Crystal structure of the M-fragment of alpha-catenin: implications for modulation of cell adhesion

Abstract

The cytoskeletal protein alpha-catenin, which shares structural similarity with vinculin, is required for cadherin-mediated cell adhesion, and functions to modulate cell adhesive strength and to link the cadherins to the actin-based cytoskeleton. Here we describe the crystal structure of a region of alpha-catenin (residues 377-633) termed the M-fragment. The M-fragment is composed of a tandem repeat of two antiparallel four-helix bundles of virtually identical architectures that are related in structure to the dimerization domain of alpha-catenin and the tail region of vinculin. These results suggest that alpha-catenin is composed of repeating antiparallel helical domains. The region of alpha-catenin previously defined as an adhesion modulation domain corresponds to the C-terminal four-helix bundle of the M-fragment, and in the crystal lattice these domains exist as dimers. Evidence for dimerization of the M-fragment of alpha-catenin in solution was detected by chemical cross-linking experiments. The tendency of the adhesion modulation domain to form dimers may explain its biological activity of promoting cell-cell adhesiveness by inducing lateral dimerization of the associated cadherin molecule.

Fig. 1. Schematic of α-catenin indicating that the protein is composed of tandem repeats of antiparallel α-helical bundles with defined biological functions. The positions of the VH1, VH2, VH3 and the α-catenin VR regions, together with sites of proteolysis are indicated. The domains of α-catenin for which structural data are available (β-catenin/dimerization domain; M-fragment of α-catenin; α-catenin tail) and the position of the predicted VR are shown.

Fig. 2. Detection of protease-resistant domains of α-catenin using limited proteolysis. Incubation of α-catenin with increasing concentra tions of trypsin for 20 min reveals the formation of a partially resistant 63 kDa fragment.

Fig. 3. Structure of α-catenin M-fragment. (A) Overall view of the structure with two four-helix domains, coloured from the N-terminus (blue) to the C-terminus (red). (B) Stereo-view showing the two crystallographically independent molecules (A and B) superimposed using equivalent C_α atoms of domain 2. Red denotes molecule A and blue denotes molecule B. The figure was prepared using BOBSCRIPT (Esnouf, 1997), Raster3D (Merrit and Murphy, 1994) and PREPI (S.Islam and M.Sternberg).

Fig. 4. Similarities between α-catenin M-fragment domains 1 and 2 and domains of other structurally related proteins. (A) Superimposition of α-catenin M-fragment domains 1 (red) and 2 (green), the vinculin tail domain (blue) and the α-catenin dimerization domain (grey). (B) A comparison of the α-catenin M-fragment domains 1 (red/orange) and 2 (green) with the vinculin tail domain (dark/light blue) and α-catenin dimerization domain (grey). Darker colours denote the structures used for alignment onto domain 2 of the α-catenin M-fragment. The figures were prepared using PREPI (S.Islam and M.Sternberg).

Fig. 5. Similarities between α-catenin M-fragment domains 1 and 2. (A) Sequence alignment of α-catenin M-fragment domains 1 and 2 and the vinculin tail domain. (B) Superimposition of α-catenin M-fragment domains 1 and 2 showing residues Val433, Pro462, Ala501 and Val502 of domain 1 and the structurally equivalent residues of domain 2 [indicated with an asterisk in (A)].

Fig. 6. View of the dimer interface of the α-catenin M-fragment domain 2. (A) Crystal packing of dimers of molecules A and B of the α-catenin M-fragment viewed perpendicular to the 2-fold non-crystallographic symmetry axis. (B) Stereo-view showing the interactions between the monomers at the dimer interface formed from domain 2. Colouring scheme as in Figure 3B.

Fig. 7. Evidence that the M-fragment of α-catenin exists as a dimer in solution. Lane 1: 0.25 mg/ml M-fragment of α-catenin in the absence of the cross-linking reagent dimethyl suberimidate (DMS). Lanes 2–5: 0.05, 0.1, 0.25 and 0.5 mg/ml of the α-catenin M-fragment incubated with a 30-fold molar access of DMS for 1.5 h at 20°C. The formation of dimers of the α-catenin M-fragment at concentrations of >0.1 mg/ml (3 µM) (lanes 3–5) is detected by the formation of dimers cross-linked with DMS visualized on an SDS–polyacrylamide gel.

Fig. 8. Sequence alignment of three internal repeats of human meta-vinculin (vinculin repeats) and the related sequence in human α(E)-catenin. The mean sequence identity between the α-catenin VR and the three VRs of vinculin is ∼10%. The positions of the four predicted α-helices are shown. The figure was produced using ESPript (Gouet et al., 1999).