The mu-opioid receptor (MOR) is the primary target of opioid analgesics and plays a central role in pain modulation and opioid pharmacology. Upon ligand binding at its extracellular side, MOR undergoes conformational changes within its transmembrane (TM) domains, initiating crucial intracellular signaling events. In this study, we employed a water-soluble MOR (wsMOR) variant engineered to retain ligand-binding activity while allowing detailed structural characterization in solution. Long-timescale all-atom molecular dynamics simulations revealed a structurally stable receptor exhibiting a diverse ensemble of conformational states. Using a combination of quasielastic neutron scattering (QENS) and single-molecule Förster fluorescence resonance energy transfer (smFRET), we captured real-time conformational dynamics, with QENS providing ensemble-averaged timescales of motion and smFRET specifically resolving the movement of TM6. Our data indicate that unliganded wsMOR is highly flexible, with agonist binding reducing this flexibility on both picosecond and millisecond timescales. Importantly, wsMOR preserves transmembrane helix 6 movements characteristic of GPCR activation, with positive allosteric modulation and G protein binding stabilizing a fully active conformation. These findings provide insights into the plasticity of MOR outside the membrane and highlight the utility of engineered soluble GPCR variants as tractable systems for structural studies and ligand screening in aqueous environments.
Agyemang et al. (Sun,) studied this question.