Interaction Mechanism Between MaltOBP12 And Odorants In Monochamus Alternatus Hope

Insect behavior regulation is currently an important way to achieve green pest control.Trapping and killing by attractants are one of the important technical measures in the prevention and control of Monochamus alternatus Hope,a primary insect vector of the major forest disease pine wood nematode.However,there are currently problems with the single active ingredient and poor stability of attractants of M.alternatus,and the development of efficient attractants of M.alternatus is still an important research direction.Odorant-binding proteins（OBPs）,the olfactory proteins in insect olfactory system,are considered to be an indispensable class of macromolecules for maintaining the normal odor detection of insects,and have been used to screen behaviourally active compounds of insects.Previously,28 MaltOBPs had been identified from the antenna transcriptome of M.alternatus,ligand-binding characteristics of MaltOBPs and their functions in olfactory reception of M.alternatus were investigated.Based on this,a behaviourally active compound,camphene,was screened.However,due to the unclear interaction mechanism between MaltOBPs and ligands,the application of MaltOBPs in screening behaviourally active compounds from M.alternatus has been limited to some extent.Therefore,MaltObp12 that was specifically and highly expressed in the antenna of M.alternatus,was selected as the target gene.On the basis of cloning,analysis of expression pattern and signal peptide activity,MaltOBP12 expressing in Escherichia coli and purifying by affinity chromatography,binding affinities of 16 pine volatiles to MaltOBP12 was determined in vitro by fluorescence competitive binding assay.The form and mode of interactions between MaltOBP12 and odorants were explored by protein interactions technology,protein comparative modeling,molecular docking,site-directed mutagenesis and ligand-binding assay,with a view to clarifying the interaction mechanism between MaltOBP12and odorants,and providing theoretical support for the efficient screening and structural optimization of behaviourally active compounds of M.alternatus.The main findings are summarized as follows:1.MaltObp12 cloning,analysis of expression pattern and activity of protein’s signal peptideThe CDS sequences of full-length MaltObp12（Gen Bank:KF977565.1）was accurately cloned from M.alternatus,and spatiotemporal expression mode of this gene were analyzed.It was found that MaltObp12 was specifically and highly expressed in the antenna of male M.alternatus,and reached a peak in the antenna of M.alternatus at 5 days after eclosion.Amino acid sequence analysis found that MaltOBP12 has 144 amino acids,15.92 k Da molecular weight and 4.5 isoelectric point in prediction with a putative signal peptide in the 1-21 amino acid sequence region;the secretion function of MaltOBP12’s signal peptide was proved through secretion experiments of yeast strain YTK12 and Drosophila S2 cells.2.Ligand-binding characteristics of MaltOBP12Using Escherichia coli system to express MaltOBP12 and affinity chromatography to purify the recombinant MaltOBP12,the conformation of purified MaltOBP12 was evaluated by binding assay with the fluorescent probe 1-NPN,and the results showed that this protein could be used for subsequent ligand-binding assay.Binding affinities of MaltOBP12 to 16 pine volatiles under two p H conditions were detected by ligand-binding experiment.The results displayed that p H had a significant impact on ligand-binding of MaltOBP12.At p H=7.4,MaltOBP12 can bind 11 out of 16 tested pine volatiles.Among them,myrcene,（+）-α-longipinene,butylated hydroxytoluene had strong binding affinity to MaltOBP12;（+）-limonene oxide,（1R）-（+）-α-pinene,α-terpinene,S-（-）-limonene,（±）-camphor,3-carene,4-propyltoluene,（+）-fenchone had weak binding affinity with MaltOBP12;MaltOBP12 had no binding affinity to（R）-（+）-limonene,β-caryophyllene,camphene,（+）-β-pinene.However,compared to neutral conditions,at p H=5.0,binding affinities of（+）-limonene oxide,α-terpinene,3-carene,（+）-β-pinene,（+）-fenchone,（1R）-（+）-α-pinene with MaltOBP12 decreased significantly;using the far-uv circular dichroism scanning to further verify the effect of p H on secondary structure of MaltOBP12,it was found that the effect of p H on MaltOBP12-ligands interactions was not achieved by changing the secondary structure of MaltOBP12.3.Cooperative interactions between MaltOBP12 and other MaltOBPScreening and analysis of MaltObps were conducted with reference to the gradient expression pattern of Obps in antennae of M.alternatus.MaltObp1,MaltObp3,MaltObp4,MaltObp10,MaltObp21,which were similar to the gradient expression pattern in antennae of MaltObp12,were selected as candidates for interactions validation;detecting interactions between MaltOBP12 and candidate by yeast two-hybrid technology,it was found that only MaltOBP1 could interact with MaltOBP12;The binding assay of mixed protein to the fluorescent probe 1-NPN and GST pull-down further proved that MaltOBP1and MaltOBP12 could not interact directly in vitro,and there were no cooperative interactions between them.Therefore,MaltOBP12 interacts with ligands alone.4.The binding Mechanism between MaltOBP12 and odorantsUsing methods of homology modeling,molecular docking,architecture of MaltOBP12’s binding cavity was analyzed,and it was found that this binding cavity was mainly composed of large aromatic and hydrophobic residues,with a molecular volume of559.433?³.The effects of aromatic residues in the binding cavity on interactions between MaltOBP12 and ligands were analyzed through site-directed mutagenesis,ligand-binding assay,and principal coordinate analysis.Principal coordinate analysis showed that the binding potency of MaltOBP12^Y50A,MaltOBP12^F109A,MaltOBP12^Y112A or MaltOBP12^F122A was significantly separated from the wild-type protein,meaning 4aromatic residues play a vital role in MaltOBP12’s odorant-binding.However,4 aromatic residues had different effects on interactions between ligands and MaltOBP12,alanine substitution of Phe109 caused more than a 2-fold change in the binding affinity of all tested ligands to protein,with the highest accumulated value of binding affinity change.Therefore,Phe109 is one of the most critical residues in the binding cavity of MaltOBP12,and most of ligands interact with it.In addition,in the binding assay of MaltOBP12^F109A and MaltOBP12^V15M to the large volume ligand butylated hydroxytoluene,it was found that the size of the side chain of Phe109 and Val15 in the binding pocket of MaltOBP12 had a significant impact on the binding of MaltOBP12 to butylated hydroxytoluene.Finally,the mode of interactions between MaltOBP12 and ligands was analyzed by combining ligand-protein interactions analysis,site-directed mutagenesis of nonpolar residue in binding cavity to polar residue,ligand-binding assay and linear regression analysis,these results support the conclusion that MaltOBP12 binds different odorants predominantly though non-directional hydrophobic interactions,which contribute to a flexible odorant-binding.In summary,this study analyzed the expression pattern,secretion characteristics,and ligand-binding characteristics of MaltOBP12,supporting the"classic"transporter function of MaltOBP12 in olfactory reception;clarified widely existing hydrophobic interactions between MaltOBP12 binding pocket and ligands.These results provide a theoretical basis for analyzing the interaction mechanism between OBPs and ligands,as well as the efficient screening and structural optimization of behaviourally active compounds of M.alternatus by OBPs.