Binding Characterization Andmolecular Modelingof Odorant Binding Protein AquaOBP4 In Ambrostoma Quadriimpressum

Ambrostoma quadriimpressum is one of the major forest pest feeds exclusively on elms,which causes great damage to wood industry and the afforestation of city,however,the knowledge of the molecular basis in olfactory sensing from A.quadriimpressum was comparatively lacking.Based on our previous RNA sequencing data,the most abundent and antennal specific OBP in A.quadriimpressum,AquaOBP4,was expressed and purified.In order to explore the binding mode and establish a reliable prediction and screening model,we conducted fluorescence binding assays and molecular modeling.the selectivity of AquiOBP4 was investigated by screening against a panel of 40 habitat relevent compounds.Five ligands showed binding to AquaOBP4,which were Dibutyl phthalate,Diisobutyl phthalate,β-Ionone,cis-3-Hexenyl benzoate and Cinnamaldehyde.Based on these fluorescence binding results,a homologous model of AquaOBP4 was established,as a result,AquaOBP4 includes six α-helices and two main binding sites,Site 1 and Site 2,docking results showed four binding ligands Dibutyl phthalate,Diisobutyl phthalate,β-Ionone,and cis-3-Hexenyl benzoate were found to be located in the same Site1 while another binding ligand Cinnamaldehyde was situated on the other Site2,further 2 new ligands were also docking into the model,the fluorescence binding results of these two compound were highly consistent with the binding mode and binding affinity predicted by our model,which concluded that the binding model of AquaOBP4 showed a good prediction ability to evaluate the binding ability of different ligand in our study.Besides,this model helped us identify a best ligands towards AquaOBP4 ever tested,and the possible biological function of AquaOBP4 is briefly discussed that AquaOBP4 is more likely to be linked with the foraging behaviour of A.quadriimpressum.The resultsof binding assaysat PH=5 showed,the lowerpH will decreased binding affinity of AquaOBP4,which might caused by theconformation change of AquaOBP4.This study give us a better understand of the structure and binding mode of insect OBPs and provide us a new reliable tool for future large-scale compounds screening by computational chemistry methods.Futher exploration on the behavioural effect of these identified active odorants is needed,which will guide us for a better pest management on A.quadriimpressum.