Visualizing agonist-induced M2 receptor activation regulated by aromatic ring dynamics

Despite the growing number of G protein-coupled receptor (GPCR) structures being resolved, the dynamic process of how GPCRs transit from the inactive toward the active state remains unclear. In this study, comprehensive molecular dynamics simulations were performed to explore how ligand binding modulates the conformational dynamics of the M2 muscarinic acetylcholine receptor (M2R). We observed a sequential occurrence of structural changes in the inactive-to-active transition of M2R induced by a superagonist iperoxo, which includes the orthosteric binding site contraction, the TM6 opening into an intermediate conformation, and a further structural change toward full activation upon binding to G protein or a G protein mimetic nanobody. Two activation intermediates were identified, which show structural features different from those reported for apo-GPCRs. Moreover, our results suggest that stabilization of a specific W400^6.48 conformation and enhanced F396^6.44 dynamics are crucial for activation, whereas distinct side-chain rotamer equilibriums of Y206^5.58 in the cytoplasmic cavity are correlated with agonist efficacies. Our work provides atomic-level structural insights into the agonist-induced M2R activation pathway and highlights a mechanism by which ligand efficacy can be encoded and transduced in the form of aromatic ring dynamics.