Structural mechanism for recognition of host receptor by botulinum neurotoxin A

 描述（由申请人提供）：肉毒杆菌神经毒素识别宿主受体的结构机制 摘要肉毒杆菌神经毒素（BoNT）由肉毒杆菌产生，是神经麻痹疾病肉毒杆菌中毒的病原体。 BoNT 的非凡毒性依赖于神经元细胞对 BoNT 的高度特异性摄取。一种广为接受的双受体模型表明，BoNT 的细胞表面结合和摄取过程是由特定蛋白质受体和神经节苷脂协同介导的。然后，BoNT 利用突触小泡回收途径进入由蛋白质受体介导的神经末端。然而，目前尚不清楚各种 BoNT 如何形成蛋白质受体识别的血清型特异性机制，这被认为是 BoNT 生物活性差异的原因。我们的研究重点是 BoNT/A 血清型，因为它是生物恐怖主义的主要关注点，也是七种 BoNT 血清型 (BoNT/A-G) 中最常用的药物。目标是了解 BoNT/A 通过具有三种亚型（SV2A、2B 和 2C）的突触小泡糖蛋白 2 特异性靶向运动神经元的分子机制。我们的初步研究表明，BoNT/A 和 SV2C 通过相对较小的蛋白质-蛋白质界面相互结合，主要涉及骨架-骨架相互作用，这不足以提供 BoNT/A 所需的高受体结合亲和力和特异性。值得注意的是，我们发现 BoNT/A 利用 SV2 糖基化（突触膜蛋白翻译后修饰的主要形式）来补偿蛋白质介导的识别的“不足”。 BoNT/A 直接与 SV2 的 N 连接聚糖结合，该聚糖在 SV2A、2B 和 2C 中保守，并且在不同脊椎动物中高度保守，从而显着增强受体结合亲和力和特异性。这代表了复杂的宿主-病原体相互作用的新范例。具体目标是（1）了解BoNT/A识别SV2C聚糖的结构基础； (2) 了解 BoNT/A-SV2 识别的亲和力和特异性要求。我们将使用一种结合 X 射线晶体学、定点诱变和结合测定的综合方法。我们目标的实现将指导设计新的治疗方法，通过阻止 BoNT/A 进入细胞来预防和治疗肉毒杆菌中毒，为基于 BoNT/A 的药物的活性和副作用提供新的见解，帮助提高其临床疗效，并提出新的应用。此外，这项研究将影响我们对永恒的宿主-病原体军备竞赛的基本生物学理解，这可能会激发治疗开发的新思路。

 DESCRIPTION (provided by applicant): Structural mechanism for recognition of host receptor by botulinum neurotoxin An Abstract Botulinum neurotoxins (BoNTs), produced by the bacterium Clostridium botulinum, are the causative agents of neuroparalytic disease botulism. The extraordinary toxicity of BoNTs relies on the highly specific uptake of BoNTs by neuronal cells. A well-accepted dual-receptor model suggests that the cell surface binding and uptake process of BoNTs is mediated synergistically by specific protein receptors and gangliosides. BoNTs then exploit the synaptic vesicle recycling pathway entering the nerve terminus mediated by the protein receptors. However, it is largely unknown how various BoNTs develop serotype-specific mechanisms for protein receptor recognition, which is believed to account for the differences in BoNTs' biological activity. Our study is focused on BoNT/A serotype because it is a major concern for bioterrorism and is also the most commonly used medicine among the seven BoNT serotypes (BoNT/A-G). The goal is to understand the molecular mechanism by which BoNT/A specifically targets motoneurons through synaptic vesicle glycoprotein 2 that has three isoforms (SV2A, 2B, and 2C). Our preliminary studies show that BoNT/A and SV2C bind to each other through a relatively small protein-protein interface mostly involving backbone-backbone interactions, which is not sufficient to provide the high receptor binding affinity and specificity that BoNT/A needs. Remarkably, we found that BoNT/A takes advantage of SV2 glycosylation, a major form of post- translational modification of synaptic membrane proteins, to compensate for the "shortfall" on protein-mediated recognition. BoNT/A directly binds to an N-linked glycan of SV2, which is conserved in SV2A, 2B, and 2C and highly conserved across different vertebrates, to significantly enhance receptor binding affinity and specificity. This represents a new paradigm of intricate host-pathogen interactions. The specific aims are (1) to understand the structural basis for recognition of SV2C glycans by BoNT/A; and (2) to understand the affinity and specificity requirements for the BoNT/A-SV2 recognition. We will use an integrated approach that combines X-ray crystallography, site-directed mutagenesis, and binding assays. The achievement of our goal will guide the design of novel therapeutic approaches to prevent and treat botulism by preventing cell entry of BoNT/A, provide new insights into activity and side- effects of BoNT/A-based drugs, help improve their clinical efficacy, and suggest novel applications. Furthermore, this study will have impact on our basic biological understanding of the everlasting host-pathogen arms race, which may stimulate new ideas for therapeutic development.