Research Article

Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F1-Fo coupling

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Science  21 Jun 2019:
Vol. 364, Issue 6446, eaaw9128
DOI: 10.1126/science.aaw9128
  • Cryo-EM structure of the Polytomella ATP synthase dimer.

    The F1 head (green) is linked to the c-ring rotor (yellow) by the central stalk and the peripheral stalk. Insets (beginning at top right) show the flexible OSCP hinge (orange); F1 rotary substates with subunits β (green), γ (blue), and c (yellow); a coordinated metal ion in the proton access channel (light blue); and the dimer-forming subunit ASA10 (red).

  • Fig. 1 High-resolution structure of the mitochondrial F1Fo ATP synthase dimer from Polytomella sp.

    (A) Composite cryo-EM map of the 62-subunit, 1.58-MDa Polytomella dimer; the right side is colored by subunit. The c10 rotor ring and subunit a make up the Fo motor complex. Proton translocation through Fo causes rotation of the c ring and the attached central stalk subunits γ, δ, and ε. The F1 head consists of three catalytic β subunits (light green) and three α subunits (dark green). Long C-terminal extensions of β wrap around the α subunits on the outside of F1 (see Movie 5). The two-domain OSCP subunit links the three α subunits to the peripheral stalk subunits ASA1 to ASA10 (see Fig. 4). Subunit ASA10 connects the two F1Fo monomers in the membrane (see figs. S9 and S11). The local map resolution is 2.7 Å for the peripheral stalk, Fo, and c ring, and 2.8 to 2.9 Å for the F1 head and central stalk (see fig. S2). Inset: Three primary rotary states (1, 2, and 3) of the F1Fo monomer are related by ~120° rotation of the central stalk within the α3β3 assembly. Unless otherwise specified, subunit coloring is consistent throughout all figures. (B) Section through F1 at the level of bound nucleotides. βDP and βTP sites contain ADP (red) and βE is empty. The three α subunits bind a structural ATP (orange). Nucleotide-coordinating Mg2+ ions are violet. The central stalk subunit γ (blue) engages with the catch loop of the βE subunit (see fig. S7). (C and D) Catalytic sites of subunits βDP and βTP. A well-defined Arg side chain of subunit α (the “arginine finger”) extends toward the nucleotide phosphate in the βDP site. ATP in the βTP site has hydrolyzed to ADP during protein isolation. Amino acid abbreviations: D, Asp; E, Glu; K, Lys; N, Asn; R, Arg; T, Thr.

  • Fig. 2 Rotary substates of mitochondrial ATP synthase.

    The three primary rotary states are subdivided into a total of 13 rotary substates at resolutions between 2.8 and 4.2 Å (see figs. S2 and S3 and table S2). The number of resolved substates and the angular increments between them differ for each of the three primary rotary states (fig. S5). (A) Composite cryo-EM map of rotary substate 1A at 2.9-Å resolution. (B to D) Overlays of substate maps indicate concerted movement of the N-terminal domain of OSCP (B), the F1 head with the central γ subunit (C), and the c ring (D) from substate 1A to substate 1F. Projected map densities of other subunits are shown in light gray for reference.

  • Fig. 3 Concerted rotation of F1 with central stalk and c ring.

    (A) Progressing in the direction of ATP synthesis, the c10 ring (yellow) rotates counterclockwise (as seen from F1) with respect to the a subunit (light blue) by up to 32° for substates of the same primary rotary state. The primary rotary states differ by power strokes of ~120°. The position of one c subunit in the first (black outline) and last rotary substate (pink) of each primary state is indicated. (B) Between substates of a given rotary state, the F1 head rotates together with the c ring and central stalk before recoiling to its original position in the first substate of the subsequent primary rotary state. Subunits α (dark green) and β (light green) with their bound nucleotides (red and orange) are shown for the first substate in each primary state. The position of the last substate in each primary state is indicated in light blue in the background. The peripheral stalk position is shown in gray.

  • Fig. 4 Subunit OSCP connects the F1 head and peripheral stalk as a flexible hinge.

