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

Adam R Blanden,Yue Liu,Stewart N Loh,Tracy Withers,Xin Yu,Jeung-Hoi Ha,Christopher Demas,Darren R Carpizo,Alan J Blayney

doi:10.7554/elife.61487

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.

Highlights

The transcription factor p53 regulates a host of cellular responses to damage and distress (Kruiswijk et al, 2015)
Our thermodynamic model of p53 folding is derived from two measurable properties: the free energy of DNA-binding domain (DBD) folding in the absence of metal, and the binding affinity of zinc to the folded protein
The remaining three belong to the DNA-contact class. These findings suggest that loss of thermodynamic stability and/or metal-binding affinity play a dominant role in p53-related cancers, and underscore p53’s remarkable sensitivity to missense mutation at nearly every codon position

Summary

Introduction

The transcription factor p53 regulates a host of cellular responses to damage and distress (Kruiswijk et al, 2015). The X-ray crystal structure of DBD (residues 94–312) reveals a b-sandwich with a DNA-binding surface consisting of a loop-sheet-helix motif and two loops (L2 and L3) (Figure 1; Cho et al, 1994) These loops are stabilized by the tetrahedral coordination of a single zinc ion by C176 and H179 of L2 and C238 and C242 of L3. Folding free energy (Butler and Loh, 2003) This inherent malleability has been demonstrated in cells by overexpressing Zn2+-chelating proteins (metallothioneins) or adding small-molecule Zn2+ chelators, and observing reversible loss of sequence-specific DNA-binding activity and a switch in recognition by an antibody that recognizes native p53 (PAB1620) to one that binds to unfolded/misfolded p53 (PAB240) (Meplan et al, 2000). The results provide a more complete picture of the p53 activation/inactivation landscape and significantly expand the number of p53 mutants that are potentially rescuable by ZMCs

Results

Discussion

Materials and methods

Funding Funder National Institutes of Health