FIGURE 3. Site-directed mutagenesis analysis of the toxin-receptor binding site in BoNT/B and in the luminal domains of Syt-I and Syt-II.

a, Pull-down assay using GST–Syt-II wild type as bait in the presence of mixed ganglioside/Triton X-100 micelles shows drastically reduced binding of various H_CB single-site mutants. b, The corresponding single-site mutations in recombinant full-length single-chain BoNT/B confirm the drastic decrease in toxicity employing the mouse phrenic nerve as an ex vivo system. Results are plotted on a logarithmic scale. c, Sequence alignment of the membrane-proximal 22 amino acids of the luminal domain of rat Syt-I and Syt-II. The non-conserved residues are shown in black. Mutated residues of Syt-I and Syt-II are indicated with blue dots and red dots, respectively. d, Pull-down assay with H_CB wild-type and mutants of GST–Syt-II (1–61) (filled columns) and GST–Syt-I (1–53) (open columns) as bait in the presence of mixed ganglioside/Triton X-100 micelles shows the crucial role of Phe 47, Phe 54, Phe 55 and Glu 57 in Syt-II. Values in a, b and d are means  s.d.