Hydrogen Exchange Reveals the Mechanism of Stabilization of P53 Rescue Mutants N235K and N239Y

Abstract

p53 protein is a transcription factor important for regulating DNA repair and cellular apoptosis. It has been shown that the dysfunction of p53 protein is sufficient to allow for uncontrolled cell proliferation. Notably, 50% of all cancers have a mutated form of p53, most of these are simple missense mutations localized in the DNA binding domain (DBD). Previous studies have shown that the function of p53 can be restored by introducing secondary mutations known as suppressor or rescue mutations. Specifically, a cancer suppressor mutation motif consisting of residues 235-239-240 has been shown to rescue 16 common cancer mutations that are known to unfold the DBD. The individual rescue mutation N239Y is known to increase thermal stability. In contrast, the stability of N235K is similar to WT, leaving the mechanism of rescue undefined. Notably, the crystal structures of these two rescue mutants are virtually identical to the WT conformation; thus, the details of how these changes compensate for the destabilization of cancer mutants is not obvious on inspection of the static structure. We set out to gain further insight about the mechanism by which both of these mutations restore function to p53 cancer mutants using backbone amide hydrogen exchange measured by NMR spectroscopy. Protection factor analysis shows that specific residues throughout the beta-sheet scaffold increase in both mutants compared to the WT p53 revealing distinct changes in dynamics. Both rescue mutations alter the profile of positions that are most protected from exchange and, consequently, show increased stability. These data provide a model for stabilizing effects that could serve as a metric in evaluating anti-cancer therapeutics that target p53.