Transition Intermediates Encode Ligand Efficacy at the μ-Opioid Receptor-Gi Protein Complex

How ligands with different efficacies regulate G protein-coupled receptor signaling remains incompletely understood. Here, we combine Markov state modeling of nearly one millisecond of aggregate all-atom molecular dynamics simulations with time-resolved cryogenic-electron microscopy (cryo-EM) to reconstruct the most probable transition pathways connecting recently resolved structures of guanosine triphosphate (GTP)-bound μ-opioid receptor (MOR)–G _i1 complexes with the full agonist lofentanil (LFT) or partial agonist mitragynine pseudoindoxyl (MP). The models predict four previously unresolved intermediate conformations. Guided by these predictions, reanalysis of the cryo-EM particle ensemble identifies conformational populations consistent with all four intermediates. Remarkably, the largest ligand-dependent structural differences emerge within these intermediates rather than in the previously resolved states. LFT favors a predominantly sequential and kinetically efficient transition pathway, whereas MP stabilizes a broader intermediate ensemble along this pathway and slows progression toward later states. Together, these complementary approaches provide an enriched thermodynamic and kinetic description of MOR-mediated G protein activation, revealing that opioid efficacy is encoded, at least in part, in the differential stabilization and kinetics of transition intermediates extending beyond GTP binding but well before complete G protein dissociation.