Core domain interactions in full-length p53 in solution

Abstract

The tumor suppressor p53 consists of four 393-residue chains, each of which has two natively unfolded (N-and C-terminal) and two folded (core and tetramerization) domains. Their structural organization is poorly characterized as the protein tends to aggregate, has defied crystallization, and is at the limits of NMR studies. We first stabilized the protein by mutation to make it more suitable for extended study and then acquired NMR spectra on full-length protein and various combinations of shorter domain constructs. The NMR spectrum ( 15 N, 1 H transverse relaxation optimized spectroscopy) of full-length p53 was close to that expected from the sum of the spectra of isolated individual domains. However, patterns of changes in chemical shifts revealed unexpected interactions between the core domains. We used the NMR data as constraints in docking algorithms and found a previously uncharacterized self-complementary surface for the association of core domains into dimers within the tetrameric complex. Binding to DNA requires about a 70°rotation to break those subunit interactions and form the known protein:protein interface in the p53-DNA complex. We verified the interactions by the effects of mutation on DNA binding. Spectroscopic, biophysical, and mutational data conspired to give a picture of the p53 tetramer as a dimer of loosely tethered core dimers of appropriate symmetry to be poised to bind target DNA. modular proteins ͉ NMR ͉ protein-protein interactions T he tumor suppressor p53 is a complex multifunctional protein that acts as a transcription factor in response to oncogenic and other stresses. It is at the centre of a multitude of networks in the cell, binds to DNA and a large number of protein signaling factors that modulate its activity, and is a subject to control by extensive posttranslational modification. Its activity is crucial in the prevention of cancer by inducing cell cycle arrest and apoptosis in response to oncogenic signals (1, 2). Each of its monomeric chains of 393 residues is comprised of several functional domains: the N-terminal domain (residues 1-93) comprises a transactivation domain (residues 1-60) (3) and a proline-rich regulatory domain (residues 64-92) (4), the DNAbinding core domain (CD) (residues 94-312) (5), the tetramerization domain (residues 324-355) (6), and the C-terminal negative regulatory domain (residues 360-393) (7). It undergoes a reversible equilibrium to form tetramers (8). The crystal structures of isolated human core domain bound to DNA (5), and of several oncogenic mutants, have been solved (9). The tetramerization domain is also well characterized by NMR (6, 10) and crystallography (11). The isolated N-terminal domain is natively unfolded (11, 12), apart from the formation of a nascent helix in the region 15-30 (11), which becomes fully helical when bound to Mdm2 (13) or in a membrane environment (14). The C-terminal negative regulatory domain of p53 is also unstructured (15, 16). The unstructured regions probably prevent the full-length protein being crystalli...

Structural basis for understanding oncogenic p53 mutations and designing rescue drugs

“…The absence of structural C B changes in surface regions, especially in the DNA-binding surface, however, is key for functionality. Hence, temperaturesensitive behavior can be expected for all cancer mutations that destabilize the core domain without compromising the surface complementarity that is crucial to the function of p53, not only for binding to specific promoter sequences but also for interactions with regulatory proteins and for correct domain organization in tetrameric full-length p53 (9,(30)(31)(32)(33).…”

“…Each chain in the p53 tetramer is composed of several domains. There are well defined DNA-binding and tetramerization domains and highly mobile, largely unstructured regions (5)(6)(7)(8)(9). Most p53 cancer mutations are located in the DNA-binding core domain of the protein (3).…”

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

“…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 (8,18,22,23). There is an increasing number of proteins being discovered to have globular domains linked by intrinsically disordered regions (24), and so the determination of such structures will be a recurring problem.…”

“…Structural studies on full-length p53 have been impeded both by its intrinsic instability and the presence of disordered regions (8,18,22,23). There is an increasing number of proteins being discovered to have globular domains linked by intrinsically disordered regions (24), and so the determination of such structures will be a recurring problem.…”

“…We have solved the instability problem of human p53 by designing a biologically active mutant with a superstable core domain (26,27) that is suitable for NMR studies. NMR studies have given a picture of the p53 tetramer as a dimer of loosely tethered core dimers of appropriate symmetry to be poised to bind target DNA with well resolved transverse relaxation optimized spectroscopy (TROSY) NMR spectra (23). But, on addition of DNA, the protein rigidifies with poorly resolved spectra (23).…”