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

Saturday, February 13, 2016
Erick Maglalang, University of California, Irvine, Irvine, CA
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 (HN) and receptor domain (HC). The HC target receptors of presynaptic motoneurons, where the HN 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 VH (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 HC which is the major focus of this study. To understand the mechanisms of VHH binding to HC of serotype B (HCB), we expressed and purified various VHHs and HCB using size exclusion chromatography. We found that two VHHs binds to HCB 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.