Conformation and Affinity Modulations by Multiple Phosphorylation Occurring in the BIN1 SH3 Domain Binding Site of the Tau Protein Proline-Rich Region.

An increase in phosphorylation of the Tau protein is associated with Alzheimer's disease (AD) progression through unclear molecular mechanisms. In general, phosphorylation modifies the interaction of intrinsically disordered proteins, such as Tau, with other proteins; however, elucidating the structural basis of this regulation mechanism remains challenging. The bridging integrator-1 gene is an AD genetic determinant whose gene product, BIN1, directly interacts with Tau. The proline-rich motif recognized within a Tau(210-240) peptide by the SH3 domain of BIN1 (BIN1 SH3) is defined as 216PTPP219, and this interaction is modulated by phosphorylation. Phosphorylation of T217 within the Tau(210-240) peptide led to a 6-fold reduction in the affinity, while single phosphorylation at either T212, T231, or S235 had no effect on the interaction. Nonetheless, combined phosphorylation of T231 and S235 led to a 3-fold reduction in the affinity, although these phosphorylations are not within the BIN1 SH3-bound region of the Tau peptide. Using nuclear magnetic resonance (NMR) spectroscopy, these phosphorylations were shown to affect the local secondary structure and dynamics of the Tau(210-240) peptide. Models of the (un)phosphorylated peptides were obtained from molecular dynamics (MD) simulation validated by experimental data and showed compaction of the phosphorylated peptide due to increased salt bridge formation. This dynamic folding might indirectly impact the BIN1 SH3 binding by a decreased accessibility of the binding site. Regulation of the binding might thus not only be due to local electrostatic or steric effects from phosphorylation but also to the modification of the conformational properties of Tau.

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.

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.

Single Domain Antibody Fragments as New Tools for the Detection of Neuronal Tau Protein in Cells and in Mice Studies.

Tau is a neuronal protein linked to pathologies called tauopathies, including Alzheimer's disease. In Alzheimer's disease, tau aggregates into filaments, leading to the observation of intraneuronal fibrillary tangles. Molecular mechanisms resulting in tau aggregation and in tau pathology spreading through the brain regions are still not fully understood. New tools are thus needed to decipher tau pathways involved in the diseases. In this context, a family of novel single domain antibody fragments, or VHHs, directed against tau were generated and characterized. Among the selected VHHs obtained from screening of a synthetic library, a family of six VHHs shared the same CDR3 recognition loop and recognized the same epitope, located in the C-terminal domain of tau. Affinity parameters characterizing the tau/VHHs interaction were next evaluated using surface plasmon resonance spectroscopy. The equilibrium constants KD were in the micromolar range, but despite conservation of the CDR3 loop sequence, a range of affinities was observed for this VHH family. One of these VHHs, named F8-2, was additionally shown to bind tau upon expression in a neuronal cell line model. Optimization of VHH F8-2 by yeast two-hybrid allowed the generation of an optimized VHH family characterized by lower KD than that of the F8-2 wild-type counterpart, and recognizing the same epitope. The optimized VHHs can also be used as antibodies for detecting tau in transgenic mice brain tissues. These results validate the use of these VHHs for in vitro studies, but also their potential for in-cell expression and assays in mouse models, to explore the mechanisms underlying tau physiopathology.