Prediction To The Key Binding Site Of Odorant Binding Protein Of Holotrichia Oblita Faldermann (Coleoptera: Scarabaeida)

Insects use their antennaes to sense chemical signals(semiochemicals) that guide their interactionswithin and between species, including interactions with hosts such as plants and vertebrates. The scarabbeetle Holotrichia oblita Faldermann (Coleoptera:Scarabaeidae)possess predominance in theunderground pests in the northern parts of China and cause great economical loss because of there widehost plants and covert habitats. Environmentally friendly strategies for controlling Holotrichia oblitawould have new and broader prospect. It is a potential pest management measure by regulatingolfactory chemoreception to control target insect pests. In order to get a better understanding of therelationship between OBP structures and their ligands, homology modeling and molecular docking havebeen conducted on HoblOBP1and HoblOBP2with Hexyl benzoate in the present work. Based on theresults, site-directed mutagenesis and binding experiments were combined to describe binding sites ofHoblOBP1, and explore a ligand-binding mechanism. The primary results are as follows:(1) Using the bioinformatics, Essential properties of HoblOBP1and HoblOBP2were studyed includingisoelectric point, signal peptide, transmembrane domain, and the content of20kinds of amic acids.After homology modeling of HoblOBP1and HoblOBP2, it is found that the three-dimensionalstructures of two proteins have the same and diferent points, they both consist of six α helix andthree disulfide bridges which connect the helices; the hydrophobic pockets are both composed offive helix. However, with the different amino acid composition, the numbers and varieties of amicacids in the hydrophobic pockets are different.(2) To further explore the common characteristic of the HoblOBP1binding site，Hexyl benzoate waschosen to dock with the predicted HoblOBP1and HoblOBP2model. Based on the docking study,we found that van der Waals interactions and hydrophobic interactions are both important betweenthe HoblOBP1and HoblOBP2with Hexyl benzoate. The hydrogen bonds in the C terminus of theprotein were formed by intramolecular residues is crucial for the ligand-binding specificity. Finally,MET48、ILE80and TYR111are binding sites predicted for HoblOBP1While LEU73、THR54andVAL137are binding sites predicted for HoblOBP2.(3) Using site-directed mutagenesis, three residues (Met48, Ile80and Tyr111) of HoblOBP1werereplaced with alanine, generating the three mutants M48A, I80A and Y111A. After mutagenesis, theexpression levels of the mutated proteins did not appear to be largely altered relative to thewild-type. After purification by column chromatography and removed His-tag, the purity of theproteins was examined by SDS-PAGE, and single bands at16kDa, as expected for a monomer ofHoblOBP1.(4) The ability of differently organic compounds, Hexyl benzoate, β-ionone, Myrcene andCinnamaldehyde which had high binding affinities to HoblOBP1(Deng et al.,2012) wereinvestigated to displace1-NPN. The result is that Tyr111from HoblOBP1is a key binding site, Apossible explanation is that this compounds could not be recognized by the mutant and remain inthe binding cavity because of the loss of the intramolecular hydrogen bonding which act as a holder. Ile80A is a binding site but not the key one, perhaps because of the specific vander Waalsinteractions. Met48is not the binding site of HoblOBP1, it may just impact on the conformation ofHexyl benzoate.