Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases

Abstract

F-ATP synthases use proton flow through the F_O domain to synthesize ATP in the F₁ domain. In Escherichia coli, the enzyme consists of rotor subunits γεc₁₀ and stator subunits (αβ)₃δab₂. Subunits c₁₀ or (αβ)₃ alone are rotationally symmetric. However, symmetry is broken by the b₂ homodimer, which together with subunit δa, forms a single eccentric stalk connecting the membrane embedded F_O domain with the soluble F₁ domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αβ)₃ catalyzes the same set of partial reactions in the time average, they might not be fully equivalent at any moment, because the structural symmetry is broken by contact with b₂δ in F₁ and with b₂a in F_O. We monitored the enzyme's rotary progression during ATP hydrolysis by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a polarization assay using gold nanorods. We found that one dwell in the three-stepped rotary progression lasting longer than the other two by a factor of up to 1.6. This effect of the structural asymmetry is small due to the internal elastic coupling.

Keywords: Escherichia coli; FOF1 ATP synthase; cryo-EM structure; elasticity; single-molecule fluorescence; subunit rotation; symmetry.

Figure 1

Front view (left) and side view (right) of a surface representation of the cryo-EM EcF_OF₁ structure (PDB-id: 5T4O) [12] in the membrane. One αβ-pair is omitted in the front view to reveal the conformation of the central stalk. The structure shows the asymmetric features, i.e., the peripheral stalk that is connected to subunit δ and one subunit α, the interface of the c-ring and subunit a with its two half-channels, and the curved central shaft composed of subunits γε. The subunits are colored in red (α), yellow (β), blue (γ), cyan (δ) green (ε), orange (a), pink, and mauve (b₂), and ice blue/lime (c-ring).

Figure 2

Single-molecule experiments with EcF_OF₁. (a) EcF_OF₁ was attached via His-tags in each β subunit to a cover glass. A fluorescently labelled actin filament, which was attached via streptactin and Strep-tags to the c-ring, served as a reporter for rotation during ATP hydrolysis [58]. (b) Fluorescently labelled EcF_OF₁ reconstituted in a liposome was diffusing through the confocal laser focus in a smFRET setup. Rotation of the central shaft during ATP hydrolysis was observed after addition of ATP, or ATP synthesis was powered by pmf [130]. (c) Immobilized EcF_OF₁ with an attached AuNR is rotating in 120° steps at saturating ATP concentrations, and intensities of the scattered light from the nanorod that were observed through a polarizer progressed sinusoidal and reached a maximum after 90° rotation [108].

Figure 3

Properties of rotating actin filament-EcF_OF₁ complexes during ATP hydrolysis. (a) Rotational rate versus ATP. For short actin filaments (≤1 µm), the rotational rate is independent of the ATP concentration and the filament length. (b) Trajectory of a typical rotating complex at 100 µM ATP in steps of 120°. The inset shows the endpoints of the 0.8 µm actin filament. (c) Normalized histogram of the angular probability distribution of the complex in b, where the three peaks, separated by 120°, were fitted with a Gaussian. (d) Dwell time histograms of each peak in c fitted with monoexponential decay. The dwell times (τ) are given for each histogram. (e) Combined normalized histogram of the angular distribution of rotating actin filament from 13 single molecules at 50–5000 µM ATP with 513 to 2174 frames per molecule. Each dataset was fitted with three Gaussians to determine the area of each peak. The datasets were aligned by positioning the largest peak at 60°.

Figure 4

FRET histograms and dwell time histograms of subunit ε rotation in single FRET-labeled EcF_OF₁ during catalysis. (a) Dwell time histogram for all FRET levels during ATP hydrolysis. The insets show the individual dwell times for each of the three FRET levels. (b) Dwell time histogram for all FRET levels during ATP synthesis. The insets show the individual dwell times for each of the three FRET levels. (c) FRET histograms showing the three FRET levels L*, M*, H* (orange), and levels L, M, H (green) from measurements at different conditions at pH 8. See text for details. (Modified from [130]).

Figure 5

Method and results of single-molecule rotation experiments with AuNR attached to the c-ring of immobilized EcF_OF₁ during ATP hydrolysis. (a) Theoretical plot of the intensity of scattered red light from a nanorod during one complete revolution that involves three consecutive power strokes and three consecutive catalytic dwells separated by exactly 120°. The nanorod is initially positioned almost—but not exactly—perpendicular to the orientation of the polarizer, such that the scattered light intensity goes through a minimum then a maximum prior to catalytic dwell 1. A transition includes the data between the minimum and maximum intensities representing 90° of the 120° of rotation. For analysis transitions from power stroke-1 to dwell 1 were selected. (Modified from [56]) (b) Examples of time-dependent changes in rotational position of n-EcF_OF₁ during F₁-ATPase–dependent power strokes at pH 5.0, where transient dwells, either present (top) or absent (bottom), are shown. Transient dwells where rotation was halted or contained CW rotation are colored green and red, respectively. (c) The distribution of single-molecule n-EcF_OF₁ power stroke data sets as a function of the percentage of the occurrence of transient dwells per data set, which were binned to each 10% (gray bar graphs). Each data set contained ~300 power strokes, derived from the indicated number of molecules. The data were fitted to the sum of three Gaussians (black line), where the probability of forming transient dwells was low (blue line), medium (red line), and high (green line). (d) The pH dependencies of the average percent of transient dwells per data set, with standard errors derived from the three Gaussians. (Modified from [108]).

Figure 6

Side view (top row) and view from the membrane side with the c-ring and subunit a omitted of the three cryo-EM structural states of EcF_OF₁ [12]. State 1 (PDB-id: 5T4O, left), state 2 (PDB-id: 5T4P, middle), and state 3 (PDB-id: 5T4Q, right) correspond to FRET levels M, L, and H, respectively (as in Figure 4). In each image, the FRET-donor position at ε56 is marked with a green sphere and the FRET-acceptor position at b64 is marked with a red sphere. The portions of subunit ε and γ that extends beyond the diameter of the c-ring must pass through the narrow gap created by the peripheral stalk during rotation from state 3 to state 1, and during rotation from state 1 to state 2, respectively. The subunit coloring is the same as in Figure 1.

Figure 7

Asymmetry between the three F₁-ATPase power strokes of EcF_OF₁, as shown by the rotary positions of residue cD61 in each c subunit (blue) relative to aR210 (red) in the three states of the EcF_OF₁ cryo-EM structures (viewed from the membrane side as in Figure 6) designated by their PDB-ids [12]. In structure 5T4Q, cD61 of c subunit chain P is closely aligned with aR210 (black line designate 0° of rotation). Assuming that this state shows the alignment with a catalytic dwell position in F₁, red lines show the other two 120° rotational positions of the catalytic dwells in F₁ during subsequent power strokes. Green lines show the 108° rotations that result from the three proton-dependent CCW rotational steps (green arrows) of the c-ring that occur during the power strokes, which results in strain between the catalytic dwell positions of F_O and F₁. From state 3 to state 1 (3 c subunits, purple), and from state 2 to state 3, the c-ring rotates by 108° (3 c subunits, ice blue), whereas from state 1 to state 2, the c-ring rotates by 144° (4 c subunits, lime).