Abstract
Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.
Highlights
Botulinum neurotoxins (BoNTs) are extremely potent toxins to humans and cause flaccid paralysis
The catalytic light chain (LC) of BoNTs is the warhead of the toxin, which is mainly responsible for BoNT’s neurotoxic effects
Prior reports have described the characterization of several variable heavy-chain (VHH) targeting BoNT/A and BoNT/B [6,8], some of which recognize the light chain protease domains of these toxins (LC/A and LC/B)
Summary
Botulinum neurotoxins (BoNTs) are extremely potent toxins to humans and cause flaccid paralysis. These toxins are widely distributed in nature and relatively simple to produce in quantity. For these reasons, BoNTs are listed by the Centers of Disease Control and Prevention (CDC) as Tier 1 Select Agents. BoNTs are highly variable in nature with at least seven major serotypes reported (A-G), most of which forms a group with multiple variant subtypes. This extreme natural variability seriously complicates the development of botulism treatments. While antitoxin treatments prevent further intoxication, they do not promote recovery from paralysis that has already occurred
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