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The utrophin actin-binding domain binds F-actin in two different modes: implications for the spectrin superfamily of proteins.

Galkin VE, Orlova A, VanLoock MS, Rybakova IN, Ervasti JM, Egelman EH - J. Cell Biol. (2002)

Bottom Line: The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments.When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin.The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.

ABSTRACT
Utrophin, like its homologue dystrophin, forms a link between the actin cytoskeleton and the extracellular matrix. We have used a new method of image analysis to reconstruct actin filaments decorated with the actin-binding domain of utrophin, which contains two calponin homology domains. We find two different modes of binding, with either one or two calponin-homology (CH) domains bound per actin subunit, and these modes are also distinguishable by their very different effects on F-actin rigidity. Both modes involve an extended conformation of the CH domains, as predicted by a previous crystal structure. The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments. When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin. The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.

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Electron micrographs of negatively stained β-actin filaments alone (A) and decorated with the utrophin ABD (B). Two different forms of decoration can be seen (B). Lightly stained, rigid filaments (white arrow) and darkly stained, flexible filaments (black arrow). The darkly stained filaments tend to aggregate extensively, whereas such aggregation is not seen with the lightly stained filaments. Bar, 2,000 Å.
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fig1: Electron micrographs of negatively stained β-actin filaments alone (A) and decorated with the utrophin ABD (B). Two different forms of decoration can be seen (B). Lightly stained, rigid filaments (white arrow) and darkly stained, flexible filaments (black arrow). The darkly stained filaments tend to aggregate extensively, whereas such aggregation is not seen with the lightly stained filaments. Bar, 2,000 Å.

Mentions: Electron micrographs show two different filament states after the incubation of β-actin filaments (Fig. 1 A) with the ut261 utrophin fragment (Fig. 1 B). Relatively rigid filaments or segments of filaments can be seen (Fig. 1 B, white arrow) that appear different from undecorated filaments. But more massive, flexible filaments or segments of filaments (Fig. 1 B, black arrow) can also be seen. These massive (dark) filaments are more likely to aggregate, whereas the light filaments are less likely to aggregate than the undecorated control filaments. Under the conditions that we are using, we expect rather complete saturation of the actin by ut261, as the binding constant that we measure for ut261 by cosedimentation is 7.1 ± 4.1 μM with a stoichiometry of 1:1 (unpublished data). These values are consistent with most previously published results (Winder, 1996; Keep et al., 1999; Moores and Kendrick-Jones, 2000; Zuellig et al., 2000), except Winder et al. (1995), where a stoichiometry of 2:1 (ut261:actin) was reported.


The utrophin actin-binding domain binds F-actin in two different modes: implications for the spectrin superfamily of proteins.

Galkin VE, Orlova A, VanLoock MS, Rybakova IN, Ervasti JM, Egelman EH - J. Cell Biol. (2002)

Electron micrographs of negatively stained β-actin filaments alone (A) and decorated with the utrophin ABD (B). Two different forms of decoration can be seen (B). Lightly stained, rigid filaments (white arrow) and darkly stained, flexible filaments (black arrow). The darkly stained filaments tend to aggregate extensively, whereas such aggregation is not seen with the lightly stained filaments. Bar, 2,000 Å.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199260&req=5

fig1: Electron micrographs of negatively stained β-actin filaments alone (A) and decorated with the utrophin ABD (B). Two different forms of decoration can be seen (B). Lightly stained, rigid filaments (white arrow) and darkly stained, flexible filaments (black arrow). The darkly stained filaments tend to aggregate extensively, whereas such aggregation is not seen with the lightly stained filaments. Bar, 2,000 Å.
Mentions: Electron micrographs show two different filament states after the incubation of β-actin filaments (Fig. 1 A) with the ut261 utrophin fragment (Fig. 1 B). Relatively rigid filaments or segments of filaments can be seen (Fig. 1 B, white arrow) that appear different from undecorated filaments. But more massive, flexible filaments or segments of filaments (Fig. 1 B, black arrow) can also be seen. These massive (dark) filaments are more likely to aggregate, whereas the light filaments are less likely to aggregate than the undecorated control filaments. Under the conditions that we are using, we expect rather complete saturation of the actin by ut261, as the binding constant that we measure for ut261 by cosedimentation is 7.1 ± 4.1 μM with a stoichiometry of 1:1 (unpublished data). These values are consistent with most previously published results (Winder, 1996; Keep et al., 1999; Moores and Kendrick-Jones, 2000; Zuellig et al., 2000), except Winder et al. (1995), where a stoichiometry of 2:1 (ut261:actin) was reported.

Bottom Line: The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments.When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin.The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.

ABSTRACT
Utrophin, like its homologue dystrophin, forms a link between the actin cytoskeleton and the extracellular matrix. We have used a new method of image analysis to reconstruct actin filaments decorated with the actin-binding domain of utrophin, which contains two calponin homology domains. We find two different modes of binding, with either one or two calponin-homology (CH) domains bound per actin subunit, and these modes are also distinguishable by their very different effects on F-actin rigidity. Both modes involve an extended conformation of the CH domains, as predicted by a previous crystal structure. The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments. When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin. The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.

Show MeSH
Related in: MedlinePlus