Structure of tumor suppressor p53 and its intrinsically disordered N-terminal transactivation domain

Abstract

Proteins with intrinsically disordered domains are implicated in a vast range of biological processes, especially in cell signaling and regulation. Having solved the quaternary structure of the folded domains in the tumor suppressor p53 by a multidisciplinary approach, we have now determined the average ensemble structure of the intrinsically disordered N-terminal transactivation domain (TAD) by using residual dipolar couplings (RDCs) from NMR spectroscopy and small-angle x-ray scattering (SAXS). Remarkably, not only were we able to measure RDCs of the isolated TAD, but we were also able to do so for the TAD in both the full-length tetrameric p53 protein and in its complex with a specific DNA response element. We determined the orientation of the TAD ensemble relative to the core domain, found that the TAD was stiffer in the proline-rich region (residues 64-92), which has a tendency to adopt a polyproline II (PPII) structure, and projected the TAD away from the core. We located the TAD in SAXS experiments on a complex between tetrameric p53 and four Taz2 domains that bind tightly to the TAD (residues 1-57) and acted as "reporters." The p53-Taz2 complex was an extended cross-shaped structure. The quality of the SAXS data enabled us to model the disordered termini and the folded domains in the complex with DNA. The core domains enveloped the response element in the center of the molecule, with the Taz2-bound TADs projecting outward from the core.

Fig. 1.

NH^N RDCs from p53(1–93) in phage measured at 800 MHz compared with predicted values. NH^N RDCs from p53(1–93) (blue) were compared with predicted values (red) from amino acid-specific statistical coil predictions including the presence of a single-turn helix at residues 22–24, populated at a level of 30% and an increased level of polyproline II sampling for each amino acid in the sequence 58–91. Helical values were centered on the conformations present in the x-ray crystal structure of the α-helix formed when p53(1–93) binds to the ubiquitin ligase MDM2. All simulated values are scaled by the same prefactor to best reproduce the experimental data.

Fig. 2.

Comparison of experimental and predicted SAXS data of p53(1–93). (a) Experimental SAXS data from p53(1–93) (blue) compared with the average simulated data using the FM ensemble calculated by using CRYSOL (red). (b) Kratky plots of the experimental data (black) compared with Kratky plots of the sampling by using enhanced PPII populations (red). The residuals between experiment and simulation are shown at the bottom of the plot.

Fig. 3.

RDCs from the N-terminal domain of flp53 in free and DNA-bound forms. (a) NH^N RDCs from residues 1–93 of p53(1–393) (open circles), p53(1–393)–DNA (blue circles), and p53(1–93) (red circles) aligned in phage at 800 MHz. (b) Comparison of experimental NH^N RDCs from p53(1–393)–DNA (blue circles) with predictions from model of DNA bound form (red circles).

Fig. 4.

Modeling the N-terminal domain in the p53–DNA complex. Shown is the N-terminal domain ensemble in our model with one representative full-length p53 molecule included for illustration. p53CTetD (gray) and DNA (magenta) are shown in space fill mode. The flexible C-terminal domain is not shown for reasons of clarity. N-terminal domains forming the four difference monomers are shown in different colors for clarity. Twenty copies are shown for each monomer. Left contains the entire N-terminal domain (TAD+PRR); Right shows only the PRR.

Fig. 5.

SAXS models of p53–Taz2 complex and p53–Taz2–DNA complex. (a) SAXS analysis of p53–Taz2 complexes. Shown are the experimental intensities and fits computed from the structural models by rigid body modeling: p53+Taz2 (blue) and p53+Taz2+DNA (red). The scattering profiles are displaced along the ordinate for better visualization. (b) SAXS model of full-length p53–Taz2 complex in solution from rigid-body analysis and addition of missing fragments. Core domains (green and cyan), tetramerization domain (red), and Taz2 (blue) are displayed in a cartoon representation, connecting linkers (gray), N termini (salmon), and C termini (yellow) in semitransparent space fill mode. (c and d) Rigid-body model of a p53–Taz2–DNA complex from SAXS data. Core domains (green and cyan), tetramerization domain (red), Taz2 (blue), and DNA (magenta) are shown in a cartoon representation, connecting linkers (gray) and N termini (salmon) in semitransparent space fill mode. c and d represent two orthogonal views.