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Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase

Nature volume 521, pages 241–245 (2015)Cite this article

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Abstract

Eukaryotic vacuolar H+-ATPases (V-ATPases) are rotary enzymes that use energy from hydrolysis of ATP to ADP to pump protons across membranes and control the pH of many intracellular compartments. ATP hydrolysis in the soluble catalytic region of the enzyme is coupled to proton translocation through the membrane-bound region by rotation of a central rotor subcomplex, with peripheral stalks preventing the entire membrane-bound region from turning with the rotor. The eukaryotic V-ATPase is the most complex rotary ATPase: it has three peripheral stalks, a hetero-oligomeric proton-conducting proteolipid ring, several subunits not found in other rotary ATPases, and is regulated by reversible dissociation of its catalytic and proton-conducting regions1,2. Studies of ATP synthases, V-ATPases, and bacterial/archaeal V/A-ATPases have suggested that flexibility is necessary for the catalytic mechanism of rotary ATPases3,4,5, but the structures of different rotational states have never been observed experimentally. Here we use electron cryomicroscopy to obtain structures for three rotational states of the V-ATPase from the yeast Saccharomyces cerevisiae. The resulting series of structures shows ten proteolipid subunits in the c-ring, setting the ATP:H+ ratio for proton pumping by the V-ATPase at 3:10, and reveals long and highly tilted transmembrane α-helices in the a-subunit that interact with the c-ring. The three different maps reveal the conformational changes that occur to couple rotation in the symmetry-mismatched soluble catalytic region to the membrane-bound proton-translocating region. Almost all of the subunits of the enzyme undergo conformational changes during the transitions between these three rotational states. The structures of these states provide direct evidence that deformation during rotation enables the smooth transmission of power through rotary ATPases.

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Figure 1: Rotational states of the V-ATPase.
Figure 2: The membrane-bound VO region.
Figure 3: Symmetry mismatch between the V1 and VO regions.
Figure 4: Flexibility of subunits.

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Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

Cryo-EM maps have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-6284, EMD-6285, and EMD-6286. Atomic models have been deposited in the Protein Data Bank under accession numbers 3J9T, 3J9U, and 3J9V.

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Acknowledgements

We thank P. Rosenthal, R. Henderson, V. Kanelis, and L. Kay for comments on the manuscript. J.Z. was supported by a Doctoral Postgraduate Scholarship from the Natural Sciences and Engineering Research Council of Canada and a Mary Gertrude l’Anson Scholarship. J.L.R. is the Canada Research Chair in Electron Cryomicroscopy. This work was supported by operating grant MOP 81294 from the Canadian Institutes of Health Research.

Author information

Author notes

  1. Jianhua Zhao and Samir Benlekbir: These authors contributed equally to this work.

Authors and Affiliations

  1. Molecular Structure and Function Program, The Hospital for Sick Children Research Institute, 686 Bay Street, Toronto, M5G 0A4, Ontario, Canada

    Jianhua Zhao, Samir Benlekbir & John L. Rubinstein

  2. Department of Medical Biophysics, The University of Toronto, Toronto Medical Discovery Tower, MaRS Centre, 101 College Street, Toronto, M5G 1L7, Ontario, Canada

    Jianhua Zhao & John L. Rubinstein

  3. Department of Biochemistry, The University of Toronto, 1 King’s College Circle, Medical Sciences Building, Toronto, M5S 1A8, Ontario, Canada

    John L. Rubinstein

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  1. Jianhua Zhao
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Contributions

S.B. and J.L.R. initiated the project. J.Z. and S.B. collected images and performed pre-processing steps. J.Z. performed the image analysis. J.Z. and J.L.R. interpreted the data, prepared figures, and wrote the manuscript. J.L.R. and J.Z. contributed new computer algorithms used in image analysis.

