MECHANISM OF BOTULINUM NEUROTOXIN TARGET, SUBSTRATE, AND INHIBITOR INTERACTIONS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 梭状芽胞杆菌神经毒素(CNTs)，如肉毒毒素(BoNT)和破伤风神经毒素(TENT)，是引起神经麻痹疾病破伤风和肉毒中毒的病原体。一旦SNARE蛋白进入神经元内部，碳纳米管就会通过特异性的蛋白分解来损害神经元的胞吐功能，导致松弛和痉挛运动瘫痪的临床表现。碳纳米管结合在神经肌肉接头处具有高度的特异性。毒素细胞识别的分子细节一直难以捉摸。我们报道了一种BONT与其蛋白受体的复合体的结构：B型肉毒神经毒素受体结合域(BONT/B)与突触素II的管腔结构域结合，分辨率为2.15°。在结合时，在与BONT/B远端的鞍状缝隙结合的管腔结构域中诱导出一个螺旋，该缝隙毗邻BONT/B的非重叠的神经节苷脂结合部位。对基于结构的突变的生化和神经元体外研究表明，这种相互作用具有高的特异性和亲和力，并且在突触素I和II亚型中具有高的选择性。突触素和神经节苷脂的协同结合对细胞吞噬后启动BONT/B转位施加了几何限制。一旦进入神经元，CNT正确识别和切割其目标陷阱的机制涉及远离活性部位(外显子)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不同靶点特异性的修饰神经毒素用于临床应用提供了基础。此外，这项工作是开发蛋白酶抑制剂的一个范例，因为由于这些酶固有的灵活性，蛋白酶是药物开发的主要挑战。

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.