Abstract
The botulinum neurotoxin serotype A light chain (BoNT/A LC) protease is the catalytic component responsible for the neuroparalysis that is characteristic of the disease state botulism. Three related peptide-like molecules (PLMs) were designed using previous information from co-crystal structures, synthesized, and assayed for in vitro inhibition against BoNT/A LC. Our results indicate these PLMS are competitive inhibitors of the BoNT/A LC protease and their Ki values are in the nM-range. A co-crystal structure for one of these inhibitors was determined and reveals that the PLM, in accord with the goals of our design strategy, simultaneously involves both ionic interactions via its P1 residue and hydrophobic contacts by means of an aromatic group in the P2′ position. The PLM adopts a helical conformation similar to previously determined co-crystal structures of PLMs, although there are also major differences to these other structures such as contacts with specific BoNT/A LC residues. Our structure further demonstrates the remarkable plasticity of the substrate binding cleft of the BoNT/A LC protease and provides a paradigm for iterative structure-based design and development of BoNT/A LC inhibitors.
Highlights
Botulinum neurotoxins (BoNTs), secreted by Clostridium botulinum [1], provide invaluable treatments for a range of medical conditions [2,3,4,5,6,7,8,9,10,11] and cosmetic purposes [12,13,14,15,16]
The holotoxin is composed of a 100 kDa heavy chain (HC) subunit and a 50 kDa light chain (LC) subunit; these two components are tethered by a disulfide bridge [1,19,20]
We restrained the conformation of I1, as it is bound to the BoNT/A LC [35], and attempted to reduce the inhibitor’s binding entropy, by introducing an aminoisobutyric acid (Aib) residue (Figure 1), which is known to favor the type II’ b-turn repeat in a 310 helix [37,38]
Summary
Botulinum neurotoxins (BoNTs), secreted by Clostridium botulinum [1], provide invaluable treatments for a range of medical conditions [2,3,4,5,6,7,8,9,10,11] and cosmetic purposes [12,13,14,15,16]. BoNTs are the most potent biological toxins known by causing the disease state botulism. The seven known BoNT serotypes are designated A – G Post secretion, they undergo proteolytic processing to provide the bioactive (i.e., poisonous) holotoxin [1]. The BoNT/LC mediated proteolytic cleavage of any one of the three SNARE proteins prevents acetylcholine-filled vesicles in the neuron from fusing with the active zone at the synaptic cleft [1]. This inhibits the transmission of motor nerve impulses, and as indicated above, results in the flaccid paralysis that is characteristic of botulism [29]
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