Dataset Information

K+ binding and proton redistribution in the E2P state of the H+, K+-ATPase.

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.

SUBMITTER: Dubey V

PROVIDER: S-EPMC6109069 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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K+ binding and proton redistribution in the E2P state of the H+, K+-ATPase.

Dubey Vikas V Han Minwoo M Kopec Wojciech W Solov'yov Ilia A IA Abe Kazuhiro K Khandelia Himanshu H

Scientific reports 20180824 1

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 E34 ...[more]

PMID: 30143663

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Project description:The catalytic alpha-subunits of Na,K- and H,K-ATPase require an accessory beta-subunit for proper folding, maturation, and plasma membrane delivery but also for cation transport. To investigate the functional significance of the beta-N terminus of the gastric H,K-ATPase in vivo, several N-terminally truncated beta-variants were expressed in Xenopus oocytes, together with the S806C alpha-subunit variant. Upon labeling with the reporter fluorophore tetramethylrho da mine-6-maleimide, this construct can be used to determine the voltage-dependent distribution between E(1)P/E(2)P states. Whereas the E(1)P/E(2)P conformational equilibrium was unaffected for the shorter N-terminal deletions betaDelta4 and betaDelta8, we observed significant shifts toward E(1)P for the two larger deletions betaDelta13 and betaDelta29. Moreover, the reduced DeltaF/F ratios of betaDelta13 and betaDelta29 indicated an increased reverse reaction via E(2)P --> E(1)P + ADP --> E(1) + ATP, because cell surface expression was completely unaffected. This interpretation is supported by the reduced sensitivity of the mutants toward the E(2)P-specific inhibitor SCH28080, which becomes especially apparent at high concentrations (100 microm). Despite unaltered apparent Rb(+) affinities, the maximal Rb(+) uptake of these mutants was also significantly lowered. Considering the two putative interaction sites between the beta-N terminus and alpha-subunit revealed by the recent cryo-EM structure, the N-terminal tail of the H,K-ATPase beta-subunit may stabilize the pump in the E(2)P conformation, thereby increasing the efficiency of proton release against the million-fold proton gradient of the stomach lumen. Finally, we demonstrate that a similar truncation of the beta-N terminus of the closely related Na,K-ATPase does not affect the E(1)P/E(2)P distribution or pump activity, indicating that the E(2)P-stabilizing effect by the beta-N terminus is apparently a unique property of the H,K-ATPase.

Project description:Based on the crystal structure of lactose permease (LacY) open to the cytoplasm, a hybrid molecular simulation approach with self-guided Langevin dynamics is used to describe conformational changes that lead to a periplasmic-open state. This hybrid approach consists of implicit (IM) and explicit (EX) membrane simulations and requires self-guided Langevin dynamics to enhance protein motions during the IM simulations. The pore radius of the lumen increases by 3.5 Å on the periplasmic side and decreases by 2.5 Å on the cytoplasmic side (relative to the crystal structure), suggesting a lumen that is fully open to the periplasm to allow for extracellular sugar transport and closed to the cytoplasm. Based on our simulations, the mechanism that triggers this conformational change to the periplasmic-open state is the protonation of Glu269 and binding of the disaccharide. Then, helix packing is destabilized by breaking of several side chains involved in hydrogen bonding (Asn245, Ser41, Glu374, Lys42, and Gln242). For the periplasmic-open conformations obtained from our simulations, helix-helix distances agree well with experimental measurements using double electron-electron resonance, fluorescence resonance energy transfer, and varying sized cross-linkers. The periplasmic-open conformations are also in compliance with various substrate accessibility/reactivity measurements that indicate an opening of the protein lumen on the periplasmic side on sugar binding. The comparison with these measurements suggests a possible incomplete closure of the cytoplasmic half in our simulations. However, the closure is sufficient to prevent the disaccharide from transporting to the cytoplasm, which is in accordance with the well-established alternating access model. Ser53, Gln60, and Phe354 are determined to be important in sugar transport during the periplasmic-open stage of the sugar transport cycle and the sugar is found to undergo an orientational change in order to escape the protein lumen.

Dataset Information

K+ binding and proton redistribution in the E2P state of the H+, K+-ATPase.

Publications

K<sup>+</sup> binding and proton redistribution in the E<sub>2</sub>P state of the H<sup>+</sup>, K<sup>+</sup>-ATPase.

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