Direct measurements of the coordination of lever arm swing and the catalytic cycle in myosin V

Abstract

Myosins use a conserved structural mechanism to convert the energy from ATP hydrolysis into a large swing of the force-generating lever arm. The precise timing of the lever arm movement with respect to the steps in the actomyosin ATPase cycle has not been determined. We have developed a FRET system in myosin V that uses three donor-acceptor pairs to examine the kinetics of lever arm swing during the recovery and power stroke phases of the ATPase cycle. During the recovery stroke the lever arm swing is tightly coupled to priming the active site for ATP hydrolysis. The lever arm swing during the power stroke occurs in two steps, a fast step that occurs before phosphate release and a slow step that occurs before ADP release. Time-resolved FRET demonstrates a 20-Å change in distance between the pre- and postpower stroke states and shows that the lever arm is more dynamic in the postpower stroke state. Our results suggest myosin binding to actin in the ADP.Pi complex triggers a rapid power stroke that gates the release of phosphate, whereas a second slower power stroke may be important for mediating strain sensitivity.

Keywords: FRET; actin; force generation; kinetics; myosin.

Scheme 1.

The actomyosin ATPase cycle.

Fig. 1.

Fluorescent probe location. The crystal structure of MV (PDB 1W7J) is shown with the probe locations. A tetracysteine motif (CCPGCC) was inserted at the N terminus which is depicted by labeling (red) the N-terminal residue (E5). Two labeling sites on calmodulin, T110C and T5C, are shown by labeling the corresponding residue in the essential light chain. T110C was labeled with QSY-9, and T5C was labeled with the QSY-9 or IAANS probe. FlAsH acted as a donor in the QSY-9–labeled T110C or T5C constructs and as an acceptor in the IAANS-labeled T5C construct.

Scheme 2.

Kinetic steps associated with the myosin recovery stroke.

Fig. 2.

Kinetics of the recovery stroke. (A and B) The kinetics of lever arm swing during the recovery stroke was measured by mixing 0.25 µM MV-F.QSY (A) or MV-F.IAANS (B) with different concentrations of ATP. A biphasic fluorescence increase (FlAsH–QSY pair with 500 µM ATP) (A, Inset) or a decrease (IAANS–FlAsH pair with 500 µM ATP) (B, Inset) was observed. The fast and slow phases are plotted as a function of ATP concentration, and the fast phase was fit to a hyperbola. The maximum rate of the fast phase was 330 ± 7⋅s⁻¹ (MV-F.QSY) and 312 ± 12⋅s⁻¹ (MV-F.IAANS). The observed rate of ATP-induced enhancement in tryptophan fluorescence in MV is plotted in A and fit to a hyperbola to determine the rate constant for the formation of the hydrolysis-competent state (332 ± 28⋅s⁻¹). Control traces of the respective donor-alone and acceptor-alone are also shown in the Insets. (C) The relative amplitudes of the fast and slow phases are plotted as a function of ATP concentration.

Scheme 3.

Kinetic steps associated with the myosin power stroke.

Fig. 3.

Kinetics of the power stroke. The kinetics of lever arm swing during the power stroke were measured by sequential mix single-turnover experiments (final concentrations: 0.15–0.25 µM MV, 0.1–0.2 µM ATP, and varying actin concentrations). (A) In the MV-F.QSY construct we observed a biphasic decrease in FlAsH fluorescence [Inset: representative traces of the FRET signal (red, k_Fast = 269 ± 18⋅s⁻¹) and phosphate release signal (green, k_obs = 97 ± 1⋅s⁻¹) at 30 µM actin]. (B) In the MV-F.IAANS construct we observed a biphasic increase in FlAsH fluorescence (Inset: representative traces at 10 µM actin). In both constructs the observed rate of the fast phase increased as a function of actin concentration, but the slow phase remained unchanged. For the FlAsH–QSY construct in A, the maximum rate of the fast phase was 352 ± 33⋅s⁻¹, and the average rate of the slow phase was 18 ± 9⋅s⁻¹. For the IAANS–FlAsH construct in B, the maximum rate of the fast phase was 493 ± 119⋅s⁻¹, and the average rate of the slow phase was 20 ± 13⋅s⁻¹. The observed rate of phosphate release was also measured with the MV-F.QSY construct and was plotted as a function of actin concentration in A (same concentrations as above and 4.5 µM PBP). The hyperbolic fit of the data allowed determination of the phosphate release rate constant (201 ± 11⋅s⁻¹). (C) The relative amplitudes of the fast and slow phases in the FRET signal were plotted as a function of actin concentration.

Fig. 4.

Interprobe distances measured by TR-FRET. Postpower stroke (green) and prepower stroke (red) distances and distance distributions measured by TR-FRET (solid lines) in the MV-F.QSY construct (SI Appendix, Table S2). Dotted lines correspond to the distances determined in the crystal structure of the pre- and postpower stroke states, respectively, as shown in SI Appendix, Fig. S11.