Structural And Functional Study Of Ryanodine Receptors And Pharmacological Study Of Targeting Compounds

Ryanodine Receptors(RyRs)are calcium-release channels expressed in the endoplasmic reticulum(ER)and sarcoplasmic reticulum(SR)membrane.They play a central role in excitation-contraction coupling of muscles.RyRs are primary targets for insecticides critical in pest control,and human drugs to treat myopathies.Therefore,the understanding of the molecular mechanism of RyRs and how they are regulated by modulatory compouds is of great value on the theory and practice.Insect RyRs are the target of diamide insecticides,which are among the top-sellers globally.However,no Ry R structure in complex with any diamide insecticides has been resolved,which hinders the mechanistic understanding of existing diamide insecticides and the rational design of new diamide insecticides.In recent years,mutations in pest RyRs have been discovered in many countries around the world,causing thousand-fold resistance to diamide insecticides,posing a threat on food safety in many countries.In my Ph.D.project,I successfully solved cryo-electron microscopy(EM)structures of diamide insecticides in complex with mammalian Ry R at resolution up to 3.8 (?).Chlorantraniliprole(CHL),a representative diamide insecticide,binds to the pocket located in the pseudo-voltage-sensor domain(p VSD)of Ry R.The binding of CHL induces a conformational change that affects the S4-S5 linker and triggers channel opening.This leads to uncontrolled release of calcium ions from ER store,which subsequently causes muscle paralysis,and ultimately the death of treated larvae.The key residues from the binding site involved in diamide binding are further validated by the data from cellular,genome-edited Drosophila,and in silico studies,which reveals the molecular mechanism for their high species-selectivity against the Lepidoptera and the Coleoptera.Our cryo-EM structure shows although the four known resistance mutations are all located surrounding the insecticide binding pocket,they cause resistance via two different mechanisms,either by increasing the size of residue side chain which induces steric hindrance or by reducing the contacts between CHL and key coordinating residues which weakens the binding.We also predicted the mechanism of action of another important diamide insecticide,flubendiamide(FLU),through homology modeling.FLU shares a similar binding site with CHL,but their binding modes are different.Up to now,more than 500 mutations in human RyRs are associated with inherited myopathies,including catecholaminergic polymorphic ventricular tachycardia(CPVT),idiopathic ventricular fibrillation(IVF),malignant hyperthermia(MH),and central core disease(CCD).So far,there is no effective drugs to treat RyRs-related myopathies.I also investigated the molecular mechanism of Ry R-related myopathies and explored new potential therapeutic methods.Our collaborators from France found a mutation in SPRY1 domain of Ry R2(cardiac isoform)is strongly correlated with the phenotype of IFV in a patient.We solved a high resolution crystal structure of mouse Ry R2-SPRY1 domain containing this IFV mutation,I784 F.The structure combining with the results from other biophysical methods reveal the impacts of the mutation on the structure,function,and thermal stability of SPRY1 domain.Based on that,I propose that I784 F alters channel gating by affecting inter-domain interactions in Ry R2.In addition,I also propose a strategy to design new therapeutic molecules to treat Ry R-related myopathies by targeting the diamide-binding site.The potential therapeutic effects have been confirmed on two CCD models.Furthermore,high-throughput screening of diamide derivatives was carried out against rabbit Ry R1(isoform of skeletal muscle)containing CCD mutations,from which 14 candidate compounds with potential therapeutic effects have been successfully obtained.In summary,my findings provide not only accurate structural templates,but also important theoretical basis for the development of highly selective insecticides and human drugs targeting insect and human RyRs,respectively,which is of important theoretical and practical value.