Ligand binding properties of three odorant-binding proteins in striped flea beetle Phyllotreta striolata towards two phthalate esters.

Odorant-binding proteins (OBPs) initiate insect olfactory perception and mediate specific binding and selection of odorants via uncertain binding mechanisms. We characterized the binding characteristics of four OBPs from the striped flea beetle Phyllotreta striolata (SFB), a major cruciferous crop pest. Tissue expression analysis revealed that the two ABPII OBPs (PstrOBP12 and PstrOBP19) were highly expressed mainly in the antenna, whereas the two minus-C OBPs (PstrOBP13 and PstrOBP16) showed a broad expression pattern. Competitive binding assays of cruciferous plant volatiles showed that PstrOBP12, PstrOBP16 and PstrOBP19 had very strong binding capacities for only two phthalate esters (Ki  < 20 μM), and PstrOBP13 specifically bound to four aromatic volatiles (Ki  < 11 μM). Fluorescence quenching assays displayed that two phthalate esters bound to three PstrOBPs via different quenching mechanisms. PstrOBP12/PstrOBP16-diisobutyl phthalate and PstrOBP19-bis(6-methylheptyl) phthalate followed static quenching, while PstrOBP12/PstrOBP16-bis(6-methylheptyl) phthalate and PstrOBP19-diisobutyl phthalate followed dynamic quenching. Homology modelling and molecular docking displayed that PstrOBP12-diisobutyl phthalate was driven by H-bonding and van der Waals interactions, while PstrOBP16-diisobutyl phthalate and PstrOBP19-bis(6-methylheptyl) phthalate followed hydrophobic interactions. Finally, behavioural activity analysis demonstrated that phthalate esters exhibited different behavioural activities of SFB at different doses, with low doses attracting and high doses repelling. Overall, we thus revealed the different binding properties of the three PstrOBPs to two phthalate esters, which was beneficial in shedding light on the ligand-binding mechanisms of OBPs.