Long-range regulation of p53 DNA binding by its intrinsically disordered N-terminal transactivation domain

Abstract

Atomic resolution characterization of the full-length p53 tetramer has been hampered by its size and the presence of extensive intrinsically disordered regions at both the N and C termini. As a consequence, the structural characteristics and dynamics of the disordered regions are poorly understood within the context of the intact p53 tetramer. Here we apply trans-intein splicing to generate segmentally ¹⁵N-labeled full-length p53 constructs in which only the resonances of the N-terminal transactivation domain (NTAD) are visible in NMR spectra, allowing us to observe this region of p53 with unprecedented detail within the tetramer. The N-terminal region is dynamically disordered in the full-length p53 tetramer, fluctuating between states in which it is free and fully exposed to solvent and states in which it makes transient contacts with the DNA-binding domain (DBD). Chemical-shift changes and paramagnetic spin-labeling experiments reveal that the amphipathic AD1 and AD2 motifs of the NTAD interact with the DNA-binding surface of the DBD through primarily electrostatic interactions. Importantly, this interaction inhibits binding of nonspecific DNA to the DBD while having no effect on binding to a specific p53 recognition element. We conclude that the NTAD:DBD interaction functions to enhance selectivity toward target genes by inhibiting binding to nonspecific sites in genomic DNA. This work provides some of the highest-resolution data on the disordered N terminus of the nearly 180-kDa full-length p53 tetramer and demonstrates a regulatory mechanism by which the N terminus of p53 transiently interacts with the DBD to enhance target site discrimination.

Keywords: DNA recognition; intein; intrinsically disordered protein; segmental isotope labeling; transcription factor.

Fig. 1.

(A) Domain structure of p53. The domains colored orange are intrinsically disordered. (B) Strategy for intein-based segmental labeling of the p53 NTAD. (C) ¹H-¹⁵N HSQC spectra of uniformly ¹⁵N-labeled full-length p53 (Left, black), ¹⁵N_NTAD–p53 (Center, red), and overlay of the two spectra (Right). The circled resonance is perturbed by the intein splice site. Spectra in C were collected in NMR buffer at 25 °C.

Fig. 2.

Comparison of NTAD NMR resonances in an isolated peptide and in the full-length p53 tetramer. (A) ¹H-¹⁵N HSQC spectrum of an isolated ¹⁵N-labeled NTAD peptide [p53(1–61), black] and ¹⁵N_NTAD–p53 tetramer (red) with selected residues labeled. The sample conditions were the same as in Fig. 1. (B) Differences in weighted averaged ¹H and ¹⁵N NTAD chemical shifts between the p53(1–61) peptide and full-length p53. The amphipathic AD1 and AD2 interaction motifs are labeled. (C) Cross-peak intensities in ¹⁵N_NTAD–p53 relative to p53(1–61) peptide. The intensity ratios shown are normalized to the resonance for Glu3. Weighted average chemical shift differences were calculated using the equation $\mathrm{\Delta }{\delta }_{\text{av}}=\sqrt{{\left(\mathrm{\Delta }{\delta }_{\text{H}}\right)}^2+{\left(\mathrm{\Delta }{\delta }_{\text{N}}/5\right)}^2}$.

Fig. 3.

The p53 NTAD interacts with the DBD. (A) Truncation constructs used to identify intramolecular NTAD interactions. (B) Overlay of selected regions of the ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled NTAD peptide [p53(1–61), black], ¹⁵N-labeled NTAD-PRD peptide [p53(1–95), orange], the ¹⁵N-labeled NTAD–PRD–DBD construct [p53(1–312), green], and the ¹⁵N_NTAD–p53 tetramer (purple). The sample conditions were the same as in Fig. 1. The full HSQC spectra are shown in SI Appendix, Fig. S1.

Fig. 4.

Addition of a p53 cognate DNA sequence disrupts the NTAD:DBD interaction. (A) Selected cross-peaks from ¹H-¹⁵N HSQC spectra of ¹⁵N_NTAD–p53 before (purple) and after (blue) the addition of a 0.25× molar equivalent of a 20-bp cognate DNA sequence. The corresponding cross-peaks in the spectrum of the isolated ¹⁵N-labeled NTAD peptide [p53(1–61)] are shown in black. Since the p53 recognition element binds four DBDs, a molar ratio of 0.25:1.0 DNA:p53 monomer gives rise to a 1:1 complex. (B) Weighted average NTAD ¹H and ¹⁵N chemical shift differences (Left) and ratio of cross-peak intensities (Right, normalized to the resonance for Glu3) between isolated ¹⁵N-labeled NTAD peptide and ¹⁵N_NTAD–p53 before (purple circles) and after (blue circles) the addition of DNA. The sample conditions are as in Fig. 1.

Fig. 5.

The specificity of DNA binding to monomeric p53(1–312) is affected by the NTAD. Regions of the ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled p53(1–312) (A and C) and ¹⁵N-labeled p53(62–312) (B and D) in the absence (black) and presence (gold) of a 0.25× equivalent of a 20-bp cognate DNA containing specific binding sites for four p53 DBDs (A and B) and of a 20-bp nonspecific DNA (C and D). For both constructs, the dispersed cross-peaks arising from residues in the DBD are broadened beyond detection upon formation of the cognate DNA complex. The dispersed DBD resonances of the nonspecific complex are broadened in the absence (D) but not in the presence (C) of the NTAD. Full ¹H-¹⁵N HSQC spectra are shown in SI Appendix, Fig. S3. Sample conditions were the same as in Fig. 1.

Fig. 6.

Measurement of DNA-binding affinity by fluorescence anisotropy. Plot of changes in fluorescence anisotropy upon the addition of increasing amounts of full-length p53 (residues 1–393) (black data points) and ∆NTAD p53 (residues 62–393) (red data points) to the 20-bp p53 recognition element from the p21 gene (circles) and to a 20-bp nonspecific DNA (squares). Fits are shown as solid lines for nonspecific DNA and as dashed lines for the cognate p21 DNA. All DNA samples were labeled with cyanine5. K_d values for the binding of full-length p53 and ∆NTAD p53 to the two DNA sequences are reported as a per-dimer K_d. Uncertainties are the SD of three independent measurements. The anisotropy data for binding to the specific DNA were fit to a 2:1 p53:DNA model, and binding to nonspecific DNA was fit to a 1:1 model.

Fig. 7.

NMR PRE experiments identify the NTAD:DBD interaction interface. (A and B) Structure of the p53 DBD (from Protein Data Bank ID code 3Q05) showing the location of residues whose HSQC cross-peaks are broadened by paramagnetic nitroxide spin labels at P58C (A) and S15C (B). The location of the DNA-binding surface is indicated by the black curved line. Spheres indicate residues that experience a >60% decrease in cross-peak intensity (red) or a 30–60% loss of intensity (yellow) in the presence of the paramagnetic spin label. (C) Magnitude of the PRE effects on NTAD resonances from a spin label attached at S121C on the DBD. The AD1 and AD2 regions are highlighted.