MECHANISM OF BOTULINUM NEUROTOXIN TARGET, SUBSTRATE, AND INHIBITOR INTERACTIONS

肉毒杆菌神经毒素靶标、底物和抑制剂相互作用的机制

基本信息

批准号：
7954183
负责人：
AXEL T BRUNGER
金额：
$ 0.91万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-03-01 至 2010-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7954183
关键词：
Active Sites Affinity Binding Binding Sites Biochemical Bontoxilysin Botulism Cells Cleaved cell Clinical Clostridial Neurotoxin Complex Computer Retrieval of Information on Scientific Projects Database Development Disease Distal Endocytosis Enzymes Exocytosis Funding Gangliosides Grant Institution Molecular Motor Mutation Neuromuscular Junction Neurons Neurotoxins Paralysed Peptide Hydrolases Preventive Protease Inhibitor Protein Isoforms Proteins Proteolysis Reporting Research Research Personnel Resolution Resources SNAP receptor Serotyping Shapes Source Spastic Specificity Structure Substrate Interaction Tetanus Toxin United States National Institutes of Health Vaccines Work base botulinum clinical application design drug development enzyme substrate flexibility inhibitor/antagonist receptor receptor binding structural biology synaptotagmin synaptotagmin I synaptotagmin II synchrotron radiation

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Clostridial neurotoxins (CNTs), such as botulinum (BoNT) and tetanus (TeNT) neurotoxins, are the causative agents of the neuroparalytic diseases tetanus and botulism. CNTs impair neuronal exocytosis by specific proteolysis of SNARE proteins once inside the neuron, resulting in the clinical manifestations of flaccid and spastic motor paralysis. CNTs bind with high specificity at neuromuscular junctions. The molecular details of the toxin-cell recognition have been elusive. We reported the structure of a BoNT in complex with its protein receptor: the receptor-binding domain of botulinum neurotoxin serotype B (BoNT/B) bound to the luminal domain of synaptotagmin II, determined at 2.15-¿ resolution. On binding, a helix is induced in the luminal domain that binds to a saddle-shaped crevice on a distal tip of BoNT/B. This crevice is adjacent to the non-overlapping ganglioside-binding site of BoNT/B. Biochemical and neuronal ex vivo studies of structure-based mutations indicate high specificity and affinity of the interaction, and high selectivity of BoNT/B among synaptotagmin I and II isoforms. Synergistic binding of both synaptotagmin and ganglioside imposes geometric restrictions on the initiation of BoNT/B translocation after endocytosis. The mechanism by which a CNT properly identifies and cleaves its target SNARE once inside the neuron involves one or more regions of enzyme-substrate interaction remote from the active site (exosites). Our studies provide the basis for the development of preventive vaccines or inhibitors against these neurotoxins for bio defense, as well as design of modified neurotoxins with different target specificities for clinical applications. In addition, this work is a paradigm for protease inhibitor development in general since proteases represent major challenges for drug development due to the inherent flexibility of these enzymes.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以出现在其他 CRISP 条目中列出的机构是。对于中心来说，它不一定是研究者的机构。梭菌神经毒素 (CNT)，例如肉毒杆菌 (BoNT) 和破伤风 (TeNT) 神经毒素，是神经麻痹性疾病破伤风和肉毒杆菌中毒的病原体，一旦进入神经元，CNT 就会通过 SNARE 蛋白的特异性蛋白水解作用损害神经元胞吐作用，从而导致临床症状。弛缓性和痉挛性运动麻痹的表现 CNT 具有高特异性结合。我们报道了 BoNT 与其蛋白受体复合物的结构：与管腔结构域结合的肉毒杆菌神经毒素 B 型受体结合结构域 (BoNT/B)。突触结合蛋白 II，测定于 2.15-¿结合时，在管腔结构域中诱导出一个螺旋，该螺旋与 BoNT/B 远端的鞍形缝隙结合，该缝隙与 BoNT/B 的非重叠神经节苷脂结合位点相邻。基于结构的突变的生化和神经元离体研究表明，突触结合蛋白 I 和 II 亚型之间的相互作用具有高特异性和亲和力，并且 BoNT/B 具有高选择性。突触结合蛋白和神经节苷脂对胞吞后 BoNT/B 易位的启动施加几何限制。一旦进入神经元，CNT 就会正确识别和切割其目标 SNARE，该机制涉及远离活性位点的一个或多个酶-底物相互作用区域。我们的研究为开发针对这些神经毒素的生物防御预防性疫苗或抑制剂以及设计具有不同靶点特异性的修饰神经毒素以用于临床应用提供了基础。此外，这项工作总体上是蛋白酶抑制剂开发的范例，因为由于这些酶固有的灵活性，蛋白酶代表了药物开发的主要挑战。