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Correspondence to John L. Rubinstein.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 V-ATPase subunits and rotation.

a, The V-ATPase from S. cerevisiae consists of subunits A3B3CDE3FG3Hacxc′yc′′zde, where x, y, and z denote unknown stoichiometries. Subunits with upper-case letter names correspond to components of the soluble V1 region while lower-case names denote components of the membrane-bound VO region. The e-subunit is not found in the detergent-solubilized S. cerevisiae V-ATPase. During rotary catalysis, ATP hydrolysis drives rotation of the rotor, consisting of subunits DFcxc′yc′′zd (outlined in black), which rotates relative to the rest of the enzyme. Upper inset, the three different nucleotide-binding sites of the V1 region can be found in three different conformations: ‘tight’ (where ATP is expected to be bound), ‘loose’ (where ADP is expected to be bound), and ‘open’ (where no nucleotide is bound). Lower inset, rotation of the cxc′yc′′z-ring against the a-subunit leads to proton translocation from the cytoplasmic side of the membrane to the luminal side of the membrane. Proton translocation occurs via two half-channels through the membrane. b, V-ATPase activity is regulated by reversible dissociation where the V1 region separates from the VO region. The H-subunit inhibits ATP hydrolysis in the dissociated V1 region. Proton translocation in the dissociated VO region is blocked by an unknown mechanism.

Extended Data Figure 2 Data collection.

a, A representative micrograph; examples of V-ATPase particle images are shown circled in red. These particle images were selected from the 200 candidate particle images identified automatically from template matching. b, Tracking of particle and other image feature trajectories with the alignparts_lmbfgs algorithm31. Trajectories are exaggerated by a factor of 5 to allow visualization.

Extended Data Figure 3 Three-dimensional maps from rotational states.

a, Surface rendered views of the three three-dimensional maps are shown. Scale bars, 25 Å. b, Fourier shell correlation (FSC) curves after a ‘gold standard’ refinement of the three maps are shown. The resolutions measured from these curves at a Fourier shell correlation of 0.143 are the same as the resolutions measured after correcting for masking effects by high-resolution noise-substitution calculations51. c, Local resolution estimation shows that features in the V1 region are better resolved than in the VO region.

Extended Data Figure 4 Map segmentation and molecular dynamics flexible fitting.

Different subunits are shown fitted into their corresponding map densities in rotational states 1, 2, and 3, including AB pair 3 (a), the N-terminal domain of subunit a (b), the central rotor DFd subcomplex (c), subunit C (d), and peripheral stalk 1 (e). Scale bar, 25 Å.

Extended Data Figure 5 C-terminal domain of the a-subunit.

a, The membrane-bound C-terminal domain of the a-subunit appears similar in all three rotational states. b, The density from the C-terminal domain of the Thermus thermophilus subunit I, equivalent of the a-subunit, at 9.7 Å resolution3 is consistent with the structure of the a-subunit from S. cerevisiae (left). However, the transmembrane α-helical densities identified previously in that map (right) are not entirely consistent with the current maps.

Extended Data Figure 6 Flexibility in V-ATPase subunits.

a–c, Each AB pair in the A3B3 hexamer goes through ‘open’, ‘loose’, and ‘tight’ conformations as the enzyme passes between the three rotational states. d–f, Overlay of all three open, all three loose, and all three tight structures shows that the conformations are nearly the same for each AB pair. g–i, Each of the three EG peripheral stalk structures undergoes similar bending motions between the three rotational states. Scale bar, 25 Å.

Supplementary information

Cross sections through the three maps, each showing a different rotational state of the V-ATPase.

Cross sections through the three maps, each showing a different rotational state of the V-ATPase. (MOV 12001 kb)

Interpolation between the three observed rotational states of the V-ATPase

Interpolation between the three observed rotational states of the V-ATPase. (MOV 32484 kb)

Exploded view of subunits when interpolating between the three observed rotational states of the V-ATPase

Exploded view of subunits when interpolating between the three observed rotational states of the V-ATPase. (MOV 30321 kb)

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Zhao, J., Benlekbir, S. & Rubinstein, J. Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase. Nature 521, 241–245 (2015). https://doi.org/10.1038/nature14365

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