Decision letter: Structure of a mitochondrial ATP synthase with bound native cardiolipin

Article Figures and data Abstract eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The mitochondrial ATP synthase fuels eukaryotic cells with chemical energy. Here we report the cryo-EM structure of a divergent ATP synthase dimer from mitochondria of Euglena gracilis, a member of the phylum Euglenozoa that also includes human parasites. It features 29 different subunits, 8 of which are newly identified. The membrane region was determined to 2.8 Å resolution, enabling the identification of 37 associated lipids, including 25 cardiolipins, which provides insight into protein-lipid interactions and their functional roles. The rotor-stator interface comprises four membrane-embedded horizontal helices, including a distinct subunit a. The dimer interface is formed entirely by phylum-specific components, and a peripherally associated subcomplex contributes to the membrane curvature. The central and peripheral stalks directly interact with each other. Last, the ATPase inhibitory factor 1 (IF1) binds in a mode that is different from human, but conserved in Trypanosomatids. eLife digest Every living thing uses the energy-rich molecule called adenosine triphosphate, or ATP, as fuel. It is the universal molecular currency for transferring energy. Cells trade it, mitochondria make it, and the energy extracted from it is used to drive chemical reactions, transport molecules across cell membranes, energize nerve impulses and contract muscles. ATP synthase is the enzyme that makes ATP molecules. It is a multi-part complex that straddles the inner membrane of mitochondria, the energy factories in cells. The enzyme complex interacts with fatty molecules in the mitochondrial inner membrane, creating a curvature that is required to produce ATP more efficiently. The mitochondrial ATP synthase has been studied in many different organisms, including yeast, algae, plants, pigs, cows and humans. These studies show that most of these ATP synthases are similar to each other, but obtaining a high resolution structure has been a challenge. Some single-cell organisms have unusual ATP synthases, which provide clues about how the enzyme evolved in pursuit of the most energy efficient arrangement. One such organism is the photosynthetic Euglena gracilis, which is closely related to the human parasites that cause sleeping sickness and Chagas disease. Now, Mü̈hleip et al. have extracted ATP synthase from E. gracilis and reconstructed its structure using electron cryo-microscopy. The high resolution of this reconstruction allowed for the first time to examine the fatty molecules associated with ATP synthase, called cardiolipins. This is important, because cardiolipins are thought to modulate the rotating motor of the enzyme and affect how the complex sits in the membrane. The analysis revealed that the ATP synthase in E. gracilis has 29 different protein subunits, 13 of which are only found in organisms of the same family. Some of the newly discovered subunits are glued together by fatty molecules and extend into the surrounding mitochondrial membrane. This distinctive structure suggests an adaptation which likely evolved independently in E. gracilis for efficiency. These results represent an important advance in the field, and provide direct evidence for the functional roles of cardiolipin. This information will be used to reconstruct the evolution of this mighty molecule and to further study the roles of cardiolipin in energy conversion. Moreover, the analysis identified similarities between the ATP synthase in E. gracilis and human parasites, which could provide new therapeutic targets in disease-causing parasites. Introduction The mitochondrial ATP synthase is a membrane protein complex that generates most of the ATP in eukaryotic cells. The synthesis of ATP from ADP and inorganic phosphate proceeds via rotary catalysis, which uses the energy of the electrochemical gradient across the mitochondrial inner membrane. The translocation of protons through the membrane-bound Fo part, mediated by subunit a, drives the rotation of a membrane-embedded c-ring and the attached central stalk, which together form the rotor. The torque of the rotor against the stator subunits induces conformational changes in the (αβ)3 headpiece, thereby