Vol. 62, Issue 4, 778-787, October 2002

Conformation of Ligands Bound to the Muscarinic Acetylcholine Receptor

Hiroyasu Furukawa, Toshiyuki Hamada, Mariko K. Hayashi, Tatsuya Haga, Yutaka Muto, Hiroshi Hirota, Shigeyuki Yokoyama, Kazuo Nagasawa, and Masaji Ishiguro

Department of Neurochemistry, Faculty of Medicine (H.F., M.K.H., T.Hag.), Department of Biophysics and Biochemistry, Faculty of Science (S.Y.), and Institute of Molecular and Cellular Biosciences, the University of Tokyo, Tokyo, Japan (K.N.); Core Research for Evolutional Science and Technology (H.F., H.H., T.Ham.); Institute for Biomolecular Science, Gakushuin University, Tokyo, Japan (T.Hag.); Genomic Sciences Center, RIKEN Yokohama Institute, Yokohama, Japan (T. Ham., Y.M., H.H., S.Y.); and Suntory Institute for Bioorganic Research, Osaka, Japan (M.I.)

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.