Stable Structural Analog of Ca^<2+>-ATPase ADP-insensitive Phosphoenzyme with Occluded Ca^<2+> Formed by Elongation of A-domain/M1′-linker and Beryllium Fluoride Binding
著者
大保, 貴嗣
(Daiho, Takashi)
Danko, Stefania
Yamasaki, Kazuo
Suzuki, Hiroshi
上位タイトル
Journal of Biological Chemistry
Vol.285,
No.32
(2010.
8)
,p.24538-
24547
識別番号
ISSN
0021-9258
DOI
10.1074/jbc.M110.144535
抄録
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 (BeFx) to the phosphorylation site of the Ca^<2+>-bound ATPase (E1Ca_2). The complex E2Ca_2·BeF_3− was also produced by lumenal Ca^<2+> binding to E2·BeF_3− (E2P ground state analog) of the elongated linker mutant. The complex was stable for at least 1 week at 25℃. Only BeFx, but not AlFx or MgFx, produced the E2PCa_2 structural analog. Complex formation required binding of Mg^<2+>, Mn^<2+>, or Ca^<2+> at the catalytic Mg^<2+> 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 Ca^<2+> 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 Mg^<2+> at the catalytic site in these processes.
