The consequence of ATP synthase dimer angle on mitochondrial morphology studied by cryo-electron tomography

Abstract

Mitochondrial ATP synthases form rows of dimers, which induce membrane curvature to give cristae their characteristic lamellar or tubular morphology. The angle formed between the central stalks of ATP synthase dimers varies between species. Using cryo-electron tomography and sub-tomogram averaging, we determined the structure of the ATP synthase dimer from the nematode worm C. elegans and show that the dimer angle differs from previously determined structures. The consequences of this species-specific difference at the dimer interface were investigated by comparing C. elegans and S. cerevisiae mitochondrial morphology. We reveal that C. elegans has a larger ATP synthase dimer angle with more lamellar (flatter) cristae when compared to yeast. The underlying cause of this difference was investigated by generating an atomic model of the C. elegans ATP synthase dimer by homology modelling. A comparison of our C. elegans model to an existing S. cerevisiae structure reveals the presence of extensions and rearrangements in C. elegans subunits associated with maintaining the dimer interface. We speculate that increasing dimer angles could provide an advantage for species that inhabit variable-oxygen environments by forming flatter more energetically efficient cristae.

Keywords: alphafold; atp synthase; cryo-electron microscopy; mitochondria; sub-tomogram averaging.

Figure 1.. ATP synthase dimer rows, and sub-tomogram average of the ATP synthase dimer from C. elegans.

(A) Tomographic slice through a whole C. elegans mitochondrion (top) and corresponding segmentation (bottom; outer membrane green, inner membrane light blue, multi-colour crista membranes). The boxed region shows an enlarged image of a single crista membrane, with green, blue and orange arrowheads indicating the outer, inner and crista membranes, respectively, and yellow arrowheads indicating ATP synthase F₁ heads. The crista membrane is coloured light blue in the corresponding segmentation; each ATP synthase dimer pair is coloured differently. (B) Tomographic slice through C. elegans isolated crista membranes with boxed region showing enlarged image of a single crista membrane (left, arrowheads indicating ATP synthase F₁ heads), and corresponding segmentation (right) coloured as in (A). Scale bars, 100 nm for tomograms, and 50 nm for enlarged views of crista membranes. (C) Sub-tomogram average of the C. elegans ATP synthase dimer. Upper panel shows side view with central and peripheral stalks indicated by red and purple arrows respectively, lower panel shows top-down view.

Figure 2.. The C. elegans ATP synthase compared with other species.

(A) Structures depicting the range of average dimer angles observed in S. cerevisiae (EMD-7067) [28], bovine heart (EMD-11436) [27], and C. elegans (this work (EMD-18991)), using the highest resolution structures available. (B) Direct comparison between S. cerevisiae (EMD-2161) [11] and C. elegans ATP synthase sub-tomogram averages, with the angle between F₁ dimer heads, the angle of crista membrane curvature, and distance between the central stalks of each monomer indicated. A bracket highlights the extra mass at the C. elegans dimer interface not apparent in S. cerevisiae. Black, transparent blue and dark green mesh represent decreasing threshold levels for the averages. (C) Cartoon detailing the occurrence of ATP synthase subunits in S. cerevisiae and C. elegans, each labelled with corresponding nomenclature for the species (details in Supplementary Table S1).

Figure 3.. Morphology of mitochondria isolated from C. elegans and S. cerevisiae.

(A) Tomographic segmentations of C. elegans and S. cerevisiae mitochondria are displayed (green, outer mitochondrial membrane; blue, inner mitochondrial membrane; multi-colour, crista membranes). See Supplementary Movie S1 (C. elegans) and Supplementary Movie S2 (S. cerevisiae). (B) The mean surface area to volume ratio per crista (n = 3 mitochondria for each organism, with n = 47 cristae for C. elegans and n = 63 cristae for S. cerevisiae) was calculated from the segmentations shown in (A). (C) A single tomographic segmentation from each organism is shown with all crista coloured blue. Pink dots indicate distances used to measure width. (D) Close up of a single crista membrane from each organism (location indicated by asterisks in (D)) to highlight the flatter crista morphology in C. elegans mitochondria compared with S. cerevisiae. (E) The mean crista width (n = 63 crista tips for C. elegans and n = 61 for S. cerevisiae) was calculated from the segmentations shown in (A). Error bars in (B) and (E) show the standard deviation of the mean and significance values were calculated using Welch's t-test for (B) or using the Mann–Whitney U-test for (E). ****P ≤ 0.0001. Scale bars in (A) and (C), 200 nm; in (D), 20 nm.

