LIG_Vh1_VBS_1

Vinculin was initially identified as a protein highly enriched in cell-to-cell contact and underlying substratum. It has a crucial role in the maintenance and regulation of cell adhesion and migration. In the cytosol, vinculin exists in an inactivated/auto inhibited state and becomes activated when recruited to cell–cell and cell–matrix adherens-type junctions and it anchors these adhesion complexes to the actin cytoskeleton by binding to talin in integrin complexes or to α-actinin in cadherin junctions. Vinculin is made up of a globular head domain (Vh) linked to a tail domain (Vt) by a short proline rich sequence. An intramolecular interaction between head and tail domains masks numerous ligand binding sites in all three regions (Ziegler,2006). The interactions between head and tail domains are so strong that vinculin activation generally requires two or more ligands (combinatorial activation pathway) to relieve the intramolecular head–tail interaction though, in some cases, exposure to a high affinity vinculin binding site present in talin or α-actinin might be sufficient (Bays,2017).

The interaction between the head domain of vinculin and some of its interacting proteins such as talin, α-actinin, IpaA of Shigella flexneri, chlamydial TarP and Rickettsial sca4 antigen induces a marked conformational change in Vh, creating a novel helical bundle structure that displaces Vt from Vh (Izard,2004). These vinculin activators are characterized by vinculin binding sites (VBSs). In the mechanosensory protein Talin, VBSs are buried amphipathic α-helices of about 19 residues in length that reside within four- or five-helix bundle domains (Goult,2013). The key interacting residues are buried inside the core of the helical bundles and are released from their buried locales by mechanical force, enabling interaction with the Vh domain. VBSs activate vinculin by burying within and inducing helix bundle conversion in the N-terminal four-helix subdomain of Vh, which displaces Vt from a distance and exposes its cryptic F-actin binding sites. Eleven potential vinculin binding sites have been described in the talin rod domain, most of them showing low affinity and were not able to induce helix bundle conversion (Gingras,2005). The functional VBS helices must bind with low nanomolar affinity as the complex is dependent on force-induced activation of the talin rod domain (del Rio,2009). The VBS of α-actinin binds to Vh1 in an inverted orientation compared to the binding of talin’s VBSs and provokes distinct structural changes in vinculin (Bois,2005). Thus, vinculin’s Vh1 domain acts as an actin filament-linking molecular switch that undergoes distinct structural changes provoked by talin and α-actinin binding in focal adhesions versus adherens junctions, respectively.

Bacterial pathogens like Shigella flexneri and Rickettsia spp. have developed protein structures that resemble the architecture of the VBSs in talin with the same biological function and co-localization with talin that can induce actin polymerization without the need of integrin activation (Hamiaux,2006, Park,2011). The IpaA, invasin protein of Shigella harbors three tandem high affinity VBS motifs which are readily available and act in a redundant manner to bind and activate vinculin. The IpaA VBSs efficiently mimic the talin VBS-induced vinculin activation and actin filament hijack by IpaA is required for maximal entry of Shigella into host cells. It can simultaneously bind three Vh1 molecules and thus stabilize the focal adhesion-like complexes seen at pathogen entry sites (Park,2011). Surface cell antigen 4 (sca4) is a protein from R. rickettsii that contains two VBSs, sca4-VBS-N and sca4-VBS-C, where the former resembles the binding of the talin or IpaA VBSs, while the later contains a Pro at position +14 causing a kink in the helix structure. The crystal structure of sca4-VBS-C with vinculin shows that the position of the α-helix α1 in the Vh1 domain of vinculin has a dramatic movement compared to its corresponding position in other structures containing non-kinked helices (Park,2011), therefore exemplifying a third variant Vh1 binding-motif. Chlamydial TarP proteins also have two or three VBSs (Whitewood,2018) and so far no bacterial effector with only a single VBS has been reported.