In this study, we annotated 49 odorant-binding proteins (OBPs) in Papilio xuthus, with four novel genes and seven improved sequences. Expression profiles identified numerous OBPs in antennae or reproductive tissues. Using two antenna-enriched general OBPs (PxutGOBP1 and PxutGOBP2) as targets, we screened three key compounds by a reverse chemical ecology strategy. Of these, an oviposition stimulant vicenin-2 could strongly interact with PxutGOBP1, representing a dissociation constant (Ki) value of 10.34 ± 0.07 μM. Molecular simulations and site-directed mutagenesis revealed the importance of His66, Thr73, and Phe118 between PxutGOBP1 and vicenin-2 interactions. Two other compounds, an ordinary floral scent β-ionone and a widely used insecticide chlorpyrifos, exhibited high affinities to PxutGOBPs (Ki < 13 μM). Furthermore, two mutations His66Ala and Thr73Ala of PxutGOBP1 significantly reduced the binding to chlorpyrifos. Our study provides insights into the putative roles of PxutGOBPs in odorant perception and identifies key binding sites of PxutGOBP1 to vicenin-2 and chlorpyrifos.
Keywords: binding property; gene structure; general odorant-binding protein; molecular simulation; site-directed mutagenesis; swallowtail butterfly; vicenin-2.