How Actin Initiates the Motor Activity of Myosin

Llinas, P.; Isabet, T.; Song, Lin; Ropars, Virginie; Zong, Bin; Benisty, Hannah; Sirigu, Serena; Morris, Carl; Kikuti, Carlos; Safer, Daniel; Sweeney, H. Lee; Houdusse, Anne

doi:10.1016/j.devcel.2015.03.025

Cited by 127 publications

(386 citation statements)

References 53 publications

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“…The full powerstroke extrapolated from the myosin V PPS structure that we crystallized is also comparable (27 vs. 25 nm) to what has been measured (18). It is possible that our slightly larger extrapolated swing is attributable to the initial movement that is produced in the transition from the PPS to the phosphate release state on actin (P i R state), which we have proposed forms the initial stereospecific interface with actin and thus represents the true beginning of the powerstroke (29). Note that the PPS cannot be the true beginning of the powerstroke on actin, because its interaction is weak, nonstereo-specific and likely not well-constrained.…”

Section: Discussionmentioning

confidence: 48%

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Force-producing ADP state of myosin bound to actin

Wulf

Ropars

Fujita‐Becker

et al. 2016

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

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133

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Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the β-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5°rotation of the myosin lever arm, coupled to a β-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V.T he actin-based molecular motor, myosin, generates force and movement via a series of structural transitions when bound to filamentous actin (F-actin). Among these structural states, the most important contribution to force production comes from the myosin ADP states that bind strongly to actin. Release of ADP from the motor is rapidly followed by MgATP binding, which then leads to myosin dissociation from actin. Once the dissociation occurs, myosin can undergo a structural change that allows it to prime its mechanical element (known as the lever arm; Fig. 1) and hydrolyze ATP. Myosin rebinding to actin triggers release of phosphate and reentry into the force-generating states on actin. Thus, force is produced by MgADP-bound states of myosin bound to actin.In the absence of strain, a strong-binding MgADP state bound to actin can exist for varying durations, depending on the type of myosin. Myosins that are designed to spend the majority of their catalytic cycle bound to actin in force-generating states in the absence of load, such as myosin V a , primarily occupy a state that is characterized by having both a high affinity for MgADP and a high affinity for actin. The myosin motor cycle is summarized in Fig. 1. By not rapidly releasing MgADP, the myosin motors can dwell in a force-generating state on actin that cannot be detached from actin by ATP binding. This is the primary force-generating state for all myosins, and its lifetime on actin is prolonged under load (for a review, see ref. 1).Although the nucleotide-free myosin V X-ray structure (2) (Rigor-like state) provides an atomic level model for the actomyosin state that is formed after ADP is released, and to which ATP can rapidly bind (2), we only have low-resolution EM reconstructions of myosin bound to actin in a MgADP state. Visualization of this state at the EM level was first performed for smooth muscle myosin II (3) and provided the first structural evidence that the myosin lever arm swings as the motor progresses through its actinbound, force-generating states. It was postulated at the time that th...

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Section: Discussionmentioning

confidence: 48%

“…However, we have recently suggested that the true powerstroke on actin is initiated when a different conformation of the motor domain, which we refer as the phosphate release (P i R) state (29), forms following the rearrangement of the PPS upon actin binding. It appears that this rearrangement may involve a small movement of the lever arm.…”

Section: Resultsmentioning

confidence: 99%

Force-producing ADP state of myosin bound to actin

Wulf

Ropars

Fujita‐Becker

et al. 2016

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

Self Cite

133

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“…Only in combination with other findings, like length-step-induced rapid force recovery and low duty ratio at v max , a rate-limiting P i -release preceding a fast force-generating step (model version 5) is the most consistent sequential model. Recently, a novel crystal structure of myosin was found that possesses a fully formed actin interface and opened a backdoor for P irelease with minimal rotation of the lever arm (20). This structure implies that the P i -release can occur before the power stroke, different from kinetic analysis of P i liberated from myosin and structural changes by FRET that indicate the power stroke can occur before P i -release (38).…”

Section: Synthesismentioning

confidence: 99%

“…Analysis of force transients induced by photolytic release of caged-P i led to the perception that the first force-generating step is an isomerization of an ADP.P i -binding state that precedes the P i -release (13,14,16). However, other models and recent evidence from novel crystal structures support a more direct coupling of the first force-generating step to P i -release (3,(18)(19)(20). Furthermore, force transients induced by length steps were often interpreted in terms of this force-generating step occurring independently or after P i -release (9,10).…”

Section: Introductionmentioning

confidence: 99%

Force Responses and Sarcomere Dynamics of Cardiac Myofibrils Induced by Rapid Changes in [Pi]

