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：
8362050
负责人：
AXEL T BRUNGER
金额：
$ 0.85万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-03-01 至 2012-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8362050
关键词：
Active Sites Affinity Binding Binding Sites Biochemical Bontoxilysin Botulism Cells Cleaved cell Clinical Clostridial Neurotoxin Complex Development Disease Distal Endocytosis Enzymes Exocytosis Funding Gangliosides Grant Molecular Motor Mutation National Center for Research Resources Neuromuscular Junction Neurons Neurotoxins Paralysed Peptide Hydrolases Preventive Principal Investigator Protease Inhibitor Protein Isoforms Proteins Proteolysis Radiation Reporting Research Research Infrastructure 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 cost design drug development enzyme substrate flexibility inhibitor/antagonist receptor receptor binding structural biology synaptotagmin synaptotagmin I synaptotagmin II

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

该副本是利用资源的众多研究子项目之一由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持而且，副投影的主要研究员可能是其他来源提供的包括其他NIH来源。列出的总费用可能代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。梭状芽胞杆菌毒素（CNT），例如肉毒杆菌（BONT）和破伤风（帐篷）神经毒素，是神经分析疾病的灾难性药物，破伤风和肉毒杆菌毒素。 CNTS通过在神经元内的SNARE蛋白的特异性蛋白解会损害神经元胞毒性，从而导致脆性和痉挛性运动瘫痪的临床表现。 CNT在神经肌肉连接处具有高特异性。毒素细胞识别的分子细节是难以捉摸的。我们报道了与其蛋白受体中的复合物的结构：肉毒杆菌神经毒素血清型B（BONT/B）的受体结合结构域结合在2.15-分辨率下确定的Synaptototagmin II的腔内结构域。在结合上，在腔内域中诱导螺旋，该螺旋在不同的BONT/B尖端上与鞍形缝隙结合。该缝隙毗邻Bont/b的非重叠神经节结合部位。基于结构的突变的生化和神经外生体研究表明，相互作用的高特异性和亲和力，以及在突触中I和II同工型中BONT/B的高选择性。突触神经毒素和神经节的协同结合对内吞作用后BONT/B易位的主动性不可能几何限制。 CNT正确识别并切割其靶标的机制在神经吸收卷中曾经在一个或多个区域的酶 - 基底相互作用区域远离活性位点（外观）。我们的研究为开发针对这些神经毒素的预防性疫苗或抑制剂开发了用于生物防御的基础，以及针对临床应用具有不同靶标规范的改良神经毒素的设计。此外，这项工作通常是蛋白质抑制剂发育的范式，因为蛋白质代表了这些酶的柔韧性，这是药物发育的主要挑战。