Thermodynamics of the high-affinity interaction of TCF4 with beta-catenin

The formation of a complex between beta -catenin and members of the TCF/LEF family of high-mobility group proteins is a key regulatory event in the wn t-signaling pathway, essential for embryonal development as well as the gro wth of normal and malignant colon epithelium. We have characterized the bin ding of TCF4 to human beta -catenin by steady-state intrinsic fluorescence quenching experiments, surface plasmon resonance (SPR) and isothermal titra tion calorimetry (TTC). Binding studies in solution and in heterogeneous ph ase showed that TCF4 binds reversibly to beta -catenin with an affinity (K- B) of 3 (+/-1) 10(8) M-1 Site-directed mutagenesis together with calorimetr ic measurements, revealed that residue D16 in TCF4 plays a crucial role in high-affinity binding. Mutation of this residue to alanine resulted in a de crease of K-B by two orders of magnitude as well as a significant reduction in binding enthalpy. Binding of TCF4 to beta -catenin gave rise to a large negative enthalpy change at 25 degreesC (-29.7 kcal/mol). Binding enthalpi es were strongly temperature dependent, which resulted in the determination of a large heat capacity change upon binding of -1.5 kcal/(mol K). The mol ecular events that take place upon complex formation are discussed using th e measured thermodynamic data together with the crystal structure of the be ta -catenin arm repeat region/TCF complex. (C) 2001 Academic Press.