triggering catalysis (Abrahams et al., 1994; Noji et al., 1997). The mitochondrial ATP synthase forms dimers, which in turn associate into dimer rows along the high-curvature membrane regions of the cristae (Davies et al., 2012; Paumard et al., 2002; Strauss et al., 2008). Loss of ATP synthase dimers results in aberrant cristae morphology, indicating that dimers are required for membrane bending and proper cristae formation in mitochondria (Davies et al., 2012; Paumard et al., 2002). Previous biochemical and mass spectrometry analysis showed that a highly divergent ATP synthase with numerous new subunits is found in the phylum of Euglenozoa, belonging to the Excavata supergroup (Perez et al., 2014; Yadav et al., 2017; Zíková et al., 2009). A combination of transcriptome analysis and sequencing of nuclear and mitochondrial genomes showed the phylogenetic relationship between the protozoan Euglena gracilis and Kinetoplastids, which include human parasites such as Trypanosoma and Leishmania that cause sleeping sickness, Chagas disease, and Leishmaniasis (Dobáková et al., 2015; Ebenezer et al., 2019). However, while the reported recruitment of additional subunits in the Euglenozoan ATP synthase implies a distinct architecture, neither the complete composition, nor the functions or structures of the specific subunits have been reported. Previous attempts were limited by resolution (Mühleip et al., 2017) or restricted to the conserved F1 subcomplex (Montgomery et al., 2018). In addition, lipids are known to be important to the structure and function of mitochondrial ATP synthases (Eble et al., 1990; Kühlbrandt, 2019; Laage et al., 2015; Srivastava et al., 2018). Proper cristae formation specifically depends on cardiolipin (CL) (Mileykovskaya and Dowhan, 2009), an anionic lipid that accounts for 20% of total lipids in the inner mitochondrial membrane (Calvayrac and Douce, 1970; Daum and Vance, 1997; Zinser and Daum, 1995). Cardiolipin is also essential for the activity of isolated mitochondrial ATP synthases (Laird et al., 1986; Pitotti et al., 1972; Santiago et al., 1973), and promotes the formation of dimer rows (Acehan et al., 2011). Although bound lipids have previously been observed in structures of rotary ATPases (Klusch et al., 2017; Murphy et al., 2019; Vasanthakumar et al., 2019), the atomic models of recently reported cryo-EM structures of mitochondrial ATP synthases do not include cardiolipin (Gu et al., 2019; Guo et al., 2017; Klusch et al., 2017; Murphy et al., 2019; Srivastava et al., 2018). To reveal how the mitochondrial ATP synthase is modulated by lipids and organised into a dimer shaping discoid cristae in Euglenozoan mitochondria, we determined the structure of the ATP synthase dimer from E. gracilis. The atomic model of the entire 2-MDa complex contains 29 different subunits (eight newly identified) and provides a comprehensive description of a markedly distinct mitochondrial ATP synthase dimer, including a previously unseen binding mode of its natural inhibitor protein IF1. The membrane region was determined to 2.8 Å resolution, enabling the identification of a structurally divergent subunit a and visualisation of 37 associated native lipids. Importantly, cardiolipin binding sites are found at the rotor-stator interface, dimer interface, and in a peripheral Fo cavity. These data provide insight into protein-lipid interactions in the mitochondrial ATP synthase and its evolution, suggesting functional roles of lipids in proton translocation, dimerization and stabilisation. Results and discussion Overall structure The mitochondrial ATP synthase dimer was purified natively from E. gracilis and subjected to cryo-EM structure determination (Supplementary file 1). Symmetry expansion of the pseudo-C2-symmetric dimer particles was used to classify individual F1/c-ring monomers into rotational states 1, 2 and 3 (named according to bovine nomenclature; Zhou et al., 2015) and resolve structures at 3.0- to 3.9 Å resolution. Using masked refinement, maps of the membrane region, the rotor and the peripheral stalk tip were refined to 2.8 and 3.3 and 3.8 Å resolution, respectively, enabling the construction of atomic models (Figure 1—figure supplements 1–2; Video 1; Supplementary file 2). The model of the complete mitochondrial ATP synthase contains 29 different proteins, of