Direct observation of stepping rotation of V-ATPase reveals rigid component in coupling between Vo and V1 motors

Abstract

V-ATPases are rotary motor proteins that convert the chemical energy of ATP into the electrochemical potential of ions across cell membranes. V-ATPases consist of two rotary motors, V_o and V₁, and Enterococcus hirae V-ATPase (EhV_oV₁) actively transports Na⁺ in V_o (EhV_o) by using torque generated by ATP hydrolysis in V₁ (EhV₁). Here, we observed ATP-driven stepping rotation of detergent-solubilized EhV_oV₁ wild-type, aE634A, and BR350K mutants under various Na⁺ and ATP concentrations ([Na⁺] and [ATP], respectively) by using a 40-nm gold nanoparticle as a low-load probe. When [Na⁺] was low and [ATP] was high, under the condition that only Na⁺ binding to EhV_o is rate limiting, wild-type and aE634A exhibited 10 pausing positions reflecting 10-fold symmetry of the EhV_o rotor and almost no backward steps. Duration time before the forward steps was inversely proportional to [Na⁺], confirming that Na⁺ binding triggers the steps. When both [ATP] and [Na⁺] were low, under the condition that both Na⁺ and ATP bindings are rate limiting, aE634A exhibited 13 pausing positions reflecting 10- and 3-fold symmetries of EhV_o and EhV₁, respectively. The distribution of duration time before the forward step was fitted well by the sum of two exponential decay functions with distinct time constants. Furthermore, occasional backward steps smaller than 36° were observed. Small backward steps were also observed during three long ATP cleavage pauses of BR350K. These results indicate that EhV_o and EhV₁ do not share pausing positions, Na⁺ and ATP bindings occur at different angles, and the coupling between EhV_o and EhV₁ has a rigid component.

Keywords: V-ATPase; molecular motors; single-molecule analysis.

Fig. 1.

(A) Overall architecture of EhV_oV₁. The dotted circular arcs represent the rotation direction driven by ATP hydrolysis. (B) (Top) Top view of the a subunit (cyan) and c₁₀ ring (brown) of EhV_o and (Bottom) A (yellow), B (orange), D (green), and F subunits (pink) of EhV₁. The black arrow in Top indicates the path of Na⁺ movement during ATP-driven rotation. The arcs in Bottom represent the catalytic AB pairs. (C) Side view of the a subunit viewed from the c subunit. This structure was constructed by the SWISS-MODEL server (36) using a structure of the a subunit of V-ATPase from T. thermophilus. The black arrows represent the path of Na⁺ movement during ATP-driven rotation. The mutated residue, aGlu634, is located on the surface of the entry half-channel of the a subunit as highlighted in red letters and a circle.

Fig. 2.

(A) Schematic model of the single-molecule rotation assay of EhV_oV₁ probed with AuNP. Each letter represents the name of the subunits. EhV_oV₁ was fixed on Ni²⁺-NTA–coated cover glass via His₃ tags added to the C terminus of the c subunit. A streptavidin-coated gold nanoparticle (40 nm in diameter) was attached to the N terminus of the A subunit, which was biotinylated by adding an Avi-tag. Solid and dotted red lines indicate the center axis of EhV_oV₁ and the centroid of the attached AuNP, respectively. Because the rotor c₁₀ ring was fixed on a glass surface, the stator subunits rotate counterclockwise against the rotor subunits as shown by red arrows. (B) [ATP] dependence of the rotation rate of wild type and aE634A at 300 mM NaCl. Red open circles and blue open squares indicate the data from individual molecules of wild type and aE634A, respectively. The closed symbols are averages, and error bars represent SDs. Data were fitted with the Michaelis–Menten equation: V = V_max^ATP · [ATP]/(K_m^ATP + [ATP]). The obtained kinetic parameters are summarized in Table 1. (C) [Na⁺] dependence of rotation rate of wild type and aE634A at 5 mM ATP. The correspondence of colored symbols is the same as in B. The black lines show the fit with the sum of two independent Michaelis–Menten equations: V = V_max1^Na · [Na⁺]/(K_m1^Na + [Na⁺]) + V_max2^Na · [Na⁺]/(K_m2^Na + [Na⁺]). The obtained kinetic parameters are summarized in Table 2. The contaminating Na⁺ in the observation buffers was taken into account as shown in SI Appendix, Fig. S6. Namely, 50 mM Bis-Tris (pH 6.5) was used for 0.09 mM Na⁺, and 20 mM potassium phosphate (pH 6.5) was used for the other [Na⁺] values as buffers.

Fig. 3.

(A) Typical trajectory of ATP-driven rotation of wild-type EhV_oV₁ at 5 mM ATP and 0.3 mM Na⁺ recorded at 3,000 fps (0.33 ms time resolution). (Right) Enlarged view of one revolution (360°). Pink, red, and black traces represent raw, median-filtered (current ± 4 frames), and fitted trajectories, respectively. (Inset) The corresponding x–y trajectory. Pink lines and red dots represent the raw and median-filtered (current ± 4 frames) coordinates, respectively. We collected 1,352 steps from 15 molecules. Other examples of trajectories are shown in SI Appendix, Fig. S7. (B) Distribution of the step size fitted with a single Gaussian assuming a single peak. The values at the top right are the fitted parameter (peak ± SD) and average. The ratio of backward steps was 0.8%. (C) Distribution of the duration time before the forward step fitted with a single exponential decay function. The value at the top right is the obtained time constant (fitted value ± SE of the fit).

