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Evidence for a conformational change in actin induced by fimbrin (N375) binding.

Hanein D, Matsudaira P, DeRosier DJ - J. Cell Biol. (1997)

Bottom Line: The binding of N375 changes actin, which we interpret as a movement of subdomain 1 away from the bound N375.This change in actin structure may affect its affinity for other actin-binding proteins and may be part of the regulation of the cytoskeleton itself.Difference maps between actin and actin decorated with other proteins provides a way to look for novel structural changes in actin.

View Article: PubMed Central - PubMed

Affiliation: The W.M. Keck Institute for Cellular Visualization and The Rosenstiel Basic Medical Sciences Research Center, Department of Biology, Brandeis University, Waltham, Massachusetts 02254, USA.

ABSTRACT
Fimbrin belongs to a superfamily of actin cross-linking proteins that share a conserved 27-kD actin-binding domain. This domain contains a tandem duplication of a sequence that is homologous to calponin. Calponin homology (CH) domains not only cross-link actin filaments into bundles and networks, but they also bind intermediate filaments and some signal transduction proteins to the actin cytoskeleton. This fundamental role of CH domains as a widely used actin-binding domain underlines the necessity to understand their structural interaction with actin. Using electron cryomicroscopy, we have determined the three-dimensional structure of F-actin and F-actin decorated with the NH2-terminal CH domains of fimbrin (N375). In a difference map between actin filaments and N375-decorated actin, one end of N375 is bound to a concave surface formed between actin subdomains 1 and 2 on two neighboring actin monomers. In addition, a fit of the atomic model for the actin filament to the maps reveals the actin residues that line, the binding surface. The binding of N375 changes actin, which we interpret as a movement of subdomain 1 away from the bound N375. This change in actin structure may affect its affinity for other actin-binding proteins and may be part of the regulation of the cytoskeleton itself. Difference maps between actin and actin decorated with other proteins provides a way to look for novel structural changes in actin.

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Sections through the F-actin reconstruction (F-actin)  and the Actin-N375 reconstruction (Actin-N375). Sections are  taken from the barbed end towards the pointed end. The sections  are spaced 4.3 Å apart. The difference map between the two reconstructions is presented in the right column. The positive differences are contoured using black lines. Peak 1 corresponds to  the N375 portion of the map, and peak 2 corresponds to the conformational change in subdomain 1. Contours corresponding to  the actin map are superimposed on the difference contours. Contours start at the same density (4.0) in all maps. Contours are  plotted at intervals of 6.3 in the F-actin and Actin-N375 maps and  at intervals of 1.1 in the difference map. Bar, 50 Å.
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Figure 3: Sections through the F-actin reconstruction (F-actin) and the Actin-N375 reconstruction (Actin-N375). Sections are taken from the barbed end towards the pointed end. The sections are spaced 4.3 Å apart. The difference map between the two reconstructions is presented in the right column. The positive differences are contoured using black lines. Peak 1 corresponds to the N375 portion of the map, and peak 2 corresponds to the conformational change in subdomain 1. Contours corresponding to the actin map are superimposed on the difference contours. Contours start at the same density (4.0) in all maps. Contours are plotted at intervals of 6.3 in the F-actin and Actin-N375 maps and at intervals of 1.1 in the difference map. Bar, 50 Å.

Mentions: Two positive difference peaks (bold contours) are seen in transverse sections through the difference map superimposed on the actin map (Fig. 3). The sections are spaced 4.3 Å apart in one asymmetric unit of the filament. For comparison, a surface view of the two positive peaks is shown in a stereo presentation in Fig 4 d. The first difference peak (1 in Fig. 3) sits at the outer surface of the actin map. The density is kidney shaped with dimensions corresponding to ∼50 × 25 × 25 Å. The dimensions are determined by the volume over which the difference is significant; it is not set to fit the expected volume of N375, for example. Peak 1 appears in Fig. 3, a–c, and it reappears on the other side of the actin filament in Fig. 3, e, f, and g. This region of extra density extends from actin subdomains 1 and 2. The variance corresponding to peak 1 is low (data not shown).


