Inhibitor and ion binding sites on the gastric H,K-ATPase

Abstract

The gastric H,K-ATPase catalyzes electroneutral exchange of H+ for K+ as a function of enzyme phosphorylation and dephosphorylation during transition between E1/E1-P (ion site in) and E 2-P/E2 (ion site out) conformations. Here we present homology modeling of the H,K-ATPase in the E2-P conformation as a means of predicting the interaction of the enzyme with two known classes of specific inhibitors. All known proton pump inhibitors, PPIs, form a disulfide bond with cysteine 813 that is accessible from the luminal surface. This allows allocation of the binding site to a luminal vestibule adjacent to Cys813 enclosed by part of TM4 and the loop between TM5 and TM6. K+ competitive imidazo-1,2α-pyridines also bind to the luminal surface of the E2-P conformation, and their binding excludes PPI reaction. This overlap of the binding sites of the two classes of inhibitors combined with the results of site-directed mutagenesis and cysteine cross-linking allowed preliminary assignment of a docking mode for these reversible compounds in a position close to Glu795 that accounts for the detailed structure/activity relationships known for these compounds. The new E2-P model is able to assign a possible mechanism for acid secretion by this P2-type ATPase. Several ion binding side chains identified in the sr Ca-ATPase by crystallography are conserved in the Na,K- and H,K-ATPases. Poised in the middle of these, the H,K-ATPase substitutes lysine in place of a serine implicated in K+ binding in the Na,K-ATPase. Molecular models for hydronium binding to E1 versus E2-P predict outward displacement of the hydronium bound between Asp824, Glu820, and Glu795 by the R-NH3+ of Lys791 during the conformational transition from E1P and E2P. The site for luminal K+ binding at low pH is proposed to be between carbonyl oxygens in the nonhelical part of the fourth membrane span and carboxyl oxygens of Glu795 and Glu820. This site of K + binding is predicted to destabilize hydrogen bonds between these carboxylates and the -NH3+ group of Lys791, allowing the Lys791 side chain to return to its E1 position. © 2005 American Chemical Society.