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The structure of the C-terminal actin-binding domain of talin.

Gingras AR, Bate N, Goult BT, Hazelwood L, Canestrelli I, Grossmann JG, Liu H, Putz NS, Roberts GC, Volkmann N, Hanein D, Barsukov IL, Critchley DR - EMBO J. (2007)

Bottom Line: Mutagenesis shows that dimerisation is essential for filamentous actin (F-actin) binding and indicates that the dimerisation helix itself contributes to binding.We have used these structures together with small angle X-ray scattering to derive a model of the entire domain.Electron microscopy provides direct evidence for binding of the dimer to F-actin and indicates that it binds to three monomers along the long-pitch helix of the actin filament.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Leicester, Leicester, UK.

ABSTRACT
Talin is a large dimeric protein that couples integrins to cytoskeletal actin. Here, we report the structure of the C-terminal actin-binding domain of talin, the core of which is a five-helix bundle linked to a C-terminal helix responsible for dimerisation. The NMR structure of the bundle reveals a conserved surface-exposed hydrophobic patch surrounded by positively charged groups. We have mapped the actin-binding site to this surface and shown that helix 1 on the opposite side of the bundle negatively regulates actin binding. The crystal structure of the dimerisation helix reveals an antiparallel coiled-coil with conserved residues clustered on the solvent-exposed face. Mutagenesis shows that dimerisation is essential for filamentous actin (F-actin) binding and indicates that the dimerisation helix itself contributes to binding. We have used these structures together with small angle X-ray scattering to derive a model of the entire domain. Electron microscopy provides direct evidence for binding of the dimer to F-actin and indicates that it binds to three monomers along the long-pitch helix of the actin filament.

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The C-terminal actin-binding site in talin binds to the sides of actin filaments. (A) The talin fragment binds to the side of actin filaments at specific sites (arrowheads). This binding does not follow helical symmetry. The scale bar represents 50 nm. (B) Two orthogonal views of the dimer model (monomers in blue and green cartoon representation) and the envelope derived by SAXS (transparent grey). The small grey arrows indicate the direction of the twist that can be used to improve the fit of the SAXS model into the 3D reconstruction. (C) Surface representation of the 3D reconstruction of F-actin decorated with the talin C-terminal domain. The three views perpendicular to the filament axis are related by successive 90° anticlockwise rotations around the axis. The pointed end of the filament is to the top of the figure for these views. The rightmost view is along the filament axis from the pointed end towards the barbed end. The two connected densities are indicated (1 and 2) (D) Docked atomic models of F-actin (pink) and a dimer of the talin C-terminal domain (monomers in blue and green) inside the 3D reconstruction (transparent grey). Views as in (C). (E) Molecular surface of the docked models. Views and colours as in (D).
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f5: The C-terminal actin-binding site in talin binds to the sides of actin filaments. (A) The talin fragment binds to the side of actin filaments at specific sites (arrowheads). This binding does not follow helical symmetry. The scale bar represents 50 nm. (B) Two orthogonal views of the dimer model (monomers in blue and green cartoon representation) and the envelope derived by SAXS (transparent grey). The small grey arrows indicate the direction of the twist that can be used to improve the fit of the SAXS model into the 3D reconstruction. (C) Surface representation of the 3D reconstruction of F-actin decorated with the talin C-terminal domain. The three views perpendicular to the filament axis are related by successive 90° anticlockwise rotations around the axis. The pointed end of the filament is to the top of the figure for these views. The rightmost view is along the filament axis from the pointed end towards the barbed end. The two connected densities are indicated (1 and 2) (D) Docked atomic models of F-actin (pink) and a dimer of the talin C-terminal domain (monomers in blue and green) inside the 3D reconstruction (transparent grey). Views as in (C). (E) Molecular surface of the docked models. Views and colours as in (D).

Mentions: To determine where the C-terminal domain of talin binds on the actin filament, we used the talin 2334–2541 construct lacking the USH, since it has a higher affinity for F-actin. Differential scanning calorimetry (DSC) showed that this construct is properly folded and stable under the conditions used, and formation of a complex with F-actin was verified by co-sedimentation and DSC (Supplementary Figure S9). Using electron microscopy, extra density was visible decorating the actin filaments at both pH 7.0 and 7.5 in the presence of the talin construct (Figure 5A arrows), clearly indicating binding. Two complementary image-reconstruction approaches for helically symmetric structures were applied to two independent data sets. The analysis of the resulting three-dimensional (3D) reconstructions shows that the helical symmetry is consistent with the values reported for decorated actin filaments by us and others. However, difference mapping of reconstructions of F-actin with and without talin 2334–2541 did not provide any clear difference peaks that might correspond to the talin domain.


