Ligand Binding Kinetics, Thermodynamics, and Gating of Insect Odorant Receptor

Insect olfactory receptors (ORs) are unique ligand-gated ion channels specialized in detecting volatile molecules. The ancestral M. hrabei MhOR5 operates independently of the Orco co-receptor and exhibits broad ligand-binding capacity, yet the molecular underpinnings of its ligand specificity and gating dynamics have long remained unclear. To resolve this knowledge gap, we applied an integrated molecular dynamics enhanced sampling approach, combining collective variable machine learning, alchemical free energy calculations, and kinetic calculations, to dissect the ligand recognition and gating mechanisms of MhOR5 in complex with two key ligands: eugenol (EOL) and DEET. Our findings reveal striking differences in the binding modes of EOL and DEET, identify dual ligand binding/release pathways (solvent-facing and membrane-facing), and pinpoint two critical molecular determinants: W158 as a central gating residue regulating ligand dissociation, and a proline-disrupted S4 helix that modulates ligand specificity via water-mediated hydrogen bonding with DEET. Thermodynamic and kinetic calculations further demonstrate that DEET binds MhOR5 substantially more stably (-11.2 kcal/mol vs. -8.4 kcal/mol for EOL) and associates marginally faster, while EOL dissociates two to three orders of magnitude more rapidly. Collectively, these findings elucidate the atomic-level mechanisms of MhOR5s ligand recognition and gating, providing a foundational framework for understanding insect OR function.