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Horizontal membrane-intrinsic α-helices in the stator a-subunit of an F-type ATP synthase

Nature volume 521, pages 237–240 (2015)Cite this article

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Abstract

ATP, the universal energy currency of cells, is produced by F-type ATP synthases, which are ancient, membrane-bound nanomachines. F-type ATP synthases use the energy of a transmembrane electrochemical gradient to generate ATP by rotary catalysis. Protons moving across the membrane drive a rotor ring composed of 8–15 c-subunits1. A central stalk transmits the rotation of the c-ring to the catalytic F1 head, where a series of conformational changes results in ATP synthesis2. A key unresolved question in this fundamental process is how protons pass through the membrane to drive ATP production. Mitochondrial ATP synthases form V-shaped homodimers in cristae membranes3. Here we report the structure of a native and active mitochondrial ATP synthase dimer, determined by single-particle electron cryomicroscopy at 6.2 Å resolution. Our structure shows four long, horizontal membrane-intrinsic α-helices in the a-subunit, arranged in two hairpins at an angle of approximately 70° relative to the c-ring helices. It has been proposed that a strictly conserved membrane-embedded arginine in the a-subunit couples proton translocation to c-ring rotation4. A fit of the conserved carboxy-terminal a-subunit sequence places the conserved arginine next to a proton-binding c-subunit glutamate. The map shows a slanting solvent-accessible channel that extends from the mitochondrial matrix to the conserved arginine. Another hydrophilic cavity on the lumenal membrane surface defines a direct route for the protons to an essential histidine–glutamate pair5. Our results provide unique new insights into the structure and function of rotary ATP synthases and explain how ATP production is coupled to proton translocation.

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Figure 1: Electron cryomicroscopy map of the Polytomella F-type ATP synthase dimer.
Figure 2: a-Subunit structure.
Figure 3: Two aqueous half-channels.
Figure 4: Proton translocation through F-type ATP synthases.

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Electron Microscopy Data Bank

Protein Data Bank

Referenced accessions

Protein Data Bank

Data deposits

The electron cryomicroscopy map of the Polytomella sp. proton-driven ATP synthase dimer has been deposited in the Electron Microscopy Data Bank under accession number EMD-2852. Raw image data have been deposited in the European Bioinformatics Institute Electron Microscopy Pilot Image Archive (http://www.ebi.ac.uk/pdbe/emdb/empiar/) under accession number EMPIAR-10023.

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Acknowledgements

We thank T. Meier and J. D. Faraldo-Gómez for discussions and reading the manuscript. Ö. Yildiz and J. F. Castillo-Hernandez provided computer support. This work was funded by the Max Planck Society (M.A., N.K., D.J.M., J.V., K.M.D., W.K.) and the Deutsche Forschungsgemeinschaft Cluster of Excellence Frankfurt ‘Macromolecular Complexes’ (K.M.D., W.K.).

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Authors and Affiliations

  1. Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany,

    Matteo Allegretti, Niklas Klusch, Deryck J. Mills, Janet Vonck, Werner Kühlbrandt & Karen M. Davies

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Contributions

K.M.D., M.A. and W.K. designed the experiments. N.K. purified the protein. M.A., K.M.D., N.K. and D.J.M. collected images. M.A., N.K., K.M.D., J.V. and D.J.M. processed data. K.M.D., M.A., J.V. and W.K. analysed the data and wrote the paper.

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Correspondence to Werner Kühlbrandt or Karen M. Davies.

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Extended data figures and tables

Extended Data Figure 1 Oligomycin-sensitive ATPase activity.

ATPase assays27 performed with the Polytomella ATP synthase dimers used for electron cryomicroscopy data collection indicated high activity of 6–8 units per milligram protein, close to the reported activity for this complex27. ATPase activity was ∼90% inhibited by oligomycin, indicating that the F1Fo complex is coupled. The vertical lines indicate addition of dodecyl maltoside detergent to initiate the reaction. Measurements were performed in triplicates for each purification.

Extended Data Figure 2 Projection images of the Polytomella F-type ATP synthase dimer.

a, Typical electron cryo-micrograph of dimers in vitrified buffer recorded at 2.5 µm underfocus. b, Two-dimensional class averages and (c) corresponding projection images calculated from the final three-dimensional volume. d, Angular distribution of projection images used for three-dimensional reconstruction. ATP synthase dimers are oriented randomly in the thin layer of vitrified buffer. Scale bar in a, 50 nm.

