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Conformational ensemble of yeast ATP synthase at low pH reveals unique intermediates and plasticity in F1–Fo coupling

Nature Structural & Molecular Biology volume 31, pages 657–666 (2024)Cite this article

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Abstract

Mitochondrial adenosine triphosphate (ATP) synthase uses the proton gradient across the inner mitochondrial membrane to synthesize ATP. Structural and single molecule studies conducted mostly at neutral or basic pH have provided details of the reaction mechanism of ATP synthesis. However, pH of the mitochondrial matrix is slightly acidic during hypoxia and pH-dependent conformational changes in the ATP synthase have been reported. Here we use single-particle cryo-EM to analyze the conformational ensemble of the yeast (Saccharomyces cerevisiae) ATP synthase at pH 6. Of the four conformations resolved in this study, three are reaction intermediates. In addition to canonical catalytic dwell and binding dwell structures, we identify two unique conformations with nearly identical positions of the central rotor but different catalytic site conformations. These structures provide new insights into the catalytic mechanism of the ATP synthase and highlight elastic coupling between the catalytic and proton translocating domains.

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Fig. 1: Structure of yeast ATP synthase in Conf-1 and its comparison with Conf-0.
Fig. 2: Comparison of Conf-1 with the binding dwell of F1-ATPase from Bacillus PS3.
Fig. 3: Conf-2 and its comparison with Conf-1.
Fig. 4: Conf-3 and its comparison with Conf-1 and Conf-0.
Fig. 5: Conformations of ATP synthase resolved in this study, in context of the reaction cycle.

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Data availability

Three-dimensional cryo-EM density maps of the yeast mitochondrial ATP synthase in nanodiscs have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-29270, EMD-29278, EMD-28685, EMD-28687, EMD-28689, EMD-28809, EMD-28811, EMD-28813, EMD-28814, EMD-28835, EMD-28836, EMD-28837, EMD-29250 and EMD-29251. The corresponding atomic coordinates for the atomic models have been deposited in the PDB under accession numbers 8FL8, 8F29, 8F39 and 8FKJ (Table 1). PDB 6CP6 was used as an initial model for model building. Source data are provided with this paper.

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Acknowledgements

The project was supported by a grant from the National Institutes of Health, no. R35GM131731, to D.M.M. Cryo-EM data for ATP synthase at pH 6 were collected at Stanford-SLAC Cryo-EM Center (S2C2), which is supported by the National Institutes of Health Common Fund Transformative High-Resolution Cryo-Electron Microscopy program (grant no. U24 GM129541). Cryo-EM data for ATP synthase in presence of Mg:ATP were collected at the National Center for Cryo-EM Access and Training and the Simons Electron Microscopy Center located at the New York Structural Biology Center, supported by the National Institutes of Health Common Fund Transformative High-Resolution Cryo-Electron Microscopy program (grant no. U24 GM129539) and by grants from the Simons Foundation (grant no. SF349247) and NY State Assembly. M. Liao is an investigator of SUSTech Institute for Biological Electron Microscopy.

Author information

Author notes

  1. Stuti Sharma

    Present address: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA

  2. Min Luo

    Present address: Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Singapore

  3. Maofu Liao

    Present address: Department of Chemical Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China

  4. Maofu Liao

    Present address: Institute for Biological Electron Microscopy, Southern University of Science and Technology, Shenzhen, China

Authors and Affiliations

  1. Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Stuti Sharma, Min Luo & Maofu Liao

  2. Center for Genetic Diseases, The Chicago Medical School, Rosalind Franklin University, North Chicago, IL, USA

    Hiral Patel & David M. Mueller

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Contributions

D.M.M. and M. Liao conceived and supervised the project. H.P. expressed, purified and reconstituted ATP synthase into lipid nanodiscs. M. Luo and S.S. prepared cryo-EM samples and collected data. S.S. processed cryo-EM data and built models. S.S., M. Liao and D.M.M. analyzed the data and wrote the paper with input from all authors.

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Correspondence to Stuti Sharma, David M. Mueller or Maofu Liao.

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Nature Structural & Molecular Biology thanks Alain Dautant and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Cryo-EM of ATP synthase in presence of Mg:ATP.

A, Representative micrograph (from a dataset of 4219 images) of ATP synthase in lipid nanodiscs under continuous ATP hydrolysis. B, Two-dimensional class averages of F1Fo. C, Image processing workflow of the dataset (see Methods for details). D, Local resolution (left) and angular distribution (right) of the consensus and focused maps. E, Fourier shell correlation (FSC) between the half maps with resolution at FSC = 0.143 indicated. F, Model for yeast ATPase in the rotational state 2 (pdb ID 6CP6) fit into the map for the minor conformation (20% of particle images), with sectional view showed in G. Consensus map (H) and model (I) of the major conformation. J, Sectional view (as observed from the membrane) of the major conformation (ribbon) overlaid with the structure of bovine ATP synthase in rotational state 1 (pdb ID 5ARA) depicted as transparent surface. In panels G and J, the rotational angle of the central rotor is designated by a black arrow. “CR” refers to central rotor.

