Talin contains three similar vinculin-binding sites predicted to form an amphipathic helix

Abstract

Using recombinant talin polypeptides and an SDS/PAGE-blot overlay assay, we have previously identified three regions of talin that are involved in binding to vinculin [Gilmore, Wood, Ohanian, Jackson, Patel, Rees, Hynes and Critchley (1993) J. Cell Biol. 122, 337-347]. We have confirmed these observations by using a yeast two-hybrid assay and shown that talin residues 498-656, 852-950 and 1929-2029 are each capable of binding to vinculin residues 1-258. We have further defined the three vinculin-binding sites in talin to residues 607-636, 852-876 and 1944-1969; alignment of these sequences shows 59% similarity, although there are only two identical residues. Predictions of secondary structure indicate that this vinculin-binding motif forms an amphipathic alpha-helix. The hydrophobic face of helix 607-636 contains three aligned leucines (residues 608, 615 and 622), which show conservative substitutions in the other two sites. To test the possibility that this might constitute a leucine zipper involved in vinculin binding, we mutated each leucine residue to an alanine. The results showed that this leucine repeat is not essential to the interaction between talin and vinculin. We also used the yeast two-hybrid system to define further the talin-binding site within vinculin residues 1-258. C-terminal deletions made in accordance with exon boundaries showed that vinculin residues 1-167 are capable of interacting with each of the three vinculin-binding sites in talin. However, all N-terminal deletions abolished binding. The results suggest that the talin-binding site in vinculin has a relatively complex fold, whereas the vinculin-binding motif in talin is contained within a short linear peptide sequence that is repeated three times in the talin rod domain.

Structural Basis for Amplifying Vinculin Activation by Talin

“…3A and Ref. 17). This similarity in structure also occurs even though VBS3 has three additional ordered residues that add an extra turn to its N terminus and that VBS1 has six additional ordered residues on its C terminus which bend away at ϳ40 o from its position in the Vh⅐VBS3 structure.…”

“…Talin is unique among the vinculin-binding partners because it harbors, curiously, three noncontiguous VBSs, VBS1 (residues 607-636), VBS2 (residues 852-879), and VBS3 (1944 -1969; Refs 15,17,and 23). Based upon their ␣-helical amphipathic nature (17) and in part from competition studies using larger portions of talin harboring VBS1 (residues 498 -636; Ref.…”

“…For example, an NPXY/F variant of the canonical phosphotyrosine binding domain of integrin receptors mediates its interaction with the FERM 1 (four point one, ezrin, radixin, and moesin) motif present in the head domain of talin (13,14). In turn, talin binds to vinculin through vinculin binding sites (VBSs) present in its central rod domain, and this interaction contributes to reorganization of the actin cytoskeleton (15)(16)(17).…”

“…15, 17, and 23). Each of the talin VBSs have been predicted to form an amphipathic ␣-helix (17), and larger portions of talin containing these VBSs can bind individually to Vh (17) and act as competitive inhibitors that prevent the binding of Vt to Vh (23). However, it was not known whether these interactions were indeed mediated by the refined sequences of talin VBSs.…”

Vinculin Is a Dually Regulated Actin Filament Barbed End-capping and Side-binding Protein

“…The D1-Vt interaction is disrupted in the presence of a saturating amount of one of the numerous VBSs of talin (36,37). However, a protein that disrupts specifically the contacts between Vt and the other subdomains, D3 and D4, has never been reported to our knowledge.…”