Structural Basis for Amplifying Vinculin Activation by Talin

Abstract

Talin interactions with vinculin are essential for focal adhesions. Curiously, talin contains three noncontiguous vinculin binding sites (VBS) that can bind individually to the vinculin head (Vh) domain. Here we report the crystal structure of the human Vh⅐VBS1 complex, a validated model of the Vh⅐VBS2 structure, and biochemical studies that demonstrate that all of talin VBSs activate vinculin by provoking helical bundle conversion of the Vh domain, which displaces the vinculin tail (Vt) domain. Thus, helical bundle conversion is a structurally conserved response in talin-vinculin interactions. Furthermore, talin VBSs bind to Vh in a mutually exclusive manner but do differ in their affinity for Vh and in their ability to displace Vt, suggesting that the strengths of these interactions could lead to differences in signaling outcome. These findings support a model in which talin binds to and activates multiple vinculin molecules to provoke rapid reorganization of the actin cytoskeleton.

Comparative Biochemical Analysis Suggests That Vinculin and Metavinculin Cooperate in Muscular Adhesion Sites

“…As can be concluded from recent structural analyses (23), the metavinculin insert, positioned between helices 1 and 2 of VT, is probably not directly involved in binding to VH. It may, however, affect the architecture of the helical bundle and, thus, slightly alter the VH-VT interface, which has been shown to be sensitive to conformational changes in VH (23,24). Although the reduced affinity of MVT for VH is not sufficient to constantly keep the metavinculin molecule in the open conformation, as shown by the cosedimentation experiments (Fig.…”

“…First, recruitment of vinculin to adhesion sites may be mediated either by binding of VH to talin (24) or through association of the C-terminal hydrophobic finger with acidic phospholipids (PIP 2 ) in the plasma membrane (25), causing the release of the intramolecular association VH and VT, thus allowing binding of VH to talin, ␣-actinin, or ␣-catenin of adhesion complexes (9,10,17,20). Second, the C terminus of VT, acting as a clamp at the base of the helical bundle, may undergo a conformational change upon binding to PIP 2 , releasing the helical bundle to unfold (25).…”

“…Domains for the Head Considerably Differ-As mentioned above, the initial step in vinculin activation is a conformational switch in which VH is released from the VT, thus unmasking binding sites for both head and tail ligands (12,20,21,24). To elucidate whether the large acidic insert might alter the ability to change conformational shape, we analyzed the intramolecular association of VH with MVT and VT (Fig.…”

“…First it was shown that binding of acidic phospholipids, especially phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to VT, causes a release from VH (20,21). In addition, talin peptides corresponding to each of the three vinculin binding domains in talin also prevent VH-VT interactions (22)(23)(24).…”

Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues

“…Although talin binding by itself has been proposed to be sufficient to cause vinculin activation (29,30), most biochemical and structural analyses are consistent with the view that the activation of vinculin requires the simultaneous interaction of vinculin with at least two of its binding partners, actin and talin, which may promote the open conformation of vinculin by generating internal molecular stretching forces (25,28,31,32). The interaction of vinculin with acidic phospholipids and ␣-actinin may also contribute to the conversion of vinculin to an open conformation (33)(34)(35).…”