which 14 are phylum-specific, displaying a distinct architecture, particularly in the membrane region. The interactions between the monomers are expanded, resulting in a 45° dimer angle, compared to ~100° in yeast and mammals (Figure 1A) (Davies et al., 2012; Gu et al., 2019; Hahn et al., 2016). The membrane-bound Fo region is composed of 22 proteins, of which 13 have no homologs in animals and fungi. The well-resolved membrane region allowed the identification of 37 native lipids, leading to the assignment of 25 cardiolipins, whereas the 12 remaining phophoslipids could not be unambiguously identified and were modelled as phosphatidic acid. Figure 1 with 2 supplements see all Download asset Open asset Structure of the E. gracilis ATP synthase dimer. (A) Atomic model of the complete E. gracilis ATP synthase dimer with both subcomplexes in rotational state-1. The 2-MDa dimeric F1Fo-complex contains 29 different proteins. Dashed lines indicate C2-symmetry axis and 45° dimer angle. (B) OSCP/F1/c-ring subcomplex in rotational state-1, bound to its natural inhibitor protein IF1 (cyan), remaining Fo transparent. (C) Density map showing the lumen-exposed Fo region. Detergent belt shown in yellow; c-ring β-barrel in dark grey, Fo subunits as in (A). (D) Close-up of the lumenal interface of ATPEG1 (blue) with the c-ring (grey). The interaction occurs mostly via hydrophobic interactions (blue and grey sticks). (E–G) Euglenozoa-specific Fo-subunits with known folds. (E) ATPTB1 in blue superposed with Mdm38 (PDB ID: 3SKQ) (Lupo et al., 2011), six conserved helices coloured yellow, rest grey. (F) ATPTB3 in blue superposed with a bacterial homoisocitrate dehydrogenase in orange (PDB ID: 4YB4)(Takahashi et al., 2016), adopts a Rossman-fold. (G) ATPEG5 in yellow is a structurally conserved ortholog of the cytochrome c oxidase subunit VIb superfamily; bovine subunit VIb in purple (PDB ID: 2Y69) (Kaila et al., 2011). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Density map of E. gracilis ATP synthase dimer with regions corresponding to protein shown in grey and the detergent belt coloured gold. Following reports of mitochondrial ATP synthases from various organisms, different names have previously been given to subunits performing the same role in F-type ATP synthases (Kühlbrandt, 2019). For the mitochondrial ATP synthase from E. gracilis, we adopt a nomenclature that is consistent with the conserved Fo subunits from yeast (subunits a-d, f, i/j, k, 8, OSCP) (Guo et al., 2017) and euglenozoa-specific subunits previously identified in Trypanosoma brucei (ATPTB1, 3, 4, 6, 12) (Perez et al., 2014; Zíková et al., 2009), whereas the additional subunits are named ATPEG1 to 8, according to their molecular weight (Supplementary file 3). Identification of the transmembrane subunits All known mitochondrial ATP synthases form dimers in the membrane through transmembrane Fo subunits. However, in E. gracilis, only subunit c and a set of phylum-specific subunits were identified (Perez et al., 2014; Yadav et al., 2017; Zíková et al., 2009), suggesting a divergent Fo composition. The core subunit a mediates proton translocation and contains the strictly conserved R176 (Saccharomyces cerevisiae numbering). Despite its functional importance, subunit a was identified neither in the E. gracilis genome project (Ebenezer et al., 2019), nor in the mitochondrial genome and transcriptome analysis (Dobáková et al., 2015). Our cryo-EM map allowed tracing of subunit a, for which we identified six structurally conserved membrane-embedded helices (H1a to H6a) directly from side-chain densities (Figure 2—figure supplement 1A to C). Using this information, we then found the matching sequence in the available genomic data and mapped it to a mitochondrial contig, which also contains subunit 8 and nad1 in a single open reading frame (Figure 2—figure supplement 1E). Thus, by combining the information from cryo-EM and sequencing, we report the most divergent subunit a found up to date. In all previously reported ATP synthase structures, the horizontal helix H5a bends around the c-ring, following its curvature, thereby contacting four of the ten subunits in the c-rings of yeast and algae (Figure 2B,D) (Allegretti et al., 2015; Guo et al., 2017; Hahn et al., 2016). By contrast, in the E. gracilis structure, the N-terminal part of