The Crystal Structure of the β-Catenin/ICAT Complex Reveals the Inhibitory Mechanism of ICAT  Thomas A Graham, Wilson K Clements, David Kimelman, Wenqing Xu  Molecular Cell  Volume 10, Issue 3, Pages 563-571 (September 2002) DOI: 10.1016/S1097-2765(02)00637-8

Figure 1 Overall Structure of ICAT Bound to the Armadillo Repeat Region of β-Catenin The core of β-catenin is made up of the armadillo repeat region which consists of 12 repeats (labeled in the figure). Each repeat consists of three helices colored in blue, green, and yellow, except for repeat 7, which only contains the second and third helices. The third helices of repeats 5–12 form the platform upon which ICAT binds to β-catenin in an anti-parallel fashion. ICAT binds to β-catenin with two discrete modules, an N terminus 3-helix bundle (shown in fuchsia) and a C terminus extended β strand-like tail (shown in red). The 3-helix bundle domain binds to β-catenin armadillo repeats 10–12 and the tail binds along repeats 5–9. This figure was generated with GRASP (Nicholls et al., 1991), Molscript (Kraulis, 1991), and RASTER3D (Merritt and Murphy, 1994). Molecular Cell 2002 10, 563-571DOI: (10.1016/S1097-2765(02)00637-8)

Figure 2 ICAT 3-Helix Bundle Domain Bound to β-Catenin (A) Stereo 2Fo-Fc map of the 3-helix bundle domain of ICAT bound to β-catenin. The ICAT residues are denoted in fuchsia, and the β-catenin residues are denoted in yellow. The map is contoured at 1σ. (B) Ribbon and ball-and-stick diagram showing the specific interactions between the 3-helix bundle domain of ICAT and the third helix of β-catenin armadillo repeat 12. The color scheme is the same as that described in Figure 1. (C) Superposition of the β-catenin/E-cadherin and the β-catenin/ICAT structures was carried out by superimposing β-catenin repeats 10–12. ICAT is shown in fuchsia and E-cadherin in turquoise. Molecular Cell 2002 10, 563-571DOI: (10.1016/S1097-2765(02)00637-8)

Figure 3 ICAT Tail Domain Bound to β-Catenin (A) Molecular model of the ICAT tail domain bound to β-catenin. ICAT is shown in the ball-and-stick format (residues labeled in black) and β-catenin as cylinders. The two charged buttons of β-catenin, residues Lys312 and Lys435, are also shown in the ball-and-stick format. The color scheme is the same as described in Figure 1. (B) Structural alignment of the β-catenin-bound forms of ICAT (red), XTcf3 (green), and E-cadherin (turquoise). Three conserved residues are labeled and color coded with the respective sequential positions given in the structures. The structures of these three complexes were superimposed using armadillo repeats 5–8 of β-catenin. (C) Sequence alignment of the charged-button-recognition region of ICAT together with representative Tcf and cadherin family members. LZIC, a recently discovered member of the ICAT family, is also shown. The conserved aspartic acid that has been shown to interact with β-catenin Lys435 for ICAT, hTcf4, XTcf3, and E-cadherin is shown in red, and the conserved hydrophobic residue is shown in yellow. The cluster of glutamic acids, denoted in green, can form a salt bridge with Lys312, using various conformations (Graham et al., 2001). Molecular Cell 2002 10, 563-571DOI: (10.1016/S1097-2765(02)00637-8)

Figure 4 β-Catenin Phe660 and Arg661 Are Required for ICAT Binding β-catenin carrying the mutations F660A/R661A (mut βCat) was tested for its ability to bind ICAT in vitro. Wild-type (lane 1) but not mutant (lane 2) β-catenin is able to coprecipitate ICAT. All of the proteins were 35S-labeled and mixed as described. β-catenin was immunoprecipitated via its HA-epitope tag. Lanes 4–6 show protein levels prior to immunoprecipitation. Molecular Cell 2002 10, 563-571DOI: (10.1016/S1097-2765(02)00637-8)

Figure 5 The ICAT Tail Domain Is Necessary to Block the Binding of Tcf to β-Catenin β-catenin, precipitated via its HA-epitope tag, was tested for its ability to coprecipitate XTcf3ΔC (A) or C-cadherin (B) in the presence of ICAT (lane 2), ICATΔC (lane 3), or BSA (lane 4). 35S-labeled β-catenin, XTcf3ΔC, and C-cadherin were produced in vitro and mixed with 100 ng unlabeled ICAT, ICATΔC, or BSA. Lanes 5–8 show protein levels present prior to immunoprecipitation. XTcf3ΔC was used instead of full-length XTcf3 for convenience (Graham et al., 2000). (A) Wild-type ICAT (lane 2) but not ICATΔC (lane 3) blocks coprecipitation of XTcf3ΔC. (B) Either wild-type ICAT (lane 2) or ICATΔC (lane 3) blocks the coprecipitation of C-cadherin in vitro. Molecular Cell 2002 10, 563-571DOI: (10.1016/S1097-2765(02)00637-8)

Figure 6 ICAT Selectively Blocks Tcf Binding to β-Catenin In Vivo The ability of β-catenin to bind either XTcf3-HA or C-cadherin-HA in the presence or absence of ICAT was tested in Xenopus embryos. Equivalent levels of GFP RNA were injected into embryos that did not receive ICAT RNA. Embryo lysates were immunoprecipitated (lanes 6–10) for the FLAG epitope tag on β-catenin and probed with an anti-HA antibody. Lanes 1–5 correspond to lanes 6–10 and show the levels of proteins in total lysates prior to immunoprecipitation. Lanes 1 and 6 show results from uninjected embryos. ICAT prevents coprecipitation of XTcf3 (lane 8) but not C-cad (lane 10) with β-catenin. Molecular Cell 2002 10, 563-571DOI: (10.1016/S1097-2765(02)00637-8)