The N-terminal interferon-binding domain (IBiD) homology domain of p300 binds to peptides with homology to the p53 transactivation domain

Two high affinity Ser-20-phospho-LXXLL p53-binding domains of p300 map to the C-terminal interferon-binding domain (IBiD) and N-terminal IBiD homology domain (IHD) regions. Purified fractions of a recombinant IHD miniprotein are active in a set of in vitro assays highlighting its affinity to the N-terminal LXXLL domain of p53 including (i) dose-dependent binding to Ser-20-phosphorylated p53 tetramers; (ii) DNA-stimulated binding to p53 tetramers; and (iii) inhibition of MDM2-mediated p53 ubiquitination. The active component of the IHD miniprotein was localized to a 75-amino-acid fragment corresponding to amino acids 401-475 on human p300. This minimal IHD miniprotein can function in vivo as a p53-binding polypeptide in assays including: (i) complex formation with VP16-LXXLL peptide motifs in the two-hybrid assay; (ii) action as a dominant negative inhibitor of p53 from p21 luciferase templates; and (iii) attenuation of endogenous p21 protein levels. Further, we show here that the IRF-1-dependent stabilization and reactivation of p53DeltaPRO protein (LXXLL+/PXXP-) can be neutralized by the minimal IHD miniprotein, suggesting that IHD can bind to the p53 LXXLL domain in vivo. Phage-peptide display to the IHD miniprotein gave rise to an LSQXTFSXLXXLL consensus binding site that displays significant homology to the LXXLL transactivation domain of p53. These data validate the IHD scaffold as an independent LXXLL peptide-binding domain within the p300 protein, complementing the known peptide-binding domains including IBiD, C/H1, and C/H3.     

On the mechanism of sequence-specific DNA-dependent acetylation of p53: the acetylation motif is exposed upon DNA binding

P53 acetylation requires p300-docking to two contiguous sites in the activation domain that in turn mediates DNA-dependent acetylation of the tetramer. In an attempt to further define the mechanism of DNA-dependent acetylation of p53, an in vitro system has been reconstituted with distinct p53 isoforms and has been used to reveal conformational constraints on p53 acetylation. Two native p53 tetrameric isoforms purified from Sf9 cells differing by the extent of phosphorylation within the C-terminal acetylation site are both acetylated in a sequence-specific DNA-dependent manner. By contrast, p53 purified from an Escherichia coli expression system is in a largely denatured conformation and its acetylation is DNA-independent. Heating native p53 to destroy the folded structure restores DNA-independent acetylation similar to that seen with bacterially expressed p53. There are at least two sites of conformational flexibility in the p53 tetramer: the first in the flexible S10 beta-sheet within the MDM2 ubiquitination sequence and the second in the C-terminal regulatory domain. We analysed therefore whether DNA-dependent acetylation correlated with conformational changes in either of these two regions. DNA-dependent acetylation of p53 is maintained in a dose-dependent manner by low concentrations of consensus site DNA under conditions where flexibility in the S10 beta-sheet region is maintained. Oligonucleotide DNAs that promote acetylation stimulate the binding of monoclonal antibodies PAb421 and ICA-9; two antibodies whose contiguous epitopes overlap the C-terminal acetylation motif. By contrast, bent oligonucleotide DNAs that conceal both the S10 beta-sheet from binding of the monoclonal antibody DO-12 and attenuate binding of the monoclonal antibody PAb421 can preclude acetylation. These data suggest that, in the absence of DNA, the acetylation motif of p53 is in a cryptic state, but after DNA binding, allosteric effects mediate an exposure of the acetylation motif to allow DNA-dependent acetylation of the tetramer.     

