Understanding the interaction of 14-3-3 proteins with hDMX and hDM2: 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 for 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 localisation, 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.