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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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Structure of the talin dimerisation domain. (A) Cartoon representation of the crystal structure of the dimerisation helix (2496–2529) showing the antiparallel coiled-coil dimer. (B) Surface electrostatic potential of the dimer. (C) Map of conserved residues: magenta—invariant residues; yellow—highly conserved residues. (D) Sequence of residues 2494–2541, which includes the dimerisation helix—two antiparallel peptide sequences are shown.
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f2: Structure of the talin dimerisation domain. (A) Cartoon representation of the crystal structure of the dimerisation helix (2496–2529) showing the antiparallel coiled-coil dimer. (B) Surface electrostatic potential of the dimer. (C) Map of conserved residues: magenta—invariant residues; yellow—highly conserved residues. (D) Sequence of residues 2494–2541, which includes the dimerisation helix—two antiparallel peptide sequences are shown.

Mentions: Using secondary structure prediction and NMR, the optimal domain boundaries of the talin dimerisation domain were shown to be residues 2494–2541 (see Supplementary Results and Supplementary Figure S3). Large crystals that diffracted X-rays to 2.2-Å resolution were readily obtained using sparse matrix screening, and the crystal structure was determined using single-wavelength anomalous diffraction from a selenomethionine derivative (Supplementary Figure S4A, Supplementary Table SII and Supplementary Results). The two monomers in the asymmetric unit superimpose well onto one another (average r.m.s.d. for main-chain atoms 0.34 Å, and for all heavy atoms 1.08 Å), the main differences between the monomers being the orientations of long side chains of solvent-exposed residues. Each monomer is composed of a long straight helix approximately 48 Å in length (Figure 2A). The helices form an antiparallel coiled-coil dimer with a small angle between the helices. The formation of the dimer buries approximately 30% (1539 Å2) of the total surface area. Both ends of the dimer are nonpolar with a highly charged belt in the middle (Figure 2B). There is a salt bridge cluster in the centre of the dimer formed by the side chains of K2511 and E2514 from each monomer, with the four side chains making intra- and intermolecular contacts within the cluster (Supplementary Figure S4B). Additionally, intramolecular salt bridges are observed for E2516/R2519 in both monomers, stabilising the helix (Supplementary Figure S4C). Interestingly, E2507 from one of the monomers forms an intermolecular salt bridge with K2521, while in the other monomer this residue forms an intramolecular salt bridge with R2510 (Supplementary Figure S4B). In all other cases, the charged groups are too far apart to make a salt bridge and some of the side chains are hydrogen-bonded to nearby solvent molecules.


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)

Structure of the talin dimerisation domain. (A) Cartoon representation of the crystal structure of the dimerisation helix (2496–2529) showing the antiparallel coiled-coil dimer. (B) Surface electrostatic potential of the dimer. (C) Map of conserved residues: magenta—invariant residues; yellow—highly conserved residues. (D) Sequence of residues 2494–2541, which includes the dimerisation helix—two antiparallel peptide sequences are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Structure of the talin dimerisation domain. (A) Cartoon representation of the crystal structure of the dimerisation helix (2496–2529) showing the antiparallel coiled-coil dimer. (B) Surface electrostatic potential of the dimer. (C) Map of conserved residues: magenta—invariant residues; yellow—highly conserved residues. (D) Sequence of residues 2494–2541, which includes the dimerisation helix—two antiparallel peptide sequences are shown.
Mentions: Using secondary structure prediction and NMR, the optimal domain boundaries of the talin dimerisation domain were shown to be residues 2494–2541 (see Supplementary Results and Supplementary Figure S3). Large crystals that diffracted X-rays to 2.2-Å resolution were readily obtained using sparse matrix screening, and the crystal structure was determined using single-wavelength anomalous diffraction from a selenomethionine derivative (Supplementary Figure S4A, Supplementary Table SII and Supplementary Results). The two monomers in the asymmetric unit superimpose well onto one another (average r.m.s.d. for main-chain atoms 0.34 Å, and for all heavy atoms 1.08 Å), the main differences between the monomers being the orientations of long side chains of solvent-exposed residues. Each monomer is composed of a long straight helix approximately 48 Å in length (Figure 2A). The helices form an antiparallel coiled-coil dimer with a small angle between the helices. The formation of the dimer buries approximately 30% (1539 Å2) of the total surface area. Both ends of the dimer are nonpolar with a highly charged belt in the middle (Figure 2B). There is a salt bridge cluster in the centre of the dimer formed by the side chains of K2511 and E2514 from each monomer, with the four side chains making intra- and intermolecular contacts within the cluster (Supplementary Figure S4B). Additionally, intramolecular salt bridges are observed for E2516/R2519 in both monomers, stabilising the helix (Supplementary Figure S4C). Interestingly, E2507 from one of the monomers forms an intermolecular salt bridge with K2521, while in the other monomer this residue forms an intramolecular salt bridge with R2510 (Supplementary Figure S4B). In all other cases, the charged groups are too far apart to make a salt bridge and some of the side chains are hydrogen-bonded to nearby solvent molecules.

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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