Abstract
Protein–protein interactions involving 14‐3‐3 proteins regulate various cellular activities in normal and pathological conditions. These interactions have mostly been reported to be phosphorylation‐dependent, but the 14‐3‐3 proteins also interact with unphosphorylated proteins. In this work, we investigated whether phosphorylation is required, or, alternatively, whether negative charges are sufficient for 14‐3‐3ε binding. We substituted the pThr residue of pT(502–510) peptide by residues with a varying number of negative charges and investigated the binding of the peptides to 14‐3‐3ε using MD simulations and biophysical methods. We demonstrated that at least one negative charge is required for the peptides to bind 14‐3‐3ε, although phosphorylation is not necessary, and that two negative charges are preferable for high affinity binding. This discovery opens up new approaches for designing peptide‐based 14‐3‐3 protein inhibitors.