C. Botulinum Type E Catalytic Domain-Substrate Complex

描述(由申请人提供)：肉毒梭菌引起肉毒杆菌中毒，一种神经元疾病，是一个公共卫生问题，也是一个潜在的生物武器威胁。虽然有一种实验性疫苗可用，但在受到这种疾病的困扰后，没有治疗方法。尽管这是一个潜在的生物武器威胁，但可能不可能为所有人口接种疫苗。此外，这种实验性疫苗的副作用还不是很清楚。基于抗体的治疗方法正在出现，但可能需要不止一种抗体来中和单一血清型。在这种情况下，有必要开发第二道防线，开发用于治疗的抗毒素，以补充疫苗或抗体治疗。因此，这一建议的长远目标是了解肉毒神经毒素(BoNT)的结构-功能关系，特别是其催化机制，从而引导基于结构的合理药物设计用于治疗。 短期目标是测定和分析肉毒杆菌神经毒素E-轻链(BoNT/E-Lc)及其相关突变体的三维结构，了解突变残基在催化作用中的作用，确定BoNT/H-Lc与其底物SNAP-25及其片段的复合体的结构，研究底物结合模式，并利用所获得的信息设计底物肽抑制剂。个人的具体目标是：A.1。克隆、表达和纯化ONT/E-LC及其相关残基单独和联合突变，并评价其对催化活性的影响。A.2.确定突变体的晶体结构，并分析和研究这些突变体引起的构象变化和/或活性中心环境的其他变化。A.3.用重组SNAP-25测定BONT/E-LC(失活状态)复合体的晶体结构。A.4.用SNAP-25及其相关片段的SNARE基序和/或剪切区的合成肽来确定BONT/E-LC的晶体结构。

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.