ICAT Inhibits β-Catenin Binding to Tcf/Lef-Family Transcription Factors and the General Coactivator p300 Using Independent Structural Modules  Danette L Daniels, William I Weis  Molecular Cell  Volume 10, Issue 3, Pages 573-584 (September 2002) DOI: 10.1016/S1097-2765(02)00631-7

Figure 1 Overall View of the β-Catenin/ICAT Complex (A) Schematic of ICAT primary structure showing major secondary structure and β-catenin interaction regions. The residues corresponding to the three helices of ICAT, termed A, B, and C, and the extended region are shown. Dashed lines indicate residues of ICAT not observed in the crystal. (B) Ribbon diagram of ICAT (purple) bound to the β-catenin arm repeat domain. The H3 helices of the arm repeats are shown in blue, and the remainder of β-catenin is shown in gray. The dashed line between the helical domain and the extended region of ICAT is based upon electron density visible at low contour levels that likely corresponds to residues 55–59. These residues could not be modeled reliably and were not included in the final model. (C) Ribbon diagram showing the overlay of β-catenin ligands. The coordinates of β-catenin from the crystal structures bound to ICAT (purple), xTcf-3 (Graham et al., 2000) (green), and E-cadherin (Huber and Weis, 2001) (yellow), were superimposed, then removed to show the relative position of each ligand. The orientation is the same as in part (B) and the N and C termini of each ligand are marked in their respective color. (B) and (C), as well as Figures 2, 3A, and 3C were made with MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt and Bacon, 1997). Molecular Cell 2002 10, 573-584DOI: (10.1016/S1097-2765(02)00631-7)

Figure 2 The ICAT Helical Domain (A) Structure of the helical domain showing key hydrophobic core packing interactions. Helices A, B, and C are indicated. (B) Hydrophobic interactions between helix A and residues of β-catenin. The color scheme is the same as Figure 1B, with β-catenin arm repeats 11 and 12 termed R11 and R12, respectively. Side chains from each protein that participate in hydrophobic interactions are shown. Also shown is the superposition of the region II helix of E-cadherin (Huber and Weis, 2001) (yellow; residue numbers shown in parentheses). The superposition was performed as described in Figure 1C. The hydrogen bond between Tyr 654 of β-catenin and Asp 665 of E-cadherin is shown with a solid line. (C) Electrostatic interactions (dashed lines) of ICAT glutamate residues 37, 38, and 39 with arginine residues in β-catenin or within ICAT, labeled as in (B). Mutation of these three residues to alanine abolishes ICAT binding to β-catenin. Molecular Cell 2002 10, 573-584DOI: (10.1016/S1097-2765(02)00631-7)

Figure 3 Overlap of Extended Region with Other β-Catenin Ligands (A) Comparison of the backbone in the extended region after superimposing β-catenin. Purple, ICAT; yellow, E-cadherin; red, hTcf-4 (Graham et al., 2001); and blue, APC 15-mer repeat A (Spink et al., 2001). Arm repeats 5–10 of β-catenin are labeled R5–R10, and the color scheme is the same as Figure 1B. (B) Comparison of extended region sequences. Conserved residues involved in interactions with β-catenin are boxed. The two acidic residues form salt bridges with β-catenin. The spacing of the first four conserved residues is preserved in all structures, whereas the position of the second acidic residue varies. For example, the sequence of hTcf-4 can adopt two conformations, using different acidic residues to bind to the same residue, Lys312, of β-catenin (see [C]). (C) Detailed comparison of ICAT with the two hTcf-4/β-catenin complex structures that adopt different conformations in the extended region. Purple, ICAT; red, hTcf-4 (Graham et al., 2001); and cyan, hTcf-4 (Poy et al., 2001). The view is rotated 180° about the vertical axis relative to that shown in (A). Interactions with β-catenin residues are shown schematically. ICAT residues are labeled and β-catenin residues are shown in solid boxes. Polar side chains and backbone atoms of β-catenin which form electrostatic interactions (dashed lines) with residues of ICAT are depicted by red- and blue-filled circles corresponding to oxygen and nitrogen atoms, respectively. The black half circles depict hydrophobic interactions. Side chains of residues highly conserved in the extended region among all ligands (see [B]) are shown for ICAT and both Tcf structures. Residues of β-catenin with altered conformations or which are unique to the ICAT interaction compared to other ligands, are marked with a star. Molecular Cell 2002 10, 573-584DOI: (10.1016/S1097-2765(02)00631-7)

Figure 4 β-Catenin Complexes with Lef-1 and p300 and Their Inhibition by ICAT (A) Schematic diagram of β-catenin and the binding regions for Tcf/Lef, CBP/p300, and ICAT. The N- and C-terminal domains of β-catenin are indicated as NT and CT, respectively, and the arm repeats are labeled 1–12. Residue numbers indicate domain boundaries. (B) Schematic diagrams of the constructs used in the binding experiments. Labeling for the β-catenin constructs is similar to that used in part (A). Lef-1 and Tcf-4 β-catenin binding domains are termed βBD. The constructs of ICAT include the full-length protein (ICAT) and the helical domain (ICAT-61). Constructs containing the p300 CH3 domain and CBP KIX (or CREB binding) domain are also shown. Residue numbers indicate domain boundaries. (C) Overlays of Superdex 200 gel filtration elution profiles for the indicated complexes, as well as unliganded Lef-1 βBD. (D) SDS-PAGE profile showing the peak Superdex 200 fraction of the β-cat ARM-CT/Lef-1 βBD/p300 GST-CH3 ternary complex. Sizes of molecular weight standards are indicated. CH3 and GST-CH3 behave similarly in terms of complex formation (Table 2A), but the free p300 CH3 domain stains poorly with Coomassie blue, so the gel in this and the next panel show experiments in which the GST-CH3 fusion construct was used. (E) Disruption of the binary β-cat ARM-CT/p300 GST-CH3 complex by ICAT-61. SDS-PAGE profile of fractions across an S200 gel filtration column in the order of increasing elution volume. The S200 purified β-cat ARM-CT/GST-CH3 complex was mixed with an excess of purified ICAT-61 and applied to the column. The separation of β-cat ARM-CT from p300 GST-CH3, and the coelution of the β-cat ARM-CT/ICAT-61 complex are readily apparent. Sizes of molecular weight standards are indicated. Molecular Cell 2002 10, 573-584DOI: (10.1016/S1097-2765(02)00631-7)

Figure 5 Model of ICAT as a Bipartite Transcriptional Inhibitor On the left, transcription activation complexes consisting of β-catenin, Tcf/Lef, and CBP/p300 are bound to cognate DNA sites through interaction of the Tcf/Lef HMG box. Binding of ICAT to β-catenin simultaneously displaces CBP/p300 and Tcf/Lef, allowing for Groucho/TLE repressor proteins to bind to Tcf/Lef. Molecular Cell 2002 10, 573-584DOI: (10.1016/S1097-2765(02)00631-7)