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

S. Knapp, M. Zamai, D. Volpi, V. Nardese, N. Avanzi, J. Breton, S. Plyte, M. Flocco, M. Marconi, A. Isacchi, V. R. Caiolfa

Research output: Contribution to journalArticlepeer-review


The formation of a complex between β-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 β-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 β-catenin with an affinity (KB) of 3 (± 1) 108 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 β-catenin gave rise to a large negative enthalpy change at 25°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 β-catenin arm repeat region/TCF complex.

Original languageEnglish
Pages (from-to)1179-1189
Number of pages11
JournalJournal of Molecular Biology
Issue number5
Publication statusPublished - Mar 9 2001


  • Cancer
  • Isothermal titration calorimetry
  • TCF4, β-catenin
  • Wnt-signaling

ASJC Scopus subject areas

  • Virology


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