Andrey A. Bobkov

Structural basis for vinculin activation at sites of cell adhesion

Vinculin is a highly conserved intracellular protein with a crucial role in the maintenance and regulation of cell adhesion and migration. In the cytosol, vinculin adopts a default autoinhibited conformation. On recruitment to cell–cell and cell–matrix adherens-type junctions, vinculin becomes activated and mediates various protein–protein interactions that regulate the links between F-actin and the cadherin and integrin families of cell-adhesion molecules. Here we describe the crystal structure of the full-length vinculin molecule (1,066 amino acids), which shows a five-domain autoinhibited conformation in which the carboxy-terminal tail domain is held pincer-like by the vinculin head, and ligand binding is regulated both sterically and allosterically. We show that conformational changes in the head, tail and proline-rich domains are linked structurally and thermodynamically, and propose a combinatorial pathway to activation that ensures that vinculin is activated only at sites of cell adhesion when two or more of its binding partners are brought into apposition.

Recreation of the terminal events in physiological integrin activation

In vitro analysis confirms talin binding is sufficient for activation and extension of membrane-embedded integrin.

Structural and Functional Analysis of the Actin Binding Domain of Plectin Suggests Alternative Mechanisms for Binding to F-Actin and Integrin β4

Plectin is a widely expressed cytoskeletal linker. Here we report the crystal structure of the actin binding domain of plectin and show that this region is sufficient for interaction with F-actin or the cytoplasmic region of integrin alpha6beta4. The structure is formed by two calponin homology domains arranged in a closed conformation. We show that binding to F-actin induces a conformational change in plectin that is inhibited by an engineered interdomain disulfide bridge. A two-step induced fit mechanism involving binding and subsequent domain rearrangement is proposed. In contrast, interaction with integrin alpha6beta4 occurs in a closed conformation. Competitive binding of plectin to F-actin and integrin alpha6beta4 may rely on the observed alternative binding mechanisms and involve both allosteric and steric factors.

αE-catenin is an autoinhibited molecule that coactivates vinculin

αE-catenin, an essential component of the adherens junction, interacts with the classical cadherin–β-catenin complex and with F-actin, but its precise role is unknown. αE-catenin also binds to the F-actin-binding protein vinculin, which also appears to be important in junction assembly. Vinculin and αE-catenin are homologs that contain a series of helical bundle domains, D1–D5. We mapped the vinculin-binding site to a sequence in D3a comprising the central two helices of a four-helix bundle. The crystal structure of this peptide motif bound to vinculin D1 shows that the two helices adopt a parallel, colinear arrangement suggesting that the αE-catenin D3a bundle must unfold in order to bind vinculin. We show that αE-catenin D3 binds strongly to vinculin, whereas larger fragments and full-length αE-catenin bind approximately 1,000-fold more weakly. Thus, intramolecular interactions within αE-catenin inhibit binding to vinculin. The actin-binding activity of vinculin is inhibited by an intramolecular interaction between the head (D1–D4) and the actin-binding D5 tail. In the absence of F-actin, there is no detectable binding of αE-catenin D3 to full-length vinculin; however, αE-catenin D3 promotes binding of vinculin to F-actin whereas full-length αE-catenin does not. These findings support the combinatorial or “coincidence” model of activation in which binding of high-affinity proteins to the vinculin head and tail is required to shift the conformational equilibrium of vinculin from a closed, autoinhibited state to an open, stable F-actin-binding state. The data also imply that αE-catenin must be activated in order to bind to vinculin.

Structural Determinants of Integrin Binding to the Talin Rod

The adaptor protein talin serves both to activate the integrin family of cell adhesion molecules and to couple integrins to the actin cytoskeleton. Integrin activation has been shown to involve binding of the talin FERM domain to membrane proximal sequences in the cytoplasmic domain of the integrin β-subunit. However, a second integrin-binding site (IBS2) has been identified near the C-terminal end of the talin rod. Here we report the crystal structure of IBS2 (residues 1974-2293), which comprises two five-helix bundles, “IBS2-A” (1974-2139) and “IBS2-B” (2140-2293), connected by a continuous helix with a distinct kink at its center that is stabilized by side-chain H-bonding. Solution studies using small angle x-ray scattering and NMR point to a fairly flexible quaternary organization. Using pull-down and enzyme-linked immunosorbent assays, we demonstrate that integrin binding requires both IBS2 domains, as does binding to acidic phospholipids and robust targeting to focal adhesions. We have defined the membrane proximal region of the integrin cytoplasmic domain as the major binding region, although more membrane distal regions are also required for strong binding. Alanine-scanning mutagenesis points to an important electrostatic component to binding. Thermal unfolding experiments show that integrin binding induces conformational changes in the IBS2 module, which we speculate are linked to vinculin and membrane binding.

