Dataset Information

Direct real-time detection of the actin-activated power stroke within the myosin catalytic domain.

ABSTRACT: We have used transient kinetics, nanosecond time-resolved fluorescence resonance energy transfer (FRET), and kinetics simulations to resolve a structural transition in the Dictyostelium myosin II relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with the force-transducing rotation of the myosin light-chain domain. Previous research in the absence of actin showed that ATP binding to myosin induces a dynamic equilibrium between a bent prepower stroke state of the relay helix and a straight postpower stroke state, which dominates in the absence of ATP or when ADP is bound. We now ask whether actin binding reverses this transition and if so, how this reversal is coordinated with actin-activated phosphate release. We labeled a Cys-lite Dictyostelium myosin II motor domain with donor and acceptor probes at two engineered Cys residues designed to detect relay helix bending. We then performed transient time-resolved FRET following stopped-flow mixing of actin with labeled myosin, preincubated with ATP. We determined the kinetics of actin-activated phosphate release, using fluorescent phosphate-binding protein. The results show that actin binding to the myosin.ADP.P complex straightens the relay helix before phosphate dissociation. This actin-activated relay helix straightening is reversible, but phosphate irreversibly dissociates from the postpower stroke state, preventing reversal of the power stroke. Thus, relay helix straightening gates phosphate dissociation, whereas phosphate dissociation provides the thermodynamic driving force underlying force production.

SUBMITTER: Muretta JM

PROVIDER: S-EPMC3645529 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Direct real-time detection of the actin-activated power stroke within the myosin catalytic domain.

Muretta Joseph M JM Petersen Karl J KJ Thomas David D DD

Proceedings of the National Academy of Sciences of the United States of America 20130415 18

We have used transient kinetics, nanosecond time-resolved fluorescence resonance energy transfer (FRET), and kinetics simulations to resolve a structural transition in the Dictyostelium myosin II relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with the force-transducing rotation of the myosin light-chain domain. Previous research in the absence of actin showed that ATP bindin ...[more]

PMID: 23589853

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Project description:Inherited mutations in human beta-cardiac myosin (M2β) can lead to severe forms of heart failure. The E525K mutation in M2β is associated with dilated cardiomyopathy (DCM) and was found to stabilize the interacting heads motif (IHM) and autoinhibited super-relaxed (SRX) state in dimeric heavy meromyosin. However, in monomeric M2β subfragment 1 (S1) we found that E525K enhances (3-fold) the maximum steady-state actin-activated ATPase activity ( k cat ) and decreases (6-fold) the actin concentration at which ATPase is one-half maximal ( K ATPase ). We also found a 3 to 4-fold increase in the actin-activated power stroke and phosphate release rate constants at 30 μM actin, which overall enhanced the duty ratio 3-fold. Loaded motility assays revealed that the enhanced intrinsic motor activity translates to increased ensemble force in M2β S1. Glutamate 525, located near the actin binding region in the so-called activation loop, is highly conserved and predicted to form a salt-bridge with another conserved residue (lysine 484) in the relay helix. Enhanced sampling molecular dynamics simulations predict that the charge reversal mutation disrupts the E525-K484 salt-bridge, inducing conformations with a more flexible relay helix and a wide phosphate release tunnel. Our results highlight a highly conserved allosteric pathway associated with actin activation of the power stroke and phosphate release and suggest an important feature of the autoinhibited IHM is to prevent this region of myosin from interacting with actin. The ability of the E525K mutation to stabilize the IHM likely overrides the enhanced intrinsic motor properties, which may be key to triggering DCM pathogenesis.Significance statementHeart disease can be caused by inherited mutations in beta-cardiac myosin, the molecular motor that powers systolic contraction in the ventricles of the heart. However, it remains unclear how these mutations lead to contractile dysfunction and pathogenic remodeling of the heart. We investigated a unique dilated cardiomyopathy mutation (E525K) that dramatically stabilizes the autoinhibited state while enhancing intrinsic motor function. Thus, we examined how this mutation impacts transient kinetic steps of the ATPase cycle, motile properties, and structural changes associated with the power stroke and phosphate release. Our results provide a kinetic and structural basis for how beta-cardiac myosin mutations may disrupt molecular-level contractile function in complex ways, which may inform the development of targeted therapeutics.

Project description:Myosins are ATP-driven linear molecular motors that work as cellular force generators, transporters, and force sensors. These functions are driven by large-scale nucleotide-dependent conformational changes, termed "strokes"; the "power stroke" is the force-generating swinging of the myosin light chain-binding "neck" domain relative to the motor domain "head" while bound to actin; the "recovery stroke" is the necessary initial motion that primes, or "cocks," myosin while detached from actin. Myosin Va is a processive dimer that steps unidirectionally along actin following a "hand over hand" mechanism in which the trailing head detaches and steps forward ?72 nm. Despite large rotational Brownian motion of the detached head about a free joint adjoining the two necks, unidirectional stepping is achieved, in part by the power stroke of the attached head that moves the joint forward. However, the power stroke alone cannot fully account for preferential forward site binding since the orientation and angle stability of the detached head, which is determined by the properties of the recovery stroke, dictate actin binding site accessibility. Here, we directly observe the recovery stroke dynamics and fluctuations of myosin Va using a novel, transient caged ATP-controlling system that maintains constant ATP levels through stepwise UV-pulse sequences of varying intensity. We immobilized the neck of monomeric myosin Va on a surface and observed real time motions of bead(s) attached site-specifically to the head. ATP induces a transient swing of the neck to the post-recovery stroke conformation, where it remains for ?40 s, until ATP hydrolysis products are released. Angle distributions indicate that the post-recovery stroke conformation is stabilized by ? 5 k(B)T of energy. The high kinetic and energetic stability of the post-recovery stroke conformation favors preferential binding of the detached head to a forward site 72 nm away. Thus, the recovery stroke contributes to unidirectional stepping of myosin Va.

Dataset Information

Direct real-time detection of the actin-activated power stroke within the myosin catalytic domain.

Publications

Direct real-time detection of the actin-activated power stroke within the myosin catalytic domain.

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