Actin Residue Glu93 Is Identified as an Amino Acid Affecting Myosin Binding

Razzaq, Azam; Schmitz, Stephan; Veigel, Claudia; Molloy, Justin E.; Geeves, Michael A.; Sparrow, John C.

doi:10.1074/jbc.274.40.28321

Cited by 54 publications

(36 citation statements)

References 49 publications

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“…If this is true, then one might also expect tropomyosin binding to be weakened, but instead it is increased 3-fold by the mutation. A second possibility, more likely in our view, is that these results are supporting structural and kinetic data demonstrating interactions between myosin and actin subdomain 2 (25,46,47), and it is these interactions that are inhibited by the mutation. In favor of this, both myosin binding to actin and the MgATPase rate remain suppressed by the mutation when troponin-tropomyosin and calcium are added, despite the absence of bundling.…”

Section: Discussionsupporting

confidence: 67%

An Actin Subdomain 2 Mutation That Impairs Thin Filament Regulation by Troponin and Tropomyosin

Korman¹,

Hatch²,

Dixon³

et al. 2000

Journal of Biological Chemistry

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Striated muscle thin filaments adopt different quaternary structures, depending upon calcium binding to troponin and myosin binding to actin. Modification of actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting that this region of actin may contain important protein-protein interaction sites. We used yeast actin mutant D56A/E57A to examine this issue. The mutation increased the affinity of tropomyosin for actin 3-fold. The addition of Ca 2؉ to mutant actin filaments containing troponin-tropomyosin produced little increase in the thin filament-myosin S1 MgATPase rate. Despite this, three-dimensional reconstruction of electron microscope images of filaments in the presence of troponin and Ca 2؉ showed tropomyosin to be in a position similar to that found for muscle actin filaments, where most of the myosin binding site is exposed. Troponin-tropomyosin bound with comparable affinity to mutant and wild type actin in the absence and presence of calcium, and in the presence of myosin S1, tropomyosin bound very tightly to both types of actin. The mutation decreased actin-myosin S1 affinity 13-fold in the presence of troponin-tropomyosin and 2.6-fold in the absence of the regulatory proteins. The results suggest the importance of negatively charged actin subdomain 2 residues 56 and 57 for myosin binding to actin, for tropomyosinactin interactions, and for regulatory conformational changes in the actin-troponin-tropomyosin complex.Cardiac and skeletal muscle contraction is controlled by a complex allosteric system in which myosin-actin interactions are regulated by tropomyosin and troponin. Tropomyosin is an extended, dimeric coiled-coil and spans seven actin monomers on the thin filament. Troponin is composed of three subunits: troponin T connects the other two subunits and tropomyosin to the actin filament, troponin I inhibits myosin binding to actin, and troponin C serves as a Ca 2ϩ sensor for the system (for reviews see Refs. 1 and 2). Structural studies have shown that tropomyosin can bind to three different regions of the actin filament (3-7). In the absence of Ca 2ϩ , tropomyosin appears to bind to subdomain 1 of actin and to bridge over subdomain 2 to the next actin monomer (4). In the presence of Ca 2ϩ , tropomyosin moves toward subdomains 3 and 4 (3) and moves even further in the presence of myosin (8). Whereas these and other results support a three state model of muscle regulation involving tropomyosin movement (9 -12), information defining the position and interactions of troponin on the filament and actin residues that specifically interact with the regulatory proteins is limited.One strategy to examine muscle protein-protein interactions has been to exploit functionally altered mutants (13-15). For example, a Drosophila melanogaster actin subdomain 2 mutant, E93K, which inhibits tropomyosin-actin sliding in isolated protein preparations, has been utilized to investigate tropomyosin-based regulation (16). These and other data (17)(18)(19)(20) suggested that actin subdomain 2 may be ...

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Section: Discussionsupporting

confidence: 67%

An Actin Subdomain 2 Mutation That Impairs Thin Filament Regulation by Troponin and Tropomyosin

Korman¹,

Hatch²,

Dixon³

et al. 2000

Journal of Biological Chemistry

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“…In our model, Asp-374 at the so-called ''loop 4'' of myosin also fits well to Lys-326 of the first actin. Many recent kinetic studies with actin mutants (18)(19)(20) or myosin mutants (5,6,21,22) also show that the foregoing residues are involved in forming the weakly bound complex. Interestingly, our model suggests that before cleft closure, both Trp-546 and Phe-547 in the hydrophobic triplet of myosin are already close to Ile-345 and Leu-349 of the first actin, although a surface loop bearing Pro-534 and Pro-535 (''prolinerich loop'') is not as yet close to either Leu-349 or Phe-352 of the first actin.…”

Section: Resultsmentioning

confidence: 99%

A closer look at energy transduction in muscle

Onishi

Morales

2007

Proc. Natl. Acad. Sci. U.S.A.

