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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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Identification of residues in the C-terminal actin-binding site of talin, which contributes to binding. (A–C) Ribbon diagrams highlighting the mutations introduced in talin 2300–2541. (A) F-actin-binding surface on the core five-helix bundle, (B) the dimerisation domain and (C) the USH. Residues are colour coded according to the effects of the mutation on F-actin binding compared to wild type: red—increase in binding; green—no change; blue—decrease in binding. Residue Q2388 is shown in yellow. (D–F) Quantitative analysis of the effects of talin mutations on F-actin binding (means of three independent experiments) as determined using the actin-co-sedimentation assay described in Materials and methods. Bars represent standard deviations. The data for all the mutants analysed are shown in Supplementary Figures S5, S7 and S8.
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f3: Identification of residues in the C-terminal actin-binding site of talin, which contributes to binding. (A–C) Ribbon diagrams highlighting the mutations introduced in talin 2300–2541. (A) F-actin-binding surface on the core five-helix bundle, (B) the dimerisation domain and (C) the USH. Residues are colour coded according to the effects of the mutation on F-actin binding compared to wild type: red—increase in binding; green—no change; blue—decrease in binding. Residue Q2388 is shown in yellow. (D–F) Quantitative analysis of the effects of talin mutations on F-actin binding (means of three independent experiments) as determined using the actin-co-sedimentation assay described in Materials and methods. Bars represent standard deviations. The data for all the mutants analysed are shown in Supplementary Figures S5, S7 and S8.

Mentions: The five-helix bundle that comprises the core of the C-terminal actin-binding domain of talin contains a number of conserved surface-exposed residues that are predominantly clustered on the face made up of helices 3 and 4 (Figure 1C). This part of the surface consists of an extensive hydrophobic patch surrounded by positively charged groups (Figure 1D), characteristics that make it a good candidate for a region involved in F-actin binding. Indeed, the actin-binding site in the HIP1R THATCH domain has been mapped to the equivalent surface (Brett et al, 2006). However, this surface is on the face of the domain opposite helix 1, which has been shown to negatively regulate actin binding (Senetar et al, 2004). In addition, actin binding depends on the presence of the C-terminal dimerisation helix (see below). To map the residues in talin 2300–2541 directly or indirectly involved in actin binding, we tested the effects of a series of mutations on its affinity for F-actin. In the presence of a six-fold molar excess of F-actin, ∼30% of wild-type talin 2300–2541 co-sedimented with F-actin (Figure 3D), a result similar to that previously observed for talin and HIP1R (Senetar et al, 2004). The single mutations Q2388D, Q2437E, K2443D, V2444D and K2445D (equivalent to those analysed in the HIP1R THATCH domain; Brett et al, 2006) all caused a significant reduction in F-actin binding (Table I), and the triple mutant K2443D/V2444D/K2445D reduced binding to 27% of wild type. Interestingly, although the Q2437E mutant reduced binding affinity, the equivalent mutation in HIP1R (Q916E) increased binding. While this difference is puzzling, our results clearly demonstrate that incorporating acidic amino acids into the actin-binding surface of talin reduces binding. Both the Q2388D mutation in talin (Supplementary Figure S5D and Table I) and the equivalent R867D mutation in HIP1R reduce binding to F-actin; interestingly, substituting talin Q2388 with arginine had no effect on binding, indicating that either a basic or an uncharged residue at this position can be tolerated. Talin D2447, located close to the conserved basic residues K2443 and K2445, is homologous to N926 in HIP1R and a talin D2447N mutant increased F-actin binding to 144% that of wild type (Figure 3D and Table I). Thus, reducing the acidic charge in the proximity of the conserved basic residues (Figure 1C and D) increases the ability of talin to bind F-actin.


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)

Identification of residues in the C-terminal actin-binding site of talin, which contributes to binding. (A–C) Ribbon diagrams highlighting the mutations introduced in talin 2300–2541. (A) F-actin-binding surface on the core five-helix bundle, (B) the dimerisation domain and (C) the USH. Residues are colour coded according to the effects of the mutation on F-actin binding compared to wild type: red—increase in binding; green—no change; blue—decrease in binding. Residue Q2388 is shown in yellow. (D–F) Quantitative analysis of the effects of talin mutations on F-actin binding (means of three independent experiments) as determined using the actin-co-sedimentation assay described in Materials and methods. Bars represent standard deviations. The data for all the mutants analysed are shown in Supplementary Figures S5, S7 and S8.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Identification of residues in the C-terminal actin-binding site of talin, which contributes to binding. (A–C) Ribbon diagrams highlighting the mutations introduced in talin 2300–2541. (A) F-actin-binding surface on the core five-helix bundle, (B) the dimerisation domain and (C) the USH. Residues are colour coded according to the effects of the mutation on F-actin binding compared to wild type: red—increase in binding; green—no change; blue—decrease in binding. Residue Q2388 is shown in yellow. (D–F) Quantitative analysis of the effects of talin mutations on F-actin binding (means of three independent experiments) as determined using the actin-co-sedimentation assay described in Materials and methods. Bars represent standard deviations. The data for all the mutants analysed are shown in Supplementary Figures S5, S7 and S8.
Mentions: The five-helix bundle that comprises the core of the C-terminal actin-binding domain of talin contains a number of conserved surface-exposed residues that are predominantly clustered on the face made up of helices 3 and 4 (Figure 1C). This part of the surface consists of an extensive hydrophobic patch surrounded by positively charged groups (Figure 1D), characteristics that make it a good candidate for a region involved in F-actin binding. Indeed, the actin-binding site in the HIP1R THATCH domain has been mapped to the equivalent surface (Brett et al, 2006). However, this surface is on the face of the domain opposite helix 1, which has been shown to negatively regulate actin binding (Senetar et al, 2004). In addition, actin binding depends on the presence of the C-terminal dimerisation helix (see below). To map the residues in talin 2300–2541 directly or indirectly involved in actin binding, we tested the effects of a series of mutations on its affinity for F-actin. In the presence of a six-fold molar excess of F-actin, ∼30% of wild-type talin 2300–2541 co-sedimented with F-actin (Figure 3D), a result similar to that previously observed for talin and HIP1R (Senetar et al, 2004). The single mutations Q2388D, Q2437E, K2443D, V2444D and K2445D (equivalent to those analysed in the HIP1R THATCH domain; Brett et al, 2006) all caused a significant reduction in F-actin binding (Table I), and the triple mutant K2443D/V2444D/K2445D reduced binding to 27% of wild type. Interestingly, although the Q2437E mutant reduced binding affinity, the equivalent mutation in HIP1R (Q916E) increased binding. While this difference is puzzling, our results clearly demonstrate that incorporating acidic amino acids into the actin-binding surface of talin reduces binding. Both the Q2388D mutation in talin (Supplementary Figure S5D and Table I) and the equivalent R867D mutation in HIP1R reduce binding to F-actin; interestingly, substituting talin Q2388 with arginine had no effect on binding, indicating that either a basic or an uncharged residue at this position can be tolerated. Talin D2447, located close to the conserved basic residues K2443 and K2445, is homologous to N926 in HIP1R and a talin D2447N mutant increased F-actin binding to 144% that of wild type (Figure 3D and Table I). Thus, reducing the acidic charge in the proximity of the conserved basic residues (Figure 1C and D) increases the ability of talin to bind F-actin.

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