    (A) Overview of F1Fo monomer indicating the position of OSCP (orange) on top of the F1 head (green) relative to the peripheral stalk (gray). (B) The two-domain OSCP interacts with the helical C-terminal extensions of subunits α1, α2, and α3 (green) (see Movie 4). Extensions of α2 and α3 form short helix bundles with helices of the globular N-terminal OSCP domain, whereas α1 binds to the elongated C-terminal OSCP domain that is attached to the peripheral stalk via the α1 extension. The two OSCP domains are connected by a flexible peptide link that facilitates ~30° back-and-forth rotation of the N-terminal domain with F1 (curved black arrow) relative to the C-terminal OSCP domain and peripheral stalk. (C) Between substates of a given primary rotary state, the N-terminal OSCP domain rotates with F1 by up to 28° (straight dashed lines) in synthesis direction (orange arrows) and then recoils to its starting position in the first substate of the next primary rotary state (red arrows). The OSCP position in the first substate of each primary state is shown in orange; the OSCP position in the last resolved rotary state in each primary state is indicated in light blue in the background.

  • Fig. 5 Metal ion and ordered water molecules in the Fo proton access and release channels.

    (A) The H5/H6 helix hairpin of subunit a (light blue) forms the bearing against which the c10 ring (yellow) rotates, shown here for c-ring position 1. (B) Sectional view of H5 and outer-ring c-subunit helices with proton-binding cGlu111, c-ring position 1. (C and D) Water molecules (purple density) coordinated by the ion-binding cGlu111 and adjacent cSer112 in the matrix channel for c-ring positions 1 (C) and 2 (D). Maps are displayed at density thresholds of 0.035 for subunit a, 0.025 for the c ring, and 0.025 (C) or 0.018 (D) for water. (E) The strictly conserved aArg239 and aGln295 that separate the proton access and release channels in the membrane bind two water molecules, shown in the consensus C2-refined map of the Fo region. (F to H) Conserved residues aHis248 and aHis252 in H5 of subunit a coordinate a metal ion (green). Bond distances change depending on c-ring position. The predominant c-ring position [position 1, full color, and (H)] accounts for 58% of particles; a second position, differing by rotation of roughly 13° [position 2, faint yellow, and (G)] accounts for 33% of particles. Except where specified, all maps in a given panel are rendered at the same density threshold. E, Glu; H, His; Q, Gln; R, Arg; S, Ser.

  • Movie 1. Three-dimensional map of the Polytomella ATP synthase dimer.

    One monomer is gray; the other monomer map is colored by subunit, as in Fig. 1.

  • Movie 2.

    Morph of 13 rotary substates of the Polytomella ATP synthase monomer. The c ring (yellow) and central stalk (subunits: γ, blue; δ, cyan; ε, pale blue) together rotate in three roughly equal ~120° steps. The F1 head moves with them for the first ~30° of each step. α, dark green; β, bright green. The two-domain OSCP subunit (orange) works as a hinge between the moving F1 head and the stationary peripheral stalk (gray).

  • Movie 3. Section through movie 2 at the level of nucleotide-binding sites in the F1 head.

    Catalytic sites of the β subunits (bright green) are red. Binding sites for the structural ATP in the three α subunits (dark green) are orange. The β catch loop is drawn in purple. Central stalk subunit γ, blue; peripheral stalk, gray.

  • Movie 4. Hinge movement of the two-domain OSCP subunit (orange).

    The proximal α-helical OSCP domain at the right is attached to the F1 head by the N-terminal extensions of two of the three α subunits (dark green). The distal β-sheet OSCP domain is attached to the peripheral stalk (gray) by interaction of one OSCP helix and the N-terminal extension of one of the three α subunits.

  • Movie 5. Extensions of subunits α and β in Polytomella ATP synthase.

    The polypeptide sequences of subunits α and β in mitochondrial F1Fo ATP synthases of chlorophycean algae (including Polytomella sp.) have characteristic extensions at their N and C termini. The ~15-residue N-terminal extension of subunit α interacts with OSCP (see Fig. 4 and movie 4). The ~60-residue C-terminal extension of the β subunit wraps around the outside of the neighboring subunit α in the F1 head. Both extensions would render the chlorophycean F1Fo complex more stable than that of mammalian or fungal mitochondrial ATP synthases.

Supplementary Materials

  • Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F1-Fo coupling

    Bonnie J. Murphy, Niklas Klusch, Julian Langer, Deryck J. Mills, Özkan Yildiz, Werner Kühlbrandt

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Figs. S1 to S12 
    • Tables S1 to S3
    • References 

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