Figure 5.

Mechanism of graded activation of the M₂ muscarinic GPCR: The M₂ receptor (ribbons) samples a large conformational space with significant structural rearrangements, especially for the TM6 helix. Binding of the inverse agonist QNB (green spheres) confines the receptor in the inactive state. Without the G protein or mimetic nanobody, the partial agonist ARC (yellow spheres) biases the M₂ receptor to visit an intermediate state “I1” (orange ribbons). ARC is able to dissociate completely to the bulk solvent via the extracellular vestibule and rebinds to the receptor repeatedly during a 2030 ns GaMD simulation. In comparison, the full agonist IXO (red spheres) biases the receptor further, sampling both intermediate “I1” (orange ribbons) and “I2” (purple ribbons). IXO escapes out of the orthosteric pocket and visits the extracellular vestibule in one of the GaMD simulations. By adding the G protein mimetic nanobody (purple surface), the M₂ receptor is stabilized in the fully active state (red ribbons) as bound by IXO or ARC, although ARC adopts two alternative conformations in the orthosteric pocket, ARC-P1 (yellow spheres) and ARC-P1′ (cyan spheres).