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Muscular force is the sum of unitary force interactions generated as filaments of myosins move forcibly along parallel filaments of actins, understanding that the free energy required comes from myosin-catalyzed ATP hydrolysis. Using results from conventional biochemistry, our own mutational studies, and diffraction images from others, we attempt, in molecular detail, an account of a unitary interaction, i.e., what happens after a traveling myosin head, bearing an ADP-P i, reaches the next station of an actin filament in its path. We first construct a reasonable model of the myosin head and actin regions that meet to form the ''weakly bound state''. Separately, we consider Holmes' model of the rigor state [Holmes, K. C., Angert, I., Kull, F. J., Jahn, W. & Schrö der, R. R. actin ͉ ATPase ͉ mutation ͉ myosin H ere, we attempt a molecular account of the central interactions that occur when muscle contracts or sustains tension. Because of a peculiar structural organization of muscle, the contractile apparatus can be thought of as effectively a linear filament of myosin molecules forcibly advancing along a parallel filament of actin molecules, the process being thermodynamically paid for by losing free energy of myosin-bound ATP hydrolysis (1). In this sense, contraction is an instance of transducing chemical (free) energy into mechanical work. For our purposes, however, it is simpler to consider our task to be explaining how a traveling myosin head, bearing a partly hydrolyzed ATP, on reaching interaction distance to an actin pair, fastens to it, first weakly and later strongly, and then, how it, in a remarkable cooperation with the actins, casts off the terminal phosphate of ATP and delivers a mechanical impulse to the actins.The account that follows is put together by using reports from many laboratories. These are of three general kinds, conventional biochemical [e.g., Scheme 1, § showing the timedependence of forming complexes of myosin and actin (2-4)]; mutational [e.g., recent works identifying myosin surface loops engaged in complexing with actin (5, 6)]; and x-ray diffraction [e.g., studies of Rayment (7-10), of Cohen (11), and of their pioneering associates]. Application of diffraction techniques has dramatically improved resolution and has shown that a globular head of myosin has specialized ''organs,'' like an enzyme pocket in which to conduct ATP hydrolysis, a long, stiff ␣-helix (''relay helix'') to transmit linear force, a converter to turn it into a rotation, and a lever arm to deliver a mechanical impulse to (at the time attached) actins (Fig. 1). In addition, from fitting crystal structures of actin and the myosin head to the 3D structure reconstructed from cryoEM pictures of actin filaments decorated with S1 (an isolated single myosin head), Rayment et al. (7) have suggested that ionic and hydrophobic residues, which are possibly involved in actin binding, are localized at one end of the myosin head, which is far from either the enzyme pocket or the converter (Fig. 1). At the end of the head, th...

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“…The Bradford assay (Coomassie Plus-200, Pierce) and semiquantitative SDS-PAGE gave similar results when rabbit myosin was used as the protein standard. Actin Isolation-Actin was isolated from dissected DLMs as described by Razzaq et al (22). Purified F-actin was centrifuged at 150,000 ϫ g for 1.5 h, and resuspended in actin buffer (25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl 2, 1 mM EGTA, and 1 mM DTT) (23).…”

Section: Methodsmentioning

confidence: 99%

Alternative Exon-encoded Regions of Drosophila Myosin Heavy Chain Modulate ATPase Rates and Actin Sliding Velocity

Swank¹,

Bartoo

Knowles

et al. 2001

Journal of Biological Chemistry

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137

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To investigate the molecular functions of the regions encoded by alternative exons from the single Drosophila myosin heavy chain gene, we made the first kinetic measurements of two muscle myosin isoforms that differ in all alternative regions. Myosin was purified from the indirect flight muscles of wild-type and transgenic flies expressing a major embryonic isoform. The in vitro actin sliding velocity on the flight muscle isoform (6.4 m⅐s ؊1 at 22°C) is among the fastest reported for a type II myosin and was 9-fold faster than with the embryonic isoform. With smooth muscle tropomyosin bound to actin, the actin sliding velocity on the embryonic isoform increased 6-fold, whereas that on the flight muscle myosin slightly decreased. No difference in the step sizes of Drosophila and rabbit skeletal myosins were found using optical tweezers, suggesting that the slower in vitro velocity with the embryonic isoform is due to altered kinetics. Basal ATPase rates for flight muscle myosin are higher than those of embryonic and rabbit myosin. These differences explain why the embryonic myosin cannot functionally substitute in vivo for the native flight muscle isoform, and demonstrate that one or more of the five myosin heavy chain alternative exons must influence Drosophila myosin kinetics.

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Actin Residue Glu93 Is Identified as an Amino Acid Affecting Myosin Binding

Cited by 54 publications

References 49 publications

An Actin Subdomain 2 Mutation That Impairs Thin Filament Regulation by Troponin and Tropomyosin

An Actin Subdomain 2 Mutation That Impairs Thin Filament Regulation by Troponin and Tropomyosin

A closer look at energy transduction in muscle

Alternative Exon-encoded Regions of Drosophila Myosin Heavy Chain Modulate ATPase Rates and Actin Sliding Velocity

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