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. 2018 Aug 24;8(1):12732.
doi: 10.1038/s41598-018-30885-w.

K + Binding and Proton Redistribution in the E 2 P State of the H +, K +-ATPase

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Free PMC article

K + Binding and Proton Redistribution in the E 2 P State of the H +, K +-ATPase

Vikas Dubey et al. Sci Rep. .
Free PMC article

Abstract

The H+, K+-ATPase (HKA) uses ATP to pump protons into the gastric lumen against a million-fold proton concentration gradient while counter-transporting K+ from the lumen. The mechanism of release of a proton into a highly acidic stomach environment, and the subsequent binding of a K+ ion necessitates a network of protonable residues and dynamically changing protonation states in the cation binding pocket dominated by five acidic amino acid residues E343, E795, E820, D824, and D942. We perform molecular dynamics simulations of spontaneous K+ binding to all possible protonation combinations of the acidic amino acids and carry out free energy calculations to determine the optimal protonation state of the luminal-open E2P state of the pump which is ready to bind luminal K+. A dynamic pKa correlation analysis reveals the likelihood of proton transfer events within the cation binding pocket. In agreement with in-vitro measurements, we find that E795 is likely to be protonated, and that E820 is at the center of the proton transfer network in the luminal-open E2P state. The acidic residues D942 and D824 are likely to remain protonated, and the proton redistribution occurs predominantly amongst the glutamate residues exposed to the lumen. The analysis also shows that a lower number of K+ ions bind at lower pH, modeled by a higher number of protons in the cation binding pocket, in agreement with the 'transport stoichiometry variation' hypothesis.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(A) The E2P-like H+, K+-ATPase model system embedded in POPC bilayer membrane. Water molecules and the lipid chains are omitted for clarity. Orange and blue beads represent the position of phosphorus atom in POPC lipids and bound K+ in the binding pocket, respectively. The red box shows the ion binding pocket (B) Five key acidic residues and one positively charged K791 residue in the cation binding pocket. E820 and D824 are protonated in this image.
Figure 2
Figure 2
Three observed K+ binding conformations (A) 1 K+ is bound in site I/II (E820+D824+). (B) 2 K+ are bound in site I/II (E795+D824+D942+). (C) 1 K+ is bound in site I/II and another K+ is bound in site III (E343+D824+). (D) Average number of spontaneously bound K+ in 20 different protonation states with either 2 or 3 protonated residues in the binding pocket. Each bar represents the average number of bound K+ from three MD trajectories with different initial velocity distribution. The average number was calculated from the number of bound ions at the end of the each simulation. Red and blue bars represent the number of bound K+ ions in site I/II and site III, respectively. Dashed and solid lines on the bars show the standard error at site I/II and site III respectively. Sites I and II could not be distinguished in the simulations. Green and brown dashed horizontal lines show the averages of all the 2 protonated and 3 protonated states respectively.
Figure 3
Figure 3
(A,C) and (E) pKa fluctuations of the 5 key acidic residues in the cation binding pocket of the HKA during the 250 ns trajectories of the E795+D824+D942+, E795+E820+D942+ and E343+E795+D824+ simulations. (B,D) and (F) Pearson’s correlation coefficients (r) between pairs of acidic residues. The three highest values of r have been shown with 99% confidence intervals. The pKa values are calculated every 1 ns. The red (r = −1) and green (r = 1) colours represent perfect negative and perfect positive linear correlation. The dashed line in (E) shows the time when K+ binds to the cation binding site (near t = 126 ns). In case of (A) and (C) K+ binds at the very beginning of the simulation (near t = 0 ns).
Figure 4
Figure 4
Diagrams of proton transfer pathways from pKa correlation coefficient analysis in Table 1. The direction of the arrows represents the proton transfer direction and the thickness represent the likelihood of a proton transfer event calculated from pKa correlation matrices. The transparent residues in the background are a guide to the eye, and do not represent a specific simulation.
Figure 5
Figure 5
(A) The pulling pathway of the bound K+ ion from the binding site to the gastric lumen for the free energy calculations. The pathway is shown in blue and the arrow represents the pulling direction. (B) Potential of mean force along the z-axis computed for the three different protonation states as calculated with umbrella sampling. The error bars are obtained from bootstrapping analysis.
Figure 6
Figure 6
K+-dependence on ATPase activities of wild-type and indicated mutants. Data plotted were corrected for background values in the absence of K+ and in the presence of the specific inhibitor SCH28080, and normalized to their maximum velocity as 100% The affinities for K+ (Km) in our measurement showed 1.2 mM, 5.5 mM and 0.4 mM for wild-type, E343Q and E795Q respectively. The affinity for K+ of E820Q mutant could not be determined as this mutant showed K+-independent ATPase activity as reported previously, see text for details.

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References

    1. Berg, J. M., Tymoczko, J. L. & Stryer, L. Biochemistry (7th ed.) (2012).
    1. Shin JM, Munson K, Vagin O, Sachs G. The gastric H, K-ATPase: structure, function, and inhibition. Pflügers. Arch.- Eur. J. Physiol. 2009;457:609–622. doi: 10.1007/s00424-008-0495-4. - DOI - PMC - PubMed
    1. Munson K, Garcia R, Sachs G. Inhibitor and ion binding sites on the gastric H, K-ATPase. Biochemistry. 2005;44:5267–5284. doi: 10.1021/bi047761p. - DOI - PubMed
    1. Post RL, Kume S, Tobin T, Orcutt B, Sen AK. Flexibility of an active center in sodium-plus-potassium adenosine triphosphatase. J. Gen. Physiol. 1969;54:S306. doi: 10.1085/jgp.54.1.306. - DOI - PMC - PubMed
    1. Albers RW. Biochemical aspects of active transport. Annu. Rev. Biochem. 1967;36:727–756. doi: 10.1146/annurev.bi.36.070167.003455. - DOI - PubMed

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