Identification Of The Crystal Structure Of Diamondback Moth Ryanodine Receptor N-Terminal Domain And The Virtual Screening Of Diamide Lead Compounds

Ryanodine receptors(RyRs)are large calcium-release channels located in sarcoplasmic reticulum membrane.They play a central role in excitation-contraction coupling of muscle cells.Three novel commercialized diamide insecticides targeting pest RyRs generate worldwide annual sales of over 2 billion U.S.dollars due to their characteristics of high selectively and efficiency and low toxicity.To date,there has been no structural study of RyRs in insects,which hinders ourunderstanding of the molecular mechanism of insecticidal action and the possibility of rationally modified insecticides.In my Master’s research,I analyzed the high-resolution(2.84 (?))crystal structure of the first insect RyR,taken from the important agricultural pest.known as the diamondback moth(DBM).This study revealed the protein structure of the structure domain of the RyR N-terminal.The N-terminal structure plays an important role in the switch regulation of the RyR channel.The crystal structure showed that the N-terminal domain structure of Plutella xylostella is similar to the corresponding domain of mammalian RyRs.Similar to its mammalian homologue,the DBM RyR N-terminal domain(NTD)is a trefoil structure consisting of twelve beta-folding and one flanking alpha helix;two regions in the NTD interacting with neighboring domains showed distinct conformations in the DBM RyRs relative to those in mammals.Using homology modeling and molecular dynamics simulation,we created a structural model of the N-terminal three domains showing two unique binding pockets that potentially could be targeted by species-specific insecticides.In addition,this will provide an important template for the virtual screening of novel insecticides in the next step.A thermal melt experiment showed that the stability of the DBM RyR NTD was higher than that of mammalian RyRs,probably due to a stable intradomain disulfide bond observed in the crystal structure.Previously,the DBM NTD was shown to be one of the two critical regions interacting with the insecticide flubendiamide,but isothermal titration calorimetry experiments negated the DBM NTD alone as a major binding site for flubendiamide.At the same time,we used structural homology modeling in the computational software Schr?dinger to construct the DBM RyR transmembrane domain.We obtained the wild type and different resistance types(I4790M,G4946E,and I4790M/G4946E)of the RyR transmembrane domain structure.Based on the model results,the four alpha helices in the transmembrane domain are likely to form a binding site of the diamide insecticides.In addition,the two mutations I4790M and G4946E are in the pocket,and they are very closely positioned in three-dimensional space.Analyzing the structures,we found the G4946E mutation has a far greater influence on the binding with diamide insecticides than does the I4790M mutation,and this result is consistent with the results of previous functional experiments.Then,we performed docking between the three kinds of commercialized diamide insecticides and the wild-type and different resistance types of transmembrane domain structures,and the molecular docking experiments confirmed our hypothesis that the binding between the wild-type of the DBM RyR transmembrane domains and diamide insecticides is stronger than that for the mutation types(G4946E,I4790M,and G4946E/I4790M)and that the binding between the G4946E DBM RyR transmembrane domains and the diamide insecticides is weaker than that for the I4790M mutation type.Finally,we used the Schr?dinger software to modify the chlorantraniliprole rationally and obtained the diamide derivatives.Then,we used the molecular docking experiment to virtually screen high-selectivity diamide derivatives.The insecticidal activity of these compounds will be tested by bioactivity tests at the protein,cell and insect levels.