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Originally published In Press as doi:10.1074/jbc.M010813200 on February 5, 2001

J. Biol. Chem., Vol. 276, Issue 18, 15232-15239, May 4, 2001
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The Role of the M6-M7 Loop (L67) in Stabilization of the Phosphorylation and Ca2+ Binding Domains of the Sarcoplasmic Reticulum Ca2+-ATPase (SERCA)*

Zhongsen Zhang, David Lewis, Carlota Sumbilla, and Giuseppe InesiDagger

From the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201

Chikashi Toyoshima

From the Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

The amino acid sequence (L67) intervening between the M6 and M7 transmembrane segments of the Ca2+ transport ATPase was subjected to mutational analysis. Mutation of Pro820 to Ala interferes with protein expression even though transcription occurs at normal levels. Single mutations of Lys819 or Arg822 to Ala, Phe, or Glu allow good expression, but produce strong inhibition of ATPase activity. The main defect produced by these mutations is strong interference with enzyme phosphorylation by ATP in the presence of Ca2+, and also by Pi in the absence of Ca2+. The Lys819 and Arg822 mutants undergo slight and moderate reduction of Ca2+ binding affinity, respectively. Reduction of overall steady state ATPase velocity is then due to inhibition of phosphorylated intermediate formation. On the other hand, a cluster of conservative mutations of Asp813, Asp815, and Asp818 to Asn interferes strongly with enzyme activation by Ca2+ binding and formation of phosphorylated enzyme intermediate by utilization of ATP. Enzyme phosphorylation by Pi in the absence of Ca2+ undergoes slight or no inhibition by the triple aspartate mutation. Therefore, the triple mutation interferes mainly with the calcium-dependent activation of the ATPase. The effect of the triple mutation can be to a large extent reproduced by single mutation of Asp813 (but not of Asp815 or Asp818) to Asn. Functional and structural analysis of the experimental data demonstrates that the L67 loop plays an important role in protein folding and function. This role is sustained by linking the cytosolic catalytic domain and the transmembrane Ca2+ binding domain through a network of hydrogen bonds.

* This work was supported by National Institutes of Health Program Project HL27867 and by grants-in-aid for scientific research and for international scientific research from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 410-706-3220; Fax: 410-706-8297; E-mail: ginesi@umaryland.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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