Structural basis of the Axin-adenomatous polyposis coli interaction

Abstract

Axin and the adenomatous polyposis coli (APC) tumor suppressor protein are components of the Wnt/Wingless growth factor signaling pathway. In the absence of Wnt signal, Axin and APC regulate cytoplasmic levels of the proto-oncogene beta-catenin through the formation of a large complex containing these three proteins, glycogen synthase kinase 3beta (GSK3beta) and several other proteins. Both Axin and APC are known to be critical for beta-catenin regulation, and truncations in APC that eliminate the Axin-binding site result in human cancers. A protease-resistant domain of Axin that contains the APC-binding site is a member of the regulators of G-protein signaling (RGS) superfamily. The crystal structures of this domain alone and in complex with an Axin-binding sequence from APC reveal that the Axin-APC interaction occurs at a conserved groove on a face of the protein that is distinct from the G-protein interface of classical RGS proteins. The molecular interactions observed in the Axin-APC complex provide a rationale for the evolutionary conservation seen in both proteins.

Fig. 1. Primary structure of the Axin and APC proteins. (A) Schematic of APC primary structure. The conserved oligomerization (olig.), armadillo repeat (arm.), basic and discs large interaction (dlg) domains are indicated. The 15 amino acid β-catenin-binding repeats are labeled A, B and C (white boxes). The 20 amino acid β-catenin-binding repeats are labeled 1–7 (black boxes). The Axin-binding repeats are labeled SAMP1–3 (gray boxes). Truncations in the midpoint cluster region (MCR) account for >60% of oncogenic mutations in APC (Miyoshi et al., 1992). The APC 7L, APC 7S and APC SAMP3 constructs used for binding assays are shown below the schematic and their boundaries are indicated. (B) Schematic of Axin primary structure showing regions identified by deletion experiments to be important for protein–protein interactions with APC, GSK3β, β-catenin and protein phosphatase 2a (PP2a). A region of homology to the DIX domain of Dishevelled has been implicated in Axin homodimerization. The region of sequence homology to RGS proteins is indicated. Bars indicate the regions corresponding to the thrombin-defined p38 fragment and the elastase-defined Axin-RGS fragment.

Fig. 2. Identification of minimal interaction domains of APC and Axin. (A) Binding of the Axin-RGS and p38 fragments to the GST–APC 7S fusion protein. Lane 1, 1 nmol Axin-RGS; lane 2, 1 nmol p38; lanes 3–8, GST pull-down assays containing Axin-RGS, p38, GST and GST–APC 7S as indicated. The GST in lane 7 is a result of cleavage of the fusion protein by a thrombin contaminant in the Axin-RGS preparation. (B) Binding of Axin-RGS to the GST–APC SAMP3, GST–APC 7S and GST–APC 7L constructs.

Fig. 3. Conservation of SAMP repeats and RGS domains. (A) Alignment of the SAMP repeats of Drosophila APC (dAPC), Drosophila APC2 (eAPC), Xenopus and human APC. Starting residue numbers for within the full-length proteins are indicated. The SAMP3 sequence used in crystallization is boxed. Residues that contact Axin-RGS in the RGS–SAMP3 complex structure are indicated by a diamond (contacts by side chain atoms) or an asterisk (contacts by main chain atoms only) below the alignment. A consensus sequence is given (h, hydrophobic; b, basic), and the residues that form the α-helical portion of the peptide are indicated. (B) Structure-based alignment of Axin-RGS and RGS4 with other RGS family members. Conserved hydrophobic core residues are highlighted in gray, residues determined to contact G_iα in the RGS4–G_iα complex structure (Tesmer et al., 1997) are in pink. Conserved Axin subfamily residues are light blue. Residues that contact SAMP3 in the RGS–SAMP3 complex structure are indicated by symbols above the alignment, as in (A). Those residues changed in human Axin in the mutagenesis experiments are boxed. Secondary structure elements observed in Axin-RGS are indicated above the alignment (α1–α9), and tick marks indicate every 10 residues in human Axin. Axin family members shown are: human Axin (crystal structure in this paper, DDBJ/EMBL/GenBank accession No. AAC51624), human conductin (NM_004655), chicken Axin (AF009012), Drosophila Axin (AF086811) and Xenopus Axin (AF097313). Other RGS family members are Caenorhabditis elegans Egl10 (CEU32326), C05B5 (Z32679) and F16H9 (Z50005), human GAIP (NM_005873), human RGS 1 (NM_002922), 2 (NM_002923), 3 (HSU27655) and 7 (U32439) and rat RGS4 (AF117211).

Fig. 4. Structure of Axin-RGS. (A) Final Axin-RGS 2F_o – F_c α-calc electron density map in the region of surface-exposed, conserved residues Phe156 and Gly160. The map is contoured at 1.2σ. (B) Comparison of Axin-RGS with RGS4. Axin-RGS is red, RGS4 is gray. Helices are labeled as in Figure 3B. The additional helix of Axin-RGS (α5a) and the turn of the π-helix (π) are indicated.

Fig. 5. Structure of the RGS–SAMP3 complex. (A) Final RGS–SAMP3 2F_o – F_c α-calc electron density map in the region of SAMP3 residues Cys2043–Pro2049. The map is contoured at 1σ. (B) The SAMP3-binding site of Axin-RGS is distinct from the G_iα-binding site of RGS4. The Axin-RGS–SAMP3 complex is superimposed on the structure of the RGS4–G_iα complex. Axin-RGS is red, SAMP3 is blue, RGS4 is light gray and G_iα is dark gray. The complex is rotated 90° perpendicular to the page, then 180° around the vertical relative to the orientation of Axin-RGS in Figure 4B. (C) Conservation of the APC-binding surface of Axin-RGS. Surface representation of Axin-RGS, colored by conservation of residues within Axin family members. White indicates that a residue is not significantly conserved, yellow and orange indicate residues that are conserved or conservatively substituted, and red indicates residues that are absolutely conserved in Axin homologs. The SAMP3 peptide C_α trace is drawn in blue. The second conserved patch referred to in the text is visible near the top of Axin-RGS, above the SAMP3-binding site. The complex is rotated 180° around the horizontal relative to its orientation in (B).

Fig. 6. The molecular interactions between Axin-RGS and SAMP3. (A) Interactions of SAMP3 Leu2039 and Leu2040 with Axin-RGS. Protein backbones are colored red for Axin-RGS or blue for SAMP3, and residue numbers are labeled using the same color scheme. Side chains are colored by atom type (carbon, gray; nitrogen, blue; oxygen, red; sulfur, yellow). Solid black lines indicate hydrophobic contacts. The orientation of the complex is as in Figure 5B. (B) Interactions of SAMP3 Cys2043 and Ile2044 with Axin-RGS. Coloring is as in (A). The complex is rotated 180° around the horizontal relative to its orientation in Figure 5B. (C) Interactions of the characteristic Ser-Ala-Met-Pro sequence of SAMP3 with Axin-RGS. Coloring is as in (A) and (B). Orientation is as in Figure 5B. (D) Effects of mutation of conserved Axin-RGS residues on SAMP3 binding. Graph shows mean ± SD of four binding assays for each mutant, plotted as a percentage of wild-type binding.