期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 397, 期 4, 页码 1106-1118出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2010.01.070
关键词
botulinum neurotoxin type A; llama VHH; single-domain antibody; alpha-exosite; naive yeast-displayed library
资金
- NIAID NIH HHS [U01 AI075502-03, U54 AI057153-06, U01 AI056493-05, U01 AI056493, U01 AI075502, U54 AI065359, U54 AI057153, U54 AI065359-056070] Funding Source: Medline
- PHS HHS [200-2006-16697] Funding Source: Medline
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 0.03 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 Kd for BoNT/A Lc of 1.47 x 10(-10) M and an IC50 (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 solyed the X-ray crystal structure of the BoNT/ A Lc-Aa1 VHH complex at 2.6 angstrom 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 a-exosite as a target for inhibitor development. (C) 2010 Published by Elsevier Ltd.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据