Significance: The p53 tumor suppressor is a sequence-specific DNA binding protein that activates gene transcription to regulate cell survival and proliferation. This report investigates the role of the p53 N-terminal transactivation domain in regulating DNA binding affinity and specificity. The results suggest the N-terminal acidic transactivation region of p53 dynamically interacts with the DNA binding domain near residues that contact DNA, thus inhibiting DNA binding and increasing sequence specificity by nucleic acid mimicry or electrostatic shielding. Furthermore, p53 N-terminal–interacting proteins and posttranslational modifications may regulate DNA binding affinity and specificity partly by modulating the N terminus–DNA binding domain interaction.

The use of peptidomimetic scaffolds is a promising strategy for inhibition of protein–protein interactions. Herein we demonstrate that sulfono-γ-AApeptides can be rationally designed to mimic the p53 α-helix and inhibit p53-MDM2 protein-protein interactions (PPIs). The best inhibitor, with Kd and IC50 values of 26 nM and 0.891 µM toward MDM2, respectively, is amongst the most potent unnatural peptidomimetic inhibitors disrupting the p53-MDM2/MDMX interaction. Using fluorescence polarization assays, circular dichroism (CD), NMR spectroscopy, and computational simulations we demonstrate that sulfono-γ-AApeptides adopt helical structures resembling p53 and competitively inhibit the p53-MDM2 interaction by binding to the hydrophobic cleft of MDM2. Intriguingly, the stapled sulfono-γ-AApeptides showed promising cellular activity by enhancing p53 transcriptional activity and inducing expression of MDM2 and p21. Moreover, sulfono-γ-AApeptides exhibited remarkable resistance to proteolysis, augmenting their biological potential. Our results suggest that sulfono-γ-AApeptides are a new class of unnatural helical foldamers that disrupt PPIs.

Appropriate integration of cellular signals requires a delicate balance of ligand–target binding affinities. Increasing the level of residual structure in intrinsically disordered proteins (IDPs), which are overrepresented in these cellular processes, has been shown previously to enhance binding affinities and alter cellular function. Conserved proline residues are commonly found flanking regions of IDPs that become helical upon interacting with a partner protein. Here, we mutate these helix-flanking prolines in p53 and MLL and find opposite effects on binding affinity upon an increase in free IDP helicity. In both cases, changes in affinity were due to alterations in dissociation, not association, rate constants, which is inconsistent with conformational selection mechanisms. We conclude that, contrary to previous suggestions, helix-flanking prolines do not regulate affinity by modulating the rate of complex formation. Instead, they influence binding affinities by controlling the lifetime of the bound complex.