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J. Biol. Chem., Vol. 280, Issue 28, 26508-26516, July 15, 2005

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Critical Hydrophobic Interactions between Phosphorylation and Actuator Domains of Ca²+-ATPase for Hydrolysis of Phosphorylated Intermediate^*

Guoli Wang, Kazuo Yamasaki, Takashi Daiho, and Hiroshi Suzuki¶

From the Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan and the Department of Nuclear Medicine, the First Clinical College of China Medical University, Shenyang 110001, China

Functional roles of seven hydrophobic residues on the interface between the actuator (A) and phosphorylation (P) domains of sarcoplasmic reticulum Ca²⁺-ATPase were explored by alanine and serine substitutions. The residues examined were Ile¹⁷⁹/Leu¹⁸⁰/Ile²³² on the A domain, Val⁷⁰⁵/Val⁷²⁶ on the P domain, and Leu¹¹⁹/Tyr¹²² on the loop linking the A domain and M2 (the second transmembrane helix). These residues gather to form a hydrophobic cluster around Tyr¹²² in the crystal structures of Ca²⁺-ATPase in Ca²⁺-unbound E2 (unphosphorylated) and E2P (phosphorylated) states but are far apart in those of Ca²⁺-bound E1 (unphosphorylated) and E1P (phosphorylated) states. The substitution-effects were also compared with those of Ile²³⁵ on the A domain/M3 linker and those of T¹⁸¹GE of the A domain, since they are in the immediate vicinity of the Tyr¹²²-cluster. All these substitutions almost completely inhibited ATPase activity without inhibiting Ca²⁺-activated E1P formation from ATP. Substitutions of Ile²³⁵ and T¹⁸¹GE blocked the E1P to E2P transition, whereas those in the Tyr¹²²-cluster blocked the subsequent E2P hydrolysis. Substitutions of Ile²³⁵ and Glu¹⁸³ also blocked EP hydrolysis. Results indicate that the Tyr¹²²-cluster is formed during the E1P to E2P transition to configure the catalytic site and position Glu¹⁸³ properly for hydrolyzing the acylphosphate. Ile²³⁵ on the A domain/M3 linker likely forms hydrophobic interactions with the A domain and thereby allowing the strain of this linker to be utilized for large motions of the A domain during these processes. The Tyr¹²²-cluster, Ile²³⁵, and T¹⁸¹GE thus seem to have different roles and are critical in the successive events in processing phosphorylated intermediates to transport Ca²⁺.

Received for publication, April 7, 2005

^* This work was supported by a grant-in-aid for Scientific Research (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. and Dr. Chikashi Toyoshima (Head Investigator of the Grant), University of Tokyo) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. I-IV.

^¶ To whom correspondence should be addressed. Tel.: 81-166-68-2350; Fax: 81-166-68-2359; E-mail: hisuzuki{at}asahikawa-med.ac.jp.

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