Cristae Là Gì ? Nghĩa Của Từ Cristae Trong Tiếng Việt Nghĩa Của Từ Cristae Trong Tiếng Việt

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Cristae Là Gì ? Nghĩa Của Từ Cristae Trong Tiếng Việt Nghĩa Của Từ Cristae Trong Tiếng Việt

Mitochondrial ATP synthase plays a key role in inducing membrane curvature to establish cristae. In Apicomplexa causing diseases such as malaria and toxoplasmosis, an unusual cristae morphology has been observed, but its structural basis is unknown. Here, we report that the apicomplexan ATP synthase assembles into cyclic hexamers, essential to shape their distinct cristae. Cryo-EM was used to determine the structure of the hexamer, which is held together by interactions between parasite-specific subunits in the lumenal region. Overall, we identified 17 apicomplexan-specific subunits, and a minimal and nuclear-encoded subunit-a. The hexamer consists of three dimers with an extensive dimer interface that includes bound cardiolipins and the inhibitor IF1. Cryo-ET and subtomogram averaging revealed that hexamers arrange into ~20-megadalton pentagonal pyramids in the curved apical membrane regions. Knockout of the linker protein ATPTG11 resulted in the loss of pentagonal pyramids with concomitant aberrantly shaped cristae. Together, this demonstrates that the unique macromolecular arrangement is critical for the maintenance of cristae morphology in Apicomplexa.

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F-type ATP synthases are energy-converting membrane protein complexes that synthesize adenosine triphosphate (ATP) from ADP and inorganic phosphate. These universal enzymes function by using the energy stored in an electrochemical potential across the bioenergetic membrane by rotary catalysis1,2. The soluble F1 subcomplex and membrane-bound Fo subcomplex together form the F1Fo ATP synthase monomer, which is found in bacteria and chloroplasts3,4. In mitochondria, F1Fo ATP synthase resides in the crista membrane where it is known to form dimers, which can further assemble into rows critical for inducing the membrane curvature and maintaining membrane potential and morphology5,6,7,8,9,10.

The main driving force for the synthesis of ATP in mitochondria is the membrane potential11, which has been shown to be higher in the cristae lumen than in the adjacent intermembrane space12. Cristae shaping has been shown to depend on the assembly of ATP synthase dimers into dimer rows, which is the basis for energy conversion in all mitochondria studied to date13. However, the molecular interactions that convey the membrane-shaping properties of the oligomeric ATP synthase are poorly understood. Furthermore, structural data has shown that cristae morphology varies between eukaryotic lineages13.

The infectious apicomplexan parasite Toxoplasma gondii14 is commonly used as a model organism for the malaria-causing agent Plasmodium spp15. These parasites have a unique bulbous cristae morphology, which differs substantially from the lamellar cristae of their mammalian hosts16,17. The underlying mechanism for the bulbous cristae is unknown. Loss of ATP synthase is accompanied by parasite death and defects in cristae abundance in the T. gondii stage responsible for acute toxoplasmosis18, and results in the death of the Plasmodium mosquito form responsible for malaria spread19. Here, we investigate the mechanism for the generation of the unique cristae in the Apicomplexa, using a combination of single-particle cryo-EM, cryo-ET and subtomogram averaging. We first report cristae-embedded ATP synthase hexamers arranged in pentagonal pyramids in the wild type, then identify a key subunit for the assembly, and finally characterise mutant cells with a generated knockout of this subunit.

