Mechanistic insights into the active site and allosteric communication pathways in human nonmuscle myosin-2C

Chinthalapudi, Krishna; Heissler, Sarah M.; Preller, Matthias; Sellers, James R.; Manstein, Dietmar J.

doi:10.7554/elife.32742

Cited by 23 publications

(21 citation statements)

References 63 publications

(95 reference statements)

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“…The response of NM2 to different levels of external load is a mechanism by which FRAP rates could be altered, as this can greatly affect the actin binding properties of NM2 motor proteins. Structural investigations have shown that external load can disrupt the allosteric communication between the subdomains of the NM2 motor domain 49 , which in turn increases the lifetime of the strong-actin binding primed-lever state of myosin with bound ADP (M.ADP state), leading to NM2 spending longer time intervals bound to actin 18,49 . Tanner et al have shown that significant regional differences are present in the stress fiber populations in regards to strain; namely, peripheral stress fibers showed greater retraction upon laser ablation compared to central fibers, suggesting higher levels of strain 17 .…”

Section: Discussionmentioning

confidence: 99%

Effect of allosteric inhibition of non-muscle myosin 2 on its intracellular diffusion

Horváth

Gyimesi

Várkuti

et al. 2020

Sci Rep

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Subcellular dynamics of non-muscle myosin 2 (NM2) is crucial for a broad-array of cellular functions. To unveil mechanisms of NM2 pharmacological control, we determined how the dynamics of NM2 diffusion is affected by NM2′s allosteric inhibitors, i.e. blebbistatin derivatives, as compared to Y-27632 inhibiting ROCK, NM2′s upstream regulator. We found that NM2 diffusion is markedly faster in central fibers than in peripheral stress fibers. Y-27632 accelerated NM2 diffusion in both peripheral and central fibers, whereas in peripheral fibers blebbistatin derivatives slightly accelerated NM2 diffusion at low, but markedly slowed it at high inhibitor concentrations. In contrast, rapid NM2 diffusion in central fibers was unaffected by direct NM2 inhibition. Using our optopharmacological tool, Molecular Tattoo, sub-effective concentrations of a photo-crosslinkable blebbistatin derivative were increased to effective levels in a small, irradiated area of peripheral fibers. These findings suggest that direct allosteric inhibition affects the diffusion profile of NM2 in a markedly different manner compared to the disruption of the upstream control of NM2. The pharmacological action of myosin inhibitors is channeled through autonomous molecular processes and might be affected by the load acting on the NM2 proteins. Stress fibers are contractile actomyosin bundles in non-muscle cells, playing fundamental roles in diverse biological functions 1-4. Stress fibers and stress fiber-like structures play central roles in cell division as key components of the cleavage furrow 5 , determine cancer cell motility during metastasis 6 , influence cancer cell growth 7 , and they have been demonstrated as crucial elements in neuronal plasticity including neurite outgrowth 4,8-11. These force-generating structures consist of actin filaments and different co-assemblies of non-muscle myosin 2 (NM2) isoforms 12,13. The two major stress fiber-forming myosin isoforms in human cells are NM2A and NM2B, that together build up mixed minifilaments of various length and thickness 14. Stress fibers may be categorized as central and peripheral, based on their subcellular localization. Central stress fibers include those localized ventrally, dorsally, in transverse arcs and those associated around the nucleus. Their main roles are in cell adhesion, motility, cell division and cell shape determination 15,16. Peripheral stress fibers are located at the edge of the cell. Laser nanosurgery experiments have shown that incision of peripheral stress fibers causes them to retract significantly, suggesting that these structures are under larger mechanical strain than their central counterparts 17. Resisting loads on NM2 heads have been shown to significantly decrease the rate of ADP release from actin-bound myosin heads, resulting in slower actomyosin dissociation 18-20. NM2 minifilaments are also activated or repressed by the phosphorylation or dephosphorylation of the myosin light chains (MLC), respectively 21-24. Rho-dependent kinase (ROCK) positively regulates...

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Section: Discussionmentioning

confidence: 99%

Effect of allosteric inhibition of non-muscle myosin 2 on its intracellular diffusion

Horváth

Gyimesi

Várkuti

et al. 2020

Sci Rep

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“…However, the substitution of leucine at this position appears to improve the efficiency of this structural transition, possibly because a smaller hydrophobic side chain allows a more rapid reorganization of the hydrophobic cluster. A recent study that mutated a nearby residue R778K in nonmuscle myosin IIC (Arg-762 in MV) found a 50% increase in the maximum rate of ATP hydrolysis, indicating that this region is important for tuning the recovery stroke, which is coupled to formation of the hydrolysis-competent state (48). Other studies in insect flight muscle found that mutations that disrupt the converter-relay interactions have a major impact on myosin and muscle function (49,50).…”

Section: Impact Of Converter Mutations On the Recovery Strokementioning

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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“…Pivotal to this allosteric communication is the central seven-stranded β-sheet (the transducer) (twisted in the rigor state, but untwisted in the pre-power stroke state) in the myosin motor domain, which couples the large actin-binding cleft (closed while strongly attached to F-actin, and open in actin-detached states) with the active site phosphate-sensing elements—the P-loop, switch-1 and switch-2—(closed in the hydrolysis-competent pre-power stroke state, but partially or fully opened in all other states) as well as through the relay helix (bent in the pre-power stroke state, while straight in the rigor state) and SH1/SH2-region with the mechanical converter and lever arm (down position in the rigor state, and up position in the pre-power stroke state). We and others have shown that mutations in these key regions disturb the motor function by interfering with chemomechanical coupling pathways [ 4 , 5 , 6 , 7 , 8 , 9 ].…”

Section: Introductionmentioning

confidence: 99%

Structural and Computational Insights into a Blebbistatin-Bound Myosin•ADP Complex with Characteristics of an ADP-Release Conformation along the Two-Step Myosin Power Stoke

Ewert

Franz

Tsiavaliaris

et al. 2020

IJMS

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The motor protein myosin drives a wide range of cellular and muscular functions by generating directed movement and force, fueled through adenosine triphosphate (ATP) hydrolysis. Release of the hydrolysis product adenosine diphosphate (ADP) is a fundamental and regulatory process during force production. However, details about the molecular mechanism accompanying ADP release are scarce due to the lack of representative structures. Here we solved a novel blebbistatin-bound myosin conformation with critical structural elements in positions between the myosin pre-power stroke and rigor states. ADP in this structure is repositioned towards the surface by the phosphate-sensing P-loop, and stabilized in a partially unbound conformation via a salt-bridge between Arg131 and Glu187. A 5 Å rotation separates the mechanical converter in this conformation from the rigor position. The crystallized myosin structure thus resembles a conformation towards the end of the two-step power stroke, associated with ADP release. Computationally reconstructing ADP release from myosin by means of molecular dynamics simulations further supported the existence of an equivalent conformation along the power stroke that shows the same major characteristics in the myosin motor domain as the resolved blebbistatin-bound myosin-II·ADP crystal structure, and identified a communication hub centered on Arg232 that mediates chemomechanical energy transduction.

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Mechanistic insights into the active site and allosteric communication pathways in human nonmuscle myosin-2C

Cited by 23 publications

References 63 publications

Effect of allosteric inhibition of non-muscle myosin 2 on its intracellular diffusion

Effect of allosteric inhibition of non-muscle myosin 2 on its intracellular diffusion

Converter domain mutations in myosin alter structural kinetics and motor function

Structural and Computational Insights into a Blebbistatin-Bound Myosin•ADP Complex with Characteristics of an ADP-Release Conformation along the Two-Step Myosin Power Stoke

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