bi201322c_si_002.pdf (500.25 kB)
Location of the Synaptosome-Binding Regions on Botulinum Neurotoxin B
journal contribution
posted on 2012-01-10, 00:00 authored by Behzod
Z. Dolimbek, Lance E. Steward, K. Roger Aoki, M. Zouhair AtassiThe 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.