Figure 6. Peripheral F_o subcomplex and the peripheral stalk.

(A) Euglenozoa-specific subunits form a peripheral F_o subcomplex. Density of the F_o with proteins of the peripheral region coloured, c-ring model shown in grey, outline of the detergent belt (yellow dashed lines) with 2 nm offset towards the lumen indicated as determined by the density (transparent gold) (B) Atomic model of the F_o periphery, cavity lipids are shown in magenta. (C to E) Attachment of the peripheral stalk to F₁. (C) E. gracilis ATP synthase with proteins constituting the peripheral stalk coloured. (D) Side view of the peripheral stalk tip and F₁ (white, crown domain light grey). C-terminal helix of OSCP (light green) extending towards the membrane attaches OSCP to the rest of the peripheral stalk via subunit d (red). (E) The N-terminal extension of OSCP (yellow) interacts with the C-terminal extension of the rotor subunit γ (conserved region purple, extension dark blue).

Figure 6—figure supplement 1. The peripheral F_o subcomplex and inverted topology structural motif.

Side view (A) and front view (B) of the F_o region. The peripheral subcomplex (pink) and the conserved F_o-subunits (white) are joined together by ATPTB1 on the matrix side and by subunits a and k in the lumen and separated by the lipid-filled F_o cavity (B). (C) Hairpin helices of ATPTB1 and ATPEG3 extend into the membrane region from opposite sides in the membrane, where they interact, forming an inverted topology motif. (D) Inverted topology motif of the prokaryotic aspartate transporter Glt_Ph consisting of two hairpin loops (HP1 and HP2, PDB ID: 1XFH) (Yernool et al., 2004).

Figure 6—figure supplement 2. Coarse-grained molecular dynamics simulations of the E. gracilis ATP synthase dimer.

(A) The 45°-angle of the E. gracilis ATP synthase dimer (grey) generates significant membrane curvature (acyl chains in light blue; phosphate groups in orange, ethanolamine groups in red, choline groups in green), resulting in a 7 nm local displacement of the membrane from the bilayer plane (marked with bar). (B) Radial distribution function g (r) of the center of mass of head groups from different lipid types. The first two maxima indicate well-defined coordination shells around the transmembrane region. Phosphatidic acid (PA) and cardiolipin (CDL) have a higher density in the annulus of the transmembrane region than phosphatidyl choline (PC) and phosphatidylethanolamine (PE). (C) View of F_o cavity with orange sphere indicating region used for lipid binding analysis. (D) Probabilities of entering and staying bound in the cavity shown for each lipid type. (E) Mean residence time of each lipid type in the cavity. Error bars indicating the 90% confidence interval obtained from bootstrapping. Nonparametric significance tests were performed for each lipid type’s residence time against cardiolipin: * p-value<0.05, ** p-value<1×10⁻⁵. (F) Side view of F_o cavity with lipids forming a bilayer-like array.

Figure 6—figure supplement 3. Interactions of OSCP extension with F₁ and the peripheral stalk.

(A, B) Peripheral stalk architectures of S. cerevisiae (A) (Guo et al., 2017) and E. gracilis (B) viewed from the F₁ (not shown) towards the peripheral stalk. The two structures have subunits b, d, 8 and OSCP in common. Unlike in S. cerevisiae, subunit f does not contribute to the peripheral stalk in E. gracilis. Subunit F6 is not found in E. gracilis. Red arrowheads indicate different heights of OSCP attachment to subunit b and subunit d respectively. (C) The C-terminal extension of E. gracilis OSCP (red rectangle) extends in between the peripheral stalk (dark grey) and F₁ (light grey). (D) Close-up of OSCP/subunit d interaction in the E. gracilis peripheral stalk. The indicated residues are conserved in Euglenozoa. (E) The E. gracilis OSCP contains conserved structural elements (light green)(Srivastava et al., 2018) with the N-terminal domain consisting of helices 1,2,5 and 6 and the C-terminal domain consisting of a four-stranded β-sheet and helices 7 and 8 (dihydrolipicolinate reductase domain 2-like fold). The N-terminal extension (light yellow) interacts with subunit γ (blue) of the rotor, whereas the C-terminal extension (orange) is anchored to the rest of the peripheral stalk. (F-H) Multiple sequence alignments of the OSCP C-terminal region (F), with the Euglenozoa-specific extension highlighted red; OSCP N-terminal region (G) with the N-terminal helix found in the E. gracilis OSCP highlighted yellow; red rectangles indicate predicted cleavage sites of mitochondrial processing peptidase. (H) Subunit γ C-terminal region, with euglenozoa-specific extension highlighted red; Bos tarus (Bt), Homo sapiens (Hs), Caenorhabditis elegans (Ce), Saccharomyces cerevisiae (Sc), the trypanosomatids Trypanosoma brucei (Tb), Leishmania major (Lm), Endotrypanum monterogeii (Em), Crithidia fasciculata (Cf), Leptomonas pyrrhocoris (Lp), Blechomonas ayalai (Ba), Phytomonas sp. (Ps) and the euglenoid Euglena gracilis (Eg).