Journal: EMBO J / Year: 2009 Title: Inter-subunit interaction of gastric H+,K+-ATPase prevents reverse reaction of the transport cycle. Authors: Kazuhiro Abe / Kazutoshi Tani / Tomohiro Nishizawa / Yoshinori Fujiyoshi / Abstract: The gastric H(+),K(+)-ATPase is an ATP-driven proton pump responsible for generating a million-fold proton gradient across the gastric membrane. We present the structure of gastric H(+),K(+)-ATPase ...The gastric H(+),K(+)-ATPase is an ATP-driven proton pump responsible for generating a million-fold proton gradient across the gastric membrane. We present the structure of gastric H(+),K(+)-ATPase at 6.5 A resolution as determined by electron crystallography of two-dimensional crystals. The structure shows the catalytic alpha-subunit and the non-catalytic beta-subunit in a pseudo-E(2)P conformation. Different from Na(+),K(+)-ATPase, the N-terminal tail of the beta-subunit is in direct contact with the phosphorylation domain of the alpha-subunit. This interaction may hold the phosphorylation domain in place, thus stabilizing the enzyme conformation and preventing the reverse reaction of the transport cycle. Indeed, truncation of the beta-subunit N-terminus allowed the reverse reaction to occur. These results suggest that the beta-subunit N-terminus prevents the reverse reaction from E(2)P to E(1)P, which is likely to be relevant for the generation of a large H(+) gradient in vivo situation.
History
Deposition
Mar 9, 2009
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Header (metadata) release
Mar 24, 2009
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Map release
May 22, 2009
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Update
Dec 26, 2012
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Current status
Dec 26, 2012
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
pH: 4.87 Details: 20 mM propionate, 1 mM MgCl2, 0.5 mM AlCl3, 4 mM NaF, 0.3 mM ADP, 3 mM DTT, 10% (w/v) glycerol
Grid
Details: molybdenum grid
Vitrification
Cryogen name: NITROGEN / Chamber temperature: 4.2 K / Instrument: REICHERT-JUNG PLUNGER Details: Vitrification instrument: Reichert plunger. vitrification carried out in cold room at 4 degrees celsius. Method: carbon sandwich preparation
Details
dialysis
Crystal formation
Details: dialysis
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Electron microscopy
Microscope
JEOL KYOTO-3000SFF
Temperature
Average: 4.2 K
Image recording
Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 346 / Average electron dose: 25 e/Å2 / Bits/pixel: 12
Tilt angle min
0
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Specimen holder: top entry / Specimen holder model: JEOL / Tilt angle max: 73.3 / Tilt series - Axis1 - Min angle: 0 ° / Tilt series - Axis1 - Max angle: 73.3 °
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Image processing
Final reconstruction
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: OTHER / Software - Name: MRC
Crystal parameters
Unit cell - A: 140.4 Å / Unit cell - B: 109.0 Å / Unit cell - C: 320.0 Å / Unit cell - γ: 90.0 ° / Unit cell - α: 90.0 ° / Unit cell - β: 90.0 ° / Plane group: P 2 21 21
CTF correction
Details: Each image
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