Figure 2.

Fyn SH3 preferentially binds the 5^th/6th PXXP motifs in Tau. (A) A portion of the proline-rich domain of Tau containing the 4^th–7^th PXXPs is shown. Residues potentially involved in polyproline helix binding are in bold and residues that were mutated for alanine scanning in panel C are underlined. Residues present in peptides spanning the 5^th/6^th or 7^th PXXP motifs used in panel B are indicated. Primary prolines in the PXXP motifs are indicated by larger size. (B) Dose-response with Tat-peptides revealed significantly more potent inhibition by a peptide spanning the 5^th/6^th PXXPs than a peptide spanning the 7^th PXXP. *** indicates p < 0.001 peptide effect by two-way ANOVA, and **** indicates p < 0.0001 by Sidak’s post hoc (n = 4 wells per group) vs. alternate peptide. Error bars indicate SEM. (C) An alanine scan revealed significant attenuation of the Tau–Fyn SH3 interaction by mutation of the proline shared by the 5^th/6^th PXXP motifs (P216A), but not by mutation of several residues in and around the 7^th PXXP motif. ANOVA, p < 0.0001; **** indicates p < 0.0001 by Dunnett’s post hoc vs. WT control (n = 3–18 independent protein preparations per group). Error bars indicate SEM. (C, Inset) Representative protein concentration gel showing no difference between the concentration of WT and P216A protein preparations.