Visualizing myosin's power stroke in muscle contraction

Mary C. Reedy () 0 0 Department of Cell Biology, Duke University Medical Center , Durham, NC 27710, 919 684 5674 , USA SUMMARY The long-standing swinging crossbridge or lever arm hypothesis for the motor action of myosin heads finds support in recent results from 3-D tomograms of insect flight muscle (IFM) fast frozen during active contraction and from both fluorescence polarization and X-ray diffraction during rapid stretches or releases of isometrically contracting fibers. The latter provide direct evidence for lever arm movements synchronous with force changes. Rebuilding the atomic model of nucleotide-free subfragment 1 (S1) to fit fast-frozen, active IFM crossbridges suggests a two-stage power stroke in which the catalytic domain rolls on actin from weak to strong binding; this is followed by a 5-nm lever arm swing of the light chain domain, which gives a total interaction distance Elucidating the structure of myosin motor proteins and the mechanism by which actin-myosin motors transduce the chemical energy of ATP hydrolysis to power the movements of animals and cells remains one of the major challenges in biological science. A host of myosins move intracellular cargo along actin filaments in non-muscle cells, produce the contractions of smooth muscle and drive cytokinesis. Myosin attains its most ambitious functional level acting in the ordered ensembles of muscle, powering such diverse functions as insect flight (at 100 beats/second) or the breaching of a whale. In this Commentary, I focus on the structure of skeletal muscle myosin II assembled in thick filaments and interacting with actin-containing thin filaments in insect flight and vertebrate striated muscles. Skeletal muscle myosin II consists of two globular heads linked by helical segments that supercoil to form a long helical rod (Fig. 1). The myosin rod segments form the shaft of the thick filaments, and the myosin heads project outward toward the actin thin filaments, forming the myosin crossbridges. Insect flight muscle (IFM) displays myosin heads, in very well-ordered arrangement, bridging between the myosin and actin filaments in 25-nm longitudinal sections that include only a single layer of alternating myosin and actin filaments (the myac layer). I shall place particular emphasis on 3-D tomographic snapshots of myosin freezetrapped during active contraction in IFM and on how these of approx. 12 nm. Comparison of S1 crystal structures with in situ myosin heads suggests that actin binding may be necessary in order to view the full repertoire of myosin motor action. The differing positions of the catalytic domains of actin-attached myosin heads in contracting IFM suggest that both the actin-myosin binding energy and the hydrolysis of ATP may be used to cock the crossbridge and drive the power stroke. Movies available on-line: (http://www.biologists.com/JCS/movies/jcs1259.html) freeze frames of a power stroke relate to the atomic structures of the myosin head and to the swinging crossbridge or lever arm hypothesis for myosin motor action. Recent reviews that address related topics not discussed here can be found elsewhere (Cooke, 1997; Spudich et al., 1995; Holmes, 1997; Highsmith, 1999; Geeves and Holmes, 1999; Vale and Milligan, 2000; Holmes and Geeves, 2000; Duke, 2000). A BRIEF HISTORY OF THE SWINGING CROSSBRIDGE OR LEVER ARM HYPOTHESIS The swinging crossbridge hypothesis for the force-producing mechanism of myosin on actin developed over decades, beginning with the founding observations and insights included in the sliding filament model for muscle contraction codiscovered by H. E. Huxley and A. F. Huxley (Huxley and Hanson, 1954; Huxley and Niedergerke, 1954). In his 1969 review, H. E. Huxley (Huxley, 1969) proposed that the myosin heads projecting from the thick filament interact with actin in the thin filament, and that a change in crossbridge angle or shape coupled to the hydrolysis of MgATP produces sliding between the actin and myosin filaments, which causes muscle force and shortening (Fig. 1). The demonstration in highly ordered insect flight muscle (IFM) that detached myosin crossbridges are at an approx. 90 angle relative to the long axis of the filaments in ATP-relaxed IFM and attached at an approx. 45 angle in rigor, the state of high tension and maximal crossbridge attachment in the absence of ATP, supported the idea that the myosin crossbridges act as lever arms during contraction i.e. that they swing from 90 to 45 following attachment to actin, thereby producing filament sliding and force (Reedy et al., 1965). Soon after, mechanical experiments on single muscle fibers by A. F. Huxley (Huxley and Simmons, 1971) specified important features of crossbridge behavior. Huxley proposed that crossbridges are independent force generators that interact with actin over a distance of approx. 12 nm in a multi-step power stroke that includes an instantaneous elastic response to a quick release step and an approx. 6 nm or larger active segment in which the myosin rolls over the actin binding site (Huxley, 1974) (Fig. 1). Early models usually treated crossbridges as unitary lever arms whose actin ends served as pivot points as the entire myosin head changed angle. However, Huxley (1974) also suggested that a crossbridge might bend around a internal fulcrum while the part of the crossbridge bound to actin remained stationary (Fig. 1). Huxley and Kress modeled the crossbridge (Huxley and Kress, 1985) as composed of three domains connected by elastic elements, and proposed that it binds to actin over a 12nm interaction distance and progresses through an evolving actin-myosin interface to end with a short, 4-nm power stroke. Holmes and collaborators (Holmes et al., 1980; Holmes and Goody, 1984) proposed, on the basis of analysis of X-ray patterns of muscle, that only part of the mass of the myosin head (the nose cone) closely follows the actin helix after binding to actin. This would cause only a small intensity increase of the reflections in the X-ray pattern that signal myosin attachment. They further proposed that the nose cone mass remains at a constant angle relative to actin following myosins initial attachment to actin. This proposal was consistent with the scarcity of a rigor-like, 45 orientation of the mass of the entire myosin head in numerous X-ray diffraction studies of actively contracting muscle. H. E. Huxley commented in a review (Huxley, 1990) that, even though in vitro assays showed that a myosin subfragment 1 (S1) head produces movement (Sheetz and Spudich, 1983; Toyoshima et al., 1987), unambiguous experimental evidence for a change in head configuration or orientation directly linked to force production had not been forthcoming. Advances in chemical fixation of IFM, combined with X-ray monitoring and 3-D electron tomography (Tregear et al., 1990; Schmitz et al., 1996; Schmitz et al., 1997), produced 3-D images of crossbridges in equilibrium states that were designed to (...truncated)