Mechanism of Ca²⁺ Pump as Revealed by Mutations, Development of Stable Analogs of Phosphorylated Intermediates, and Their Structural Analyses

Abstract

  Sarco(endo)plasmic reticulum Ca²⁺-ATPase is a representative member of P-type cation transporting ATPases and catalyzes Ca²⁺ transport coupled with ATP hydrolysis. The ATPase possesses three cytoplasmic domains (N, P, and A) and ten transmembrane helices (M1-M10). Ca²⁺ binding at the transport sites in the transmembrane domain activates the ATPase and then the catalytic aspartate is auto-phosphorylated to form the phosphorylated intermediate (EP). Structural and functional studies have shown that, during the isomerization of EP in the Ca²⁺ transport cycle, large motions of the three cytoplasmic domains take place, which then rearranges the transmembrane helices thereby destroying the Ca²⁺ binding sites, opening the lumenal gate, and thus releasing the Ca²⁺ into lumen. Stable structural analogues for the Ca²⁺-occluded and -released states of phosphorylated intermediates and for the transition and product states of the phosphorylation and dephosphorylation reactions were developed for biochemical and atomic-level structural studies to reveal the coupled changes in the catalytic and transport sites. Mutation studies identified the residues and structural regions essential for the structural changes and Ca²⁺ transport function. Genetic dysfunction of Ca²⁺-ATPase causes various isoform-specific diseases. In this manuscript, recent understanding of the Ca-ATPase will be reviewed.