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.

项目概要 梭菌神经毒素（由破伤风和肉毒杆菌神经毒素组成）是毒性最强的毒素之一 人类已知的病原体，会导致危及生命的麻痹性疾病，对重大公共健康具有潜在影响。 故意释放造成的影响，加上批准的疫苗或疗法的缺乏，导致 将 BoNT 分类为第 1 级、A 类精选药物，但矛盾的是，BoNT 的高度特异性作用。 A 型和 B 型 BoNT 使其成为优秀的药物，适用于越来越多的不同种类的患者 尽管最近有许多以周围神经末梢过度活跃为特征的人类疾病。 对梭菌神经毒素结构与功能关系、分子事件的理解取得进展 通过这种方式，神经毒素重链（HC）能够将其酶结构域转移（易位） 膜双层的定义仍然不明确，在当前的应用中，我们将采用单颗粒冷冻。 电子显微镜测定破伤风神经毒素 (TeNT) 的中等分辨率 (4-10 Å) 结构 在目标 1 中，我们将确定与不同状态的脂质纳米盘相互作用。 含有神经元受体神经节苷脂 GT1b 的小约 100 Å 纳米圆盘不会产生这种结果。 不仅揭示了毒素如何在膜环境中与 GT1b 相互作用，而且还提供了新的细节 关于目标 2 中酶域和易位域的空间排列，界面和插入。 使用具有足够双层表面积的较大 (~170 Å) 纳米圆盘将生成 TeNT 的有效形式 允许 TeNT 在低 pH 条件下从结合态转变为界面/插入 TeNT 构象。 这些快照将提供直接的第一个证据来测试酶转运的假设 跨双层的域是通过穿过易位子孔的内腔来介导的。 目标对于增进我们对易位机制的结构和结构理解至关重要。 确定 TeNT 从水转变时的初始结构构象。 可溶性膜插入蛋白对于未来的设计和验证非常有用 独特的定向小分子毒素转化抑制剂，可快速阻止内体下的毒素转化 pH 条件，从而防止或延迟毒素衍生的细胞毒性事件。