Rotation mechanism of Enterococcus hirae V1-ATPase based on asymmetric crystal structures

Journal name:
Nature
Volume:
493,
Pages:
703–707
Date published:
DOI:
doi:10.1038/nature11778
Received
Accepted
Published online
Corrected online

In various cellular membrane systems, vacuolar ATPases (V-ATPases) function as proton pumps, which are involved in many processes such as bone resorption and cancer metastasis, and these membrane proteins represent attractive drug targets for osteoporosis and cancer1. The hydrophilic V1 portion is known as a rotary motor, in which a central axis DF complex rotates inside a hexagonally arranged catalytic A3B3 complex using ATP hydrolysis energy, but the molecular mechanism is not well defined owing to a lack of high-resolution structural information. We previously reported on the in vitro expression, purification and reconstitution of Enterococcus hirae V1-ATPase from the A3B3 and DF complexes2, 3. Here we report the asymmetric structures of the nucleotide-free (2.8Å) and nucleotide-bound (3.4Å) A3B3 complex that demonstrate conformational changes induced by nucleotide binding, suggesting a binding order in the right-handed rotational orientation in a cooperative manner. The crystal structures of the nucleotide-free (2.2Å) and nucleotide-bound (2.7Å) V1-ATPase are also reported. The more tightly packed nucleotide-binding site seems to be induced by DF binding, and ATP hydrolysis seems to be stimulated by the approach of a conserved arginine residue. To our knowledge, these asymmetric structures represent the first high-resolution view of the rotational mechanism of V1-ATPase.

At a glance

Figures

  1. Structure of the A3B3 complex.
    Figure 1: Structure of the A3B3 complex.

    a, Side view of the nucleotide-free A3B3 structure (eA3B3). b, c, Superimposed structures at the N-terminal β-barrel (white) of three structures of Eh-A (b) and Eh-B (c) in eA3B3. Open (O and O’) and closed (C) conformations of Eh-A and Eh-B are shown in light and dark colours, respectively. The P-loop and arm are shown in yellow and white, respectively. d, Top view of the C-terminal domain (shown in a as transparent surface) of eA3B3 from the N-terminal β-barrel side. Red arrows indicate the nucleotide-binding sites. eh, Structures of the AMP-PNP-bound A3B3 complex (bA3B3) viewed and coloured as in ad. il, Magnified nucleotide-binding sites with conserved residues, corresponding to red (b) and green (f) boxes. Right panels show the A–B interfaces rotated 90° around a vertical axis from the left panels.

  2. Comparison of the asymmetric structures of nucleotide-free A3B3DF and A3B3 complexes.
    Figure 2: Comparison of the asymmetric structures of nucleotide-free A3B3DF and A3B3 complexes.

    a, Side view of the nucleotide-free A3B3DF structure (eV1). b, Top views of the C-terminal domain of eV1 as in Fig. 1d, which is superimposed at the empty form onto that of transparent eA3B3 (grey). Open (O and O’), closed (C) and closer (CR) conformations of Eh-A and -B are shown in light, dark and darker colours, respectively. ch, Protein–protein interactions between A3B3 and DF in eV1. The BC (c), AO (d), BO’ (e), AC (f), BCR (g) and ACR (h) with DF complex in eV1 are shown in side-viewed ribbon representation, which are compared with corresponding subunits (grey) of eA3B3 superimposed as in b. The P-loop is shown in cyan. The residues with buried surface area >10 Å2, as calculated by PDBePISA (http://pdbe.org/pisa/), are shown in yellow.

  3. Comparison of the nucleotide-binding sites.
    Figure 3: Comparison of the nucleotide-binding sites.

    ad, The viewing position, colours and representations of the binding site correspond to those of the right columns in Fig. 1i–l. These structures were superimposed at Eh-A (residues 67–593) of the compared AB pairs. a, Tight form in eV1 (colour) compared with bound form in eA3B3 (grey). b, Bound form in bV1 (colour) compared with bound form in eV1 (grey). c, Tight form in bV1 (colour) compared with tight form in eV1 (grey). d, Tight form in bV1 (colour) compared with bound form in bV1 (grey). The distances (Å) between atoms are shown with dotted lines. e, A schematic representation of the nucleotide-binding sites of bV1. The distances (Å) between atoms in the bound form or tight form (shown in parentheses) are shown with dotted lines. The distances (Å) between Cαs in the superimposed structure (d) are shown in blue brackets.

