Critical Role of Glu^<40>-Ser^<48> Loop Linking Actuator Domain and First Transmembrane Helix of Ca^<2+>-ATPase in Ca^<2+> Deocclusion and Release from ADP-insensitive Phosphoenzyme
著者
大保, 貴嗣
(Daiho, T)
Yamasaki, K
Danko, S
Suzuki, H
上位タイトル
Journal of Biological Chemistry
Vol.282,
No.47
(2007.
11)
,p.34429-
34447
Functional importance of the length of the A/M1-linker (Glu^<40>-Ser^<48>) connecting the Actuator domain and 1st transmembrane helix of sarcoplasmic reticulum Ca^<2+>-ATPase was explored by its elongation with glycines-insertion at Pro^<42>/Ala^<43> and Gly^<46>/Lys^<47>. Two or more glycines-insertion at each site completely abolished ATPase activity. The isomerization of phosphoenzyme intermediate (EP) from the ADP-sensitive form (E1P) to ADP-insensitive form (E2P) was markedly accelerated but the decay of EP was completely blocked in these mutants. E2P thus accumulated was demonstrated to be E2PCa_2 possessing two occluded Ca^<2+> ions at the transport sites, and the Ca^<2+> deocclusion and release into lumen was blocked in the mutants. By contrast, the hydrolysis of theCa^<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 the E1PCa_2 and Ca^<2+>-released E2P states. 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 2nd transmembrane helix is not formed properlyyet. This is consistent with our previous finding that these interactions at Tyr^<122> are critical for formation of Ca^<2+>-released E2P structure. Results showed that the EP isomerization/Ca^<2+>-release process consists of 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.
