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. 2007 Jul 24;104(30):12324-9.
doi: 10.1073/pnas.0705069104. Epub 2007 Jul 9.

Quaternary structures of tumor suppressor p53 and a specific p53 DNA complex

Henning Tidow  1 , Roberto Melero, Efstratios Mylonas, Stefan M V Freund, J Guenter Grossmann, José María Carazo, Dmitri I Svergun, Mikel Valle, Alan R Fersht
Affiliations

Quaternary structures of tumor suppressor p53 and a specific p53 DNA complex

Henning Tidow et al. Proc Natl Acad Sci U S A. .

Abstract

The homotetrameric tumor suppressor p53 consists of folded core and tetramerization domains, linked and flanked by intrinsically disordered segments that impede structure analysis by x-ray crystallography and NMR. Here, we solved the quaternary structure of human p53 in solution by a combination of small-angle x-ray scattering, which defined its shape, and NMR, which identified the core domain interfaces and showed that the folded domains had the same structure in the intact protein as in fragments. We combined the solution data with electron microscopy on immobilized samples that provided medium resolution 3D maps. Ab initio and rigid body modeling of scattering data revealed an elongated cross-shaped structure with a pair of loosely coupled core domain dimers at the ends, which are accessible for binding to DNA and partner proteins. The core domains in that open conformation closed around a specific DNA response element to form a compact complex whose structure was independently determined by electron microscopy. The structure of the DNA complex is consistent with that of the complex of four separate core domains and response element fragments solved by x-ray crystallography and contacts identified by NMR. Electron microscopy on the conformationally mobile, unbound p53 selected a minor compact conformation, which resembled the closed conformation, from the ensemble of predominantly open conformations. A multipronged structural approach could be generally useful for the structural characterization of the rapidly growing number of multidomain proteins with intrinsically disordered regions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
SAXS analysis of p53 constructs. (a) Experimental intensities (dots with error bars) and fits computed from the structural models by rigid body modeling: CTetD (cyan), CTetCD (magenta), flp53 (red), CTetD+DNA (dark green). For the flp53–DNA complex the fit from the ab initio model (SI Fig. 9) is displayed in blue. The scattering profiles are displaced along the ordinate for better visualization. The fit to the CTetD data from the model proposed by Okorokov et al. (25) and the fit to flp53 data from the EM-map by Okorokov et al. (25) are shown in dashed orange and dashed light green, respectively. The fit to our EM-based model is shown in dashed yellow. (b) The distance distribution plots computed from the experimental data and normalized to the maximum value of unity.
Fig. 2.
Fig. 2.
Overlay of TROSY spectra acquired for p53 core domain (black) and p53 CTetD (blue) of 100-μM samples. Chemical shift deviations for the p53 CTetD spectra were estimated from the overlay and partly confirmed with TROSY-based triple resonance spectra (data not shown). Most of the core resonances showed insignificant chemical shift deviations between the two spectra. The expansions a, b, and c show signals that are strong and well isolated in the spectrum of isolated core domains but are not seen in the spectrum of CTetD most likely because of substantial line broadening associated with residues in a protein–protein interface. In a 181 and in b 244 are lost, and in c 243 is severely diminished. Spectra were plotted close to the noise level; additional signals in a are from the linker region between core and tetramerization domain.
Fig. 3.
Fig. 3.
SAXS models of free p53 in solution from rigid body analysis and addition of missing fragments. (a) CTetD portion of flp53 shown in b. Core domains and tetramerization domain are displayed in cartoon representation, connecting linkers (gray), N termini (salmon), and C termini (yellow) in semitransparent spacefill mode. Both models are presented in two orthogonal views. The model of flp53 and its CTetD and CTetCD portions fit simultaneously the experimental SAXS data from appropriate constructs in Fig. 1a.
Fig. 4.
Fig. 4.
Rigid body model of a p53–DNA complex from SAXS data. Core domains, tetramerization domain and DNA are shown in cartoon representation, connecting linkers in semitransparent spacefill mode. The model is displayed in two orthogonal views.
Fig. 5.
Fig. 5.
3D-EM map for flp53–DNA complex. The 3D map for the p53–DNA complex is rendered with a volume that covers 100% of the expected volume for a p53 tetramer in solid (a–c) and semitransparent (d–f) views. The fitted coordinates place the representation for the dsDNA in the see-through channel of one of the side views (see a and d).
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