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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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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).
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fig3: 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).

Mentions: Analysis of a total of ∼22,500 segments, each containing ∼14 actin subunits, revealed that three different populations could be found within the decorated filaments. These are two different modes of decoration by ut261, and a third category which contained undecorated, partially decorated actin, or disordered binding. The IHRSR approach (Figs. 2, 3, and 4) was key to the separation of these modes, since conventional helical analysis (DeRosier and Klug, 1968) would tend to average these states together. Although we find that there is a cooperativity in the mode of binding, as evidenced by the fact that different filament types can be differentiated by eye in electron micrographs (Fig. 1), detailed analysis shows that this cooperativity is far from complete. Two different independent methods were employed to sort and classify the filament segments used for three-dimensional reconstruction. One approach is based upon cross-correlations with projections of reference volumes (Fig. 2), whereas the second approach is based upon using differences in the two-dimensional radial density distributions within images for sorting (Fig. 3).


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)

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).
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fig3: 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).
Mentions: Analysis of a total of ∼22,500 segments, each containing ∼14 actin subunits, revealed that three different populations could be found within the decorated filaments. These are two different modes of decoration by ut261, and a third category which contained undecorated, partially decorated actin, or disordered binding. The IHRSR approach (Figs. 2, 3, and 4) was key to the separation of these modes, since conventional helical analysis (DeRosier and Klug, 1968) would tend to average these states together. Although we find that there is a cooperativity in the mode of binding, as evidenced by the fact that different filament types can be differentiated by eye in electron micrographs (Fig. 1), detailed analysis shows that this cooperativity is far from complete. Two different independent methods were employed to sort and classify the filament segments used for three-dimensional reconstruction. One approach is based upon cross-correlations with projections of reference volumes (Fig. 2), whereas the second approach is based upon using differences in the two-dimensional radial density distributions within images for sorting (Fig. 3).

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