The p53-Zn2+ Energy Landscape and Metallochaperone Hypothesis

Abstract

p53 is a tumor suppressor protein found mutated in essentially half of human cancers, and dysfunctional in nearly all human cancers. Each DNA-binding domain of the protein contains a critical tetrahedrally coordinated Zn2+. In this work, we present a quantitative thermodynamic model describing the energetics of the p53-Zn2+ interaction, as well as the mechanism of action of a new class of therapeutic compounds we call synthetic zinc metallochaperones (ZMC) that restore proper structure and function to many mutant p53s by delivering Zn2+ to the protein in the cell. We combine recombinant protein expression and in vitrobiophysical characterization with cell biology, molecular biology, medicinal chemistry, and live cell imaging to address these issues. Our model for both the mechanism of action of ZMCs and the p53-Zn2+interaction are broadly based on the Metallochaperone Hypothesis originally proposed by our group in 2010. We find that the core tenants of the Metallochaperone Hypothesis are accurate, and have expanded that model to quantitatively describe the link between p53-Zn2+ binding and protein stability noted for decades in the field. We find that at physiological temperature and Zn2+ concentrations, wild-type p53 has a folding energy of ~0 kcal mol-1, and as such is exquisitely sensitive to inactivation by mutation, and rapidly changes the fraction folded in response to changes in Zn2+ concentration. We demonstrate that ZMCs are ionophores, transport Zn2+ from the extracellular space into cells, and rescue mutant p53 by increasing the intracellular free Zn2+ concentration. This increase in Zn2+ stabilizes the mutant proteins via the same mechanism previously described for substrate stabilization of enzymes, and is only seen in a "Goldilocks Zone" of Zn2+ concentrations and ZMC Kds. This presents a fundamentally new way to interact with and reactivate mutant p53s, and raises questions about the potential for biological exploitation of this interaction for signaling or other functions.