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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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Solution structure of the C-terminal actin-binding domain of talin (residues 2300–2482). (A) Sequence alignment of mouse talin1 with human HIP1R THATCH domain. Symbols denote the degree of conservation: (*) identical, (:) conservative substitution and (.) semi-conservative substitutions. Secondary structures of mouse talin and human HIP1R THATCH core are shown above and below the alignment, respectively—the position of the putative dimerisation domain is indicated. N.D.—structure not determined. Numbering is from mouse talin (P26039). The talin residues mutated are highlighted depending on their effects on F-actin binding: red—increased binding; green—binding similar to wild type; blue—decreased binding. Residue Q2388 is highlighted in yellow. The residues mutated in HIP1R that are equivalent to those analysed in talin are also highlighted for comparison. (B) Ribbon drawing of a representative low-energy structure showing the overall topology of the five-helix bundle of the C-terminal actin-binding domain of talin. (C) Map of conserved surface residues. Magenta—invariant residues; yellow—residues that are highly conserved. (D) Map of surface charge.
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f1: Solution structure of the C-terminal actin-binding domain of talin (residues 2300–2482). (A) Sequence alignment of mouse talin1 with human HIP1R THATCH domain. Symbols denote the degree of conservation: (*) identical, (:) conservative substitution and (.) semi-conservative substitutions. Secondary structures of mouse talin and human HIP1R THATCH core are shown above and below the alignment, respectively—the position of the putative dimerisation domain is indicated. N.D.—structure not determined. Numbering is from mouse talin (P26039). The talin residues mutated are highlighted depending on their effects on F-actin binding: red—increased binding; green—binding similar to wild type; blue—decreased binding. Residue Q2388 is highlighted in yellow. The residues mutated in HIP1R that are equivalent to those analysed in talin are also highlighted for comparison. (B) Ribbon drawing of a representative low-energy structure showing the overall topology of the five-helix bundle of the C-terminal actin-binding domain of talin. (C) Map of conserved surface residues. Magenta—invariant residues; yellow—residues that are highly conserved. (D) Map of surface charge.

Mentions: Initially, we used secondary structure prediction and NMR spectroscopy of a range of constructs to identify a talin polypeptide containing the C-terminal actin-binding site suitable for NMR structure determination (see Supplementary Results and Supplementary Figure S1). These studies demonstrated the presence of a stable globular domain (residues 2300–2482) connected by a flexible linker to a helical dimerisation domain (residues 2496–2529). The structure of talin 2300–2482 comprises five antiparallel α-helices (Figure 1A and B; see also Supplementary Results and Supplementary Figure S2D), as described for the homologous HIP1R actin-binding domain (referred to as the THATCH core) (Brett et al, 2006). The helical bundle is stabilised by hydrophobic interactions. There are hydrophobic cores at each end of the bundle separated by a set of small hydrophilic side chains (Thr 2356, 2404, 2435 and Ser 2467) reminiscent of the ‘threonine belt' observed in the structure of talin 782–889 (Fillingham et al, 2005). The hydrophobic core at the N-terminal end of the bundle is arranged around the aromatic ring of Trp 2389 (Supplementary Figure S2C) and incorporates the side chain of the conserved Gln 2367, which points into the bundle. The core at the C-terminal end is made up of the hydrophobic side chains of Leu, Ile and Val residues, and is capped by the aromatic ring of Phe 2341. Helices 2 and 3 are the longest (32 residues), while the other three are approximately two turns shorter (22–27 residues). Helix 2 is connected to the neighbouring helices by long loops (14 and 9 residues), while the other two loops are relatively short (4–5 residues). The long loop between helices 1 and 2 is unstructured and highly dynamic, as indicated by sharp NMR resonances and the lack of NOEs (Figure 1B and Supplementary Figure S2A). The loop between helices 2 and 3 has restricted mobility due to the hydrophobic contacts made by the Val 2376 side chain and the presence of Pro 2380.


