Understanding how ligands with different efficacies modulate μ-opioid receptor (MOR) signaling requires dissecting the thermodynamics and kinetics of MOR-G protein activation at atomic resolution. Recent time-resolved cryo-electron microscopy (cryo-EM) studies of MOR-G i1 complexes bound to the full agonist lofentanil or the partial agonist mitragynine pseudoindoxyl revealed four distinct intermediate states of MOR-G i1 activation upon GTP binding. Analysis of these data suggests ligand-dependent differences in state occupancy, raising the possibility that partial agonists induce a “kinetic trap” that hinders efficient G-protein activation. Here, we extend these findings using millisecond-scale molecular dynamics simulations of each cryo-EM structure to capture the dynamic evolution of GTP-bound MOR-G i1 complexes toward dissociation. Markov state models built from these simulations quantify state-to-state transitions and identify the molecular determinants that govern the ligand-specific conformational and kinetic landscape of MOR-G i1 activation. By bridging static structural snapshots with dynamic transition pathways, our framework establishes a quantitative link between ligand efficacy and receptor-G protein activation kinetics. In particular, we quantify the differential stabilization of MOR-G i1 intermediate states by lofentanil and mitragynine pseudoindoxyl, where variation in G i1 α-helical domain opening kinetics correlates with efficacy. Together, these findings provide a kinetic and dynamic framework for understanding MOR-G i1 signaling and suggest new opportunities for the rational design of safer opioid therapeutics.
Konovalov et al. (Sun,) studied this question.