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 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.
Highlights
Phosphoenzyme (E1P) that can react with ADP to regenerate ATP
E1P formation results in Ca2ϩ occlusion at the transport sites (E1PCa2)
We have recently developed an E1Ca21⁄7BeF3Ϫ complex as a stable analog of E1PCa21⁄7Mg2ϩ (E1PCa2 with bound Mg2ϩ at the catalytic site) [27]
Summary
Mutagenesis and Expression—The pMT2 expression vector [30] carrying the mutant rabbit SERCA1a cDNA with four glycine residues inserted between Gly and Lys (4Gi-46/47) was constructed as described previously [14]. Expression levels of wild type SERCA1a and the mutants were 2–3% that of total microsomal proteins. Metal Fluoride Treatment—Microsomes expressing the wild type or 4Gi-46/47 were treated at 25 °C for 30 min with BeFx, AlFx, and MgF42Ϫ as described previously [14, 23,24,25, 27, 33,34,35,36] and in the legends for Figs. The amount of Ca2ϩ bound to the transport sites of EP in the expressed SERCA1a was obtained by subtracting the amount of nonspecific Ca2ϩ-binding, which was determined as described in the legends for Figs. Limited Proteolysis and Western Blot Analysis—Major intermediates of the Ca2ϩ-ATPase and their stable analogs were produced and subjected to structural analysis by limited proteolysis with trypsin and proteinase K (prtK) as described in the legends for supplemental Figs. Three-dimensional models of the enzyme were reproduced by the program VMD [41]
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