Stable structural analog of Ca2+-ATPase ADP-insensitive phosphoenzyme with occluded Ca2+ formed by elongation of A-domain/M1'-linker and beryllium fluoride binding.

ABSTRACT: We have developed a stable analog for the ADP-insensitive phosphoenzyme intermediate with two occluded Ca(2+) at the transport sites (E2PCa(2)) of sarcoplasmic reticulum Ca(2+)-ATPase. This is normally a transient intermediate state during phosphoenzyme isomerization from the ADP-sensitive to ADP-insensitive form and Ca(2+) 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 (BeF(x)) to the phosphorylation site of the Ca(2+)-bound ATPase (E1Ca(2)). The complex E2Ca(2)xBeF(3)(-) was also produced by lumenal Ca(2+) binding to E2xBeF(3)(-) (E2P ground state a

SUBMITTER: Daiho T 

PROVIDER: S-EPMC2915690 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature