Study On Cydia Pomonella Pheromone Binding Proteins And Screening Of Potentially Active Semiochemicals

As an economic pest, the codling moth Cydia pomonella could cause great harm to fruit production throughout the world.Even though much attention is paid to the behavioral disruption of codling moth, due progresses have not been made. Up till now, only the sex pheromone E,E-8,10-dodecadienol is commercially produced. The screening of active odorants traditionally depends on the behavioral action of insects, not only is this method labor-consuming, but also limits the range and number of candidate molecules. By reference to the strategies of drug development, the notion of “Reverse Chemical Ecology”was proposed. According to this conception, odorants are screened by detecting their affinity to olfaction related proteins. This involves the interaction between odorants and proteins which can be simulated by computational methods, thus allowing the high-throughput screening of odorants.Of all the known proteins related to insect olfaction, OBPs are the best tudied, and are suitable to odorants searching. Based on the biochemical characterization and functional analysis of PBPs from codling moth, we developed novel methods to screen semiochemicals efficiently by combining computational technology and the idea of “Reverse Chemical Ecology”,the key interactions and key amino acids between odorants and CpomPBPs were analyzed as well.(1) Structural insights into CpomPBP2 mediated screening of odorantsIn the present study, 31 candidate odorants composed of pear/apple semiochemicals and Cydia pomonella pheromones were prepared. Virtual screening and in vitro binding assay were utilized in combination to study their affinity to CpomPBP2, the agreement of the ligands affinity sequence obtained from the two dependent experiments suggested the rationality of the method we applied to screen potentially active odorants. To better understand the interactions between CpomPBP2 and ligands, the analysis of binding mode and binding free energy were performed as well. The hydrophobic interaction and hydrogen bond were reported to be two key factors in determining the ligands affinity. The roles of CpomPBP2 C-terminal tail were also studied, by comparing the affinity changes of CpomPBP2 and TPBP2(C-terminus truncated CpomPBP2), we pointed out that the C-terminus of CpomPBP2 not only impeded the binding of ligands under low pH, but also affected the “uploading” of ligands.(2) Molecular characterization and functional analysis of CpomPBP1The full-length ORF of CpomPBP1, one typical lepidopteran PBP, was cloned and analyzed by RACE PCR and other molecular biology methods. By combining the methods like RT-PCR, Western blot and immunofluorescence assay, the tissue distribution of CpomPBP1 were identified to be antenna-specific protein at the gene and protein level. 16 semiochemicals were subjected to competitive binding assay and 3 of them exhibited relatively high affinity to CpomPBP1. The molecule similarity of the screened odorants could be regarded as a reflection of the olfaction sensitivity. To study the role of the C-terminus of CpomPBP1, we compared the affinity changes of CpomPBP1 and TPBP1(C-terminus truncated CpomPBP1) under different pH. It was found that the affinity of TPBP1 became insensitive to pH variation, even though its binding ability was not affected by C-terminus deletion. It could be concluded that the C-terminus of CpomPBP1 tended to block the binding of ligands under low pH.(3) Key amino acids associated with the interaction between CpomPBP1 and CodlemoneIn this study, homology modelling and molecular docking were applied to construct the 3D structures of CpomPBP1 and CpomPBP1/Codlemone complex, the stability of these two constructed structures were identified by molecular dynamics simulation and binding free energy calculation. To better understand the interaction between CpomPBP1 and Codlemone, the contributions of each free energy item were analyzed. We found that the hydrophobic interaction and the electrostatic interaction were the two most important energy contributors, whereas the hydrogen bond between –OH and the sidechain of Trp37 was vital to the stability of CpomPBP1/Codlemone complex. By decomposing energy contribution of each amino acid, 6 amino acids(Phe12, Phe36, Trp37, Ile52, Ile94 and Phe118), whose sidechain energy contributions were above 0.5kcal/mol, were finally subjected to computational alanine mutagenesis scanning and site-directed mutagenesis. Phe12 and Trp37 were identified as the key amino acids in the interaction between CpomPBP1 and Codlemone.