Three-Dimensional Reconstruction of Tarantula Myosin Filaments Suggests How Phosphorylation May Regulate Myosin Activity

Departamento de Biología Estructural, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela.
Journal of Molecular Biology (Impact Factor: 4.33). 11/2008; 384(4):780-97. DOI: 10.1016/j.jmb.2008.10.013
Source: PubMed

ABSTRACT Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLCs). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the free head and the blocked head. Three-dimensional reconstruction of frozen-hydrated specimens revealed that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal in this study was to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction revealed intra- and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC and fitted an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 (subfragment 2) crystal structure to the reconstruction. The fitting suggests one intramolecular interaction, between the cardiomyopathy loop of the free head and its own S2, and two intermolecular interactions, between the cardiac loop of the free head and the essential light chain of the blocked head and between the Leu305-Gln327 interaction loop of the free head and the N-terminal fragment of the RLC of the blocked head. These interactions, added to those previously described, would help switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament.

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    • "Recently AL-Khayat et al.24 determined, for the first time, the 3D reconstruction of relaxed myosin filaments from human heart muscle by isolating the myosin filaments under relaxing conditions, performing negatively stained TEM and then applying single particle 3D image analysis (Figures 15, 17 & 19). It was found that the myosin heads densities on the reconstructed crown levels could be fitted quite well using the atomic models of the myosin heads crystal structures (Figure 19).34,74 It was also possible to define densities attributed to the domains of both titin and MyBP-C (Figure 17). "
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    ABSTRACT: High resolution information about the three-dimensional (3D) structure of myosin filaments has always been hard to obtain. Solving the 3D structure of myosin filaments is very important because mutations in human cardiac muscle myosin and its associated proteins (e.g. titin and myosin binding protein C) are known to be associated with a number of familial human cardiomyopathies (e.g. hypertrophic cardiomyopathy and dilated cardiomyopathy). In order to understand how normal heart muscle works and how it fails, as well as the effects of the known mutations on muscle contractility, it is essential to properly understand myosin filament 3D structure and properties in both healthy and diseased hearts. The aim of this review is firstly to provide a general overview of the 3D structure of myosin thick filaments, as studied so far in both vertebrates and invertebrate striated muscles. Knowledge of this 3D structure is the starting point from which myosin filaments isolated from human cardiomyopathic samples, with known mutations in either myosin or its associated proteins (titin or C-protein), can be studied in detail. This should, in turn, enable us to relate the structure of myosin thick filament to its function and to understanding the disease process. A long term objective of this research would be to assist the design of possible therapeutic solutions to genetic myosin-related human cardiomyopathies.
    11/2013; 2013(3):280-302. DOI:10.5339/gcsp.2013.36
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    • "The last few years have seen significant advances in structural studies of thick myosin filaments from various types of muscles under resting conditions by (cryo-)electron microscopy (cryoEM) and three-dimensional (3D)-image reconstruction using single particle approaches [1], [2]. Using an atomic structure of myosin molecule, these studies have revealed the structure of thick filaments to nanometer-scale resolution, suggesting that interactions between myosin heads resulting in the formation of a so-called “interacting head motif” are responsible for switching off the myosin molecules in vertebrate smooth muscles [3], invertebrate striated muscles with phosphorylation-dependent regulation such as tarantula [4], [5] and limulus [6] muscles and in scallop muscles [7] with Ca2+-dependent (dual) regulation. Recent studies showed that similar head-head interactions of myosin crossbridges occurred in isolated thick filaments from vertebrate fish skeletal [8] and cardiac striated [9], [10] muscles and also in heavy meromyosin (HMM) molecules (comprising of the two heads and part of the rod) from vertebrate cardiac and skeletal muscles when they were treated with blebbistatin, a known inhibitor of actin-binding and ATPase (catalysis of the hydrolysis of adenosine triphosphate (ATP)) activity of myosin molecules, although those muscles are not thought to be intrinsically regulated by the myosin molecules [11]. "
