Patel Bipin, Gingras Alexandre R, Bobkov Audrey A, Fujimoto L Miya, Zhang Man, Liddington Robert C, Mazzeo Daniela, Emsley Jonas, Roberts Gordon C K, Barsukov Igor L, Critchley David R
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom.
J Biol Chem. 2006 Mar 17;281(11):7458-67. doi: 10.1074/jbc.M508058200. Epub 2006 Jan 9.
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.
踝蛋白杆包含大约11个纽蛋白结合位点（VBS），每个位点由一系列两亲性螺旋中的疏水残基定义，这些螺旋通常埋藏在构成杆的螺旋束内。与此一致的是，踝蛋白无法竞争纽蛋白Vd1结构域与含有组成型活性VBS的固定化踝蛋白多肽的结合。然而，在下拉试验中，踝蛋白确实与GST-Vd1结合，等温滴定量热法测量表明解离常数（K(d)）约为9 μM。有趣的是，Vd1结合暴露了踝蛋白杆中898和899位残基之间的一个胰蛋白酶切割位点，表明在踝蛋白杆的N端部分存在一个或多个活性VBS。该区域包括一个五螺旋束（482 - 655位残基），后面跟着一个七螺旋束（656 - 889位残基），并包含五个VBS（螺旋4、6、9、11和12）。482 - 655内的单个VBS在室温下是隐蔽的。相比之下，踝蛋白482 - 889与Vd1具有高亲和力结合（K(d)约为0.14 μM），表明656 - 889内的四个VBS中的一个或多个是活性的，这可能代表完整踝蛋白中的纽蛋白结合区域。支持这一点的是，血凝素标记的踝蛋白482 - 889有效地定位于粘着斑，而482 - 655则没有。差示扫描量热法显示Vd1结合与螺旋束稳定性之间存在强烈的负相关，并且具有更稳定折叠的755 - 889突变体与野生型相比结合Vd1的能力要差得多。我们得出结论，构成踝蛋白杆的螺旋束的稳定性是决定单个VBS活性的一个重要因素。
