Core domain interactions in full-length p53 in solution.
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 (15N,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 degrees 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.
Veprintsev
Dmitry B
DB
Medical Research Council Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom.
Freund
Stefan M V
SM
Andreeva
Antonina
A
Rutledge
Stacey E
SE
Tidow
Henning
H
Cañadillas
José Manuel Pérez
JM
Blair
Caroline M
CM
Fersht
Alan R
AR
D003461:Crystallography; D004247:DNA / Q000737:chemistry; D019281:Dimerization; D006801:Humans; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D012996:Solutions / Q000737:chemistry; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics
D012996:Solutions; D016159:Tumor Suppressor Protein p53; D004247:DNA