Tropomyosin Inhibits the Glutaraldehyde-Induced Cross-Link between the Central 48-kDa Fragment of Myosin Head and Segment 48-67 in Actin Subdomain 2

Bonafé, Nathalie; Mathieu, Magali; Kassab, Ridha; Chaussepied, Patrick

doi:10.1021/bi00175a031

Cited by 17 publications

(21 citation statements)

References 85 publications

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“…However, Squire and Morris (12) raised the possibility that changes seen in the structural studies of regulated thin filaments may be due not only to shifts of the Tm/Tn complex, but also to subdomain 2 movements within each actin protomer. The observations that selected subdomain 2 alterations impair regulation by troponin-tropomyosin (13,14) and that the presence of Tm and Tm/Tn interferes with cross-linking between subdomain 2 and S1 (15) are consistent with this hypothesis. Moreover, the recent paper of Luo et al (16), reporting the cross-linking of Tn-I to Met 47 on subdomain 2 of actin, suggests that TnI interacts with this region of actin.…”

supporting

confidence: 64%

The Regulation of Subtilisin-cleaved Actin by Tropomyosin/Troponin

Pavlov¹,

Gerson²,

Yu³

et al. 2003

Journal of Biological Chemistry

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Vertebrate striated muscle contraction is regulated in a Ca 2؉ -dependent fashion by tropomyosin (Tm) and troponin (Tn). This regulation involves shifts in the position of Tm and Tn on actin filaments and may include conformational changes in actin that are then communicated to myosin subfragment 1 (S1). To determine whether subdomain 2 of actin plays a role in this regulation, the DNase-I loop 38 -52 of this subdomain was cleaved by subtilisin between residues Met 47 and Gly 48 . Despite impaired unregulated function, the potentiation and regulation of cleaved actin movement in the in vitro motility assay was not significantly different from that of uncleaved actin. Stopped-flow measurements of ADP release from regulated and unregulated cleaved acto-S1 showed a marked increase in ADP release from acto-S1 in the presence of the regulatory complex. The enhancement of the actin affinity for S1 in the presence of regulatory proteins was greater for uncleaved than for cleaved F-actin. Finally, both cleaved and uncleaved actins protect myosin loop 1 from papain cleavage equally well. Our results suggest that the potentiation of actin function in the in vitro motility assay by regulatory proteins stems from changes in cross-bridge cycle kinetics. In addition, the unimpaired calcium-sensitive regulation of cleaved actin indicates that subdomain 2 conformation does not play an essential role in the regulation process.

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supporting

confidence: 64%

The Regulation of Subtilisin-cleaved Actin by Tropomyosin/Troponin

Pavlov¹,

Gerson²,

Yu³

et al. 2003

Journal of Biological Chemistry

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“…2 B and D) could supplant the originally proposed secondary binding site. There are (tropomyosin-dependent) cross-linking data supporting this suggestion (29). Actin residue 43, which is always hydrophobic (30), may interact with S1 residue 554 (Ͼ97% hydrophobic).…”

Section: Discussionmentioning

confidence: 88%

The structure of the acto-myosin subfragment 1 complex: Results of searches using data from electron microscopy and x-ray crystallography

Mendelson

Morris

1997

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

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Surmises of how myosin subfragment 1 (S1) interacts with actin filaments in muscle contraction rest upon knowing the relative arrangement of the two proteins. Although there exist crystallographic structures for both S1 and actin, as well as electron microscopy data for the acto-S1 complex (AS1), modeling of this arrangement has so far only been done ''by eye.'' Here we report fitted AS1 structures obtained using a quantitative method that is both more objective and makes more complete use of the data. Using undistorted crystallographic results, the best-fit AS1 structure shows significant differences from that obtained by visual fitting. The best fit is produced using the F-actin model of Holmes et al. . S1 residues at the AS1 interface are now found at a higher radius as well as being translated axially and rotated azimuthally. Fits using S1 plus loops missing from the crystal structure were achieved using a homology search method to predict loop structures. These improved fits favor an arrangement in which the loop at the 50-to 20-kDa domain junction of S1 is located near the N terminus of actin. Rigid-body movements of the lower 50-kDa domain, which further improve the fit, produce closure of the large 50-kDa domain cleft and bring conserved residues in the lower 50-kDa domain into an apparently appropriate orientation for close interaction with actin. This finding supports the idea that binding of ATP to AS1 at the end of the ATPase cycle disrupts the actin binding site by changing the conformation of the 50-kDa cleft of S1.The contraction of muscle occurs when the heads of thickfilament-based myosin interact with F-actin (FA) in adjacent thin filaments. This interaction is preceded by the hydrolysis of MgATP by detached myosin heads. The crystallography of actin (1, 2), the generation of a plausible model for FA (3-6), and the crystallography of the myosin head (subfragment 1; S1) (7) have led to the visual ''docking'' of S1 on FA using these structures and electron microscopy (EM) data (8, 9). This docking led to the first detailed model of the acto-S1 (AS1) interface in the ''rigor'' (no-ATP) state.Here we report the quantitative docking of S1 on FA using an extension of an approach used to study FA structure (5). In this method quantitative fitting is done computationally in reciprocal space rather than visually in real space. Thus the fitting is sensitive to all of the electron density in the EM data, not just the more readily visualized surface. Reciprocal space fitting also allows the removal of the least-well determined portion of the AS1 Fourier-Bessel transform-the equator. This removal accentuates higher spatial frequencies making the fitting more sensitive to small changes in the model. Our model resembles, but also significantly differs from, that obtained visually. These differences have consequences for the understanding of the molecular mechanism of muscle contraction. METHODSFitting Procedure. The fitting of AS1 model structures to the cryo-EM data of Milligan and colleagues (8) was...

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“…in the absence of Ca 2ϩ ) indicate that tropomyosin may bridge over subdomain 2 without extensive contact (4 (17)(18)(19)(20). Actin mutants provide an alternative approach to assess specific interactions between tropomyosin and actin.…”

Section: Discussionmentioning

confidence: 99%

“…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 important for tropomyosin and troponin function. Recently, we have shown that Saccharomyces cerevisiae actin can serve as a model system for examining troponin-tropomyosin-mediated regulation (21).…”

mentioning

confidence: 99%

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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Tropomyosin Inhibits the Glutaraldehyde-Induced Cross-Link between the Central 48-kDa Fragment of Myosin Head and Segment 48-67 in Actin Subdomain 2

Cited by 17 publications

References 85 publications

The Regulation of Subtilisin-cleaved Actin by Tropomyosin/Troponin

The Regulation of Subtilisin-cleaved Actin by Tropomyosin/Troponin

The structure of the acto-myosin subfragment 1 complex: Results of searches using data from electron microscopy and x-ray crystallography

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

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