Myosin complexed with ADP and blebbistatin reversibly adopts a conformation resembling the start point of the working stroke

Takács, Balázs; Billington, Neil; Gyimesi, Máté; Kintses, Bálint; Málnási‐Csizmadia, András; Knight, Peter J.; Kovács, Mihály

doi:10.1073/pnas.0907585107

Cited by 43 publications

(51 citation statements)

References 32 publications

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“…Crystal structures of myosin bound by blebbistatin with ADP and vanadate (26) or ADP and vanadate without blebbistatin (27) show switch-2 in a closed conformation. Furthermore, spectroscopic studies suggest that blebbistatin (28) and vanadate (13) stabilize the closed state of switch-2 in the presence of ADP without P i or P i analogs. Thus, switch-2 is likely predominantly closed in the presence of blebbistatin and vanadate, and because actin-induced LCD rotation is inhibited by both, we infer that the power stroke requires acceleration of switch-2 opening just as predicted by previous studies (5,14) and by crystallographic models (4,16).…”

Section: Discussionmentioning

confidence: 99%

Direct real-time detection of the structural and biochemical events in the myosin power stroke

Muretta

Rohde

Johnsrud

et al. 2015

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

156

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A principal goal of molecular biophysics is to show how protein structural transitions explain physiology. We have developed a strategic tool, transient time-resolved FRET [(TR) 2 FRET], for this purpose and use it here to measure directly, with millisecond resolution, the structural and biochemical kinetics of muscle myosin and to determine directly how myosin's power stroke is coupled to the thermodynamic drive for force generation, actin-activated phosphate release, and the weak-to-strong actin-binding transition. We find that actin initiates the power stroke before phosphate dissociation and not after, as many models propose. This result supports a model for muscle contraction in which power output and efficiency are tuned by the distribution of myosin structural states. This technology should have wide application to other systems in which questions about the temporal coupling of allosteric structural and biochemical transitions remain unanswered.FRET | myosin | power stroke | phosphate release | structural kinetics M yosin family proteins use ATP hydrolysis to generate force and movement required for normal physiology. They drive muscle contraction, help control cell division and cellular motility, move organelles through the cytoplasm, and are important elements of the cellular mechanical-sensing machinery (1, 2). The key to understanding how myosin and related enzymes function in cells, and how to modulate their activity to treat disease, is to determine how the protein's structural dynamics and biochemical kinetics are coupled. Although high-resolution crystal structures provide best-guess snapshots of protein structure over a range of biochemical states, determining the physiological relevance of these snapshots remains one of the central challenges of structural biophysics.How myosin generates force remains debated despite more than 50 y of intense research (1-3). The most popular current model (2, 4) proposes that after ATP hydrolysis, myosin interacts weakly with actin and this interaction initiates an ordered series of structural and biochemical transitions that culminate in the dissociation of hydrolyzed phosphate, followed by the isomerization of the actin-binding interface to a state that binds actin with nanomolar affinity and then the rotation of the myosin lightchain domain (LCD) toward the plus end of the actin filament. This rotation converts the thermodynamic energy of phosphate release and actin binding into mechanical energy that performs work. A number of results question this model, however, including spectroscopic data showing that a structural transition in the myosin relay helix, hypothesized to be coupled to LCD rotation, precedes P i release (5) and force development precedes P i release in muscle fibers (6).Determining how these events take place in solution and in cells is an important question, because (i) differences in the mechanics of different myosins likely reflect differences in how the biochemical and structural transitions described above are coordinated (4); (ii) disea...

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Section: Discussionmentioning

confidence: 99%

Direct real-time detection of the structural and biochemical events in the myosin power stroke

Muretta

Rohde

Johnsrud

et al. 2015

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

156

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“…2,3-butanedione monoxime (BDM), N -benzyl-p-toluenesulphonamide (BTS) and blebbistatin are inhibitors of class-2 myosins [193–198]. The inhibitory effect of the low-affinity compound BDM is skeletal-muscle myosin-2 specific (IC 50 ~5 mM) and associated with the allosteric inhibition of P i release [197].…”

Section: Regulation By Small Moleculesmentioning

confidence: 99%

“…2). The M·ADP·blebbistatin complex has a very low actin affinity and adopts a structural state that resembles the starting point of the powerstroke [193, 196, 200]. Structural studies show the blebbistatin binding site in the apex of the myosin cleft [201].…”

Section: Regulation By Small Moleculesmentioning

confidence: 99%

Various Themes of Myosin Regulation

Heissler

Sellers

2016

Journal of Molecular Biology

117

129

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Members of the myosin superfamily are actin-based molecular motors that are indispensable for cellular homeostasis. The vast functional and structural diversity of myosins accounts for the variety and complexity of the underlying allosteric regulatory mechanisms that determine the activation or inhibition of myosin motor activity and enable precise timing and spatial aspects of myosin function at the cellular level. This review focuses on the molecular basis of posttranslational regulation of eukaryotic myosins from different classes across species by allosteric intrinsic and extrinsic effectors. First, we highlight the impact of heavy and light chain phosphorylation. Second, we outline intramolecular regulatory mechanisms such as autoinhibition and subsequent activation. Third, we discuss diverse extramolecular allosteric mechanisms ranging from actin-linked regulatory mechanisms to myosin:cargo interactions. At last, we briefly outline the allosteric regulation of myosins with synthetic compounds.

