Contemporary biophysical approaches for studying 14-3-3 protein-protein interactions

Abstract

14-3-3 proteins are a family of regulatory hubs that function through a vast network of protein-protein interactions. Their dysfunction or dysregulation is implicated in a wide range of diseases, and thus they are attractive drug targets, especially for molecular glues that promote protein-protein interactions for therapeutic intervention. However, an incomplete understanding of the molecular mechanisms that underpin 14-3-3 function hampers progress in drug design and development. Biophysical methodologies are an essential element of the 14-3-3 analytical toolbox, but in many cases have not been fully exploited. Here, we present a contemporary review of the predominant biophysical techniques used to study 14-3-3 protein-protein interactions, with a focus on examples that address key questions and challenges in the 14-3-3 field.

Characterising the Protein-Protein Interaction Between MDM2 and 14-3-3σ; Proof of Concept for Small Molecule Stabilisation

“…This is interesting because the interactions of di-phosphorylated peptides that span the 14-3-3 dimer and simultaneously engage both binding grooves are typically enthalpy-dominated. 34 The data seem to suggest that the MDM2 peptide binds to the 14-3-3 dimer in more than one way, and/or that MDM2 peptide binding displaces previously ordered water molecules in the binding groove.…”

“…Peptides mimicking this di-phosphorylated MDM2 motif bind via an entropy-driven process which is unusual in the context of other 14-3-3 PPIs with di-phosphorylated motifs. 34 This process might be explained by a significant hydrophobic effect, or a change in protein secondary structure upon peptide binding. In contrast to a previous study, 17 our data show that a single MDM2 peptide occupies both binding grooves present on a 14-3-3σ dimer.…”

Characterizing the protein–protein interaction between MDM2 and 14-3-3σ; proof of concept for small molecule stabilization

“…Overall however, the interaction of the MDM2 161-191 pS166/pS186 peptide with both 14-3-3 isoforms was entropically driven ( Table 1 ). This is interesting because the interactions of di-phosphorylated peptides that span the 14-3-3 dimer and simultaneously engage both binding grooves are typically enthalpy-dominated ( 37 ). The data seem to suggest that the MDM2 peptide binds to the 14-3-3 dimer in more than one way, binding induces a conformational change, and/or that MDM2 peptide binding displaces previously ordered water molecules in the binding groove.…”

“…This is unusual in the context of other 14-3-3 PPIs whereby binding of di-phosphorylated motifs tend to be enthalpically driven when a ‘multivalent’ binding mechanism is invoked ( i.e. whereby the peptide spans the 14-3-3 dimer with simultaneous engagement of both phospho-sites) ( 37 ). Therefore, this finding strongly suggests that the MDM2-derived di-phosphorylated peptide is not engaged in ‘multivalent binding’.…”