Zinc shapes the folding landscape of p53 and establishes a new pathway for reactivating structurally diverse p53 mutants

Missense mutations in the DNA binding domain (DBD) of the p53 tumor suppressor contribute to approximately half of new cancer cases each year worldwide. A primary goal in cancer therapy is to develop drugs that rescue the transcription function of mutant p53. Here we present a thermodynamic model that quantifies and links the major pathways by which mutations inactivate p53. The model is constructed by measuring folding free energies, zinc dissociation constants, and DNA dissociation constants of 20 of the most common DBD mutations in the p53 database. We report here that DBD possesses two unusual properties——one of the highest zinc binding affinities of any eukaryotic protein and extreme instability in the absence of zinc—which are predicted to cause p53 to be poised on the edge of folding/unfolding in the cell, with a major determinant being the concentration of available zinc. Eighty percent of the mutations examined impair either thermodynamic stability, zinc binding affinity, or both. Using a combination of biophysical experiments, cell based assays, and murine cancer models, we demonstrate for the first time that a synthetic zinc metallochaperone not only rescues mutants with decreased zinc affinities, but also mutants that destabilize DBD without impairing zinc binding. The latter is a broad class of p53 mutants of which only one member (Y220C) has been successfully targeted by small molecules. The results suggest that zinc metallochaperones have the capability to treat 120,500 patients per year in the U.S.