Latent and active p53 are identical in conformation.

p53 is a nuclear phosphoprotein that regulates cellular fate after genotoxic stress through its role as a transcriptional regulator of genes involved in cell cycle control and apoptosis. The C-terminal region of p53 is known to negatively regulate sequence specific DNA-binding of p53; modifications to the C-terminus relieve this inhibition. Two models have been proposed to explain this latency: (i) an allosteric model in which the C-terminal domain interacts with another domain of p53 or (ii) a competitive model in which the C-terminal and the core domains compete for DNA binding. We have characterized latent and active forms of dimeric p53 using gel mobility shift assays and NMR spectroscopy. We show on the basis of chemical shifts that dimeric p53 both containing and lacking the C-terminal domain are identical in conformation and that the C-terminus does not interact with other p53 domains. Similarly, NMR spectra of isolated core and tetramerization domains confirm a modular p53 architecture. The data presented here rule out an allosteric model for the regulation of p53. Inactivation of p53 through either deletion, mutation or interaction with cellular or viral proteins is a key step in over half of human cancers1,2. The main role of p53 in normal cells is the induction of cell cycle arrest or apoptosis in response to cellular stress, particularly DNA damage3. The central role that p53 plays in determining the fate of a cell mandates that its functions be tightly regulated (reviewed in refs 4,5). At the DNA binding level, several aspects of the regulation of p53 activity remain ellusive, most notably the mechanism by which the C-terminal domain inhibits specific DNA binding6,7. p53 binds sequence-specifically as a tetramer to DNA targets with a consensus sequence consisting of either a two 10-base pair repeat of 5′-PuPuPu-C(A/T)(T/A)GPyPyPy-3′ (where Pu is a purine and Py is a pyrimidine) or a palindromic site comprised of four five-base pair inverted repeats with a similar sequence8,9. Full length p53 has been shown to be inactive for specific DNA binding; activation can be achieved by covalent and noncovalent modifiers of the basic C-terminal domain5. p53 constructs lacking the C-terminal domain exhibit enhanced specific DNA binding activity, confirming the inhibitory effect of this domain on p53 activity10. Murine p53 has an alternatively spliced form, which lacks the basic C-terminal domain and exhibits enhanced DNA binding, strengthening the case for a regulatory role for the C-terminal domain in vivo11,12. The p53 protein can be divided functionally and structurally into five regions: an acidic, N-terminal transactivation domain (residues 1–70); a proline-rich stretch containing five copies of the sequence PXXP (residues 60–97); a hydrophobic DNA binding domain highly conserved among p53 from different species and within the p53 family (100–300); an oligomerization domain (320–360); and a basic C-terminal domain (360–393). Several three-dimensional structures of individual domains of p53 have revealed important determinants of p53–DNA and p53–protein interactions, and the consequences of oncogenic mutations and posttranslational modifications on these interactions13–20. However, these studies have not provided any insight into the overall architecture of oligomeric p53, especially whether the different domains of p53 interact with one another. Of particular interest is whether or not the C-terminal regulatory domain interacts with any of the other p53 domains, as proposed as a mechanism for inhibition of the specific DNA binding of p53 (refs 6,7,21). In this study we used NMR spectroscopy to detect possible conformational differences between the latent and active forms of p53.