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

Abstract

. (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.

Arginine substitution of a cysteine in transmembrane helix M8 converts Na + ,K + -ATPase to an electroneutral pump similar to H + ,K + -ATPase

“…The lysine of transmembrane helix M5 replacing S777 of Na + , K + -ATPase in the H + ,K + -ATPases has previously been attributed a role as an internal cation similar to the present proposal for the arginine of M8 (7,9). In favor of this function of the M5 lysine is the finding that alanine substitution confers electrogenicity to the nongastric H + ,K + -ATPase, thus raising the number of cations transported toward the extracellular side per cycle (7).…”

“…In favor of this function of the M5 lysine is the finding that alanine substitution confers electrogenicity to the nongastric H + ,K + -ATPase, thus raising the number of cations transported toward the extracellular side per cycle (7). Although a similar gain of electrogenicity was not observed for the corresponding mutant of gastric H + ,K + -ATPase (10), the conformational change from E 1 P to E 2 P was proposed to result in the formation of a salt bridge between the M5 lysine and the M6 glutamate in the gastric H + ,K + -ATPase, thereby expelling the bound H + toward the extracellular side (9,10). In combination with the present results, the previous findings with the M5 lysine suggest that both the lysine and the M8 arginine contribute internal cations that, together, ensure the electroneutrality and ability of H + , K + -ATPases to work against a steep concentration gradient.…”

Cryo-EM structure of gastric H + ,K + -ATPase with a single occupied cation-binding site

“…The density is likely to be surrounded by several amino acids involved in cation coordination (SI Appendix, Fig. S11), as determined by mutagenesis (17)(18)(19)(20)(21)(22), and most of the homologous residues in Na + ,K + -ATPase or SERCA are also involved in cation coordination (21,23). Among them, E343 is in close proximity to the strong density located at site II in our homology model (SI Appendix, Fig.…”

“…4C). Together with previously reported findings regarding the E2 or E2P preference of H + ,K + -ATPase (19)(20)(21)(22)28), the proposed vectorial transport model (Fig. 4) describes how gastric H + ,K + -ATPase can generate the highly acidic condition in the gastric lumen.…”

Analysis of the Gastric H,K ATPase for Ion Pathways and Inhibitor Binding Sites

“…An important goal is accurate description of the high-affinity binding site of these reversible inhibitors as the foundation for structure-based drug design. The active conformation of SCH28080 has been identified (9), and the rotationally restricted naphthyridine, Byk99, that mimics this structure was therefore used in combination with homology modeling to define inhibitor access and binding in a new E 2 P model of the H,K ATPase.The homology modeling approach has been used to construct heuristic models of the catalytic subunits of the H,K and Na,K ATPases by using the peptide backbone of the srCa ATPase as the initial template since this is the only P 2 -type ATPase that has been amenable to highresolution crystal analysis (10)(11)(12). Although the derived structures are unlikely to be correct in detail because of their sequence differences and the absence of the β subunit and lipid, they nevertheless can provide important insights and predictions, many of which have been substantiated by empirical results.…”