Conformation of ligands bound to the muscarinic acetylcholine receptor

Many biogenic amines evoke a variety of physiological responses by acting on G protein-coupled receptors. We have determined the conformation of two acetylcholine analogs, (S)-methacholine and (2S,4R,5S)-muscarine, bound to the M₂ muscarinic acetylcholine receptor (M₂ mAChR) by NMR spectroscopy. The analysis of the transferred nuclear Overhauser effect indicated that the receptor selectively recognized the conformers of (S)-methacholine and (2S,4R,5S)-muscarine with the gauche O-C2-C1-N dihedral angle at +60°. This is distinct from the predominant conformations of these ligands in solution with O-C2-C1-N dihedral angle (+80∼85°) in the absence of the M₂ mAChR, as assessed by analyses of the coupling constants and nuclear Overhauser effect spectroscopy. We have also built a molecular model of the M₂ mAChR-(S)-methacholine complex, based on the X-ray crystallographic structure of rhodopsin. This model indicated that the conformation with the gauche O-C2-C1-N dihedral angle at +55.5°, which is similar to the one determined by NMR measurement, is energetically favored in the binding of (S)-methacholine to the receptor. We suggest that this conformation represents the binding of the agonist to the M₂ mAChR in the absence of G protein.