Abstract
The functional importance of the length of the A/M1 linker (Glu(40)-Ser(48)) connecting the actuator domain and the first transmembrane helix of sarcoplasmic reticulum Ca(2+)-ATPase was explored by its elongation with glycine insertion at Pro(42)/Ala(43) and Gly(46)/Lys(47). Two or more glycine insertions at each site completely abolished ATPase activity. The isomerization of phosphoenzyme (EP) intermediate from the ADP-sensitive form (E1P) to the ADP-insensitive form (E2P) was markedly accelerated, but the decay of EP was completely blocked in these mutants. The E2P accumulated was therefore demonstrated to be E2PCa(2) possessing two occluded Ca(2+) ions at the transport sites, and the Ca(2+) deocclusion and release into lumen were blocked in the mutants. By contrast, the hydrolysis of the Ca(2+)-free form of E2P produced from P(i) without Ca(2+) was as rapid in the mutants as in the wild type. Analysis of resistance against trypsin and proteinase K revealed that the structure of E2PCa(2) accumulated is an intermediate state between E1PCa(2) and the Ca(2+)-released E2P state. Namely in E2PCa(2), the actuator domain is already largely rotated from its position in E1PCa(2) and associated with the phosphorylation domain as in the Ca(2+)-released E2P state; however, in E2PCa(2), the hydrophobic interactions among these domains and Leu(119)/Tyr(122) on the top of second transmembrane helix are not yet formed properly. This is consistent with our previous finding that these interactions at Tyr(122) are critical for formation of the Ca(2+)-released E2P structure. Results showed that the EP isomerization/Ca(2+)-release process consists of the following two steps: E1PCa(2) --> E2PCa(2) --> E2P + 2Ca(2+); and the intermediate state E2PCa(2) was identified for the first time. Results further indicated that the A/M1 linker with its appropriately short length, probably because of the strain imposed in E2PCa(2), is critical for the correct positioning and interactions of the actuator and phosphorylation domains to cause structural changes for the Ca(2+) deocclusion and release.
Highlights
Our results indicated that the A/M1 linker with its correct length critically contributes to the EP isomerization/Ca2ϩ release and to the E2P hydrolysis, and we pointed out the possible importance of this linker in the proper positioning of the A and P domains for their motions and association during these processes
We explored the functional importance of the length of the A/M1 linker (Glu40–Ser48 loop) and found that its elongation markedly accelerates the loss of the ADP sensitivity in EP but blocks almost completely the decay of the ADP-insensitive EP accumulated
The results indicate that the EP isomerization/Ca2ϩ-release process described as a single step, E1PCa2 3 E2P ϩ 2Ca2ϩ, consists of or can be dissected into the two successive steps E1PCa2 3 E2PCa2 3 E2P ϩ 2Ca2ϩ, i.e. the loss of ADP sensitivity at the catalytic site and the subsequent Ca2ϩ release into lumen
Summary
Mutagenesis and Expression—The QuikChangeTM site-directed mutagenesis method (Stratagene, La Jolla, CA) was utilized for the insertions and substitutions of residues in the rabbit SERCA1a cDNA. Ca2ϩ Occlusion in EP—As described in Fig. 8 legend, the expressed mutant SERCA1a in microsomes was phosphorylated with ATP and 45CaCl2, and the mixture was diluted by a “washing solution” containing excess EGTA and immediately filtered through a 0.45-m nitrocellulose membrane filter (Millipore). 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 by including 1 M thapsigargin (TG) in the phosphorylation mixture, otherwise as above. This background subtraction is ensured by the fact that TG inhibits the Ca2ϩ binding at the transport sites and the EP formation [30]. E2P was first formed from Pi in the absence of Ca2ϩ, and 45Ca2ϩ was added to E2P otherwise as described in Fig. 10 legend, and the amount of occluded 45Ca2ϩ was determined as above
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