How does p53 work? Regulation by the intrinsically disordered domains

Abstract

Defects in the tumor suppressor protein p53 are found in the majority of cancers. The p53 protein (393 amino acids long) contains the folded DNA-binding domain (DBD) and tetramerization domain (TET), with the remainder of the sequence being intrinsically disordered. Since cancer-causing mutations occur primarily in the DBD, this has been the focus of most of the research on p53. However, recent reports show that the disordered N-terminal activation domain (NTAD) and C-terminal regulatory domain (CTD) function synergistically with the DBD to regulate p53 activity. We propose a mechanistic model in which intermolecular and intramolecular interactions of the disordered regions, modulated by post-translational modifications, perform a central role in the regulation and activation of p53 in response to cellular stress.

Keywords: DNA binding; DNA damage; intrinsically disordered domains; post-translational modifications; transcription factor.

Figure 1.

A. Domain structure of the p53 monomer. NTAD: N-terminal transactivation domain; PRD: proline-rich domain; NLS: nuclear localization signal; TET: tetramerization domain; CTD: C-terminal regulatory domain. Disordered domains are shown in yellow, the folded DBD and TET (folded in the tetramer) domains are shown in green. Phosphorylation sites are shown as red dots and sequence numbers. A sequence alignment of the NTAD (residues 1–61) is shown, together with the locations of the AD1 and AD2 amphipathic sequence motifs and the sites of interaction of the MDM2 and CBP/p300 partners. B. Domain structure of CBP. NRID: nuclear receptor interaction domain; TAZ1: transcriptional activator zinc finger 1; KIX: kinase inducible interaction domain; CRD1: sumoylation site; BRD: bromodomain; CH2: Cys-His rich domain 1; HAT: histone acetyl transferase; ZZ: zinc containing domain; TAZ2: transcriptional activator zinc finger 2; NCBD: nuclear coactivator binding domain. The names of domains that form complexes with p53 NTAD are identified in red. C. Structures of the p53 NTAD (yellow) in complex with MDM2 (blue) (PDB 1YCR [64]), TAZ1 (green) (PDB 5HOU [65]), TAZ2 (purple) (PDB 5HPD [65]) and NCBD (orange) (PDB 2L14 [66]). Numbers indicate the N-and C-terminal residues of the p53 structures shown.

Figure 2.

A. Superposition of the ¹H-¹⁵N HSQC spectrum of a ¹⁵N-labeled peptide p53(1–61) (black) with that of full-length p53 segmentally labeled with ¹⁵N in residues 1–61 (red). (adapted from reference [28] with permission). B. DNA binding to the CTD. Weighted average ¹H and ¹⁵N chemical shift change (Δδ_avg) upon addition of a 60 basepair DNA to p53 segmentally labeled with ¹⁵N at residues 304–393 with (orange circles) and without (black circles) acetylation. The red dashed line represents the average chemical shift change for the unacetylated CTD after addition of DNA. Vertical dashed lines connect data points for the same residue. (adapted from reference [29] with permission).

Figure 3.

Schematic representation of p53-DNA interactions. (A) Non-specific DNA. The AD2 domain of the NTAD interacts with the DBD and competes with DNA binding. [28] Only two NTADs are shown for clarity. The CTD makes dynamic contacts with the DNA outside the immediate vicinity of the DBD, keeping p53 close to the DNA while allowing sliding and hopping for target site search. (B) When p53 encounters a specific recognition element (p53RE), the NTAD is displaced and is accessible for post-translational modification and binding of transcriptional coactivators such as CBP/p300. The CTD continues to interact with DNA flanking the p53RE. (C) The CTD is acetylated by CBP/p300, weakening its interactions with flanking DNA and thereby removing competing interactions that could facilitate diffusion away from the p53RE. (reproduced from reference [29] with permission)

Figure 4.. Phosphorylation of p53.

A. A rheostat model for stabilization and activation of p53 by phosphorylation. In unstressed cells, p53 forms a ternary complex with MDM2 and CBP/p300, promoting polyubiquitination and degradation [47]. p53 must compete with cellular transcription factors (designated TF1 and TF2) for binding to the limited amounts of CBP/p300. Genotoxic stress results in phosphorylation of Thr18, which lowers the affinity of p53 NTAD for MDM2 and slightly increases its affinity for CBP/p300. Prolonged or severe stress leads to phosphorylation of additional sites in the NTAD, further increasing its affinity for CBP/p300 and enhancing its ability to compete with other cellular transcription factors. (figure adapted from reference [50] with permission). B. Changes in fluorescence anisotropy upon titration of p53 into Cy5-labeled p21 (left panel) and DINP1 (right panel) DNA. Increases in fluorescence anisotropy (Δr) upon binding of non-phosphorylated T55-p53^A3 (a mutant where serines 6, 46 and 392 are changed to alanine, yielding a single phosphorylation site at T55) (blue) and S46/T55-p53^A2 (a mutant where serines 6 and 392 are changed to alanine, yielding two phosphorylation sites at S46 and T55) (green) are fitted to a 2:1 dimer:DNA binding model [55], while the bell-shaped curves associated with binding of pT55-p53^A3 (black) and pS46/pT55-p53^A2 (red) are fitted to a biphasic model [54]. (figure reproduced from reference [54] with permission). C. Cartoon illustrating the closed (autoinhibited) and open (accessible to DNA) states of p53 (adapted from reference [54]). The NTAD-PRD region is indicated by black lines and the DBDs as ovals. For clarity, interactions between the NTAD and DBD are shown for only two of the p53 subunits. Thr55 functions as a phosphorylation-dependent switch that modulates the population of the open and closed states and activates or terminates p53-mediated transcriptional processes.