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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 wnt-signaling pathway, essential for embryonal development as well as the growth of normal and malignant colon epithelium. We have characterized the binding of TCF4 to human beta-catenin by steady-state intrinsic fluorescence quenching experiments, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Binding studies in solution and in heterogeneous phase showed that TCF4 binds reversibly to beta-catenin with an affinity (KB) of 3(+/-1) 10(8) M(-1). Site-directed mutagenesis, together with calorimetric measurements, revealed that residue D16 in TCF4 plays a crucial role in high-affinity binding. Mutation of this residue to alanine resulted in a decrease of KB 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 degrees C (-29.7 kcal/mol). Binding enthalpies were strongly temperature dependent, which resulted in the determination of a large heat capacity change upon binding of -1.5 kcal/(mol K). The molecular events that take place upon complex formation are discussed using the measured thermodynamic data together with the crystal structure of the beta-catenin arm repeat region/TCF complex.

Original publication

DOI

10.1006/jmbi.2001.4463

Type

Journal article

Journal

J Mol Biol

Publication Date

09/03/2001

Volume

306

Pages

1179 - 1189

Keywords

Binding Sites, Circular Dichroism, Cloning, Molecular, Cytoskeletal Proteins, DNA Primers, Fluorescence, Glutathione Transferase, Humans, Models, Molecular, Polymerase Chain Reaction, Protein Structure, Secondary, Recombinant Fusion Proteins, Surface Plasmon Resonance, TCF Transcription Factors, Thermodynamics, Trans-Activators, Transcription Factor 7-Like 2 Protein, Transcription Factors, beta Catenin