Understanding the interaction of 14‐3‐3 proteins with h DMX and h DM2: a structural and biophysical study

p53 plays a critical role in regulating diverse biological processes: DNA repair, cell cycle arrest, apoptosis and senescence. The p53 pathway has therefore served as the focus of multiple drug‐discovery efforts. p53 is negatively regulated by h DMX and h DM2; prior studies have identified 14‐3‐3 proteins as h DMX and h DM2 client proteins. 14‐3‐3 proteins are adaptor proteins that modulate localization, degradation and interactions of their targets in response to phosphorylation. Thus, 14‐3‐3 proteins may indirectly modulate the interaction between h DMX or h DM2 and p53 and represent potential targets for modulation of the p53 pathway. In this manuscript, we report on the biophysical and structural characterization of peptide/protein interactions that are representative of the interaction between 14‐3‐3 and h DMX or h DM2. The data establish that proximal phosphosites spaced ~20–25 residues apart in both h DMX and h DM2 co‐operate to facilitate high‐affinity 14‐3‐3 binding and provide structural insight that can be utilized in future stabilizer/inhibitor discovery efforts.