ATP synthase hexamer assemblies shape cristae of Toxoplasma mitochondria

Abstract

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.

Fig. 1. Overall architecture of T. gondii ATP synthase dimer and hexamer.

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.

Fig. 2. Conserved and apicomplexan-specific F_o components of hexameric ATP synthase.

a The canonical F_o subunits b, d, f, i/j, k, and 8 contain apicomplexan-specific extensions contributing to a large F_o. Subunit-a contains only the conserved H5-6_a, and ATPTG16, ATPTG17 partially replace the missing H1-4_a. 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 H1-4_a separates subunit-a from several canonical F_o-subunits. Resulting lipid-filled F_o void outlined (black dash). c Cross section through the F_o region of the map. ATPTG11 extends from the lumenal region to plug the c-ring through interactions with H1_TG11.

Fig. 3. Parasite-specific subunits and resolved lipids at the dimer interface.

a F_o 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 F_o 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 F_o (dark grey density) and F₁ of both monomers (transparent grey), linking them together. Close-up inset shows IF₁ interactions with subunit-b (violet).

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) or according to the Moon-Fleming (MF) or Wimley-White (WW) 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 encoded 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.

Fig. 5. Structure of T. gondii ATP synthase hexamer reveals two lumenal F_o contact sites between neighbouring dimers.

a First contact site is shown on the ATP synthase hexamer composite map viewed from the lumen with central lipid bilayer and surrounding detergent belt (gold). The lumenal regions of the three dimers interact to form a triangular complex (bottom panel). Three copies of the CHCHD protein ATPTG9 form homotypic interactions. b Second contact site is shown from the side view: tilted helix hairpin (H2, H3) of ATPTG11 mediates interactions with ATPTG5, ATPTG8 and ATPTG10, whereas ATPTG6 interacts with ATPTG8.

Fig. 6. T. gondii ATP synthase arranges into pentagonal pyramids with icosahedral symmetry to induce membrane curvature.

a Cryo-ET of the mitochondrial membranes (blue) and subtomogram averaging of dimers reveals their macromolecular arrangement into pentagonal pyramids held together by proteins in the lumen. b Schematic representation shows five hexamers (coloured) arranged around a C₅-axis. Red and blue arrows indicate cross-sections shown in the other panels. Inner and outer dimers are shown as red and black ellipses, respectively. c Schematic of interactions between luminal subunits involved in the assembly of the pentagonal pyramid. d Neighbouring hexamer planes (blue and red) are arranged at a ~45°-angle around the shared dimer (grey). e Cross section through the pentagonal ATP synthase pyramid showing two 40°-angles between hexamer (yellow, red) and pentamer (grey) planes.

Fig. 7. Pentagonal pyramids are required for maintenance of the native cristae morphology.

a, b Parental strain (a) and ATPTG11-KO (b) cross sections of tomograms of mitochondrial membranes decorated with ATP synthase (yellow arrows). c, d Segmentation of mitochondrial membranes (blue) with repositioned subtomogram averages of the dimers (yellow). Whereas the parental strain forms pentagonal pyramids that cap the bulbous membrane protrusions, hexamer and pyramid formation is disrupted in ATPTG11-KO, and ATP synthase dimers arrange in row-like or disordered arrays along elongated or tubular membranes. Close-up views show the pentagonal pyramid in the parental strain and row-like arrangements in the mutant strain. e, f Relative abundances of parental (e) and ATPTG11-KO (f) of the mixed-culture growth competition assay as determined by qPCR of total gDNA, normalized to t0. Each passage represents 3–8 biological replicates; error bars are SD; p-values were determined by one-way ANOVA followed by Dunnett’s multiple comparisons test comparing each passage to P1.

Fig. 8. Pentagonal ATP synthase pyramid arrays decorate bulbous cristae in T. gondii mitochondria.

a Cryo-ET of a T. gondii tachyzoite mitochondrion (IBM, inner boundary membrane, blue; OM, outer mitochondrial membrane, grey) and subtomogram averaging of ATP synthase dimers (yellow). Cristae are connected to the inner boundary membrane via circular cristae junctions and decorated with pentagonal ATP synthase pyramids. b Close-up view of a crista membrane containing three bulbous protrusions, each decorated with an ATP synthase array containing ten ATP synthase dimers.