Structural basis for recognition of SV2 by type E botulinum neurotoxin

E型肉毒杆菌神经毒素识别SV2的结构基础

• 批准号: 10281936
• 负责人: Rongsheng Jin
• 金额: $ 23.55万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-09 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10281936
• 关键词: Acetylcholine; Achievement; Affect; Affinity; Alpaca; Amino Acid Substitution; Antibodies; Bacteria; Bacterial Toxins; Binding; Binding Sites; Biological; Biological Assay; Bontoxilysin; Botox; Botulism; Cell Surface Receptors; Cell surface; Cells; Chimeric Proteins; China; Cleaved cell; Clinic; Clinical; Clostridium botulinum; Complex; Cosmetics; Crystallization; Custom; Data; Disease; Drug or chemical Tissue Distribution; Esthetics; GTP-Binding Protein alpha Subunits, Gs; Gangliosides; Glycoproteins; Goals; Human; Knowledge; Korea; Link; Mediating; Medicine; Modeling; Molecular; Motor Neurons; Muscle; Mutagenesis; Neuromuscular Junction; Neurons; Paralysed; Pathogenicity; Peptides; Pharmaceutical Preparations; Pharmacology; Polysaccharides; Process; Property; Protein Isoforms; Proteins; Receptor Cell; Resolution; SNAP receptor; Series; Serotyping; Site-Directed Mutagenesis; Specificity; Structure; Synaptic Vesicles; Syndrome; Therapeutic; Toxic effect; Toxin; X-Ray Crystallography; anthrax lethal factor; base; botulinum toxin type B; botulinum toxin type E; clinical application; clinical development; design; ganglioside receptor; improved; in vivo; innovation; nanobodies; novel; novel therapeutics; protein complex; public health relevance; receptor; receptor binding; uptake

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.

摘要 肉毒杆菌神经毒素（BoNT）由细菌肉毒梭菌（Clostridium Botulinum）产生，其是肉毒杆菌毒素的致病因子。 神经麻痹性疾病肉毒杆菌中毒的病原体。然而，A型和B型肉毒杆菌神经毒素（BoNT/A和 BoNT/B）已经成功地用于临床上的各种美容和治疗应用。高 BoNTs的效力依赖于运动神经元特异性和有效地结合和摄取BoNTs， 神经肌肉接头在神经元内，BoNTs切割SNARE复合物并阻断 乙酰胆碱，导致受影响的肌肉麻痹。人们普遍认为，大多数BONT协同 结合运动神经元上的特异性蛋白受体和神经节苷脂以进入细胞。值得注意的是， 与保守的神经节苷脂结合模式相反，蛋白质受体识别的多种结合模式。在 在这项研究中，我们将重点关注E型毒素（BoNT/E），这是研究最少的人类致病性BoNT， 与充分表征的BoNT/A和BoNT/B进行比较。巧合的是，BoNT/E目前正作为一种新的 治疗和美容产品，显示出独特的药理和临床特点时，比较 到BoNT/A和BoNT/B。在分子水平上，BoNT/E识别突触囊泡的三种亚型中的两种 糖蛋白2（SV 2A和SV 2B）作为其神经元受体进入细胞，但不是密切相关的SV 2C亚型。 由于SV 2A、2B和2C在体内具有不同的组织分布，因此更好地理解BoNT/E如何识别 SV 2A、2B和2C的差异对于理解基于BoNT/E的药物的治疗特征也至关重要 开发新的适应症。为此，我们提出了两个具体目标，采用综合方法， 结合了X射线晶体学、定点诱变和结合分析。在目标1中，我们建议研究 BoNT/E识别SV 2A的结构基础。在目标2中，我们的目标是了解亲和力和特异性 BoNT/E识别三种SV 2亚型的要求，而所有结构发现将通过 基于结构的诱变研究。我们目标的实现将有助于理解 BoNT/E的药理学和临床概况，以及促进新对策的设计 针对BoNT/E。