Key site residues of Cnaphalocrocis medinalis odorant-binding protein 13 CmedOBP13 involved in interacting with rice plant volatiles

Abstract

Odorant binding proteins (OBPs) play key roles in the insect olfactory system by assisting the neuronal response to hydrophobic odor molecules, understanding their interaction with ligands will facilitate the virtual screening of behaviorally active compounds in insects. Here, we successfully cloned and confirmed CmedOBP13, an antennae-biased OBP from the rice leaffolder Cnaphalocrocis medinalis, as a secreted protein. Recombinant CmedOBP13 was obtained using the Escherichia coli system, and its binding affinities to 35 volatile compounds emitted by rice plants and three sex pheromone components from female moths were assessed by a competitive binding assay. The results revealed that CmedOBP13 exhibited binding affinity to 23 rice volatiles, while no binding affinity for sex pheromone components. Furthermore, the stability of its conformation was found to be dependent on the pH level. Finally, the interaction between CmedOBP13 and odorants was predicted and confirmed by molecular docking and mutation functional assays, respectively. The combination of multiple hydrophobic residues created an adequate hydrophobic setting for ligands, and three residues (Glu13, Arg34, and Tyr115) might form hydrogen bonds with 15 odorants. Single mutations of Glu13, Arg34, Leu72, and Tyr115 diminished the binding affinities of CmedOBP13 to corresponding odorants, respectively. These findings provided valuable insights into the mode of action of CmedOBP13 interacting with the volatiles of rice plants and will guide the screening of behaviorally active compounds against C. medinalis in future.

Keywords: Cnaphalocrocis medinalis; Fluorescence competitive binding assays; Molecular docking; Odorant-binding proteins; Site-directed mutagenesis.