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)

Solution structure of the C-terminal actin-binding domain of talin (residues 2300–2482). (A) Sequence alignment of mouse talin1 with human HIP1R THATCH domain. Symbols denote the degree of conservation: (*) identical, (:) conservative substitution and (.) semi-conservative substitutions. Secondary structures of mouse talin and human HIP1R THATCH core are shown above and below the alignment, respectively—the position of the putative dimerisation domain is indicated. N.D.—structure not determined. Numbering is from mouse talin (P26039). The talin residues mutated are highlighted depending on their effects on F-actin binding: red—increased binding; green—binding similar to wild type; blue—decreased binding. Residue Q2388 is highlighted in yellow. The residues mutated in HIP1R that are equivalent to those analysed in talin are also highlighted for comparison. (B) Ribbon drawing of a representative low-energy structure showing the overall topology of the five-helix bundle of the C-terminal actin-binding domain of talin. (C) Map of conserved surface residues. Magenta—invariant residues; yellow—residues that are highly conserved. (D) Map of surface charge.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Solution structure of the C-terminal actin-binding domain of talin (residues 2300–2482). (A) Sequence alignment of mouse talin1 with human HIP1R THATCH domain. Symbols denote the degree of conservation: (*) identical, (:) conservative substitution and (.) semi-conservative substitutions. Secondary structures of mouse talin and human HIP1R THATCH core are shown above and below the alignment, respectively—the position of the putative dimerisation domain is indicated. N.D.—structure not determined. Numbering is from mouse talin (P26039). The talin residues mutated are highlighted depending on their effects on F-actin binding: red—increased binding; green—binding similar to wild type; blue—decreased binding. Residue Q2388 is highlighted in yellow. The residues mutated in HIP1R that are equivalent to those analysed in talin are also highlighted for comparison. (B) Ribbon drawing of a representative low-energy structure showing the overall topology of the five-helix bundle of the C-terminal actin-binding domain of talin. (C) Map of conserved surface residues. Magenta—invariant residues; yellow—residues that are highly conserved. (D) Map of surface charge.
Mentions: Initially, we used secondary structure prediction and NMR spectroscopy of a range of constructs to identify a talin polypeptide containing the C-terminal actin-binding site suitable for NMR structure determination (see Supplementary Results and Supplementary Figure S1). These studies demonstrated the presence of a stable globular domain (residues 2300–2482) connected by a flexible linker to a helical dimerisation domain (residues 2496–2529). The structure of talin 2300–2482 comprises five antiparallel α-helices (Figure 1A and B; see also Supplementary Results and Supplementary Figure S2D), as described for the homologous HIP1R actin-binding domain (referred to as the THATCH core) (Brett et al, 2006). The helical bundle is stabilised by hydrophobic interactions. There are hydrophobic cores at each end of the bundle separated by a set of small hydrophilic side chains (Thr 2356, 2404, 2435 and Ser 2467) reminiscent of the ‘threonine belt' observed in the structure of talin 782–889 (Fillingham et al, 2005). The hydrophobic core at the N-terminal end of the bundle is arranged around the aromatic ring of Trp 2389 (Supplementary Figure S2C) and incorporates the side chain of the conserved Gln 2367, which points into the bundle. The core at the C-terminal end is made up of the hydrophobic side chains of Leu, Ile and Val residues, and is capped by the aromatic ring of Phe 2341. Helices 2 and 3 are the longest (32 residues), while the other three are approximately two turns shorter (22–27 residues). Helix 2 is connected to the neighbouring helices by long loops (14 and 9 residues), while the other two loops are relatively short (4–5 residues). The long loop between helices 1 and 2 is unstructured and highly dynamic, as indicated by sharp NMR resonances and the lack of NOEs (Figure 1B and Supplementary Figure S2A). The loop between helices 2 and 3 has restricted mobility due to the hydrophobic contacts made by the Val 2376 side chain and the presence of Pro 2380.

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