The Predicted 3D Structures of the Human M1 Muscarinic Acetylcholine Receptor with Agonist or Antagonist Bound

The muscarinic acetylcholine G‐protein‐coupled receptors are implicated in diseases ranging from cognitive dysfunctions to smooth‐muscle disorders. To provide a structural basis for drug design, we used the MembStruk computational method to predict the 3D structure of the human M1 muscarinic receptor. We validated this structure by using the HierDock method to predict the binding sites for three agonists and four antagonists. The intermolecular ligand–receptor contacts at the predicted binding sites agree well with deductions from available mutagenesis experiments, and the calculated relative binding energies correlate with measured binding affinities. The predicted binding site of all four antagonists is located between transmembrane (TM) helices 3, 4, 5, 6, and 7, whereas the three agonists prefer a site involving residues from TM3, TM6, and TM7. We find that Trp 157(4) contributes directly to antagonist binding, whereas Pro 159(4) provides an indirect conformational switch to position Trp 157(4) in the binding site (the number in parentheses indicates the TM helix). This explains the large decrease in ligand binding affinity and signaling efficacy by mutations of Trp 157(4) and Pro 159(4) not previously explained by homology models. We also found that Asp 105(3) and aromatic residues Tyr 381(6), Tyr 404(7), and Tyr 408(7) are critical for binding the quaternary ammonium head group of the ligand through cation–π interactions. For ligands with a charged tertiary amine head group, we suggest that proton transfer from the ligand to Asp 105(3) occurs upon binding. Furthermore, we found that an extensive aromatic network involving Tyr 106(3), Trp 157(4), Phe 197(5), Trp 378(6), and Tyr 381(6) is important in stabilizing antagonist binding. For antagonists with two terminal phenyl rings, this aromatic network extends to Trp 164(4), Tyr 179(extracellular loop 2), and Phe 390(6) located at the extracellular end of the TMs. We find that Asn 382(6) forms hydrogen bonds with selected antagonists. Tyr381(6) and Ser 109(3) form hydrogen bonds with the ester moiety of acetylcholine, which binds in the gauche conformation.