NMR Hydrogen Exchange and Relaxation Reveal Positions Stabilized by p53 Rescue Mutants N239Y and N235K

Abstract

AbstractInactivation of p53 is found in over 50% of all cancers; p53 disfunction is often caused by a single missense mutation localized in the DNA binding domain (DBD). Rescue mutants N235K and N239Y stabilize and restore function to multiple p53 cancer mutants. Here, we use NMR to compare protein dynamics between WT and rescue mutants to understand the mechanism of stabilization. We measured and compared folding dynamics by calculating protection factors (PFs) from NMR hydrogen exchange rates of backbone amides. We find that both rescue mutants impose a global stabilizing effect that dampens their motions compared to WT DBD, predominantly in the β-sandwich. However, a few regions become more flexible in rescue mutants. Notably, positions that have increased PFs map to cancer mutants rescued by each mutant. We also compared relaxation results to obtain flexibility information in the ps to ns timescale regime. Protein sequence analysis was used to determine the occurrence of these rescue mutants in nature and showed that 235K is found in mice and rats, but there is no evidence of 239Y occurring naturally in any species. Understanding the mechanism by which stabilizing mutants rescue p53 may reveal novel avenues for the development of cancer therapeutics. Our findings suggest that cancer therapeutics aimed at restoring p53 function could consider protein dynamics as a metric of drug efficacy.Statement of SignificanceTwo mutations (N235K and N239Y) within the DNA binding domain of p53 are known to reverse the effects of multiple cancer mutants. However, the mechanism of rescue is not clear since the crystal structures of these mutants are virtually identical to that of the WT. Here we use NMR methods to show that the protein dynamics of the rescue mutants are significantly different compared to WT, allowing us to describe the stability on a per-residue basis. Although these mutations are only four residues apart, they stabilize the structure distinctly in different regions, consistent with the specific cancer mutations they to which they restore function.