C. Botulinum Type E Catalytic Domain-Substrate Complex

C. E 型肉毒杆菌催化域-底物复合物

• 批准号: 7111195
• 负责人: SUBRAMANYAM SWAMINATHAN
• 金额: $ 43.2万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7111195
• 关键词: Clostridium botulinum; X ray crystallography; active sites; bacterial proteins; bioterrorism /chemical warfare; botulinum toxins; botulism; catalyst; conformation; molecular site; nerve /myelin protein; neurotoxins; protein binding; protein purification; protein structure function; recombinant proteins; site directed mutagenesis; structural biology; synaptosomes

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.

描述（由申请人提供）：引起肉毒杆菌中毒（一种神经元疾病）的肉毒杆菌是一个公共卫生问题，并且也正在成为潜在的生物战威胁。虽然有实验性疫苗可用，但患有这种疾病后没有治疗方法。尽管这是一种潜在的生物战威胁，但不可能为全体人口接种疫苗来预防这种威胁。此外，实验疫苗的副作用尚不清楚。基于抗体的疗法正在出现，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）的结构与功能关系，特别是催化机制，从而实现基于结构的合理治疗药物设计。 近期目标是确定和分析E型肉毒神经毒素-轻链（BoNT/E-LC）及其相关突变体的三维结构，以了解突变残基在催化作用中的作用，并确定其催化作用。 BoNT/H-L.C 与其底物 SNAP-25 及其片段的复合物结构，以研究底物结合模式并使用所得信息设计底物肽抑制剂。各个具体目标是： A.1．克隆、表达和纯化具有单独和组合突变的相关残基的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（灭活形式）与重组 SNAP-25 复合物的晶体结构。 A.4.确定 BoNT/E-LC 的晶体结构，其中合成肽对应于 SNARE 基序和/或 SNAP-25 的易裂键区域及其相关片段。