Structures of Membrane bound and Inserted Tetanus Toxin

膜结合和插入破伤风毒素的结构

• 批准号: 9387099
• 负责人: MICHAEL R BALDWIN
• 金额: $ 24.17万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-26 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9387099
• 关键词: Adopted; Anti-Bacterial Agents; Antitoxins; Area; Bacteria; Bacterial Toxins; Binding; Bontoxilysin; Botulinum Toxins; Categories; Cells; Cholates; Classification; Clostridial Neurotoxin; Clostridium difficile; Complex; Cryoelectron Microscopy; Crystallization; Cytoplasm; Dimensions; Diphtheria Toxin; Disease; Electron Microscope; Electron Microscopy; Endosomes; Environment; Event; Foundations; Future; GT1b ganglioside; Gangliosides; Glycolipids; Goals; Human; Hyperactive behavior; Image; Imagery; Immobilization; Intoxication; Kinetics; Knowledge; Laboratories; Life; Lipid Bilayers; Lipids; Mediating; Medical; Membrane; Membrane Proteins; Methods; Midwestern United States; Modeling; Molecular; Molecular Conformation; Nature; Negative Staining; Neurons; Neurotoxins; Paralysed; Peripheral Nerves; Pharmaceutical Preparations; Pharmacologic Substance; Phase Transition; Physiological; Poison; Positioning Attribute; Procedures; Process; Proteins; Public Health; Recombinants; Research Personnel; Resolution; Sampling; Structure; Structure-Activity Relationship; Surface; Temperature; Testing; Tetanus Toxin; Toxin; Universities; Vaccines; Water; alpha Toxin; anthrax toxin; base; cold temperature; conformer; cytotoxic; design; electron tomography; extracellular; ganglioside receptor; human disease; inhibitor/antagonist; innovation; insight; interfacial; nanodisk; neuromuscular; novel; particle; prevent; prophylactic; protein complex; receptor; reconstruction; response; skills; small molecule; tetanospasmin; translocase

PROJECT SUMMARY The clostridial neurotoxins (composed of the tetanus and botulinum neurotoxins) are among the most toxic agents known to humans and cause life-threatening, paralytic disorders. The potential for major public health impact resulting for an intentional release, combined with the paucity of approved vaccines or therapies has led to the classification of BoNTs as Tier 1, Category A Select Agents. Paradoxically, the highly specific action of BoNT types A and B make them excellent pharmaceuticals for a growing and heterogeneous number of human diseases that are characterized by a hyperactivity of peripheral nerve terminals. Despite many recent advances in understanding the structure-function relationship of clostridial neurotoxins, the molecular events by which the neurotoxin heavy chain (HC) is able to transfer (translocate) its enzymatic domain across the membrane bilayer remains poorly defined. In the current application, we will employ single-particle cryo- electron microscopy to determine medium resolution (4-10 Å) structures of tetanus neurotoxin (TeNT) interacting with lipid nanodiscs in various states. In aim 1 we will determine the structure of TeNT bound to small ~100 Å nanodiscs containing the neuronal receptor ganglioside GT1b. The resulting structure will not only expose how the toxin interacts with GT1b within a membrane environment, but also provide new details on the spatial arrangement of the enzymatic and translocation domains. In aim 2, interfacial and insertion competent forms of TeNT will be generated using larger (~170 Å) nanodiscs of sufficient bilayer surface area to allow TeNT to transition at low pH from the bound state to the interfacial/inserted TeNT conformations. These snap shots will provide the direct first evidence testing the assumption that transport of the enzymatic domain across the bilayer is mediated by transit through the lumen of the translocon pore. Realizing these goals is crucial for advancing our understanding of the translocation mechanism from both a structural and kinetic standpoint. Determining the initial structural conformations of TeNT as it transitions from a water soluble to membrane inserted protein will be extremely useful in future efforts for designing and validating unique directed small molecule toxin transition inhibitors to rapidly prevent toxin transitions under endosomal pH conditions, thus preventing or delaying toxin derived cytotoxic events.

项目摘要 梭菌神经毒素（由破伤风和肉毒神经毒素组成）是最有毒的 人类已知的特工并引起威胁生命，麻痹性疾病。大型公共卫生的潜力 有意释放产生的影响，结合批准的疫苗或疗法的缺乏，已导致 将BONTS分类为第1层，类别A选择代理。矛盾的是，高度具体的动作 Bont类型A和B使它们成为出色的药品，可增长和异质数量 以周围神经末端过度活跃的特征的人类疾病。尽管最近有很多 理解梭菌神经毒素的结构 - 功能关系的进步，分子事件 神经毒素重链（HC）能够通过其转移（转移）酶促域越过 膜双层的定义较差。在当前应用中，我们将采用单粒子冷冻 电子显微镜确定破伤风神经毒素（帐篷）的中分辨率（4-10Å）结构 在各个州与脂质纳米盘相互作用。在AIM 1中，我们将确定帐篷结构与 小〜100Å纳米散发，含有神经元受体神经节苷脂GT1b。最终的结构不会 仅揭示毒素在膜环境中与GT1B相互作用的方式，但也提供了新的细节 关于酶和易位域的空间排列。在AIM 2，界面和插入 使用足够的双层表面积的较大（〜170Å）纳米盘生成胜任的帐篷形式 为了使帐篷在低pH下从结合状态过渡到界面/插入的帐篷构象。 这些快照将提供直接的第一个证据，以测试酶促运输的假设 跨双层的结构域是通过转运孔的腔介导的。意识到这些 目标对于促进我们对结构性和结构的易位机制的理解至关重要 动力学的立场。确定帐篷从水过渡时的初始结构构象 可溶于膜插入的蛋白质将在未来的设计和验证方面非常有用 独特的定向小分子毒素过渡抑制剂，以迅速防止内体内的毒素过渡 pH条件，从而防止或延迟毒素衍生的细胞毒性事件。