Binding of ciA-C2 does not cause obvious conformational change to HCA1 when compared to HCA1 in its apo state (PDB: 3FUO) (root-mean-square (RMS) deviation of ~0

Binding of ciA-C2 does not cause obvious conformational change to HCA1 when compared to HCA1 in its apo state (PDB: 3FUO) (root-mean-square (RMS) deviation of ~0.35 over 367 aligned C pairs, calculated by Pymol)21. peptide-moiety of SV2. Interestingly, this neutralization mechanism is similar to that of a monoclonal antibody in clinical Val-cit-PAB-OH trials, despite that ciA-C2 is more than 10-occasions smaller. Taken together, these results enlighten our understanding of BoNT/A1 interactions with its neuronal receptor, and further demonstrate that inhibiting toxin binding to the host receptor is an efficient countermeasure strategy. == Introduction == Botulinum neurotoxins (BoNTs) are among the most poisonous natural substances and are categorized as Tier 1 select agents by the Centers for Disease Control and Prevention (CDC). BoNTs cause botulism in humans and other mammals, birds, and fish; and could be misused as biological weapons1. BoNTs function as potent proteases, which cleave the neuronal members of solubleN-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) complex. As SNAREs are indispensable for the release of acetylcholine at the neuromuscular junctions (NMJs), cleavage of SNAREs by BoNT results in paralysis of muscles2,3. Among the four major human pathogenic BoNTs (BoNT/A, B, E and F), BoNT/A poses the most serious challenge for medical treatment due to its extremely high potency and remarkable persistence in human patients4. Botulism is usually primarily a consequence of the flaccid paralysis of respiratory muscles, and for patients exposed to BoNT/A, recovery commonly requires several months on a ventilator within an intensive care unit5. To date, there is no small-molecule drug approved for either prevention or post-intoxication treatment of botulism. Antitoxin therapy is currently the only available treatment for botulism patients. The first FDA-approved antitoxin that neutralizes all seven known BoNT serotypes, BAT (Botulism Antitoxin Heptavalent (A, B, C, D, E, F, G) (Equine)), is able to neutralize circulating BoNTs thereby preventing further disease progression. However, BAT consists of equine-derived polyclonal IgG antibodies largely processed to F(ab)2, and its use can cause various side effects6. An Investigational New Drug (IND) XOMA 3AB, which contains three human monoclonal antibodies, is effective in neutralizing BoNT/A7. However, monoclonal antibody drugs are generally expensive, require intravenous administration, and IL18 antibody have limited shelf lives. Therefore, alternative therapeutic antitoxin approaches that reduce costs and improve convenience remain an important goal. Recently, we and other laboratories found that the antigen-binding region (VH) of the heavy-chain-only antibodies (VHHs, also referred to as nanobodies) produced by camelids show strong anti-BoNT activities in animal models810. VHHs possess full antigen-binding capacity with high affinity and specificity, and are advantageous in their small size, Val-cit-PAB-OH remarkable stability, low immunogenicity to humans, as well as ease of production11. Furthermore, VHHs usually present convex paratopes that allow improved opportunities to identify brokers that bind otherwise inaccessible conformational epitopes, which often represent enzyme active sites and receptor-binding domains12,13. Thus VHHs have excellent promise as components of improved anti-BoNT therapeutic agents. In this study, we focused on an alpaca VHH (named ciA-C2) that potently neutralizes BoNT/A1, one of the eight known BoNT/A subtypes (A1-A8)10. To further characterize ciA-C2 and understand the molecular basis of BoNT/A1 neutralization, we mapped the ciA-C2-binding region to the C-terminal receptor-binding domain name of BoNT/A1 (HCA1) (Fig.1A) using both a neuron surface binding assay and biochemical binding assays. The extreme toxicity of BoNT/A1 depends to a large degree on its highly specific recognition of motoneurons in NMJs by HCA11419. We decided a high-resolution co-crystal structure of HCA1 in complex with ciA-C2, and found that ciA-C2 occupies a strategic site on BoNT/A1 that is needed for the toxin to recognize its receptor SV2, therefore preventing BoNT/A1 from attaching to motoneurons. == Physique 1. == ciA-C2 neutralizes BoNT/A1. (A) Overall architecture of BoNT/A1 (PDB: 3BTA). The light chain Val-cit-PAB-OH (LC/A), the translocation domain name (HNA), and the receptor-binding domain name (HCA) are colored violet, green, and sand/orange, respectively. (B) ciA-C2 inhibits SNAP-25 cleavage in cultured primary neurons. BoNT/A1 (2.5 nM) was pre-incubated with indicated VHHs (50 nM) for 40 min at 4 C and the mixtures were added to neuron culture media for 7 hrs. Neurons were then harvested and the cell lysates were subjected to Val-cit-PAB-OH immunoblot analysis to examine SNAP-25 and Synaptobrevin (Syb). Cleavage of SNAP-25 by BoNT/A1 generates a smaller fragment, which is usually indicated by an asterisk. Syb serves as an internal control. (C) The neurotoxicity of BoNT/A1 (1.63 pM) in the presence of various concentrations of ciA-C2 or ciA-F12 was assessed in the MPN hemidiaphragm assay with 35 technical replicates. == Results == == ciA-C2 potently neutralizes BoNT/A1 == In earlier studies, we found that ciA-C2 binds tightly to BoNT/A1.