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

authorFunctional importance of the length of the A/M1-linker (Glu^-Ser^) connecting the Actuator domain and 1st transmembrane helix of sarcoplasmic reticulum Ca^-ATPase was explored by its elongation with glycines-insertion at Pro^/Ala^ and Gly^/Lys^. 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^ ions at the transport sites, and the Ca^ deocclusion and release into lumen was blocked in the mutants. By contrast, the hydrolysis of theCa^-free form of E2P produced from P_I without Ca^ 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^-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^-released E2P state,however in E2PCa_2 the hydrophobic interactions among these domains and Leu^/Tyr^ on the top of 2nd transmembrane helix is not formed properlyyet. This is consistent with our previous finding that these interactions at Tyr^ are critical for formation of Ca^-released E2P structure. Results showed that the EP isomerization/Ca^-release process consists of two steps; E1PCa_2 → E2PCa_2 → E2P + 2Ca^, 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^ deocclusion and release

Remarkable stability of solubilized and delipidated sarcoplasmic reticulum Ca2+-ATPase with tightly bound fluoride and magnesium against detergent-induced denaturation

American Society for Biochemistry and Molecular Biology, Yamasaki, Kazuo ; Daiho, Takashi ; Suzuki, Hiroshi, Journal of Biological Chemistry, 277(16), 2002, 13615-13619 authorConditions were developed in the absence of Ca(2+) for purification, delipidation, and long term stabilization of octaethylene glycol monododecyl ether (C(12)E(8))-solubilized sarcoplasmic reticulum Ca(2+)-ATPase with tightly bound Mg(2+) and F(-), an analog for the phosphoenzyme intermediate without bound Ca(2+). The Ca(2+)-ATPase activity to monitor denaturation was assessed after treatment with 20 mm Ca(2+) to release tightly bound Mg(2+)/F(-). The purification and delipidation was successfully achieved with Reactive Red-agarose affinity chromatography. The solubilized Mg(2+)/F(-)-bound Ca(2+)-ATPase was very rapidly denatured at pH 8, but was perfectly stabilized at pH 6 against denaturation for over 20 days at 4 degrees C even without exogenously added phospholipid and at a high C(12)E(8)/enzyme weight ratio (10:1). The activity was not restored unless the enzyme was treated with 20 mm Ca(2+), showing that tightly bound Mg(2+)/F(-) was not released during the long term incubation. The perfect stability was attained with or without 0.1 mm dithiothreitol, but inactivation occurred with a half-life of 10 days in the presence of 1 mm dithiothreitol, possibly due to reduction of a specific disulfide bond(s). The remarkable stability is likely conferred by intimate gathering of cytoplasmic domains of Ca(2+)-ATPase molecule induced by tight binding of Mg(2+)/F(-). The present study thus reveals an essential property of the Mg(2+)/F(-)/Ca(2+)-ATPase complex, which will likely provide clues to understanding structure of the Ca(2+)-released form of phosphoenzyme intermediate at an atomic level