Abstract
Muscle contraction is driven by cyclic association and dissociation of myosin head of the thick filament with thin actin filament coupled with ATP binding and hydrolysis by myosin. However, because of the absence of actomyosin rigour structure at high resolution, it still remains unclear how the strong binding of myosin to actin filament triggers the release of hydrolysis products and how ATP binding causes their dissociation. Here we report the structure of mammalian skeletal muscle actomyosin rigour complex at 5.2 Å resolution by electron cryomicroscopy. Comparison with the structures of myosin in various states shows a distinctly large conformational change, providing insights into the ATPase-coupled reaction cycle of actomyosin. Based on our observations, we hypothesize that asymmetric binding along the actin filament could function as a Brownian ratchet by favouring directionally biased thermal motions of myosin and actin.
Highlights
Muscle contraction is driven by cyclic association and dissociation of myosin head of the thick filament with thin actin filament coupled with ATP binding and hydrolysis by myosin
Upon binding of MgATP, myosin hydrolyses ATP relatively quickly but the hydrolysis products ADP and Pi stay in the nucleotide-binding pocket, and its ATPase cycle does not proceed until myosin head binds to actin filament
Structural studies by X-ray crystallography on the head domains of various myosins, such as myosin II, V and VI, in different nucleotide states have suggested that myosin undergoes large conformational changes during ATPase cycle in its lever arm domain to be in largely different angles within the plane of actin filament axis and that such changes represent a power stroke that drives the unidirectional movement of myosin against actin filament[1,2]
Summary
CryoEM data collection and helical image analysis. For sample preparation of the actomyosin rigour complex, we used apyrase to completely remove residual ATP in solution to stabilize the rigour complex. The structure of the skeletal actomyosin rigour complex is shown in Fig. 1 (Supplementary Movies 1 and 2) together with a typical cryoEM image of F-actin fully decorated with myosin heads (Fig. 1a). We used a homology model of rabbit skeletal muscle myosin based on the crystal structure of squid muscle myosin S1 fragment in the rigour-like state (Protein Data Bank (PDB): 3I5G)[16] and a cryoEM structure of F-actin from rabbit skeletal muscle (PDB: 3MFP)[23] for docking and refinement by flexible fitting[25,26]. The homology model of myosin head in the rigour-like conformation did not fit well into the map (Supplementary Figs 2 and 3), and significant rearrangements of domains and secondary structures by flexible fitting were necessary to refine the model against the map. Myosin head can be roughly divided into the following five subdomains: the N-terminal 25 kDa domain (N25D: 1–205); the upper 50 kDa domain (U50D: 206–466; 603–627); the lower 50 kDa domain (L50D: 467–602; 628–680); the converter domain (CD: 681–770); and the lever arm domain (LAD: 771–845)
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