Magnesium Modulates Actin Binding and ADP Release in Myosin Motors

Swenson, Anja M.; Trivedi, Darshan V.; Rauscher, Anna Á.; Yuan, Wang; Takagi, Yasuharu; Palmer, Bradley M.; Málnási‐Csizmadia, András; Debold, Edward P.; Yengo, Christopher M.

doi:10.1074/jbc.m114.562231

Cited by 32 publications

(40 citation statements)

References 69 publications

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“…Thus, the high duty ratio of NtMD-2R is the product of 2 essential rate constants, one that determines the acto-myosin dissociation and a second that defines the rate of ADP release. Variations in the concentration of ATP and ADP, as well as of bivalent cations such as Ca 2+ and Mg 2+ , significantly impact the functional properties of a number of myosins by modulating ATP turnover, motor activity, and processivity (44,(69)(70)(71)(72). Particularly, the influence of Ca 2+ ions on the mechanical properties of myosin-11 is believed to be a key factor for the regulation of cytoplasmic streaming in plant cells (73).…”

Section: Discussionmentioning

confidence: 99%

Kinetic mechanism ofNicotiana tabacummyosin‐11 defines a new type of a processive motor

et al. 2014

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The 175-kDa myosin-11 from Nicotiana tabacum (Nt 175kDa myosin-11) is exceptional in its mechanical activity as it is the fastest known processive actin-based motor, moving 10 times faster than the structurally related class 5 myosins. Although this ability might be essential for long-range organelle transport within larger plant cells, the kinetic features underlying the fast processive movement of Nt 175kDa myosin-11 still remain unexplored. To address this, we generated a single-headed motor domain construct and carried out a detailed kinetic analysis. The data demonstrate that Nt 175kDa myosin-11 is a highduty ratio motor, which remains associated with actin most of its enzymatic cycle. However, different from other processive myosins that establish a high duty ratio on the basis of a rate-limiting ADP-release step, Nt 175kDa myosin-11 achieves a high duty ratio by a prolonged duration of the ATP-induced isomerization of the actin-bound states and ADP release kinetics, both of which in terms of the corresponding time constants approach the total ATPase cycle time. Molecular modeling predicts that variations in the charge distribution of the actin binding interface might contribute to the thermodynamic fine-tuning of the kinetics of this myosin. Our study unravels a new type of a high duty ratio motor and provides important insights into the molecular mechanism of processive movement of higher plant myosins.-Diensthuber, R. P., Tominaga, M., Preller, M., Hartmann, F. K., Orii, H., Chizhov, I., Oiwa, K., Tsiavaliaris, G. Kinetic mechanism of Nicotiana tabacum myosin-11 defines a new type of a processive motor. FASEB J. 29, 81-94 (2015). www.fasebj.org

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Section: Discussionmentioning

confidence: 99%

Kinetic mechanism ofNicotiana tabacummyosin‐11 defines a new type of a processive motor

et al. 2014

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“…Similar results were obtained by measuring the absorbance and using the predicted extinction coefficient (ε 280 = 1.38 × 10 5 M −1 ·cm −1 ). Skeletal muscle heavy meromyosin (Sk HMM) was prepared from rabbit psoas muscle as described (Swenson et al, 2014). Actin was purified from rabbit skeletal muscle using an acetone powder method (Pardee and Spudich, 1982).…”

Section: Methodsmentioning

confidence: 99%

“…The M2β-S1 experiments were performed in conditions (buffer and temperature) that were similar to the muscle mechanic studies described below. The actin filament sliding assay was performed as previously described (Trivedi et al, 2013; Swenson et al, 2014) except for the method of adhering the myosin to the surface in the case of M2β-S1. Microscope cover slips were coated with 1% nitrocellulose in amyl acetate (Ladd Research).…”

