Decision letter: A single K+-binding site in the crystal structure of the gastric proton pump (2019)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The gastric proton pump (H+,K+-ATPase), a P-type ATPase responsible for gastric acidification, mediates electro-neutral exchange of H+ and K+ coupled with ATP hydrolysis, but with an as yet undetermined transport stoichiometry. Here we show crystal structures at a resolution of 2.5 Å of the pump in the E2-P transition state, in which the counter-transporting cation is occluded. We found a single K+ bound to the cation-binding site of the H+,K+-ATPase, indicating an exchange of 1H+/1K+ per hydrolysis of one ATP molecule. This fulfills the energy requirement for the generation of a six pH unit gradient across the membrane. The structural basis of K+ recognition is resolved and supported by molecular dynamics simulations, establishing how the H+,K+-ATPase overcomes the energetic challenge to generate an H+ gradient of more than a million-fold—one of the highest cation gradients known in mammalian tissue—across the membrane. https://doi.org/10.7554/eLife.47701.001 Introduction The highly acidic environment (pH 1) in the stomach is generated by the gastric proton pump, H+,K+-ATPase, which mediates an exchange of H+ and K+ across the parietal cell membrane that is coupled with ATP hydrolysis (Figure 1) (Ganser and Forte, 1973). Like other P-type ATPases, the vectorial cation transport of gastric H+,K+-ATPase is accomplished by cyclical conformational changes in the enzyme (abbreviated as ‘E’) (Rabon and Reuben, 1990), generally described using E1/E2 nomenclature based on the Post-Albers scheme for Na+,K+-ATPase (Figure 2A) (Post et al., 1969). During the transport cycle, a conserved aspartate is reversibly auto-phosphorylated to form phosphoenzyme intermediates (EPs), a hallmark of members of the P-type ATPase family (Post and Kume, 1973). The H+,K+-ATPase consists of two subunits. The 110 kDa catalytic α-subunit is homologous to other related P2-type ATPases such as Na+,K+-ATPase, with which it shares 65% identity (Morth et al., 2007), and the serco(endo)plasmic reticulum Ca2+-ATPase (SERCA) (Toyoshima et al., 2000), with which it shares 35% sequence identity (Palmgren and Axelsen, 1998). The α-subunit is composed of 10 transmembrane (TM) helices, in which the cation-binding sites are located, and three cytoplasmic domains (the nucleotide (N), phosphorylation (P) and actuator (A) domains) that catalyze ATP hydrolysis and the auto-phosphorylation reaction. The β-subunit is a single-span membrane protein with a short N-terminal cytoplasmic tail and a large C-terminal ectodomain, and it is involved in correct membrane integration and targeting of the complex to the cell surface (Chow and Forte, 1995). Figure 1 Download asset Open asset Transport stoichiometry and free energy from ATP hydrolysis. The free energy that is derived from ATP hydrolysis, ΔGATP—calculated from ΔG’0 and the measured intracellular concentrations of ATP, ADP and Pi in the parietal cell—is about −13 kcal/mol (Durbin et al., 1974). Under physiological conditions in intact parietal cells with an internal pH of approximately 7, a pH gradient of at least 6 pH units must be created. The maximum electrochemical gradient, Δμi, that can be formed by an ion-transporting ATPase is a function of the free energy of ATP hydrolysis. Taking reasonable values of pH 7 and 120 mM K+ for intracellular conditions and the measured pH (1.0) and K+ concentration (10 mM) of the gastric juice, we can calculate concentration gradients across the parietal cell membrane of 106 and 12 times for H+ and K+, respectively. For an electro-neutral H+/K+ exchange pump for which NH+=NKK+=1, the sum of chemical potentials is about −10 kcal/mol, and is within the range of ATP free energy (Reenstra and Forte, 1981). However, if the exchange of two cations is assumed, in which case NH+=NKK+=2, the reaction is thermodynamically disallowed at pH 1. Therefore, the ratio of H+ transported to ATP hydrolyzed must be approximately 1, and cannot be as large as 2, when gastric pH is around 1. However, this cannot be the case when luminal pH is neutral to weakly acidic (e.g., at pH 4). A different postulate is that two H+ are transported for each ATP molecule hydrolyzed under these conditions (Rabon et al., 1982), and that the number of transported H+, and therefore K+ as well, changes from 2 to 1 as luminal pH decreases. On the basis of two different results for H+/ATP ratio, two hypotheses have been proposed. Hypothesis 1: transport stoichiometry remains constant (1H+:1K+) regardless of luminal pH. Hypothesis 2: 2H+:2K+ ions are exchanged when luminal pH is neutral to weakly acidic, and it returns to 1H+:1K+ transport mode when the luminal solution becomes highly acidic. https://doi.org/10.7554/eLife.47701.002 Figure 2 Download asset Open asset Characterization of the Tyr799Trp mutant of H+,K+-ATPase. (A) Post-Albers type reaction scheme for H+,K+-ATPase. The K+-occluded E2-P transition state is highlighted in red. (B) ATPase activities of the wild-type enzyme with the indicated cations, showing a Hill coefficient (n) close to 1. K+-dependent ATPase activity of (C) wild-type or (D) Tyr799Trp mutant H+,K+-ATPase in the absence or presence of indicated concentrations of the K+-competitive inhibitor vonoprazan. Thermal stability of (E) wild-type or (F) Try799Trp mutant H+,K+-ATPase in the presence of the indicated ligands, evaluated by fluorescence size-exclusion chromatography. https://doi.org/10.7554/eLife.47701.003 Although the closely related Na+,K+-ATPase mediates electrogenic transport of three Na+ and two K+ ions coupled with the hydrolysis of one ATP molecule (Jorgensen et al., 2003), the number of transported cations in the electroneutral operation (Sachs et al., 1976; van der Hijden et al., 1990; Burnay et al., 2001; Burnay et al., 2003) of the gastric H+,K+-ATPase remains unclear. Under physiological conditions, intact parietal cells with an internal pH of approximately seven must generate a pH gradient of at least six pH units (Wolosin, 1985). The secretion of a relatively voluminous flow of gastric acid requires the expenditure of considerable cellular energy. Taking reasonable values of pH 7 and 120 mM K+ for the intracellular condition, together with measured pH (1.0) and K+ concentration (10 mM) of the gastric juice, we can calculate concentration gradients across the membrane of 106 and 12 times for H+ and K+, respectively. The sum of chemical potentials is about −10 kcal/mol. The reported free energy derived from ATP hydrolysis in the parietal cell is about −13 kcal/mol (Durbin et al., 1974). Therefore, the ratio of H+ transported to ATP hydrolyzed must be approximately 1, and cannot be as large as 2, when the gastric pH approaches 1 (Figure 1). Accordingly, a previous investigation of H+ transport shows a 1:1 stoichiometry of H+ transport and ATP hydrolysis at pH 6.1–6.9 (Reenstra and Forte, 1981). However, this ratio might change when the luminal pH is neutral to weakly acidic, in which case a 2H+:1ATP stoichiometry would be feasible in principle (Figure 1). A separate investigation, on the other hand, showed the transport of two H+'s for each ATP hydrolyzed at pH 6.1 (Rabon et al., 1982). It was speculated that the number of H+ transported may change from two (at neutral pH) to one as the luminal pH decreases (Shin et al., 2009; Abe et al., 2012). The electroneutrality of the transport cycle of the H+,K+-ATPase over a wide pH range is indisputable based on the electrophysiological studies (Sachs et al., 1976; van der Hijden et al., 1990; Burnay et al., 2001; Burnay et al., 2003). Therefore, whether the H+,K+-ATPase constantly exchanges 1H+:1K+ regardless of luminal pH (Figure 1, Hypothesis 1) or switches its transport mode from 1H+:1K+ to 2H+:2K+ depending on conditions (Figure 1, Hypothesis 2) has long time been the subject of debate in the membrane transport field. The reason for the discrepancy probably lies in the difficulty in measuring the transport stoichiometry, namely, one H+ or two H+’s per ATP hydrolysis. These measurements are very sensitive because the loss of H+ from the external space of the vesicle must be measured with a glass electrode in vitro, and scalar proton production resulting from ATP hydrolysis must be corrected for. The corrected rate of H+ transport is then compared with that of ATP hydrolyzed under the same conditions as an independent measurement. The inside-out vesicles used in these experiments are sedimented from the natural source (i.e., pig stomach), and contain H+,K+-ATPase as