Movie S1.

Binding of the agonist IXO and G-protein mimetic nanobody Nb9-8 to the M2 muscarinic GPCR in a 4,500-ns GaMD simulation. Although the agonist reached only the receptor extracellular vestibule, the nanobody was observed to bind the receptor intracellular G-protein-coupling site with a minimum rmsd of 2.48 Å in the core domain compared with the X-ray structure. The nanobody approached the M2 receptor on the intracellular side and formed initial contacts with the ICL2. The TM6 cytoplasmic end became disordered in the C-terminal region of ICL3 and facilitated binding of the nanobody. Then, the nanobody rearranged its conformation and fit into the receptor intracellular pocket. The nanobody interacted dynamically with the receptor flexible ICLs and the TM7–H8 hinge. The nanobody rmsd dropped <5 Å during ∼3,880−4,000 ns. In the absence of the agonist IXO at the orthosteric site, the nanobody deviated from the target G-protein-coupling site during ∼4,000–4,500 ns while maintaining interactions with the receptor intracellular domains.