Multiple conformations of full-length p53 detected with single-molecule fluorescence resonance energy transfer

Abstract

The tumor suppressor p53 is a member of the emerging class of proteins that have both folded and intrinsically disordered domains, which are a challenge to structural biology. Its N-terminal domain (NTD) is linked to a folded core domain, which has a disordered link to the folded tetramerization domain, which is followed by a disordered C-terminal domain. The quaternary structure of human p53 has been solved by a combination of NMR spectroscopy, electron microscopy, and small-angle X-ray scattering (SAXS), and the NTD ensemble structure has been solved by NMR and SAXS. The murine p53 is reported to have a different quaternary structure, with the N and C termini interacting. Here, we used single-molecule FRET (SM-FRET) and ensemble FRET to investigate the conformational dynamics of the NTD of p53 in isolation and in the context of tetrameric full-length p53 (flp53). Our results showed that the isolated NTD was extended in solution with a strong preference for residues 66 -86 forming a polyproline II conformation. The NTD associated weakly with the DNA binding domain of p53, but not the C termini. We detected multiple conformations in flp53 that were likely to result from the interactions of NTD with the DNA binding domain of each monomeric p53. Overall, the SM-FRET results, in addition to corroborating the previous ensemble findings, enabled the identification of the existence of multiple conformations of p53, which are often averaged and neglected in conventional ensemble techniques. Our study exemplifies the usefulness of SM-FRET in exploring the dynamic landscape of multimeric proteins that contain regions of unstructured domains.natively disordered ͉ domain-domain interaction ͉ quaternary structure ͉ FRET ͉ time-resolved T he tumor suppressor p53 is a tetrameric, multidomain transcription factor that plays key roles in maintaining the integrity of the human genome and in DNA repair machinery (1, 2). p53 is a partly intrinsically disordered protein, containing two folded domains: the DNA-binding core domain (CD; residues 94-294) and the tetramerization domain (TetD; residues 323-360) (3, 4). The intrinsically disordered N-terminal domain (NTD; residues 1-94) and C-terminal domain (CTD; residues 360-393) (5, 6) mediate interactions with several proteins such as p300/CBP, MDM2, 14-3-3, and S100 family that in turn regulate the activity of p53. Moreover, the NTD and CTD are the target sites of numerous posttranslational modificiations that modulate the activity of p53.High-resolution structures of the CD and the TetD have been solved by using X-ray crystallography and NMR spectroscopy (3,4,7,8). But, the intrinsic instability and the presence of highly disordered regions in p53 have impeded the application of conventional structural studies on full-length p53 (flp53). A combination of NMR spectroscopy and small-angle X-ray scattering (SAXS) in solution with electron microscopy on immobilized samples was recently used to solve the quaternary structures of a mutationally stabilized human flp53 and its DNA complex (...

Electron microscopy studies on the quaternary structure of p53 reveal different binding modes for p53 tetramers in complex with DNA

“…Atomic coordinates for DBD · DNA complex [pdb code: 2ATA; (19)] and Tet domains [pdb code: 3SAK; (44)] were fitted as rigid bodies in Chimera (45), which was also used to produce figures. SM-FRET experiments were carried out as described previously (37). In the gene encoding for the fragment p53CTC (94-393) of human p53 all the surface-exposed cysteines (124, 182, 229, 275, and 277) were mutated to alanine.…”

“…We therefore investigated the dynamics of the C-ter domain by single-molecule fluorescence resonance energy transfer (SM-FRET). All surfaceexposed cysteines in p53CTC (p53 residues 94-393 containing DBD, Tet, and C-ter) were mutated to alanine, and two new cysteines, one in the DBD (residue 292) and the other in the C-ter (residue 371) were introduced and labeled in a random manner with Alexa fluor 546 and 647 as previously described (37). The mutant protein exhibited the same DNA-binding properties as the wild type.…”

A single-molecule characterization of p53 search on DNA

“…Intriguingly, while the C terminus can provide sliding, it is the core domain that recognizes the cognate sequence (12)(13)(14). While structural studies have ruled out allosteric models of direct interactions between C terminus and core domains (15,16), interplay between the two domains remains a subject of great interest. Aiming to understand the role of individual domains and to investigate the molecular mechanism underlying one-dimensional diffusion of p53 protein on DNA, we visualized and quantitatively characterized the motion of individual p53 proteins in vitro along flow-stretched DNA.…”

Molecular dynamics of the full-length p53 monomer

“…[76][77][78] p53 is active as a tetramer and its structure bound to DNA has been resolved in the truncated core domain form, 79 even though only the full-length protein produces the maximum bending and twisting of the consensus DNA RE. 80 The full-length p53 has been resolved only in its tetrameric form by a combination of NMR, small-angle X-ray scattering, electron microscopy, and FRET, [81][82][83][84][85][86] showing that in absence of DNA, an open cross-shaped structure is formed, with loosely coupled dimers interacting via the core domain, whereas the structure rigidifies upon DNA binding and becomes more compact.…”