Solution structure of the p53 regulatory domain of the p19(Arf) tumor suppressor protein

Arf is a tumor suppressor that regulates p53 function and is a frequent tar get for loss in human cancers. Through two novel mechanisms, Arf inhibits t he oncoprotein Hdm2, a negative regulator of p53. (1) Arf inhibits the E3 u biquitin ligase activity of Hdm2 that leads to p53 degradation, and (2) Arf sequesters Hdm2 within nucleoli, These activities of Arf promote p53-media ted cell cycle arrest and apoptosis. Fundamental to these processes are int eractions between Arf and Hdm2. Here we show that a peptide containing the 37 N-terminal amino acids of mouse Arf (mArfN37) localizes to nucleoli, seq uesters Hdm2 within nucleoli, and causes cell cycle arrest. Circular dichro ism and NMR spectroscopy show that mArfN37 is largely unstructured under aq ueous conditions; however, the peptide adopts two a-helices (helix 1, resid ues 4-14; and helix 2, residues 20-29) in 2,2,2-trifluoroethanol (TFE). Eac h helix contains an amino acid motif that is repeated twice in mArfN37, onc e in each helix. The two helices, however, do not interact but are connecte d by an apparently flexible linker. The repeated motif contains Arg residue s spaced by a hydrophobic segment that may be involved in Hdm2 recognition and binding. The RRPR nucleolar localization signal, contained within resid ues 31-34, appears to be disordered under all conditions. The identificatio n of two Arf structural modules suggests that short peptides containing the repeated motif may function as Arf mimics and may allow the design of smal l molecule Arf mimics in the future.