Three-dimensional structure of vertebrate cardiac muscle filaments

Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2008; 105(7):2386-90. DOI: 10.1073/pnas.0708912105
Source: PubMed

ABSTRACT

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.

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    • "In relaxed thick filaments, pairs of myosin heads are helically organized on the filament backbone, forming asymmetric ''J motifs " in which the two heads of each myosin molecule interact, inhibiting each other's activity. This motif is conserved across multiple vertebrate and invertebrate species (Woodhead et al., 2005; Zhao et al., 2009; Pinto et al., 2012; Woodhead et al., 2013; Zoghbi et al., 2008). In the tarantula, these motifs are axially separated by 145 Å (i.e. "
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    ABSTRACT: Single particle analysis is widely used for three-dimensional reconstruction of helical filaments. Near-atomic resolution has been obtained for several well-ordered filaments. However, it is still a challenge to achieve high resolution for filaments with flexible subunits and a large axial rise per subunit relative to pixel size. Here, we describe an approach that improves the resolution in such cases. In filaments with a large axial rise, many segments must be shifted a long distance along the filament axis to match with a reference projection, potentially causing loss of alignment accuracy and hence resolution. In our study of myosin filaments, we overcame this problem by pre-determining the axial positions of myosin head crowns within segments to decrease the alignment error. In addition, homogeneous, well-ordered segments were selected from the raw data set by checking the assigned azimuthal rotation angle of segments in each filament against those expected for perfect helical symmetry. These procedures improved the resolution of the filament reconstruction from 30 Å to 13 Å. This approach could be useful in other helical filaments with a large axial rise and/or flexible subunits.
    No preview · Article · Nov 2015 · Journal of Structural Biology
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    • "Hence, MyBP-C appears essential for normal cardiac functioning. In cardiac thick filaments from knockout mice, the interaction between some of the myosin heads was disrupted [32] [38] and the intensity ratio of the X-ray equatorial reflections suggested that the myosin heads lay further out from the thick filament backbone [40]. Thus, one function of MyBP-C might be to interact with myosin heads near the head–tail junction and tether them closer to the backbone. "

    Preview · Article · Nov 2014 · Journal of Molecular Biology
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    • "Much of the biochemistry can be explained by an asymmetric, intramolecular interaction between the two-myosin heads, first visualized by cryoelectron microscopy (cryoEM) of 2-D arrays of dephosphorylated smHMM (Wendt et al., 2001). Subsequently, this motif was also identified in thick filaments from three striated muscles (Woodhead et al., 2005; Zhao et al., 2009; Zoghbi et al., 2008), as well as in electron micrographs of negatively stained single molecules of myosin II isoforms from several species (Burgess et al., 2007; Jung et al., 2008a). The near ubiquitous presence of this intramolecular myosin head–head interaction has led to the suggestion that it is both an ancient and general mechanism for myosin II inhibition (Jung et al., 2008a,b). "
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    ABSTRACT: The activity of smooth and non-muscle myosin II is regulated by phosphorylation of the regulatory light chain (RLC) at serine 19. The dephosphorylated state of full-length monomeric myosin is characterized by an asymmetric intramolecular head-head interaction that completely inhibits the ATPase activity, accompanied by a hairpin fold of the tail, which prevents filament assembly. Phosphorylation of serine 19 disrupts these head-head interactions by an unknown mechanism. Computational modeling (Tama et al., 2005. J. Mol. Biol.345, 837-854) suggested that formation of the inhibited state is characterized by both torsional and bending motions about the myosin heavy chain (HC) at a location between the RLC and the essential light chain (ELC). Therefore, altering relative motions between the ELC and the RLC at this locus might disrupt the inhibited state. Based on this hypothesis we have derived an atomic model for the phosphorylated state of the smooth muscle myosin light chain domain (LCD). This model predicts a set of specific interactions between the N-terminal residues of the RLC with both the myosin HC and the ELC. Site directed mutagenesis was used to show that interactions between the phosphorylated N-terminus of the RLC and helix-A of the ELC are required for phosphorylation to activate smooth muscle myosin.
    Full-text · Article · Dec 2013 · Journal of Structural Biology
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