Stable Structural Analog of Ca2+-ATPase ADP-insensitive Phosphoenzyme with Occluded Ca2+ Formed by Elongation of A-domain/M1′-linker and Beryllium Fluoride Binding

We have developed a stable analog for the ADP-insensitive phosphoenzyme intermediate with two occluded Ca2+ at the transport sites (E2PCa(2)) of sarcoplasmic reticulum Ca2+-ATPase. This is normally a transient intermediate state during phosphoenzyme isomerization from the ADP-sensitive to ADP-insensitive form and Ca2+ deocclusion/ release to the lumen; E1PCa(2) -> E2PCa(2) -> E2P + 2Ca(2+). Stabilization was achieved by elongation of the Glu(40)-Ser(48) loop linking the Actuator domain and M1 (1st transmembrane helix) with four glycine insertions at Gly(46)/Lys(47) and by binding of beryllium fluoride (BeFx) to the phosphorylation site of the Ca2+-bound ATPase (E1Ca(2)). The complex E2Ca(2)center dot BeF3- was also produced by lumenal Ca2+ binding to E2 center dot BeF3- (E2P ground state analog) of the elongated linker mutant. The complex was stable for at least 1 week at 25 degrees C. Only BeFx, but not AlFx or MgFx, produced the E2PCa(2) structural analog. Complex formation required binding of Mg2+, Mn2+, or Ca2+ at the catalytic Mg2+ site. Results reveal that the phosphorylation product E1PCa(2) and the E2P ground state (but not the transition states) become competent to produce the E2PCa(2) transient state during forward and reverse phosphoenzyme isomerization. Thus, isomerization and lumenal Ca2+ release processes are strictly coupled with the formation of the acylphosphate covalent bond at the catalytic site. Results also demonstrate the critical structural roles of the Glu(40)-Ser(48) linker and of Mg2+ at the catalytic site in these processes.