Roger Craig

The structural basis of thin filament-linked regulation of muscle contraction is not yet understood. Here we have used electron microscopy and three-dimensional image reconstruction to observe the effects of Ca2+ and myosin head binding on thin filament structure, especially on the position of tropomyosin. Thin filaments isolated in EGTA were treated with Ca2+ or myosin heads (S-1) and negatively stained. Tropomyosin strands were directly visualized in electron micrographs, and distinct EGTA, Ca2+ and S-1-dependent positions were apparent in reconstructions. By fitting reconstructions to the atomic model of F-actin, clusters of amino acids on actin lying beneath tropomyosin were defined under each set of conditions. In the presence of Ca2+, tropomyosin moved 25 degrees away from its low Ca2+ position, exposing most, but not all, of the previously blocked myosin-binding sites. Saturation of filaments with myosin heads produced a further 10 degrees shift in tropomyosin position, thereby exposing the entire myosin-binding site. Our results thus suggest that full switching-on of thin filaments by reversal of steric-blocking requires both Ca2+ and the binding of myosin heads, acting in sequence. By using filaments which were partially decorated with heads, tropomyosin movement was shown to be cooperative, and the size of the actin-tropomyosin cooperative unit was estimated directly. Our results provide direct structural support for previous models of thin filament activation based on kinetics of actin-myosin interaction.

Contraction of the heart results from interaction of the myosin and actin filaments. Cardiac myosin filaments consist of the molecular motor myosin II, the sarcomeric template protein, titin, and the cardiac modulatory protein, myosin binding protein C (MyBP-C). Inherited hypertrophic cardiomyopathy (HCM) is a disease caused mainly by mutations in these proteins. The structure of cardiac myosin filaments and the alterations caused by HCM mutations are unknown. We have used electron microscopy and image analysis to determine the three-dimensional structure of myosin filaments from wild-type mouse cardiac muscle and from a MyBP-C knockout model for HCM. Three-dimensional reconstruction of the wild-type filament reveals the conformation of the myosin heads and the organization of titin and MyBP-C at 4 nm resolution. Myosin heads appear to interact with each other intramolecularly, as in off-state smooth muscle myosin [Wendt T, Taylor D, Trybus KM, Taylor K (2001) Proc Natl Acad Sci USA 98:4361-4366], suggesting that all relaxed muscle myosin IIs may adopt this conformation. Titin domains run in an elongated strand along the filament surface, where they appear to interact with part of MyBP-C and with the myosin backbone. In the knockout filament, some of the myosin head interactions are disrupted, suggesting that MyBP-C is important for normal relaxation of the filament. These observations provide key insights into the role of the myosin filament in cardiac contraction, assembly, and disease. The techniques we have developed should be useful in studying the structural basis of other myosin-related HCM diseases.