Discovering Key Activation Hotspots in the M₂ Muscarinic Receptor

The M₂ muscarinic receptor (M₂R) is a prototypical G protein-coupled receptor (GPCR) that serves as a model system for understanding ligand recognition and GPCR activation. Here, using vibrational spectroscopy, we identify the mechanisms governing M₂R activation by its native agonist, acetylcholine. Combined with mutagenesis, computational chemistry, and organic synthetic chemistry, our analyses found that the precise distance between acetylcholine and Asn404, one of the amino acids constituting the ligand-binding site, is important for M₂R activation and that the N404Q mutant undergoes partial active state-like conformational changes. We discovered that a water molecule bridging acetylcholine and Asn404 forms a precise and flexible hydrogen bond network, triggering the outward movement of transmembrane helix 6 in M₂R. Consistent with this observation, disruptions in this hydrogen bond network via chemical modification at the α- or β-position of acetylcholine failed to activate M₂R. Collectively, our findings pinpoint Asn404 as a critical residue that both senses acetylcholine binding and induces M₂R activation.