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 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.
Knapp
S
S
Department of Structural Chemistry, Pharmacia Corporation, Discovery Research Oncology, Nerviano, Italy. stefan.knapp@eu.pnu.com
Zamai
M
M
Volpi
D
D
Nardese
V
V
Avanzi
N
N
Breton
J
J
Plyte
S
S
Flocco
M
M
Marconi
M
M
Isacchi
A
A
Caiolfa
V R
VR
D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D005453:Fluorescence; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods; D051820:TCF Transcription Factors; D013816:Thermodynamics; D015534:Trans-Activators; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051176:beta Catenin
C495270:CTNNB1 protein, human; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D051820:TCF Transcription Factors; C544894:TCF7L2 protein, human; D015534:Trans-Activators; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors; D051176:beta Catenin; D005982:Glutathione Transferase