Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC) and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS). Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand.

[1]  Arthur Christopoulos,et al.  Critical Role for the Second Extracellular Loop in the Binding of Both Orthosteric and Allosteric G Protein-coupled Receptor Ligands* , 2007, Journal of Biological Chemistry.

[2]  B. Imbimbo,et al.  Medicinal chemistry and therapeutic potential of muscarinic M3 antagonists , 2009, Medicinal research reviews.

[3]  J W Saldanha,et al.  Transmembrane Domains 4 and 7 of the M1Muscarinic Acetylcholine Receptor Are Critical for Ligand Binding and the Receptor Activation Switch* , 2001, The Journal of Biological Chemistry.

[4]  R. Eglen.  Muscarinic receptor subtype pharmacology and physiology. , 2005, Progress in medicinal chemistry.

[5]  J. Wess,et al.  Functional role of proline and tryptophan residues highly conserved among G protein‐coupled receptors studied by mutational analysis of the m3 muscarinic receptor. , 1993, The EMBO journal.

[6]  J. Wess,et al.  Site‐directed mutagenesis of the m3 muscarinic receptor: identification of a series of threonine and tyrosine residues involved in agonist but not antagonist binding. , 1991, The EMBO journal.

[7]  Tsutomu Kouyama,et al.  Crystal structure of squid rhodopsin , 2008, Nature.

[8]  N. Birdsall,et al.  International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. , 1998, Pharmacological reviews.

[9]  Alan Grossfield,et al.  Recent progress in the study of G protein-coupled receptors with molecular dynamics computer simulations. , 2011, Biochimica et biophysica acta.

[10]  R. Stevens,et al.  High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein–Coupled Receptor , 2007, Science.

[11]  T. Darden,et al.  Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems , 1993 .

[12]  R. Stevens,et al.  GPCR Engineering Yields High-Resolution Structural Insights into β2-Adrenergic Receptor Function , 2007, Science.

[13]  Charles L. Brooks,et al.  Community-wide assessment of GPCR structure modelling and ligand docking: GPCR Dock 2008 , 2009, Nature Reviews Drug Discovery.

[14]  Gebhard F. X. Schertler,et al.  Structure of a β1-adrenergic G-protein-coupled receptor , 2008, Nature.

[15]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[16]  M. Brann,et al.  The Second Intracellular Loop of the m5 Muscarinic Receptor Is the Switch Which Enables G-protein Coupling* , 1998, The Journal of Biological Chemistry.

[17]  Marta Filizola,et al.  Modern homology modeling of G-protein coupled receptors: which structural template to use? , 2009, Journal of medicinal chemistry.

[18]  E C Hulme,et al.  Phenotypic classification of mutants: a tool for understanding ligand binding and activation of muscarinic acetylcholine receptors. , 2007, Biochemical Society transactions.

[19]  M. Burghammer,et al.  Crystal structure of the human β2 adrenergic G-protein-coupled receptor , 2007, Nature.

[20]  J. Wess,et al.  Role of conserved threonine and tyrosine residues in acetylcholine binding and muscarinic receptor activation. A study with m3 muscarinic receptor point mutants. , 1992, The Journal of biological chemistry.

[21]  Exploring the binding site of the human muscarinic M3 receptor: Homology modeling and docking study , 2007 .

[22]  J. van Durme,et al.  Repulsive separation of the cytoplasmic ends of transmembrane helices 3 and 6 is linked to receptor activation in a novel thyrotropin receptor mutant (M626I). , 2006, Molecular endocrinology.

[23]  J. Andrew McCammon,et al.  Mapping the Druggable Allosteric Space of G-Protein Coupled Receptors: a Fragment-Based Molecular Dynamics Approach , 2010, Chemical biology & drug design.

[24]  E C Hulme,et al.  Scanning mutagenesis studies of the M1 muscarinic acetylcholine receptor. , 2003, Receptors & channels.

[25]  O. Berger,et al.  Molecular dynamics simulations of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature. , 1997, Biophysical journal.

[26]  A. Krejci,et al.  Changes of cooperativity between N-methylscopolamine and allosteric modulators alcuronium and gallamine induced by mutations of external loops of muscarinic M(3) receptors. , 2001, Molecular pharmacology.

[27]  E. Hulme,et al.  Muscarinic acetylcholine receptors. Peptide sequencing identifies residues involved in antagonist binding and disulfide bond formation. , 1990, The Journal of biological chemistry.

[28]  Jonathan A. Javitch,et al.  Structure of the Human Dopamine D3 Receptor in Complex with a D2/D3 Selective Antagonist , 2010, Science.

[29]  J. Ramachandran,et al.  Structure and Function of G Protein Coupled Receptors , 1990, Pharmaceutical Research.

[30]  J. Saldanha,et al.  Seven-transmembrane receptors: crystals clarify. , 2002, Trends in pharmacological sciences.

[31]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[32]  P. Escribá,et al.  Lipid-protein interactions in GPCR-associated signaling. , 2007, Biochimica et biophysica acta.

[33]  B. Testa,et al.  Muscarinic Receptors: A Comparative Analysis of Structural Features and Binding Modes through Homology Modelling and Molecular Docking , 2006, Chemistry & biodiversity.

[34]  K. Page,et al.  Scanning mutagenesis identifies amino acid side chains in transmembrane domain 5 of the M(1) muscarinic receptor that participate in binding the acetyl methyl group of acetylcholine. , 2000, Molecular pharmacology.

