Molecular mechanism of cardiolipin-mediated assembly of respiratory chain supercomplexes

Abstract

Mitochondria produce most of the ATP consumed by cells through the respiratory chain in their inner membrane. This process involves protein complexes assembled into larger structures, the respiratory supercomplexes (SCs). Cardiolipin (CL), the mitochondrial signature phospholipid, is crucial for the structural and functional integrity of these SCs, but it is as yet unclear by what mechanism it operates. Our data disclose the mechanism for bulk CL in gluing SCs, steering their formation, and suggest how it may stabilize specific interfaces. We describe self-assembly molecular dynamics simulations of 9 cytochrome bc₁ (CIII) dimers and 27 cytochrome c oxidase (CIV) monomers from bovine heart mitochondria embedded in a CL-containing model lipid bilayer, aimed at mimicking the crowdedness and complexity of mitochondrial membranes. The simulations reveal a large diversity of interfaces, including those of existing experimental CIII/CIV SC models and an alternative interface with CIV rotated by 180°. SC interfaces enclose 4 to 12 CLs, a ∼10 fold enrichment from the bulk. Half of these CLs glue complexes together using CL binding sites at the surface of both complexes. Free energy calculations demonstrate a larger CL binding strength, compared to other mitochondrial lipids, that is exclusive to these binding sites and results from non-additive electrostatic and van der Waals forces. This study provides a key example of the ability of lipids to selectively mediate protein-protein interactions by altering all ranges of forces, lubricate protein interfaces and act as traffic control agents steering proteins together.

Fig. 1. Molecular system. (A) Bovine cytochrome bc₁ (CIII) and cytochrome c oxidase (CIV). The models of CIII and CIV are identical to the ones used in our previous studies. Briefly, CIII’s dimer was built from a combination of four experimental structures (PDB entries: 1 l0l,1sqb/1sqq and 2a06 (ref. 55)), excluding the six hemes and two iron-sulfur clusters. CIV's monomer was built from the PDB entries 1occ and 2occ, also excluding two deeply buried hemes. Details are given in the ESI Methods section. (B) Matrix view of the membrane-exposed CL binding sites on both complexes. The detail of the CIII and CIV subunits, their nomenclature (Fig. S1†) and the comparison of predicted CL binding sites to experimental data have been described previously. The location of the non-CL-binding surface on CIV is indicated by a star. In the simulated system the complexes are embedded in a POPC bilayer containing CL at a 1:15 CL:POPC molar ratio; side (C) and top (D) views. The system shown contains 9 CIII dimers, 27 CIV, 17 462 POPCs, 1175 CLs (∼32 000 beads) and the aqueous phase (∼1 116 000 water beads and ∼2600 sodium ions), thus a total of slightly less than 1 400 000 CG beads. To ease visualization, the aqueous phase is omitted and to emphasize the relative orientation of the complexes two subunits of each complex are highlighted (A and B in red for CIII and D and G in yellow for CIV). CL topology and parameters were taken from the work of Dahlberg et al. (E) View of the CL-containing system after 20 μs of self-assembly simulation.

Fig. 2. Self-assembly process and architecture of respiratory chain supercomplexes. The time evolution of properties of the system with (+CL; blue) and without (-CL; cyan) cardiolipins are reported: (A) protein burial, a_b, with contributions from the CIII/CIV and CIV/CIV interfaces, (B) numbers of interfaces, (C) numbers of CIII and CIV monomers, (D) the mean square displacement (MSD) of the CIII and CIV monomers in isolation in both membrane environments (+CL and -CL), and (E and F) average protein burial during CIII/CIV and CIV/CIV interface maturation with intra- and extra-membraneous contributions.

