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J. Biol. Chem., Vol. 282, Issue 47, 34429-34447, November 23, 2007

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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^*

From the Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan

The functional importance of the length of the A/M1 linker (Glu⁴⁰-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/Ala⁴³ 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 P_i without Ca²⁺ 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 E1PCa₂ and associated with the phosphorylation domain as in the Ca²⁺-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 Tyr¹²² 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 E2PCa₂ E2P + 2Ca²⁺; and the intermediate state E2PCa₂ 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.

Received for publication, September 12, 2007

^* This work was supported by grants-in-aid for Scientific Research C (to T. D.) and B (to H. S.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and in part by a Creative Science Project Grant (to H. S.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Head Investigator of the Grant, Dr. Chikashi Toyoshima, University of Tokyo). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, Asahikawa Medical College, Midorigaoka-higashi, Asahikawa, 078-8510, Japan. Tel.: 81-166-68-2350; Fax: 81-166-68-2359; E-mail: daiho{at}asahikawa-med.ac.jp.

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