Essential Role of the ε Subunit for Reversible Chemo-Mechanical Coupling in F1-ATPase

Abstract

F₁-ATPase is a rotary motor protein driven by ATP hydrolysis. Among molecular motors, F₁ exhibits unique high reversibility in chemo-mechanical coupling, synthesizing ATP from ADP and inorganic phosphate upon forcible rotor reversal. The ε subunit enhances ATP synthesis coupling efficiency to > 70% upon rotation reversal. However, the detailed mechanism has remained elusive. In this study, we performed stall-and-release experiments to elucidate how the ε subunit modulates ATP association/dissociation and hydrolysis/synthesis process kinetics and thermodynamics, key reaction steps for efficient ATP synthesis. The ε subunit significantly accelerated the rates of ATP dissociation and synthesis by two- to fivefold, whereas those of ATP binding and hydrolysis were not enhanced. Numerical analysis based on the determined kinetic parameters quantitatively reproduced previous findings of two- to fivefold coupling efficiency improvement by the ε subunit at the condition exhibiting the maximum ATP synthesis activity, a physiological role of F₁-ATPase. Furthermore, fundamentally similar results were obtained upon ε subunit C-terminal domain truncation, suggesting that the N-terminal domain is responsible for the rate enhancement.

Figure 1

Rotary catalysis of F₁-ATPase. (a) Crystal structure of the α₃β₃γε complex, F₁^+ε, from thermophilic Bacillus PS3 (PDB: 4XD7). The α, β, γ, and ε subunits are colored in blue, green, yellow, and red, respectively. The black arrowheads indicate the ε subunit. (b) Chemo-mechanical coupling scheme of F₁ at low ATP concentration. The circles and yellow arrows represent the catalytic state of the β subunits and the angular positions of the γ subunit. Each β subunit completes one turnover of ATP hydrolysis in a turn of the γ subunit, where the three β subunits vary in their catalytic phase by 120°. Regarding the catalytic state of the top β subunit (green), ATP binding, hydrolysis, ADP release, and inorganic phosphate (P_i) release occur at 0, 200, 240, and 320°, respectively. (c) The rotational velocity (V) of F₁^+ε (red, left panel), F₁^−ε (gray, left panel), F₁^+ε_(βE190D) (red, right panel), and F₁^−ε_(βE190D) (gray, right panel) obtained using magnetic beads at various ATP or ATPγS concentrations. The curves represent Michaelis–Menten fits with V = V_max[ATP]/([ATP] + K_m), where V_max = 5.5 and 5.6 s⁻¹, K_m = 2.0 and 1.2 μM, and the corresponding ATP-binding rate, k_on = 3 × V_max/K_m; 0.8 × 10⁷ and 1.4 × 10⁷ M⁻¹·s⁻¹ for F₁^+ε and F₁^−ε, respectively. To see this figure in color, go online.

Figure 2

Single-molecule manipulation with magnetic tweezers. (a) Schematic of manipulation procedures. When F₁ paused at the ATP-binding or hydrolysis dwell, the magnetic tweezers were turned on to stall F₁ at the target angle then turned off to release the motor after the set time. Released F₁ either steps forward (ON) or returns to the original pause angle (OFF). These behaviors indicate that the reaction under investigation has completed or not, respectively. (b) Examples of stall-and-release traces for ATP binding of F₁^+ε at 200 nM ATP. During a pause, F₁^+ε was stalled for 2 s and then released. After release, F₁^+ε stepped to the next binding angle without moving back (red), indicating that ATP had already bound to F₁^+ε before release. When stalled for 2 s, F₁^+ε rotated back to the original binding angle (blue), indicating that no ATP binding had occurred. To see this figure in color, go online.

Figure 3

Angle dependence of ATP binding and release. (a and b) Time courses of p_ON of F₁^+ε (a) and F₁^−ε (b) at 200 nM ATP after stalling at −30° (cyan), −10° (blue), 0° (red), +10° (green), +30° (black), or +50° (yellow) from the original ATP-binding angle. k_on^ATP and k_off^ATP were determined by fitting to a single exponential function: p_ON = (k_on^ATP·[ATP]/(k_on^ATP[ATP] + k_off^ATP)) × (1 − exp(−(k_on^ATP[ATP] + k_off^ATP) × t)), according to the reversible reaction scheme, F₁ + ATP ⇄ F₁∙ATP. Each data point was obtained from 29 to 80 trials using more than five molecules. The error in p_ON is given as $\sqrt{p_{\text{ON}}\left(100-p_{\text{ON}}\right)/N}$, where N is the number of trials for each stall measurement. (c–e) Angle dependence of k_on^ATP, k_off^ATP, and K_d^ATP plotted against the arrest angle. Zero degrees corresponds to the ATP-binding angle in Fig. 1b. The open symbols represent the k_on^ATP determined from freely rotating F₁. Red and gray symbols represent the values for F₁^+ε and F₁^−ε, respectively. deg, degree. To see this figure in color, go online.

