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Crystal structure of a membrane-embedded H+-translocating pyrophosphatase


H+-translocating pyrophosphatases (H+-PPases) are active proton transporters that establish a proton gradient across the endomembrane by means of pyrophosphate (PPi) hydrolysis1,2. H+-PPases are found primarily as homodimers in the vacuolar membrane of plants and the plasma membrane of several protozoa and prokaryotes2,3. The three-dimensional structure and detailed mechanisms underlying the enzymatic and proton translocation reactions of H+-PPases are unclear. Here we report the crystal structure of a Vigna radiata H+-PPase (VrH+-PPase) in complex with a non-hydrolysable substrate analogue, imidodiphosphate (IDP), at 2.35 Å resolution. Each VrH+-PPase subunit consists of an integral membrane domain formed by 16 transmembrane helices. IDP is bound in the cytosolic region of each subunit and trapped by numerous charged residues and five Mg2+ ions. A previously undescribed proton translocation pathway is formed by six core transmembrane helices. Proton pumping can be initialized by PPi hydrolysis, and H+ is then transported into the vacuolar lumen through a pathway consisting of Arg 242, Asp 294, Lys 742 and Glu 301. We propose a working model of the mechanism for the coupling between proton pumping and PPi hydrolysis by H+-PPases.

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Figure 1: The crystal structure of the Vr H + -PPase-IDP complex.
Figure 2: The substrate-binding site.
Figure 3: The proton transport pathway of Vr H + -PPase.
Figure 4: A working model for proton pumping in Vr H + -PPase.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates and structure factors of VrH1-PPase are deposited in the Protein Data Bank under the accession code 4A01.


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We thank M. F. Tam, P. C. Huang and H. J. Kung for their critical reading of the manuscript and for useful comments. The X-ray diffraction data were collected from the in-house X-ray facility at National Tsing Hua University and from beamlines BL13B1/BL13C1 at the National Synchrotron Radiation Research Center, Taiwan, and BL44XU/BL12B2 at SPring-8, Japan. This work was supported by grants from the National Science Council of Taiwan (NSC 99-2311-B-007-007-MY3 to Y.-J.S.; NSC 100-2311-B-007-001-MY3 and NSC 100-2627-M-007-012 to R.-L.P.) and National Tsing Hua University, Taiwan (99N82416E1 to Y.-J.S.).

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Authors and Affiliations



S.-M.L. isolated the VrH+-PPase, grew the crystals and determined the structure. J.-Y.T. assisted with structural determination and completed the structural refinement. C.-D.H. assisted with structural phase analysis. C.-L.C. performed the data collection and data processing. J.-Y.T. and M.-H.L. assisted with the data collection. Y.-T.H. and T.-H.L. assisted in protein isolation. All authors participated in discussions of the results and in preparing the manuscript. Y.-J.S. and R.-L.P. supervised the project and wrote the manuscript.

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Correspondence to Rong-Long Pan or Yuh-Ju Sun.

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The authors declare no competing financial interests.

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Lin, SM., Tsai, JY., Hsiao, CD. et al. Crystal structure of a membrane-embedded H+-translocating pyrophosphatase. Nature 484, 399–403 (2012).

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