Asn792 Participates in the Hydrogen Bond Network Around the K+-binding Pocket of Gastric H,K-ATPase

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

doi:10.1074/jbc.m412321200

Herman G.P. Swarts, Elmar Krieger + Show 3 more

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https://doi.org/10.1074/jbc.m412321200

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Abstract

Asn792 present in M5 of gastric H,K-ATPase is highly conserved within the P-type ATPase family. A direct role in K+ binding was postulated for Na,K-ATPase but was not found in a recent model for gastric H,K-ATPase (Koenderink, J. B., Swarts, H. G. P., Willems, P. H. G. M., Krieger, E., and De Pont, J. J. H. H. M. (2004) J. Biol. Chem. 279, 16417-16424). Therefore, its role in K+ binding and E1/E2 conformational equilibrium in gastric H,K-ATPase was studied by site-directed mutagenesis and expression in Sf9 cells. N792Q and N792A, but not N792D and N792E, had a markedly reduced K+ affinity in both the ATPase and dephosphorylation reactions. In addition, N792A shifted the conformational equilibrium to the E1 form. In double mutants, the effect of N792A on K+ sensitivity was overruled by either E820Q (K(+)-independent activity) or E343D (no dephosphorylation activity). Models were made for the mutants based on the E2 structure of Ca(2+)-ATPase. In the wild-type model the acid amide group of Asn792 has hydrogen bridges to Lys791, Ala339, and Val341. Comparison of the effects of the various mutants suggests that the hydrogen bridge between the carbonyl oxygen of Asn792 and the amino group of Lys791 is essential for the K+ sensitivity and the E2 preference of wild-type enzyme. Moreover, there was a high positive correlation (r = 0.98) between the in silico calculated energy difference of the E2 form (mutants versus wild type) and the experimentally measured IC50 values for vanadate, which reflects the direction of the E2<-->E1 conformational equilibrium. These data strongly support the validity of the model in which Asn792 participates in the hydrogen bond network around the K(+)-binding pocket.

Highlights

Charged and other polar residues present in transmembrane helices in P-type ATPases have long been considered to be important for cation binding
Comparison of the effects of the various mutants suggests that the hydrogen bridge between the carbonyl oxygen of Asn792 and the amino group of Lys791 is essential for the K؉ sensitivity and the E2 preference of wild-type enzyme
Each Kϩ ion was surrounded by six oxygen atoms that are responsible for cation binding

Summary

Introduction

Charged and other polar residues present in transmembrane helices in P-type ATPases have long been considered to be important for cation binding. In Na,K-ATPase, the analogous residues Glu334 (8), Glu786 (5), and Asp811 (9) were demonstrated to be involved in Kϩ-stimulated dephosphorylation In the latter enzyme, direct binding studies with the Kϩ analogue, 204Tlϩ, supported these findings (10). Jorgensen (13) postulated a roughly similar structure for the Kϩ-binding sites in Na,KATPase but suggested an additional role of Glu334 (M4) in Kϩ-binding site II The latter role was based on equilibrium binding studies with expressed Na,K-ATPase mutants showing that mutation of Glu334 in either an Asp or a Gln residue completely abolished the 204Tlϩ occlusion (10). Role of Asn792 in Gastric H,K-ATPase in gastric H,K-ATPase originated from the side chains of Glu795 (M5) and Glu820 (M6) as well as from the main chain residues of Val338 and Val341 (M4) Both oxygen atoms of Glu343 (M4), which is analogous to Glu334 in Na,K-ATPase, bound the Kϩ ion. We examine the putative role of Asn792 in the

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