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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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Related in: MedlinePlus

The crystal structure of the utrophin ABD (Keep et al., 1999) can be used to completely explain both modes of binding observed. The half decoration (A) can be fit with one ABD for every two actin subunits along the same long-pitch helical strand. In this mode of binding, there is either a CH1 or CH2 domain bound to every actin subunit, and the density in the reconstruction additional to actin results from an average of both the CH1 and the CH2 domains. The binding in this mode is between subdomain 2 of one subunit and subdomain 1 of the subunit above it on the same long pitch strand. In the single-decoration mode (B), there is one ABD bound to each actin subunit. The actin-binding surfaces of utrophin (Keep et al., 1999) are shown in yellow. In B, three of the inserts in the actin sequence that are not present in bacterial MreB (Egelman, 2001b; van den Ent et al., 2001) are shown in red. These inserts are residues 40–48 (subdomain 2, DNase I-binding loop), 228–235 (subdomain 4), and 353–375 (subdomain 1, C terminus). Each is involved in a contact with the utrophin ABD. The 40–48 and 353–375 inserts appear to be involved in both modes of binding. In contrast, the 228–235 insert makes a contact with the CH2 domain of an ABD, whose CH1 domain is bound to an actin subunit on the opposite strand (black arrows). The surface in B is at 140% of the expected molecular volume to show these contacts, but the model fits extremely well at 100% volume. Steric clashes exist between the DNase I-binding loop of actin's subdomain 2 and CH1 in B, and both CH1 and CH2 in A. An extensive literature on the mobility of this loop (Egelman, 2001a) suggests that it might be repositioned in the complex. The regions in actin that are likely to be involved in contacts with utrophin are shown in either green (223–230) or red (C). Both models for utrophin binding (A and B) involve relative domain shifts between CH1 and CH2 of the utrophin ABD from the crystal structure (Keep et al., 1999). The shifts of the CH2 domains that have been used are shown in D, where the unperturbed crystal structure is in green, the fit to the half-decorated state is in yellow (∼145° rotation from crystal structure), and the fit to the singly decorated state is in blue (∼110° rotation from the crystal structure), after the CH1 domains have been aligned. The magnitude of these shifts is similar to what is observed when the homologous ABD from dystrophin (Norwood et al., 2000), shown in red, is aligned with the CH1 from utrophin.
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fig6: The crystal structure of the utrophin ABD (Keep et al., 1999) can be used to completely explain both modes of binding observed. The half decoration (A) can be fit with one ABD for every two actin subunits along the same long-pitch helical strand. In this mode of binding, there is either a CH1 or CH2 domain bound to every actin subunit, and the density in the reconstruction additional to actin results from an average of both the CH1 and the CH2 domains. The binding in this mode is between subdomain 2 of one subunit and subdomain 1 of the subunit above it on the same long pitch strand. In the single-decoration mode (B), there is one ABD bound to each actin subunit. The actin-binding surfaces of utrophin (Keep et al., 1999) are shown in yellow. In B, three of the inserts in the actin sequence that are not present in bacterial MreB (Egelman, 2001b; van den Ent et al., 2001) are shown in red. These inserts are residues 40–48 (subdomain 2, DNase I-binding loop), 228–235 (subdomain 4), and 353–375 (subdomain 1, C terminus). Each is involved in a contact with the utrophin ABD. The 40–48 and 353–375 inserts appear to be involved in both modes of binding. In contrast, the 228–235 insert makes a contact with the CH2 domain of an ABD, whose CH1 domain is bound to an actin subunit on the opposite strand (black arrows). The surface in B is at 140% of the expected molecular volume to show these contacts, but the model fits extremely well at 100% volume. Steric clashes exist between the DNase I-binding loop of actin's subdomain 2 and CH1 in B, and both CH1 and CH2 in A. An extensive literature on the mobility of this loop (Egelman, 2001a) suggests that it might be repositioned in the complex. The regions in actin that are likely to be involved in contacts with utrophin are shown in either green (223–230) or red (C). Both models for utrophin binding (A and B) involve relative domain shifts between CH1 and CH2 of the utrophin ABD from the crystal structure (Keep et al., 1999). The shifts of the CH2 domains that have been used are shown in D, where the unperturbed crystal structure is in green, the fit to the half-decorated state is in yellow (∼145° rotation from crystal structure), and the fit to the singly decorated state is in blue (∼110° rotation from the crystal structure), after the CH1 domains have been aligned. The magnitude of these shifts is similar to what is observed when the homologous ABD from dystrophin (Norwood et al., 2000), shown in red, is aligned with the CH1 from utrophin.

