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.

CNRS ERL9002 Integrative Structural Biology Lille France

Department of Physical and Macromolecular Chemistry Faculty of Science Charles University Prague Czech Republic

Department of Structural Biology of Signaling Proteins Division BIOCEV Institute of Physiology of the Czech Academy of Sciences Vestec Czech Republic

Inserm CHU Lille Institut Pasteur de Lille U1167 RID AGE Risk Factors and Molecular Determinants of Aging Related Diseases Univ Lille Lille France

Laboratory of Chemical Biology Department of Biomedical Engineering and Institute for Complex Molecular Systems Eindhoven University of Technology Eindhoven The Netherlands

Medicinal Chemistry Research and Early Development Cardiovascular Renal and Metabolism BioPharmaceuticals R and D AstraZeneca Gothenburg Sweden

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Structural insights into the functional roles of 14-3-3 proteins