The structure of p53 tumour suppressor protein reveals the basis for its functional plasticity

Abstract

p53 major tumour suppressor protein has presented a challenge for structural biology for two decades. The intact and complete p53 molecule has eluded previous attempts to obtain its structure, largely due to the intrinsic flexibility of the protein. Using ATP-stabilised p53, we have employed cryoelectron microscopy and single particle analysis to solve the first three-dimensional structure of the full-length p53 tetramer (resolution 13.7 A). The p53 molecule is a D2 tetramer, resembling a hollow skewed cube with node-like vertices of two sizes. Four larger nodes accommodate central core domains, as was demonstrated by fitting of its X-ray structure. The p53 monomers are connected via their juxtaposed N- and C-termini within smaller N/C nodes to form dimers. The dimers form tetramers through the contacts between core nodes and N/C nodes. This structure revolutionises existing concepts of p53's molecular organisation and resolves conflicting data relating to its biochemical properties. This architecture of p53 in toto suggests novel mechanisms for structural plasticity, which enables the protein to bind variably spaced DNA target sequences, essential for p53 transactivation and tumour suppressor functions.

Figure 1

EM of p53. (A) Schematic representation of p53 major domains. TA, transcription activation domain; Pro, proline-rich domain; core, core domain; NLS, nuclear localisation sequence; 4x, oligomerisation domain; Ct, C-terminus. (B) p53 protein sample used for EM. Lane 1, molecular weight markers (kDa); lane 2, Coomassie-stained protein before tag removal; lane 3, silver-stained final p53 sample after tag removal and gel filtration; lanes 4–6, the immunoblot with PAb248 (anti-Nt), PAb240 (anti-core) and PAb421 (anti-Ct) antibodies. (C) Part of a micrograph of p53 in ice. Arrows point at encircled p53 particles. The bar is 300 Å. (D) Image analysis of p53. (i) Representative class averages of p53 particles. (ii) Reprojections of p53 structure in the orientations found for the class averages in (i).

Figure 2

3D reconstruction of p53. The skewed-cube-shaped p53 molecule viewed at different angles. Side views (A, C, D) and the top view (B). The surface rendering is shown at 1σ density threshold. LN and SN indicate positions of the large and small vertices/nodes, respectively. (E) Stereo view of the p53 molecule.

Figure 3

Identification of the core domain position within the p53 map. (A) A representation of core domains occupying all four core nodes (surfaces are shown at 1σ in blue and 5σ in green). (B) The large node with the core structure fitted into the EM map. Zn atoms are represented as cyan spheres. (C) Atomic coordinates of the oligomerisation domain and two α-helices representing N-terminus were fitted into the N/C node of the 3D map (3σ). Ct (yellow) and N1 and N2 (red) indicate positions of the C- and N-terminal helices, respectively.

Figure 4

Structural organisation of p53. (A) The upper density layer of the p53 3D map at threshold 1σ, with fitted structures of two core domains shown in green and orange. N and C represent positions of the N- (blue) and C-termini (magenta), respectively. (B) Schematic representations of the corresponding monomer interactions. N/C nodes are represented as blue/magenta joints and the linkers representing N-termini are in blue and those for C-termini are in magenta. The core nodes are shown as spheres coloured similar to their corresponding core domains. Cut away view (C) and schematic representation (D) of the lower density layer with the second pair of core domains (coloured red and yellow) fitted in. (E) Domain structures are fitted into the corresponding nodes of the tetramer. (F) A schematic model of p53's quaternary organisation. The core nodes are coloured similar to their corresponding core domains. Grey linkers represent core node to N/C node contacts.

Figure 5

Biochemical and structural characterisation of the N/C nodes. (A) GST or GST-p53 proteins N and dN, spanning residues 1–100 and 1–63, respectively, were immobilised on glutathione–Sepharose and incubated with the His-tagged C-terminal fragment 323 (left panel) or oligomerisation-defective C-terminal mutant KEEK (right panel). N-terminus-bound fractions were analysed by Western blot using PAb421 antibody, and after C-terminal fragments were detected with this antibody, the membrane was stripped and re-blotted using anti-GST antibody to visualise GST and GST-fusion p53 proteins (lower panels). (B) His-tagged N-terminal and FLAG-tagged C-terminal fragments of p53 (1–186 and 187–393, respectively) were coexpressed in p53-negative HCT116 cells and their binding in vivo was analysed using co-immunoprecipitations. The panels on the left show the presence of the expressed p53 fragments in total cell lysates. The C-terminal p53 fragment was found to interact with the N-terminal fragment, as it co-immunoprecipitated with the anti-N-terminal antibody DO1 (IP with DO1; upper right panel) and, accordingly, the N-terminus was co-immunoprecipitated with the C-terminal fragment (IP with FLAG; lower right panel). The immunoprecipitates were identified by Western blotting using PAb122 (upper right panel) or DO1 (lower right panel) antibodies. (C) Complexes of p53 and PAb421 (anti-C-terminus) were studied by cryoEM and single particle analysis. Particles of antibody alone (i1), p53 in complex with one antibody (ii1) and p53 in complex with two antibodies (iii1) were selected into separate groups for statistical analysis. The subsets of images were aligned and those with the highest correlation were averaged (shown as i2, ii2 and iii2). To verify and interpret the resulting averages, p53 complexes with one or two antibodies were modelled (ii3 and iii3) and projections of those complexes were calculated (ii4 and iii4). i3 and i4 are the model and projection of the antibody molecule (PDB: 1igt). Bar is 150 Å.

Figure 6

Model of p53–DNA complex formation. Views of p53 bound to DNA on one side via one dimer (A, B) and on both sides with the whole p53 molecule involved in specific DNA binding (C). N and C represent positions of the N- and C-termini, respectively. (D) A schematic model for p53 re-adjustment during specific DNA binding. The semi-transparent spheres depict the p53 molecule bound to one half-site and the solid spheres represent re-adjusted p53 bound to both half-sites. The arrow indicates the general direction of the corresponding core node movement.