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

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Krishna Chinthalapudi ◽  
Sarah M Heissler ◽  
Matthias Preller ◽  
James R Sellers ◽  
Dietmar J Manstein
Keyword(s):  
Active Site ◽  
Structural Changes ◽  
Motor Domain ◽  
Dynamics Simulation ◽  
Actin Binding ◽  
Nonmuscle Myosin ◽  
Cortical Actin ◽  
Allosteric Communication ◽  
Myosin Motor Domain ◽  
Communication Pathway

Despite a generic, highly conserved motor domain, ATP turnover kinetics and their activation by F-actin vary greatly between myosin-2 isoforms. Here, we present a 2.25 Å pre-powerstroke state (ADP⋅VO4) crystal structure of the human nonmuscle myosin-2C motor domain, one of the slowest myosins characterized. In combination with integrated mutagenesis, ensemble-solution kinetics, and molecular dynamics simulation approaches, the structure reveals an allosteric communication pathway that connects the distal end of the motor domain with the active site. Disruption of this pathway by mutation of hub residue R788, which forms the center of a cluster of interactions connecting the converter, the SH1-SH2 helix, the relay helix, and the lever, abolishes nonmuscle myosin-2 specific kinetic signatures. Our results provide insights into structural changes in the myosin motor domain that are triggered upon F-actin binding and contribute critically to the mechanochemical behavior of stress fibers, actin arcs, and cortical actin-based structures.

scholarly journals Mechanistic Insights into the Active Site and Allosteric Communication Pathways in Human Nonmuscle Myosin-2C

2017 ◽  
Author(s):  
Krishna Chinthalapudi ◽  
Sarah M. Heissler ◽  
Matthias Preller ◽  
James R. Sellers ◽  
Dietmar J. Manstein
Keyword(s):  
Active Site ◽  
Structural Changes ◽  
Release Kinetics ◽  
Motor Domain ◽  
Actin Binding ◽  
Nonmuscle Myosin ◽  
Cortical Actin ◽  
Allosteric Communication ◽  
Dynamics Simulations ◽  
Communication Pathway

AbstractThe cyclical interaction of myosin with F-actin and nucleotides is the basis for contractility of the actin cytoskeleton. Despite a generic, highly conserved motor domain, ATP turnover kinetics and their activation by F-actin vary greatly between myosins-2 isoforms. Here, we present a 2.25 Å crystal structure of the human nonmuscle myosin-2C motor domain, one of the slowest myosins characterized. In combination with integrated mutagenesis, ensemble-solution kinetics, and molecular dynamics simulations approaches, this study reveals an allosteric communication pathway that connects the distal end of the motor domain with the active site. Genetic disruption of this pathways reduces nucleotide binding and release kinetics up to 85-fold and abolishes nonmuscle myosin-2 specific kinetic signatures. These results provide insights into structural changes in the myosin motor domain that are triggered upon F-actin binding and contribute critically to the mechanochemical behavior of stress fibers, actin arcs, and cortical actin-based structures.

scholarly journals Structure-function studies of the myosin motor domain: importance of the 50-kDa cleft.

1996 ◽  
Vol 7 (7) ◽  
pp. 1123-1136 ◽  
Author(s):  
K M Ruppel ◽  
J A Spudich
Keyword(s):  
Active Site ◽  
Three Dimensional ◽  
Point Mutations ◽  
Random Mutagenesis ◽  
Motor Domain ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Null Cell ◽  
Myosin Motor Domain ◽  
Cell Phenotypes

We used random mutagenesis to create 21 point mutations in a highly conserved region of the motor domain of Dictyostelium myosin and classified them into three distinct groups based on the ability to complement myosin null cell phenotypes: wild type, intermediate, and null. Biochemical analysis of the mutated myosins also revealed three classes of mutants that correlated well with the phenotypic classification. The mutated myosins that were not fully functional showed defects ranging from ATP nonhydrolyzers to myosins whose enzymatic and mechanical properties are uncoupled. Placement of the mutations onto the three-dimensional structure of myosin showed that the mutated region lay along the cleft that separates the active site from the actin-binding domain and that has been shown to move in response to changes at the active site. These results demonstrate that this region of myosin plays a key role in transduction of chemical energy to mechanical displacement.

scholarly journals Dynamics of actomyosin interactions in relation to the cross-bridge cycle

