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Quantitative analysis of approaches to measure cooperative phosphate release in polymerized actin.

ABSTRACT: We use stochastic simulations that treat several experimental probes of actin dynamics to explore the extent to which phosphate dissociation in filamentous actin may be cooperative. Phosphate time-courses from polymerization and copolymerization experiments of ATP- and ADP-actin are studied, including the effects of variations in filament-number concentration as well as single-filament depolymerization time-courses. We find that highly cooperative models are consistent with the treated experimental data. We also find that some types of experiments that are believed to provide strong constraints on the cooperativity of actin hydrolysis models do not provide such constraints.

SUBMITTER: Burnett MM

PROVIDER: S-EPMC3514521 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Quantitative analysis of approaches to measure cooperative phosphate release in polymerized actin.

Burnett Mark M MM Carlsson Anders E AE

Biophysical journal 20121201 11

We use stochastic simulations that treat several experimental probes of actin dynamics to explore the extent to which phosphate dissociation in filamentous actin may be cooperative. Phosphate time-courses from polymerization and copolymerization experiments of ATP- and ADP-actin are studied, including the effects of variations in filament-number concentration as well as single-filament depolymerization time-courses. We find that highly cooperative models are consistent with the treated experimen ...[more]

PMID: 23283236

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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.

Dataset Information

Quantitative analysis of approaches to measure cooperative phosphate release in polymerized actin.

Publications

Quantitative analysis of approaches to measure cooperative phosphate release in polymerized actin.

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