Structural basis for recognition of SV2 by type E botulinum neurotoxin

摘要 肉毒神经毒素(BoNTs)是由肉毒杆菌产生的，这种细菌是引起肉毒杆菌 神经麻痹疾病的毒剂肉毒杆菌中毒。然而，A型和B型肉毒神经毒素(BONT/A和 BONT/B)已经成功地在临床上用于各种美容和治疗应用。高潮 BoNTs的效力依赖于运动神经元对BoNTs的特异和有效的结合和摄取 神经肌肉接头。在神经元内部，BoNTs裂解SNARE复合体并阻止释放 乙酰胆碱，导致受影响肌肉瘫痪。人们普遍认为，大多数BoNTs具有协同作用 将特定的蛋白受体和神经节苷脂结合在运动神经元上，以便于细胞进入。值得注意的是，BoNTs的发展 蛋白质受体识别的多种结合模式，而不是保守的神经节苷脂结合模式。在……里面 在这项研究中，我们将集中在E型毒素(BONT/E)，它是目前研究最少的人类致病BONT 与特征良好的BONT/A和BONT/B的比较矛盾的是，BONT/E目前作为一种新的临床试验 治疗和美容产品，比较时显示出明显的药理和临床特征 在分子水平上，BoNT/E识别突触小泡的三种亚型中的两种 糖蛋白2(SV2A和SV2B)是其进入细胞的神经元受体，但不是密切相关的SV2C亚型。 由于SV2A、2B和2C在体内的组织分布不同，因此更好地理解了BONT/E是如何识别的 SV2A、2B和2C的不同对于理解基于BONT/E的药物的治疗概况也是至关重要的 以开发新的适应症。为此，我们使用一种综合方法提出了两个具体目标 结合了X射线结晶学、定点突变和结合分析。在目标1中，我们建议研究 ONT/E识别SV2A的结构基础。在目标2中，我们旨在了解其亲和力和特异性。 要求BONT/E识别三种SV2亚型，而所有结构发现将通过 基于结构的突变研究。我们目标的实现将有助于理解独特的 BONT/E的药理学和临床概况，以及促进新对策的设计 抗BONT/E

Abstract Botulinum neurotoxins (BoNTs) are produced by the bacterium Clostridium botulinum, which are the causative agents of neuroparalytic disease botulism. Nevertheless, type A and type B botulinum neurotoxins (BoNT/A and BoNT/B) have been successfully used in clinic for a variety of aesthetic and therapeutic applications. The high potency of BoNTs relies on the specific and efficient binding and uptake of BoNTs by motor neurons at neuromuscular junctions. Inside the neurons, BoNTs cleave SNARE complex and block the release of acetylcholine, resulting in paralysis of the affected muscles. It is well accepted that most BoNTs synergistically bind specific protein receptors and gangliosides on motor neurons for cell entry. Remarkably, BoNTs develop diverse binding modes for protein receptor recognition, in contrast to a conserved ganglioside-binding mode. In this study, we will focus on the type E toxin (BoNT/E) that is the least studied human pathogenic BoNT in comparison to well-characterized BoNT/A and BoNT/B. Paradoxically, BoNT/E is currently in clinic trial as a new therapeutic and aesthetic product, which displays distinct pharmacological and clinic features when compared to BoNT/A and BoNT/B. At the molecular level, BoNT/E recognizes two of the three isoforms of synaptic vesicle glycoprotein 2 (SV2A and SV2B) as its neuronal receptors for cell entry, but not the closely related SV2C isoform. As SV2A, 2B, and 2C have different tissue distribution in vivo, a better understanding of how BoNT/E recognizes SV2A, 2B, and 2C differentially is crucial to understand the therapeutic profiles of BoNT/E-based drugs as well as to develop new indications. To this end, we propose two Specific Aims using an integrated approach that combines X-ray crystallography, site-directed mutagenesis, and binding assays. In Aim 1, we propose to study the structural basis for recognition of SV2A by BoNT/E. In Aim 2, we aim to understand the affinity and specificity requirements for BoNT/E to recognize three SV2 isoforms, while all the structural findings will be verified by structure-based mutagenesis studies. The achievement of our goals will help to understand the unique pharmacological and clinical profiles of BoNT/E, as well as to facilitate the design of new countermeasures against BoNT/E.