approximately 70% of their total protein (Abe and Olesen, 2016). This system may introduce a different estimate of ATPase activity, especially with regards to the interpretation of the basal Mg2+-sensitive ATPase fraction in the absence of K+. Therefore, we reason that the best way to resolve the disputed transport stoichiometry of the pump is by direct observation of the number of counter-transported cations occluded in the H+,K+-ATPase at neutral pH, given that the transport is approximately electroneutral (Sachs et al., 1976; van der Hijden et al., 1990; Burnay et al., 2003; Burnay et al., 2001). To this end, we determined crystal structures at neutral pH, in order to determine the maximum capacity of K+ occlusion. Results Tyr799Trp mutant H+,K+-ATPase prefers the K+-occluded E2-P transition state According to the transport cycle of the H+,K+-ATPase (Rabon and Reuben, 1990) (Figure 2A), binding of counter-transporting K+ induces luminal gate closure and accelerates dephosphorylation of the auto-phosphorylated intermediate, E2P. Subsequently, the enzyme moves to the E2-P transition state in which counter-transported K+ is occluded (Abe et al., 2012). For crystallization, the transition state phosphate analogs (Danko et al., 2004) (magnesium fluoride (MgFx) or aluminum fluoride (AlFx)) together with counter-transporting cation (K+ or its congener Rb+, see Figure 2B) were applied to the wild-type (WT) H+,K+-ATPase. However, the crystals diffracted poorly in these conditions and the resolution was limited to 4.3 Å, which is insufficient for the precise definition of the K+-coordination. Better-resolving crystals were obtained by using a Tyr799Trp mutation on the α-subunit (Y799W). This mutant was originally obtained from the screening of the inhibitor-binding site of H+,K+-ATPase, and its unusual ATPase profile prompted us to apply this mutant to the crystallization of the K+-occluded form. Tyr799 is located at the entrance of the luminal-facing cation gate to which the specific inhibitors bind (Abe et al., 2018), 12 Å distant from the K+ binding site, and its mutation does not affect the K+ binding site directly. To our surprise, however, the Tyr799Trp mutant shows its highest ATPase activity in the absence of K+ and the activity decreases with increasing K+ concentration, in marked contrast to the K+-dependent increase in ATPase activity of the wild-type enzyme (Figure 2C,D). The observed K+-independent ATPase activity of Tyr799Trp in the absence of K+ indicates that the luminal gate of this mutant is spontaneously closed, like those of the previously reported constitutively active mutants (Abe et al., 2018). The Tyr799Trp mutation exerts a molecular signal that induces E2P dephosphorylation when K+ is bound to the cation binding site. Decreasing ATPase activity of Tyr799Trp with increasing K+ concentration (K0.5,cyto = ~5 mM) can be interpreted as the result of K+-occlusion from the cytoplasmic side, which is also observed in the wild-type enzyme, albeit with much lower affinity (K0.5,cyto =~200 mM) (Ljungström et al., 1984). Furthermore, in the presence of an inhibiting concentration of the K+-competitive blocker vonoprazan, Tyr799Trp shows high-affinity K+-activation of ATPase activity. These data indicate that, despite the unique properties of luminal gate closure, the cation-binding site of Tyr799Trp is intact and capable of high-affinity K+-binding. The thermal stability (Hattori et al., 2012) of Tyr799Trp is significantly higher in the presence of K+ and AlFx than in the presence of AlFx alone (Figure 2F), qualitatively indicating K+-occlusion in the Tyr799Trp mutant. This K+-occluded E2-P transition state is the most stable conformation of all of the evaluated conditions for Tyr799Trp, whereas for the wild-type enzyme, inhibitor-bound and luminal-open E2BeF is the most stable condition (Figure 2E). We therefore conclude that Tyr799Trp prefers the luminal-closed K+-occluded state, (K+)E2-P, and is thus suitable for the structural analysis of the K+-occluded form. As a note, a similar mutant (Phe788Leu in Na+,K+-ATPase, which corresponds to Tyr799 in H+,K+-ATPase) that shows K+-independent dephosphorylation has been reported for the Na+,K+-ATPase (Vilsen, 1999), suggesting the conservation of a luminal gating mechanism between the two related