Structure of the hexameric ATP synthase and its herein identified elements

A large-scale preparation of T. gondii tachyzoite mitochondria and subsequent mild solubilisation with digitonin resulted in the isolation of intact ATP synthase complexes, which we identified as native hexamers. We then performed solubilisation with n-dodecyl-β-D-maltoside (β–DDM) that resulted in dissociation of the hexamers into dimers. Both oligomeric forms were subjected to cryo-EM structure determination (Fig. 1, Supplementary Figs. 1 and 2). Masked refinements of the ATP synthase dimer resulted in maps of the membrane region, the OSCP/F1/c-ring complex, the rotor and the peripheral stalk, ranging in resolution from 2.8 to 3.5 Å (Supplementary Figs. 1 and 3), thus allowing de novo modelling of the respective regions. Refinement into a 2.9-Å resolution consensus map allowed model construction of the entire ATP synthase dimer (Fig. 1a, b and Supplementary Table 1). The 1.85-MDa complex consists of 32 different subunits, of which only 15 are canonical with structural equivalents in other phyla. Homolog searches of 17 noncanonical subunits revealed them to be largely conserved in mitochondriate Apicomplexa including Plasmodium parasites, and in the related phyla of chromerids and perkinsozoa, suggesting that the herein described architecture is likely representative of myzozoans (Supplementary Fig. 4). Thus, following a species-specific nomenclature established in protozoan ATP synthases20,21,22, we term the 17 apicomplexan-conserved T. gondii subunits ATPTG1-17 (TG for T. gondii), with ATPTG1, ATPTG7, and ATPTG16 identified directly from the cryo-EM map (Supplementary Fig. 5a and Supplementary Table 2).


a The composite cryo-EM map of the dimer highlights a small dimer angle and large lumenal region. b The atomic model of the dimer with highlighted apicomplexan-specific structural components responsible for the specific mode of dimerization. c Cryo-EM map of the hexamer showing an assembly as trimer of dimers. d Atomic model of the hexamer with individually coloured subunits.

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The apicomplexan-conserved subunits and extensions of the canonical subunits constitute a membrane-embedded Fo subcomplex, which ties the two F1/c-ring subcomplexes together at the angle of 19° (Fig. 1a, b). This is in stark contrast to ~100° found in the yeast and mammalian ATP synthase dimers23,24, suggesting the narrow-angle parasite dimer induces substantially less membrane curvature. The enlarged T. gondii Fo subcomplex differs markedly in its overall architecture from other ATP synthase structures. It displays distinct structural features including a peripheral matrix-exposed part that we term ‘wing’ region and a 360-kDa lumenal region (Fig. 1b). The Fo periphery contains several compact folds, including three coiled-coil-helix-coiled-coil-helix domain (CHCHD) containing proteins ATPTG7-9; a thioredoxin-like fold in ATPTG4; and ubiquitin-like fold in subunit-k (Supplementary Fig. 5b).

The apicomplexan-conserved Fo-subunit ATPTG11 extends from the lumenal region and plugs the central cavity of the c-ring. This is mediated by the short N-terminal amphipathic helix of ATPTG11 (Ala9-Leu17), which is sequence-conserved in Apicomplexa (Fig. 2a, b). The interface is located on the border of the detergent belt and dominated by hydrophobic residues of ATPTG11 pointing towards the inside of the c-ring, which is unlikely to inhibit the rotation of the rotor (Fig. 2a, c). Protein density on the inside of the c-ring, as suggested for the porcine complex24, was not observed.


a The canonical Fo subunits b, d, f, i/j, k, and 8 contain apicomplexan-specific extensions contributing to a large Fo. Subunit-a contains only the conserved H5-6a, and ATPTG16, ATPTG17 partially replace the missing H1-4a. Inset shows N-terminal helix of ATPTG11 forming a parasite-specific rotor-stator interface with the lumenal side of the c-ring. b Top view of rotor-stator interface. The absence of H1-4a separates subunit-a from several canonical Fo-subunits. Resulting lipid-filled Fo void outlined (black dash). c Cross section through the Fo region of the map. ATPTG11 extends from the lumenal region to plug the c-ring through interactions with H1TG11.

Masked refinement of the hexamer membrane region resulted in a 4.8-Å resolution map (Supplementary Fig. 2). Refining three copies of the dimer model into the hexamer map resulted in a good fit and showed that it forms a cyclic trimer of dimers. No additional subunits or substantial conformational changes of the dimer units were found in the hexameric assembly (Fig. 1c, d and Supplementary Movie 1). The C2 symmetry axis through the dimer is tilted 22° with respect to the C3 axis in the hexamer, thereby bringing into proximity the lumenal regions, which extend 80 Å from the membrane.

