Mechanisms of SNAP-25 Association with Botulinum Neurotoxin Light Chain A and SNARE Proteins

Abstract

Botulism neurotoxins (BoNTs), a family of neurotoxins produced by the anaerobic bacterium Clostridium botulinum, are the most poisonous biological toxins known to man and are listed as category A bioterrorism agents by the Centers for Disease Control and Prevention. BoNTs are zinc proteases that cleave the SNARE (soluble NSF attachment protein receptor where NSF stands for N-ethylmaleimide-sensitive fusion protein) family of proteins and prevent the fusion of neurotransmitter-carrying vesicles to the plasma membrane of neurons, resulting in flaccid paralysis. The structure of BoNT/A contains a 100 kDa heavy chain linked to a 50 kDa light chain via a disulfide bond. The light chain of BoNT/A is a zinc metalloprotease that specifically cleaves SNAP-25 between residues Gln197-Arg198.

It is critical to understand the mechanism of binding and cleavage between enzyme and substrate in order to design more potent inhibitors that target key residues or substrate pockets involved during substrate binding and catalysis. Binding and kinetic studies of fluorescently labeled SNAP-25 constructs of varying length with the light chain will provide means of analyzing kinetic parameters of different fragments of SNAP-25 in its association with LC/A to identify residues/loops that are critical to substrate recognition and/or catalysis. Therefore, the first step is to express and purify the BoNT/A LC and SNARE proteins in order to be further utilized for the binding and kinetic studies.

A list of eight screened small molecule inhibitors was kindly given by Dr. Radhakrishnan Padmanabhan, Professor of Microbiology and Immunology at the Georgetown Medical Center. Dr. Padmanabhan's research focuses on understanding how the dengue virus interacts at the molecular level and developing small molecule inhibitors of the virus by study-activity relationship studies. Specifically, Dr. Padmnabhan has studied the role and structure of NS3 serine protease in the dengue virus in hopes of developing a specific inhibitor to counteract the dengue and other flaviviral proteases. The substrate interaction of the NS3 serine protease in the dengue may be similar to the substrate interaction in the zinc metalloprotease of the light chain in botulism neurotoxin, and therefore the list of HPLC based screened compounds against the protease in dengue could also serve as potential inhibitors of the protease in neurotoxin. Three of the eight compounds were tested for their ability to inhibit the protease activity of BoNT/A LC. In order to conduct future kinetic studies between enzyme and substrate, BoNT/A LC was expressed and purified. For tertiary structural studies, SNAP-25 constructs were expressed and purified where the SNAP-25 construct was labeled at two locations with two separate fluorescence probes, such as EGFP and ReAsH. GST was used as a tag for purification.

The effect of cleavage of SNAP-25 by BoNT/A LC on the assembly and disassembly of the SNARE complex is another area of interest. Components of the SNARE complex are expressed and purified for future studies.