Abstract
This study employed a combined computational and experimental approach to investigate the molecular recognition mechanism of 4-methylphenol by human olfactory receptor hOR9Q2. The strategy integrated molecular docking using BIOVIA Discovery Studio, structural modeling of hOR9Q2 based on the AlphaFold2-predicted, molecular dynamics simulations with GROMACS software employing the AMBER14SB force field, and systematic site-directed mutagenesis validation. Computational simulations revealed that the binding cavity formed by transmembrane domains TM3, TM5, and TM6 serves as the key interaction region, with van der Waals, hydrophobic, and Pi-sulfur interactions driving stable binding (ΔG = -40.173 ± 0.34 kJ/mol). Functional characterization identified six critical residues (Cys112, Val158, Met207, Phe251, Leu255, and Tyr259) as essential for receptor activation, while mutations at Ile71 and Ala108 resulted in partial functional impairment. This study reveals the structural basis for hOR9Q2's selective response to 4-methylphenol, while establishing a computational-experimental framework for precisely locating functional sites on olfactory receptors. These findings elucidate the molecular mechanism of odorant recognition and provide insights for developing odorant prediction models and designing specific olfactory receptor modulators.