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. 2016 Aug 4;63(3):445-56.
doi: 10.1016/j.molcel.2016.05.037. Epub 2016 Jun 30.

Structure of a Complete ATP Synthase Dimer Reveals the Molecular Basis of Inner Mitochondrial Membrane Morphology

Alexander Hahn  1 , Kristian Parey  1 , Maike Bublitz  2 , Deryck J Mills  1 , Volker Zickermann  3 , Janet Vonck  1 , Werner Kühlbrandt  4 , Thomas Meier  5
Affiliations

Structure of a Complete ATP Synthase Dimer Reveals the Molecular Basis of Inner Mitochondrial Membrane Morphology

Alexander Hahn et al. Mol Cell. .

Abstract

We determined the structure of a complete, dimeric F1Fo-ATP synthase from yeast Yarrowia lipolytica mitochondria by a combination of cryo-EM and X-ray crystallography. The final structure resolves 58 of the 60 dimer subunits. Horizontal helices of subunit a in Fo wrap around the c-ring rotor, and a total of six vertical helices assigned to subunits a, b, f, i, and 8 span the membrane. Subunit 8 (A6L in human) is an evolutionary derivative of the bacterial b subunit. On the lumenal membrane surface, subunit f establishes direct contact between the two monomers. Comparison with a cryo-EM map of the F1Fo monomer identifies subunits e and g at the lateral dimer interface. They do not form dimer contacts but enable dimer formation by inducing a strong membrane curvature of ∼100°. Our structure explains the structural basis of cristae formation in mitochondria, a landmark signature of eukaryotic cell morphology.

Keywords: F(1)F(o)-ATP synthase dimer; X-ray crystallography; bioenergetics; cryoelectron microscopy; inner membrane morphology; membrane protein complex; mitochondria; rotary ATPase mechanism; yeast Yarrowia lipolytica.

