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

Isolation and Biochemistry of Yarrowia lipolytica ATP Synthase Dimers

ATP synthase dimers from Y. lipolytica were purified from dodecylmaltoside (DDM)-solubilized mitochondrial membranes by centrifugation in a digitonin-containing glycerol gradient, followed by anion exchange chromatography. Two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry (LC-MS) indicated that the 2YLF₁F_o (Y. lipolytica ATP synthase dimer) fraction contained all ATP synthase subunits, including e, g, and k, which are known as dimer specific (Arnold et al., 1998) (Figure S1; Table S1, available online). The DDM-purified monomeric Y. lipolytica ATP synthase (1YLF₁F_o) lacks subunits e, g, and k. The ATP hydrolysis activity of both 1YLF₁F_o and 2YLF₁F_o is ∼2.25 U/mg. F_o is coupled to 95% and 75%, respectively, as determined by oligomycin inhibition. The lower percentage of coupled complexes in 2YLF₁F_o is most likely due to free F₁ subcomplexes and detergent in the dimer preparation (Figure S1A). The similarly high activities of 2YLF₁F_o and 1YLF₁F_o indicate that the two ATP synthase monomers within the dimer operate independently in ATP hydrolysis mode.

F₁c₁₀ Crystal Structure

Crystals of the YLF₁c₁₀ subcomplex were obtained from the 1YLF₁F_o complex. Whereas previous crystallographic studies of similar complexes (Giraud et al., 2012, Pagadala et al., 2011, Stock et al., 1999) used an excess of nucleotide substrates or inhibitors to trap functional states, we crystallized YLF₁c₁₀ without any such additives to ensure similar conditions for crystallography and cryo-EM. The 3.5 Å X-ray structure of YLF₁c₁₀ (Table 1; Figures S2A–S2D) reveals that all three non-catalytic α subunits bind Mg·ATP in their nucleotide sites. Of the three catalytic β subunits, one is empty (β_E), while both the β_DP and β_TP sites (Abrahams et al., 1994) contain Mg·ADP (Figures 1 and S2E).

Cryo-EM Structure of the Yarrowia lipolytica ATP Synthase Dimer

We determined the structure of the 2YLF₁F_o by single-particle cryo-EM (Figure 2A). After 2D and 3D classification, 38,679 particles were selected for reconstruction of a 3D map with C2 symmetry imposed. The central stalks of the two monomers include an angle of ∼100°. Masking one monomer in the dimer during 3D refinement improved the resolution to 6.9 Å for the F₁ subcomplex and masking the F_o dimer improved it to 6.2 Å, as determined by gold-standard Fourier shell correlation (Figure S4). The long helices in the peripheral stalks and the F_o part of the stator are the best-resolved features (Movie S1). The resolution of the F₁c₁₀ subcomplex in the cryo-EM map is slightly lower, due to minor variations in the dimer angle (Figures S3D and S3E) and to differences in rotational position of the rotor assembly. Further classification revealed that the position of the central stalk varies independently in both monomers (Figures S3F and S3G), confirming that the two ATP synthase assemblies in the dimer function independently, as already suggested by the similar ATPase hydrolysis activities of 1YLF₁F_o and 2YLF₁F_o.

Classification of the same dataset with one monomer in the dimer masked enabled us to distinguish three different rotational states of the F₁ head assembly, with two out of three positions favored, in which the positions of the central stalk differ by ∼120° or 240° (Figures S3H and S3I). Interaction with the central stalk affects the nucleotide binding domains and C-terminal domains of the β subunits (Figure S3I). The three conformations show the three “Boyer states,” open, loose, and tight (Boyer, 1997), of the Y. lipolytica complex as seen in the YLF₁c₁₀ crystal structure (Movies S2 and S3), similar to the crystal structure of the bovine F₁ complex (Abrahams et al., 1994). The three states are trapped in energy wells, which stall the rotor in defined positions upon dissipation of the pmf by the membrane-solubilizing detergent.

In the most populated class (45% of the particles; subclass 2 in Figures S3H and S3I), a rod-like density protrudes from the α_DPβ_DP pair close to the peripheral stalk (Figure 2D). This density superposes precisely on the inhibitor IF1 in an X-ray structure of the bovine mitochondrial F₁F_o-ATP synthase with IF1 bound (Gledhill et al., 2007). The presence of IF1 in ATP synthases prepared from large-scale yeast fermentations is not unexpected, as oxygen concentration of these cultures can decrease, which reduces the matrix pH and triggers IF1 binding, as observed with yeast grown on non-fermentable substrates (Satre et al., 1975). The fact that IF1 is found in only one of the three classes is, however, surprising.