Parasite-specific subunits form a dimer interface that includes IF1 and bound cardiolipins

The structure of the T. gondii ATP synthase reveals that the unusual architecture of the dimer is generated by the peripheral stalks that are laterally offset, extending away from the central dimer axis (Fig. 1a, b). This architecture does not allow the formation of the conventional dimerization interface of type-I ATP synthases found in animals and yeast (Supplementary Fig. 5i, j), in which peripheral stalks extend along the dimer long axis13. We therefore examined the dimerization interface, which is formed by the apicomplexan subunits and extensions of the canonical Fo subunits. Those elements involve eleven proteins from each monomer that contribute more than 7000 Å2 of buried surface area, making the interface substantially larger than in mammalian, yeast and algal ATP synthase structures (Supplementary Fig. 5c–j).

The dimerization interface in the membrane and lumenal regions is governed by homotypic interactions between symmetry-related subunits, most of which extend deep into both monomers. Subunit-b contains two transmembrane helices, each binds one of the symmetry-related subunit-a copies, which are therefore linked by four transmembrane helices (Fig. 2a, b). In addition, two cardiolipins are found on the matrix side, forming specific protein-lipid interactions bridging the two copies of subunit-b and subunit-f (Fig. 3a). This cardiolipin pair is sequestered in the Fo subcomplex with no apparent path to the bulk membrane, suggesting a structural role. An additional 15 cardiolipins and 12 other phospholipids were found to mediate a network of interactions throughout the membrane region (Supplementary Fig. 6a). These native lipids are primarily bound in two vestibules within the Fo subcomplex (Fig. 3a) with the lipid head groups mediating charged interactions between numerous subunits (Supplementary Fig. 6b–e), which indicates a contribution to the stability of the complex.


a Fo map cross-section showing apicomplexa-specific (purple) and conserved (light blue) subunits, as well as lipid vestibules (red, orange). The apicomplexa-specific subunits scaffold the Fo architecture. Close-up inset shows protein-cardiolipin (CDL) contact at dimer interface with subunits b and f interacting via a tightly bound cardiolipin. b IF1 dimer (green density) binds to Fo (dark grey density) and F1 of both monomers (transparent grey), linking them together. Close-up inset shows IF1 interactions with subunit-b (violet).

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The inhibitor protein of ATPase activity IF1 is bound to subunit-b, contributing to the Fo dimer interface with its C-terminal helix extending from F1 to interact with subunit-b’ of the neighbouring monomer (Fig. 3b). IF1 in our structure is bound exclusively to the α/β-interface facing the dimer interface (Fig. 3b and Supplementary Fig. 7a–d, f), thereby locking it in the ADP-bound state (βDP). The N-terminal IF1 region that contacts the central stalk in the bovine complex25 is absent in our structure (Supplementary Fig. 7b). Because central stalk rotation and conformational changes in the catalytic sites of F1 are interdependent, the sterically restrictive IF1 binding in T. gondii to only one of the three catalytic sites results in the trapping of the ATP synthase in a single rotational state in both the dimer and hexamer. In our cryo-EM maps, IF1 is contiguous with Fo-associated density extending to the C2-symmetry axis, thus linking the two F1 monomers (Fig. 2d, Supplementary Fig. 7f). We assign it to the unmodeled C-terminal region of IF1, which has previously been characterised as a homo-oligomerisation domain in mammals26. This bridging of two F1 monomers is intra-dimeric, which is different from the mammalian ATP synthase tetramer, where bridging occurs between the two neighbouring dimers24,27 (Supplementary Fig. 7e, f).

