative response to [Ca2+]o, is essential for the ability of the receptor to respond over a narrow physiological range of [Ca2+]o (1.1–1.3 mm) (3.Breitwieser G.E. Calcium sensing receptors and calcium oscillations. Calcium as a first messenger.Curr. Top. Dev. Biol. 2006; 73: 85-114Crossref PubMed Scopus (44) Google Scholar). CaSR has an estimated Hill coefficient of 3–4 for its regulation of processes such as activating intracellular Ca2+ signaling and inhibiting parathyroid hormone release. Under physiological conditions, l-amino acids, especially aromatic amino acids (e.g., l-Phe), as well as short aliphatic and small polar amino acids (18.Francesconi A. Duvoisin R.M. Divalent cations modulate the activity of metabotropic glutamate receptors.J. Neurosci. Res. 2004; 75: 472-479Crossref PubMed Scopus (30) Google Scholar), potentiate the high [Ca2+]o-elicited activation of the CaSR by altering the EC50 values required for [Ca2+]o-evoked [Ca2+]i responses and its functional cooperativity (19.Conigrave A.D. Quinn S.J. Brown E.M. l-Amino acid sensing by the extracellular Ca2+-sensing receptor.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 4814-4819Crossref PubMed Scopus (418) Google Scholar, 20.Wang M. Yao Y. Kuang D. Hampson D.R. Activation of family C G-protein-coupled receptors by the tripeptide glutathione.J. Biol. Chem. 2006; 281: 8864-8870Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). In aggregate, the levels of amino acids in human serum in the fed state are close to those activating the CaSR in vitro (19.Conigrave A.D. Quinn S.J. Brown E.M. l-Amino acid sensing by the extracellular Ca2+-sensing receptor.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 4814-4819Crossref PubMed Scopus (418) Google Scholar, 21.Conigrave A.D. Mun H.C. Lok H.C. Aromatic l-amino acids activate the calcium-sensing receptor.J. Nutr. 2007; 137: 1524S-1548SCrossref PubMed Google Scholar) and can further enhance functional cooperativity via positive heterotropic cooperativity. Recently, several groups have reported that the CaSR in cells within the lumen of the gastrointestinal tract is activated by l-Phe and other amino acids, which have long been recognized as activators of key digestive processes. Hence, the CaSR enables the tract to monitor events relevant to both mineral ion and protein/amino acid metabolism in addition to the sensing capability of CaSR in blood and other extracellular fluids (19.Conigrave A.D. Quinn S.J. Brown E.M. l-Amino acid sensing by the extracellular Ca2+-sensing receptor.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 4814-4819Crossref PubMed Scopus (418) Google Scholar, 22.Conigrave A.D. Brown E.M. Taste receptors in the gastrointestinal tract. II. l-amino acid sensing by calcium-sensing receptors. Implications for GI physiology.Am. J. Physiol. Gastrointest. Liver Physiol. 2006; 291: G753-G761Crossref PubMed Scopus (122) Google Scholar, 23.Liou A.P. Sei Y. Zhao X. Feng J. Lu X. Thomas C. Pechhold S. Raybould H.E. Wank S.A. The extracellular calcium-sensing receptor is required for cholecystokinin secretion in response to l-phenylalanine in acutely isolated intestinal I cells.Am. J. Physiol. Gastrointest. Liver Physiol. 2011; 300: G538-G546Crossref PubMed Scopus (157) Google Scholar). Glutathione and its γ-glutamylpeptides also allosterically modulate the CaSR at a site similar to the l-amino acid-binding pocket but with over 1,000-fold higher potencies (20.Wang M. Yao Y. Kuang D. Hampson D.R. Activation of family C G-protein-coupled receptors by the tripeptide glutathione.J. Biol. Chem. 2006; 281: 8864-8870Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 24.Broadhead G.K. Mun H.C. Avlani V.A. Jourdon O. Church W.B. Christopoulos A. Delbridge L. Conigrave A.D. Allosteric modulation of the calcium-sensing receptor by γ-glutamyl peptides. Inhibition of PTH secretion, suppression of intracellular cAMP levels, and a common mechanism of action with l-amino acids.J. Biol. Chem. 2011; 286: 8786-8797Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Thus, CaSR is essential for monitoring and integrating