Two negatively charged invariant residues influence ligand binding and conformational dynamics of 14-3-3ζ

14-3-3 proteins bind and modulate the activities of a wide variety of phosphoproteins. Crystal structures of 14-3-3 isoforms bound to phospholigands have identified several residues important for ligand binding. Here, we report the role of two invariant residues, D124 and E131, in peptide binding and peptide-induced conformational changes of the binding pocket. Surprisingly, the D124A mutation abrogates peptide binding, while the E131A mutation results in a twofold increase in peptide affinity. The mutants are less stable than the wild-type protein, and peptide binding restores native-like stability to the E131A mutant. This reversibility is lost in the more open structure of D124A. Based on these results, we infer that E131 is a regulator of protein plasticity and D124 is the guardian of the active site geometry.