αE-catenin is an autoinhibited molecule that coactivates vinculin

Abstract

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.

Fig. 1.

(A) Primary structures of αE-catenin and vinculin with domains observed in the vinculin crystal structure (Inset) and several functional binding sites indicated. (B) Trypsin digestion of full-length αE-catenin dimer, vinculin D1 and the full-length αE-catenin–vinculin D1 complex. The region of αE-catenin protected by D1 in the complex is indicated. (C) Sequence of αE-catenin protected from trypsin by vinculin D1. Residues shown in gray originate from the expression vector and remain after cleavage of the N-terminal GST affinity tag. Hydrophobic residues that interact with vinculin are underlined in red, and residues that form polar contacts are underlined in green. Italicized residues are not visible in the structure.

Fig. 2.

Crystal structure of αE-catenin^302–356 bound to vinculin D1. αE-catenin is shown in magenta and vinculin in turquoise. Also shown in gray is the bound Shigella IpaA peptide. Interacting hydrophobic side chains are shown in stick representation.

Fig. 3.

Model of vinculin D1 (cyan) binding to αE-catenin. (Left) Model of αE-catenin based on the vinculin crystal structure. The D2 domain was deleted and the flexibility between D3b and D4 in crystal structures of the αE-catenin M domain (12, 23) was modeled by superimposing the D3b bundles of vinculin and αE-catenin and considering the range of positions adopted by αE-catenin D4. For clarity, the actin-binding domain D5 has been omitted from the figure because it has no effect on the affinity for vinculin and is likely flexibly linked to the rest of the protein. (Middle) The two vinculin-binding helices (light green) have been modeled as observed in the crystal structure bound to vinculin D1 with physically plausible connections made to D1 and to D3b. (Right) The structure of the complex between vinculin D1 (cyan) and αE-catenin^302–356 (magenta) has been superimposed on the speculative “open” αE-catenin model. Domains are colored as in Fig. 1, and the D3a helices that bind to vinculin are colored as in Fig. 2.

Fig. 4.

Vinculin activation assays. Pelleting of 10 μM full-length vinculin with F-actin is shown as a function of increasing concentrations of αE-catenin D1–D4 (Left) or αE-catenin D3 (Middle). The lanes marked asterisk (*) show the background sedimentation of the αE-catenin fragment at 10 μM in the absence of F-actin. For comparison, the gel on the right shows the equivalent amount of vinculin loaded at the indicated concentrations.