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（由破伤风和肉毒杆菌神经毒素组成）是毒性最强的 人类已知的并导致危及生命的麻痹性疾病的物质。潜在的重大公共卫生 故意释放造成的影响，加上缺乏批准的疫苗或疗法， 将BoNT分类为Tier 1，Category A Select Agent。巧合的是， BoNT A型和B型使它们成为用于越来越多的和异质数量的人的优良药物。 以外周神经末梢活动过度为特征的人类疾病。尽管最近许多 梭菌神经毒素结构-功能关系的研究进展， 神经毒素重链（HC）能够通过其将其酶促结构域转移（易位）穿过 膜双分子层仍然不清楚。在目前的应用中，我们将采用单粒子低温- 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 与各种状态的脂质纳米盘相互作用。在目标1中，我们将确定TeNT的结构， 含有神经元受体神经节苷脂GT 1b的小的~100 μ m纳米盘。由此产生的结构将不会 不仅揭示了毒素如何在膜环境中与GT 1b相互作用，而且还提供了新的细节。 酶和易位结构域的空间排列。在目标2中，界面和插入 将使用具有足够双层表面积的较大（~170 μ m）纳米盘来产生TeNT的胜任形式 以允许TeNT在低pH下从结合状态转变为界面/插入的TeNT构象。 这些快照将提供直接的第一个证据，检验酶的假设 跨双层结构域的转运是通过转运子孔的内腔来介导的。实现这些 目标是至关重要的，以促进我们的理解易位机制，从结构和 动力学观点确定TeNT从水转变时的初始结构构象 可溶于膜插入蛋白质将在未来的设计和验证工作中非常有用 独特的定向小分子毒素转换抑制剂， pH条件，从而防止或延迟毒素衍生的细胞毒性事件。