Crystal structure of a full-length beta-catenin

Abstract

beta-catenin plays essential roles in cell adhesion and Wnt signaling, while deregulation of beta-catenin is associated with multiple diseases including cancers. Here, we report the crystal structures of full-length zebrafish beta-catenin and a human beta-catenin fragment that contains both the armadillo repeat and the C-terminal domains. Our structures reveal that the N-terminal region of the C-terminal domain, a key component of the C-terminal transactivation domain, forms a long alpha helix that packs on the C-terminal end of the armadillo repeat domain, and thus forms part of the beta-catenin superhelical core. The existence of this helix redefines our view of interactions of beta-catenin with some of its critical partners, including ICAT and Chibby, which may form extensive interactions with this C-terminal domain alpha helix. Our crystallographic and NMR studies also suggest that the unstructured N-terminal and C-terminal tails interact with the ordered armadillo repeat domain in a dynamic and variable manner.

Figure 1. Human β-Catenin-R1C Crystal Structure

(A) Stereoview of SIRAS experimental map of β-catenin-R1C C-terminal domain helix contoured at 1 σ. (B) β-catenin-R1C crystal structure. Each armadillo repeat of β-catenin, except repeat 7, is composed of three helices that are shown as blue (helix 1), green (helix 2), and yellow (helix 3) cylinders, whereas the C-terminal domain is colored in red. The C-terminal domain α helix is named helix C. (C) SDS-gel analysis of β-catenin-R1C crystals. Lane 1, molecular weight marker. Lane 2, purified β-catenin armadillo repeat domain protein (residues 133–665). Lane 3, purified β-catenin-R1C protein (residues 133–781). Lane 4, dissolved β-catenin-R1C crystals.

Figure 2. Interactions between β-Catenin C-Terminal Domain and Central Armadillo Repeat Domain

(A) Electrostatic surface of the β-catenin armadillo repeat domain covered by the β-catenin C-terminal domain. Residues from the armadillo repeat domain are labeled in black. β-catenin C-terminal domain is in a stick model labeled in red. Key residues of the C-terminal domain interacting with the armadillo repeat domain are highlighted in green for hydrophobic interactions and yellow for charge-charge interactions. (B) Sequence alignment of the β-catenin C-terminal domain with CLUSTALW. Invariant residues are labeled with stars in red, strongly similar residues are labeled with (:) in green, and weakly similar residues are labeled with (.) in blue. Sequences of the C-terminal domain visible in the β-catenin-R1C crystal structure are framed. The conserved hydrophobic residues (L674, L678, and L682) that docked the helix C into the armadillo repeat domain are marked with arrows. (C) Ligplot presentation of interactions between β-catenin residues in the C-terminal domain (purple) and the armadillo repeat domain (red). Hydrogen bonds and charge-charge interactions are designated with green dashed lines and distance in Å. A red starburst together with a red dashed line represents hydrophobic interactions.

Figure 3. Full-Length Zebrafish β-Catenin Crystal Structure

(A) Crystal structure of full-length zebrafish β-catenin. The color scheme of β-catenin armadillo repeats is the same as that in Figure 1. The C-terminal helix (helix C) is colored in red. The N-terminal helix is colored in blue and green with an arrow pointing to the kink. All β-catenin residue numbers shown in this figure are these of corresponding residue numbers of human β-catenin. (B) SDS-gel analysis of the dissolved full-length zebrafish β-catenin crystals. Lane 1, molecular weight marker. Lane 2, dissolved crystals. Lane 3, purified full-length zebrafish β-catenin. (C) Structural superposition between human β-catenin-R1C (red) and full-length zebrafish β-catenin (cyan) around helix C.

Figure 4. The Role of Helix C in β-Catenin Protein-Protein Interactions

(A) Potential interactions between β-catenin helix C and ICAT. β-catenin-R1C (βcat-R1C, cyan) is superimposed onto β-catenin/ICAT (βcat/ICAT, yellow and magenta), based on the Cα’s of β-catenin armadillo repeats 10–12 (residues 563–663). (B) Helix C is unlikely to affect the interaction between the phophorylated E-cadherin and β-catenin armadillo repeats. β-catenin-R1C (βcat-R1C, cyane) is superimposed with β-catenin/phospho-Ecadherin (βcat/pEcad, yellow and red, PDB code: 1I7W) based on all twelve armadillo repeats. (C) The helix C is required for β-catenin to interact with Chibby. Purified MBP-Chibby protein was immobilized to amylose beads; purified β-catenin and β-catenin fragments were tested for their binding to immobilized MBP-Chibby. The total input and bound β-catenin and β-catenin fragments were visualized by Western blot.

Figure 5. NMR Analysis of Potential Intramolecular Interactions of β-Catenin

NMR studies suggest highly dynamic interactions between the C-terminal tail and the β-catenin N-R12 fragment ¹⁵N-TROSY spectra of the ¹⁵N-labeled β-catenin(687–781) alone (green) and with unlabeled β-catenine(1–686) (red). Poor dispersion of the resonance indicates that β-catenin(687–781) is unstructured in the absence or presence of β-catenine(1–686). Some minor chemical shift perturbations, as indicated by the arrows, suggest that β-catenin(687–781) may interact with β-catenine(1–686) in a highly dynamic manner.