Peripheral Stalk

The peripheral stalk consists of several long, well-resolved α helices, which were traced without ambiguity (Figures 2A–2C). Homology models based on crystal structures of the bovine subunits b, d, and OSCP (Dickson et al., 2006, Rees et al., 2009) were fitted to the soluble sector of the Y. lipolytica peripheral stalk, which has the same subunit composition (Table S1). Subunit h has only 20% sequence identity to the equivalent bovine F6 (Fujikawa et al., 2015), accounting for the observed structural differences. The overall curvature of the peripheral stalk differs from that in the bovine crystal structure, but resembles that in the cryo-EM map of the monomeric bovine complex (Baker et al., 2012), suggesting that crystal contacts affect stalk curvature. As in the bovine complex (Zhou et al., 2015), helices 1 and 5 of OSCP on the F₁ head are in contact with the N terminus of α_E (Rees et al., 2009). Two further close contacts are found at the N terminus of α_TP, which interacts with helices 4 and 5 of OSCP, and at the N terminus of α_DP, which intercalates between the peripheral stalk helices. The N terminus of this α subunit forms a previously unrecognized four-helix bundle with b, h, and the C terminus of OSCP, which positions the F₁ head and bonds it to the peripheral stalk (Figure 2B). The contacts in this interaction are mainly hydrophobic, except for those mediated by the conserved residues αGlu33 and αArg41. The d subunit interacts with the C terminus of the α_DP subunit, displacing it toward the peripheral stalk by 5 Å relative to the YLF₁c₁₀ X-ray structure (Figure 2C). Below the F₁ head, peripheral stalk subunits d and b bend toward the central stalk. The density of subunit b, which is thought to have two trans-membrane helices at its N terminus (Figure S4), continues without interruption into the membrane.

Helix Assignment in the F_o Stator

The Y. lipolytica F_o stator subcomplex comprises the eight membrane protein subunits a, b, e, f, g, i, k, and 8. The F_o part of each monomer contains ten well-defined α-helical densities enveloped by a detergent micelle that features the characteristic ∼90° dimer membrane curvature (Davies et al., 2012) (Figure 2A). Six of these densities indicate trans-membrane α helices, numbered 1–6 in Figures 3A and 3B and assigned in Figure 3C. The loops connecting the helices are, for the most part, not visible at this resolution, but the helix segments can be identified on the basis of sequence comparison, secondary structure predictions, proximity, and known helix topology.

Rotational F₁ States

A detailed comparison of the YLF₁c₁₀ crystal structure to the bovine (Abrahams et al., 1994) and Saccharomyces cerevisiae (Kabaleeswaran et al., 2006) F₁ and F₁c₁₀ complexes (Figure S8) reveals that the three conformational states of the corresponding α/β heterodimers are very similar in the two yeast species, with root-mean-square deviation (RMSD) values below 1.7 Å, while nucleotide binding and C-terminal regions of the bovine β subunits differ (Table S2; Movies S4 and S5). In YLF₁c₁₀ both the β_E and the β_DP site are more open than in the bovine complex, while their β_TP sites are similar. Overall, the three β subunits resemble one another more closely in Y. lipolytica than in the bovine and S. cerevisiae complexes (Figure S8B).

Aligning the γ subunits in all F₁ X-ray structures and comparing the relative positions of the conserved P loop in the β_DP subunit (Figure S8A), we find that the YLF₁c₁₀ P loop is shifted to a position that, in bovine F₁ (Rees et al., 2012), would indicate a post-hydrolysis or pre-product release state. The post-hydrolysis position of the γ subunit in YLF₁c₁₀ agrees with the presence of bound Mg·ADP in the catalytic β_DP and β_TP sites. Since YLF₁c₁₀ was crystallized without added nucleotides, the ADP originates from ATP hydrolysis during isolation or crystallization (Abrahams et al., 1996, Gledhill et al., 2007). The fact that ADP is present in the Y. lipolytica β_DP at this late stage of hydrolysis without addition of nucleotide-stabilizing azide (Bowler et al., 2006) is surprising, as ADP was not found in the binding sites of other F₁ complexes crystallized under similar conditions (Bianchet et al., 1998, Stocker et al., 2007). This might indicate a higher nucleotide affinity of the Y. lipolytica β_DP site. In contrast, phosphate (P_i) was not detected in the β_DP and β_TP sites, in line with a possible alternative leaving route for P_i, as described for S. cerevisiae F₁ (Kabaleeswaran et al., 2006).

