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