A conformation-specific interhelical salt bridge in the K+ binding site of gastric H,K-ATPase

Abstract

Homology modeling of gastric H, K-ATPase based on the E-2 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 E-2 form-specific salt bridge between Glu(820) (M6) and Lys(791) (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 E-1 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, Lys(791) was mutated alone and in combination with other crucial residues. In the K791A mutant, the K+ affinity was markedly reduced without altering the E-2 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 E-1. This indicates that the salt bridge is essential for high-affinity K+ binding and the E-2 preference of H,K-ATPase. Moreover, its breakage (E820Q) can generate a K+-insensitive activity and an E-1 preference. In addition, the study gives a molecular explanation for the electroneutrality of H, K-ATPases.