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

Abstract

Missense mutations in the p53 DNA binding domain (DBD) contribute to half of new cancer cases annually. Here we present a thermodynamic model that quantifies and links the major pathways by which mutations inactivate p53. We find that DBD possesses two unusual properties-one of the highest zinc affinities of any eukaryotic protein and extreme instability in the absence of zinc-which are predicted to poise p53 on the cusp of folding/unfolding in the cell, with a major determinant being available zinc concentration. We analyze the 20 most common tumorigenic p53 mutations and find that 80% impair zinc affinity, thermodynamic stability, or both. Biophysical, cell-based, and murine xenograft experiments demonstrate that a synthetic zinc metallochaperone rescues not only mutations that decrease zinc affinity, but also mutations that destabilize DBD without impairing zinc binding. The results suggest that zinc metallochaperones have the capability to treat 120,500 patients annually in the U.S.

Selective functional inhibition of a tumor-derived p53 mutant by cytosolic chaperones identified using split-YFP in budding yeast

“…It was previously proposed (Dearth et al 2007) that the reason some misfolded p53 mutants are recessive is because their proper folding is restored via interaction with properly-folded WT p53 molecules in the context of "mixed" hetero-tetramers. The V272M mutant adopts a non-native conformation in a temperature-sensitive manner (North et al 2002;Blanden et al 2020) and acts recessively when co-expressed with WT p53 in yeast cells harboring a URA3 reporter gene containing p53 binding sites in the promoter region Dearth et al 2007) (see Figure 3). To ask if WT p53 is able to "chaperone" the proper folding of p53(V272M), we used a splitdihydrofolate reductase (split-DHFR) assay (Pittman et al 2012).…”

Defining function of wild-type and patient specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

“…Significantly, compounds like ZMC1 enhance p53-induced tumor cell apoptosis. ZMC1 is thought to stabilize mutant p53 protein through increasing Zn 2+ ions in cells and decrease tumor growth (Blanden et al, 2020;Blanden et al, 2015;Yu, Vazquez, Levine, & Carpizo, 2012). We found that ZMC1 effectively stabilizes mutant p53 C176F and selectively increases ERMS apoptosis in tp53 -/-+ TP53 C176F but not tp53 -/tumors, revealing an effective in vivo drug efficacy platform to identify therapeutic strategies for p53 mutant ERMS in the future.…”

Selective functional inhibition of a tumor-derived p53 mutant by cytosolic chaperones identified using split-YFP in budding yeast

“…It was previously proposed ( Dearth et al 2007 ) that the reason some misfolded p53 mutants are recessive is because their proper folding is restored via interaction with properly folded WT p53 molecules in the context of “mixed” hetero-tetramers. The V272M mutant adopts a non-native conformation in a TS manner ( North et al 2002 ; Blanden et al 2020 ) and acts recessively when coexpressed with WT p53 in yeast cells harboring a URA3 reporter gene containing p53 binding sites in the promoter region ( Brachmann et al 1996 ; Dearth et al 2007 ; see Figure 3 ). To ask if WT p53 is able to “chaperone” the proper folding of p53(V272M), we used a split-dihydrofolate reductase (split-DHFR) assay ( Pittman et al 2012 ).…”

“…p53(R273H) specifically abolishes contact with DNA without global conformational consequences ( Ang et al 2006 ). On the other hand, p53(V272M) causes misfolding and conformational instability ( North et al 2002 ; Blanden et al 2020 ). These observations are consistent with a model in which chaperones recognize specific, exposed sequences in p53 that are largely buried in the native fold.…”