Extended Data Figure 3 Local resolution estimates.

a, The unfiltered electron cryomicroscopy map was analysed with the programme ResMap38 to assess local resolution. The catalytic F1 head (red to green) is less well-ordered than the peripheral stalk and the membrane-embedded a-subunit (green to blue), as indicated by the rainbow colour code on the left. The insets show cross-sections through the density at the levels indicated on the right. b, Fourier shell correlation curves calculated from two independently refined data sets after soft masking. The resolution was 7.0 Å for the whole complex (red), 7.4 Å for the F1/c-ring complex (blue mask and curve) and 6.2 Å for the peripheral stalk and a-subunit (yellow mask and curve).

Extended Data Figure 4 Resolution of unsymmetrized map.

Side view (a) and top view (b) of the dimer map refined without imposing c2 symmetry (blue). c, The gold-standard Fourier shell correlation curve indicates a resolution of 8.0 Å for the unsymmetrized map, which shows all essential features of the Polytomella dimer, including c2 symmetry.

Extended Data Figure 5 Horizontal helices in the three-dimensional map and in two-dimensional projection.

a, Section of the three-dimensional map showing a side view of the c10-ring on the left and the a-subunit on the right. b, A slice of the three-dimensional map volume indicated in a shows the horizontal helices clearly. c, In a two-dimensional map generated by projecting the three-dimensional map volume in a along the indicated direction (arrow), the long horizontal helices are in effect invisible, whereas the vertical c-ring helices stand out clearly. Scale bar, 10 Å.

Extended Data Figure 6 Sequence alignment of about 120 C-terminal subunit a residues.

Sequences of bacterial (blue), mitochondrial (red) and chloroplast (green) F-type ATP synthases are compared. The interchangeable residue pairs aR239/aQ295 and aE288/aH248 are dark blue and green, respectively; sequences of helices fitted in Fig. 2b are light blue. The black arrowhead marks the strictly conserved arginine essential for coupled proton translocation. Red, prolines; magenta, solvent-exposed residues18,43; green, residues that crosslink to the c-ring10,11. Identical (★) or similar (‘.’ or ‘:’) residues are indicated.

Extended Data Figure 7 Subunit a and c crosslinking distances.

Pairs of a- and c-subunit residues crosslinked in E. coli using (a) zero-length crosslinkers10 and (b) bis-MTS reagents11. Tabulated distances were measured between beta carbons of residues in the modelled a-subunit helices and Saccharomyces c-ring helices fitted to the map. Zero-length crosslinks can be considerably shorter than crystallographic distances44,45, most probably as a result of thermal motion during the long incubation times required for crosslinking10. Nevertheless, the inter-residue distances in our model agree with the E. coli crosslinking data within reasonable margins, allowing for species differences between bacterial and mitochondrial ATP synthases.

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion. (PDF 125 kb)

Mitochondrial ATP synthase dimer map

Cryo-EM map of the Polytomella mitochondrial ATP synthase dimer, highlighting the helix-turn helix pair (blue) and Armadillo repeat-like protein (pink) at the dimer interfaces. (MOV 20962 kb)

a-subunit structure

Zoomed-in view of membrane domains of one protomer showing the structure and orientation of the a-subunit (blue) to the c10-ring (yellow). (MOV 8181 kb)

Aqueous half-channels

Zoomed-in view of membrane region showing the two aqueous cavities. c-ring (yellow); a-subunit (blue); proposed interaction site of the a- and c-subunits (dark blue); proposed position of interchangeable residue pair aE288/aH248 (light green). The 1σ density threshold indicates the detergent shell (pink mesh). (MOV 25921 kb)

Solvent-exposed residues in the matrix cavity

Residues of the E. coli a subunit equivalent to Polytomella a225, 226, 228, 229, 231, 232, 235, 303 and 306-30820,43, as well as c-ring residues equivalent to Polytomella c44-6621 have been shown to be solvent exposed by cysteine mutagenesis. In our model, residues a225-235 (red), a303-308 (orange) and c44-66 (green) line the aqueous matrix cavity observed in the EM map of the Polytomella ATP synthase dimer. Thus our model is in excellent agreement with published biochemical studies on the solvent accessibility of a-subunit residues. Blue, a-subunit; yellow, c-ring; pink mesh, detergent shell; dark blue, location of conserved arginine. (MOV 9436 kb)

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Allegretti, M., Klusch, N., Mills, D. et al. Horizontal membrane-intrinsic α-helices in the stator a-subunit of an F-type ATP synthase. Nature 521, 237–240 (2015). https://doi.org/10.1038/nature14185

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