Location of the Synaptosome-Binding Regions on Botulinum Neurotoxin B

The regions of botulinum neurotoxin B (BoNT/B) involved in binding to mouse brain synaptosomes (snps) were localized. Sixty 19-residue overlapping peptides (peptide C31 consisted of 24 residues) encompassing BoNT/B H chain (residues 442–1291) were synthesized and used to inhibit binding of <sup>125</sup>I-labeled BoNT/B to snps. Synaptosome-binding regions were noncompeting and existed on both H<sub>N</sub> and H<sub>C</sub> domains of neurotoxin. At 37 °C, inhibitory activities on H<sub>N</sub> resided, in decreasing order, in peptides 638–656 (26.7%), 596–614 (18.2%), 512–530 (13.9%), 778–796 (13.8%), and 526–544 (11.6%). On H<sub>C</sub>, activity resided in decreasing order in peptides 1170–1188 (44.6%), 1128–1146 (21.6%), 1184–1202 (18.6%), 1156–1174 (13.0%), 946–964 (11.8%), 1114–1132 (11.2%), 1100–1118 (6.2%), 876–894 (6.1%), 1268–1291 (4.6%), and 1226–1244 (4.3%). The 45 remaining H<sub>N</sub> and H<sub>C</sub> peptides had no activity. At 4 °C, peptide C24 (1170–1188) remained quite active (inhibiting, 31.2%), while activities of peptides N15, C21, and C25 were little under 10%. The snp-binding regions contained sites that bind synaptotagmin II and gangliosides. Despite the low degree of sequence homology, BoNT/B and BoNT/A display significant structural homology and appeared to bind in part to the same snp-binding regions. Binding of each labeled toxin to snps was inhibited ∼50% by the other toxin, 70–72% by its correlate H<sub>C</sub>, and by the H<sub>C</sub> of the other toxin [29% (BoNT/A by H<sub>C</sub> of B) or 32% (BoNT/B by H<sub>C</sub> of A)]. In the three-dimensional structure of BoNT/B, the greater part of H<sub>C</sub>, one H<sub>N</sub> face, and part of the belt on the same side interact with snps. Thus, BoNT/B binds to snps through the H<sub>C</sub> head and employs regions on one H<sub>N</sub> face and the belt, reserving flexibility for the belt’s unbound part to release the light chain. Most snp-binding regions coincide or overlap with blocking antibody (Ab)-binding regions explaining how such Abs prevent BoNT/B toxicity.