title: The activity of the vinculin binding sites in talin is influenced by the stability of the helical bundles that make up the talin rod.
abstract: The talin rod contains approximately 11 vinculin binding sites (VBSs), each defined by hydrophobic residues in a series of amphipathic helices that are normally buried within the helical bundles that make up the rod. Consistent with this, talin failed to compete for binding of the vinculin Vd1 domain to an immobilized talin polypeptide containing a constitutively active VBS. However, talin did bind to GST-Vd1 in pull-down assays, and isothermal titration calorimetry measurements indicate a K(d) of approximately 9 mum. Interestingly, Vd1 binding exposed a trypsin cleavage site in the talin rod between residues 898 and 899, indicating that there are one or more active VBSs in the N-terminal part of the talin rod. This region comprises a five helix bundle (residues 482-655) followed by a seven-helix bundle (656-889) and contains five VBSs (helices 4, 6, 9, 11, and 12). The single VBS within 482-655 is cryptic at room temperature. In contrast, talin 482-889 binds Vd1 with high affinity (K(d) approximately 0.14 mum), indicating that one or more of the four VBSs within 656-889 are active, and this likely represents the vinculin binding region in intact talin. In support of this, hemagglutinin-tagged talin 482-889 localized efficiently to focal adhesions, whereas 482-655 did not. Differential scanning calorimetry showed a strong negative correlation between Vd1 binding and helical bundle stability, and a 755-889 mutant with a more stable fold bound Vd1 much less well than wild type. We conclude that the stability of the helical bundles that make up the talin rod is an important factor determining the activity of the individual VBSs.
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom.
authors.forename: Bipin
authors.initials: B
authors.lastname: Patel
authors.forename: Alexandre R
authors.initials: AR
authors.lastname: Gingras
authors.forename: Audrey A
authors.initials: AA
authors.lastname: Bobkov
authors.forename: L Miya
authors.initials: LM
authors.lastname: Fujimoto
authors.forename: Man
authors.initials: M
authors.lastname: Zhang
authors.forename: Robert C
authors.initials: RC
authors.lastname: Liddington
authors.forename: Daniela
authors.initials: D
authors.lastname: Mazzeo
authors.forename: Jonas
authors.initials: J
authors.lastname: Emsley
authors.forename: Gordon C K
authors.initials: GC
authors.lastname: Roberts
authors.forename: Igor L
authors.initials: IL
authors.lastname: Barsukov
authors.forename: David R
authors.initials: DR
authors.lastname: Critchley
mesh_terms: D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D002152:Calorimetry, Differential Scanning; D002645:Chickens; D002850:Chromatography, Gel; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D022001:Focal Adhesions; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D041681:NIH 3T3 Cells; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D016608:Talin / Q000737:chemistry*; D013696:Temperature; D014357:Trypsin / Q000737:chemistry; D016596:Vinculin / Q000737:chemistry*
chemical_list: D018076:DNA, Complementary; D010455:Peptides; D011994:Recombinant Proteins; D016608:Talin; D016596:Vinculin; D005982:Glutathione Transferase; D014357:Trypsin