Llama Antibody Inhibits Botulinum Neruotoxin

From Proteopedia

Clostridium Botulinum

Clostridium botulinum is a rod-shaped microorganism. It is an obligate anaerobe, meaning that oxygen is poisonous to the cells. However, C. botulinum tolerates traces of oxygen due to the enzyme called superoxide dismutase (SOD) which is an important antioxidant defense in nearly all cells exposed to oxygen. [1] C. botulinum does not form endospores as a way to protect the viability of the organism, but rather as a mechanism to produce the neurotoxin. C. botulinum is only able to produce the neurotoxin during sporulation, which can only happen in an anaerobic environment. Other bacterial species produce spores in an unfavorable growth environment to preserve the organism's viability and permit survival in a dormant state until the spores are exposed to favorable conditions. Template:Taxobox

PubMed Abstract: Template:STRUCTURE 3k3q Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ] Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.