H5a is kinked towards the lumen, and therefore does not interact with the c-ring, and instead extends towards the lumenal membrane surface (Figure 2A,C). This structural rearrangement results in a smaller interface between subunits a and c, with only three c-subunits forming interactions, which also has implications for the formation of the matrix half-channel, as discussed below. Figure 2 with 1 supplement see all Download asset Open asset E. gracilis subunit a fold comparison. Top view (upper panel) and side view (lower panel) of the E. gracilis (left) and S. cerevisiae (right) (Guo et al., 2017) subunit a (green) and c-ring (grey). Both structures contain the conserved H1-6a, with E. gracilis displaying two helices (H4'a and H4''a) in an extension segment and a C-terminal extension. Whereas the N-terminal region of H5a (dark green) is kinked towards the c-ring in the yeast complex, it extends towards the lumen in the E. gracilis structure, thereby diminishing its interface with the c-ring. Unlike its yeast homolog, the N-terminus of E. gracilis subunit a is not involved in dimerisation, but contributes a strand to a β-sheet along the lumenal side of the detergent micelle. In the yeast mitochondrial ATP synthase, subunit a interacts with transmembrane subunits b, f, i/j, k, 8 and membrane-associated subunit d (Guo et al., 2017). Since no homologs were reported for any of these subunits in E. gracilis or T. brucei (Perez et al., 2014; Yadav et al., 2017; Zíková et al., 2009), we next investigated their putative location through superimposition of our Fo model with the yeast counterpart (Guo et al., 2017). Based on the matching position and topology of the transmembrane helices as well as conserved positions of interactions with subunit a, we identified all six associated subunits, which are structurally conserved, but display no significant sequence similarity to yeast counterparts (Figure 3). Subunits b, f, i/j, k and 8 contain a single transmembrane helix associated with subunit a, whereas subunit d forms a clamp around subunit 8 at the base of the peripheral stalk, containing a structurally conserved two-helix motif at the matrix side of the membrane (Figure 3A). Finally, subunit k is bound peripherally to subunit a, as in yeast, however the H5a kink results in a 15-Å displacement of the transmembrane helix of subunit k away from the c-ring, compared to its yeast counterpart (Figure 3B). These data show that despite sequence divergence, the assembly of the central Fo subunits around subunit a is architecturally conserved between Euglenozoa and Metazoa. Figure 3 Download asset Open asset Conserved subunits of the Fo region. (A) Side view of the conserved E. gracilis Fo subunits. Transmembrane helices with structural equivalents in yeast are labelled. (B) Top view of the superimposed conserved Fo subcomplexes from E. gracilis (coloured) and yeast (grey) PDB ID: 6B2Z (Guo et al., 2017). Although subunit k does not superimpose well, it occupies the same position relative to the H5a. The striking architectural divergence of the E. gracilis ATP synthase dimer is brought about by euglenozoa-specific subunits and extensions of the structurally conserved Fo subunits, which render them on average 2.5 times larger than in the yeast mitochondrial ATP synthase. Only subunit b is truncated. The extensions of the conserved Fo subunits are mostly involved in forming interactions with the euglenozoa-specific subunits, thus providing a platform for the observed increased molecular mass of the Fo (Figure 4—figure supplement 1A,C). The additional 13 euglenozoa-specific Fo subunits determine the architecture of the ATP synthase dimer, giving the Fo a markedly different overall shape, making it almost three times the size of its yeast counterpart (Figure 4—figure supplement 1). They contribute to the dimerization interface, the peripheral stalk and Fo periphery. The C-terminal helix of euglenozoa-specific ATPEG1 (H5EG1) extends 50 Å from the membrane region into the lumen, where it interacts with the N-terminal extensions of subunit c, which together form a ten-stranded β-barrel (Figure 1C and D) protruding ~20 Å into the lumen. The N-terminal residues of subunit c (A24, I25) form a hydrophobic interface with hydrophobic residues (M148, M152, L155, I159, L163) of the amphipathic ATPEG1 helix. The