The activity of the vinculin binding sites in talin is influenced by the stability of the helical bundles that make up the talin rod
2007-10-30T10:19:39Z (GMT) by
The talin rod contains ~11 vinculin binding sites (VBS’s) 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 immobilised talin polypeptide containing a constitutively active VBS. However, talin did bind to GST-Vd1 in pull-down assays, and ITC measurements indicate a Kd of ~9μM. Interestingly, Vd1 binding exposed a trypsin-cleavage site in the talin rod between residues 898/899, indicating that there are one or more active VBS’s 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 VBS’s (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 (Kd ~ 0.14μM), indicating that one or more of the four VBS’s within 656-889 are active, and this likely represents the vinculin-binding region in intact talin. In support of this, HA-tagged talin 482-889 localised 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 VBS’s.