Critical role of Glu40-Ser48 loop linking actuator domain and first transmembrane helix of Ca2+-ATPase in Ca2+ deocclusion and release from ADP-insensitive phosphoenzyme.

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.