FC0084
p53 TAD  -  Nuclear Coactivator Binding domains (NCBD) of CBP

Biological function
The p53 tumor suppressor acts as a hub in signal transduction networks that mediate the cellular response to stress, leading to cell-cycle arrest, senescence, or apoptosis. Owing to its role in determining cell fate, p53 is tightly controlled by numerous regulatory proteins that include MDM2, MDMX, CBP/p300, and various kinases.

Domain organization/sequence features
p53 contains an N-terminal transactivation domain (TAD), a proline-rich domain, a DNA-binding domain (DBD ), a tetramerization domain (TD), and a C-terminal regulatory domain.

Structural evidence
Upon binding to CBP p53 TAD folds into two helices: which extend from Phe19 to Leu25 and Pro47 to Trp53, whereas residues in between remain disordered in the complex, as indicated in shifts of the ¹³C resonances. residues around 27–39, which form a long loop between the Pα1 and Pα2 helices, are disordered in the structure ensemble in accord with their small heteronuclear NOEs sharp resonances, and the lack of intermolecular NOEs for these residues. Residue 27– 39 loop of p53 wraps around helix Cα3 of the NCBD but appears to form no persistent contacts since it gives rise to no detectable intermolecular NOEs. The total buried hydrophobic surface area is 880 Ų, of which 580 Ų is contributed by helix Pα2 and the neighboring residues 40–45. The hydrophobic residues in AD2 (M40, L43, M44, L45, I50, W53, and F54) are directly involved in interactions with the NCBD, in the deep groove formed between helices Cα1–Cα2 and helix Cα3. Residues 40–45 appear to be less well ordered and have a smaller heteronuclear NOE than residues in helices Pα1 and Pα2, but this region of the p53 backbone exhibits a tendency towards a distorted helical conformation in the ensemble of p53:NCBD structures. F19, W23, and L26 in AD1 region also interact with the hydrophobic surface formed between helices Cα1 and Cα3. The hydrophobic residues in AD1 and AD2 are synergistically involved in the binding to the NCBD. These data are fully consistent with the structure of the p53 TAD:NCBD complex, where both AD1 and AD2 simultaneously interact with the NCBD domain. 13 residues between AD1 and AD2 remain disordered in the bound form.

Biochemical evidence
The affinity of full p53 TAD is 1.7 μM. While binding is dominated by the AD2 motif (K_d = 5–14 μM for peptides containing only AD2, compared to K_D > 300 μM for the isolated AD1 peptide), both AD1 and AD2 contribute to the overall affinity for binding to the NCBD.

Mechanism category
tethering

Posttranslational modification
Multisite phosphorylation represents a mechanism for a graded p53 response, with each successive phosphorylation event resulting in increasingly efficient recruitment of CBP/p300 to p53- regulated transcriptional programs, in the face of competition from cellular transcription factors. Multisite phosphorylation thus acts as a rheostat to enhance binding to CBP/p300 and provides a plausible mechanistic explanation for the gradually increasing p53 response observed following prolonged or severe genotoxic stress. 13-61 AA p53 peptide KD is 9.3 μM for NCBD, of P33S 5.2 μM, P37S 5.0 μM. For TAZ1 the P33S, P37S double mutant changes the KD from 920 nM to 78 nM, for TAZ2 the affinity incerase is 10-fold (from 20 to 2 nM) and for HDM2 230 nM to 1180 nM.

Significance
Fuzziness enables simultaneous binding of the AD1 and AD2 motifs to the target protein. The presence of two interaction motifs in the intrinsically disordered TAD imparts the flexibility to form ternary complexes with MDM2 and CBP/p300 domains, or to bind with enhanced affinity through clamp-like interactions with the NCBD or other CBP/ p300 domains.

Further reading
20961098, 20962272