ATPases. Crystal structures reveal the number of K+ occluded at the cation-binding site In order to define the number of K+ that are occluded, and their coordination chemistry in the cation-binding site of the H+,K+-ATPase, we attempted to obtain a high-resolution structure using the Tyr799Trp mutant. As expected, the crystals were significantly improved when using the Tyr799Trp mutant, which provided a 2.5 Å resolution structure in the best case [Y799W(K+)E2-MgFx] (Figure 3). We analyzed several crystal structures in the presence of different combinations of K+ or Rb+, and AlFx or MgFx, all of which mimic the K+-occluded E2-P transition state and are indistinguishable in molecular conformation (Figure 3—figure supplement 1). Although the analyzed resolution is limited, the structure of the wild-type enzyme also shows almost the same molecular conformation as that of the Tyr799Trp mutant (Figure 3—figure supplement 2). We therefore use the Y799W(K+)E2-MgFx structure analyzed at the best resolution in the following discussion (Table 1). The overall structure of H+,K+-ATPase Y799W(K+)E2-MgFx (Figure 3A) is very close to the corresponding structure of Na+,K+-ATPase (Morth et al., 2007; Shinoda et al., 2009) (Figure 3—figure supplement 1). However, instead of having two K+ ions occluded in the transmembrane cation-binding site of the Na+,K+-ATPase, only a single K+ is observed in the cation-binding site of H+,K+-ATPase (Figure 3). The observed single K+-binding is confirmed by the anomalous difference Fourier maps of Y799W(Rb+)E2-MgFx (Figure 3B), Y799W(Rb+)E2-AlFx and Y799W(K+)E2-MgFx structures (Figure 3—figure supplement 1), which unambiguously show a single strong peak at the cation binding site located between TM4, TM5 and TM6 in the middle section of the membrane. The presence of saturating concentrations of cation (400 mM KCl or RbCl) in the crystallization buffer ensures high occupancy of K+ at the cation-binding site, although several other binding sites, presumably low-affinity and/or non-specific, were determined in the cytoplasmic domains (Figure 3—figure supplement 1). Figure 3 with 2 supplements see all Download asset Open asset Crystal structure of the K+-occluded E2-P transition state of H+,K+-ATPase. (A) Overall structure of the K+-occluded E2-MgFx state [Y799W(K+)E2-MgFx] in ribbon representations. For the α-subunit, the three cytoplasmic domains (A, P and N) are shown in different colors. The color of the TM helices gradually changes from purple to red (TM1–TM10). The β-subunit with a single TM helix and six N-glycosylation sites in the ecto-domain is shown in gray. Phospholipids, a cholesterol and detergent molecules are also modeled (as sticks). Red dots and purple spheres represent water molecules and K+ ions, respectively. (B) The TM K+-binding site viewed from a position parallel to membrane plane. Orange mesh represents an anomalous density map of the Rb+-bound form [(Rb+)E2-MgFx] with 8 σ contour level. Amino acids that contribute to the K+-coordination are shown in sticks. (C) The hydrophobic gate centered around Tyr799Trp (green), with surrounding hydrophobic residues (gray), is shown. The dotted line indicates a hydrogen bond between a nitrogen atom of the Trp residue and a main chain oxygen of L811. https://doi.org/10.7554/eLife.47701.004 Table 1 Data collection and refinement statistics. https://doi.org/10.7554/eLife.47701.007 Y799W(K+)E2-MgFxY799W(Rb+)E2-MgFxY799W(Rb+)E2-AlFxWT(Rb+)E2-MgFxData collectionResolution (Å)†2.7 × 2.8 × 2.5 (2.6–2.5)‡2.8 × 2.8 × 2.6 (2.7–2.6)2.8 × 3.0 × 2.5 (2.6–2.5)5.1 × 5.1 × 4.3 (4.5–4.3)Space groupP 31 2 1P 31 2 1P 31 2 1C 1 2 1Cell dimensionsa, b, c (Å)103.37, 103.37, 370.01103.38, 103.38, 369.86103.23, 103.23, 369.44191.51, 106.43, 250.96α, β, γ (°)90, 90, 12090, 90, 12090, 90, 12090, 107.79, 90Rmerge0.067 (3.685)0.080 (2.248)0.106 (3.499)0.152 (1.359)Rpim0.022 (1.179)0.030 (0.855)0.040 (1.295)0.065 (0.544)I/σI20.18 (0.93)16.19 (1.16)11.64 (0.77)7.19 (0.92)C/C1/20.99 (0.59)0.99 (0.70)0.99 (0.60)0.99 (0.75)Completeness (%)85.65 (26.79)88.93 (36.11)76.00 (18.98)94.94 (53.46)Redundancy10.2 (10.6)8.0 (7.8)8.0 (8.2)6.6 (7.2)RefinementResolution (Å)48–2.5 (2.6–2.5)48–2.6 (2.7–2.6)50–2.5 (2.6–2.5)48–4.3 (4.5–4.3)No. of reflections69,037 (2135)63,831 (2533)61,013 (1502)31,390 (1747)Rwork/Rfree (%)20.1/25.7 (37.3/48.0)20.6/26.2 (35.1/43.5)21.5/27.4 (32.7/42.6)26.3/33.9 (33.4/41.2)Wilson B-factor60.7452.6048.43158.52No. of atoms10,51310,48510,49819,823Protein97369762976319,623Ligands362362364200Average B-factor74.4062.6860.03244.07Protein (Å2)72.6260.9857.83243.40Ligands (Å2)122.80114.14103.16310.29R.m.s deviationsBond lengths (Å)0.0100.0090.0100.005Bond angles (°)1.361.511.391.18 †The diffraction data are anisotropic. The resolution limits given are for the a*, b* and c* axes, respectively. ‡Statistics for the highest-resolution shell are shown in parentheses. To exclude the possibility that the observed single K+-binding results from an artifact of the Tyr799Trp mutation, we determined the wild-type WT(Rb+)E2-MgFx structure in the same conformation (Figure 3—figure supplement 2), despite its limited resolution of 4.3 Å. The β-subunit ectodomain in protomer A, and the A- and N-domains in protomer B show relatively weak density, due to lack of tight crystal packing at these regions. The N-terminal tail of the β-subunit is partially visible in the wild-type structure, in contrast to that in the Tyr799Trp structure. We could only assign an additional nine amino acids (Tyr20–Gln28 of the β-subunit) for this region that extends along with the membrane surface. The morphology of the β-subunit N-terminus is similar to that observed in the previously reported electron crystallographic structures (Abe et al., 2009). However, the well-ordered regions, especially for the TM helices that define the luminal-closed K+-occluded state, are almost identical to those of the high-resolution Tyr799Trp structure. We therefore conclude that the molecular conformation of the wild-type H+,K+-ATPase is essentially the same as that of the Tyr799Trp mutant. At the cation-binding site of the wild-type, a single Rb+ anomalous peak is found at the position close to the bound K+ in Tyr799Trp, as well as at site II in Na+,K+-ATPase (Morth et al., 2007; Shinoda et al., 2009). An anomalous signal is hardly seen at the position corresponding to the cation-binding site I in Na+,K+-ATPase, even at the low contour level (Figure 3—figure supplement 2). These observations allow us to assert that the conclusions extracted for the Tyr799Trp mutant in regard to K+ stoichiometry can be extended to the wild-type protein. A single K+-binding in H+,K+-ATPase is also supported by a Hill coefficient for K+ of close to 1.0 (Figure 2B), in marked contrast to the Hill coefficient of 1.5 for the K+-dependence of Na+,K+-ATPase (Sweadner, 1985) in which two K+ ions are occluded at the cation-binding site. were generated at the close to neutral pH of the condition also used for previous in H+ transport measurements (Reenstra and Forte, et al., 1982). electro-neutral transport in the H+,K+-ATPase (Sachs et al., 1976; van der Hijden et al., 1990; Burnay et al., 2003; Burnay et al., if two H+’s are transported at neutral pH, as by a previous in measurements (Rabon et al., 1982), then two K+ ions must be A single bound K+ observed in the crystal structure therefore indicates that H+,K+-ATPase only one K+ at and therefore only one H+ in the for ATP hydrolyzed at neutral pH (Figure 1, Hypothesis 1) (Reenstra and Forte, 1981). for the luminal gate closure the previously reported Rb+-bound and luminal-open structure of H+,K+-ATPase (Abe et al., 2018), in the Y799W(K+)E2-MgFx state, the cation-binding site is from the luminal solution by an hydrophobic centered around Tyr799Trp (Figure at the luminal of Tyr799 to Trp hydrophobic with the surrounding amino the of which may be strong for luminal gate closure, as by the high ATPase activity of the Tyr799Trp mutant in the absence of K+ (Figure 2, Figure 4). A hydrogen bond between the nitrogen of the residue and main chain oxygen of Å, Figure Figure for the of a Trp suitable for hydrophobic with its and its dynamics the presence of this hydrogen and also show that in the wild-type enzyme, the of Tyr799 a similar hydrogen bond with the main chain oxygen of Å, Figure 1). However, the chain of is than that of and thus a hydrophobic with the surrounding These are in the stability of the luminal gate conformation in the simulations, as seen in the of Tyr799 in the wild-type when compared to that in the Tyr799Trp mutant (Figure 1). Figure with 1 supplement see all Download asset