| We have designed and executed molecular dynamic studies using the AMBER force field with models of explicitly solvated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer containing either a wild-type, Thr192Ala, Ala196Gly, or Thr192Ala/Ala196Gly human M1 muscarinic acetylcholine receptor with acetylcholine ligand or wild type receptor without ligand. Following sequence alignment of human M1 muscarinic acetylcholine receptor with other G protein-coupled receptors, including bovine rhodopsin, spacial coordinates for human and mutant M1 muscarinic acetylcholine receptor atoms were derived from the X-ray crystal structure data reported for bovine rhodopsin (PDB id 1L9H, 2.6 A resolution) using the comparative modeling program MODELLER. Receptor models of wild-type and mutant receptors thus constructed were complete including the full length amino and carboxy termini, three extracellular loops (EL), three intracellular loops, and seven transmembrane helices. Molecular dynamic simulations were carried out to at least 2 ns. Computational results were compared for self-consistency, with published computational and theoretical data, and known biological activity data. Based on analysis of the molecular dynamic studies outcomes, interaction of the acetylcholine cationic amine with receptor was observed to be mediated through Asp105 and Tyr106 of TM-III, with the Tyr106 interactions being of a cation-pi nature. The acetyl methyl group of acetylcholine was found to interact with residues of TMs-V, VI and EL-II. Within the wild-type receptor, Ala196 in concert with Va1385 and Ile180 formed a hydrophobic pocket for the acetyl methyl group. Absence of Thr192 or Ala196 was associated with the counter-clockwise rotation of the extracellular segment of TM-V, crowding residues from the protein/lipid interface into the binding pocket. While Thr192 was not observed to hydrogen bond directly with acetylcholine, it was observed to stabilize TMs-IV and V through a water bridge involving Gln165, Tyr179 and Tyr106. Acetylcholine was observed to adopt a single preferred conformation (trans/-gauche) within the wild-type receptor, comprising 97% of the captured states, within the mutants multiple conformations were observed in addition to a significant reduction in the wild-type binding mode. Results from analysis of acetylcholine binding within the computational models were thus predictive of physical experimental observations made in both wild-type and mutant receptors. The methodology employed is applicable to the development of virtual models for other proteins, especially G protein-coupled receptors, in a lipid bilayer environment. The molecular model of the human M1 muscarinic acetylcholine receptor developed will be useful for improving the understanding of ligand binding to receptor as well as the potential for exogenous molecule binding. |
