Characterization of mutant myosins of Dictyostelium discoideum equivalent to human familial hypertrophic cardiomyopathy mutants. Molecular force level of mutant myosins may have a prognostic implication.

Fujita, Hideo; Sugiura, Seiryo; Momomura, Shin‐ichi; Omata, Masao; Sugi, Haruo; Sutoh, Kazuo

doi:10.1172/jci119228

Cited by 70 publications

(44 citation statements)

References 44 publications

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“…For example, maximum actin-activated ATPase activity in solution, which is thought to correspond to k redev in muscle fibers (25), was found to increase (42) or to decrease (41), whereas in our fiber measurements k redev was not affected by the mutation. Force measured with a laser trap and in vitro sliding velocity were both reduced in the Dictyostelium myosin (41), but were unchanged when the equivalent mutation was introduced in smooth muscle myosin (42).…”

Section: Relation To Other Studies On Mutations In the Converter Domaincontrasting

confidence: 67%

Mutation of the myosin converter domain alters cross-bridge elasticity

Köhler

Winkler²,

Schulte³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Elastic distortion of a structural element of the actomyosin complex is fundamental to the ability of myosin to generate motile forces. An elastic element allows strain to develop within the actomyosin complex (cross-bridge) before movement. Relief of this strain then drives filament sliding, or more generally, movement of a cargo. Even with the known crystal structure of the myosin head, however, the structural element of the actomyosin complex in which elastic distortion occurs remained unclear. To assign functional relevance to various structural elements of the myosin head, e.g., to identify the elastic element within the cross-bridge, we studied mechanical properties of muscle fibers from patients with familial hypertrophic cardiomyopathy with point mutations in the head domain of the ␤-myosin heavy chain. We found that the Arg-719 3 Trp (Arg719Trp) mutation, which is located in the converter domain of the myosin head fragment, causes an increase in force generation and fiber stiffness under isometric conditions by 48 -59%. Under rigor and relaxing conditions, fiber stiffness was 45-47% higher than in control fibers. Yet, kinetics of active cross-bridge cycling were unchanged. These findings, especially the increase in fiber stiffness under rigor conditions, indicate that cross-bridges with the Arg719Trp mutation are more resistant to elastic distortion. The data presented here strongly suggest that the converter domain that forms the junction between the catalytic and the light-chain-binding domain of the myosin head is not only essential for elastic distortion of the cross-bridge, but that the main elastic distortion may even occur within the converter domain itself. It is widely accepted that active force and movement generated by muscle fibers result from structural changes in the head domain of the myosin molecule (the cross-bridge) while it is attached to the actin filament. These changes are thought to involve a tilting of the light-chain-binding domain of the myosin head relative to its catalytic domain (1-7). As a result of such structural changes distortion of an elastic element within the actin-attached myosin head allows strain to develop before movement (8). Relief of this strain drives sliding of actin filaments past the myosin filaments, or alternatively, if filament sliding is prevented, active force is generated because of continued strain of the elastic element. Despite the central significance of this concept, however, it remained unclear which part of the actomyosin complex represents the elastic element-i.e., the element that experiences the main elastic distortion while other parts act more like rigid bodies. Neither the known crystal structures of the myosin head nor cryo-electron microscopy with reconstruction of the actomyosin complex have resolved this question. Some authors considered elastic bending of the long, light-chain-binding ␣-helix an obvious candidate (4, 9, 10). The actin-myosin interface (11) or the junction between the lightchain domain and the catalytic domain of the...

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Section: Relation To Other Studies On Mutations In the Converter Domaincontrasting

confidence: 67%

Mutation of the myosin converter domain alters cross-bridge elasticity

Köhler

Winkler²,

Schulte³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…The myopathy loop is a structured surface loop containing an unusual clustering of heart disease-implicated mutations, implying a functional significance for the loop (20,21,59). The myopathy loop is probably an actin binding site (13, 16); however, the role of its disease-implicated point mutations in motor functions remains unclear (20,21,25,60). The C-loop is the second structured surface loop to be implicated in cardiac myosin motor function.…”

Section: Discussionmentioning

confidence: 99%

“…The fundamental assumption of myosin structure/function research is that the functional significance of key structural elements of myosin are preserved across myosin isoforms, suggesting that chimeric substitutions into dicty or smooth muscle myosin hosts are useful model systems when expression for the original myosin (in this case ␤S1) is problematic (19,25,63). We apply the fundamental assumption to the C-loop and the smooth muscle myosin isoform model, although we acknowledge that in some cases mutagenized ␤S1 behaves unlike its mutagenized model (20,21,25). Recently, various arguments have been advanced to rationalize these differences while maintaining the validity of the fundamental assumption for the myopathy loop (60).…”

Section: Discussionmentioning

confidence: 99%

“…In acto-S1, S1 contacts two adjacent actin monomers in a filament (12). Protein-protein contacts in actomyosin were identified by experimental structural studies combined with simulated docking of the myosin and actin crystal structures (13)(14)(15)(16) and by detecting changes in actin binding strength, actin-activated myosin ATPase, and in vitro motility caused by the mutation of small peptide segments or individual residues in myosin (17)(18)(19)(20)(21)(22)(23)(24)(25). Primary actin contacts on S1, aa 529 -558 2 and aa 647-659, are helices maintaining hydrophobic interactions with actin.…”

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confidence: 99%

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The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction

