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

Abstract

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.

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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.

References

  1. Rea, P. A. & Poole, R. J. Vacuolar H+-translocating pyrophosphatase. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 157–180 (1993)

    Article  CAS  Google Scholar 

  2. Maeshima, M. Vacuolar H+-pyrophosphatase. Biochim. Biophys. Acta 1465, 37–51 (2000)

    Article  CAS  Google Scholar 

  3. Serrano, A., Perez-Castineira, J. R., Baltscheffsky, H. & Baltscheffsky, M. Proton-pumping inorganic pyrophosphatases in some archaea and other extremophilic prokaryotes. J. Bioenerg. Biomembr. 36, 127–133 (2004)

    Article  CAS  Google Scholar 

  4. Maeshima, M. Tonoplast transporters: organization and function. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 469–497 (2001)

    Article  CAS  Google Scholar 

  5. Baltscheffsky, H., Von Stedingk, L. V., Heldt, H. W. & Klingenberg, M. Inorganic pyrophosphate: formation in bacterial photophosphorylation. Science 153, 1120–1122 (1966)

    Article  ADS  CAS  Google Scholar 

  6. Chanson, A., Fichmann, J., Spear, D. & Taiz, L. Pyrophosphate-driven proton transport by microsomal membranes of corn coleoptiles. Plant Physiol. 79, 159–164 (1985)

    Article  CAS  Google Scholar 

  7. Rea, P. A. & Poole, R. J. Proton-translocating inorganic pyrophosphatase in red beet (Beta vulgaris L.) tonoplast vesicles. Plant Physiol. 77, 46–52 (1985)

    Article  CAS  Google Scholar 

  8. Britten, C. J., Turner, J. C. & Rea, P. A. Identification and purification of substrate binding subunit of higher plant H+-translocating inorganic pyrophosphatase. FEBS Lett. 256, 200–206 (1989)

    Article  CAS  Google Scholar 

  9. Maeshima, M. & Yoshida, S. Purification and properties of vacuolar membrane proton-translocating inorganic pyrophosphatase from mung bean. J. Biol. Chem. 264, 20068–20073 (1989)

    CAS  PubMed  Google Scholar 

  10. Sarafian, V., Kim, Y., Poole, R. J. & Rea, P. A. Molecular cloning and sequence of cDNA encoding the pyrophosphate-energized vacuolar membrane proton pump of Arabidopsis thaliana . Proc. Natl Acad. Sci. USA 89, 1775–1779 (1992)

    Article  ADS  CAS  Google Scholar 

  11. Kim, E. J., Zhen, R. G. & Rea, P. A. Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport. Proc. Natl Acad. Sci. USA 91, 6128–6132 (1994)

    Article  ADS  CAS  Google Scholar 

  12. Rea, P. A. et al. Vacuolar H+-translocating pyrophosphatases: a new category of ion translocase. Trends Biochem. Sci. 17, 348–353 (1992)

    Article  CAS  Google Scholar 

  13. Belogurov, G. A. & Lahti, R. A lysine substitute for K+. A460K mutation eliminates K+ dependence in H+-pyrophosphatase of Carboxydothermus hydrogenoformans . J. Biol. Chem. 277, 49651–49654 (2002)

    Article  CAS  Google Scholar 

  14. Li, J. et al. Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ development. Science 310, 121–125 (2005)

    Article  ADS  CAS  Google Scholar 

  15. Ferjani, A. et al. Keep an eye on PPi: the vacuolar-type H+-pyrophosphatase regulates postgerminative development in Arabidopsis . Plant Cell 23, 2895–2908 (2011)

    Article  CAS  Google Scholar 

  16. Park, S. et al. Up-regulation of a H+-pyrophosphatase (H+-PPase) as a strategy to engineer drought-resistant crop plants. Proc. Natl Acad. Sci. USA 102, 18830–18835 (2005)

    Article  ADS  CAS  Google Scholar 

  17. Guo, S. et al. Molecular cloning and characterization of a vacuolar H+-pyrophosphatase gene, SsVP, from the halophyte Suaeda salsa and its overexpression increases salt and drought tolerance of Arabidopsis . Plant Mol. Biol. 60, 41–50 (2006)

    Article  CAS  Google Scholar 

  18. Nakanishi, Y., Saijo, T., Wada, Y. & Maeshima, M. Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase. J. Biol. Chem. 276, 7654–7660 (2001)

    Article  CAS  Google Scholar 

  19. Hirono, M., Nakanishi, Y. & Maeshima, M. Essential amino acid residues in the central transmembrane domains and loops for energy coupling of Streptomyces coelicolor A3(2) H+-pyrophosphatase. Biochim. Biophys. Acta 1767, 930–939 (2007)