Fig. 4.

Number of detected pauses per single turn of (A) wild type at 5 mM ATP and 0.3 mM Na⁺ corresponding to Fig. 3 and SI Appendix, Fig. S7; (B) aE634A at 5 mM ATP and 0.3 mM Na⁺ corresponding to Fig. 5 and SI Appendix, Fig. S8; and (C) aE634A at 1 μM ATP and 0.3 mM Na⁺ corresponding to Fig. 7 and SI Appendix, Fig. S10.

Fig. 5.

(A) Typical trajectory of ATP-driven rotation of aE634A at 5 mM ATP and 0.3 mM Na⁺ recorded at 1,000 fps (1 ms time resolution). (Right) Enlarged view of one revolution (360°). Pink, red, and black traces represent raw, median-filtered (current ± 7 frames), and fitted trajectories, respectively. (Inset) The corresponding x–y trajectory. Pink lines and red dots represent the raw and median-filtered (current ± 7 frames) coordinates, respectively. We collected 1,718 steps from 23 molecules. Other examples of trajectories are shown in SI Appendix, Fig. S9. (B) Distribution of the step size fitted with a single Gaussian assuming a single peak. The values at the top right are the fitted parameter (peak ± SD) and average. The ratio of backward steps was 1.2%. (C) Distribution of the duration time before the forward step fitted with a single exponential decay function. The value at the top right is the obtained time constant (fitted value ± SE of the fit).

Fig. 6.

Single-molecule analysis of aE634A at saturated [ATP] (5 mM). Experimental conditions are described on the left. Examples of trajectories are shown in SI Appendix, Fig. S10. (A and B) Distribution of the step size at 0.09 and 1.3 mM Na⁺. Black lines represent fitting with single Gaussians. The values at the top right are the fitted parameters (peak ± SD) and averages. (C and D) Distribution of the duration time before the forward step. Black lines represent fitting with single exponential decay functions. The value at the top right is the obtained time constant (fitted value ± SE of the fit). (E) Plot between [Na⁺] (0.09, 0.3, and 1.3 mM) and time constant obtained by the fitting at 5 mM ATP. The solid red line represents a straight line connecting two data points at 0.09 and 0.3 mM Na⁺, and its slope is −1.1. The solid blue line is the result of linear fitting among all three data points. The obtained slope is −1.0.

Fig. 7.

(A) Typical trajectory of ATP-driven rotation of aE634A at 1 μM ATP and 0.3 mM Na⁺ recorded at 1,000 fps (1 ms time resolution). (Right) Enlarged view of one revolution (360°). Pink, red, and black traces represent raw, median-filtered (current ± 7 frames), and fitted trajectories, respectively. (Inset) The corresponding x–y trajectory. Pink lines and red dots represent the raw and median-filtered (current ± 7 frames) coordinates, respectively. We collected 920 steps from 21 molecules. Other examples of trajectories are shown in SI Appendix, Fig. S11. (B) Distribution of the step size fitted with the sum of three Gaussians: one peak in backward (minus) direction and two peaks in forward (plus) direction, one of which was fixed at 36°, assuming that it was the step of EhV_o. The ratio of backward steps was 6.1%. (C) Distribution of the duration time before the forward step fitted with the sum of two exponential decay functions. The values at the top right are obtained time constants (fitted value ± SE of the fit) and the coefficient of determination (R²) of fitting.

Fig. 8.

Backward steps of aE634A observed at 1 μM ATP and 0.3 mM Na⁺ with 1,000 fps (1 ms time resolution). (A) Examples of trajectories showing the backward steps. The pink, red, and black traces represent the raw, median-filtered (current ± 7 frames), and fitted trajectories of the median-filtered data identified by the algorithm, respectively. The green, cyan, and purple lines indicate forward steps just before backward steps, backward steps, and forward steps just after backward steps (recovery steps), respectively. An asterisk indicates a backward step that is not detected as a step by the algorithm (underfitting). (B–D) Distributions of step size for forward steps just before backward steps, backward steps, and recovery steps, respectively. In B and D, the distributions seemed to show two peaks at <36° and 36°. In C, the peak position was larger than −36°, and the average value was −18.8°.

Fig. 9.

Schematic models of the stepping rotation and rigid coupling of EhV_oV₁. The orange circles and dark green squares indicate the pausing positions waiting for Na⁺ binding to EhV_o and ATP binding to EhV₁, respectively. The red arrows indicate the 36° steps between adjacent pausing positions for the EhV_o. The blue arrows indicate the backward and forward steps smaller than 36° between adjacent pausing positions for EhV_o and EhV₁. (A) Condition in which only Na⁺ binding to EhV_o is rate-limiting. In this condition, the pauses waiting for ATP binding to EhV₁ are too short to be detected, and EhV_oV₁ rotates unidirectionally without backward steps. (B) Condition in which both Na⁺ and ATP bindings are rate-limiting. The pausing positions waiting for ATP binding are visualized, and then 13 pausing positions are detected per single turn. Because no torque is generated during the pauses waiting for ATP binding to EhV₁, EhV_oV₁ rotates to the backward and forward pausing positions of EhV_o driven by Brownian motion. As a result, backward and forward steps smaller than 36° are observed. A detailed model is shown in SI Appendix, Fig. S15.