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

AbstractThe 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 H+,K+-ATPase, indicating an exchange of 1H+/1K+ per hydrolysis of one ATP molecule. This fulfils the energy requirement for the generation of a six pH unit gradient across the membrane. The structural basis of K+recognition is resolved, supported by molecular dynamics simulations, and this establishes how H+,K+-ATPase overcomes the energetic challenge to generate an H+ gradient of more than a million-fold – the highest cation gradient known in any mammalian tissue – across the membrane.

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A single K+-binding site in the crystal structure of the gastric proton pump

eLife ◽

10.7554/elife.47701 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 7

Author(s):

Kenta Yamamoto ◽

Vikas Dubey ◽

Katsumasa Irie ◽

Hanayo Nakanishi ◽

Himanshu Khandelia ◽

...

Keyword(s):

Binding Site ◽

Proton Pump ◽

Atp Hydrolysis ◽

Energy Requirement ◽

Cation Binding ◽

Structural Basis ◽

Cation Binding Site ◽

Gastric Proton Pump ◽

Dynamics Simulations ◽

P Type

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.

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High diagnostic performance of a novel assay for autoantibodies against ATP4A and ATP4B subunits of gastric proton pump H+,K+-ATPase in atrophic body gastritis patients

10.26226/morressier.57c5383cd462b80296c9c6f0 ◽

2016 ◽

Author(s):

Edith Lahner

Keyword(s):

Proton Pump ◽

Diagnostic Performance ◽

Gastric Proton Pump

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The Crystal Structure of Calmodulin Bound to the Cardiac Ryanodine Receptor (RyR2) at Residues Phe4246–Val4270 Reveals a Fifth Calcium Binding Site

Biochemistry ◽

10.1021/acs.biochem.1c00152 ◽

2021 ◽

Author(s):

Qinhong Yu ◽

David E. Anderson ◽

Ramanjeet Kaur ◽

Andrew J. Fisher ◽

James B. Ames

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Ryanodine Receptor ◽

Calcium Binding ◽

Calcium Binding Site ◽

Cardiac Ryanodine Receptor

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The crystal structure of poplar apoplastocyanin at 1.8-A resolution. The geometry of the copper-binding site is created by the polypeptide.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)43220-0 ◽

1984 ◽

Vol 259 (5) ◽

pp. 2822-2825 ◽

Cited By ~ 6

Author(s):

T P Garrett ◽

D J Clingeleffer ◽

J M Guss ◽

S J Rogers ◽

H C Freeman

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Copper Binding

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Luminescent Immunoprecipitation System (LIPS) for Detection of Autoantibodies Against ATP4A and ATP4B Subunits of Gastric Proton Pump H+,K+-ATPase in Atrophic Body Gastritis Patients

Clinical and Translational Gastroenterology ◽

10.1038/ctg.2016.71 ◽

2017 ◽

Vol 8 (1) ◽

pp. e215 ◽

Cited By ~ 13

Author(s):

Edith Lahner ◽

Cristina Brigatti ◽

Ilaria Marzinotto ◽

Marilia Carabotti ◽

Giulia Scalese ◽

...

Keyword(s):

Proton Pump ◽

Gastric Proton Pump

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Characterization of the Acetate Binding Pocket in the Methanosarcina thermophila Acetate Kinase

Journal of Bacteriology ◽

10.1128/jb.187.7.2386-2394.2005 ◽

2005 ◽

Vol 187 (7) ◽

pp. 2386-2394 ◽

Cited By ~ 36

Author(s):

Cheryl Ingram-Smith ◽

Andrea Gorrell ◽

Sarah H. Lawrence ◽

Prabha Iyer ◽

Kerry Smith ◽

...

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Binding Pocket ◽

Acetate Kinase ◽

Phosphoryl Group ◽

Acetyl Phosphate ◽

Hydrophobic Pocket ◽

Methanosarcina Thermophila ◽

Methyl Group ◽

Substrate Binding Sites

ABSTRACT Acetate kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer of the ATP γ-phosphoryl group to acetate. Inspection of the crystal structure of the Methanosarcina thermophila enzyme containing only ADP revealed a solvent-accessible hydrophobic pocket formed by residues Val93, Leu122, Phe179, and Pro232 in the active site cleft, which identified a potential acetate binding site. The hypothesis that this was a binding site was further supported by alignment of all acetate kinase sequences available from databases, which showed strict conservation of all four residues, and the recent crystal structure of the M. thermophila enzyme with acetate bound in this pocket. Replacement of each residue in the pocket produced variants with Km values for acetate that were 7- to 26-fold greater than that of the wild type, and perturbations of this binding pocket also altered the specificity for longer-chain carboxylic acids and acetyl phosphate. The kinetic analyses of variants combined with structural modeling indicated that the pocket has roles in binding the methyl group of acetate, influencing substrate specificity, and orienting the carboxyl group. The kinetic analyses also indicated that binding of acetyl phosphate is more dependent on interactions of the phosphate group with an unidentified residue than on interactions between the methyl group and the hydrophobic pocket. The analyses also indicated that Phe179 is essential for catalysis, possibly for domain closure. Alignments of acetate kinase, propionate kinase, and butyrate kinase sequences obtained from databases suggested that these enzymes have similar catalytic mechanisms and carboxylic acid substrate binding sites.

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Crystal structure of the DENR-MCT-1 complex revealed zinc-binding site essential for heterodimer formation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809688116 ◽

2018 ◽

Vol 116 (2) ◽

pp. 528-533 ◽

Cited By ~ 6

Author(s):

Ivan B. Lomakin ◽

Sergey E. Dmitriev ◽

Thomas A. Steitz

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Translation Initiation ◽

Zinc Ion ◽

Zinc Binding ◽

Ion Binding ◽

Binding Interface ◽

Zinc Binding Site ◽

Reading Frames ◽

Zinc Ion Binding

The density-regulated protein (DENR) and the malignant T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein support noncanonical translation initiation, promote translation reinitiation on a specific set of mRNAs with short upstream reading frames, and regulate ribosome recycling. DENR and MCT-1 form a heterodimer, which binds to the ribosome. We determined the crystal structure of the heterodimer formed by human MCT-1 and the N-terminal domain of DENR at 2.0-Å resolution. The structure of the heterodimer reveals atomic details of the mechanism of DENR and MCT-1 interaction. Four conserved cysteine residues of DENR (C34, C37, C44, C53) form a classical tetrahedral zinc ion-binding site, which preserves the structure of the DENR’s MCT-1–binding interface that is essential for the dimerization. Substitution of all four cysteines by alanine abolished a heterodimer formation. Our findings elucidate further the mechanism of regulation of DENR-MCT-1 activities in unconventional translation initiation, reinitiation, and recycling.

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