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. 2007 Apr 24;104(17):7009-14.
doi: 10.1073/pnas.0702010104. Epub 2007 Apr 16.

Four domains of p300 each bind tightly to a sequence spanning both transactivation subdomains of p53

Daniel P Teufel  1 , Stefan M Freund, Mark Bycroft, Alan R Fersht
Affiliations

Four domains of p300 each bind tightly to a sequence spanning both transactivation subdomains of p53

Daniel P Teufel et al. Proc Natl Acad Sci U S A. .

Abstract

The transcriptional coactivator p300 binds to and mediates the transcriptional functions of the tetrameric tumor suppressor p53. Both proteins consist of independently folded domains linked by intrinsically disordered sequences. A well studied short sequence of the p53 transactivation domain, p53(15-29), binds weakly to four folded domains of p300 [Taz1/cysteine-histidine-rich region 1 (CH1), Kix, Taz2/CH3, IBiD], with dissociation constants (K(D)) in the 100 muM region. However, we found that a longer N-terminal transactivation domain construct p53(1-57) bound tightly to each p300 domain. Taz2/CH3 had the greatest affinity (K(D) = 27 nM) and competes with the N-terminal domain of Mdm2 for the p53 N terminus. p300 thus can protect the N terminus of p53 against the binding of other proteins. Mutations of p53 that abrogate transactivation (L22Q/W23S, W53Q/F54S) greatly weakened binding to each p300 domain, linking phenotypic defects to weakened coactivator binding. We propose a complex between tetrameric p53 and p300 in which four domains of p300 wrap around the four transactivation domains of p53.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Domain organization of p300 and p53. (A) Domain structure of human p53. TAD1/2 corresponds to the transactivation subdomains 1 and 2. Its precise boundaries are subject to debate. (B) Domain structure of human p300. Taz1, PHD, and ZZ-Taz2 are also described as CH1, CH2, and CH3, respectively. The approximate domain boundaries were taken from the p300 Pfam database entry (identifier Q094720) and sources from text.
Fig. 2.
Fig. 2.
Binding of N-terminal fragments of p53 with p300 and Mdm2 domains. (A) Fluorescence anisotropy titrations of short p53(15–29) TAD1 peptide with Taz2, Taz1, Kix, and IBiD of p300. (B) Fluorescence anisotropy titrations of the full p53(1–57) peptide with CH3 and Taz2. (C) As above, but with Taz1, Kix, and IBiD, respectively. (D) Fluorescence anisotropy titrations of mutant p53(1–57)QS2 with CH3, Kix, Taz1, and IBiD of p300. (E) Titrations of mutant p53(1–57)QS1/QS2 with p300 domains. (F) Competition experiment of Mdm2 and CH3 for labeled p53(1–57). CH3 was titrated into a preformed complex of 100 nM WT p53(1–57) and 1,000 nM Mdm2. Note the higher anisotropy value at the beginning of the titration, which indicates the presence of the Mdm2–p53 complex. (G) Mdm2 was titrated into a mixture of 100 nM WT p53(1–57) and 300 nM CH3. The decrease in anisotropy indicates displacement of CH3 from p53. The upward slope that follows is because of linear drift.
Fig. 3.
Fig. 3.
NMR HSQC spectra of free 1H 15N-labeled p53(1–93) N terminus (150 μM) in the absence and presence of a 1.2-fold excess of unlabeled p300 domain. Resonances that display clearly assignable chemical shifts are labeled with the residue to which they correspond. Labels for resonances that are absent from the bound state remain at their original positions. (A) 15N p53(1–93) (black) overlaid with the Taz2-bound spectrum (blue) and with the CH3-bound spectrum (red). (B) 15N p53(1–93) (blue) overlaid with the Kix-bound spectrum (red).
Fig. 4.
Fig. 4.
Sequence-based chemical shift map of p53(1–61) in the presence of p300 domains (A) and Mdm2 (B). Residues 62–93 are not shown because few or no chemical shifts can be observed within that region. Peaks that remain unaffected in the bound state are shown as boxes in white. Prolines, which do not produce resonances in an HSQC, are shown in light gray. Resonances that shift >0.02 ppm in both dimensions are shown in orange. Resonances that disappear in their bound states are shown in red. The extended binding site on p53 is seen with all binding partners.
Fig. 5.
Fig. 5.
Model of the p300–p53 tetramer interaction. Four TADs of a p53 tetramer tightly contact four p300 domains. The bromodomain of p300 may further stabilize this complex by weakly interacting with Lys-382-acetylated p53 (55). For clarity, the C-terminal regulatory domain of p53 and HAT, PHD, and ZZ of p300 are not shown.
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