Mentions: Sorting of images based on projected density. Image segments were initially aligned (shifted in x and y, and rotated in plane) against reference projections of a pure F-actin reconstruction (Orlova et al., 2001). Segments with shifts of >5 pixels, or with rotations from 0° or 180° greater than 10° were discarded. This reduced the 16,070 light segments to 11,698, and reduced the 6,240 dark segments to 3,533. The projected density within columns of pixels corresponding to a particular radial band was then integrated. This radial band was 40–56 Å from the helical axis for the light segments A, and 45–55 Å from the helical axis for the dark segments B. These radial limits were chosen based upon analysis of where the greatest differences occurred from pure F-actin, and these bands are indicated by the bars under the inset images (A and B). The resulting histograms of density within these radial bands are shown for the light segments A and the dark segments B. Images were then sorted into groups based upon this density distribution. Reconstructions are shown for four groups in A: #1, n = 858, density from 11 to 40; #2, n = 4,369, density from 41 to 60; #3, n = 1,396, density from 81 to 100; and #4, n = 694, density from 91 to 110. The red arrows indicate the feature due to the bound ut261 that becomes progressively stronger from #1 to #3. However, in #4, a new feature appears (blue arrow) that is due to the second mode of binding, showing that the segments initially selected as light are predominantly the half-bound complex (Fig. 4), but contain some regions with single decoration. The same approach was done for the dark segments, which have been divided into three groups in (B): #1, n = 767, density from 21 to 60; #2, n = 1,811, density from 61 to 100; #3, n = 721, density from 101 to 140. It can be seen that the dark segments contain both modes of binding, as group #1 can be explained quite well by the half decoration, whereas only group #3 shows the saturated single binding that we interpret (Fig. 6) as CH1 (red arrow) and CH2 (blue arrow).


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)

