Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors

Abstract

The angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor A₃B₃DF, from the Methanosarcina mazei Gö1 A-ATP synthase, and the thermophilic motor α₃β₃γ, from Geobacillus stearothermophilus (formerly known as Bacillus PS3) F-ATP synthase, are resolved at 5 μs resolution for the first time. Unexpectedly, the angular velocity profile of the A-type was closely similar in the angular positions of accelerations and decelerations to the profiles of the evolutionarily distant F-type motors of thermophilic and mesophilic origins, and they differ only in the magnitude of their velocities. M. mazei A₃B₃DF power strokes occurred in 120° steps at saturating ATP concentrations like the F-type motors. However, because ATP-binding dwells did not interrupt the 120° steps at limiting ATP, ATP binding to A₃B₃DF must occur during the catalytic dwell. Elevated concentrations of ADP did not increase dwells occurring 40° after the catalytic dwell. In F-type motors, elevated ADP induces dwells 40° after the catalytic dwell and slows the overall velocity. The similarities in these power stroke profiles are consistent with a common rotational mechanism for A-type and F-type rotary motors, in which the angular velocity is limited by the rotary position at which ATP binding occurs and by the drag imposed on the axle as it rotates within the ring of stator subunits.

Keywords: A-ATP synthase; ATP synthase; F-ATP synthase; F1FO-ATPase; angular velocity; bioenergetics; molecular motor; power stroke; single molecule biophysics; single molecule rotation.

FIGURE 1.

Comparison of the related lever and foot domains of the MmA₃B₃DF and F-type α₃β₃γ complexes. A, composite structure of subunits A, B, D, and F in the MmA₃B₃DF complex of the A-ATP synthase. The catalytic domain (gray ribbon) and the lever domain (orange) of subunit A (PDB code 3I4L (37) in the A3B3 hexamer that surrounds the central stalk subunits was formed by subunit F (magenta, PDB code 2OV6 (34)) and subunit D (yellow, PDB code 3AON (38)). B, structural components of the α₃β₃γ complex of bovine mitochondrial F-ATP synthase (PDB code 1E79) (39) showing the catalytic domain (gray ribbon) and lever domain (purple) of β subunits that surround the coiled-coil (green) and foot (blue) domains of the γ-subunit. Subunits B and α are omitted for clarity.

FIGURE 2.

Rotary mechanism of bacterial F ATPases at saturating ATP concentrations. The (α₃β₃)-ring (viewed form the membrane side) is designated as a ring of α-subunit (gray) and β-subunit (white) hexagons that surround the γ-subunit composed of the coiled-coil (green) and foot (red) domains. During the catalytic dwell ATP hydrolysis occurs at β_D (a), followed by P_i release at β_E (b) that initiates the power stroke (c). At saturating ATP concentrations, the power stroke is continuous for 120° (c) during which time ATP binding to β_E and ADP release from β_D occur. Upon formation of the next catalytic dwell (d), β_T changed conformation to β_D to induce ATP hydrolysis.

FIGURE 3.

Purification and ATPase activity of MmA₃B₃DF complexes. A, SDS-17% polyacrylamide gel (40) of the purified recombinant protein complexes with the respective molecular weight marker. B, specific ATPase activities of protein complexes as determined from the initial slope of the decrease in NADH absorbance in the coupled ATPase assay.

FIGURE 4.

Single molecule assay of rotating protein-gold nanorod complexes. A, schematic model of the microscope setup with a protein-gold nanorod complex. The MmA₃B₃DF complex (subunits A, B, D, and F in orange, green, yellow, and purple, respectively) is attached via its His₈ tags in subunit A to a coverslip, although a neutravidin-coated nanorod is attached to the biotinylated cysteine (red) in subunit D. Gold-nanorods were illuminated by a dark-field condenser. Red scattered polarized light was recorded by an APD after passing through a polarizer and a bandpass filter to block shorter wavelengths of light. B, consecutive histograms of light intensity scattered from a nanorod attached to a rotating MmA₃B₃DF complex collected at 1 kHz as a function of the polarization angle during ATPase-powered rotation. C, SDS-9% polyacrylamide gel of purified recombinant Gsα₃β₃γ and molecular mass markers. D, consecutive histograms of light intensity scattered from a nanorod attached to a rotating Gsα₃β₃γ complex collected at 1 kHz as a function of the polarization angle during ATPase-powered rotation. The approximate courses of the three sinusoidal curves that resulted from the three catalytic dwells are indicated in gray.

FIGURE 5.

Power stroke analysis. A, light intensities as a function of time collected at 200 kHz during a single power stroke event scattered from a rotating nanorod attached to a single MmA₃B₃DF complex in the presence of 1 mm ATP. Starting from a catalytic dwell where the nanorod was aligned with the polarizer such that the scattered light intensity was at a minimum, the intensity increased through a maximum during the power stroke (red) until it reached the subsequent catalytic dwell. B, rotational position of the power stroke from A as a function of time calculated from the arcsine^1/2 function.

FIGURE 6.

