Negative Charges, Not Necessary Phosphorylation, are Required for Ligand Recognition by 14-3-3 Proteins

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 14-3-3{varepsilon} binding. We substituted the pThr residue of pT(502-510) peptide by residues with varying number of negative charges, and investigated binding of the peptides to 14-3-3{varepsilon} using MD simulations and biophysical methods. We demonstrated that at least one negative charge is required for the peptides to bind 14-3-3{varepsilon} while phosphorylation is not necessary, and that two negative charges are preferable for high affinity binding.