Fig. 6. VHH H3-2 binds with high affinity to its tau C-terminal epitope and dimerizes in vitro.
a Sensorgrams (reference subtracted data) of multi-cycle kinetics (MCK) analysis performed on immobilized biotinylated VHH E2-2 and H3-2, with injections of increasing concentrations of tau[369–381] C-terminal peptide (from 3 nM to 100 µM and from 3 nM to 32 µM, respectively; n = 3 independent experiments). Black lines correspond to the fitted curves, red lines correspond to the measurements. KDs were obtained using the kinetic model and are presented as mean values ± standard deviation (b). The maximum response (RU) observed for each peptide concentration was plotted and represented as a concentration-response curve (CRC). Black lines correspond to the fitted curves, red dots to the mean of the maximum response (in RU), from experiment (a) for each peptide concentration. Error bars represent standard deviation. The KD and stoichiometric ratio (SR) values extracted from these data are shown. KDs were obtained using the steady-state fitting model and are presented as mean values ± standard deviation. The SR have been calculated from the Rmax of the biological replicate represented here. c Ribbon representation of the crystal structure of the complex between VHH H3-2 and the tau[369–381] C-terminal peptide. The CDR1, CDR2, and CDR3 loops of VHH H3-2 are colored in pink, in dark blue, and in red, respectively. The framework regions of the VHHs are shown in green for one subunit and yellow for the other. The tau[369–381] peptides are shown in cyan and light blue.