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“…For example, crosslinking of myosin with azidoblebbistatin in different nucleotide-and actin-bound states may stabilize important intermediates of force generation. Such a possibility is substantiated by our recent finding that the complex of myosin with ADP and blebbistatin adopts a previously inaccessible structural state resembling the start point of the powerstroke (13). In this as well as many other cases, the relatively low affinity of the ligands for the enzyme is the most severe technical hurdle in the way of detailed structural characterization, which can be overcome by covalent crosslinking to azidoblebbistatin.…”

Section: Discussionmentioning

confidence: 96%

“…Furthermore, we recently showed that myosin populates a previously inaccessible conformational state when bound to ADP and blebbistatin. This conformational state, characterized by a primed lever and high actin affinity, resembles the start point of the powerstroke (13).…”

mentioning

confidence: 91%

Azidoblebbistatin, a photoreactive myosin inhibitor

Képiró¹,

Várkuti²,

Bodor³

et al. 2012

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

Self Cite

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Photoreactive compounds are important tools in life sciences that allow precisely timed covalent crosslinking of ligands and targets. Using a unique technique we have synthesized azidoblebbistatin, which is a derivative of blebbistatin, the most widely used myosin inhibitor. Without UV irradiation azidoblebbistatin exhibits identical inhibitory properties to those of blebbistatin. Using UV irradiation, azidoblebbistatin can be covalently crosslinked to myosin, which greatly enhances its in vitro and in vivo effectiveness. Photo-crosslinking also eliminates limitations associated with the relatively low myosin affinity and water solubility of blebbistatin. The wavelength used for photo-crosslinking is not toxic for cells and tissues, which confers a great advantage in in vivo tests. Because the crosslink results in an irreversible association of the inhibitor to myosin and the irradiation eliminates the residual activity of unbound inhibitor molecules, azidoblebbistatin has a great potential to become a highly effective tool in both structural studies of actomyosin contractility and the investigation of cellular and physiological functions of myosin II. We used azidoblebbistatin to identify previously unknown low-affinity targets of the inhibitor (EC 50 ≥ 50 μM) in Dictyostelium discoideum, while the strongest interactant was found to be myosin II (EC 50 = 5 μM). Our results demonstrate that azidoblebbistatin, and potentially other azidated drugs, can become highly useful tools for the identification of strong-and weak-binding cellular targets and the determination of the apparent binding affinities in in vivo conditions. interactom profile | azidation | photoactivation V arious biological processes, including muscle contraction, cell migration, differentiation, and cytokinesis require the activity of myosin II, a class of actin-based ATP-driven motor proteins. Cell-permeable small molecules that can perturb myosin functions have greatly aided the dissection and understanding of molecular processes underlying the above phenomena. Myosin II inhibitors described to date include 2,3-butanedione monoxime (BDM) (1, 2), N-benzyl-p-toluenesulphonamide (BTS) (3), and blebbistatin (4-6). As BDM has turned out to have a broad effect on many other proteins (7) and the inhibitory effect of BTS is limited to fast skeletal muscle myosin, until now blebbistatin has been the only potent tool for specific blocking of myosin II-dependent processes in various species and cell types. Recent data have shown that halogenated pseudilins also have nonspecific inhibitory effects on myosin II isoforms (8-10).In vitro, blebbistatin inhibits vertebrate striated muscle and nonmuscle myosin II isoforms as well as Dictyostelium discoideum (Dd) myosin II by about 95%, with an IC 50 of 0.5-5 μM (11). Vertebrate smooth muscle and Acanthamoeba myosin II are incompletely inhibited even at high blebbistatin concentrations. In vivo experiments performed with Dd showed that the effective inhibition of myosin II-dependent processes, including growth in...

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Myosin complexed with ADP and blebbistatin reversibly adopts a conformation resembling the start point of the working stroke

Cited by 43 publications

References 32 publications

Direct real-time detection of the structural and biochemical events in the myosin power stroke

Direct real-time detection of the structural and biochemical events in the myosin power stroke

Various Themes of Myosin Regulation

Azidoblebbistatin, a photoreactive myosin inhibitor

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