Phosphorylated full‐length Tau interacts with 14‐3‐3 proteins via two short phosphorylated sequences, each occupying a binding groove of 14‐3‐3 dimer

Pharmacological modulation of the 14‐3‐3/Tau interaction has interest in drug discovery projects related to Alzheimer’s disease mitigation. Biophysical methods were used to define the interface, stoichiometry, and affinity characterizing the binding of 14‐3‐3 to full‐length phosphorylated Tau protein. We concluded that the small binding interface and the interaction of moderate affinity make the interface likely druggable for inhibition. Protein–protein interactions (PPIs) remain poorly explored targets for the treatment of Alzheimer’s disease. The interaction of 14‐3‐3 proteins with Tau was shown to be linked to Tau pathology. This PPI is therefore seen as a potential target for Alzheimer’s disease. When Tau is phosphorylated by PKA (Tau‐PKA), several phosphorylation sites are generated, including two known 14‐3‐3 binding sites, surrounding the phosphorylated serines 214 and 324 of Tau. The crystal structures of 14‐3‐3 in complex with peptides surrounding these Tau phosphosites show that both these motifs are anchored in the amphipathic binding groove of 14‐3‐3. However, in the absence of structural data with the full‐length Tau protein, the stoichiometry of the complex or the interface and affinity of the partners is still unclear. In this work, we addressed these points, using a broad range of biophysical techniques. The interaction of the long and disordered Tau‐PKA protein with 14‐3‐3σ is restricted to two short sequences, containing phosphorylated serines, which bind in the amphipathic binding groove of 14‐3‐3σ. Phosphorylation of Tau is fundamental for the formation of this stable complex, and the affinity of the Tau‐PKA/14‐3‐3σ interaction is in the 1–10 micromolar range. Each monomer of the 14‐3‐3σ dimer binds one of two different phosphorylated peptides of Tau‐PKA, suggesting a 14‐3‐3/Tau‐PKA stoichiometry of 2 : 1, confirmed by analytical ultracentrifugation. These results contribute to a better understanding of this PPI and provide useful insights for drug discovery projects aiming at the modulation of this interaction.