Structurally Constrained Residues Outside the Binding Motif Are Essential in the Interaction of 14-3-3 and Phosphorylated Partner

14-3-3 proteins participate in many key cellular processes after binding to disordered phospho-partners. Usually, the phosphorylated state is an essential target for the binding. Here, we show for the first time residues other than those in the 14-3-3 binding motif that are essential for the binding between 14-3-3 and a phosphorylated partner. Results support that phosphorylation, although necessary, is not sufficient for 14-3-3′s complex formation, as structurally constrained anchor residues play a critical function in stabilizing the protein–protein interaction.