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

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.