We found the inhibitor protein IF1 bound to one of the three different rotational F₁ states in the cryo-EM maps, but not in the YLF₁c₁₀ X-ray structure, indicating that it was lost during crystallization. By contrast, IF1 was present in all seven rotary states in the cryo-EM maps of monomeric bovine F₁F_o-ATP synthase (Zhou et al., 2015), as would be expected since the complex was purified by IF1 affinity chromatography. Apart from the absence of IF1 in the β_DP region (Figure 2D), the 3.5 Å YLF₁c₁₀ crystal structure matches the dimer map closely (Figures 2A, S3H, and S3I). Like the X-ray structure, the cryo-EM map therefore shows a post-hydrolysis state.

Structure of the F_o Stator

The subunit a structure is remarkably conserved in F₁F_o-ATP synthases. Densities for all six helices of our Y. lipolytica structure are also present in the same orientations in the cryo-EM map of the bovine heart monomer (Zhou et al., 2015), while four helices are present in the recent 4 Å X-ray structure of the bacterial complex (Morales-Rios et al., 2015) (Figure S7B). The Polytomella cryo-EM map (Allegretti et al., 2015) has elements that correspond to each of the six a subunit helices in Y. lipolytica, even though the polypeptide sequences diverge. The other Polytomella stator subunits do not resemble those of yeasts, metazoans, or bacteria.

The cryo-EM map of the bovine monomer shows four trans-membrane helices, two of which were identified as belonging to the peripheral stalk subunit b, and one each to subunits a and A6L (subunit 8 in fungi) (Zhou et al., 2015). All helices superpose well on our map and the assignment agrees with ours, except that there is no density for the f subunit in the bovine map (Zhou et al., 2015). Although the bovine complex prepared according to Runswick et al. (Runswick et al., 2013) should contain the f subunit, this subunit was not identified by Zhou et al. Subunit f may have dissociated during isolation of the bovine monomer, suggesting that it is not firmly attached. As the f subunit is responsible for direct dimer contacts in our assignment (Figure 6), its dissociation from the bovine complex may explain why dimers from mammalian mitochondria are, in our experience, less stable.

The X-ray structure of the bacterial F₁F_o complex from Paracoccus denitrificans (Morales-Rios et al., 2015) shows only two of the trans-membrane helices in the F_o stator, which superpose well on the trans-membrane helix of subunit 8 and aH1 in the Y. lipolytica dimer (panel (iv) in Figure S7B). Therefore, these helix densities, which were unassigned in the Paracoccus map, belong to one of the two bacterial b subunits and the trans-membrane helix of subunit a (aH1), lending strong support to our conclusion that mitochondrial subunit 8 derives from a bacterial b subunit. Surprisingly, the trans-membrane helix of the second b subunit seems to be completely absent in the Paracoccus structure, indicating that it is flexible or disordered in the 4 Å X-ray map.

Proton Translocation through F_o

Ion translocation through F_o is mediated by the a subunit and the c-ring (Figure 7). A number of high-resolution X-ray structures of ATPase c- or K-rings have shown that the conserved glutamate residues in the c subunits lock the protons (or Na⁺) in the hydrophobic environment of the lipid bilayer (Meier et al., 2005, Murata et al., 2005, Pogoryelov et al., 2009) but open to release the ions in a hydrophilic environment (Mizutani et al., 2011, Pogoryelov et al., 2010, Symersky et al., 2012). The recent cryo-EM structures of the Polytomella ATP synthase and S. cerevisiae V-type ATPase indicate two aqueous half-channels at the subunit a/c interface that are thought to conduct protons to and from the c-ring protonation sites (Allegretti et al., 2015). We find similar aqueous half-channels in equivalent positions of the Y. lipolytica dimer map (Figures 7A and 7B). Conserved hydrophilic residues of aH5 and aH6 line the aqueous cavity on the matrix side (Figures 7C and 7D). The conserved charged and polar residues of aH5, starting with Glu168 four helix turns upstream of Arg182, create the hydrophilic environment to release the proton from the opposing c subunit glutamate into the matrix. The ∼20° tilt of the aH5/aH6 hairpin places the hairpin loop close to the IMS surface, and the C terminus of aH6 on the matrix side (Figure 7A). Consequently, the lumenal half-channel near the hairpin loop and the matrix half-channel at the C terminus of subunit a are laterally offset, as anticipated (Junge et al., 1997, Vik and Antonio, 1994). The proton entrance channel on the IMS side is likely to include the conserved Asn186 in aH5, Asn106 in aH3, and the exchangeable pair His191/Glu229 in aH5 and aH6, one of which is Glu or Asp in all ATP synthases (Figure S5). Rather than mediating proton release, the essential Arg182 would allow only deprotonated c subunit glutamate side chains to pass (Figure 7D). Removal of this arginine by mutagenesis uncouples ion translocation from ATP synthesis (Mitome et al., 2010), as it results in futile proton translocation without c-ring rotation.

