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 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 analog) of the elongated linker mutant. The complex was stable for at least 1 week at 25 degrees C. Only BeF(x), but not AlF(x) or MgF(x), 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.
Department of Biochemistry, Asahikawa Medical University, Asahikawa 078-8510, Japan. daiho@asahikawamed.ac.jp
Takashi
T
Daiho
Stefania
S
Danko
Kazuo
K
Yamasaki
Hiroshi
H
Suzuki
D000244:Adenosine Diphosphate / Q000737:chemistry*; D000818:Animals; D001608:Beryllium / Q000737:chemistry; D019556:COS Cells; D002118:Calcium / Q000737:chemistry; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D005459:Fluorides / Q000737:chemistry; D007700:Kinetics; D008861:Microsomes / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry
D010710:Phosphates; C035982:beryllium fluoride; D000244:Adenosine Diphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D001608:Beryllium; D005459:Fluorides; D002118:Calcium