2004 ◽  
Vol 359 (1452) ◽  
pp. 1843-1855 ◽  
Author(s):  
K. C. Holmes ◽  
D. R. Trentham ◽  
R. Simmons ◽  
Wei Zeng ◽  
Paul B. Conibear ◽  
...  
Keyword(s):  
Motor Domain ◽  
Actin Binding ◽  
Kinetic Measurements ◽  
Product Release ◽  
Myosin Motor ◽  
Cross Bridge ◽  
Lever Arm ◽  
Arm Position ◽  
Myosin Motor Domain ◽  
The Cross

Transient kinetic measurements of the actomyosin ATPase provided the basis of the Lymn–Taylor model for the cross–bridge cycle, which underpins current models of contraction. Following the determination of the structure of the myosin motor domain, it has been possible to introduce probes at defined sites and resolve the steps in more detail. Probes have been introduced in the Dicytostelium myosin II motor domain via three routes: (i) single tryptophan residues at strategic locations throughout the motor domain; (ii) green fluorescent protein fusions at the N and C termini; and (iii) labelled cysteine residues engineered across the actin–binding cleft. These studies are interpreted with reference to motor domain crystal structures and suggest that the tryptophan (W501) in the relay loop senses the lever arm position, which is controlled by the switch 2 open–to–closed transition at the active site. Actin has little effect on this process per se . A mechanism of product release is proposed in which actin has an indirect effect on the switch 2 and lever arm position to achieve mechanochemical coupling. Switch 1 closing appears to be a key step in the nucleotide–induced actin dissociation, while its opening is required for the subsequent activation of product release. This process has been probed with F239W and F242W substitutions in the switch 1 loop. The E706K mutation in skeletal myosin IIa is associated with a human myopathy. To simulate this disease we investigated the homologous mutation, E683K, in the Dictyostelium myosin motor domain.

scholarly journals Tropomyosin isoforms bias actin track selection by vertebrate myosin Va

2016 ◽  
Vol 27 (19) ◽  
pp. 2889-2897 ◽  
Author(s):  
Maria Sckolnick ◽  
Elena B. Krementsova ◽  
David M. Warshaw ◽  
Kathleen M. Trybus
Keyword(s):  
Full Length ◽  
Actin Binding ◽  
Myosin Isoforms ◽  
Cortical Actin ◽  
Actin Binding Domain ◽  
Myosin Motor Domain ◽  
Myosin Va ◽  
Temporal Localization

Tropomyosin (Tpm) isoforms decorate actin with distinct spatial and temporal localization patterns in cells and thus may function to sort actomyosin processes by modifying the actin track affinity for specific myosin isoforms. We examined the effect of three Tpm isoforms on the ability of myosin Va (myoVa) to engage with actin in vitro in the absence or presence of the cargo adapter melanophilin (Mlph), which links myoVa to Rab27a-melanosomes for in vivo transport. We show that both the myosin motor domain and the cargo adapter Mlph, which has an actin-binding domain that acts as a tether, are sensitive to the Tpm isoform. Actin–Tpm3.1 and actin–Tpm1.8 were equal or better tracks compared to bare actin for myoVa-HMM based on event frequency, run length, and speed. The full-length myoVa-Mlph complex showed high-frequency engagement with actin-Tpm3.1 but not with actin-Tpm1.8. Actin–Tpm4.2 excluded both myoVa-HMM and full-length myoVa-Mlph from productive interactions. Of importance, Tpm3.1 is enriched in the dendritic protrusions and cortical actin of melanocytes, where myoVa-Mlph engages in melanosome transport. These results support the hypothesis that Tpm isoforms constitute an “actin–Tpm code” that allows for spatial and temporal sorting of actomyosin function in the cell.

scholarly journals Author response: Mechanistic insights into the active site and allosteric communication pathways in human nonmuscle myosin-2C

2017 ◽  
Author(s):  
Krishna Chinthalapudi ◽  
Sarah M Heissler ◽  
Matthias Preller ◽  
James R Sellers ◽  
Dietmar J Manstein
Keyword(s):  
Active Site ◽  
Author Response ◽  
Nonmuscle Myosin ◽  
Allosteric Communication

scholarly journals Abl Tyrosine Kinase Phosphorylates Nonmuscle Myosin Light Chain Kinase to Regulate Endothelial Barrier Function