Evolutionary and functional aspects of a minimal and nuclear-encoded subunit-a

We assigned subunit-a by locating topologically conserved transmembrane helices of the canonical subunits b, d, f, i/j, k, and 8 (Fig. 2a, b and Supplementary Fig. 8d). Based on the sequence identified directly from the cryo-EM map, we found that T. gondii subunit-a is encoded in the nucleus, and not in mitochondria as in most other organisms. Thus, in T. gondii, all ATP synthase subunits are nuclear-encoded (Supplementary Table 2). In addition, unlike in the canonical six-helix (H1-6a) fold, which is conserved in bacteria, chloroplasts and other mitochondria4,28,29,30, the subunit-a in T. gondii lacks H1-4a, and only the horizontal H5a and H6a are found. They interact with the c-ring at the rotor-stator interface (Fig. 2a, b; Supplementary Fig. 8a, b). This is the smallest subunit-a structure reported to date.

The unmodelled sequence that would make up the canonical transmembrane H1-4a, corresponds to a mitochondrial targeting sequence with a predicted cleavage site located N-terminally of H5a, thereby causing the truncation (Supplementary Fig. 8c). Thus, compared to its mitochondria-encoded homologs, T. gondii subunit-a displays a reduced overall hydrophobicity, which we found to be conserved in Apicomplexa (Fig. 4a). A similar observation for different mitochondrial membrane proteins has been proposed to enable mitochondrial protein targeting following gene transfer to the nucleus31,32,33.

Fig. 4: The minimal subunit-a is parasite-conserved and forms a salt bridge at the rotor-stator interface.


a Heat map indicating the average hydrophobicity of subunit-a in divergent organisms, calculated as the grand average of hydropathy (KD)84 or according to the Moon-Fleming (MF)85 or Wimley-White (WW)86 hydrophobicity scales. The nuclear-encoded subunit-a of apicomplexan parasites, as well as the related chromerid alveolates C. velia and V. brassicaformis show a reduced hydrophobicity compared to the mitochondria-encoded subunit-a homologs of animals and fungi. Reduced hydrophobicity is also found in subunit-a of the green alga P. parva, which is also nuclear encoded87 and lacks TM helix 1. b Top view of the subunit-a/c interfaces. The central arginine/glutamate pair is within interaction distance and enclosed by six aromatic residues. c Close-up view of the matrix half-channel (blue) with hydrophilic residues of subunits a, d and the C-terminus of ATPTG16 indicated. d Lumenal half-channel (burgundy) with proposed proton path to c-ring (black arrows). The channel entrance is occupied by a β-DDM molecule. e Proton half-channels shown in red (lumenal) and blue (matrix) colours and compared to gaps (dotted black circles) in detergent density (dark gold) of the cryo-EM density map of the dimer.

The missing interactions of truncated H1-4a are compensated by lipids and apicomplexan subunits and extensions surrounding the canonical subunits, anchoring them to the enlarged Fo region and the wing region (Figs. 2b and 3a). Thus, the minimal T. gondii subunit-a exemplifies an evolutionary mechanism that combines subunit truncation and reduced hydrophobicity with structural compensation that allowed gene transfer. Together, our analyses illustrate how the substantial mitochondrial genome reduction occurred in apicomplexan parasites, retaining only three mitochondrial genes, while maintaining functional mitochondrial energy conversion.

The IF1-locked rotational state reveals salt bridge formation at the rotor-stator interface

In addition to minimal architecture and evolutionary insight, the subunit-a structure also reveals its interactions with the c-ring. The IF1-arrested structure, in which ATP synthases are locked in a single rotational state, allowed us to obtain a map of the rotor-stator interface at 3.5 Å (Supplementary Fig. 1d), resolving both the c-ring and H5-6 of subunit-a, where proton transfer occurs. Mechanistically, the essential arginine on H5a (Arg166 in T. gondii) is thought to be responsible for deprotonation of the conserved glutamate on the c-ring (Glu150 in T. gondii)29. Translocating protons enter Fo via a lumenal access channel, are transferred to the protonatable glutamate on the c-ring and released via a matrix channel34,35. While our cryo-EM map does not display unambiguous density for Glu150, previous X-ray crystal structures have shown that this side chain can adopt an open unprotonated or closed proton-locked rotamer36,37. Both formation and absence of a salt bridge between the arginine and glutamate have been observed in different structures29,38,39, including a suggestion of a bridging water molecule40.