Structural Effects of Cofilin on Longitudinal Contacts in F-actin

Structural effects of yeast cofilin on skeletal muscle and yeast actin were examined in solution. Cofilin binding to native actin was non-cooperative and saturated at a 1:1 molar ratio, with K(d)<or=0.05 microM for both CaATP-G-actin and F-actin. Cofilin binding enhanced the fluorescence of dansyl ethylenediamine (DED) attached to Gln41 on the DNase I binding loop of skeletal muscle F-actin and decreased the fluorescence of AEDANS at Cys41 on yeast Q41C/C374S mutant F-actin. However, cofilin had no effect on the spectral properties of DED or AEDANS on CaATP-G-actin. Fluorescence energy transfer (FRET) from tryptophan residues to DED at Gln41 on skeletal muscle actin and to AEDANS at Cys41 on yeast Q41C/C374S actin was decreased by cofilin binding to F- but not to G-actin. Cofilin inhibited strongly the rate of interprotomer disulfide cross-linking of Cys41 to Cys374 on yeast Q41C mutant F-actin. Binding of cofilin enhanced excimer formation between pyrene probes attached to Cys41 and Cys374 on Q41C F-actin. These results indicate that cofilin alters the interface between subdomains 1 and 2 and shifts the DNase I binding loop away from subdomain 1 of an adjacent actin protomer. Cofilin reduced FRET from tryptophan residues to 4-azido-2-nitrophenyl-putrescine (ANP) at Gln41 in skeletal muscle F-but not in G-actin. However, following the interprotomer cross-linking of Gln41 to Cys374 in F-actin by ANP, cofilin binding did not change FRET from the tryptophan residues to ANP. This suggests that cofilin binding and the conformational effect on F-actin are not coupled tightly. Overall, this study provides solution evidence for the weakening of longitudinal, subdomain 2/1 contacts in F-actin by cofilin.

Vaccinia Virus Virulence Factor N1L is a Novel Promising Target for Antiviral Therapeutic Intervention

The 14 kDa homodimeric N1L protein is a potent vaccinia and variola (smallpox) virulence factor. It is not essential for viral replication, but it causes a strong attenuation of viral production in culture when deleted. The N1L protein is predicted to contain the BH3-like binding domain characteristic of Bcl-2 family proteins, and it is able to bind the BH3 peptides. Its overexpression has been reported to prevent infected cells from committing apoptosis. Therefore, interfering with the N1L apoptotic blockade may be a legitimate therapeutic strategy affecting the viral growth. By using in silico ligand docking and an array of in vitro assays, we have identified submicromolar (600 nM) N1L antagonists belonging to the family of polyphenols. Their affinity is comparable to that of the BH3 peptides (70-1000 nM). We have also identified the natural polyphenol resveratrol as a moderate N1L inhibitor. Finally, we show that our ligands efficiently inhibit growth of vaccinia virus.

The Activity of the Vinculin Binding Sites in Talin Is Influenced by the Stability of the Helical Bundles That Make Up The Talin Rod

The talin rod contains ∼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 Kd of ∼9 μm. 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 (Kd ∼ 0.14 μm), 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.

A mechanism of Rap1-induced stabilization of endothelial cell--cell junctions.

Rap1 stabilizes cell–cell junctions by directly binding to KRIT1, displacing it from microtubules and enabling localization at the junctions.

Structural basis of membrane targeting by the Dock180 family of Rho family guanine exchange factors (Rho-GEFs)

The Dock180 family of atypical Rho family guanine nucleotide exchange factors (Rho-GEFs) regulate a variety of processes involving cellular or subcellular polarization, including cell migration and phagocytosis. Each contains a Dock homology region-1 (DHR-1) domain that is required to localize its GEF activity to a specific membrane compartment where levels of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) are up-regulated by the local activity of PtdIns 3-kinase. Here we define the structural and energetic bases of phosphoinositide specificity by the DHR-1 domain of Dock1 (a GEF for Rac1), and show that DHR-1 utilizes a C2 domain scaffold and surface loops to create a basic pocket on its upper surface for recognition of the PtdIns(3,4,5)P3 head group. The pocket has many of the characteristics of those observed in pleckstrin homology domains. We show that point mutations in the pocket that abolish phospholipid binding in vitro ablate the ability of Dock1 to induce cell polarization, and propose a model that brings together recent mechanistic and structural studies to rationalize the central role of DHR-1 in dynamic membrane targeting of the Rho-GEF activity of Dock180.