  4. A model of the rotation mechanism of V1-ATPase.
    Figure 4: A model of the rotation mechanism of V1-ATPase.

    ad, The structure models are on the basis of the crystal structures of bV1 (a and d), eA3B3 (b) and bA3B3 (c) in this study. ATP with yellow ‘P’ in a and d represents an ATP molecule that is committed to hydrolysis. The blue ‘P’ in b represents a Pi molecule after hydrolysis of ATP. See text for further details.

Videos

  1. conformational changes of A3B3 complex induced by AMP-PNP:Mg binding
    Video 1: conformational changes of A3B3 complex induced by AMP-PNP:Mg binding
    The colouring and viewing are consistent with Fig. 1. The video was generated by morphing between X-ray crystal structures of nucleotide-free (eA3B3) and nucleotide-bound (bA3B3) A3B3 complexes using PyMOL.
  2. conformational changes of nucleotide-free eA3B3 complex induced by DF binding
    Video 2: conformational changes of nucleotide-free eA3B3 complex induced by DF binding
    The colouring and viewing are consistent with Fig. 2. The video was generated by morphing between X-ray crystal structures of nucleotide-free A3B3 (eA3B3) and A3B3DF (eV1) complexes using PyMOL.
  3. conformational changes of nucleotide-bound A3B3 complex induced by DF binding
    Video 3: conformational changes of nucleotide-bound A3B3 complex induced by DF binding
    The colouring and viewing are consistent with Fig. 2. The video was generated by morphing between X-ray crystal structures of nucleotide-bound A3B3 (bA3B3) and A3B3DF (bV1) complexes using PyMOL.
  4. conformational difference of the nucleotide-binding sites of nucleotide-bound V1-ATPase
    Video 4: conformational difference of the nucleotide-binding sites of nucleotide-bound V1-ATPase
    The colouring and viewing are consistent with Fig. 3. The video was generated by morphing between X-ray crystal structures of the ACBO’ pair (bound form) and ACRBCR pair (tight form) in nucleotide-bound A3B3DF (bV1) complex using PyMOL.

Accession codes

Primary accessions

Change history

Corrected online 30 January 2013
The structure in Fig. 1e was corrected.