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Our structure indicates that in the open conformation Glu150 is within 2.3 Å distance from the juxtaposed Arg166, allowing the formation of a salt bridge (Fig. 4b). The rotor-stator interface surrounding the Arg166/Glu150 pair is more hydrophobic compared to other structures, with subunits a and c contributing a total of eight aromatic residue side chains (Fig. 4b, Supplementary Fig. 8e). Thus, the tight hydrophobic interface between the decameric c-ring and subunit-a in T. gondii is consistent with a direct, rather than water-mediated Arg/Glu interaction.

We traced two cavities in the Fo subcomplex corresponding to the proton half-channels on the lumenal and matrix sides (Fig. 4c, d, Supplementary Fig. 8g). The lumenal proton half-channel displays a hydrophilic entrance between subunit-a and ATPTG2 facing towards the c-ring (Fig. 4d, Supplementary Fig. 8f). Inside the membrane, the lumenal channel is lined by membrane-inserted loops of ATPTG2 and ATPTG3 and the C-terminal transmembrane helix of subunit-b (Fig. 4d). The channel extends through the only acidic patch between H5a and H6a near a conserved glutamate (Glu201), which is thought to mediate proton transfer to the c-ring (Supplementary Fig. 8f)29. The matrix half-channel locates to a hydrophilic region between subunits a, d, ATPTG16, ATPTG17 and extends into the membrane region towards R159 of H5a, which is widely conserved (Fig. 4c). Remarkably, the C-terminus of ATPTG16 contributes the only nearby carboxylate group, likely serving an equivalent role to acidic side chains thought to mediate proton release in ATP synthases of other organisms (Fig. 4c)24,28,29,38,41.

The lateral offset between the two proton half-channels is also evident in the density map, where discontinuation of the detergent belt matches the positions of the two half-channels in support of an aqueous environment for proton translocation (Fig. 4e, Supplementary Fig. 8g). Taken together, both half-channels are partially lined by apicomplexan-specific subunits resulting in a divergent structure and likely the involvement of different residues in proton translocation compared to structures from other organisms.

Peripheral stalk subunit-b contains a structural motif found in the mammalian subunit F6

The peripheral stalk extends from the membrane-embedded part of Fo and attaches to the tip of F1, holding it stationary against the torque of the central stalk. In T. gondii the peripheral stalk is composed of subunit-b, d, ATPTG12 and OSCP (Fig. 1a, Supplementary Fig. 9b). The attachment to F1 is mediated through OSCP, which adopts a fold conserved in prokaryotic and eukaryotic homologs (Supplementary Fig. 9a). Subunit-b displays structural similarity with its bacterial, algal and mammalian counterparts, engaging in conserved interactions with the C-terminal domain of OSCP as observed in other structures. Compared to the yeast and mammalian ATP synthases, T. gondii displays an augmented peripheral stalk structure with extensions in subunit-b, subunit-d and the additional ATPTG12 (Supplementary Fig. 9b). Unlike yeast and porcine24,39, neither subunit-f nor 8 (A6L in mammals) contribute to the peripheral stalk. Instead, the apicomplexan-conserved subunit ATPTG12 forms extensive interactions with subunit-b and d throughout the peripheral stalk structure (Supplementary Fig. 9b).

Interestingly, peripheral stalk subunit F6 (subunit h in yeast) is not found in T. gondii ATP synthase. Instead, the C-terminal extension of subunit-b, adopts a fold that structurally resembles subunit F6/h and provides supporting interactions with the long subunit-b helix (Supplementary Fig. 9c). Both, the yeast subunit-h (on non-fermentable carbon sources) and the augmented T. gondii subunit-b are essential18,42, suggesting a critical role in peripheral stalk assembly.

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Formation of the ATP synthase hexamer involves two contact sites in the lumenal region

Next, we asked how ATP synthase dimers interact in the hexamer structure to form the cyclic trimer of dimers. The hexamer model shows that each of the three dimer-dimer interfaces contributes ~1211 Å2 to hexamer contacts. Those contacts holding the hexamer together are found in two separate sites in the lumenal regions, which form a triangular subcomplex (Fig. 5a).

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