1. Volume 15, Issue 4, Pages 523-533 (August 2004)
Crystal Structure of a β-Catenin/APC Complex Reveals a Critical Role for APC Phosphorylation in APC Function 
Yi Xing, Wilson K. Clements, Isolde Le Trong, Thomas R. Hinds, Ronald Stenkamp, David Kimelman, Wenqing Xu 
Molecular Cell 
Volume 15, Issue 4, Pages (August 2004)
DOI: /j.molcel

2. Figure 1
Overall Structure of the β-Catenin/Phospho-APC 20 aa Repeat Complex
(A) Domain structure of APC and sequence of the APC-2,3 fragment. A schematic representation of the APC primary structure shows, from N to C terminus, the oligomerization domain (olig.), armadillo repeats (arm), 15 amino acid (15 aa) β-catenin binding repeats (A-C), 20 amino acid (20 aa) β-catenin binding repeats (1-7), Axin binding repeats (SAMP1-3), basic region (basic), and discs large interaction domain (dlg). A schematic representation and amino acid sequence of the human APC fragment, APC-2,3 (amino acids ), used in the crystallographic study is also shown. APC-2,3 includes the second and third 20 aa repeats (highlighted in yellow). Black bars are marked underneath the APC-2,3 sequence at ten residue intervals with the first mark at residue K1370. The secondary structure of the regions of APC-2,3 visible in the crystal structure is illustrated in red on top of the sequence with α-helical regions indicated. Residues not visible in the structure are marked in black dashes. Phosphorylated APC residues in the crystal structure are marked with red stars. Two charged residues essential for the binding of the extended region of APC-2,3 to β-catenin are marked with vertical red bars.
(B) Two views of the overall β-catenin/phospho-APC 20 aa repeat complex. Each armadillo repeat of β-catenin, except repeat 7, is composed of three helices that are shown as blue, green, and yellow cylinders, whereas the phospho-APC is shown as a red ribbon. Of the APC fragment, only the third 20 aa repeat and its flanking regions are visible in the crystal structure.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Interactions between the Extended Region of Phospho-APC and β-Catenin
(A) Electrostatic surface of β-catenin (armadillo repeats 10-12) bound to the α-helix of APC-2,3 (shown in stick form) N-terminal to the extended region. The surface of β-catenin is colored according to its relative electrostatic potential with blue representing positively charged residues and red representing negatively charged residues. β-catenin and APC amino acids are labeled in yellow and red, respectively.
(B) Electrostatic surface of β-catenin (armadillo repeats 5-9) bound to the extended region of phospho-APC.
(C) Critical contacts in the interface between the phospho-APC extended region and the β-catenin groove of armadillo repeats 5-9. APC is shown in a red ball-and-stick representation with red labels, and β-catenin (with helices colored as in Figure 1B) side chains are yellow with black labels. Phosphorylated Thr1487 of APC is labeled in italics. The hydrogen bonding and charge-charge interactions are designated with pink lines. Water molecules in the interface are shown as blue balls. For clarity, hydrogen bonds bridged by water molecules between APC and β-catenin residues, R474, R386, D390, and K354, are not shown.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
Interactions between the Third Phospho-20 aa Repeat of APC and β-Catenin
(A) Stereo 2Fo - Fc simulated annealed omit map of the phosphorylated third 20 aa repeat of APC (labeled in red) bound to β-catenin. The map is contoured at 1σ.
(B) Electrostatic surface of β-catenin (armadillo repeats 1-5) bound to the phosphorylated third 20 aa repeat and its C-terminal residues. APC and β-catenin are colored as in Figure 2A.
(C) Critical contacts in the interface of the phospho-20 aa repeat of APC bound to the β-catenin groove formed by armadillo repeats 1-5. APC and β-catenin are colored as in Figure 2C.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Comparison of APC Binding to β-Catenin with Other β-Catenin Binding Partners
(A) Sequence alignments of the β-catenin binding regions of human Tcf, E-cadherin, the seven 20 aa repeat regions of APC, and the five 20 aa repeat regions of APC2. Residues included in the alignment are numbered in parentheses. Key conserved residues are colored in red. The 20 aa repeats of APC and APC2 are framed by a red rectangle. Residues phosphorylated in the β-catenin/phospho-APC 20 aa repeat and the β-catenin/phospho-E-cadherin complex structure (Huber and Weis, 2001) are boxed in black. Residues interacting with the two charged buttons of β-catenin are well conserved and highlighted in green. Serine residues absolutely conserved and phosphorylated in the β-catenin/phospho-APC 20 aa repeat crystal structure are highlighted in cyan.
(B) Structural comparison of different protein partners upon binding to the β-catenin groove. Phospho-APC third 20 aa repeat fragment, red; Tcf3-Catenin binding domain, green; APC 15 aa repeat, yellow; cytoplasmic domain of phospho-E-cadherin, blue.
(C) Superposition of the extended region of the phospho-APC-2,3 fragment and the corresponding region of Tcf3. APC and Tcf3 residues are labeled in red and green, respectively. Phosphorylated T1487 is in italics. Green arrows identify residues that make critical contacts with the two charged buttons of β-catenin.
(D) Structural comparison of the third phospho-20 aa repeat of APC (red) and the N-terminal helix of the Tcf3-Catenin binding domain (green) bound to the β-catenin groove of armadillo repeats 2-5 (gray with yellow side chains).
(E) Structural comparison of the third phospho-20 aa repeat of APC (red) and the corresponding region of phospho-E-cadherin (blue) bound to the β-catenin groove of armadillo repeats 2-5 (gray with yellow side chains).
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
The 20 Amino Acid Repeat Region of APC Competes with Tcf for Binding to β-Catenin when Phosphorylated
HA-tagged β-catenin was tested for its ability to coprecipitate Tcf3 in the presence of various competitors. The phosphorylated 20 aa repeat fragment APC-2,3 (lanes 6–8, 0.5 μg pAPC-2,3, 10- and 100-fold dilutions) specifically and dose-dependently blocks coprecipitation of Tcf3 by HA-tagged β-catenin. Unphosphorylated APC-2,3 (lane 5, 0.5 μg APC-2,3), the 15 aa repeat fragment APC-B,C (lane 4, 0.5 μg APC-B,C) and a mock phosphorylation reaction containing no peptide (lane 3), all do not block Tcf3 binding to β-catenin.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
Calorimetric Analysis of the Binding of APC-2,3 to the Full-Length Human β-Catenin
The raw heat data obtained upon each injection was measured in μcal/sec and plotted in the inset windows over the time course of the experiment. The binding isotherms were graphed and normalized to the concentration of the injected protein. (A) For phosphorylated APC-2,3 (pAPC-2,3), the ITC measurement resulted in a binding constant (Kb) of 4.71 (±2.3) × 108 M−1 or a dissociation constant Kd of 2.1 nM, binding stoichiometry N = 1.05 (±0.01), enthalpy change ΔH = −20.7 (±0.4) kcal/mol and entropy change TΔS = −9.0 kcal/mol. (B) For unphosphorylated APC-2,3, we obtained a binding constant (Kb) of 1.69 (±0.08) × 106 M−1 or a dissociation constant Kd of 0.6 μM, binding stoichiometry N = 0.94 (±0.006), enthalpy change ΔH = −14.2 (±0.1) kcal/mol and entropy change TΔS = −6.0 kcal/mol.
Molecular Cell  , DOI: ( /j.molcel )