Mapping the interactions of the p53 transactivation domain with the KIX domain of CBP

Biochemistry. 2009 Mar 17;48(10):2115-24. doi: 10.1021/bi802055v.


Molecular interactions between the tumor suppressor p53 and the transcriptional coactivators CBP/p300 are critical for the regulation of p53 transactivation and stability. The transactivation domain (TAD) of p53 binds directly to several CBP/p300 domains (TAZ1, TAZ2, NCBD, and KIX). Here we map the interaction between the p53 TAD and the CBP KIX domain using isothermal titration calorimetry and NMR spectroscopy. KIX is a structural domain in CBP/p300 that can simultaneously bind two polypeptide ligands, such as the activation domain of MLL and the kinase-inducible activation domain (pKID) of CREB, using distinct interaction surfaces. The p53 TAD consists of two subdomains (AD1 and AD2); peptides corresponding to the isolated AD1 and AD2 subdomains interact with KIX with relatively low affinity, but a longer peptide containing both subdomains binds KIX tightly. In the context of the full-length p53 TAD, AD1 and AD2 bind synergistically to KIX. Mapping of the chemical shift perturbations onto the structure of KIX shows that isolated AD1 and AD2 peptides bind to both the MLL and pKID sites. Spin-labeling experiments show that the complex of the full-length p53 TAD with KIX is disordered, with the AD1 and AD2 subdomains each interacting with both the MLL and pKID binding surfaces. Phosphorylation of the p53 TAD at Thr18 or Ser20 increases the KIX binding affinity. The affinity is further enhanced by simultaneous phosphorylation of Thr18 and Ser20, and the specificity of the interaction is increased. The p53 TAD simultaneously occupies the two distinct sites that have been identified on the CBP KIX domain and efficiently competes for these sites with other known KIX-binding transcription factors.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites / physiology
  • Binding, Competitive / physiology
  • CREB-Binding Protein / chemistry*
  • CREB-Binding Protein / genetics
  • CREB-Binding Protein / metabolism
  • Calorimetry
  • Cyclic AMP Response Element-Binding Protein / chemistry
  • Cyclic AMP Response Element-Binding Protein / genetics
  • Cyclic AMP Response Element-Binding Protein / metabolism
  • Humans
  • Mice
  • Models, Chemical
  • Models, Molecular
  • Myeloid-Lymphoid Leukemia Protein / chemistry
  • Myeloid-Lymphoid Leukemia Protein / genetics
  • Myeloid-Lymphoid Leukemia Protein / metabolism
  • Nuclear Magnetic Resonance, Biomolecular
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Phosphorylation / physiology
  • Protein Binding / physiology
  • Protein Interaction Domains and Motifs / physiology*
  • Protein Structure, Secondary
  • Proto-Oncogene Proteins c-myb / chemistry
  • Proto-Oncogene Proteins c-myb / genetics
  • Proto-Oncogene Proteins c-myb / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Tumor Suppressor Protein p53 / chemistry*
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism


  • Cyclic AMP Response Element-Binding Protein
  • Peptide Fragments
  • Proto-Oncogene Proteins c-myb
  • Recombinant Proteins
  • Tumor Suppressor Protein p53
  • Myeloid-Lymphoid Leukemia Protein
  • CREB-Binding Protein
  • Crebbp protein, mouse