[35]  J. Ballesteros,et al.  [19] Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors , 1995 .

[36]  W. L. Jorgensen,et al.  Development and Testing of the OPLS All-Atom Force Field on Conformational Energetics and Properties of Organic Liquids , 1996 .

[37]  R. Abagyan,et al.  Structures of the CXCR4 Chemokine GPCR with Small-Molecule and Cyclic Peptide Antagonists , 2010, Science.

[38]  John Ellis,et al.  Mutational Disruption of a Conserved Disulfide Bond in Muscarinic Acetylcholine Receptors Attenuates Positive Homotropic Cooperativity between Multiple Allosteric Sites and Has Subtype-Dependent Effects on the Affinities of Muscarinic Allosteric Ligands , 2007, Molecular Pharmacology.

[39]  A. Sali,et al.  Modeller: generation and refinement of homology-based protein structure models. , 2003, Methods in enzymology.

[40]  A. Cordomí,et al.  Molecular dynamics simulations of rhodopsin in different one-component lipid bilayers. , 2007, Journal of Physical Chemistry B.

[41]  L. Pardo,et al.  Ligand-specific regulation of the extracellular surface of a G protein coupled receptor , 2009, Nature.

[42]  T. Schwartz,et al.  Allosteric enhancers, allosteric agonists and ago-allosteric modulators: where do they bind and how do they act? , 2007, Trends in pharmacological sciences.

[43]  P. Garriga,et al.  Molecular Dynamics Simulations of Rhodopsin Point Mutants at the Cytoplasmic Side of Helices 3 and 6 , 2008, Journal of biomolecular structure & dynamics.

[44]  Manfred Burghammer,et al.  Structure of bovine rhodopsin in a trigonal crystal form. , 2003, Journal of molecular biology.

[45]  Kirstin Jöhren,et al.  A model of the human M2 muscarinic acetylcholine receptor , 2002, J. Comput. Aided Mol. Des..

[46]  Sid Topiol,et al.  Use of the X-ray structure of the Beta2-adrenergic receptor for drug discovery. , 2008, Bioorganic & medicinal chemistry letters.

[47]  E C Hulme,et al.  The Functional Topography of Transmembrane Domain 3 of the M1 Muscarinic Acetylcholine Receptor, Revealed by Scanning Mutagenesis* , 1999, The Journal of Biological Chemistry.

[48]  J. Wess,et al.  Muscarinic acetylcholine receptors: structural basis of ligand binding and G protein coupling. , 1995, Life sciences.

[49]  R. Stevens,et al.  FoldGPCR: Structure prediction protocol for the transmembrane domain of G protein‐coupled receptors from class A , 2010, Proteins.

[50]  Darrell R. Abernethy,et al.  International Union of Pharmacology: Approaches to the Nomenclature of Voltage-Gated Ion Channels , 2003, Pharmacological Reviews.

[51]  Willem Soudijn,et al.  Allosteric modulation of G-protein-coupled receptors , 2001 .

[52]  S. Lazareno,et al.  Detection, quantitation, and verification of allosteric interactions of agents with labeled and unlabeled ligands at G protein-coupled receptors: interactions of strychnine and acetylcholine at muscarinic receptors. , 1995, Molecular pharmacology.

[53]  M. Ishii,et al.  Muscarinic acetylcholine receptors. , 2006, Current pharmaceutical design.

[54]  R. Stevens,et al.  The 2.6 Angstrom Crystal Structure of a Human A2A Adenosine Receptor Bound to an Antagonist , 2008, Science.

[55]  Thomas M Frimurer,et al.  Ligand binding and micro-switches in 7TM receptor structures. , 2009, Trends in pharmacological sciences.

[56]  Hans-Dieter Höltje,et al.  Molecular dynamics simulations of bovine rhodopsin: influence of protonation states and different membrane-mimicking environments , 2005, Journal of molecular modeling.

[57]  K. Palczewski,et al.  Crystal Structure of Rhodopsin: A G‐Protein‐Coupled Receptor , 2002, Chembiochem : a European journal of chemical biology.

[58]  Berk Hess,et al.  GROMACS 3.0: a package for molecular simulation and trajectory analysis , 2001 .

[59]  P. Garriga,et al.  Critical Role of Electrostatic Interactions of Amino Acids at the Cytoplasmic Region of Helices 3 and 6 in Rhodopsin Conformational Properties and Activation* , 2007, Journal of Biological Chemistry.

[60]  C. Langmead,et al.  Roof and Floor of the Muscarinic Binding Pocket: Variations in the Binding Modes of Orthosteric Ligands , 2007, Molecular Pharmacology.

[61]  Catherine L. Worth,et al.  Comparative Sequence and Structural Analyses of G-Protein-Coupled Receptor Crystal Structures and Implications for Molecular Models , 2009, PloS one.

[62]  Jessica Sallander,et al.  Conformational Toggle Switches Implicated in Basal Constitutive and Agonist-Induced Activated States of 5-Hydroxytryptamine-4 Receptors , 2009, Molecular Pharmacology.

[63]  S. Colowick,et al.  Methods in Enzymology , Vol , 1966 .

[64]  Francesca Fanelli,et al.  Computational modeling approaches to structure-function analysis of G protein-coupled receptors. , 2005, Chemical reviews.