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

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.DNA binding ͉ intrinsically unfolded ͉ modular ͉ natively disordered ͉ protein T he tumor suppressor p53 is a tetrameric, multidomain transcription factor that plays a central role in the cell cycle and maintaining genomic integrity (1, 2). It binds to specific DNA response elements, is integrated in various signaling networks by a multitude of protein-protein interactions, and is controlled by extensive posttranslational modifications (1, 3, 4). p53 protein is a homotetramer of 4 ϫ 393 residues. Each chain consists of two folded domains [the core, and the tetramerization domain (323-360)] that are linked by an intrinsically disordered sequence. The transactivation domain (1-67) (5), proline-rich region (67-94), nuclear localization signal (NLS)-containing region (303-323) (6), and C-terminal negative regulatory domain (360-393) are also intrinsically disordered (7-9) (see refs. 10 and 11 for reviews). The DNAbinding core domain (residues 94-294) binds to sequencespecific response elements associated with p53 target gene promoters (12)(13)(14). The structures of the core domain complexes with DNA have been solved by crystallography (15)(16)(17), and in solution in the absence of DNA by NMR (18). The structure of the tetramerization domain has been solved by both NMR and x-ray crystallography (19-21).Structural studies on full-length p53 have been impeded both by its intrinsic instability and the presence of disordered regions (...

Ultraslow oligomerization equilibria of p53 and its implications

“…These experiments would not have been possible using wildtype p53 but required the superstable quadruple mutant that has been key to our structural studies (22,24,25,(30)(31)(32): a half-life of 40 min at 37°C for formation of tetramers is longer than the half-life of 9-16 min for the spontaneous denaturation of wild-type p53 at 37°C (28). The combination of the high instability of wild-type p53 and its slow oligomerization has profound implications: unless there are special factors within the cell, p53 will spontaneously denature faster than it will form active tetramers.…”

“…Isopropyl 1-thio-␤-D-galactopyranoside (IPTG) was added to a final concentration of 1 mM to induce expression. The cells were incubated for 15 h at 25°C before they were harvested and the proteins were purified as described (24,30) except expression of 13 C-and 15 N-labeled p53 was in M9 minimal medium supplemented with a vitamin mix together with 1 g/L 15 NH4Cl and 13 C D-glucose (30). Protein concentration was determined spectrophotometrically using a molar extinction coefficient 280 ϭ 20,400 M Ϫ1 cm Ϫ1 .…”

“…Here, we have used ESI-MS to determine the dissociation and assembly kinetics of the subunits of the wt p53 tetramer in a framework, stabilized by four mutations in the core domain that do not perturb the structure (22,23) that allows the protein to be studied over extended periods (22,23). All experiments were performed on the core plus tetramerization domain construct (residues 94-356) which has the same quaternary structure as full-length protein (24). Parallel experiments were done on the R273H contact mutant (25), which has the same stability as the other construct (26).…”

Aggregation and Prion-Like Properties of Misfolded Tumor Suppressors: Is Cancer a Prion Disease?

“…Active p53 is a tetrameric protein consisting of an oligomerization domain (OD), a DNA-binding domain (DBD), and a transactivation domain (TAD). Schematic representation (left) and crystal structures (right; PDB IDs: OD, 2J0Z; DBD, 2XWR; and TAD, 2L14) of p53 monomers (green, red, blue, and gold) in the predicted active conformation based on electron microscopy and small-angle X-ray-scattering reconstructions (Tidow et al 2007;Melero et al 2011). block the p300/CBP-mediated acetylation of p53 (Sabbatini and McCormick 2002), a modification that is critical for the tumor-suppressor functions of p53 (Brooks and Gu 2011).…”

Single-Molecule Studies of Unlabeled Full-Length p53 Protein Binding to DNA

“…The corresponding populations for the diameter measurements, corrected for tip effects, gave population 1 and 2, as shown in Table 2. We compared the dimensions obtained from our measurements with those in the literature (see Figure 1a.), 25,26,27 and found that most likely population 1 corresponds to dimeric flp53 protein, whilst population 2 corresponds to tetrameric flp53 protein.…”