HCMV IE2-mediated inhibition of HAT activity downregulates p53 function

Targeting of cellular histone acetyltransferases (HATs) by viral proteins is important in the development of virus-associated diseases. The immediate-early 2 protein (IE2) of human cytomegalovirus (HCMV) binds to the tumor suppressor, p53, and inactivates its functions by unknown mechanisms. Here, we show that IE2 binds to the HAT domain of the p53 coactivators, p300 and CREB-binding protein (CBP), and blocks their acetyltransferase activity on both histones and p53. The minimal HAT inactivation region on IE2 involves the N-terminal 98 amino acids. The in vivo DNA binding of p53 and local histone acetylation on p53-dependent promoters are all reduced by IE2, but not by mutant IE2 proteins that lack the HAT inhibition region. Furthermore, the p53 acetylation site mutant, K320/373/382R, retains both DNA binding and promoter transactivation activity in vivo and these effects are repressed by IE2 as well. Together with the finding that only wild-type IE2 exerts an antiapoptotic effect, our results suggest that HCMV IE2 downregulates p53-dependent gene activation by inhibiting p300/CBP-mediated local histone acetylation and that IE2 may have oncogenic activity.     

Binding of p53 to the central domain of Mdm2 is regulated by phosphorylation

The Mdm2 protein is the major regulator of the tumor suppressor protein p53. We show that the p53 protein associates both with the N-terminal and with the central domain of Mdm2. The central p53-binding site of Mdm2 encompasses amino acids 235-300. Binding of p53 to the central domain is significantly enhanced after phosphorylation of the central domain of Mdm2. The N-terminal and central domains of Mdm2 act synergistically in binding to p53. p53 mutants that have mutations in the tetramerization domain and that fail to oligomerize do not show such an enhancement of binding in the presence of the other binding site.     

p53 binds to cisplatin-damaged DNA

We have previously shown that bacterially expressed p53 protein or p53 protein isolated from cis-diamminedichloroplatinum II (cisplatin)-damaged cells is capable of binding to double-stranded platinated DNA molecules lacking any p53 DNA binding sites. Here we report using various p53 mutants that two separate domains of p53 protein affect p53 binding to platinated DNA. Mutations within the central core of p53, the domain responsible for sequence-specific DNA binding activity, completely eliminated p53 binding to platinated DNA. Based on competition experiments p53 preferred binding to sequence-specific DNA molecules over platinated DNA molecules. However, p53 binding to platinated DNA molecules was significantly stronger than p53 interactions with DNA molecules lacking damage and a p53 consensus site. Finally, an antibody specific to the C-terminal domain of p53 (pAb421) which activates sequence-specific DNA binding activity inhibited p53 binding to platinated DNA. Taken together, these results suggest that in addition to binding to p53 DNA binding sites, p53 also interacts with cisplatin-damaged DNA molecules.     

Investigations of the supercoil-selective DNA binding of wild type p53 suggest a novel mechanism for controlling p53 function.

The tumor suppressor protein, p53, selectively binds to supercoiled (sc) DNA lacking the specific p53 consensus binding sequence (p53CON). Using p53 deletion mutants, we have previously shown that the p53 C-terminal DNA-binding site (CTDBS) is critical for this binding. Here we studied supercoil-selective binding of bacterially expressed full-length p53 using modulation of activity of the p53 DNA-binding domains by oxidation of cysteine residues (to preclude binding within the p53 core domain) and/or by antibodies mapping to epitopes at the protein C-terminus (to block binding within the CTDBS). In the absence of antibody, reduced p53 preferentially bound scDNA lacking p53CON in the presence of 3 kb linear plasmid DNAs or 20 mer oligonucleotides, both containing and lacking the p53CON. Blocking the CTDBS with antibody caused reduced p53 to bind equally to sc and linear or relaxed circular DNA lacking p53CON, but with a high preference for the p53CON. The same immune complex of oxidized p53 failed to bind DNA, while oxidized p53 in the absence of antibody restored selective scDNA binding. Antibodies mapping outside the CTDBS did not prevent p53 supercoil-selective (SCS) binding. These data indicate that the CTDBS is primarily responsible for p53 SCS binding. In the absence of the SCS binding, p53 binds sc or linear (relaxed) DNA via the p53 core domain and exhibits strong sequence-specific binding. Our results support a hypothesis that alterations to DNA topology may be a component of the complex cellular regulatory mechanisms that control the switch between latent and active p53 following cellular stress.