Myosin Filament Structure
We are interested in how myosin filaments are constructed from myosin molecules, and whether the structural organization of myosin can account for the switching off of the filament in relaxed muscle. We focus on invertebrate muscle filaments – which are especially amenable to structural analysis – but our results have direct implications for vertebrate muscle. Using cryo-EM to preserve native structure, and single particle techniques to carry out 3D reconstruction, we have shown that the heads of each myosin molecule interact with each other in relaxed muscle (collaboration with Dr. R. Padron). The interactions, similar to those occurring in isolated myosin molecules, physically block sites needed for actin interaction and ATP hydrolysis, thus causing contraction to be switched off (Woodhead et al., 2005, Nature). Our current research is aimed at improving the detail of the reconstruction using the highest resolution EM techniques.
Left: reconstruction of tarantula skeletal myosin filament. “J” indicates repeating motif consisting of a pair of myosin heads. Right: 3D fitting of myosin head atomic structure into J motif shows how the two heads (yellow and green outline) point back towards the tail (red) and interact with each other. From Woodhead et al., 2005.
Head-head interaction is a common motif across the animal kingdom
The head-head interaction that appears to switch off activity in tarantula myosin filaments is very similar to that in isolated molecules from vertebrate smooth muscle. The evolutionary distance between these muscles and species suggests that this is a common motif for switching off contraction. We are testing this in two ways. Comparison of isolated molecules from several species, including vertebrates and invertebrates and nonmuscle myosin, shows that the same motif can be found with all, although its prevalence varies (less common in vertebrate; Jung et al., 2008). We have also reconstructed other filaments, and find the same motif in all species so far studied, including Limulus (the horseshoe crab; Zhao et al., 2009), scallop, and vertebrate filaments (see below). The conservation of this motif implies that it arose early in evolution and suggests that it is crucial to the ability of muscles to relax. We are currently carrying out mutational studies to determine which residues of the head are required for the formation of this interaction.
Limulus thick filament reconstruction (left) with atomic fit of heads (middle); from Zhao et al., 2009. Molecule images (with interpretation in terms of interacting heads (pink and green)) are from Limulus, tarantula and nonmuscle myosin (left, top to bottom) and vertebrate cardiac and skeletal muscle (right, top and bottom; from Jung et al., 2008.