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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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Results of the IHRSR method can be seen for singly decorated (bottom) and half- decorated filament segments (top). An atomic model of F-actin (Holmes et al., 1990) has been filtered to low resolution (left), and this is used as the starting point for subsequent cycles of the procedure. Images of filament segments, each containing about 14 actin subunits, have been sorted based upon both differences in twist and in the binding of ut261 (Figs. 2 and 3). The morphing of the half decorated filaments from the starting actin model (left) during this procedure is shown after 2, 5, and 10 cycles using 1,396 filament segments. The green arrow indicates the single density due to ut261 that emerges. For the 772 segments classified as singly decorated, a stable solution requires more iterations. The results after 5, 10, and 20 cycles are shown. The blue arrow indicates the density that we have interpreted as being due to CH2 (Fig. 6).
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fig4: Results of the IHRSR method can be seen for singly decorated (bottom) and half- decorated filament segments (top). An atomic model of F-actin (Holmes et al., 1990) has been filtered to low resolution (left), and this is used as the starting point for subsequent cycles of the procedure. Images of filament segments, each containing about 14 actin subunits, have been sorted based upon both differences in twist and in the binding of ut261 (Figs. 2 and 3). The morphing of the half decorated filaments from the starting actin model (left) during this procedure is shown after 2, 5, and 10 cycles using 1,396 filament segments. The green arrow indicates the single density due to ut261 that emerges. For the 772 segments classified as singly decorated, a stable solution requires more iterations. The results after 5, 10, and 20 cycles are shown. The blue arrow indicates the density that we have interpreted as being due to CH2 (Fig. 6).

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)

Results of the IHRSR method can be seen for singly decorated (bottom) and half- decorated filament segments (top). An atomic model of F-actin (Holmes et al., 1990) has been filtered to low resolution (left), and this is used as the starting point for subsequent cycles of the procedure. Images of filament segments, each containing about 14 actin subunits, have been sorted based upon both differences in twist and in the binding of ut261 (Figs. 2 and 3). The morphing of the half decorated filaments from the starting actin model (left) during this procedure is shown after 2, 5, and 10 cycles using 1,396 filament segments. The green arrow indicates the single density due to ut261 that emerges. For the 772 segments classified as singly decorated, a stable solution requires more iterations. The results after 5, 10, and 20 cycles are shown. The blue arrow indicates the density that we have interpreted as being due to CH2 (Fig. 6).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199260&req=5

fig4: Results of the IHRSR method can be seen for singly decorated (bottom) and half- decorated filament segments (top). An atomic model of F-actin (Holmes et al., 1990) has been filtered to low resolution (left), and this is used as the starting point for subsequent cycles of the procedure. Images of filament segments, each containing about 14 actin subunits, have been sorted based upon both differences in twist and in the binding of ut261 (Figs. 2 and 3). The morphing of the half decorated filaments from the starting actin model (left) during this procedure is shown after 2, 5, and 10 cycles using 1,396 filament segments. The green arrow indicates the single density due to ut261 that emerges. For the 772 segments classified as singly decorated, a stable solution requires more iterations. The results after 5, 10, and 20 cycles are shown. The blue arrow indicates the density that we have interpreted as being due to CH2 (Fig. 6).
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