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J. Biol. Chem., Vol. 279, Issue 30, 31629-31637, July 23, 2004

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Ca²⁺ Occlusion and Gating Function of Glu³⁰⁹ in the ADP-Fluoroaluminate Analog of the Ca²⁺-ATPase Phosphoenzyme Intermediate^*

Giuseppe Inesi, Hailun Ma, David Lewis, and Cheng Xu

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

In the absence of ATP the sarcoplasmic reticulum ATPase (SERCA) binds two Ca²⁺ with high affinity. The two bound Ca²⁺ rapidly undergo reverse dissociation upon addition of EGTA, but can be distinguished by isotopic exchange indicating fast exchange at a superficial site (site II), and retardation of exchange at a deeper site (site I) by occupancy of site II. Site II mutations that allow high affinity binding to site I, but only low affinity binding to site II, show that retardation of isotopic exchange requires higher Ca²⁺ concentrations with the N796A mutant, and is not observed with the E309Q mutant even at millimolar Ca²⁺. Fluoroaluminate forms a complex at the catalytic site yielding stable analogs of the phosphoenzyme intermediate, with properties similar to E2-P or E1-P·Ca₂. Mutational analysis indicates that Asp³⁵¹, Lys³⁵², Thr³⁵³, Asp⁷⁰³, Asn⁷⁰⁶, Asp⁷⁰⁷, Thr⁶²⁵, and Lys⁶⁸⁴ participate in stabilization of fluoroaluminate and Mg²⁺ at the phosphorylation site. In the presence of fluoroaluminate and Ca²⁺, ADP (or AMP-PCP) favors formation of a stable ADP·E1-P·Ca₂ analog. This produces strong occlusion of Ca²⁺ bound to both sites (I and II), whereby dissociation occurs very slowly even following addition of EGTA. Occlusion by fluoraluminate and ADP is not observed with the E309Q mutant, suggesting a gating function of Glu³⁰⁹ at the mouth of a binding cavity with a single path of entry. This phenomenon corresponds to the earliest step of the catalytic cycle following utilization of ATP. Experiments on limited proteolysis reveal that a long range conformational change, involving displacement of headpiece domains and transmembrane helices, plays a mechanistic role.

Received for publication, March 23, 2004 , and in revised form, May 17, 2004.

^* This work was supported by the National Institutes of Health NHLBI Grant RO1 HL69830 and the Human Frontier Science Program. 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.

To whom correspondence should be addressed: Dept. of Biochemistry, University of Maryland School of Medicine, Baltimore, MD 21201-1503. Tel.: 410-706-3220; Fax: 410-706-8297; E-mail: ginesi{at}umaryland.edu.

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