Figure 4.. AlphaFold homology model of the C. elegans ATP synthase dimer.

(A) Two ATP synthase monomers from the C. elegans homology model (helical representation) fitted into the sub-tomogram average of the C. elegans ATP synthase dimer. (B) Surface view of S. cerevisiae and C. elegans ATP synthase dimer models coloured by chain in side (top) and top-down (bottom) views. Subunits are annotated and shown as α, red; β, gold; γ, indigo; δ, magenta; ε, coral; c, grey; a, purple; b, blue; d, turquoise; F₆, navy; OSCP, orange; e, pale blue; f, pink; g, yellow; j, brown; k, dark green; 8, lime. All subunits are labelled in the side views apart from subunit 8 which is buried. Only the dimer interface subunits are labelled in the top-down views. (C) Top-down view of the C. elegans ATP synthase dimer homology model fitted to the sub-tomogram average showing sequential dimer pairs (coloured differently) in a row. (D) As per (C), but exclusively showing dimer interface subunits e, f and g, labelled and coloured by chain as per (B). (E) and (F) show the same interactions as in (C) and (D), respectively, but viewed from the side of a dimer row.

Figure 5.. Comparison of the dimer interface and peripheral stalk in C. elegans vs S. cerevisiae.

(A) Overlays of individual subunits at the dimer interface and peripheral stalk, where there are extensions in C. elegans subunits (AlphaFold predictions, blue) compared with S. cerevisiae (PDB 6B8H, pink) [28]. C. elegans subunit extensions are highlighted in orange. Since the S. cerevisiae atomic model for the ATP synthase dimer (PDB 6B8H) [28] does not contain complete density for subunit F₆, the S. cerevisiae monomeric atomic model (PDB 6CP6) [70] was used to display a more complete S. cerevisiae chain for the overlay. (B) Left, dimer interface subunits in the S. cerevisiae atomic model (6B8H) [28] coloured by chain and fitted into an S. cerevisiae sub-tomogram average (EMD-2161) [11]. Right, dimer interface subunits in the C. elegans homology model coloured by chain fitted to the C. elegans sub-tomogram average. An alpha helix projecting from the dimer interface towards the peripheral stalk (subunit 8 in S. cerevisiae and subunit f in C. elegans) is boxed, and the sharp angle induced by subunits e and g in C. elegans indicated by a black arrowhead. (C) As per (B), but with all subunits coloured black, highlighting subunits missing in C. elegans relative to S. cerevisiae (j, k and 8) in red (left) and extensions in C. elegans subunits e, f and g relative to S. cerevisiae in orange (right). (D) Left, peripheral stalk subunits b, d and OSCP in the S. cerevisiae atomic model (PDB 6B8H) [28], and F₆ from the monomeric atomic model (PDB 6CP6) [70], fitted to the S. cerevisiae sub-tomogram average (EMD-2161) [11]. Right, peripheral stalk subunits in the C. elegans homology model coloured by chain fitted to the C. elegans sub-tomogram average. (E) As per (D), but with all subunits coloured black, highlighting extensions in C. elegans subunits b, d and F₆ relative to S. cerevisiae in orange. Subunits in (B–E) are annotated and shown as b, blue; d, turquoise; F₆, navy; OSCP, orange; e, pale blue; f, pink; g, yellow; j, brown; k, dark green; 8, lime.

Figure 6.. Schematic to illustrate the effect of crista shape on efficiency of proton diffusion from the proton pumping complexes (CI, III and IV) to ATP synthase.

Dashed arrows indicate the direction of proton travel; black represents diffusion along membrane surfaces from ‘source’ to ‘sink’, red represents dissipation away from the membrane surface. Cartoons representing respirasomes and ATP synthase are derived from PDB depositions 5J4Z [73] and 6ZPO [23], respectively. The figure was created with Biorender.com. (A) Narrower cristae. In C. elegans, a wide angle between ATP synthase dimer heads (105°) induces a sharp angle of membrane curvature, producing lamellar cristae with the potential for increased protein packing and little opportunity for proton dissipation. (B) Wider cristae. In S. cerevisiae, and most other type I dimer species, a shallower angle is formed between the ATP synthase dimer heads (86°). This induces a less pronounced membrane curvature and therefore less lamellar-shaped cristae compared with C. elegans. There is a greater potential for protons to dissipate, and thus a greater distance to travel from source to sink.