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Figures

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Graphical abstract
Figure 1
Figure 1
Nucleotide Binding Sites in Yarrowia lipolytica F1c10 Cross section through the F1 X-ray structure shows the six nucleotide binding sites at the α/β subunit interfaces viewed from the matrix. Subunits α (dark green, dark gray) and β (light green, light gray) are arranged around the central γ subunit (blue). Green and red mesh indicates unbiased mFobs-DFcalc nucleotide difference densities contoured at 3.0σ and −3.0σ, respectively. Boxed, close-up views of Walker A nucleotide binding motifs (cartoon) with αThr202, βThr195, and nucleotides in stick representation. Mg2+ ions with coordinated water molecules are shown as spheres. Atoms of C, N, O, P, and Mg are colored yellow, blue, red, orange, and magenta, respectively. Positive difference densities match Mg·ATP [·3 H2O] in all three α sites, and Mg·ADP[·4 H2O] in βDP and βTP. The βE site is empty. The conformational flexibility of the βE subunit is a possible cause of the weaker adenosine density of the ATP in the adjacent αTP site. See Figure S3 for further difference maps after modeling various nucleotides in the α and β sites.
Figure 2
Figure 2
Cryo-EM Structure of the Yarrowia lipolytica F1Fo-ATP Synthase Dimer (A) Side view of the map (gray surface and volume). The monomer on the right was fitted in Coot (Emsley et al., 2010) with the X-ray structure of the Y. lipolytica F1c10 complex and homology models of peripheral stalk subunits based on atomic models from the B. taurus outer stalk structures (PDB: 2WSS and 2CLY) (Figures 1 and S2E). Cross sections shown in Figures 3A and 3B and viewing directions for (B)–(D) are indicated. (B and C) Detailed views of peripheral stalk subunit interactions as indicated in (A). (B) Upper and (C) lower section. Dark and light green, α and β subunits, respectively; light blue, subunit a; yellow, c10 ring; blue, subunit γ; cyan, subunit δ; light gray, subunit ε; purple, subunit b; orange, subunit d; salmon, subunit h; red, OSCP. (D) The intrinsic inhibitor protein IF1 (light blue) binds in the α/βDP site proximal to the peripheral stalk. The overall map resolution of 7.8 Å in (A) improved upon masking to 6.9 Å for the F1 complex (B–D). Movie S1 is a video of the rotating complete ATP synthase dimer.
Figure 3
Figure 3
Assignment of the Yarrowia lipolytica F1Fo-ATP Synthase Stator Region (A and B) Cross sections through the Fo stator region as indicated in Figure 2A. (A) View from the matrix and (B) from the membrane. Four horizontal and six vertical helix densities (labeled 1–6) next to the c10 ring rotor (yellow) in the detergent micelle (gray) were assigned to stator subunits. Blue, subunit a; purple, subunit b; green, subunit 8; lavender, subunit f; dark orange, subunit i. (C) Overview of peripheral stalk and stator subunits in the cryo-EM map. The overall map resolution of 7.8 Å in Figure 2A improved upon masking to 6.2 Å for the Fo dimer (A and B) and 6.9 Å for the F1 complex (C).
Figure 4
Figure 4
Subunit a View (A) from the dimer interface, (B) along the a/c interface, and (C) from the matrix. The hydrophilic N terminus on the IMS side (region (i) in the text) is not resolved. Helix aH1 (blue, region (ii)) is the only vertical trans-membrane helix of subunit a. The amphipathic helix aH2 (cyan, region (iii)) runs along the matrix membrane surface. The membrane-intrinsic helices aH3 (green) and aH4 (yellow) of region (iv) form a hairpin. Regions (v) and (vi) are the unresolved connection between helices aH4 and aH5. The membrane-intrinsic helices aH5 (orange) and aH6 (red) form a second, longer hairpin (region (vii)), tilted by 20°–25° relative to the membrane plane. aH5 follows the curve of the c-ring (light gray) closely. aH5 and aH6 are ∼70 Å long and in contact with 3–4 c subunits, while the helices in the distal hairpin, aH3 and aH4, are ∼45 and ∼35 Å long; neither is in direct contact with the c-ring. The aH4/aH5 hairpin loop is on the IMS side, while the C terminus is exposed on the matrix surface. All six a subunit helices are highly conserved (Figure S5).
Figure 5
Figure 5
Dimer Interface (A) Matrix view of the Y. lipolytica ATP synthase cryo-EM dimer map (gray) with superposed monomer maps (yellow and blue). (B) Vertical slice through monomer maps as indicated in (A). Dimer contacts are mediated by protein densities outside the detergent micelle of the monomer (boxed). (C) Side view of superposed maps in (A). (D) Horizontal slice through the dimer interface as indicated in (C). The white density (boxed) belongs to membrane subunits present in the dimer, but not in the monomer. LC-MS analysis of monomer and dimer subunit composition identifies these subunits as e and g (Table S1). (E) Detailed view of the lumenal dimer contacts as indicated in (B). (F) Detailed view of the density assigned to the dimer-specific subunits e and g as indicated in (D). (G) Central slice of the dimer. A sheet-like density (arrowhead) connecting subunits a and 8 may be the yeast-specific subunit k, the only unassigned protein in the dimer map.
Figure 6
Figure 6
The Fo Stator Membrane protein densities in the two Fo stator complexes of the Y. lipolytica ATP synthase dimer. Subunits a, b, f, i, and 8 are colored as in Figure 2. Subunits e and g are ivory. (A) Side view of the dimer interface. The IMS extensions of subunit e (arrowhead) were segmented at a lower contour level. Fo subunits of the two ATP synthase monomers interact on the IMS side. A 40 Å gap on the matrix side contains lipid or detergent. (B) View from the IMS. The C-terminal segment of subunit f mediates a direct protein contact between the two monomers in the dimer. Densities on either side of the protein contact are assigned to subunits e and g (see Figure 6). (C) View from the c-ring. (D) Oblique view in the direction indicated in (A). For an evaluation of Cys-Cys crosslinks, see Figure S6 and Table S3.
Figure 7
Figure 7
Ion Translocation through Fo (A) IMS surface of the Y. lipolytica dimer. Subunits are color coded as in Figure 2. The c-ring and the dimer contact region are solvent exposed. aH5 of subunit a (light blue) is visible from the IMS side. The central plug on the c-rings consists of lipid or detergent (Meier et al., 2001). (B) Oblique view from the matrix shows the gap between the c-ring (yellow) and subunit a (light blue). (C) Fitted aH5 and aH6 hairpin of subunit a next to the c-ring rotor with modeled positions of conserved positive (blue), negative (red), and polar residues (green). The helical wheel projection (inset) indicates alternating polar, charged, and hydrophobic residues at the start of aH5, with polar or charged residues oriented toward the c-ring. The transparent pink bar indicates the level at which the protonated c-ring glutamates rotate past the strictly conserved aArg182. (D) Section through the c10-ring (gray) and subunit a (gray mesh) with a cartoon model (blue) of the c-ring at the level of protonated glutamates in the locked (green) or open (orange) conformation (Pogoryelov et al., 2010, Symersky et al., 2012). aH6 peels away from the c-ring, accounting for the observed gap at the a/c interface. Dashed arrows in (C) and (D) indicate the proton pathway from the IMS (P side) to the matrix (N side), as proposed for the Polytomella ATP synthase (Allegretti et al., 2015).
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