Talin contains three similar vinculin-binding sites predicted to form an amphipathic helix.
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.
Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, U.K.
M D
MD
Bass
B J
BJ
Smith
S A
SA
Prigent
D R
DR
Critchley
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D016608:Talin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016596:Vinculin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D016608:Talin; D016596:Vinculin