information from both mineral ions/nutrients/polyamines in blood and related extracellular fluids. Nevertheless, we still lack a thorough understanding of the molecular mechanisms by which CaSR is activated by [Ca2+]o and amino acids, which, in turn, regulate CaSR functional positive cooperativity. In addition, in a clinical setting, the molecular basis for the alterations in this cooperativity caused by disease-associated mutations is largely unknown because of the lack of knowledge of the structure of this receptor and its weak binding affinities for [Ca2+]o and amino acids (13.Miedlich S. Gama L. Breitwieser G.E. Calcium sensing receptor activation by a calcimimetic suggests a link between cooperativity and intracellular calcium oscillations.J. Biol. Chem. 2002; 277: 49691-49699Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 15.Hannan F.M. Nesbit M.A. Zhang C. Cranston T. Curley A.J. Harding B. Fratter C. Rust N. Christie P.T. Turner J.J. Lemos M.C. Bowl M.R. Bouillon R. Brain C. Bridges N. Burren C. Connell J.M. Jung H. Marks E. McCredie D. Mughal Z. Rodda C. Tollefsen S. Brown E.M. Yang J.J. Thakker R.V. Identification of 70 calcium-sensing receptor mutations in hyper- and hypo-calcaemic patients. Evidence for clustering of extracellular domain mutations at calcium-binding sites.Hum. Mol. Genet. 2012; 21: 2768-2778Crossref PubMed Scopus (131) Google Scholar, 25.Huang Y. Zhou Y. Yang W. Butters R. Lee H.W. Li S. Castiblanco A. Brown E.M. Yang J.J. Identification and dissection of Ca2+-binding sites in the extracellular domain of Ca2+-sensing receptor.J. Biol. Chem. 2007; 282: 19000-19010Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 26.Huang Y. Zhou Y. Castiblanco A. Yang W. Brown E.M. Yang J.J. Multiple Ca2+-binding sites in the extracellular domain of the Ca2+-sensing receptor corresponding to cooperative Ca2+ response.Biochemistry. 2009; 48: 388-398Crossref PubMed Scopus (105) Google Scholar). In the present study, we use two complementary approaches—monitoring [Ca2+]i oscillations in living cells and performing molecular dynamics (MD) simulations—to provide important insights into how the CaSR functions and the behavior of the receptor at the atomic level. We first demonstrate that the molecular connectivity between [Ca2+]o-binding sites that is encoded within key Ca2+-binding site 1 in the hinge region of the CaSR ECD is responsible for the functional positive homotropic cooperativity in the CaSR response to [Ca2+]o. We further identify an l-Phe-binding pocket adjacent to Ca2+-binding site 1. We show that occupancy of this binding pocket by l-Phe is essential for functional positive heterotropic cooperativity by virtue of its having a marked impact on all five of the predicted Ca2+-binding sites in the ECD with regard to [Ca2+]o-evoked [Ca2+]i signaling. Furthermore, with MD simulations, we show that the simulated motions of Ca2+-binding site 1 are correlated with those of the other predicted Ca2+-binding sites. Finally, the dynamic communication of l-Phe at its predicted binding site in the hinge region with the CaSR Ca2+-binding sites not only influences the adjacent [Ca2+]o binding site 1 but also globally (i.e., by exerting effects widely over the ECD) enhances cooperative activation of the receptor in response to alterations in [Ca2+]o. The structure of the extracellular domain of CaSR (residues 25–530) was modeled based on the crystal structure of metabotropic glutamate receptor 1 (mGluR1) (Protein Data Bank codes 1EWT, 1EWK, and 1ISR), and the potential Ca2+-binding sites in the CaSR ECD were predicted using MetalFinder (25.Huang Y. Zhou Y. Yang W. Butters R. Lee H.W. Li S. Castiblanco A. Brown E.M. Yang J.J. Identification and dissection of Ca2+-binding sites in the extracellular domain of Ca2+-sensing receptor.J. Biol. Chem. 2007; 282: 19000-19010Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 27.Jiang Y. Huang Y. Wong H.C. Zhou Y. Wang X. Yang J. Hall R.A. Brown E.M. Yang J.J. Elucidation of a novel extracellular calcium-binding site on metabotropic glutamate receptor 1α (mGluR1α) that controls receptor activation.J. Biol. Chem. 2010; 285: 33463-33474Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Prediction of the l-Phe-binding site was performed by AutoDock-Vina (28.Trott O. Olson A.J. AutoDock Vina. Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading.J. Comput. Chem. 2010; 31: 455-461Crossref PubMed Scopus (0) Google Scholar). In brief, the docking center and grid box of the model structure and the rotatable bonds of l-Phe were defined by AutoDock tools 1.5.4. The resultant l-Phe coordinates were combined back to the Protein Data Bank file of the model structure for input into the ligand-protein contacts and contacts of structural units (LPC/CSU) server to analyze interatomic contacts between the ligand and receptor (29.Sobolev V. Sorokine A. Prilusky J. Abola E.E. Edelman M. Automated analysis of interatomic contacts in proteins.Bioinformatics. 1999; 15: 327-332Crossref PubMed Scopus (707) Google Scholar). The residues within 5 Å around l-Phe were considered as l-Phe-binding residues. Measurement of intracellular free Ca2+ was assessed as described by Huang et al. (30.Huang Y. Zhou Y. Wong H.C. Castiblanco A. Chen Y. Brown E.M. Yang J.J. Calmodulin regulates Ca2+-sensing receptor-mediated Ca2+ signaling and its cell surface expression.J. Biol. Chem. 2010; 285: 35919-35931Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Briefly, wild type CaSR or its mutants were transiently transfected into HEK293 cells grown on coverslips and cultured for 48 h. The cells were subsequently loaded for 15 min using 4 μm Fura-2 AM in 2 ml of physiological saline buffer (10 mm HEPES, 140 mm NaCl, 5 mm KCl, 1.0 mm MgCl2, 1 mm CaCl2, pH 7.4). The coverslips were mounted in a bath chamber on the stage of a Leica DM6000 fluorescence microscope, and the cells were incubated in calcium-free physiological saline buffer for 5 min. The cells were then alternately illuminated with 340- or 380-nm light, and the fluorescence at an emission wavelength 510 nm was recorded in real time as the concentration of extracellular Ca2+ was increased in a stepwise manner in the presence or absence of 5 mm l-Phe. The ratio of the emitted fluorescence intensities resulting from excitation at both wavelengths was utilized as a surrogate for changes in [Ca2+]i and was further plotted and analyzed as a function of [Ca2+]o. All experiments were performed at room temperature. The signals from 30–60 single cells were recorded for each measurement. Oscillations were defined as three successive fluctuations in [Ca2+]i after the initial peak. The [Ca2+]i responses of wild type CaSR and its mutants were measured as described by Huang et al. (25.Huang Y. Zhou Y. Yang W. Butters R. Lee H.W. Li S. Castiblanco A. Brown E.M. Yang J.J. Identification and dissection of Ca2+-binding sites in the extracellular domain of Ca2+-sensing receptor.J. Biol. Chem. 2007; 282: 19000-19010Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Briefly, CaSR-transfected HEK293 cells were grown on 13.5 × 20-mm coverslips. After the cells reached 90% confluence, they were loaded by incubation with 4 μm Fura-2 AM in 20 mm HEPES, containing 125 mm NaCl, 5 mm KCl, 1.25 mm CaCl2, 1 mm MgCl2, 1 mm NaH2PO4, 1% glucose, and 1% BSA (pH 7.4) for 1 h at 37 °C and then washed once with 20 mm HEPES (pH 7.4) containing 125 mm NaCl, 5 mm KCl, 0.5 mm CaCl2, 0.5 mm MgCl2, 1% glucose, and 1% BSA (bath buffer). The coverslips with transfected, Fura-2-loaded HEK293 cells were placed diagonally in 3-ml quartz cuvettes containing bath buffer. The fluorescence spectra at 510 nm were measured during stepwise increases in [Ca2+]o with alternating excitation at 340 or 380 nm. The ratio of the intensities of the emitted light at 510 nm when excited at 340 or 380 nm was used to monitor changes in [Ca2+]i. The EC50 and Hill constants were fitted using the following Hill equation,ΔS=[M]nKdn+[M]n(Eq. 1) where ΔS is the total signal change in the equation and [M] is the free ligand concentration. MD simulation provides an approach complementary to the experiments in live cells for understanding biomolecular structure, dynamics, and function. The initial coordinates for all the simulations were modeled from the 2.20 Å resolution x-ray crystal structure of mGluR1 with Protein Data Bank code 1EWK (31.Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor.Nature. 2000; 407: 971-977Crossref PubMed Scopus (1108) Google Scholar). The AMBER 10 suite of programs (32.Case D.A. Darden T.A. Cheatham I.T.E. Simmerling C.L. Wang J. Duke R.E. Luo R. Crowley M. Walker R.C. Zhang W. Merz K.M. Wang B. Hayik S. Roitberg A. Seabra G. Kolossva′ry I. Wong K.F. Paesani F. Vanicek J. Wu X. Brozell S.R. Steinbrecher T. Gohlke H. Yang L. Tan C. Mongan J. Hornak V. Cui G. Mathews D.H. Seetin M.G. Sagui C. Babin V. Kollman P.A. AMBER 10. University of California, San Francisco2008Google Scholar) was used to carry out all of the simulations in an explicit TIP3P water model (33.Jorgensen W.L. Chandrasekhar J. Madura J.D. Impey R.W. Klein M.L. Comparison of simple potential functions for simulating liquid water.J. Chem. Phys. 1983; 79: 926-935Crossref Scopus (29869) Google Scholar), using the modified version of the all-atom Cornell et al. (34.Cornell W.D. Cieplak P. Christopher I.B. Gould I.R. Merz J.K. Ferguson D.M. Spellmeyer D.C. Fox T. Caldwell J.W. Kollman P.A. A second generation force field for the simulation of proteins, nucleic acids, and organic molecules.J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11548) Google Scholar) force field and the reoptimized dihedral parameters for the peptide ω-bond (35.Urmi D. Hamelberg D. Reoptimization of the AMBER force field parameters for peptide bond (Omega) torsions using accelerated molecular dynamics.J. Phys. Chem. 2009; 113: 16590-16595Crossref Scopus (65) Google Scholar). An initial 2-ns simulation was performed using NOE restraint during the equilibration to reorient the side chains residues in the Ca2+-binding site, but no restraints were used during the actual simulation. A total of three MD simulations were carried out for 50 ns each on the apo-form and ligand-loaded forms. During the simulations, an integration time step of 0.002 ps was used to solve Newton's equation of motion. The long range electrostatic interactions were calculated using the particle mesh Ewald method (36.Darden T. York D. Pedersen L. Particle mesh Ewald. An N log(N) method for Ewald sums in large systems.J. Chem. Phys. 1993; 98: 10089-10092Crossref Scopus (20807) Google Scholar), and a cutoff of 9.0 Å was applied for nonbonded interactions. All bonds involving hydrogen atoms were restrained using the SHAKE algorithm (37.Ryckaert J.P. Ciccotti G. Berendsen H.J. Numerical integration of the cartesian equations of motion of a system with constraints. Molecular dynamics of n-alkanes.J. Comput. Phys. 1977; 23: 327-341Crossref Scopus (16896) Google Scholar). The simulations were carried out at a temperature of 300 K and a pressure of 1 bar. A Langevin thermostat was used to regulate the temperature with a collision frequency of 1.0 ps−1. The trajectories were saved every 500 steps (1 ps). The trajectories were analyzed using the ptraj module in Amber 10. Accelerated MD was carried out on the free CaSR ECD using the Rotatable accelerated Molecular Dynamics, (RaMD) method (38.Doshi U. Hamelberg D. Improved statistical sampling and accuracy with accelerated molecular dynamics on rotatable torsions.J. Chem. Theory Comput. 2012; 8: 4004-4012Crossref PubMed Scopus (30) Google Scholar) implemented in a pmemd module of AMBER on the rotatable torsion. A boost energy, E, of 2,000 kcal/mol was added to the average dihedral energy, and a tuning parameter, α, of 200 kcal/mol was used. The dual boost was also applied to accelerate the diffusive and solvent dynamics as previously described (39.Hamelberg D. de Oliveira C.A. McCammon J.A. Sampling of slow diffusive conformational transitions with accelerated molecular dynamics.J. Chem. Phys. 2007; 127: 155102Crossref PubMed Scopus (189) Google Scholar). The simulation conditions were similar to that of the normal MD simulations above. Principal component analysis was carried out on the trajectories using the ptraj module in AMBER. The directions of the eigenvectors for the slowest modes were visualized using the Interactive Essential Dynamics plugin (40.Mongan J. Interactive essential dynamics.J. Comput. Aided Mol. Des. 2004; 18: 433-436Crossref PubMed Scopus (78) Google Scholar). The binding energies for the ligands were calculated using an ensemble-docking method and Autodock vina (28.Trott O. Olson A.J. AutoDock Vina. Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading.J. Comput. Chem. 2010; 31: 455-461Crossref PubMed Scopus (0) Google Scholar). The ensemble of conformations of CaSR was generated using molecular dynamics simulations as described above. Gasteiger charges were assigned to the ligands and CaSR using the Autodock ADT program. The ligands were flexible during docking to each conformation of CaSR using the following parameters: the grid spacing was 1.0 Å; the box size was 25 Å in each dimension, and the center of the box was chosen as the center of the active site of CaSR, with a large enough space to sample all possible ligand conformations within the box. The maximum number of binding modes saved was set to 10. The conformation with the lowest binding energy was used and assumed to be the best binder. Distributions of the binding energies for each ligand were calculated based on the lowest binding energy of each ligand to each conformation in the ensemble of CaSR conformations. Using the ptraj module of AMBER 10, principal component analysis (PCA) (41.Levy R.M. Srinivasan A.R. Olson W.K. McCammon J.A. Quasi-harmonic method for studying very low frequency modes in proteins.Biopolymers. 1984; 23: 1099-1112Crossref PubMed Scopus (187) Google Scholar, 42.Jolliffe I.T. Principal Component Analysis. Springer, New York2002Google Scholar) was performed on all the atoms of the residues that are 5 Å away from site 1 of CaSR ECD. The covariance matrix of the x, y, and z coordinates of all the atoms obtained from each snapshot of the combined trajectories of the ligand-free CaSR ECD, the Ca2+-loaded form, the form loaded with only l-Phe, and the form loaded with both Ca2+ and l-Phe were calculated. The covariance matrix was further diagonalized to produce orthonormal eigenvectors and their corresponding eigenvalues, ranked on the basis of their corresponding variances. The first three eigenvectors, the principal components that contributed the majority of all the atomic fluctuations, were used to project the conformational space onto them, i.e., along two dimensions. The data are presented as means ± S.E. for the indicated number of experiments. Statistical analyses were carried out using the unpaired Student's t test when two groups were compared. A p value of < 0.05 was considered to indicate a statistically significant difference. It has been documented that in several regions of the CaSR and mGluRs, the amino acid residues are highly conserved (43.Bai M. Structure-function relationship of the extracellular calcium-sensing receptor.Cell Calcium. 2004; 35: 197-207Crossref PubMed Scopus (56) Google Scholar). Those conserved elements provide a structural framework for the modeling of the CaSR ECD. Among all the available crystal structures of the mGluRs, studies on mGluR1 give concrete structural information about ligand-free as well as various ligand-bound forms of the receptor. Moreover, CaSR and mGluR1 share similar signaling pathways and can form heterodimers with one another either in vivo or in vitro (44.Gama L. Wilt S.G. Breitwieser G.E. Heterodimerization of calcium sensing receptors with metabotropic glutamate receptors in neurons.J. Biol. Chem. 2001; 276: 39053-39059Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Thus, the cr" @default.
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• W2021552011 date "2014-02-01" @default.
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• W2021552011 title "Identification of an l-Phenylalanine Binding Site Enhancing the Cooperative Responses of the Calcium-sensing Receptor to Calcium" @default.
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