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

Abstract

The multidomain homotetrameric tumor suppressor p53 has two modes of binding dsDNA that are thought to be responsible for scanning and recognizing specific response elements (REs). The C termini bind nonspecifically to dsDNA. The four DNA-binding domains (DBDs) bind REs that have two symmetric 10 base-pair sequences. p53 bound to a 20-bp RE has the DBDs enveloping the DNA, which is in the center of the molecule surrounded by linker sequences to the tetramerization domain (Tet). We investigated by electron microscopy structures of p53 bound to DNA sequences consisting of a 20-bp RE with either 12 or 20 bp nonspecific extensions on either end. We found a variety of structures that give clues to recognition and scanning mechanisms. The 44-and 60-bp sequences gave rise to three and four classes of structures, respectively. One was similar to the known 20-bp structure, but the DBDs in the other classes were loosely arranged and incompatible with specific DNA recognition. Some of the complexes had density consistent with the C termini extending from Tet to the DNA, adjacent to the DBDs. Single-molecule fluorescence resonance energy transfer experiments detected the approach of the C termini towards the DBDs on addition of DNA. The structural data are consistent with p53 sliding along DNA via its C termini and the DNA-binding domains hopping on and off during searches for REs. The loose structures and posttranslational modifications account for the affinity of nonspecific DNA for p53 and point to a mechanism of enhancement of specificity by its binding to effector proteins.protein | recognition | specificity

Domain–domain interactions in full-length p53 and a specific DNA complex probed by methyl NMR spectroscopy

“…The line widths of the signals indicated that binding of DNA immobilized the p53C domains and constrained them rotationally more than the TET domain (Table S1). Those observations combined with tetrameric state of the TET domain and unstructured character of TAD and CT support the quaternary structure model based on combination of crystal structures of p53C-DNA complex and TET domain (4,5,33). In our experiments, TET appeared loosely attached to the p53C-DNA conglomerate and, because of its flexibility, is unlikely to contribute directly structurally to the formation of the complex.…”

“…This construct did not allow us to observe unspecific interactions of the CT domain with DNA segments flanking the p53 recognition site, as reported elsewhere (5,34). It is, however, possible that in the presence of longer DNA, interaction with additional bases might act as an additional constraint and bring the TET domain closer to p53C domains.…”

“…S1); however, their arrangement in the full-length protein has been a subject of controversy, and alternative models of quaternary structural arrangement have been postulated. One model of the human protein obtained from electron microscopy and small-angle X-ray scattering (SAXS) is consistent with structural studies on the isolated TET domain whereby the full-length tetramer and truncated constructs associate via the TET domain (4)(5)(6). But in a model of the murine protein, it is proposed that oligomerization occurs via contacts between N and C termini of the protein, as well as the core domains, and the subunits of the TET domain do not oligomerize (7,8).…”

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).…”

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.…”