Figure 4

Angle dependence of ATPγS hydrolysis, synthesis, and Thio-Pi release. (a and b) Time courses of p_ON of F₁^+ε_(βE190D) (a) and F₁^−ε_(βE190D) (b) at 1 mM ATPγS after stalling at −50° (blue), +10° (red) or +50° (green) from the original hydrolysis waiting angle (200° in Fig. 1b). k_hyd^ATPγS, k_syn^ATPγS, K_E^ATPγS, and k_off^Thio-Pi were determined by fitting to a consecutive reaction model. Each data point was obtained from 12 to 78 trials using more than five molecules. The error in p_ON is given as $\sqrt{p_{\text{ON}}\left(100-p_{\text{ON}}\right)/N}$, where N is the number of trials for each stall measurement. (c–f) Angle dependence of k_hyd^ATPγS, k_syn^ATPγS, K_E^ATPγS, and k_off^Thio-Pi plotted against arrest angle. Zero degrees corresponds to the hydrolysis angle in Fig. 1b. Red and gray symbols represent the values for F₁^+ε_(βE190D) and F₁^−ε_(βE190D), respectively. deg, degree. To see this figure in color, go online.

Figure 5

Buffer exchange experiments. (a) Schematic of buffer exchange experiments: p_ON measured for a target F₁ molecule, exchanged with the buffer including 50 nM ε subunits for the reconstitution of F₁^+ε or F₁^+ε_(βE190D) complex, and then p_ON measured again. (b) Left panel shows the time course of p_ON of F₁^+ε (red) and F₁^−ε (gray) at 200 nM ATP determined from the ensemble average at ± 0°. p_ON at 3 s was highlighted using a yellow bar. Right panel shows p_ON determined by buffer exchange experiments, with a stall at ± 0° for 3 s for the same rotating molecules before (gray) and after reconstitution of the ε subunit (red). The p_ON for each molecule is displayed using different symbols. The open circles represent the averages of seven molecules. (c) Left panel shows the time course of p_ON of F₁^+ε_(βE190D) (red) and F₁^−ε_(βE190D) (gray) at 1 mM ATPγS determined from the ensemble average at ± 0°. p_ON at 20 s was highlighted using a yellow bar. Right panel shows P_ON determined by buffer exchange experiments, with a stall at ± 0° for 20 s for the same rotating molecules before (gray) and after reconstitution of the ε subunit (red). The p_ON for each molecule is displayed using different symbols. The open circles represent the averages of four molecules. w/o, without; w/, with. To see this figure in color, go online.

Figure 6

C-terminal domain of the ε subunit. (a) Cross section view of F₁^+ε (left) and F₁^+εΔc (right) from thermophilic Bacillus PS3 (PDB: 4XD7). The C-terminal α-helices of the ε subunit are illustrated using cylindrical diagrams. The ε subunits with (ε) or without C-terminal α-helices (ε^Δc) are colored in red and orange, respectively. (b) p_ON at 200 nM ATP for F₁^−ε (gray), F₁^+ε (red), and F₁^+εΔc (orange) were plotted against the arrest angle. (c) p_ON at 1 mM ATPγS for F₁^−ε_(βE190D) (gray), F₁^+ε_(βE190D) (red), and F₁^+εΔc_(βE190D) (orange) were plotted against the arrest angle. deg, degree; w/o, without; w/, with. To see this figure in color, go online.

Figure 7

Chemo-mechanical coupling efficiency during ATP synthesis. (a) The coupling efficiency of forcible rotation with ATP release in the absence of the ε subunit. The gray lines represent the results of numerical calculation based on k_off^ATP(θ) = 0.12 × exp(−0.043·θ) determined for F₁^−ε in Fig. 3d at θ_off = 120° (light gray), 140° (gray), and 160° (thick gray). The gray circle represents the net coupling efficiency of F₁^−ε during ATP synthesis, as experimentally determined in the previous study (31). (b) The coupling efficiency of forcible rotation with ATP release in the presence of the ε subunit. The colored lines represent the results of numerical calculation based on k_off^ATP(θ) = 0.58 × exp(−0.042·θ) determined for F₁^+ε in Fig. 3 d at θ_off = 120° (orange), 140° (pink), and 160° (red). The red circle represents the net coupling efficiency of F₁^+ε as determined in the previous study (31). w/o, without; w/, with. To see this figure in color, go online.