The Binding And Releasing Mechanism Between Minus-C OBPs From Batocera Horsfieldi(Hope) And Ligands

Batocera horsfieldi(Hope)is widely distributed in China and is an important stem borer in forestry.B.horsfieldi has a sensitive olfactory system that is essential for host location and mating.Odorant binding proteins(OBPs)transport hydrophobic odorants from the environment to odorant receptors and play an important role in specific recognition of chemical substances.However,the binding and releasing mechanism of Bhor OBPs to ligands remains unclear.Here,we measured the binding characteristics with 17 host volatiles using a fluorescence competition-binding assay.Homology modeling,molecular docking and site-directed mutagenesis experiments were performed to investigate the 3D structure of Bhor OBPm2 and interactions between Bhor OBPm2 and volatiles.The main results are as follows:1.Cloning,expression and purification of Minus-C OBPs from B.horsfieldiRecombinant plasmids p GEX-6P-1-Bhor OBPm1,p ET30a-Bhor OBPm2 and p ET17b-Bhor OBPm4 were successfully constructed.p GEX-6P-1-Bhor OBPm1 mainly expressed in supernatant,we obtained purified Bhor OBPm1 through the GST Multi TrapTM 4B chromatography column.p ET30a-Bhor OBPm2 and p ET17b-Bhor OBPm4 mainly expressed in inclusion bodies,we obtained purified Bhor OBPm2 and Bhor OBPm4 through Ni sepharose high performanc column and DE52 affinity columns.2.The binding property of Minus-C OBPs from B.horsfieldi1-NPN was selected as a fluorescent probe,the binding properties of three Minus-C OBPs from B.horsfieldi with host plant volatiles were measured at neutral and acidic p H.Bhor OBPm1 showed weak binding affinities with salicylaldehyde at neutral p H and high binding affinities with(-)-verbenone at acidic p H.Bhor OBPm2 showed high binding affinities with three compounds at neutral p H,including trans-2-hexenal,hexanal and trans-2-hexen-1-ol.However,Bhor OBPm2 showed weak binding affinities with trans-2-hexen-1-ol,hexanal and 2-methyl-butanal at acidic p H.At neutral p H,Bhor OBPm4 had no binding affinities with all volatiles,but had weak binding affinities with(-)-limonene oxide,salicylaldehyde and cis-3-hexen-1-ol.3.Molecular modeling and docking of Bhor OBPm2 from B.horsfieldi2ERB was selected as the template of Bhor OBPm2 for homology modeling.Bhor OBPm2 has six ?-helices and two disulfide bridges according to the predicted 3D structure,which belongs to the minus-C insect OBPs family.Similar to Amel OBP1,Cqui OBP1 and Aaeg OBP1,residues Phe123 and Tyr50 in the C terminus of Bhor OBPm2 formed a hydrogen bond,acting like a wall over the binding pocket.The docking result of 56 ligands showed a tunnel formed by six ?-helices in the Bhor OBPm2 core,with two different solvent-exposed openings.Notably,most regions of the binding pocket were hydrophobic,except for the C-terminal wall.The ligands depend upon hydrophobic interactions inside the cavity.Residues Ile121 and Phe123 in the C-terminal region formed the only polar surfaces in the pocket.4.Site-directed mutagenesis and binding assays of Bhor OBPm2The fluorescence competition-binding assay showed that Bhor OBPm2 exhibited high binding affinity with chain volatiles and that ligands were selected based on chain length.Bhor OBPm2 exhibited better binding affinities with ligands at p H 7.4 relative to p H 5.0.Bhor OBPm2 which belonged Minus-C OBPs showed one continuous channel for chain ligands binding,similar to classic OBPs Agam OBP1 and Cqui OBP1.Unexpectedly,we observed a larger binding pocket for Bhor OBPm2 and broader specificity for ligands than classic OBPs due to the expansive flexibility of Bhor OBPm2 resulting from a lack of disulfide bonds.These findings suggested that Bhor OBPm2 might present an intermediate structure in the evolution of OBPs.Furthermore,we designed two mutant proteins C-ter113 and Y50 F to simulate and verify functions of the C-terminal region.The changes in binding affinity observed here indicated a novel action differing from that of the “lid” described in previous studies.