The working stroke upon myosin–nucleotide complexes binding to actin

Abstract

Evidence from electron micrographs for different conformations of myosin led to the rowing model of actomyosin (AM) crossbridge action (1, 2) in which myosin binds to actin in one conformation, undergoes a working stroke, and detaches in a second conformation. The Lymn–Taylor scheme (3) describing the biochemical kinetics of AM provided a natural match to the mechanical model (Fig. 1). The detached myosin products (M.ADP.P_i) state bound to actin, and the working stroke occurred in association with product release. The binding of ATP facilitated dissociation of actin, and its hydrolysis took place while myosin was dissociated. The model postulated that the hydrolysis step is associated with repriming the head from the postpower-stroke structure back to the prepower-stroke form, although there was no evidence on this point at the time. Based on the observation that the detached states, myosin-ATP and M.ADP.P_i, behaved in a similar manner with respect to actin binding, Eisenberg and collaborators (4) favored a model in which there was a single detached conformation of myosin.

Fig. 1.

The Lymn–Taylor alignment of biochemical states with the crossbridge cycle. Pr = Products, ADP.P.

Measurement of the ATP binding constant (5) and the actin binding constant (6) were crucial steps in allowing the basic energetics of the AM mechanism to be elucidated. Much of the free energy of ATP hydrolysis is associated with actin binding to the M.ADP.P_i state and forming the rigor AM state (7), the part of the scheme that must encompass the working stroke. The myosin state in the absence of nucleotide binds even more tightly to actin, and if it is the energy of AM binding that powers the mechanical action, it would suggest that a working stroke would also result from myosin binding to actin. This idea is consistent with the views of Eisenberg (4) and the “3G” model of contraction (8). In the latter model all myosin states exist in a single myosin conformation and actin binding occurs in two steps, the first being the binding and formation of a weakly bound complex (A state) and the second being the conversion to a tightly bound complex (R state). It was natural to assume that the weak-to-strong (or A-to-R) transition corresponded to the working stroke.

In recent years, a scheme, closely related to that of Lymn and Taylor (3), has been developed based on the crystal structures of myosin subfragment 1 (S1). When crystallized in a variety of nucleotide states, two forms are common, which differ in a number of regions, including the nucleotide binding pocket, the switch 2 region, and the cleft between the upper and lower 50-kDa domains. The two forms generally are labeled based on the state of the nucleotide binding pocket as closed and open. The transition from the closed to the open form appears to be linked with movement of the lever arm and has been associated with the working stroke. The transition from open to closed occurs before the hydrolysis step and has been associated with repriming (9).

These models can be tested directly at the level of a single myosin molecule by using optical tweezers to hold an actin filament taut between two trapped beads and presenting it to a myosin molecule on a third fixed bead (10). Displacements of the actin beads “dumbbell” are monitored and used to detect attachment events; the difference between the mean displacements in free and bound periods gives the myosin working stroke, which under conditions of ATP turnover is ≈5–6 nm. To isolate the working stroke event in the biochemical cycle, we have investigated the binding of myosin and myosin–nucleotide complexes in the absence of ATP, where transitions are restricted to actin binding and loss of nucleotide. Although these transitions are reversible and no net work can be extracted from the dumbbell, any associated working stroke would still be detected as described in Materials and Methods. It is important to emphasize that the observed working stroke reflects the difference in orientations between the bound state and the dissociated state at the moment of attachment. If there is a mixture of different dissociated conformations, the relative rates of actin binding become important.

For each nucleotide, data from a single dumbbell are summarized to avoid combining working strokes from actin filaments of possibly different polarities. Errors are SEM. * indicates pooled data sets (absolute values of the working stroke averaged) using different dumbbells of unknown polarity (except for the case of ATP). BDTC-RLC S1 was used throughout. NA, not applicable.

Acknowledgments

Notes

Abbreviations: AM, actomyosin; S1, subfragment 1; M.ADP, myosin-ADP; M.PP_i, myosin-pyrophosphate; AMPPNP, adenyl-5′yl imidodiphosphate; M.AMPPNP, myosin-AMPPNP; RLC, regulatory light chain; BDTC, biotin-dependent transcarboxylase; QD, quadrant detector.