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.

Grant Number: 5R01GM151334-03
NIH Institute/Center: NIH
Principal Investigator: Mark Bowen