Extended Data Fig. 2 Cryo-EM image processing workflow.

A, Representative micrograph (from a dataset of 19,744 images) of ATP synthase in lipid nanodiscs at pH 6. B. 2D class averages of F1Fo C, Image processing workflow resulting in cryo-EM maps for Conf-0 (green), Conf-1 (red). D, Image processing workflow focused on F1 resulting in cryo-EM maps for Conf-1 (red) and Conf-2 (blue). E, Fo-centered image processing workflow resulting in cryo-EM maps for Conf-0 (green) and Conf-3 (pink). “CR” refers to central rotor.

Source data

Extended Data Fig. 3 Analysis and Statistics of Cryo-EM maps.

Local resolution (left) and angular distribution (right) of Conf-1 consensus map (A), Conf-1 F1 focused map (B), Conf-1 Fo + CR (central rotor) focused map (C) and Conf-1 Fo-focused map (D). E, Fourier shell correlation (FSC) between the half maps for Conf-1 with resolution at FSC = 0.143 indicated. Local resolution (left) and angular distribution (right) of Conf-2 consensus map (F), Conf-2 F1-focused map (G) and Conf-1 Fo + CR focused map (H). I, Fourier shell correlation (FSC) between the half-maps for Conf-2 with resolution at FSC = 0.143 indicated. Local resolution (left) and angular distribution (right) of Conf-3 consensus map (J), and Conf-3 Fo + CR focused map (K). L, Fourier shell correlation (FSC) between the half-maps for Conf-3 with resolution at FSC = 0.143 indicated.

Extended Data Fig. 4 Representative density from high-resolution maps.

Cryo-EM density from high-resolution maps with the corresponding model fit into the density for Conf-1 (A) and Conf-2 (B).

Extended Data Fig. 5 Comparison of observed conformations at pH 6 with existing catalytic and binding dwell structures.

A, Sectional view across F1 of Conf-0 (pdb ID 6CP6) fit into the map of bovine ATP synthase in State 2 (EMD-3166). B, Sectional view across F1 of Conf-0 showing catalytic site conformations and rotational angle of the central rotor (indicated with gray arrow) similar to catalytic dwell structures of Bacillus PS3 (pdb ID 7LIR) (C) and Sc (pdb ID 7TKR) (D). E. Sectional view across F1 of Conf-3 showing catalytic site conformations and rotational angle of the central rotor similar to binding dwell structures of Bacillus PS3 (pdb ID 7L1Q) (F) and Sc (pdb ID 7TKM) (G). H, Sectional view across F1 of Conf-1 with catalytic sites conformations consistent with binding dwell but rotational angle of the central rotor is not. I, Sectional view across F1 of Conf-2 with catalytic sites conformatio consistent with catalytic dwell but rotational angle of the central rotor is not. Rotational angle of the central rotor in each structure is indicated by gray arrows and the rotation from catalytic dwell to binding dwell to Conf-1 and Conf-2, is indicated in blue. Sc refers to Saccharomyces cerevisiae.

Extended Data Fig. 6 Comparison of the a/c interface between Conf-1 and Conf-2.

Cryo-EM map and model from Fo domain’s a/c interface in Conf-1 (A) and Conf-0 (pdb ID 6CP6 fit into the map for Conf-0) (B). The pdb chain IDs of individual c-subunits have been labeled (letters K-T). C, Overlay of Conf-1 (blue) and Conf-0 (green).

Extended Data Fig. 7 Structure of the F1 domain from Conf-1 docked into the map for Conf-3.

A, Cryo-EM map for conf-3. B, Model for F1 domain and peripheral stalk in Conf-1 docked into the corresponding cryo-EM density for Conf-3 (transparent), shown here as a sectional view.

Extended Data Fig. 8 Comparison of the a/c interface between Conf-1 and Conf-3.

Comparison of the model from Fo domain’s a/c interface between conf-1 (A) and conf-3 (B). The pdb chain IDs for individual c-subunits have been denoted as letters (K-T).

Supplementary information

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Supplementary Video 1

Conformational transition from Conf-0 to Conf-1. The animation shows a morph from Conf-0 to Conf-1 with subunits color coded as in the main text figures.

Supplementary Video 2

Conformational transition from Conf-1 to Conf-2. The animation shows a morph from Conf-1 to Conf-2 with subunits color coded as in the main text figures.

Supplementary Video 3

Conformational transition from Conf-1 to Conf-3. The animation shows a morph from Conf-1 to Conf-3 with subunits color coded as in the main text figures.

Source data

Source Data Extended Data Fig. 2

Source data for Extended Data Fig. 2a: uncropped micrograph of ATP synthase in lipid nanodiscs.

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Sharma, S., Luo, M., Patel, H. et al. Conformational ensemble of yeast ATP synthase at low pH reveals unique intermediates and plasticity in F1–Fo coupling. Nat Struct Mol Biol 31, 657–666 (2024). https://doi.org/10.1038/s41594-024-01219-4

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