Stehle

2017

Biophysical Journal

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The second phase of the biphasic force decay upon release of phosphate from caged phosphate was previously interpreted as a signature of kinetics of the force-generating step in the cross-bridge cycle. To test this hypothesis without using caged compounds, force responses and individual sarcomere dynamics upon rapid increases or decreases in concentration of inorganic phosphate [P i ] were investigated in calcium-activated cardiac myofibrils. Rapid increases in [P i ] induced a biphasic force decay with an initial slow decline (phase 1) and a subsequent 3-5-fold faster major decay (phase 2). Phase 2 started with the distinct elongation of a single sarcomere, the so-called sarcomere ''give''. ''Give'' then propagated from sarcomere to sarcomere along the myofibril. Propagation speed and rate constant of phase 2 (k þPi(2) ) had a similar [P i ]-dependence, indicating that the kinetics of the major force decay (phase 2) upon rapid increase in [P i ] is determined by sarcomere dynamics. In contrast, no ''give'' was observed during phase 1 after rapid [P i ]-increase (rate constant k þPi (1) ) and during the single-exponential force rise (rate constant k ÀPi ) after rapid [P i ]-decrease. The values of k þPi(1) and k ÀPi were similar to the rate constant of mechanically induced force redevelopment (k TR ) and Ca 2þ -induced force development (k ACT ) measured at same [P i ]. These results indicate that the major phase 2 of force decay upon a P i -jump does not reflect kinetics of the force-generating step but results from sarcomere ''give''. The other phases of P i -induced force kinetics that occur in the absence of ''give'' yield the same information as mechanically and Ca 2þ -induced force kinetics (k þPi(1)~k-Pi~kTR~kACT ). Model simulations indicate that P i -induced force kinetics neither enable the separation of P i -release from the rate-limiting transition f into force states nor differentiate whether the ''forcegenerating step'' occurs before, along, or after the P i -release.

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“…It has been suggested that loop-2 of myosin initiates weak binding of myosin to the TF (21,22). Subsequent binding of the lower L50 domain of myosin to F-actin through the helix-turn-helix motif (23) promotes P i release (24) and completes the initial strong binding of myosin to F-actin. We approximated the initial strong binding mode of myosin using the same approach described by von der Ecken et al (23) (SI Appendix, Fig.…”

Section: Significancementioning

confidence: 99%

Ca ²⁺ -induced movement of tropomyosin on native cardiac thin filaments revealed by cryoelectron microscopy

Risi

Eisner

Belknap

et al. 2017

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

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Muscle contraction relies on the interaction of myosin motors with F-actin, which is regulated through a translocation of tropomyosin by the troponin complex in response to Ca 2+ . The current model of muscle regulation holds that at relaxing (low-Ca 2+ ) conditions tropomyosin blocks myosin binding sites on F-actin, whereas at activating (high-Ca 2+ ) conditions tropomyosin translocation only partially exposes myosin binding sites on F-actin so that binding of rigor myosin is required to fully activate the thin filament (TF). Here we used a single-particle approach to helical reconstruction of frozen hydrated native cardiac TFs under relaxing and activating conditions to reveal the azimuthal movement of the tropomyosin on the surface of the native cardiac TF upon Ca 2+ activation. We demonstrate that at either relaxing or activating conditions tropomyosin is not constrained in one structural state, but rather is distributed between three structural positions on the surface of the TF. We show that two of these tropomyosin positions restrain actomyosin interactions, whereas in the third position, which is significantly enhanced at high Ca 2+ , tropomyosin does not block myosin binding sites on F-actin. Our data provide a structural framework for the enhanced activation of the cardiac TF over the skeletal TF by Ca 2+ and lead to a mechanistic model for the regulation of the cardiac TF.thin filament | cardiac muscle regulation | cryoelectron microscopy

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How Actin Initiates the Motor Activity of Myosin

Cited by 127 publications

References 53 publications

Force-producing ADP state of myosin bound to actin

Force-producing ADP state of myosin bound to actin

Force Responses and Sarcomere Dynamics of Cardiac Myofibrils Induced by Rapid Changes in [Pi]

Ca ²⁺ -induced movement of tropomyosin on native cardiac thin filaments revealed by cryoelectron microscopy

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How Actin Initiates the Motor Activity of Myosin

Cited by 127 publications

References 53 publications

Force-producing ADP state of myosin bound to actin

Force-producing ADP state of myosin bound to actin

Force Responses and Sarcomere Dynamics of Cardiac Myofibrils Induced by Rapid Changes in [Pi]

Ca 2+ -induced movement of tropomyosin on native cardiac thin filaments revealed by cryoelectron microscopy

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Product

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Ca ²⁺ -induced movement of tropomyosin on native cardiac thin filaments revealed by cryoelectron microscopy