Muscle is formed by two sets of overlapping filaments: the thin actin-containing and the thick myosin-containing filaments. The thick filaments of striated muscle are polymers of myosin II. The tails of myosin molecules are packed together to form a backbone, whereas the two heads of each myosin molecule protrude from the molecule's surface. Muscle contraction occurs when the filament sets actively slide against each other, shortening the sarcomere (see Figure 1c and d). Nerve stimulation causes the release of calcium, which can initiate muscle contraction by acting on actin filaments (thin-filament regulation) or myosin filaments (thick-filament regulation).
As a result of advances in x-ray diffraction and electron microscopy (EM) studies of thin filaments, the molecular basis of actin-linked regulation is well understood. Thin-filament regulation is mediated by calcium binding to troponin C and involves—via the troponin complex—movement of tropomyosin toward the thin filament groove, removing the steric block that prevents myosin heads from attaching to actin molecules. Thin filaments have two structural states: the switched-off state, in which tropomyosin sits on the actin filament in such a way that it blocks actin's myosin binding site; and the switched-on state, in which tropomyosin releases the myosin binding site by moving away from it and into the actin groove.
By contrast, the molecular basis of myosin-linked regulation is not well understood. The regulation occurs either by direct calcium binding to myosin light chains—as in scallops—or by phosphorylation of myosin regulatory light chains (RLCs)—as in arthropod chelicerates and vertebrate striated and smooth muscle. The limiting step to advancing our understanding of the molecular mechanism of myosin-linked regulation is in determining the molecular structure of thick filaments. The species used is an important factor; the thick filaments from tarantula leg muscles have proven to be the most easily preserved.
Structural studies on the myosin-linked regulation mechanism have been hampered by the relatively low resolution of maps. A combination of adequate specimens, a negative staining method that preserves structure, and improved reconstruction techniques allowed us to calculate a 5-nm–resolution, three-dimensional map of tarantula striated muscle myosin. We interpreted this map with available structural information about the myosin head, but resolution was not adequate to unambiguously fit the atomic structures. Cryoelectron microscopy of frozen hydrated tarantula thick filaments increased resolution to 2.5 nm, and by using single-particle averaging techniques we were able to calculate a three-dimensional map. Fitting the atomic structure of heavy meromyosin (HMM) to the map removed the ambiguity in the map, allowing us to propose an atomic model of the relaxed thick filament. (HHM is the proteolytic cleavage product of myosin II and contains the two head domains of myosin II, also known as motor domains, connected by a portion of their coiled tails, and two pairs of light chains—myosin essential light chain [ELC] and RLC—attached to the myosin heads.) The model revealed the intra- and intermolecular interactions that retain myosin heads in a helical configuration close to the backbone surface of the thick filament.
Why Do Myosin Heads Form Helices in the Relaxed State?
The map's similarity to the interacting-heads atomic model of HMM reported in the literature—obtained by EM of crystals of smooth muscle HHM—facilitated interpretation of the map. The new map showed a detailed repeating motif on the filament surface representing myosin head pairs in the switched-off state, with dephosphorylated RLC. The specific interactions that, in this conformation, occur between both heads—three intramolecular interactions that stabilize the head pairs and two intermolecular interactions between the head pairs—cause the helical arrangement of the myosin heads in the thick filament and their packing down on the filament surface away from the thin filaments (Figure 1b and c). Figure 1c shows our current concept of the structure of thick filaments of tarantula striated muscle in the relaxed state. Myosin molecules, with heads in the asymmetric interacting-heads configuration, are packed together to form four helices of myosin head pairs, which constitute the relaxed thick filament.
The strong similarity between structures of myosin molecules isolated from smooth chicken muscle and striated tarantula muscle suggests that such an interacting-heads structure underlies the relaxed state of thick filaments in a wide range of species.
Molecular Mechanism of Myosin-Linked Regulation of Muscle Contraction and Thick Filament Activation
Determining the molecular structure of the thick filaments can help us understand phosphorylation-based, myosin-linked regulation. For a myosin head to interact with a thin filament to produce force, thick filaments must first become activated. EM and x-ray evidence suggests that RLC phosphorylation is involved in breaking the interactions between the RLCs of the myosin heads, leading to disordering and release of the heads, which allows them to interact with thin filaments. We envisage the activation of thick filaments as the physiological release of heads from myosin's surface before development of force, leading to an interaction between activated heads and thin filaments, and contraction and shortening of the sarcomere. In tarantula muscle, it is still not clear whether RLC phosphorylation directly regulates (as in smooth muscle and Limulus) or merely modulates the number of heads that become activated; the latter mechanism could be a way to control the number of heads actively involved in producing force after a series of twitches in tetanus.
Isolated myosin molecules of scallop striated muscle in the switched-off (relaxed) state have their two heads held down pointing toward the myosin tail, forming a rigid structure with low ATPase activity. When they are switched on by calcium binding, the heads become flexed randomly around the junction with the tail. In tarantula thick filament, a similar process can take place, suggesting that intra- and intermolecular interactions are diminished after RLCs are phosphorylated. Further experiments in which the resolution of the tarantula three-dimensional map is increased by using field emission gun cryoelectron microscopy or tomography should help to define more precisely the specific nature of the intra- and intermolecular interactions involved in myosin's activation mechanism.
In summary, we have revealed the molecular structure of thick filaments of striated muscle in the relaxed state. The structure reveals intra- and intermolecular interactions that keep heads together, thus forming helices close to the surface of the thick filament. RLC phosphorylation weakens these interactions, allowing thick filaments to be activated, producing disordering and release of heads, and enabling them to interact with the thin filaments. These results have opened the way to understanding the molecular mechanism of myosin-linked regulation of muscle contraction and are particularly important given that mutations associated with midventricular cardiomyopathy occur in the RLC near the phosphorylation site.
Last updated August 2008