A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic α-exosite binding region

Jianbo Dong, Aaron A. Thompson, Yongfeng Fan, Jianlong Lou, Fraser Conrad, Mengfei Ho, Melissa Pires-Alves, Brenda A. Wilson, Raymond C. Stevens, James D. Marks

Research output: Contribution to journalArticlepeer-review

Abstract

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 (Kd) from 230 to 0.03nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution Kd for BoNT/A Lc of 1.4710-10M and an IC50 (50% inhibitory concentration) of 4.710-10M 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 2.6Å resolution. The structure reveals that the Aa1 VHH binds in the α-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 α-exosite as a target for inhibitor development.

Original languageEnglish (US)
Pages (from-to)1106-1118
Number of pages13
JournalJournal of Molecular Biology
Volume397
Issue number4
DOIs
StatePublished - Apr 2010

Keywords

  • Botulinum neurotoxin type A
  • Llama VHH
  • Naïve yeast-displayed library
  • Single-domain antibody
  • α-exosite

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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