Systematic peptide array-based delineation of the differential beta-catenin interaction with Tcf4, E-cadherin, and adenomatous polyposis coli

Nuclear accumulation of the complex between beta-catenin and proteins of the T-cell factor (Tcf) family is a hallmark of many cancers. Targeting this interaction for drug development is complicated by the fact that E-cadherin and adenomatous polyposis coli (APC) bind to overlapping sites on beta-catenin. Inhibiting their interactions might actually promote tumor growth. To identify selective beta-catenin binding hot spots of Tcf4, E-cadherin, and APC, array technology with peptides of up to 53 amino acids length was used. Interactions were monitored by a quantitative fluorescent readout, which was shown to represent a monitor of true equilibrium binding constants. We identified minimal binding motifs in the beta-catenin ligands and showed that most of the 15-mer and 20-mer repeats of APC did not interact, at least when non-phosphorylated, and defined a consensus binding motif also present in APC. We confirmed previously found hot spots and identified new ones. The method allowed us to locate a hydrophobic pocket that was relevant for the Tcf, but not the E-cadherin interaction, and would thus constitute an ideal drug target site.     

Hot spots in Tcf4 for the interaction with beta-catenin

The interaction of beta-catenin with T-cell factor (Tcf) 4 plays a central role in the Wnt signaling pathway and has been discussed as a possible site of intervention for the development of anti-cancer drugs. In this study, we performed Ala-scanning mutagenesis of all Tcf4 residues in the Tcf-beta-catenin interface and studied the binding energetics of these mutants using isothermal titration calorimetry. Binding of Tcf4 was found to be highly cooperative. Single site mutations of most Tcf4 residues resulted in a significant reduction in binding enthalpies but in similar binding constants as compared with wild type Tcf4. Interestingly, this was also true for residues that are disordered in the reported crystal structures. The mutation D16A caused the largest reduction in binding constant (50-fold) accompanied by a large unfavorable enthalpy change (DeltaDeltaHobs) of +8 kcal/mol at 25 degrees C. In contrast, the mutation of the Tcf residues Glu24 and Glu28, which have been proposed as an interaction hot spot due to their location in a field of strong positive electrostatic potential on the beta-catenin surface (charge button), resulted only in a significant reduction of binding enthalpies, which were largely compensated for by unfavorable entropic contributions to the binding. Other mutations that significantly reduced Tcf binding constants were D11A and alanine mutations of the hydrophobic residues Leu41, Val44, and Leu48. The measured thermodynamic data are discussed with the available structural information of Tcf-beta-catenin crystal structures and allow us to propose possible sites for development of Tcf antagonists.