    Article  CAS  Google Scholar 

  20. Lee, C. H. et al. Identification of essential lysines involved in substrate binding of vacuolar H+-pyrophosphatase. J. Biol. Chem. 286, 11970–11976 (2011)

    Article  CAS  Google Scholar 

  21. Gordon-Weeks, R., Steele, S. H. & Leigh, R. A. The role of magnesium, pyrophosphate, and their complexes as substrates and activators of the vacuolar H+-pumping inorganic pyrophosphatase: studies using ligand protection from covalent inhibitors. Plant Physiol. 111, 195–202 (1996)

    Article  CAS  Google Scholar 

  22. Samygina, V. R. et al. Reversible inhibition of Escherichia coli inorganic pyrophosphatase by fluoride: trapped catalytic intermediates in cryo-crystallographic studies. J. Mol. Biol. 366, 1305–1317 (2007)

    Article  CAS  Google Scholar 

  23. Tzeng, C. M. et al. Subunit structure of vacuolar proton-pyrophosphatase as determined by radiation inactivation. Biochem. J. 316, 143–147 (1996)

    Article  CAS  Google Scholar 

  24. Zhen, R. G., Kim, E. J. & Rea, P. A. Acidic residues necessary for pyrophosphate-energized pumping and inhibition of the vacuolar H+-pyrophosphatase by N,N′-dicyclohexylcarbodiimide. J. Biol. Chem. 272, 22340–22348 (1997)

    Article  CAS  Google Scholar 

  25. Pan, Y. J. et al. The transmembrane domain 6 of vacuolar H+-pyrophosphatase mediates protein targeting and proton transport. Biochim. Biophys. Acta 1807, 59–67 (2011)

    Article  ADS  CAS  Google Scholar 

  26. Luecke, H., Richter, H. T. & Lanyi, J. K. Proton transfer pathways in bacteriorhodopsin at 2.3 angstrom resolution. Science 280, 1934–1937 (1998)

    Article  ADS  CAS  Google Scholar 

  27. Van, R. C. et al. Role of transmembrane segment 5 of the plant vacuolar H+-pyrophosphatase. Biochim. Biophys. Acta 1709, 84–94 (2005)

    Article  CAS  Google Scholar 

  28. Buch-Pedersen, M. J., Pedersen, B. P., Veierskov, B., Nissen, P. & Palmgren, M. G. Protons and how they are transported by proton pumps. Pflugers Arch. 457, 573–579 (2009)

    Article  CAS  Google Scholar 

  29. Pedersen, B. P., Buch-Pedersen, M. J., Morth, J. P., Palmgren, M. G. & Nissen, P. Crystal structure of the plasma membrane proton pump. Nature 450, 1111–1114 (2007)

    Article  ADS  CAS  Google Scholar 

  30. Huang, Y. T. et al. Distance variations between active sites of H+-pyrophosphatase determined by fluorescence resonance energy transfer. J. Biol. Chem. 285, 23655–23664 (2010)

    Article  CAS  Google Scholar 

  31. Hsu, S. H. et al. Purification, characterization, and spectral analyses of histidine-tagged vacuolar H+-pyrophosphatase expressed in yeast. Bot. Stud. (Taipei, Taiwan) 50, 291–301 (2009)

    CAS  Google Scholar 

  32. Kirsch, R. D. & Joly, E. An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes. Nucleic Acids Res. 26, 1848–1850 (1998)

    Article  CAS  Google Scholar 

  33. McPherson, A. Current approaches to macromolecular crystallization. Eur. J. Biochem. 189, 1–23 (1990)

    Article  CAS  Google Scholar 

  34. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  35. Potterton, E., Briggs, P., Turkenburg, M. & Dodson, E. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994)

    Article  Google Scholar 

  36. Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861 (1999)

    Article  CAS  Google Scholar 

  37. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002)

    Article  Google Scholar 

  38. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010)

    Article  CAS  Google Scholar 

  39. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010)

    Article  CAS  Google Scholar 

  40. Vagin, A. A. et al. REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr. D Biol. Crystallogr. 60, 2184–2195 (2004)

    Article  Google Scholar 

  41. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283–291 (1993)

    Article  CAS  Google Scholar 

  42. DeLano, W. L. The PyMOL Molecular Graphics System (DeLano Scientific) (2002)

  43. Ho, B. K. & Gruswitz, F. HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures. BMC Struct. Biol. 8, 49 (2008)

    Article  Google Scholar 

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Acknowledgements

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

Authors

Contributions

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.

Corresponding authors

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). https://doi.org/10.1038/nature10963

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