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 125I-labeled BoNT/B to snps. Synaptosome-binding regions were noncompeting and existed on both HN and HC domains of neurotoxin. At 37 °C, inhibitory activities on HN 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 HC, 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 HN and HC 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 HC, and by the HC of the other toxin [29% (BoNT/A by HC of B) or 32% (BoNT/B by HC of A)]. In the three-dimensional structure of BoNT/B, the greater part of HC, one HN face, and part of the belt on the same side interact with snps. Thus, BoNT/B binds to snps through the HC head and employs regions on one HN 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.