3-D Structure Prediction And Binding Characterization Of Odorant Binding Protein AlinOBP5in Adelphocoris Lineolatus

Olfaction plays an important role for Insects’ survival and reproduction.Odorant-binding proteins(OBPs) are considered to be one of the crucial proteins in insects olfaction. In this study, a novel OBPgene (AlinOBP5) in the alfalfa plant bug, Adelphocoris lineolatus, was cloned, expressed and a purifiedrecombinant protein was obtained. The binding affinities of AlinOBP5with mirids sex pheromoneanalogs and cotton volatiles were measured by fluorescence competitive binding assays. Binding sitesof AlinOBP5were predicted by3-dimensional structure modeling and molecular docking, and then theprediction was verified by sitedirected mutagenesis tests. The main results were as follow:1Sequence analysis showed that AlinOBP5was a classical OBP, and belonged to medium-chainOBPs. From the results of phylogenetic tree analysis, the AlinOBP5and mosquito might have thesame ancestral gene.2A purified recombinant AlinOBP5was obtained through prokaryotic expression, and the bindingproperties of this protein was investigated by the fluorescence competition binding assays. Theresults revealed that AlinOBP5cannot effectively bind with the miridae sex pheromone analogs. Ofthe cotton volatiles, nerolidol had the strongest binding affinity with AlinOBP5, ethyl laurate,β-ionone, β-caryophyllene,2,3-dimethylbenzoic acid and farnesol also showed certain affinities.From the results mentioned above, we suspect that AlinOBP5is a general odorant binding protein(GOBP) and can selectively bind with particular cotton volatiles and participate in thechemoreception process of A. lineolatus.3Based on the3-dimensional (3D) modeling and molecular docking, we find that the binding pocketof AlinOBP5was mainly formed by hydrophobic amino acids which may play some important rolesin AlinOBP5binding with nerolidol. The Lys74and Pro121might be the key binding sites.4Site-directed mutagenesis assays and followed measurement of binding affinities revealed thatLys74and Pro121play crucial roles in the ligands binding of AlinOBP5. The hydrophilic Lys74participates in specificity recognition of ligands, and Pro121takes part in the ligands binding andreleasing.