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

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.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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).