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. 2010 Sep 17;285(38):29577-87.
doi: 10.1074/jbc.M109.095455. Epub 2010 Jul 7.

Central region of talin has a unique fold that binds vinculin and actin

Alexandre R Gingras  1 , Neil Bate, Benjamin T Goult, Bipin Patel, Petra M Kopp, Jonas Emsley, Igor L Barsukov, Gordon C K Roberts, David R Critchley
Affiliations

Central region of talin has a unique fold that binds vinculin and actin

Alexandre R Gingras et al. J Biol Chem. .

Abstract

Talin is an adaptor protein that couples integrins to F-actin. Structural studies show that the N-terminal talin head contains an atypical FERM domain, whereas the N- and C-terminal parts of the talin rod include a series of α-helical bundles. However, determining the structure of the central part of the rod has proved problematic. Residues 1359-1659 are homologous to the MESDc1 gene product, and we therefore expressed this region of talin in Escherichia coli. The crystal structure shows a unique fold comprised of a 5- and 4-helix bundle. The 5-helix bundle is composed of nonsequential helices due to insertion of the 4-helix bundle into the loop at the C terminus of helix α3. The linker connecting the bundles forms a two-stranded anti-parallel β-sheet likely limiting the relative movement of the two bundles. Because the 5-helix bundle contains the N and C termini of this module, we propose that it is linked by short loops to adjacent bundles, whereas the 4-helix bundle protrudes from the rod. This suggests the 4-helix bundle has a unique role, and its pI (7.8) is higher than other rod domains. Both helical bundles contain vinculin-binding sites but that in the isolated 5-helix bundle is cryptic, whereas that in the isolated 4-helix bundle is constitutively active. In contrast, both bundles are required for actin binding. Finally, we show that the MESDc1 protein, which is predicted to have a similar fold, is a novel actin-binding protein.

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Figures

FIGURE 1.
FIGURE 1.
Domain structure and binding partners of talin. Schematic diagram of the talin molecule indicating the regions involved in binding to various ligands. The talin head (residues 1–400) contains a FERM domain (comprising F1, F2, and F3 subdomains) preceded by a domain region referred to as F0 (16). The rod domain contains 62 predicted α-helices (ovals) organized into a series of amphipathic helical bundles. Domain boundaries based on structural determination are indicated by solid lines. Dashed lines indicate boundaries that are tentative. The ∼11 VBS are shown in red. The last α-helix (blue) contains the dimerization domain (DD) (27).
FIGURE 2.
FIGURE 2.
Crystal structure of talin rod residues 1359–1659. A, schematic representation of the talin(1359–1659) crystal structure. This region of talin encodes nine α-helices forming a 5-helix bundle and a 4-helix bundle with an unusual domain linkage. The helix numbers shown in brackets are for full-length talin. B, surface electrostatic potential of the molecule shown in the same orientation as A. There is no evidence of hydrophobic or electrostatic interactions between the two domains. C, this talin module contains nine α-helices (ovals) organized into two different amphipathic helical bundles with an unusual topology. The first three α-helices and the last two helices (α1, α2, α3, α8, and α9) form a 5-helix bundle, whereas helix 4–7 form a 4-helix bundle (α4, α5, α6, and α7). The two vinculin-binding sites (VBS) are shown in red. D, diagram showing the organization of the helices into 5-helix (left) and 4-helix (right) bundles. Solid and dashed lines represent connecting loops on opposite ends of the helices. The coloring is as in A.
FIGURE 3.
FIGURE 3.
Structural details of the linker region between the two bundles. A, view of the hydrophobic interactions at the bottom of the 4-helix bundle. The short helix α4 leaves a gap that is filled with a cluster of four phenylalanines and two hydrophobic residues from the linker region at the N-terminal end of helix α4, i.e. Leu-1461 and Val-1462. B, view of the linker region that forms a two-stranded anti-parallel β-sheet-like structure. The complementary backbone hydrogen bonds are highlighted by the dashed red lines. C, hydrogen bond networks observed at the bottom of the 5-helix bundle and the linker region.
FIGURE 4.
FIGURE 4.
Vinculin Vd1 binding analyzed by gel filtration. Vinculin Vd1 was incubated using a 1:1 ratio with the talin 5-helix (A), the 4-helix (B), or the 9-helix (C) polypeptides at various temperatures, and complex formation was analyzed on a Superdex-75 (10/300) GL gel filtration column at room temperature (RT). Incubation of the 4-helix (B) or 9-helix polypeptides (C) with Vd1 resulted in complex formation. Preincubation of the proteins at 37 °C did not increase significantly the formation of a talin-Vd1 complex (data not shown). No binding was observed with the 5-helix bundle alone. D, denaturation profiles for the talin rod polypeptides were measured by monitoring the change in circular dichroism at 222 nm with increasing temperatures. Profiles are shown for the 9-helix module (squares), the 5-helix bundle (circles), and the 4-helix bundle (triangles). The melting temperature (Tm) for each domain is indicated.
FIGURE 5.
FIGURE 5.
Both the talin 9-helix module and MESDc1 bind F-actin. The talin 9-helix, 5-helix, and 4-helix polypeptides were incubated with F-actin, and binding was determined using a cosedimentation assay. After centrifugation, supernatant (S) and pellet (P) fractions were analyzed by SDS-PAGE. A, talin 9-helix module binds F-actin, but not the individual subdomains, i.e. the 5- and 4-helix bundles. B, MESDc1 proteins binds F-actin with higher affinity than the talin 9-helix module. C, images of NIH3T3 cells transfected with cDNAs encoding either GFP-talin residues 1359–1659 or GFP-MESDc1 after Triton X-100 extraction. The F-actin cytoskeleton was stained with phalloidin Alexa 594. Scale bar, 10 μm. Both the GFP-tagged talin 9-helix module and MESDc1 colocalize with actin stress fibers.
FIGURE 6.
FIGURE 6.
Biochemical characterization of MESDc1. A, secondary structure analysis of MESDc1 by circular dichroism. The profile suggests that the protein is largely helical. B, denaturation profile for MESDc1 was measured by monitoring the change in circular dichroism at 222 nm with increasing temperatures. The melting temperature (Tm) is indicated. C, vinculin Vd1 (residues 1–258) was incubated with MESDc1 at various temperatures, and complex formation was analyzed on a Superdex-75 (10/300) GL gel filtration column at room temperature (RT). MESDc1 does not bind to Vd1 even after preincubation of the proteins at 45 °C for 30 min. D, VBS peptide sequences were aligned using ClustalW as described in Gingras et al. (28). Residues highlighted in blue align with the buried (>75%) hydrophobic side chains from the VBS1-Vd1 complex crystal structure (32). Residues that clash with the consensus VBS sequence are highlighted in red, i.e. hydrophobic residue substituted by positive residue. The residues that do not fit with the ideal consensus VBS sequence are highlighted in orange. The 50 and 90% VBS consensus sequence is shown at the top as described in Gingras et al. (28). Uppercase letters indicate conserved residues (single-letter amino acid code). Lowercase letters indicate conserved classes of amino acids as follows: h, hydrophobic residues (A, C, F, G, H, I, K, L, M, P, T, V, W, Y); p, polar residues (C, D, E, H, K, N, Q, R, S, T); c, charged resides (D, E, H, K, R); s, small residues (A, C, D, G, N, P, S, T, V); +, positive residues (H, K, R); l, aliphatic residues (I, L, V); and u, tiny (A, G, S, C).
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