Core Domain Interactions in Full-Length p53 in Solution

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 103; no. 7; pp. 2115 - 2119
• Main Authors: Veprintsev, Dmitry B., Freund, Stefan M. V., Andreeva, Antonina, Rutledge, Stacey E., Tidow, Henning, Cañadillas, José Manuel Pérez, Blair, Caroline M., Fersht, Alan R.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 14.02.2006; National Acad Sciences
• Subjects: Binding sites; Biological Sciences; Chemical equilibrium; Crystal structure; Crystallography; Deoxyribonucleic acid; Dimerization; Dimers; DNA; DNA - chemistry; Genetic mutation; Humans; Medical research; Monomers; Mutagenesis; Mutation; NMR; Nuclear magnetic resonance; Protein Structure, Tertiary; Proteins; Protons; Solutions - chemistry; Spectroscopy; Spectrum analysis; Tumor Suppressor Protein p53 - chemistry; Tumor Suppressor Protein p53 - genetics; Tumors
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0511130103

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 ($^{15}N$,¹H 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 p53DNA 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.