Clinical studies show that mutations in aH5 and aH6 impair the functionality or assembly of ATP synthase in human mitochondria (Kucharczyk et al., 2009, Xu et al., 2015), giving rise to severe neuropathological disorders (Houstek et al., 2006), such as the maternally inherited Leigh syndrome or retinitis pigmentosa (Kucharczyk et al., 2009). A molecular understanding of the exact ion translocation mechanism is essential for exploring future therapy. The structure of the genetically accessible Y. lipolytica ATP synthase now provides a basis for structural and functional studies to combat these diseases at the molecular level.

Dimer Contacts

In the Y. lipolytica F₁F_o dimer, which contains subunits e and g, the detergent belt is bent by roughly 100°. In the bovine F₁F_o monomer, which contains subunits e and g, it is bent by ∼50° (Baker et al., 2012, Zhou et al., 2015), but in the Y. lipolytica monomer, which lacks e and g, the belt is more or less straight (Figure S7A; Table S1). We conclude that e and g are chiefly responsible for inducing the membrane curvature that results in mitochondrial cristae morphology.

There is no evident function for the C-terminal IMS helix of subunit e in the dimeric complex (Figure 6A). In contrast to the bovine map (Zhou et al., 2015), this elongated density protrudes straight out of the F_o stator region into the IMS, while in the bovine map it is curved and contacts the central plug of the c-ring. A role in the formation of higher-order ATP synthase oligomers and dimer rows (Strauss et al., 2008, Davies et al., 2012) seems unlikely, as the extension is not easily accessible and appears to point in the wrong direction for interaction between adjacent dimers (Figure 5D). Moreover, the dimer spacing along the rows is irregular (Daum et al., 2013), which suggests that the inter-dimer protein contacts are dynamic. Instead, the subunit e extension with its predicted coiled-coil propensity may play a role in complex assembly.

The two F_o subcomplexes in the membrane are separated by a wedge-shaped gap that is ∼40 Å wide on the matrix side and narrows to ∼15 Å on the IMS side (Figures 5G and 6A). On the matrix side, this wedge appears to be filled by lipid or detergent, as there is no distinct protein density. The tip of the wedge on the IMS side contains the dimer contact domain assigned to the C terminus of subunit f (Figure 6). The center of the wedge-shaped gap is bridged by a conspicuous sheet of density connecting the trans-membrane helices of subunits a and 8 (Figure 5G), which may contribute to dimer formation. It is tempting to speculate that this density belongs to the so far unassigned yeast-specific subunit k, a small, partly hydrophilic protein without predicted trans-membrane helix (Figure S4D). The sheet may form a barrier between the membrane leaflet on the matrix side and hydrophilic protein domains on the IMS side of the dimer interface. The matrix half of the wedge-shaped space has the thickness of one membrane leaflet, implying a new and unusual membrane architecture in this region of the dimer.

Role of ATP Synthase Dimers in Membrane Morphology

The comparatively simple bacterial and chloroplast ATP synthases consist of eight or nine different subunits, which are sufficient for ATP production. The chloroplast ATP synthase has been shown to be monomeric (Daum et al., 2010), and no ATP synthase dimers have been reported in bacteria. By contrast, all known mitochondrial ATP synthases form dimers in the membrane that self-assemble into rows (Davies et al., 2012). Mitochondrial ATP synthases of yeasts and metazoans have eight additional subunits of so far unexplained structure and function. Our map shows how the mitochondria-specific subunits in the mitochondrial F_o subcomplex are arranged, and that most of them have a role in dimer formation: the N-terminal trans-membrane extension of b anchors e and g to the complex, and the e/g heterodimer induces local membrane curvature, which in turn appears to enable the IMS domain of f (and possibly subunit k) to establish protein-protein contacts across the dimer interface.

ATP synthase dimer rows are a prerequisite for the formation of inner membrane cristae (Davies et al., 2012), a hallmark signature of mitochondrial morphology. Cristae formation extends the membrane surface to accommodate a large number of respiratory chain complexes, making it possible to meet the high energy demands of eukaryotic cells (Lane and Martin, 2010). They also form a mitochondrial sub-compartment that supports a locally increased proton concentration in the confined cristae space. The structure of the mitochondrial ATP synthase dimer thus offers new insights into how mitochondria became the efficient power plants of eukaryotic cells.