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

Nature volume 493, pages 703–707 (2013)Cite this article

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Abstract

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 (2.8 Å) and nucleotide-bound (3.4 Å) 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 (2.2 Å) and nucleotide-bound (2.7 Å) 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.

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Figure 1: Structure of the A 3 B 3 complex.
Figure 2: Comparison of the asymmetric structures of nucleotide-free A 3 B 3 DF and A 3 B 3 complexes.
Figure 3: Comparison of the nucleotide-binding sites.
Figure 4: A model of the rotation mechanism of V 1 -ATPase.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

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).

Change history

  • 30 January 2013

    The structure in Fig. 1e was corrected.

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Acknowledgements

We thank J. E. Walker for his suggestions, especially through the structural studies of F1-ATPase. The synchrotron radiation experiments were performed at SPring-8 and Photon Factory (proposals 2008S2-001, 2011S2-005, 2009G660, 2009B1031 and 2012G132). We also thank the beamline staff at BL41XU of SPring-8 (Harima, Japan) and NE3A, NW12A and BL1A of Photon Factory (Tsukuba, Japan) for help during data collection. This work was supported by the Targeted Proteins Research Program, grants-in-aid (23370047, 23118705), Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese government.

Author information

Author notes

  1. Satoshi Arai, Shinya Saijo and Kano Suzuki: These authors contributed equally to this work.

Authors and 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

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  1. Satoshi Arai
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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.

Corresponding author

Correspondence to Takeshi Murata.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

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. (PDF 12825 kb)

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. (MOV 9918 kb)

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. (MOV 12726 kb)

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. (MOV 13168 kb)

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. (MOV 2162 kb)

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Arai, S., Saijo, S., Suzuki, K. et al. Rotation mechanism of Enterococcus hirae V1-ATPase based on asymmetric crystal structures. Nature 493, 703–707 (2013). https://doi.org/10.1038/nature11778

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