Binding Mechanism of PsauPBP3 to Sex Pheromones in Peridroma saucia: Insights from Computational and Experimental Approaches

The variegated cutworm <i>Peridroma saucia</i> Hübner, a recently emerged polyphagous pest in China's Huang-Huai River Basin, uses sex pheromones (Z)-11-hexadecenyl acetate (Z11-16: Ac) and (Z)-9-tetradecenyl acetate (Z9-14: Ac) for mate finding. Insect pheromone-binding proteins (PBPs) serve as the primary filter for detecting specific sex pheromones. Although comprehensive functional analyses of PBPs exist, their binding mechanisms remain poorly characterized. In this study, we elucidated the binding properties and mechanisms of PsauPBP3 in sex pheromone recognition by computational and experimental approaches. PsauPBP3, predominantly expressed in male <i>P. saucia</i> antennae, showed high binding affinity for both Z11-16: Ac and Z9-14: Ac, as demonstrated by binding-free-energy calculations and fluorescence binding assays. Molecular dynamics simulations and docking studies identified five key residues (Thr-10, Phe-13, Ile-53, Ile-95, and Phe-119) that consistently interact with these pheromones, indicating their critical role in ligand binding. Computational alanine scanning further demonstrated that all five residues act as binding determinants, with Phe-13 and Ile-95 making particularly significant contributions to ligand affinity. The results were further validated by site-directed mutagenesis and fluorescence binding assays. This work provides insights into the function and binding mechanisms of PBPs in sex pheromone recognition and supports the development of targeted mating disruption strategies for <i>P. saucia</i> control.