Analysis Of Crystal Structures Of The Ryanodine Receptor SPRY1 And SPRY2 Domains From The Diamondback Moth,plutella Xylostella

Ryanodine receptors(RyRs)are the largest calcium ion release channels located on the sarcoplasmic membrane.They play an important role in the excitation-contraction coupling of muscle cells.Five commercialized diamide insecticides targeting RyRs are a major class of pesticides used to control a wide range of agricultural pests,generating worldwide sales over 2 billion U.S.dollars annually,but their efficacies have been reduced dramatically by the recent emerging resistance mutations.There is a pressing need to develop novel insecticides targeting distinct and novel binding sites within insect RyRs to overcome the resistance crisis,however,the limited structural information on insect RyRs is a major roadblock to our understanding of their molecular mechanisms.RyRs are large ion channels composed of four identical subunits with molecular size over 2.2 MDa,and each subunit consists of about 20 functional domains.In this paper,we chose the diamondback moth(DBM),Plutella xylostella,a destructive agricultural pest worldwide that has developed resistance to all classes of insecticide as the research material.We solved the crystal structures of RyR SPRY1 and SPRY2 by X-ray protein crystallography technology,with resolution of 1.85 ? and2.06 ?,respectively.After analyzing the structure of DBM RyR SPRY1 and SPRY2,found that although the overall fold made of antiparallel β-sheets are similar to its mammalian homolog,there are several significant structural differences lie in the interface of adjacent domains.In order to future study the function of SPRY domain in the full-length insect RyR,and its interaction with other domain,we use the Computational biology software Schrodinger obtain the full-length RyR homology model structure of DBM,then the crystal structures were used to replace SPRY1 and SPRY2.The roles of SPRY1 and SPRY2 were analyzed in the full length RyR.It was found that SPRY1 interacts with FK506 binding protein(FKBP)that a regulatory protein of RyR.CO-IP experiments shown that the interaction between SPRY1 and FKBP is insect-specific.Therefore,the interface between SPRY1 and FKBP in DBM might be the potential targeting sites for green pesticides that disrupting the normal interaction between them to get the effect of controlling pests.The analysis of SPRY2 shown that two specific-loops interact with its intersubunit BSol,and the interface of SPRY2-BSol will shift by 2.3 ? during the opening and closing of the channel.Interestingly,the previously identified disease mutations of BSol domain also located on this interface,indicating the important role of the SPRY2-BSol interface in channel gating.Another loop interacts with its intra-subunit SPRY3 domian,and SPRY3 has potential interaction with the important phosphorylation domain Repeat34.When domain SPRY3 stays together with SPRY2 upon channel gating,Repeat34 domain undergoes 5.1 ? displacement away from SPRY2-SPRY3.So,the surface between SPRY2-BSol and SPRY2/SPRY3-Repeat34 are expected to be potential targeting sites for insect-specific insecticides.