Fig. 1
The BIN1-Tau interaction is mediated by the Tau PRD and the BIN1 SH3 domain. a. A schematic linear representation of 2N4R Tau (Tau FL) and BIN1 isoform1 (BIN1 FL) sequences and their respective domains (as used in GST pull-down assays). MBD: microtubule-binding domain; PRD: proline-rich domain; Bar: BIN1-amphiphysin-Rvs167; CLAP: clathrin and AP-2 binding; MycBD: Myc-binding domain; SH3: Src homology 3. b. GST pull-down assays performed with various Tau constructs corresponding to specific Tau subdomains (Lane 2: GST-Tau FL 2N4R; lane3: GST-Tau/Nter[1–152]; lane 4: GST-Tau/PRD[153–244]; lane 5: GST-Tau/MBD[244–441]) incubated with homogenates from BIN1-overexpressing HEK293 cells (n = 3). Upper panel: A representative immunoblot of BIN1 pull-down, revealed with a 99D anti-BIN1 antibody. c. GST pull-down assays performed either with full length BIN1 (Lane 2: GST-BIN1 FL) or the BIN1 SH3 domain (lane 3: GST-BIN1/SH3) incubated with homogenates from Tau FL-overexpressing HEK293 cells (n = 3). Upper panel: A representative immunoblot of Tau pull-down, revealed with a Dako anti-Tau antibody. d. GST pull-down assays performed with GST-Tau FL (lanes 3 and 4) or GST-Tau/PRD (lanes 5 and 6) incubated with homogenates from HEK293 cells overexpressing either BIN1 FL (lanes 3 and 5) or a BIN1 construct lacking the SH3 domain (BIN1/ΔSH3, lanes 4 and 6) (n = 3). Upper panel: A representative immunoblot of BIN1 pull-down, revealed with a 99D anti-BIN1 antibody. Lower panels (B, C, D): the corresponding Coomassie blue gels, used as loading controls for the pull-down assays. e. Two-dimensional (2D) [1H, 15N] HSQC spectra of 100 μM 15N 2N4R Tau, either free in solution (gray) or with a 1.6 molar amount of GST-BIN1/SH3 (blue, superimposed): Overlaid details of the full spectra presented in Additional file 3. f. Combined 1H, 15N CS perturbations (δ CS) in ppm, as defined in the Methods, in [1H, 15N] HSQC spectra of 15N 2N4R Tau with a 1.6 molar ratio of GST-BIN1/SH3 versus the molecule free in solution for every resonance along the sequence. The dashed box indicates the position of the Tau-F5 fragment within the Tau FL sequence. g. 2D [1H, 15N] HSQC spectra of 100 μM 15N Tau-F5 [165–245] free in solution (gray) or with a 1.2 molar amount of GST-BIN1/SH3 (red, superimposed): Overlaid details of full spectra presented in Additional file 4. h. Combined 1H, 15N CS perturbations (δ CS) in ppm, as defined in the Methods, in [1H, 15N] HSQC spectra of 15N Tau-F5 [165–245] with a 1.6 molar ratio of GST-BIN1/SH3, versus the free molecule in solution and for every resonance along the sequence. i. The a minima SH3 binding sequence in Tau. Proline residues are shown in bold, residues with resonance broadenings upon interaction are shown in red and residues with CS deviation when comparing free and bound states are shown in blue. The underlined amino acid residues fit the consensus sequence for SH3 binding (PxxPx+, where x is any residue and + is a positively charged residue) [30]