The crystal structure of the utrophin ABD (Keep et al., 1999) can be used to completely explain both modes of binding observed. The half decoration (A) can be fit with one ABD for every two actin subunits along the same long-pitch helical strand. In this mode of binding, there is either a CH1 or CH2 domain bound to every actin subunit, and the density in the reconstruction additional to actin results from an average of both the CH1 and the CH2 domains. The binding in this mode is between subdomain 2 of one subunit and subdomain 1 of the subunit above it on the same long pitch strand. In the single-decoration mode (B), there is one ABD bound to each actin subunit. The actin-binding surfaces of utrophin (Keep et al., 1999) are shown in yellow. In B, three of the inserts in the actin sequence that are not present in bacterial MreB (Egelman, 2001b; van den Ent et al., 2001) are shown in red. These inserts are residues 40–48 (subdomain 2, DNase I-binding loop), 228–235 (subdomain 4), and 353–375 (subdomain 1, C terminus). Each is involved in a contact with the utrophin ABD. The 40–48 and 353–375 inserts appear to be involved in both modes of binding. In contrast, the 228–235 insert makes a contact with the CH2 domain of an ABD, whose CH1 domain is bound to an actin subunit on the opposite strand (black arrows). The surface in B is at 140% of the expected molecular volume to show these contacts, but the model fits extremely well at 100% volume. Steric clashes exist between the DNase I-binding loop of actin's subdomain 2 and CH1 in B, and both CH1 and CH2 in A. An extensive literature on the mobility of this loop (Egelman, 2001a) suggests that it might be repositioned in the complex. The regions in actin that are likely to be involved in contacts with utrophin are shown in either green (223–230) or red (C). Both models for utrophin binding (A and B) involve relative domain shifts between CH1 and CH2 of the utrophin ABD from the crystal structure (Keep et al., 1999). The shifts of the CH2 domains that have been used are shown in D, where the unperturbed crystal structure is in green, the fit to the half-decorated state is in yellow (∼145° rotation from crystal structure), and the fit to the singly decorated state is in blue (∼110° rotation from the crystal structure), after the CH1 domains have been aligned. The magnitude of these shifts is similar to what is observed when the homologous ABD from dystrophin (Norwood et al., 2000), shown in red, is aligned with the CH1 from utrophin.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: The crystal structure of the utrophin ABD (Keep et al., 1999) can be used to completely explain both modes of binding observed. The half decoration (A) can be fit with one ABD for every two actin subunits along the same long-pitch helical strand. In this mode of binding, there is either a CH1 or CH2 domain bound to every actin subunit, and the density in the reconstruction additional to actin results from an average of both the CH1 and the CH2 domains. The binding in this mode is between subdomain 2 of one subunit and subdomain 1 of the subunit above it on the same long pitch strand. In the single-decoration mode (B), there is one ABD bound to each actin subunit. The actin-binding surfaces of utrophin (Keep et al., 1999) are shown in yellow. In B, three of the inserts in the actin sequence that are not present in bacterial MreB (Egelman, 2001b; van den Ent et al., 2001) are shown in red. These inserts are residues 40–48 (subdomain 2, DNase I-binding loop), 228–235 (subdomain 4), and 353–375 (subdomain 1, C terminus). Each is involved in a contact with the utrophin ABD. The 40–48 and 353–375 inserts appear to be involved in both modes of binding. In contrast, the 228–235 insert makes a contact with the CH2 domain of an ABD, whose CH1 domain is bound to an actin subunit on the opposite strand (black arrows). The surface in B is at 140% of the expected molecular volume to show these contacts, but the model fits extremely well at 100% volume. Steric clashes exist between the DNase I-binding loop of actin's subdomain 2 and CH1 in B, and both CH1 and CH2 in A. An extensive literature on the mobility of this loop (Egelman, 2001a) suggests that it might be repositioned in the complex. The regions in actin that are likely to be involved in contacts with utrophin are shown in either green (223–230) or red (C). Both models for utrophin binding (A and B) involve relative domain shifts between CH1 and CH2 of the utrophin ABD from the crystal structure (Keep et al., 1999). The shifts of the CH2 domains that have been used are shown in D, where the unperturbed crystal structure is in green, the fit to the half-decorated state is in yellow (∼145° rotation from crystal structure), and the fit to the singly decorated state is in blue (∼110° rotation from the crystal structure), after the CH1 domains have been aligned. The magnitude of these shifts is similar to what is observed when the homologous ABD from dystrophin (Norwood et al., 2000), shown in red, is aligned with the CH1 from utrophin.
Mentions: Sorting of images based on projected density. Image segments were initially aligned (shifted in x and y, and rotated in plane) against reference projections of a pure F-actin reconstruction (Orlova et al., 2001). Segments with shifts of >5 pixels, or with rotations from 0° or 180° greater than 10° were discarded. This reduced the 16,070 light segments to 11,698, and reduced the 6,240 dark segments to 3,533. The projected density within columns of pixels corresponding to a particular radial band was then integrated. This radial band was 40–56 Å from the helical axis for the light segments A, and 45–55 Å from the helical axis for the dark segments B. These radial limits were chosen based upon analysis of where the greatest differences occurred from pure F-actin, and these bands are indicated by the bars under the inset images (A and B). The resulting histograms of density within these radial bands are shown for the light segments A and the dark segments B. Images were then sorted into groups based upon this density distribution. Reconstructions are shown for four groups in A: #1, n = 858, density from 11 to 40; #2, n = 4,369, density from 41 to 60; #3, n = 1,396, density from 81 to 100; and #4, n = 694, density from 91 to 110. The red arrows indicate the feature due to the bound ut261 that becomes progressively stronger from #1 to #3. However, in #4, a new feature appears (blue arrow) that is due to the second mode of binding, showing that the segments initially selected as light are predominantly the half-bound complex (Fig. 4), but contain some regions with single decoration. The same approach was done for the dark segments, which have been divided into three groups in (B): #1, n = 767, density from 21 to 60; #2, n = 1,811, density from 61 to 100; #3, n = 721, density from 101 to 140. It can be seen that the dark segments contain both modes of binding, as group #1 can be explained quite well by the half decoration, whereas only group #3 shows the saturated single binding that we interpret (Fig. 6) as CH1 (red arrow) and CH2 (blue arrow).

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