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.