Rotation of the c-Ring Promotes the Curvature Sorting of Monomeric ATP Synthases

Abstract

ATP synthases are proteins that catalyse the formation of ATP through the rotatory movement of their membrane-spanning subunit. In mitochondria, ATP synthases are found to arrange as dimers at the high-curved edges of cristae. Here, a direct link is explored between the rotatory movement of ATP synthases and their preference for curved membranes. An active curvature sorting of ATP synthases in lipid nanotubes pulled from giant vesicles is found. Coarse-grained simulations confirm the curvature-seeking behaviour of rotating ATP synthases, promoting reversible and frequent protein-protein contacts. The formation of transient protein dimers relies on the membrane-mediated attractive interaction of the order of 1.5 k_B T produced by a hydrophobic mismatch upon protein rotation. Transient dimers are sustained by a conic-like arrangement characterized by a wedge angle of θ ≈ 50°, producing a dynamic coupling between protein shape and membrane curvature. The results suggest a new role of the rotational movement of ATP synthases for their dynamic self-assembly in biological membranes.

Keywords: E. coli; F1Fo ATP synthase; giant vesicles; lipid nanotubes; micromanipulation.

Figure 1

A) Structure of ATP synthase from Escherichia coli (left) and Bos taurus (right). The bacterial protein is monomeric, whereas the bovine counterpart forms dimers sustained by the additional subunits e (yellow), f (orange), g (red) and j (brown). φ is the wedge angle between monmomers that might induce the local bending of the membrane (dashed lines). B) Confocal micrograph of a tube pulled from a GUV containing reconstituted ATP synthases. The membrane (green channel) was labelled with the fluorescent lipid DOPE Atto‐488, whereas the protein was labelled with Alexa 555. A first micropipette holds a biotinylated silica beads and pulls a membrane tube from a GUV, which is seized by a second micropipette. The relative concentrations of lipid and protein in the tube and in the GUV are measured by confocal fluorescence microscopy (see Methods). C) Confocal images of different tubes pulled from pure lipid GUVs (row 1) and proteoGUVs containing ATP synthase in passive conditions (row 2), upon incubation with ATP (row 3) or in the presence of both ATP and the rotation inhibitor DCCD (row 4). Fluorescence intensiy of lipid and protein channels are normalized to the lipid value. Scale bar is 5 µm. D) Box plots comparing the sorting ratio for tubes pulled from GUVs ( r_t =  50 ± 20 nm) containing the red fluorescent lipid Rhodamine PE (Lipid), ATP synthase (ATPsyn), ATPsyn with ATP, ATPsyn in the synthesis mode, ATPsyn with ATP and DCCD, ATPsyn with ATP and FCCP and ATPsyn with DCCD and FCCP. The median is represented with a line; the box represents the 25th to 75th percentiles; and error bars show the 5th–95th percentile. The average sorting ratios (cercles) for the lipid control, ATPsyn, ATPsyn with ATP and DCCD and ATPsyn with DCCD and FCCP are similar $(S=1.1)$, whereas ATPsyn in rotating conditions is enriched in the tubes $(S>2)$. Statistical significance: *** (p ≤ 0.001); **** (p ≤ 0.0001).

Figure 2

MD simulations reveal the generation of out of equilibrium bilayer fluctuations and curvature preference of rotating transmembrane F_o domains. A) Top: overview of the structure of the F_o transmembrane domain of the ATP synthase and its subunits; Bottom: Snapshots of the simulated system at its initial configuration. The choline headgroups of the POPC are coloured in orange, the acyl chains are coloured in light grey, and F_o transmembrane domains in yellow. Solvent molecules have been removed from the visualization for clarity. B) Representative snapshots of the bilayer with passive (control) and active (rotating) F_o domains. The collective rotation induces bilayer fluctuations, which are negligible in the control. C) Probability distribution of the spreads between the lipid phosphate z‐coordinate maxima and minima, indicating that the rotating protein has a stronger tendency to generate bilayer fluctuations. D) Local curvature of the bilayer regions in the vicinity of the F_o domain versus the regions devoid of protein, indicating a strong tendency of the protein to position in regions with positive curvature. The bars represent a 95% confidence interval, obtained from bootstrap resampling with subsampling to account for time‐series autocorrelation.

Figure 3

Rotating and passive F_o domains generate distinct profiles of lipid packing and have different tendencies to cluster. A) Frequency of spatial clustering of F_o domains (within a 2 nm threshold distance of one another). Error bars represent the 95% confidence interval, estimated using a bootstrap; mean comparison (p‐value < 0.01) was done with a t‐test over sub‐averages of blocks longer than the autocorrelation time. B) Self‐weighted lifetimes of close contacts (within 0.7 nm of one another) between pairs of F_o domains (i.e., average of the lifetime of each F_o–F_o close contact event, weighted by each event's lifetime). Error bars represent the 95% confidence interval, estimated using a bootstrap method over the weighted lifetimes. Mean comparison (p‐value < 0.01) was done with an independent Welsh t‐test with Satterthwaite degrees of freedom. C) Protein‐protein interaction potential as a function of the distance between two monomeric ATP synthases. The effective interaction potential was calculated from the relative distances between pairwise proteins (see Methods for details). D) Thickness of bilayers measured by the inter‐leaflet distance between phosphate beads (P–P distance) as a function of the distance to the centre of mass (COM) of F_o domain. E) Area per acyl chain as a function of the distance from an active or passive F_o domain in the periplasmic (top) and cytoplasmic (bottom) monolayers. F) Tilt angle probability distribution of F_o proteins.

Figure 4

A) Protein sorting as a function of the lipid tube curvature, c, for GUVs containing a red fluorescent lipid (control), ATP synthase (ATPsyn), ATPsyn with ATP, ATPsyn in the synthesis mode, ATPsyn with ATP and DCCD, ATPsyn with ATP and FCCP and ATPsyn with DCCD and FCCP. Points correspond to binned sorting values. Solid lines are guides to the eye. B) Graphical model for curvature sorting of rotating ATP synthases. Top: proteins under passive conditions (no rotation) show no mismatch on lipid surroundings. Middle: upon rotation, ATP synthases promote a lipid‐protein mismatch, which produces a membrane‐mediated attractive interaction between proteins. Bottom: at close contact, proteins arrange into a conic‐like conformation with a wedged angle, φ ≈ 2θ, yielding their sorting into curved regions of the lipid membrane.