C. Botulinum Type E Catalytic Domain-Substrate Complex

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DESCRIPTION (provided by applicant): Clostridium botulinum causing botulism, a neuronal disorder, is a public health problem and is also emerging as a potential biowarfare threat. While there is an experimental vaccine available, there is no therapeutic treatment after being afflicted with this disease. Though this is a potential biowarfare threat, it may be impossible to vaccinate the entire population against this. Also, the side effects of the experimental vaccine are not precisely known. Therapies based on antibodies are emerging but more than one antibody may be needed to neutralize a single serotype. In this context, it is necessary to develop a second line of defense by developing antitoxins for therapeutic use to complement vaccines or treatment with antibodies. Accordingly, the long-range goal of this proposal is to understand the structure-function relationship of botulinum neurotoxins (BoNT), especially the catalytic mechanism, leading to structure-based rational drug design for treatment. The short-range goals are to determine and analyze the three-dimensional structure of botulinum neurotoxin type E - light chain (BoNT/E-LC) and its relevant mutants to understand the role of the mutated residues in the catalytic action and to determine the structure of the complex of BoNT/H-L.C with its substrate SNAP-25 and its fragments to study the substrate binding mode and to use the information derived in designing substrate peptide inhibitors. The individual specific aims are: A.1. To clone, express and purify BoNT/E-LC with relevant residues mutated individually and in combination, and to assess their effect on catalytic activity. A.2. To determine the crystal structures of the mutants, and to analyze and study the conformational changes and/or other changes in the active site environment caused by these mutants. A.3. To determine the crystal structure of the complex of BoNT/E-LC (in its inactivated form) with the recombinant SNAP-25. A.4. To determine the crystal structure of BoNT/E-LC with synthetic peptides corresponding to SNARE motifs and/or the scissile bond region of SNAP-25 and its relevant fragments.