Myosin cleft movement and its coupling to actomyosin dissociation

Conibear, Paul B.; Bagshaw, Clive R.; Fajer, Piotr G.; Kovács, Mihály; Málnási‐Csizmadia, András

doi:10.1038/nsb986

Cited by 84 publications

(97 citation statements)

References 27 publications

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“…Other studies, using fluorescent probes flanking the 50 kDa cleft (Yengo et al 2002;Conibear et al 2003) and high-resolution cryo-electron microscopic maps of myosindecorated actin (Holmes et al 2003), confirmed that the cleft closes tightly when myosin binds strongly to actin and that this closure opens switch I. Kinesin and GTP-binding proteins have switch I in this open position (Vale 1996), but the open position of switch I had not been observed previously in myosin crystal structures. A study of binding of ATP analogues with large groups in place of the c-P i , though, had predicted its appearance (Pate et al 1997).…”

Section: Resurrection Of Schemementioning

confidence: 83%

Coupling between phosphate release and force generation in muscle actomyosin

Takagi

Shuman

Goldman

2004

Phil. Trans. R. Soc. Lond. B

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Energetic, kinetic and oxygen exchange experiments in the mid-1980s and early 1990s suggested that phosphate (P i ) release from actomyosin-adenosine diphosphate P i (AMÁADPÁP i ) in muscle fibres is linked to force generation and that P i release is reversible. The transition leading to the force-generating state and subsequent P i release were hypothesized to be separate, but closely linked steps. P i shortens single force-generating actomyosin interactions in an isometric optical clamp only if the conditions enable them to last 20-40 ms, enough time for P i to dissociate. Until 2003, the available crystal forms of myosin suggested a rigid coupling between movement of switch II and tilting of the lever arm to generate force, but they did not explain the reciprocal affinity myosin has for actin and nucleotides. Newer crystal forms and other structural data suggest that closing of the actin-binding cleft opens switch I (presumably decreasing nucleotide affinity). These data are all consistent with the order of events suggested before: myosinÁADPÁP i binds weakly, then strongly to actin, generating force. Then P i dissociates, possibly further increasing force or sliding.

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Section: Resurrection Of Schemementioning

confidence: 83%

Coupling between phosphate release and force generation in muscle actomyosin

Takagi

Shuman

Goldman

2004

Phil. Trans. R. Soc. Lond. B

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“…Because the nucleotide-binding pocket is located at the interface between the N-terminal and 50-kDa upper subdomains, the rotation of the 50-kDa upper subdomain necessarily opens up the pocket and is believed to lead to the subsequent release of ADP (3,6,34). Conversely, in the ATPbound state, the opening of the 50-kDa cleft (during which the 50-kDa upper subdomain rotates toward the N-terminal subdomain) is accompanied by the closing of the nucleotide-binding pocket (36).…”

Section: A Connection Between the 50-kda Upper And N-terminal Subdomamentioning

confidence: 99%

Myosin subfragment 1 structures reveal a partially bound nucleotide and a complex salt bridge that helps couple nucleotide and actin binding

Risal

Gourinath

Himmel

et al. 2004

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

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Structural studies of myosin have indicated some of the conformational changes that occur in this protein during the contractile cycle, and we have now observed a conformational change in a bound nucleotide as well. The 3.1-Å x-ray structure of the scallop myosin head domain (subfragment 1) in the ADP-bound near-rigor state (lever arm Ϸ45°to the helical actin axis) shows the diphosphate moiety positioned on the surface of the nucleotide-binding pocket, rather than deep within it as had been observed previously. This conformation strongly suggests a specific mode of entry and exit of the nucleotide from the nucleotide-binding pocket through the so-called ''front door.'' In addition, using a variety of scallop structures, including a relatively high-resolution 2.75-Å nucleotide-free near-rigor structure, we have identified a conserved complex salt bridge connecting the 50-kDa upper and N-terminal subdomains. This salt bridge is present only in crystal structures of muscle myosin isoforms that exhibit a strong reciprocal relationship (also known as coupling) between actin and nucleotide affinity.M yosin is a motor protein that transduces ATP hydrolysis into mechanical work, leading to its translocation along filamentous actin. It is believed that the small conformational changes induced by enzymatic activity in the myosin ''motor domain'' (MD) are amplified by the motion of the ''lever arm.'' Three weak actin-binding (actin-detached) states have been identified crystallographically for scallop myosin subfragment 1 (S1) (1). These and other structures reveal that the motor function of myosin S1 is coordinated by three subdomains (50-kDa upper and lower subdomains and the converter) that rotate as rigid bodies around the relatively stable N-terminal subdomain (1-3). These rotations depend on conformational changes in three flexible joints (switch II, relay, and the SH1 helix) between the subdomains. Three-dimensional reconstructions of electron microscopic images of actin decorated by S1 of several myosin isoforms (4-6) have led to the suggestion that actin binds to portions of the 50-kDa upper and lower subdomains, whereas various crystal structures (see, for example, refs. 3 and 7-9) indicate that ATP binds at the opposite side of the myosin head in a pocket between the 50-kDa upper and Nterminal subdomains (Figs. 1 and 2A).One of the three structural conformations, the so-called ''prepower stroke,'' corresponds biochemically to the ADP⅐P i transition state (1,8,(10)(11)(12). Here, S1 displays a primed lever arm (Ϸ90°to the actin filament axis), closure of the 50-kDa cleft's base (which is near the nucleotide-binding pocket), and a bent switch II that interacts with both the nucleotide and switch I (a loop in the 50-kDa upper subdomain that coordinates the nucleotide). After this state, the tight binding of myosin to actin results in the so-called ''power stroke'' (after which the lever arm is Ϸ45°to the actin filament), a process believed to be initiated by the near-complete closing of the 50-kDa cleft (4-6). This...

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“…which make contact with a single actin subunit are structures which encircle a cleft in the 50 kDa fragment that is thought to undergo opening and closing motions [45]. In view of the importance of the description of both the nucleotide and actin-induced conformational changes in S1, it was decided to investigate these changes in solution at the strut location of the myosin head.…”

Section: Structural Studies Of Myosin S1mentioning

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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Myosin cleft movement and its coupling to actomyosin dissociation

Cited by 84 publications

References 27 publications

Coupling between phosphate release and force generation in muscle actomyosin

Coupling between phosphate release and force generation in muscle actomyosin

Myosin subfragment 1 structures reveal a partially bound nucleotide and a complex salt bridge that helps couple nucleotide and actin binding

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

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