Critical role of Glu40-Ser48 loop linking actuator domain and first transmembrane helix of Ca2+-ATPase in Ca 2+ deocclusion and release from ADP-insensitive phosphoenzyme

Abstract

The functional importance of the length of the A/M1 linker (Glu 40-Ser48) connecting the actuator domain and the first transmembrane helix of sarcoplasmic reticulum Ca2+-ATPase was explored by its elongation with glycine insertion at Pro42/Ala 43 and Gly46/Lys47. 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 E2PCa2 possessing two occluded Ca2+ ions at the transport sites, and the Ca2+ deocclusion and release into lumen were blocked in the mutants. By contrast, the hydrolysis of the Ca2+-free form of E2P produced from Pi 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 E2PCa2 accumulated is an intermediate state between E1PCa2 and the Ca2+-released E2P state. Namely in E2PCa2, 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 E2PCa2, the hydrophobic interactions among these domains and Leu119/Tyr122 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 Ca2+-released E2P structure. Results showed that the EP isomerization/Ca2+-release process consists of the following two steps:E1PCa2→E2PCa 2→E2P+2Ca2+; 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 E2PCa2, is critical for the correct positioning and interactions of the actuator and phosphorylation domains to cause structural changes for the Ca2+ deocclusion and release. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.