Camelid Single Domain Antibodies Bind to the HC Domain of Botulinum Neurotoxin Serotype B

Botulinum neurotoxins (BoNTs) are the most lethal toxins for humans and animals which causes botulism, a life-threatening neurological disease. BoNT carries the light chain (LC), translocation domain (H_N) and receptor domain (H_C). The H_C target receptors of presynaptic motoneurons, where the H_N displaces the LC to the cytoplasm. The LC will then cleave sensitive factor attachment protein receptor (SNARE) complex preventing the release of neurotransmitters which causes muscle paralysis. Development of heavy-chain-only Ab V_H (VHH) has demonstrated novel effects for neutralizing BoNT in a mouse model. Our collaborator identified five VHHs that neutralized BoNT serotype B (BoNT/B) in vitro. However, their mechanisms of action are poorly understood. Preliminary data showed that these VHHs does not bind to the LC, suggesting that they likely act through blocking the H_C which is the major focus of this study. To understand the mechanisms of VHH binding to H_C of serotype B (H_CB), we expressed and purified various VHHs and H_CB using size exclusion chromatography. We found that two VHHs binds to H_CB according to pull-down assay. The competitive binding assay confirmed that both VHHs are competitive and likely recognize overlapping epitopes. Here, we report the crystal structure HcB-VHH complex resolved to 2.2 angstrom (Å) resolution. For future experiments, we will perform biochemical assays to verify our structural data. This study will help to understand the antagonistic mechanisms of VHH which is essential for developing novel antitoxins against BoNTs.