References

  1. Forgac, M. Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nature Rev. Mol. Cell Biol. 8, 917929 (2007)
  2. Arai, S. et al. Reconstitution in vitro of the catalytic portion (NtpA3-B3-D-G complex) of Enterococcus hirae V-type Na+-ATPase. Biochem. Biophys. Res. Commun. 390, 698702 (2009)
  3. Saijo, S. et al. Crystal structure of the central axis DF complex of the prokaryotic V-ATPase. Proc. Natl Acad. Sci. USA 108, 1995519960 (2011)
  4. Walker, J. E. ATP synthesis by rotary catalysis (Nobel Lecture). Angew. Chem. Int. Edn Engl. 37, 23082319 (1998)
  5. Mulkidjanian, A. Y., Makarova, K. S., Galperin, M. Y. & Koonin, E. V. Inventing the dynamo machine: the evolution of the F-type and V-type ATPases. Nature Rev. Microbiol. 5, 892899 (2007)
  6. Abrahams, J. P., Leslie, A. G., Lutter, R. & Walker, J. E. Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria. Nature 370, 621628 (1994)
  7. Menz, R. I., Walker, J. E. & Leslie, A. G. Structure of bovine mitochondrial F1-ATPase with nucleotide bound to all three catalytic sites: implications for the mechanism of rotary catalysis. Cell 106, 331341 (2001)
  8. Kagawa, R., Montgomery, M. G., Braig, K., Leslie, A. G. W. & Walker, J. E. The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride. EMBO J. 23, 27342744 (2004)
  9. Bowler, M. W., Montgomery, M. G., Leslie, A. G. W. & Walker, J. E. Ground state structure of F1-ATPase from bovine heart mitochondria at 1.9 Å resolution. J. Biol. Chem. 282, 1423814242 (2007)
  10. Kabaleeswaran, V., Puri, N., Walker, J. E., Leslie, A. G. W. & Mueller, D. M. Novel features of the rotary catalytic mechanism revealed in the structure of yeast F1 ATPase. EMBO J. 25, 54335442 (2006)
  11. Kabaleeswaran, V. et al. Asymmetric structure of the yeast F1 ATPase in the absence of bound nucleotides. J. Biol. Chem. 284, 1054610551 (2009)
  12. Stock, D., Leslie, A. G. & Walker, J. E. Molecular architecture of the rotary motor in ATP synthase. Science 286, 17001705 (1999)
  13. Shirakihara, Y. et al. The crystal structure of the nucleotide-free α3β3 subcomplex of F1-ATPase from the thermophilic Bacillus PS3 is a symmetric trimer. Structure 5, 825836 (1997)
  14. Cingolani, G. & Duncan, T. M. Structure of the ATP synthase catalytic complex (F1) from Escherichia coli in an autoinhibited conformation. Nature Struct. Mol. Biol. 18, 701707 (2011)
  15. Noji, H., Yasuda, R., Yoshida, M. & Kinosita, K. Direct observation of the rotation of F1-ATPase. Nature 386, 299302 (1997)
  16. Yasuda, R., Noji, H., Yoshida, M., Kinosita, K. & Itoh, H. Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase. Nature 410, 898904 (2001)
  17. Adachi, K. et al. Coupling of rotation and catalysis in F1-ATPase revealed by single-molecule imaging and manipulation. Cell 130, 309321 (2007)
  18. Toei, M. et al. Dodecamer rotor ring defines H+/ATP ratio for ATP synthesis of prokaryotic V-ATPase from Thermus thermophilus. Proc. Natl Acad. Sci. USA 104, 2025620261 (2007)
  19. Maher, M. J. et al. Crystal structure of A3B3 complex of V-ATPase from Thermus thermophilus. EMBO J. 28, 37713779 (2009)
  20. Numoto, N., Hasegawa, Y., Takeda, K. & Miki, K. Inter-subunit interaction and quaternary rearrangement defined by the central stalk of prokaryotic V1-ATPase. EMBO Rep. 10, 12281234 (2009)
  21. Imamura, H. et al. Rotation scheme of V1-motor is different from that of F1-motor. Proc. Natl Acad. Sci. USA 102, 1792917933 (2005)
  22. Murata, T., Igarashi, K., Kakinuma, Y. & Yamato, I. Na+ binding of V-type Na+-ATPase in Enterococcus hirae. J. Biol. Chem. 275, 1341513419 (2000)
  23. Murata, T., Yamato, I., Kakinuma, Y., Leslie, A. G. W. & Walker, J. E. Structure of the rotor of the V-Type Na+-ATPase from Enterococcus hirae. Science 308, 654659 (2005)
  24. Murata, T. et al. Ion binding and selectivity of the rotor ring of the Na+-transporting V-ATPase. Proc. Natl Acad. Sci. USA 105, 86078612 (2008)
  25. Mizutani, K. et al. Structure of the rotor ring modified with N,N-dicyclohexylcarbodiimide of the Na+-transporting vacuolar ATPase. Proc. Natl Acad. Sci. USA 108, 1347413479 (2011)
  26. Murata, T., Yamato, I. & Kakinuma, Y. Structure and mechanism of vacuolar Na+-translocating ATPase from Enterococcus hirae. J. Bioenerg. Biomembr. 37, 411413 (2005)
  27. Yamamoto, M. et al. Interaction and stoichiometry of the peripheral stalk subunits NtpE and NtpF and the N-terminal hydrophilic domain of NtpI of Enterococcus hirae V-ATPase. J. Biol. Chem. 283, 1942219431 (2008)
  28. Zhou, M. et al. Mass spectrometry of intact V-type ATPases reveals bound lipids and the effects of nucleotide binding. Science 334, 380385 (2011)
  29. Liu, Q. et al. Site-directed mutagenesis of the yeast V-ATPase A subunit. J. Biol. Chem. 272, 1175011756 (1997)
  30. Dittrich, M., Hayashi, S. & Schulten, K. On the mechanism of ATP hydrolysis in F1-ATPase. Biophys. J. 85, 22532266 (2003)
  31. Kigawa, T. et al. Preparation of Escherichia coli cell extract for highly productive cell-free protein expression. J. Struct. Funct. Genomics 5, 6368 (2004)
  32. Deluca, M. & McElroy, W. D. Purification and properties of firefly luciferase. Methods Enzymol. 57, 315 (1978)
  33. Battye, T. G. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. W. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D 67, 271281 (2011)
  34. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760763 (1994)
  35. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D 66, 2225 (2010)
  36. Kabsch, W. XDS. Acta Crystallogr. D 66, 125132 (2010)
  37. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658674 (2007)
  38. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 21262132 (2004)
  39. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240255 (1997)
  40. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213221 (2010)
  41. Laskowski, R. A., MacArthur, M. W. & Thornton, J. M. Validation of protein models derived from experiment. Curr. Opin. Struct. Biol. 8, 631639 (1998)
  42. Lovell, S. C. et al. Structure validation by Cα geometry: φ, ψ and Cβ deviation. Proteins 50, 437450 (2003)