Evidence for a conformational change in actin induced by fimbrin (N375) binding.

Hanein D, Matsudaira P, DeRosier DJ - J. Cell Biol. (1997)

Sections through the F-actin reconstruction (F-actin)  and the Actin-N375 reconstruction (Actin-N375). Sections are  taken from the barbed end towards the pointed end. The sections  are spaced 4.3 Å apart. The difference map between the two reconstructions is presented in the right column. The positive differences are contoured using black lines. Peak 1 corresponds to  the N375 portion of the map, and peak 2 corresponds to the conformational change in subdomain 1. Contours corresponding to  the actin map are superimposed on the difference contours. Contours start at the same density (4.0) in all maps. Contours are  plotted at intervals of 6.3 in the F-actin and Actin-N375 maps and  at intervals of 1.1 in the difference map. Bar, 50 Å.
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Related In: Results  -  Collection

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Figure 3: Sections through the F-actin reconstruction (F-actin) and the Actin-N375 reconstruction (Actin-N375). Sections are taken from the barbed end towards the pointed end. The sections are spaced 4.3 Å apart. The difference map between the two reconstructions is presented in the right column. The positive differences are contoured using black lines. Peak 1 corresponds to the N375 portion of the map, and peak 2 corresponds to the conformational change in subdomain 1. Contours corresponding to the actin map are superimposed on the difference contours. Contours start at the same density (4.0) in all maps. Contours are plotted at intervals of 6.3 in the F-actin and Actin-N375 maps and at intervals of 1.1 in the difference map. Bar, 50 Å.
Mentions: Two positive difference peaks (bold contours) are seen in transverse sections through the difference map superimposed on the actin map (Fig. 3). The sections are spaced 4.3 Å apart in one asymmetric unit of the filament. For comparison, a surface view of the two positive peaks is shown in a stereo presentation in Fig 4 d. The first difference peak (1 in Fig. 3) sits at the outer surface of the actin map. The density is kidney shaped with dimensions corresponding to ∼50 × 25 × 25 Å. The dimensions are determined by the volume over which the difference is significant; it is not set to fit the expected volume of N375, for example. Peak 1 appears in Fig. 3, a–c, and it reappears on the other side of the actin filament in Fig. 3, e, f, and g. This region of extra density extends from actin subdomains 1 and 2. The variance corresponding to peak 1 is low (data not shown).

Bottom Line: The binding of N375 changes actin, which we interpret as a movement of subdomain 1 away from the bound N375.This change in actin structure may affect its affinity for other actin-binding proteins and may be part of the regulation of the cytoskeleton itself.Difference maps between actin and actin decorated with other proteins provides a way to look for novel structural changes in actin.

View Article: PubMed Central - PubMed

Affiliation: The W.M. Keck Institute for Cellular Visualization and The Rosenstiel Basic Medical Sciences Research Center, Department of Biology, Brandeis University, Waltham, Massachusetts 02254, USA.

ABSTRACT
Fimbrin belongs to a superfamily of actin cross-linking proteins that share a conserved 27-kD actin-binding domain. This domain contains a tandem duplication of a sequence that is homologous to calponin. Calponin homology (CH) domains not only cross-link actin filaments into bundles and networks, but they also bind intermediate filaments and some signal transduction proteins to the actin cytoskeleton. This fundamental role of CH domains as a widely used actin-binding domain underlines the necessity to understand their structural interaction with actin. Using electron cryomicroscopy, we have determined the three-dimensional structure of F-actin and F-actin decorated with the NH2-terminal CH domains of fimbrin (N375). In a difference map between actin filaments and N375-decorated actin, one end of N375 is bound to a concave surface formed between actin subdomains 1 and 2 on two neighboring actin monomers. In addition, a fit of the atomic model for the actin filament to the maps reveals the actin residues that line, the binding surface. The binding of N375 changes actin, which we interpret as a movement of subdomain 1 away from the bound N375. This change in actin structure may affect its affinity for other actin-binding proteins and may be part of the regulation of the cytoskeleton itself. Difference maps between actin and actin decorated with other proteins provides a way to look for novel structural changes in actin.

Show MeSH