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    ABSTRACT: The intensities of the myosin-based layer lines in the x-ray diffraction patterns from live resting frog skeletal muscles with full thick-thin filament overlap from which partial lattice sampling effects had been removed were analyzed to elucidate the configurations of myosin crossbridges around the thick filament backbone to nanometer resolution. The repeat of myosin binding protein C (C-protein) molecules on the thick filaments was determined to be 45.33 nm, slightly longer than that of myosin crossbridges. With the inclusion of structural information for C-proteins and a pre-powerstroke head shape, modeling in terms of a mixed population of regular and perturbed regions of myosin crown repeats along the filament revealed that the myosin filament had azimuthal perturbations of crossbridges in addition to axial perturbations in the perturbed region, producing pseudo-six-fold rotational symmetry in the structure projected down the filament axis. Myosin crossbridges had a different organization about the filament axis in each of the regular and perturbed regions. In the regular region that lacks C-proteins, there were inter-molecular interactions between the myosin heads in axially adjacent crown levels. In the perturbed region that contains C-proteins, in addition to inter-molecular interactions between the myosin heads in the closest adjacent crown levels, there were also intra-molecular interactions between the paired heads on the same crown level. Common features of the interactions in both regions were interactions between a portion of the 50-kDa-domain and part of the converter domain of the myosin heads, similar to those found in the phosphorylation-regulated invertebrate myosin. These interactions are primarily electrostatic and the converter domain is responsible for the head-head interactions. Thus multiple head-head interactions of myosin crossbridges also characterize the switched-off state and have an important role in the regulation or other functions of myosin in thin filament-regulated muscles as well as in the thick filament-regulated muscles.
    PLoS ONE 12/2012; 7(12):e52421. DOI:10.1371/journal.pone.0052421 · 3.23 Impact Factor
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    • "We expect similar structural states and relationships as with single-headed S1, but perhaps due to steric constraints of the head–head interactions of the auto-inhibited HMM (Wendt et al. 2001; Nelson et al. 2005) or interactions between neighboring RLCs (Wahlstrom et al. 2003), slightly shorter distances may result due to confinement in the unphosphorylated RLC. Unphosphorylated myosin is predicted to be compact, with its two heads asymmetric with neighboring RLCs in very close proximity (\4 nm) (Wendt et al. 2001; Li and Ikebe 2003; Alamo et al. 2008). "
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    ABSTRACT: We have used site-directed spectroscopic probes to detect structural changes, motions, and interactions due to phosphorylation of proteins involved in the regulation of muscle contraction and relaxation. Protein crystal structures provide static snapshots that provide clues to the conformations that are sampled dynamically by proteins in the cellular environment. Our site-directed spectroscopic experiments, combined with computational simulations, extend these studies into functional assemblies in solution, and reveal details of protein regions that are too dynamic or disordered for crystallographic approaches. Here, we discuss phosphorylation-mediated structural transitions in the smooth muscle myosin regulatory light chain, the striated muscle accessory protein myosin binding protein-C, and the cardiac membrane Ca(2+) pump modulator phospholamban. In each of these systems, phosphorylation near the N terminus of the regulatory protein relieves an inhibitory interaction between the phosphoprotein and its regulatory target. Several additional unifying themes emerge from our studies: (a) The effect of phosphorylation is not to change the affinity of the phosphoprotein for its regulated binding partner, but to change the structure of the bound complex without dissociation. (b) Phosphorylation induces transitions between order and dynamic disorder. (c) Structural states are only loosely coupled to phosphorylation; i.e., complete phosphorylation induces dramatic functional effects with only a partial shift in the equilibrium between ordered and disordered structural states. These studies, which offer atomic-resolution insight into the structural and functional dynamics of these phosphoproteins, were inspired in part by the ground-breaking work in this field by Michael and Kate Barany.
    Journal of Muscle Research and Cell Motility 08/2012; 33(6). DOI:10.1007/s10974-012-9317-6 · 1.93 Impact Factor
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