MECHANISM OF BOTULINUM NEUROTOXIN TARGET, SUBSTRATE, AND INHIBITOR INTERACTIONS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 梭菌神经毒素（Clostridial neurotoxins，CNT），如肉毒杆菌（botulinum，BoNT）和破伤风（tetanus，TeNT）神经毒素，是神经麻痹性疾病破伤风和肉毒中毒的病原体。一旦进入神经元，CNT通过SNARE蛋白的特异性蛋白水解损害神经元胞吐作用，导致弛缓性和痉挛性运动麻痹的临床表现。CNT在神经肌肉接头处具有高特异性结合。毒素-细胞识别的分子细节一直难以捉摸。我们报道了BoNT与其蛋白受体复合物的结构：肉毒杆菌神经毒素血清型B（BoNT/B）的受体结合结构域与突触结合蛋白II的管腔结构域结合，在2.15-分辨率下测定。在结合时，在管腔结构域中诱导螺旋，其结合BoNT/B的远端尖端上的鞍形裂缝。该裂隙邻近BoNT/B的非重叠神经节苷脂结合位点。 基于结构的突变的生物化学和神经元离体研究表明相互作用的高特异性和亲和力，以及BoNT/B在突触结合蛋白I和II同种型中的高选择性。synaptotagmin和神经节苷脂的协同结合对内吞作用后BoNT/B易位的起始施加几何限制。一旦进入神经元，CNT正确识别和切割其靶SNARE的机制涉及远离活性位点（外位点）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预防性疫苗或抑制剂以用于生物防御以及设计具有不同靶点特异性的经修饰的神经毒素以用于临床应用提供了基础。 此外，这项工作是蛋白酶抑制剂开发的一个范例，因为蛋白酶代表了药物开发的主要挑战，因为这些酶具有固有的灵活性。

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.