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Author information

  1. These authors contributed equally to this work.

    • Satoshi Arai,
    • Shinya Saijo &
    • Kano Suzuki

Affiliations

  1. Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan

    • Satoshi Arai,
    • Kano Suzuki,
    • Kenji Mizutani &
    • Takeshi Murata
  2. Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan

    • Satoshi Arai,
    • Shinya Saijo,
    • Kenji Mizutani &
    • Ichiro Yamato
  3. RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan

    • Shinya Saijo
  4. Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan

    • Kenji Mizutani &
    • So Iwata
  5. Laboratory of Molecular Physiology and Genetics, Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan

    • Yoshimi Kakinuma
  6. RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan

    • Yoshiko Ishizuka-Katsura,
    • Noboru Ohsawa,
    • Takaho Terada,
    • Mikako Shirouzu,
    • Shigeyuki Yokoyama,
    • So Iwata &
    • Takeshi Murata
  7. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

    • Shigeyuki Yokoyama
  8. Laboratory of Structural Biology, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

    • Shigeyuki Yokoyama
  9. JST, PRESTO, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan

    • Takeshi Murata

Contributions

T.M. designed the study. S.A., Y.K. and N.O. constructed DNAs. Y.I.-K., T.T. and M.S. expressed and purified the proteins. K.S. and T.M. crystallized the proteins. S.A., S.S., K.S., K.M. and T.M. collected X-ray data. S.A., S.S. and K.M. processed and refined X-ray data. S.A. and K.S. performed functional analysis. S.A., S.S., I.Y. and T.M. analysed the results. S.A. and S.S. prepared figures and videos. T.M. wrote the paper. All authors discussed the results and commented on the manuscript. The study was managed by S.Y., S.I., I.Y. and T.M.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Atomic coordinates and structure factors for the A3B3 and V1-ATPase complexes have been deposited in the Protein Data Bank under the accession codes 3VR2 (nucleotide-free A3B3 at 2.83), 3VR3 (nucleotide-bound A3B3 at 3.43), 3VR4 (nucleotide-free V1-ATPase at 2.23), 3VR5 (nucleotide-free V1-ATPase at 3.93) and 3VR6 (nucleotide-bound V1-ATPase at 2.73).

Author details

Supplementary information

Video

  1. Video 1: conformational changes of A3B3 complex induced by AMP-PNP:Mg binding (9,919 KB, Download)
    The colouring and viewing are consistent with Fig. 1. The video was generated by morphing between X-ray crystal structures of nucleotide-free (eA3B3) and nucleotide-bound (bA3B3) A3B3 complexes using PyMOL.
  2. Video 2: conformational changes of nucleotide-free eA3B3 complex induced by DF binding (12,727 KB, Download)
    The colouring and viewing are consistent with Fig. 2. The video was generated by morphing between X-ray crystal structures of nucleotide-free A3B3 (eA3B3) and A3B3DF (eV1) complexes using PyMOL.
  3. Video 3: conformational changes of nucleotide-bound A3B3 complex induced by DF binding (13,168 KB, Download)
    The colouring and viewing are consistent with Fig. 2. The video was generated by morphing between X-ray crystal structures of nucleotide-bound A3B3 (bA3B3) and A3B3DF (bV1) complexes using PyMOL.
  4. Video 4: conformational difference of the nucleotide-binding sites of nucleotide-bound V1-ATPase (2,162 KB, Download)
    The colouring and viewing are consistent with Fig. 3. The video was generated by morphing between X-ray crystal structures of the ACBO’ pair (bound form) and ACRBCR pair (tight form) in nucleotide-bound A3B3DF (bV1) complex using PyMOL.

PDF files

  1. Supplementary Information (4.6 MB)

    This file contains Supplementary Figures 1-17, Supplementary Tables 1-5, a Supplementary Discussion and Supplementary References. This file was replaced on 21 January 2013 as the original file posted online had corrupted.

Additional data