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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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SAXS data for the dimeric talin polypeptide 2300–2541. (A) Experimental scattering profile of the talin dimer (red) compared with the theoretical scattering curves from the shape reconstructed ab initio with GASBOR (blue line), and the structural model of the dimer obtained with the rigid body modelling program BUNCH (black line). The goodness of fit of GASBOR and BUNCH profiles versus experimental data is indicated by their χ2 values (χ2=2.5 and 2.2, respectively). (B) Three orthogonal views of the talin dimer model (monomers in cyan and green) deduced using BUNCH fitted within the shape envelope provided by GASBOR and derived from experimental scattering data alone (transparent grey surface). (C) The talin C-terminal dimerisation domain suggests that full-length talin may adopt a number of conformations, for example, (1) a parallel dimer (2) a V-shaped dimer or (3) an extended dimer.
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f4: SAXS data for the dimeric talin polypeptide 2300–2541. (A) Experimental scattering profile of the talin dimer (red) compared with the theoretical scattering curves from the shape reconstructed ab initio with GASBOR (blue line), and the structural model of the dimer obtained with the rigid body modelling program BUNCH (black line). The goodness of fit of GASBOR and BUNCH profiles versus experimental data is indicated by their χ2 values (χ2=2.5 and 2.2, respectively). (B) Three orthogonal views of the talin dimer model (monomers in cyan and green) deduced using BUNCH fitted within the shape envelope provided by GASBOR and derived from experimental scattering data alone (transparent grey surface). (C) The talin C-terminal dimerisation domain suggests that full-length talin may adopt a number of conformations, for example, (1) a parallel dimer (2) a V-shaped dimer or (3) an extended dimer.

Mentions: To investigate the overall shape of the talin 2300–2541 dimer, we carried out SAXS experiments. From the scattering profile (Figure 4A), the maximum linear dimension (Dmax) for the talin dimer is 124 (±6) Å, suggesting that the dimer is elongated. Similarly, the distribution of scattering mass of the dimer (as indicated by the radius of gyration, Rg) gives Rg=37.0 (±0.3) Å, a high value for a 52-kDa dimer; one would expect an Rg value of ∼25 Å for a spherical particle of similar mass. These observations clearly demonstrate that the dimer is characterised by an extended non-globular arrangement of the THATCH domain, consistent with the results from analytical gel filtration (see Supplementary Results).


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)

SAXS data for the dimeric talin polypeptide 2300–2541. (A) Experimental scattering profile of the talin dimer (red) compared with the theoretical scattering curves from the shape reconstructed ab initio with GASBOR (blue line), and the structural model of the dimer obtained with the rigid body modelling program BUNCH (black line). The goodness of fit of GASBOR and BUNCH profiles versus experimental data is indicated by their χ2 values (χ2=2.5 and 2.2, respectively). (B) Three orthogonal views of the talin dimer model (monomers in cyan and green) deduced using BUNCH fitted within the shape envelope provided by GASBOR and derived from experimental scattering data alone (transparent grey surface). (C) The talin C-terminal dimerisation domain suggests that full-length talin may adopt a number of conformations, for example, (1) a parallel dimer (2) a V-shaped dimer or (3) an extended dimer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: SAXS data for the dimeric talin polypeptide 2300–2541. (A) Experimental scattering profile of the talin dimer (red) compared with the theoretical scattering curves from the shape reconstructed ab initio with GASBOR (blue line), and the structural model of the dimer obtained with the rigid body modelling program BUNCH (black line). The goodness of fit of GASBOR and BUNCH profiles versus experimental data is indicated by their χ2 values (χ2=2.5 and 2.2, respectively). (B) Three orthogonal views of the talin dimer model (monomers in cyan and green) deduced using BUNCH fitted within the shape envelope provided by GASBOR and derived from experimental scattering data alone (transparent grey surface). (C) The talin C-terminal dimerisation domain suggests that full-length talin may adopt a number of conformations, for example, (1) a parallel dimer (2) a V-shaped dimer or (3) an extended dimer.
Mentions: To investigate the overall shape of the talin 2300–2541 dimer, we carried out SAXS experiments. From the scattering profile (Figure 4A), the maximum linear dimension (Dmax) for the talin dimer is 124 (±6) Å, suggesting that the dimer is elongated. Similarly, the distribution of scattering mass of the dimer (as indicated by the radius of gyration, Rg) gives Rg=37.0 (±0.3) Å, a high value for a 52-kDa dimer; one would expect an Rg value of ∼25 Å for a spherical particle of similar mass. These observations clearly demonstrate that the dimer is characterised by an extended non-globular arrangement of the THATCH domain, consistent with the results from analytical gel filtration (see Supplementary Results).

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
Related in: MedlinePlus