2010 ◽  
Vol 21 (22) ◽  
pp. 4042-4056 ◽  
Author(s):  
Steven M. Dudek ◽  
Eddie T. Chiang ◽  
Sara M. Camp ◽  
Yurong Guo ◽  
Jing Zhao ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Activity ◽  
Actin Binding ◽  
Nonmuscle Myosin ◽  
Cortical Actin ◽  
Abl Kinase ◽  
Barrier Regulation

Nonmuscle myosin light chain kinase (nmMLCK), a multi-functional cytoskeletal protein critical to vascular homeostasis, is highly regulated by tyrosine phosphorylation. We identified multiple novel c-Abl–mediated nmMLCK phosphorylation sites by mass spectroscopy analysis (including Y231, Y464, Y556, Y846) and examined their influence on nmMLCK function and human lung endothelial cell (EC) barrier regulation. Tyrosine phosphorylation of nmMLCK increased kinase activity, reversed nmMLCK-mediated inhibition of Arp2/3-mediated actin polymerization, and enhanced binding to the critical actin-binding phosphotyrosine protein, cortactin. EC challenge with sphingosine 1-phosphate (S1P), a potent barrier-enhancing agonist, resulted in c-Abl and phosphorylated nmMLCK recruitment into caveolin-enriched microdomains, rapid increases in Abl kinase activity, and spatial targeting of c-Abl to barrier-promoting cortical actin structures. Conversely, reduced c-Abl expression in EC (siRNA) markedly attenuated S1P-mediated cortical actin formation, reduced the EC modulus of elasticity (assessed by atomic force microscopy), reduced nmMLCK and cortactin tyrosine phosphorylation, and attenuated S1P-mediated barrier enhancement. These studies indicate an essential role for Abl kinase in vascular barrier regulation via posttranslational modification of nmMLCK and strongly support c-Abl-cortactin-nmMLCK interaction as a novel determinant of cortical actin-based cytoskeletal rearrangement critical to S1P-mediated EC barrier enhancement.

Stimulatory effects of arachidonic acid on myosin ATPase activity and contraction of smooth muscle via myosin motor domain

2010 ◽  
Vol 298 (2) ◽  
pp. H505-H514 ◽  
Author(s):  
Takeshi Katayama ◽  
Masaru Watanabe ◽  
Hideyuki Tanaka ◽  
Mizuki Hino ◽  
Takuya Miyakawa ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Smooth Muscle ◽  
Atpase Activity ◽  
Smooth Muscle Myosin ◽  
Motor Domain ◽  
Actin Binding ◽  
Smooth Muscle Tissue ◽  
Myosin Motor ◽  
Myosin Motor Domain ◽  
Muscle Myosin

We have been searching for a mechanism to induce smooth muscle contraction that is not associated with phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin (Nakamura A, Xie C, Zhang Y, Gao Y, Wang HH, Ye LH, Kishi H, Okagaki T, Yoshiyama S, Hayakawa K, Ishikawa R, Kohama K. Biochem Biophys Res Commun 369: 135–143, 2008). In this article, we report that arachidonic acid (AA) stimulates ATPase activity of unphosphorylated smooth muscle myosin with maximal stimulation (Rmax) of 6.84 ± 0.51 relative to stimulation by the vehicle and with a half-maximal effective concentration (EC50) of 50.3 ± 4.2 μM. In the presence of actin, Rmax was 1.72 ± 0.08 and EC50 was 26.3 ± 2.3 μM. Our experiments with eicosanoids consisting of the AA cascade suggested that they neither stimulated nor inhibited the activity. Under conditions that did not allow RLC to be phosphorylated, AA stimulated contraction of smooth muscle tissue with an Rmax of 1.45 ± 0.07 and an EC50 of 27.0 ± 4.4 μM. In addition to the ATPase activities of the myosin, AA stimulated those of heavy meromyosin, subfragment 1 (S1), S1 from which the RLC was removed, and a recombinant heavy chain consisting of the myosin head. The stimulatory effects of AA on these preparations were about twofold. The site of AA action was indicated to be the step-releasing inorganic phosphate (Pi) from the reaction intermediate of the myosin-ADP-Pi complex. The enhancement of Pi release by AA was supported by computer simulation indicating that AA docked in the actin-binding cleft of the myosin motor domain. The stimulatory effect of AA was detectable with both unphosphorylated myosin and the myosin in which RLC was fully phosphorylated. The AA effect on both myosin forms was suggested to cause excess contraction such as vasospasm.

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