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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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Surfaces (A–C) and cross-sections (D–F) of reconstructions from pure β-actin (A and D), half-decorated filaments (B and E), and singly decorated filaments (C and F). The half decoration corresponds to one utrophin ABD (containing the two CH domains, labeled CH1, and CH2) per two actin subunits, whereas the single decoration corresponds to one utrophin ABD for each actin subunit (Fig. 6). The actin subdomains 1–4 are labeled (A and B), with subdomains 1 and 2 from a different actin subunit labeled as 1′ and 2′. In D, subdomains 2 and 4 come from a subunit on the opposite strand, and are labeled 2′ and 4′, respectively. The nucleotide-binding cleft in the pure β-actin (*), opens up with respect to the structure of the α-actin-ATP subunit (Kabsch et al., 1990) used for the starting model (Fig. 4). In contrast, this cleft appears to be closed in both the half-decorated (B, *) and singly decorated state (C). In the half-decorated mode, the additional mass that is seen associated with each actin subunit is an average of both CH1 and CH2, as only one of these can be bound at each site. The surfaces correspond to 100% of the expected molecular volume for pure actin (A), one ut261 fragment for every two actins (B), and one ut261 for every actin (C).
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fig5: Surfaces (A–C) and cross-sections (D–F) of reconstructions from pure β-actin (A and D), half-decorated filaments (B and E), and singly decorated filaments (C and F). The half decoration corresponds to one utrophin ABD (containing the two CH domains, labeled CH1, and CH2) per two actin subunits, whereas the single decoration corresponds to one utrophin ABD for each actin subunit (Fig. 6). The actin subdomains 1–4 are labeled (A and B), with subdomains 1 and 2 from a different actin subunit labeled as 1′ and 2′. In D, subdomains 2 and 4 come from a subunit on the opposite strand, and are labeled 2′ and 4′, respectively. The nucleotide-binding cleft in the pure β-actin (*), opens up with respect to the structure of the α-actin-ATP subunit (Kabsch et al., 1990) used for the starting model (Fig. 4). In contrast, this cleft appears to be closed in both the half-decorated (B, *) and singly decorated state (C). In the half-decorated mode, the additional mass that is seen associated with each actin subunit is an average of both CH1 and CH2, as only one of these can be bound at each site. The surfaces correspond to 100% of the expected molecular volume for pure actin (A), one ut261 fragment for every two actins (B), and one ut261 for every actin (C).

Mentions: In the half-decorated mode (Fig. 5, B and E) there is one compact density added to F-actin for every actin subunit in the filament, and this density (Fig. 5, B and E, black arrows) is located between subdomain 2 of one actin subunit and subdomain 1 of the actin subunit above it on the same long-pitch helical strand. The location of this density is very similar to what has been described for the binding of a fimbrin fragment to F-actin (Hanein et al., 1997, 1998). However, in contrast to that study, in which the density attributable to the fimbrin fragment was much weaker in the reconstruction than that attributable to actin, we see the same peak density levels for both actin and the additional mass due to ut261 (Fig. 5 E). We think that this results from the IHRSR single particle sorting, where a large number of segments (∼40%) containing incomplete or nonhomogeneous binding of ut261 were eliminated.


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)

Surfaces (A–C) and cross-sections (D–F) of reconstructions from pure β-actin (A and D), half-decorated filaments (B and E), and singly decorated filaments (C and F). The half decoration corresponds to one utrophin ABD (containing the two CH domains, labeled CH1, and CH2) per two actin subunits, whereas the single decoration corresponds to one utrophin ABD for each actin subunit (Fig. 6). The actin subdomains 1–4 are labeled (A and B), with subdomains 1 and 2 from a different actin subunit labeled as 1′ and 2′. In D, subdomains 2 and 4 come from a subunit on the opposite strand, and are labeled 2′ and 4′, respectively. The nucleotide-binding cleft in the pure β-actin (*), opens up with respect to the structure of the α-actin-ATP subunit (Kabsch et al., 1990) used for the starting model (Fig. 4). In contrast, this cleft appears to be closed in both the half-decorated (B, *) and singly decorated state (C). In the half-decorated mode, the additional mass that is seen associated with each actin subunit is an average of both CH1 and CH2, as only one of these can be bound at each site. The surfaces correspond to 100% of the expected molecular volume for pure actin (A), one ut261 fragment for every two actins (B), and one ut261 for every actin (C).
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Related In: Results  -  Collection

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fig5: Surfaces (A–C) and cross-sections (D–F) of reconstructions from pure β-actin (A and D), half-decorated filaments (B and E), and singly decorated filaments (C and F). The half decoration corresponds to one utrophin ABD (containing the two CH domains, labeled CH1, and CH2) per two actin subunits, whereas the single decoration corresponds to one utrophin ABD for each actin subunit (Fig. 6). The actin subdomains 1–4 are labeled (A and B), with subdomains 1 and 2 from a different actin subunit labeled as 1′ and 2′. In D, subdomains 2 and 4 come from a subunit on the opposite strand, and are labeled 2′ and 4′, respectively. The nucleotide-binding cleft in the pure β-actin (*), opens up with respect to the structure of the α-actin-ATP subunit (Kabsch et al., 1990) used for the starting model (Fig. 4). In contrast, this cleft appears to be closed in both the half-decorated (B, *) and singly decorated state (C). In the half-decorated mode, the additional mass that is seen associated with each actin subunit is an average of both CH1 and CH2, as only one of these can be bound at each site. The surfaces correspond to 100% of the expected molecular volume for pure actin (A), one ut261 fragment for every two actins (B), and one ut261 for every actin (C).
Mentions: In the half-decorated mode (Fig. 5, B and E) there is one compact density added to F-actin for every actin subunit in the filament, and this density (Fig. 5, B and E, black arrows) is located between subdomain 2 of one actin subunit and subdomain 1 of the actin subunit above it on the same long-pitch helical strand. The location of this density is very similar to what has been described for the binding of a fimbrin fragment to F-actin (Hanein et al., 1997, 1998). However, in contrast to that study, in which the density attributable to the fimbrin fragment was much weaker in the reconstruction than that attributable to actin, we see the same peak density levels for both actin and the additional mass due to ut261 (Fig. 5 E). We think that this results from the IHRSR single particle sorting, where a large number of segments (∼40%) containing incomplete or nonhomogeneous binding of ut261 were eliminated.

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