Open asset K+-dependence on ATPase activities of and mutation of the surrounding hydrophobic amino observed in the luminal gate of (A) and (B) residues that are to contribute to the luminal gate stability are shown as sticks. (C) of the for of the Trp residue in is and the of the Tyr799 residue in the H+,K+-ATPase The to a of large indicating higher of the in the enzyme compared to those of the in the Tyr799Trp mutant also 1). (D) K+-dependence of the ATPase activities of the indicated Data values in the presence of 10 data from a at different concentrations of K+ were and results from more than three independent measurements for each mutant are shown in the To the of a hydrogen bond between Tyr799Trp and main chain oxygen of and hydrophobic with surrounding residues on luminal gate we Although the mutant is probably because it is (as evaluated by fluorescence size-exclusion the mutant in the of Tyr799Trp shows weak K+-dependence in its ATPase activity (Figure 4). This that the might the thus the with of the surrounding hydrophobic residues in the Try799Trp does not significantly change the K+-dependence profile of Tyr799Trp activity, that the Tyr799Trp mutant shows a K+-dependent increase in its ATPase activity. However, a K+-independent ATPase fraction remains in the absence of K+. In the of an additional mutation or K+-dependent ATPase activity to a level that of the wild-type These data that gate closure of the Tyr799Trp mutant is by the hydrophobic with its and that these are by the position of Tyr799Trp by the between and main K+-dependence can be by additional based on the observed structure of We therefore conclude that the luminal-closed molecular conformation that is spontaneously by the Try799Trp mutation is not an and that the for the gate closure is essentially the same as that in the wild-type 1 Download asset This cannot be in because does may the for Download as Download as Download as of the Tyr799Trp mutant and wild-type A shows the luminal gate and K+-binding site structure of for Tyr799Trp mutant and the wild-type of the luminal-open E2P state structure with bound vonoprazan, a specific inhibitor for H+,K+-ATPase (Abe et al., and the K+-occluded and luminal-closed E2-P transition state several conformational luminal gate closure (Figure supplement 1). closure the luminal of close to the cation-binding site from the luminal of the membrane. This of is coupled to the of the helix to the A to the of the A that induces dephosphorylation of the The molecular for the luminal gate closure have been in et al., et al., 2007; and the same mechanism is observed in the H+,K+-ATPase, the maps of electron (Abe et al., Abe et al., The of not only the of the cation from the luminal but also main chain oxygen that are for the high-affinity K+-coordination to their as described K+-binding site the protein its specific transport is one of the for membrane transport crystal structure a high-affinity K+-binding site of H+,K+-ATPase (Figure 2), with the coordination of the surrounding amino acids at 2.5 Å The bound single K+ in H+,K+-ATPase is located at a position corresponding to site II of the Na+,K+-ATPase state (Morth et al., 2007; Shinoda et al., 2009). The bound K+ is by oxygen located within Å (Table 2). a large to K+ coordination 3 three oxygen from and and two from and The total et al., and for the bound K+ is for K+ is indicating that the K+ is almost by the surrounding oxygen (Table 2). results were obtained for Rb+ coordination in the Y799W(Rb+)E2-MgFx and Y799W(Rb+)E2-AlFx but these results are different from those previously reported for the Rb+-bound structure (Abe et al., see Table 2). As the helix is at coordination by the main chain becomes (Figure These in to the probably determine the of luminal gate closure coupled with K+-occlusion (Figure supplement 1). It to be a natural that these K+ as is also found in Na+,K+-ATPase et al., 2009) and et al., 1998). of also a at the K+ site, which is located on the of at the helix Figure Download asset Open asset K+-binding site. (A, of the transmembrane cation-binding site in the H+,K+-ATPase Y799W(K+)E2-MgFx state in viewed approximately parallel to the membrane from the TM6 (A) or approximately to the membrane from the cytoplasmic indicate that are within Å of presumably hydrogen or The purple