Ajtai

Garamszegi

Watanabe

et al. 2004

Journal of Biological Chemistry

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The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Mediating the actomyosin interaction in myosin is an actin binding site distributed among several peptides on the myosin surface including surface loops contributing to affinity and actin regulation of myosin ATPase. A structured surface loop on ␤-cardiac myosin, the cardiac or C-loop, was recently demonstrated to affect myosin ATPase and was indirectly implicated in the actomyosin interaction. The C-loop is a conserved feature of all myosin isoforms with crystal structures, suggesting that it is an essential part of the core energy transduction machinery. It is shown here that proteolytic digestion of the C-loop in ␤-cardiac myosin eliminates actin-activated myosin ATPase and reduces actomyosin affinity in rigor more than 100-fold. Studies of C-loop function in smooth muscle myosin were also undertaken using sitedirected mutagenesis. Mutagenesis of a single charged residue in the C-loop of smooth muscle myosin alters actomyosin affinity and doubles myosin in vitro motility and actin-activated ATPase velocities, thereby involving a charged region of the loop in the actomyosin interaction. It appears likely that the C-loop is an essential electrostatic binding site for actin involved in modulation of actomyosin affinity and regulation of actomyosin ATPase velocity.The motor protein myosin is an ATPase and an actin-binding protein transducing ATP free energy into work. In muscle, myosin, actin, and ATP constitute the unitary work producer. The cyclical interaction of these components, a sequence of states each characterized as a static relation between the proteins and an intermediate in the degradation of ATP, is a contraction cycle. In a contraction cycle, myosin hydrolyzes ATP in its active site and forms a weak association with actin at a separate actin binding site. Release of phosphate from the active site initiates strong binding to actin and a large conformation change in myosin that produces work in the form of actin translation against a load. Several solved crystal structures represent intermediates in ATP hydrolysis and define myosin conformation changes in the absence of actin (1-6). Presently, we lack a detailed understanding of actomyosin structure and in particular how the elements making up the actomyosin interface contribute to mutual affinity, actin regulation of phosphate release, and the ability of myosin-bound nucleotide to modulate actomyosin affinity.Myosin consists of a globular head domain containing the enzymatic portion of the molecule and a tail involved in filament assembly. The globular head separated from its tail portion is called subfragment 1 (S1). 1 S1 interacts with filamentous actin (F-actin) consisting of polymerized actin monomers. An atomic model for F-actin built from monomeric actin crystal structures (7-9) satisfies x-ray diffraction data restraints (10) and is consistent with electron microscopic imagery (11). In...

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“…Secondary sites are an unstructured surface loop (aa 567-578) and the structured myopathy loop (aa 403-416) present on the S1 surface [36]. Myopathy loop is a structured surface loop containing an unusual clustering of heart disease-implicated mutations, implying a functional significance for the loop [37][38][39]. The myopathy loop is probably an actin binding site however, the role of its disease-implicated point mutations in motor functions remains unclear.…”

Section: Actomyosin Interfacementioning

confidence: 99%

Fluorescence labeling and computational analysis of the strut of myosin’s 50 kDa cleft

Gawalapu¹,

Root²

2006

Archives of Biochemistry and Biophysics

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Gawalapu, Ravi Kumar. Fluorescence labeling and computational analysis of the strut of myosin's 50 kDa cleft. Doctor of Philosophy (Biochemistry), August 2007, 148 pp., 3 tables, 60 illustrations, 120 references, 120 titles.In order to understand the structural changes in myosin S1, fluorescence polarization and computational dynamics simulations were used. Dynamics simulations on the S1 motor domain indicated that significant flexibility was present throughout the molecular model. The constrained opening versus closing of the 50 kDa cleft appeared to induce opposite directions of movement in the lever arm. A sequence called the "strut" which traverses the 50 kDa cleft and may play an important role in positioning the actomyosin binding interface during actin binding is thought to be intimately linked to distant structural changes in the myosin's nucleotide cleft and neck regions. To study the dynamics of the strut region, a method of fluorescent labeling of the strut was discovered using the dye CY3. CY3 served as a hydrophobic tag for purification by hydrophobic interaction chromatography which enabled the separation of labeled and unlabeled species of S1 including a fraction labeled specifically at the strut sequence. The high specificity of labeling was verified by proteolytic digestions, gel electrophoresis, and mass spectroscopy.Analysis of the labeled S1 by collisional quenching, fluorescence polarization, and actinactivated ATPase activity were consistent with predictions from structural models of the probe's location. Although the fluorescent intensity of the CY3 was insensitive to actin binding, its fluorescence polarization was notably affected. Intriguingly, the mobility of the probe increases upon S1 binding to actin suggesting that the CY3 becomes displaced from interactions with the surface of S1 and is consistent with a structural change in the strut due to cleft motions. Labeling the strut reduced the affinity of S1 for actin but did not prevent actin-activated ATPase activity which makes it a potentially useful probe of the actomyosin interface. The different conformations of myosin S1 indicated that the strut is not as flexible as several other key regions of myosin as determined by the application of force constraints to elastic portions of the myosin

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Characterization of mutant myosins of Dictyostelium discoideum equivalent to human familial hypertrophic cardiomyopathy mutants. Molecular force level of mutant myosins may have a prognostic implication.

Cited by 70 publications

References 44 publications

Mutation of the myosin converter domain alters cross-bridge elasticity

Mutation of the myosin converter domain alters cross-bridge elasticity

The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction

Fluorescence labeling and computational analysis of the strut of myosin’s 50 kDa cleft

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