Section: Methodsmentioning

confidence: 99%

Modulating Beta-Cardiac Myosin Function at the Molecular and Tissue Levels

et al. 2017

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Inherited cardiomyopathies are a common form of heart disease that are caused by mutations in sarcomeric proteins with beta cardiac myosin (MYH7) being one of the most frequently affected genes. Since the discovery of the first cardiomyopathy associated mutation in beta-cardiac myosin, a major goal has been to correlate the in vitro myosin motor properties with the contractile performance of cardiac muscle. There has been substantial progress in developing assays to measure the force and velocity properties of purified cardiac muscle myosin but it is still challenging to correlate results from molecular and tissue-level experiments. Mutations that cause hypertrophic cardiomyopathy are more common than mutations that lead to dilated cardiomyopathy and are also often associated with increased isometric force and hyper-contractility. Therefore, the development of drugs designed to decrease isometric force by reducing the duty ratio (the proportion of time myosin spends bound to actin during its ATPase cycle) has been proposed for the treatment of hypertrophic cardiomyopathy. Para-Nitroblebbistatin is a small molecule drug proposed to decrease the duty ratio of class II myosins. We examined the impact of this drug on human beta cardiac myosin using purified myosin motor assays and studies of permeabilized muscle fiber mechanics. We find that with purified human beta-cardiac myosin para-Nitroblebbistatin slows actin-activated ATPase and in vitro motility without altering the ADP release rate constant. In permeabilized human myocardium, para-Nitroblebbistatin reduces isometric force, power, and calcium sensitivity while not changing shortening velocity or the rate of force development (ktr). Therefore, designing a drug that reduces the myosin duty ratio by inhibiting strong attachment to actin while not changing detachment can cause a reduction in force without changing shortening velocity or relaxation.

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“…We examined the motor function, biochemical, and structural properties of myosin V 1IQ containing the N-terminal tetracysteine motif (MV) expressed in the baculovirus system as described (23)(24)(25)(26). To examine the impact of the mutations on the steady-state kinetic, transient kinetic, and motile properties, we co-expressed MV with WT calmodulin.…”

Section: Expression and Purification Of Myosin V Constructsmentioning

confidence: 99%

Converter domain mutations in myosin alter structural kinetics and motor function

Gunther

Rohde

Tang

et al. 2019

Journal of Biological Chemistry

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Edited by Karen G. Fleming Myosins are molecular motors that use a conserved ATPase cycle to generate force. We investigated two mutations in the converter domain of myosin V (R712G and F750L) to examine how altering specific structural transitions in the motor ATPase cycle can impair myosin mechanochemistry. The corresponding mutations in the human ␤-cardiac myosin gene are associated with hypertrophic and dilated cardiomyopathy, respectively. Despite similar steady-state actin-activated ATPase and unloaded in vitro motility-sliding velocities, both R712G and F750L were less able to overcome frictional loads measured in the loaded motility assay. Transient kinetic analysis and stopped-flow FRET demonstrated that the R712G mutation slowed the maximum ATP hydrolysis and recovery-stroke rate constants, whereas the F750L mutation enhanced these steps. In both mutants, the fast and slow power-stroke as well as actin-activated phosphate release rate constants were not significantly different from WT. Time-resolved FRET experiments revealed that R712G and F750L populate the pre-and post-power-stroke states with similar FRET distance and distance distribution profiles. The R712G mutant increased the mole fraction in the post-power-stroke conformation in the strong actin-binding states, whereas the F750L decreased this population in the actomyosin ADP state. We conclude that mutations in key allosteric pathways can shift the equilibrium and/or alter the activation energy associated with key structural transitions without altering the overall conformation of the pre-and post-powerstroke states. Thus, therapies designed to alter the transition between structural states may be able to rescue the impaired motor function induced by disease mutations. . 4 The abbreviations used are: HCM, hypertrophic cardiomyopathy; M2␤, human ␤-cardiac myosin; MV, chicken myosin Va; DCM, dilated cardiomyopathy; FlAsH, fluorescein bis-arsenical hairpin binding dye; QSY, QSY TM 9 C5-maleimide; CaM, calmodulin; mantADP, 2Ј-deoxy-ADP labeled with N-methylanthraniloy at the 3Ј-ribose position; mant, N-methylanthraniloy; PBP-MDCC, phosphate-binding protein labeled with 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin; (TR) 2 FRET, transient time-resolved FRET; ELC, essential light chain; PDB, Protein Data Bank. cro ARTICLE 1554 Figure 4. Actin-activated product release. Sequential mix single-turnover experiments were performed by mixing MV with ATP, aging the reaction to form the M.ADP.P i state (0.45 M), and then mixing with different concentrations of actin in the presence of MDCC-PBP (5 M ). A, the fluorescence transients were fit to a single-exponential function (phosphate burst) followed by a linear or slow exponential rise. B, the phosphate release rate constants were plotted as a function of actin concentration and fit to a hyperbolic function to estimate the maximum rate of phosphate release. C, acto-MV (0.25 M) in presence mantADP (5 M) was mixed with saturating ATP (1 mM) at 25°C to determine the ADP release rate constan...

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Magnesium Modulates Actin Binding and ADP Release in Myosin Motors

Cited by 32 publications

References 69 publications

Kinetic mechanism ofNicotiana tabacummyosin‐11 defines a new type of a processive motor

Kinetic mechanism ofNicotiana tabacummyosin‐11 defines a new type of a processive motor

Modulating Beta-Cardiac Myosin Function at the Molecular and Tissue Levels

Converter domain mutations in myosin alter structural kinetics and motor function

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