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.