Fig. 3. Supercomplex architectures: (A) location of CIII/CIV interfaces on CIII (top panels) and CIV (bottom panels) in +CL and -CL membranes. Each stick represents the projection of an interface onto a circle surrounding the protein. The location of an interface, γ₁ and γ₃, is determined by the position of the centers of mass of the residues contributing to it for each partner (see Fig. S14 and the ESI Methods for details). The length of a stick reports the protein burial, a_b, corresponding to the interface. Similar analysis for CIV/CIV interfaces is shown in Fig. S5. The favored interfaces found with CL (see panels B and E) are shown by a larger head stick. A triangle points towards the denser zone of contacts on CIII and CIV in the system with and without CL. (B) Contact map of CL binding sites in CIII/CIV SCs from the simulations +CL and -CL. Sites are assumed to form a contact when distant by <3.0 nm (see Methods in the ESI†). Similar analysis for CIV/CIV SCs is shown in Fig. S5. Sites on the inter-membrane space side of the bilayers (VI_CIV, VIIa_CIV and VIIb_CIV) are not reported. In all cases, the C₂ symmetry axis of the CIII dimer is used to average over both monomers. (C and E) Simulated vs. experimental and plasticity of CIII/CIV interfaces. Experimental models were derived: for bovine heart mitochondria (panel C) from Althoff et al. or Dudkina et al., (panel E) from Schäfer et al. and for yeast (panel D) Heinemeyer et al. or Mileykovskaya et al. The CL binding sites are colored when in contact (<3.0 nm) following the color code defined in Fig. 1 or left grey otherwise. The yeast model was provided by Heinemeyer et al. Althoff's bovine model was taken from the PDB entry 2ybb. Schäfer's model was built by visual fitting of CIII and CIV onto Fig. 2 and 3 from ref. 2 and thus should be considered qualitatively. The surface SCs are projected onto the membrane plane from the matrix with the orientation of CIII conserved. The distance between cytochrome c, d_Cytc, binding sites on CIII and CIV are given in (C)–(E) and detailed in Fig. S11. In (E), the compatibility of the configuration with its association with complex I is indicated by a green (possible) or red (not possible) circle, see Fig. S12 for details.

Fig. 4. Lipid content of CIII/CIV interfaces. For two CIII/CIV SCs we show: (A) a snapshot of the interfaces, t = 20 μs, which is cut open to show the lipid content (CL and POPC) at the protein surfaces. The number of shared lipids is indicated. In one case (left) the interface is formed early in the simulation and is followed by a long maturation of the interface with POPC being removed at timescales up to 14 μs. In the second case (right) the interface is formed late and POPC molecules are expelled quickly; (B) the time evolution of the number of interfacial lipids for the interface depicted in the snapshot. The vertical line denotes the formation of the interface; (C) the average number of interfacial lipids (CL and POPC) over all interfaces formed in the simulations; time relates here to interface maturation: t = 0 corresponds to the time of formation of the first contact of an interface.

Fig. 5. Lipid binding affinities. Potentials of mean force (PMFs) were determined for (A) CL vs. other lipids on a CIV CL binding site (II_CIV); (B) CL vs. other lipids on a non-CL-binding region (indicated in Fig. 1 and defined in S1, color coding as in panel A). (C) Native vs. non-native CL: full tailed CL is compared to monolyso-CL (mlCL) and dilyso-CL (dlCL) variants with different charged head groups. Shaded areas indicate the error bars. For mlCL and dlCL, the number of aliphatic tails carried by each glycerol moiety are indicated within brackets. E.g.: dl^(1:1)CL⁽²⁻⁾ is a dilyso-CL with one acyl chain on each head glycerol moiety and a −2e charge. See Table S3 for a detailed account of the respective values.

Fig. 6. Schematic model of CL implication in the formations of supercomplexes. The model shows how in the presence (right side) of CL (green dots) we observed: (i) an increased number of CIII/CIV interfaces but not of CIV/CIV, and (ii) stronger and (iii) more specific interfaces. CIII is depicted in light red and CIV in light orange. Two copies of CI are shown to illustrate its possible integration to the CIII/CIV SC formed in the simulations (Fig. S12†).