Key Amino Residues Determining Binding Activities of the Odorant Binding Protein AlucOBP22 to Two Host Plant Terpenoids of Apolygus lucorum.

Odorant binding proteins (OBPs) are considered to be highly expressed at antennae sensillum lymph and play crucial roles in detection of insect host plant volatiles. The polyphagous mirid bug Apolygus lucorum is one of a series of insect pests on many important agricultural crops that heavily rely on sophisticated olfaction to locate host plants. Previously, putative OBP genes and their tissue-related expression patterns in this pest species have been clarified. In this study, we characterized the ligand spectrum and the molecular binding mechanism of the antennae-biased AlucOBP22 to host plant volatiles of A. lucorum. Frist, the recombinant AlucOBP22 protein was constructed and purified, and its binding affinities to selected host plant volatiles were assessed. Two terpenoids, β-ionone and β-caryophyllene, could highly bind to AlucOBP22. Next, three-dimensional model prediction indicated that AlucOBP22 employed six α-helices to form a typical pocket for ligand accommodation. Molecular docking analysis suggested that both β-ionone and β-caryophyllene were located at the AlucOBP22 pocket with some hydrophobic amino acid residues close to the two chemicals, suggesting that hydrophobic interactions might be crucial for ligand-specific binding. Finally, site-directed mutagenesis combined with fluorescence binding assays revealed that mutants of five hydrophobic residues Leu5, Ile40, Met41, Val44, and Met45 displayed significantly decreased or completely abolished binding affinities to the two ligands. Our findings showed the specific binding characteristic of AlucOBP22 and suggested that hydrophobic residues and their hydrophobic interactions were involved in AlucOBP22 binding to terpenoids, which provided new insights into the molecular interaction mechanisms of hemipteran insect OBPs to host plant odors.