The structure of the C-terminal actin-binding domain of talin.

Gingras AR, Bate N, Goult BT, Hazelwood L, Canestrelli I, Grossmann JG, Liu H, Putz NS, Roberts GC, Volkmann N, Hanein D, Barsukov IL, Critchley DR - EMBO J. (2007)

The C-terminal actin-binding site in talin binds to the sides of actin filaments. (A) The talin fragment binds to the side of actin filaments at specific sites (arrowheads). This binding does not follow helical symmetry. The scale bar represents 50 nm. (B) Two orthogonal views of the dimer model (monomers in blue and green cartoon representation) and the envelope derived by SAXS (transparent grey). The small grey arrows indicate the direction of the twist that can be used to improve the fit of the SAXS model into the 3D reconstruction. (C) Surface representation of the 3D reconstruction of F-actin decorated with the talin C-terminal domain. The three views perpendicular to the filament axis are related by successive 90° anticlockwise rotations around the axis. The pointed end of the filament is to the top of the figure for these views. The rightmost view is along the filament axis from the pointed end towards the barbed end. The two connected densities are indicated (1 and 2) (D) Docked atomic models of F-actin (pink) and a dimer of the talin C-terminal domain (monomers in blue and green) inside the 3D reconstruction (transparent grey). Views as in (C). (E) Molecular surface of the docked models. Views and colours as in (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2168396&req=5

f5: The C-terminal actin-binding site in talin binds to the sides of actin filaments. (A) The talin fragment binds to the side of actin filaments at specific sites (arrowheads). This binding does not follow helical symmetry. The scale bar represents 50 nm. (B) Two orthogonal views of the dimer model (monomers in blue and green cartoon representation) and the envelope derived by SAXS (transparent grey). The small grey arrows indicate the direction of the twist that can be used to improve the fit of the SAXS model into the 3D reconstruction. (C) Surface representation of the 3D reconstruction of F-actin decorated with the talin C-terminal domain. The three views perpendicular to the filament axis are related by successive 90° anticlockwise rotations around the axis. The pointed end of the filament is to the top of the figure for these views. The rightmost view is along the filament axis from the pointed end towards the barbed end. The two connected densities are indicated (1 and 2) (D) Docked atomic models of F-actin (pink) and a dimer of the talin C-terminal domain (monomers in blue and green) inside the 3D reconstruction (transparent grey). Views as in (C). (E) Molecular surface of the docked models. Views and colours as in (D).
Mentions: To determine where the C-terminal domain of talin binds on the actin filament, we used the talin 2334–2541 construct lacking the USH, since it has a higher affinity for F-actin. Differential scanning calorimetry (DSC) showed that this construct is properly folded and stable under the conditions used, and formation of a complex with F-actin was verified by co-sedimentation and DSC (Supplementary Figure S9). Using electron microscopy, extra density was visible decorating the actin filaments at both pH 7.0 and 7.5 in the presence of the talin construct (Figure 5A arrows), clearly indicating binding. Two complementary image-reconstruction approaches for helically symmetric structures were applied to two independent data sets. The analysis of the resulting three-dimensional (3D) reconstructions shows that the helical symmetry is consistent with the values reported for decorated actin filaments by us and others. However, difference mapping of reconstructions of F-actin with and without talin 2334–2541 did not provide any clear difference peaks that might correspond to the talin domain.

Bottom Line: Mutagenesis shows that dimerisation is essential for filamentous actin (F-actin) binding and indicates that the dimerisation helix itself contributes to binding.We have used these structures together with small angle X-ray scattering to derive a model of the entire domain.Electron microscopy provides direct evidence for binding of the dimer to F-actin and indicates that it binds to three monomers along the long-pitch helix of the actin filament.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Leicester, Leicester, UK.

ABSTRACT
Talin is a large dimeric protein that couples integrins to cytoskeletal actin. Here, we report the structure of the C-terminal actin-binding domain of talin, the core of which is a five-helix bundle linked to a C-terminal helix responsible for dimerisation. The NMR structure of the bundle reveals a conserved surface-exposed hydrophobic patch surrounded by positively charged groups. We have mapped the actin-binding site to this surface and shown that helix 1 on the opposite side of the bundle negatively regulates actin binding. The crystal structure of the dimerisation helix reveals an antiparallel coiled-coil with conserved residues clustered on the solvent-exposed face. Mutagenesis shows that dimerisation is essential for filamentous actin (F-actin) binding and indicates that the dimerisation helix itself contributes to binding. We have used these structures together with small angle X-ray scattering to derive a model of the entire domain. Electron microscopy provides direct evidence for binding of the dimer to F-actin and indicates that it binds to three monomers along the long-pitch helix of the actin filament.

Show MeSH