position of the lumenal H5EG1 on the c-ring β-barrel remains largely unchanged in all three rotational states, suggesting a mechanism of transient rotor-stator interaction during c-ring rotation. A similar lumenal interaction has previously been reported in the bovine ATP synthase, where subunit e extends from the membrane to contact the c-ring (Zhou et al., 2015). In the porcine ATP synthase tetramer, subunit e has been proposed to interact with the 6.8 kDa proteolipid, which has been suggested to reside inside the c-ring (Gu et al., 2019). Other euglenozoa-specific Fo subunits contain structural domains that were shown to be functionally important in mitochondria (Figure 1E to G). ATPTB1 is a membrane-associated protein on the matrix side of the Fo periphery that adopts an Mdm38-like fold, which was shown to associate with yeast mitochondrial ribosomes at the inner mitochondrial membrane (Frazier et al., 2006). ATPTB3 is an isocitrate dehydrogenase ortholog that adopts a Rossman fold located at the tip of the peripheral stalk. ATPEG5 is a structurally conserved ortholog of the cytochrome c oxidase subunit VIb superfamily. Dimer interface and associated lipids The defining feature of mitochondrial ATP synthases is the formation of dimers in the crista membrane. In yeast, the two monomers are connected through Fo, and the dimer interface is formed on the lumenal side by the conserved Fo subunits a and i/j, as well as subunits k and e (Guo et al., 2017). Our atomic model of the E. gracilis mitochondrial ATP synthase shows that in contrast to yeast, subunits a, i/j, k do not contribute to the dimer interface, which instead is formed by species- and phylum-specific subunits and extensions of apparent homologs (Figure 4, Figure 4—figure supplement 1, Figure 4—figure supplement 2). The extensive dimer contacts are stacked across three layers: the matrix side, the transmembrane region and the lumenal side (Figure 4A and Figure 4—figure supplement 1A,C). On the matrix side, an extension of subunit d adopts an elaborated ferredoxin-like all-β fold that forms a dimer interface close to the symmetry axis (Figure 4A and Figure 4—figure supplement 1E,F). The curved H3 of ATPEG1 forms a motif along the matrix surface of the membrane contributes to the dimer interface with both a transmembrane helix that interacts with ATPEG1 and its which the two each into the rotor-stator interface of On the lumenal ATPEG1 interacts with the C-terminal extension of subunit The different Fo subunit and their in dimer formation in a 45° dimer angle, compared to ~100° in yeast (Figure 1A) (Guo et al., 2017). Thus, despite the of conserved Fo subunits and the to their extensions to the dimer interface (Figure 4—figure supplement the E. gracilis mitochondrial ATP synthase a different dimer architecture, compared to the yeast (Figure 4—figure supplement and ATP synthases (Gu et al., 2019; Murphy et al., 2019), suggesting that dimer formation evolved independently in different Figure with 3 supplements see all Download asset Open asset The dimer interface and associated lipids. and of the dimer interface along (A) and (B) to the membrane The subunits are stacked along the C2-symmetry axis and formed by two of subunit d and ATPEG1 which interacts with its as well as (green) and subunit in (B) indicate positions of to of the sites in lipids at the dimer interface identified as cardiolipin to or modelled as phosphatidic and G). residues (subunits include at Density shown as grey In to the interactions, we identified bound the dimer interface (Figure to Figure 4—figure supplement 2). of them are cardiolipin molecules subunits close to the C2-symmetry axis (Figure to two horizontal helices of the two of which extend along the matrix side of the membrane region (Figure Video 2). These protein-lipid interactions indicate a role of cardiolipin in the of the dimer which is consistent with its proposed role in subunit interactions between the transmembrane helices in mitochondrial (Mileykovskaya and Dowhan, 2014; et al., 2016). Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Atomic model of the E. gracilis ATP synthase dimer with bound lipids. The cryo-EM map of the membrane region is shown as view of cardiolipin and of the two ATPEG1 at the matrix side of the membrane region. interface and proton translocation occurs at the which is formed in the membrane by horizontal helices and the c-ring (Allegretti et al., 2015) (Figure supplement In the E. gracilis ATP synthase, the essential of H5a is conserved, and interacts with on we found two additional horizontal helices, by and subunit k (Figure and Figure supplement is bound to subunit a in the membrane, and its horizontal helix is close to the matrix side of the membrane, in to at a of Å (Figure it to interact with the same transmembrane helix of the c-ring as H5a and (Figure supplement Figure with 2 supplements see all Download asset Open asset The rotor-stator interface is by bound cardiolipin. (A) from the c-ring towards the membrane-embedded stator subunits. H5a and are by the amphipathic Cardiolipin molecules subunit a are shown in of are mostly and shown only for on the lumen and matrix side are shown in orange and subunits not shown for The conserved and the at the lumen are shown with (B) of the lumenal is by the of subunit f, subunit 8, as well as a lumenal segment of subunit the Fo, the lumen is by transmembrane helices of subunits and the of the lumen shown in orange with map as c-ring in grey. indicate proposed of proton (C) and residues between and the k contributes a horizontal helix to the rotor-stator The E. gracilis ATP synthase structure suggests a mechanism of proton translocation via two proton similar to previously in F-type ATP synthases (Allegretti et al., 2015; Guo et al., 2017; Hahn et al., 2016). the membrane region via the lumenal half-channel, which we as an of the atomic The of the lumenal is by the of subunit and extension of subunit and the N-terminal regions of and subunit 8, of which only the is structurally conserved in yeast (Figure the Fo region, the lumenal is by the conserved transmembrane helices of subunits and b and extends between as previously suggested (Guo et al., 2017). the of the lumenal half-channel, we identified a detergent with its protruding into the and the between H5a and both the and of the lumen (Figure translocation to the rotor-stator interface results in of the conserved in E. in the of the c-ring and rotation of the c-ring from the F1 to Fo et al., 1997). This proton has been suggested to be mediated by a of in S. which is with of This the lumenal is conserved in yeast, mammals and (Gu et al., 2019; Guo et al., 2017; Klusch et al., 2017) but not in E. gracilis and Figure The of an from the of the lumenal proton that it is not strictly required for proton to the c-ring in mitochondrial ATP this function to be in our structure by of which extends towards the c-ring and interacts with of thus forming an at the lumenal (Figure almost a rotation of the c-ring, the is by of subunit a. The proton is then into the matrix In ATP synthases, H5a bends around the c-ring, thereby the (Guo et al., 2017). to the kinked E. gracilis ATP synthase the interaction of the N-terminal segment of H5a following the c-ring curvature (Figure its functional role in forming the is by the of and and subunit k which forms a horizontal helix in the membrane that extends towards the c-ring (Figure Figure supplement Thus, the interface between subunits a and c is by structural forming the matrix (Figure together with the horizontal and the lumenal the E. gracilis ATP synthase and increased of c-ring interactions compared to its yeast a the E. gracilis rotor-stator interface larger surface of compared to in yeast (Figure supplement 1C and to the the E. gracilis ATP synthase structure two bound cardiolipins side of the two horizontal helices H5a and (Figure and Figure supplement to C). The of both lipids are bound around the of the membrane with their towards the rotor-stator is by of it the H5a and the matrix half-channel, is by of of and of subunit k (Figure Figure supplement to these two bound cardiolipins the two horizontal membrane helices H5a and to the Fo against proton by a high of as well as lipid and in the of the two Thus, in to studies suggesting transient interactions of the c-ring rotor with cardiolipin et al., 2016), the E. gracilis ATP synthase structure shows that of the cardiolipin is bound specifically to the indicating a functional role in proton Fo subcomplex and lipid A of phylum-specific subunits is located at the Fo away from the dimer associated

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