Structural And Functional Studies Of Calcium Transporting Proteins In Sarcoplasmic Reticulum

Sarcoplasmic reticulum Ca²⁺-ATPase（SERCA）is a calcium uptake pump located in sarcoplastic reticulum（SR）membrane.It uptakes Ca²⁺from cytosol to SR lumen at the expense of ATP hydrolysis during muscle relaxation.The activity of SERCA is regulated by a single transmembrane protein called phospholamban（PLN）.The phosphorylation of PLN by c AMP-dependent protein kinase A（PKAc）can release its inhibition of SERCA and enhances SERCA activity.There are several mutations identified on PLN,including R9C,R9H,R9L andΔR14,which are related to dilated cardiomyopathy（DCM）and heart failure（HF）.However,the molecular mechanism of how PKAc phosphorylation regulates wild type and mutant PLNs remains unclear due to the lack of complex structures.Here,we present three crystal structures of catalytic domain of PKAc in complex with WT PLN,PLN R9C and PLN A11E,respectively.The binding affinity,thermostability and phosphorylation level are measured by surface plasmon resonance（SPR）,thermal stability assay and ADP-glow assay,respectively.Our experimental results suggested that these mutations share a common disease mechanism of reducing the binding affinity to PKAc and subsequently decrease the phosphorylation level of PLN.Another-regulin（ALN）which is a newly identified more ubiquitous SERCA-regulatory peptide was also investigated and found to share a similar mechanisim of regulation by PKAc.Ryanodine receptors（RyRs）are large calcium-release channels located in SR for release of Ca²⁺from SR to cytoplasm.They play a central role in excitation contraction coupling.Diamine insecticides,which mainly target insect RyRs,have become one of the most widely used pesticide families since introduction to market recently.Despite extensive research,the molecular mechanism of action of diamide insecticides is still unclear,hindering our understanding of the molecular mechanisms of RyR-targeting insecticides and the resistance of pests.Here the crystal structure for the N-terminal domain（NTD）of RyR is reported at the resolution of 2.84?from P.xylostella,which is a destructive pest infesting the Brassica food crops all over the world.Compared with mammalian RyR,there are two regions in this domain that show unique structural characteristics.We identified two potential species-specific pockets for insecticides binding,based on the structure model of DBM RyR ABC created by molecular dynamics simulation and homologous modeling.The stability of DBM RyR NTD was higher than mammalian RyRs based on thermal melt experiment,probably due to a stable intra-domain disulfide bond observed in the crystal structure.Previous studies showed that DBM NTD is one of the two key regions interacting with the insecticide flubendiamide,but isothermal titration thermal experiment showed that DBM NTD alone cannot form the main binding site of flubendiamide.