Comparison of arcsine versus arcsine^1/2 functions to calculate the rotational position of a gold nanorod from the scattered light intensity as a function of the orientation of the polarizer. A, average light intensity (red) scattered from each of four nanorods as a function of the rotational position of the polarizer for 120° from an intensity minimum, collected at 1 kHz in successive histograms as per Fig. 4. Each nanorod was attached to a molecule of Ecα₃β₃γ that was not rotating. The data were fitted using the arcsine (black) versus the arcsine^1/2 (blue) functions assuming a perfect fit at the first data point, which was the intensity minimum. B, comparison of the total error of the fit of the data for each nanorod from A using arcsine (black) versus arcsine^1/2 (blue) functions. C, fits of the experimental data (red) from A using arcsine (black) versus arcsine^1/2 (blue) functions assuming a perfect fit at 3° after the first data point. D, comparison of the total error of the fit of the data for each nanorod from B using the arcsine (black) versus the arcsine^1/2 (blue) functions.

FIGURE 7.

Average angular velocity profiles of the 120° power stroke at 1 mm MgATP. A, average angular velocities of the ATPase-dependent power strokes of MmA₃B₃DF (blue), Gsα₃β₃γ (orange), and Ecα₃β₃γ (black). B, comparison of the angular velocity profile of Ecα₃β₃γ calculated from the arcsine (red) or the arcsine^1/2 (black) functions. Data were collected, and the rotational position was determined as described in Fig. 2. Angular velocities were determined from the moving average of the slope of three consecutive rotational positions from individual power strokes that were averaged and binned for every 3°.

FIGURE 8.

Effects of ATP concentrations that limit the rate of ATPase activity on the average angular velocity profiles. A, dwell abundance as a function of rotational position during the power stroke of Ecα₃β₃γ at 1 mm MgATP (black) versus 0.3 mm MgATP (red). B, dwell abundance as a function of rotational position during the power stroke of the MmA₃B₃ complex at 1 mm MgATP (blue) versus 0.1 mm MgATP (red). C, average angular velocity profile of the Ecα₃β₃γ power stroke at 1 mm MgATP (black) versus 0.3 mm MgATP (red). D, average angular velocity profile of the MmA₃B₃DF power stroke at 1 mm MgATP (blue) versus 0.1 mm MgATP (red).

FIGURE 9.

Effects of elevated ADP concentrations on the average angular velocity profiles. A, dwell abundance as a function of rotational position during the power stroke of Ecα₃β₃γ at 1 mm MgATP in the absence (black) versus the presence of 4 mm MgADP (red). B, dwell abundance as a function of rotational position during the power stroke of the MmA₃B₃DF complex at 1 mm MgATP in the absence (blue) versus the presence of 50 μm (red) and 250 μm (orange) MgADP. C, average angular velocity of the power stroke as a function of rotational position of EcF1 at 1 mm ATP in the absence (black) versus the presence (red) of 4 mm MgADP. D, average angular velocity of the power stroke as a function of rotational position of the MmA₃B₃DF complex at 1 mm MgATP (blue) in the absence (blue) versus the presence of 50 μm (red) and 250 μm (orange) MgADP.

FIGURE 10.

Mechanistic differences between MmA₃B₃DF and the Ecα₃β₃γ at limiting ATP and/or at elevated ADP concentrations. A, (α₃β)₃-ring of the Ecα₃β₃γ complex is designated as a ring of α-subunit (gray) and β-subunit (white) hexagons surrounding the γ-subunit composed of the coiled-coil (green), and the foot (red) domains. During the catalytic dwell ATP hydrolysis occurs at β_D (panel a), followed by P_i release at β_E (panel b) that initiates the power stroke. At limiting MgATP concentrations (panel c), an ATP-binding dwell interrupts the power stroke at 40° when ATP has not bound to β_E. At elevated ADP concentrations (panel d), an ADP-binding dwell interrupts the power stroke at 40° when ADP outcompetes ATP for binding to β_E. The power stroke resumes for 80° upon binding of ATP to β_E (panel e). Elevated ADP concentrations limit the power stroke angular velocity (yellow) by delaying release of ADP from β_D (panel f). Upon formation of the next catalytic dwell, β_T changed conformation to β_D to induce ATP hydrolysis (panel g) that will be continued by the next 120° power stroke (data not shown). B, (AB)₃-ring of the MmA₃B₃DF complex is designated as a ring of A-subunit (white) and B-subunit (gray) hexagons surrounding the central stalk composed of the D-subunit (green) and F-subunit (red). During the catalytic dwell ATP hydrolysis (panel a), phosphate release (panel b), ADP release (panel c), and ATP binding (panel d) occur in an as yet unspecified order. The 120° power strokes (panel e) are not interrupted by ATP binding or ADP-binding dwells. Upon formation of the next catalytic dwell, A_C changed conformation to A_CR to induce ATP hydrolysis (panel f) that will be continued by the next 120° power stroke (data not shown). Sites β_E, β_D, and β_T in Ecα₃β₃γ (A) correspond to sites A_O, A_CR, and A_C in MmA₃B₃DF (B) (34).