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J. Biol. Chem., Vol. 279, Issue 16, 16417-16424, April 16, 2004

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A Conformation-specific Interhelical Salt Bridge in the K⁺ Binding Site of Gastric H,K-ATPase^*

Jan B. Koenderink, Herman G. P. Swarts, Peter H. G. M. Willems, Elmar Krieger, and Jan Joep H. H. M. De Pont¶

From the Department of Biochemistry, Nijmegen Center for Molecular Life Sciences and the Center for Molecular and Biomolecular Informatics, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands

Homology modeling of gastric H,K-ATPase based on the E₂ model of sarcoplasmic reticulum Ca²⁺-ATPase (Toyoshima, C., and Nomura, H. (2002) Nature 392, 835–839) revealed the presence of a single high-affinity binding site for K⁺ and an E₂ form-specific salt bridge between Glu⁸²⁰ (M6) and Lys⁷⁹¹ (M5). In the E820Q mutant this salt bridge is no longer possible, and the head group of Lys⁷⁹¹, together with a water molecule, fills the position of the K⁺ ion and apparently mimics the K⁺-filled cation binding pocket. This gives an explanation for the K⁺-independent ATPase activity and dephosphorylation step of the E820Q mutant (Swarts, H. G. P., Hermsen, H. P. H., Koenderink, J. B., Schuurmans Stekhoven, F. M. A. H., and De Pont, J. J. H. H. M. (1998) EMBO J. 17, 3029–3035) and, indirectly, for its E₁ preference. The model is strongly supported by a series of reported mutagenesis studies on charged and polar amino acid residues in the membrane domain. To further test this model, Lys⁷⁹¹ was mutated alone and in combination with other crucial residues. In the K791A mutant, the K⁺ affinity was markedly reduced without altering the E₂ preference of the enzyme. The K791A mutation prevented, in contrast to the K791R mutation, the spontaneous dephosphorylation of the E820Q mutant as well as its conformational equilibrium change toward E₁. This indicates that the salt bridge is essential for high-affinity K⁺ binding and the E₂ preference of H,K-ATPase. Moreover, its breakage (E820Q) can generate a K⁺-insensitive activity and an E₁ preference. In addition, the study gives a molecular explanation for the electroneutrality of H,K-ATPases.

Received for publication, January 5, 2004 , and in revised form, January 28, 2004.

^* 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. E-mail: J.dePont{at}ncmls.kun.nl.

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