C. Botulinum Type E Catalytic Domain-Substrate Complex

描述（由申请人提供）：肉毒梭菌引起肉毒中毒，是一种神经元疾病，是一个公共卫生问题，也是一个潜在的生物战威胁。虽然有一种实验性疫苗，但在患有这种疾病后没有治疗方法。虽然这是一个潜在的生物战威胁，但可能不可能为整个人口接种疫苗。此外，实验性疫苗的副作用尚不清楚。基于抗体的治疗正在出现，但可能需要一种以上的抗体来中和单一血清型。在这种情况下，有必要通过开发用于治疗用途的抗毒素来开发第二道防线，以补充疫苗或抗体治疗。因此，该提案的长期目标是了解肉毒杆菌神经毒素（BoNT）的结构-功能关系，特别是催化机制，从而导致基于结构的合理药物设计用于治疗。 短期目标是确定和分析肉毒杆菌神经毒素型E-轻链的三维结构（BoNT/E-LC）及其相关突变体，以了解突变残基在催化作用中的作用，并确定BoNT/H-LC与其底物SNAP-1的复合物的结构。25及其片段来研究底物结合模式，并使用在设计底物肽抑制剂中得到的信息。具体目标如下：克隆、表达和纯化相关残基单独和组合突变的BoNT/E-LC，并评估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/或活性位点环境的其他变化。 A.3.确定BoNT/E-LC（以其失活形式）与重组SNAP-25的复合物的晶体结构。 A.4.用SNAP-25及其相关片段的SNARE基序和/或易裂键区的合成肽确定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.