Binding Mechanism Analysis Of Odorant Binding Protein CmedOBP13 From Cnaphalocrocis Medinalis(Guenée)

The sensitive olfactory system plays an important role in perception various information objects in the surrounding environment in insects.It is widely believed that odorant binding proteins（OBPs）participate in the first step of olfactory recognition,which is of great significance.In recent years,based on the selective combination of OBPs and odorants,the reverse chemical ecology of screening potential behavioral active substances of insects with OBPs as molecular targets has been developed and applied.Cnaphalocrocis medialis is the important pests in rice.In the early stage of our laboratory,several OBPs genes were identified by transcriptome sequencing.However,the binding characteristics and mechanism between CmedOBPs and host volatiles are still unclear,which makes it difficult to screen the possible behavioral active substances of Cnaphalocrocis medialis.Therefore,this paper takes CmedOBP13,which is highly expressed in the antennae of Cnaphalocrocis medialis as the object to explore its binding characteristics and mechanism with rice volatiles,in order to provide a theoretical basis for the efficient screening of potential behavioral active substances of Cnaphalocrocis medialis.The main results are as follows:1.Gene cloning of CmedOBPs in Cnaphalocrocis medinalis and its secretory characteristicsTwo genes CmedOBP13（KP975124）and CmedOBP20（KP975131）were successfully cloned.After sequence analysis,we found that CmedOBP13 has six conserved cysteine sites,belonging to Classic OBPs and CmedOBP 20 had four conserved cysteine sites,belonging to Minus-c OBPs.The results of signal peptide prediction showed that CmedOBP13 and CmedOBP20 contained 22 and 16 signal peptides respectively.The secretion of the two genes verified by yeast signal peptide screening system.Obvious color reaction could be observed by TTC staining experiment,indicating that the signal peptides of CmedOBP13 and CmedOBP20 have secretory activity.In addition,the eukaryotic expression vector pcDNA3.1-Myc-CmedOBP13 and pcDNA3.1-Myc-CmedOBP20 were constructed and transformed into HEK293T cells.The CmedOBP20was successfully detected by WB in cell lysates and culture medium,proved that CmedOBP20 can be secreted outside the cell.2.Prokaryotic expression and purification and ligand binding characteristics of CmedOBPs from Cnaphalocrocis medinalisThe vectors pET32b-CmedOBP13 and pET-32b-CmedOBP20 were successfully constructed.Through prokaryotic expression system,the CmedOBP13 and CmedOBP20were expressed and purified,and obtained high purity recombinant proteins.The affinity of CmedOBP13 with 35 rice volatiles and 3 sex pheromones at pH 7.4and pH 5.0 was determined by fluorescence competitive binding assay.The results showed that CmedOBP13 could bind to 23 volatiles such as Hexanal,Nerolidol and 2-Tridecanone（K_i<50μM）.Among them,CmedOBP13 had strong binding characteristics with 6 kinds of rice volatiles:Hexanal,2-Heptanone,Nerolidol,2-Tridecanone,Methyl salicylate and Z-Farnesene（K_i<20μM）.The binding curve with 3 sex pheromones were abnormal and could not be determined.In addition,the binding ability of CmedOBP13 with ligands was stronger at pH 7.4 than at pH 5.0.CD analysis showed that the secondary structure of CmedOBP13 was mainlyα-helix.Compared with pH 7.4,the helix content increased at pH 5.0,but decreased sharply at pH3.0.After adding 23 ligands with binding ability to recombinant CmedOBP13,it was found that 15 substances such as（-）-Limonene,Cyclohexanol and Heptanone at pH 7.4 could change theα-helix content of the protein,in which the change of（-）-Limonene more obvious than that of other substances,and with the increase of ligand concentration,α-helix increased.Five substances such as Octene,Eicosane and Z-Farnese could change theα-helix content of the protein at pH 5.0.Other substances have little effect on the secondary structure of CmedOBP13.3.Binding mechanism between CmedOBP13 and ligand of Cnaphalocrocis medinalisUsing Drosophila melanogaster Dmel OBP28a（PDB No.:6QQ4.1.A）as the template,the spatial structure of CmedOBP13 was obtained by homologous modeling.The results showed that CmedOBP13 model had three disulfide bonds and sixα-helices.A hydrophobic binding cavity formed in the middle of the protein.The docking results showed that 73 Leucine residue（Leu 73）of CmedOBP13 could form hydrogen bonds with Linalool and Heptanol,and 116 Tyrosine residue（Tyr 116）could form hydrogen bonds with trans-2-Hexen-1-ol,2-Heptanone and Hexanal.According to the molecular docking results,we speculated that hydrogen bonding is the main interaction force between ligands and CmedOBP13.Based on this,we designed5 mutant proteins of CmedOBP13,CmedOBP13^L73A、CmedOBP13^L73W、CmedOBP13^Y116A、CmedOBP13^Y116F and CmedOBP13^L73A-Y116A.The recombinant pure proteins of 5 mutants were obtained through prokaryotic expression and purification.Through fluorescence competitive binding assay,we found that compared with the original protein CmedOBP13,the binding ability of CmedOBP13^L73A、CmedOBP13^Y116F and CmedOBP13^L73A-Y116A with Linalool,Heptanol,trans-2-Hexen-1-ol,2-Heptanone and Hexanal decreased greatly,and all of them lost their binding ability（K_i>50μM）.CmedOBP13^L73W still had binding ability with Linalool and Heptanol,and CmedOBP13^Y116A still had binding ability with trans-2-Hexen-1-ol,2-Heptanone and Hexanal.Furthermore,the fluorescence competitive binding of CmedOBP13^L73A、CmedOBP13^Y116F and CmedOBP13^L73A-Y116A and the remaining 18 substances combined with CmedOBP13 were carried out.It was found that except the binding curve with 2-Tridecanone and Z-Farnesene increased abnormally and could not be measured,the binding ability of CmedOBP13^L73A to all the remaining 16 substances was significantly lower than the CmedOBP13,and 13 substances such as Cyclohexanol,R-terpinene and 3-Pentanol lost their binding ability（K_i>50μM）,it still has weak binding ability with（-）-Limonene,Nerolidol and cis-3-Hexen-1-ol.Except the binding curve with Z-Farnesene increased abnormally and could not be measured,the binding ability of CmedOBP13^Y116F to 16odorants decreased greatly,and all of them lost their binding ability（K_i>50μM）,it still has binding ability with Ionone,and the change of binding ability is small compared with CmedOBP13.Except that the binding curve with Z-Farnesene increased abnormally and could not be measured,the binding ability of CmedOBP13^L73A-Y116A with 8 substances decreased,and lost the binding ability with 6 substances such as（-）-Limonene,2-Tridecanone and 3-Pentanol（K_i>50μM）,it still has binding ability with 9 substances such as Cyclohexanol,Nerolidol and R-terpinene.Compared with CmedOBP13,the binding ability of 6 substances such as Cyclohexanol,Nerolidol and cis-3-Hexen-1-ol remains unchanged,and the binding ability of 3 substances p-Isopropyltoluene,Eicosane and Ionone were stronger.It showed that Leu 73 and Tyr 116 amino acid residues are the key sites for the binding of CmedOBP13 to ligands.In conclusion,this study cloned CmedOBP13 and CmedOBP20 of Cnaphalocrocis medinalis,identified two proteins as secretory proteins.And screened 23 substances with potential behavioral activity with CmedOBP13 as the target,and identified Leu 73 and Tyr116 as the key sites for the binding of CmedOBP13 to ligands.The results provided a reference for further clarifying the binding mechanism between OBPs and ligands.