Blebbistatin Stabilizes the Helical Order of Myosin Filaments by Promoting the Switch 2 Closed State

Zhao, Fa-Qing; Padrón, Raúl; Craig, Roger

doi:10.1529/biophysj.108.137067

Cited by 49 publications

(54 citation statements)

References 57 publications

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“…1) (7). Our findings on the blebbistatininduced conformational changes in M.ADP are in line with previous x-ray diffraction results on muscle thick filaments and overstretched fibers showing helical ordering of myosin heads in similar conditions (14,15).…”

supporting

confidence: 93%

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

Takács

Billington

Gyimesi

et al. 2010

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

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The powerstroke of the myosin motor is the basis of cell division and bodily movement, but has eluded empirical description due to the short lifetime and low abundance of intermediates during force generation. To gain insight into this process, we used wellestablished single-tryptophan and pyrene fluorescent sensors and electron microscopy to characterize the structural and kinetic properties of myosin complexed with ADP and blebbistatin, a widely used inhibitor. We found that blebbistatin does not weaken the tight actin binding of myosin.ADP, but unexpectedly it induces lever priming, a process for which the gamma-phosphate of ATP (or its analog) had been thought necessary. The results indicate that a significant fraction of the myosin.ADP.blebbistatin complex populates a previously inaccessible conformation of myosin resembling the start of the powerstroke.actomyosin | ATPase | kinetics | molecular motors | structure M yosin produces force during cyclical interaction with actin and ATP. We and others have shown that blebbistatin, a potent and selective small-molecule inhibitor of myosin 2 (1), inhibits myosin ATPase and motile activity by blocking myosin (M) in a complex with ATP hydrolysis products (M:ADP:P i ) and slowing down phosphate (P i ) release (Fig. 1) (2, 3). This feature confers the great advantage that the inhibitor blocks myosin in a weak actin-binding state and, thus, the use of blebbistatin is not associated with adverse effects that could result from inhibitorinduced actin-myosin crosslinking within the cell. Blebbistatin (B) exerts its inhibitory effect by binding deep within the actinbinding cleft of the motor (catalytic) domain of myosin, between the nucleotide and actin-binding sites (Fig. 2) (3, 4). The atomic structure of the M:ADP:V i :B complex [in which vanadate (V i ) is a P i analog] showed that the inhibitor causes only local conformational changes in this state, which is a well-characterized intermediate in the chemomechanical cycle of myosin (4). Myosin adopts this state after it detaches from actin in response to ATP binding, and hydrolyzes ATP while still detached from actin (Fig.

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supporting

confidence: 93%

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

Takács

Billington

Gyimesi

et al. 2010

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

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“…46 Blebbistatin, which inhibits myosin II ATPase activity, 67 stabilizes helical tracks in a relaxed state by promoting a closed state. 68 These two results suggest that in tarantula blebbistatin should closely hold the two heads on IHMs, diminishing the free heads that sway away and implying a very slow rate. In fact, blebbistatin stabilizes the SRX state in skeletal muscle, producing a very long-lived myosin–nucleotide complex with a very slow time constant, 65 which favors our interpretation of the very slow rate in tarantulas.…”

Section: Discussionmentioning

confidence: 94%

Conserved Intramolecular Interactions Maintain Myosin Interacting-Heads Motifs Explaining Tarantula Muscle Super-Relaxed State Structural Basis

Álamo

Wriggers

et al. 2016

Journal of Molecular Biology

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157

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Tarantula striated muscle is an outstanding system for understanding the molecular organization of myosin filaments. 3D reconstruction based on cryo-EM images and single-particle image processing revealed that in a relaxed state, myosin molecules undergo intramolecular head–head interactions, explaining why head activity switches off. The filament model obtained by rigidly docking a chicken smooth muscle myosin structure to the reconstruction was improved by flexibly fitting an atomic model built by mixing structures from different species to a tilt-corrected 2-nm 3D map of frozen-hydrated tarantula thick filament. We used heavy and light chain sequences from tarantula myosin to build a single-species homology model of two heavy meromyosin interacting-heads motifs (IHMs). The flexibly fitted model includes previously missing loops and shows five intramolecular and five intermolecular interactions that keep the IHM in a compact off structure, forming four helical tracks of IHMs around the backbone. The residues involved in these interactions are oppositely charged, and their sequence conservation suggests that IHM is present across animal species. The new model, PDB 3JBH, explains the structural origin of the ATP turnover rates detected in relaxed tarantula muscle by ascribing the very slow rate to docked unphosphorylated heads, the slow rate to phosphorylated docked heads, and the fast rate to phosphorylated undocked heads. The conservation of intramolecular interactions across animal species and presence of IHM in bilaterians suggest that a super-relaxed state should be maintained, as it plays a role in saving ATP in skeletal, cardiac, and smooth muscle.

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“…To further test this conclusion, we added 25 μM Blebbistatin, which stabilizes the OFF state of the thick filament722, to the relaxing solution at 25 °C in the presence of 5% Dextran (Fig. 2, green).…”

Section: Resultsmentioning

confidence: 99%

Thick filament mechano-sensing is a calcium-independent regulatory mechanism in skeletal muscle

et al. 2016

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Recent X-ray diffraction studies on actively contracting fibres from skeletal muscle showed that the number of myosin motors available to interact with actin-containing thin filaments is controlled by the stress in the myosin-containing thick filaments. Those results suggested that thick filament mechano-sensing might constitute a novel regulatory mechanism in striated muscles that acts independently of the well-known thin filament-mediated calcium signalling pathway. Here we test that hypothesis using probes attached to the myosin regulatory light chain in demembranated muscle fibres. We show that both the extent and kinetics of thick filament activation depend on thick filament stress but are independent of intracellular calcium concentration in the physiological range. These results establish direct control of myosin motors by thick filament mechano-sensing as a general regulatory mechanism in skeletal muscle that is independent of the canonical calcium signalling pathway.

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Blebbistatin Stabilizes the Helical Order of Myosin Filaments by Promoting the Switch 2 Closed State

Cited by 49 publications

References 57 publications

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

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

Conserved Intramolecular Interactions Maintain Myosin Interacting-Heads Motifs Explaining Tarantula Muscle Super-Relaxed State Structural Basis

Thick filament mechano-sensing is a calcium-independent regulatory mechanism in skeletal muscle

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