Mapping and Consensus Sequence Identification for Multiple Vinculin Binding Sites within the Talin Rod

Abstract

The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. Three vinculin binding sites (VBS1-3) have previously been identified in the talin rod using a yeast two-hybrid assay. To extend these studies, we spot-synthesized a series of peptides spanning all the ␣-helical regions predicted for the talin rod and identified eight additional VBSs, two of which overlap key functional regions of the rod, including the integrin binding site and C-terminal actin binding site. The talin VBS ␣-helices bind to a hydrophobic cleft in the N-terminal vinculin Vd1 domain. We have defined the specificity of this interaction by spot-synthesizing a series of 25-mer talin VBS1 peptides containing substitutions with all the commonly occurring amino acids. The consensus for recognition is LXXAAXXVAXX-VXXLIXXA with distinct classes of hydrophobic side chains at positions 1, 4, 5, 8, 9, 12, 15, and 16 required for vinculin binding. Positions 1, 8, 12, 15, and 16 require an aliphatic residue and will not tolerate alanine, whereas positions 4, 5, and 9 are less restrictive. These preferences are common to all 11 VBS sequences with a minor variation occurring in one case. A crystal structure of this variant VBS peptide in complex with the vinculin Vd1 domain reveals a subtly different mode of vinculin binding.Vinculin (1066 amino acids) is associated with integrin-containing cell-extracellular matrix and cadherin-based cell-cell junctions (1). Electron microscopy images of chicken vinculin show a trilobar head 80 Å in diameter connected to a long flexible "tail" (2), and a recent crystal structure of the full-length vinculin molecule shows a circular five-domain autoinhibited conformation in which the N-terminal head domain (Vd1) 3 forms an extensive interaction with the C-terminal tail domain (Vt) (3). The Vd1 domain contains binding sites for talin (4) and ␣-actinin (5), whereas Vt binds to paxillin (6), F-actin (7), and acidic phospholipids (8). The intramolecular Vd1-Vt interaction regulates vinculin activity by masking the binding sites for talin (9) and ␣-actinin (5) in Vd1, the VASP binding site in the proline-rich domain (10, 11), and the F-actin binding site in Vt (7).Talin (2541 amino acids) is an elongated (60 nm) flexible anti-parallel dimer, with a small globular head connected to an extended rod (2). The talin head contains a FERM domain (residues 86 -400) with binding sites for several ␤-integrin cytodomains (12) as well as the type 1␥ 661 isoform of phosphatidylinositol-4-phosphate 5-kinase (13-15), and the protein-tyrosine kinase FAK (16) both of which are important in focal adhesion dynamics. The talin rod contains a second lower affinity integrin binding site (17, 18), a highly conserved C-terminal actin binding site (residues 2345-2541) (19,20), and also several binding sites for vinculin (19). A yeast two-hybrid assay was used to map three of these vinculin binding sites (VBS1, -2, and -3) to short peptide sequences 25-30 residues in length, ...

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.…”

Subcellular Localization of Talin Is Regulated by Inter-domain Interactions

“…The talin rod contains multiple vinculin-binding sites (55), three of which are localized in VBS1/2a. VinHD contains the talin-binding site (41, 42, 44, 56).…”

A Conserved Lipid-binding Loop in the Kindlin FERM F1 Domain Is Required for Kindlin-mediated αIIbβ3 Integrin Coactivation

“…The actin-binding protein talin (270 kDa) was the first to be identified (13), and the effect of talin on integrin activation is now understood in considerable detail (9). Talin is a scaffolding protein that binds and activates the integrin, in addition to recruiting focal adhesion components such as vinculin and actin necessary for subsequent cell adhesion and spreading (13)(14)(15)(16)). There are two major closely related talin isoforms, each composed of an integrin activating N-terminal head (Ϸ50 kDa) and a flexible rod domain (Ϸ220 kDa) (17,18).…”

“…The talin head (residues 1-433) contains a FERM domain (residues 1-400) followed by an apparently unstructured linker (residues 401-481). The rod domain (residues 482-2541) harbors binding sites for actin and vinculin (14,15) as well as a dimerization site and a regulatory autoinhibitory site (19 -21). Unlike classical FERM domains, which are trilobed structures composed of three subdomains (F1, F2, and F3) (22), the talin FERM domain adopts a novel linear arrangement (23) and exhibits two other atypical features: an additional F1-like domain called F0 preceding the F1 domain, and an unstructured loop within F1 (23,24).…”