Tropomyosin-Troponin Regulation of Actin Does Not Involve Subdomain 2 Motions

Gerson, Jack H.; Kim, Eldar; Mühlrád, András; Reisler, Emil

doi:10.1074/jbc.m011070200

Cited by 9 publications

(4 citation statements)

References 49 publications

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“…Mutant A144C/C374A was chosen to test for the binding of DrABD to actin's hydrophobic cleft between subdomains 1 and 3, which is known to interact with several actin-binding proteins. All yeast actin mutants employed in this study show normal polymerization properties (20; 21). …”

Section: Resultsmentioning

confidence: 99%

Mapping of Drebrin Binding Site on F-Actin

Grintsevich

Galkin

Orlova

et al. 2010

Journal of Molecular Biology

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Drebrin is a filament binding protein involved in organizing the dendritic pool of actin. Previous in vivo studies identified the actin-binding domain of drebrin (DrABD), which causes the same rearrangements in the cytoskeleton as the full length protein. Site directed mutagenesis, electron microscopic (EM) reconstruction and chemical cross-linking combined with mass spectrometry analysis were employed here to map the DrABD binding interface on actin filaments. DrABD could be simultaneously attached to two adjacent actin protomers using the combination of 2-iminothiolane (Traut's reagent) and 1,1-methanediyl bis(methanethiosulfonate) (MTS1). Site directed mutagenesis combined with chemical cross-linking revealed that residue 238 of DrABD is located within 5.4 Å from C374 of actin protomer 1, and drebrin's native cysteine 308 is in close proximity to C374 of actin protomer 2. Mass spectrometry analysis revealed that a zero length cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), can link the N-terminal G-S extension of the recombinant DrABD, to E99 and/or E100 on actin. Efficient cross-linking of drebrin residues 238, 248, 252, 270, and 271 to actin residue 51 was achieved with reagents of different length (5.4 -19 Å). These results suggest that the 'core' DrABD is centered on actin's subdomain 2 and may adopt a folded conformation upon binding to F-actin. The results of EM reconstruction, which are in a good agreement with the cross-linking data, revealed polymorphism in DrABD binding to F-actin and suggested the existence of two binding sites. These results provide new structural insight into the previously observed competition between drebrin and several other F-actin binding proteins.

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Section: Resultsmentioning

confidence: 99%

Mapping of Drebrin Binding Site on F-Actin

Grintsevich

Galkin

Orlova

et al. 2010

Journal of Molecular Biology

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“…X-ray diffraction patterns of oriented F-actin gels revealed a small radial shift of loop 38 -52 on Tm binding to actin (32), and possible contribution of subdomain 2 movements within actin protomers to changes in x-ray diffraction patterns from muscle in response to Ca 2ϩ activation of thin filament has been discussed (33). On the other hand, fluorescence studies on both muscle actin and yeast actin mutants with probes on residues 41 or 51 indicated that transitions among the blocked, closed, and open regulatory states involve no significant subdomain 2 movement (34). A most recent study of effects of the regulatory proteins on the affinity for myosin S1 and in vitro motility of subtilisin-cleaved actin has also led to the conclusion that subdomain 2 conformation does not play an active role in the regulation of actomyosin interactions (35).…”

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confidence: 99%

The DNase-I Binding Loop of Actin May Play a Role in the Regulation of Actin-Myosin Interaction by Tropomyosin/Troponin

Moraczewska

Gruszczynska-Biegala

Redowicz

et al. 2004

Journal of Biological Chemistry

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, on its interaction with myosin S1 in the presence and absence of tropomyosin or tropomyosin-troponin. Both individual modifications reduced activation of S1 ATPase by actin to a similar extent. The effect of ECP cleavage, but not of subtilisin cleavage, was partially reversed by stabilization of interprotomer contacts with phalloidin, indicating different pathways of signal transmission from the N-and C-terminal parts of loop 38 -52 to myosin binding sites. ECP cleavage diminished the affinity to tropomyosin and reduced its inhibition of acto-S1 ATPase at low S1 concentrations, but increased the tropomyosin-mediated cooperative enhancement of the ATPase by S1 binding to actin. These effects were reversed by phalloidin. Subtilisin-cleaved actin more closely resembled unmodified actin than the ECP-modified actin. Limited proteolysis of the modified and unmodified F-actins revealed an allosteric effect of ECP cleavage on the conformation of the actin subdomain 4 region that is presumably involved in tropomyosin binding. Our results point to a possible role of the N-terminal part of loop 38 -52 of actin in communication between tropomyosin and myosin through changes in actin structure.

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“…to the closed state position (16). This regulation model does not evoke any companion dynamic changes on actin and, therefore, is not in conflict with the apparent absence of subdomain 2 motions during actin regulation (17).…”

Section: Fig 2 S1⅐adp Binding To Uncleaved and Cleaved Actins With mentioning

confidence: 99%

“…A similar conclusion was reached in an electron microscopic study of thin filaments containing mutant tropomyosin, in which troponin density was found near Met 47 (although troponin more strongly contacted subdomain 1 than subdomain 2) (7). Although previous work from our laboratory (17) did not detect any subdomain 2 movements during regulation, all of our experiments used probes attached to Gln 41 (in ␣-skeletal actin) or Cys 41 and Cys 51 (in mutant yeast actins). Because such probes may have altered the dynamics, if not the conformation, of an intrinsically dynamic subdomain 2, and because TnI cross-links to Met 47 on actin, we tested for Tm/Tn interactions with this region using subtilisin-cleaved actin (3,18).…”

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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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Tropomyosin-Troponin Regulation of Actin Does Not Involve Subdomain 2 Motions

Cited by 9 publications

References 49 publications

Mapping of Drebrin Binding Site on F-Actin

Mapping of Drebrin Binding Site on F-Actin

The DNase-I Binding Loop of Actin May Play a Role in the Regulation of Actin-Myosin Interaction by Tropomyosin/Troponin

The Regulation of Subtilisin-cleaved Actin by Tropomyosin/Troponin

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