Resolving the intoxication mechanism of botulinum neurotoxins using single molecule structural biology

用单分子结构解析肉毒神经毒素的中毒机理 生物学。 肉毒梭菌产生的毒素是目前已知的最致命的毒素之一，也是人们尊崇的 它们的药用价值。肉毒杆菌可分为七种血清型(A-G)。 它们产生的神经毒素。目前，药物开发一直依赖肉毒杆菌。 A1型神经毒素(BONT/A)。然而，E型肉毒神经毒素(BONT/E)目前正处于临床阶段 试验，因为它提供不同的药代动力学，更快的起效和更短的持续时间，从而使 新的治疗方案。BoNT蛋白是由两种成分组成的“AB毒素”家族的成员。 破伤风、霍乱和白喉毒素)，这些毒素使用 蛋白质类跨膜给药系统(B部分)。因此，它们的结构和活动一直是 研究得很好。然而，关于BONT的交付，还有几个基本的悬而未决的问题 机制，如运送货物所需的毒素数量。此外，虽然有许多 休眠毒素的结构已经被解决，关于活性毒素的结构信息很少 交付状态(S)。AB毒素通过细胞膜运送货物，通常由低pH触发， 这会导致A部分和B部分的结构变化以及插入到膜中。这个 在高蛋白浓度和膜的存在下聚集提供了许多实验 对依赖整体平均的技术的挑战。相比之下，单分子荧光可以 观察单个脂质体上的单个蛋白质以改变AB毒素结构中的这些经典问题 生物学。这些新颖的方法将回答该领域长期存在的问题，并带来新的 了解两种临床相关异构体之间的差异。

Resolving the intoxication mechanism of botulinum neurotoxins using single molecule structural biology. The toxins produced by Clostridium botulinum are some of the deadliest known yet are also revered for their pharmaceutical utility. C. botulinum is classified into seven serotypes (A-G) based on the neurotoxins that they produce. Currently, pharmaceutical development has relied on botulinum neurotoxin type A1 (BoNT/A). However, botulinum neurotoxin type E (BoNT/E) is currently in clinical trials because it provides different pharmacokinetics, faster onset and shorter duration, which enable new treatment regimes. The BoNT proteins are members of the two-component, “AB toxin” family (e.g. tetanus, cholera, and diphtheria toxins), which inject a toxic cargo enzyme (part A) using a proteinaceous transmembrane delivery system (part B). As such, their structure and activity has been well studied. However, several fundamental open questions remain regarding the BoNT delivery mechanism, such as the number of toxins required to deliver the cargo. Additionally, while numerous structures have been solved of the dormant toxins, there is little structural information on the active delivery state(s). AB toxins deliver their cargo across cellular membranes, typically triggered by low pH, which causes structural changes of both parts A and B along with insertion into the membranes. The presence of aggregation at high protein concentrations and membranes provide many experimental challenges for techniques that rely on ensemble averaging. In contrast, single molecule fluorescence can observe individual proteins on single liposomes to revist these classic problems in AB toxin structural biology. These novel approaches will answer long-standing questions in the field and lead to new understanding of the differences between two clinically relevant isoforms.