Abstract
Clostridium botulinum neurotoxins (BoNT) cause the potentially fatal paralytic disease, botulism, by inhibition of acetylcholine release at the neuromuscular junction. Many BoNTs have been identified over the years and have been categorised into unique serotypes (BoNT/A to BoNT/G, BoNT/X, BoNT/Wo, BoNT/En) with different toxicological profiles. Serotypes are further divided into subtypes (e.g., BoNT/A1, BoNT/A2…) due to minor changes in amino acid sequence. Despite the extreme toxicity of BoNTs, subtypes A1 (BoNT/A1) and B1 (BoNT/B1) are used as therapeutics to treat hyper muscular and glandular disorders.All BoNTs are comprised of the same three domains: the cell binding domain, translocation domain, and catalytic domain. The cell binding domain of BoNT/A and BoNT/B binds to both a protein and ganglioside receptor on the surface of motor neurones where BoNT is then internalised through receptor-mediated endocytosis. This is an essential step for BoNT cellular entry which facilitates the high specificity of BoNTs to motor neurones. It is therefore important to understand the precise interactions involved in receptor binding as this may be useful for the design of new BoNT-based therapeutics. The work presented in this thesis describes the resolution of six new crystal structures of BoNT/A2 to BoNT/A6 cell binding domains in complex with a receptor ganglioside. These high-resolution structures also revealed interesting features that require further investigation, such as a subdomain hinge rotation and a dynamic bridging interaction close to the ganglioside binding site.Following dual receptor recognition and subsequent endocytosis of BoNT, the translocation domain undergoes acid-induced conformational change and is believed to form channels in the endosomal membrane which grant the passage of the catalytic domain into the cytosol. This mechanism is poorly understood. In an attempt to investigate this, a protocol for the expression, purification, and crystallisation of N-terminally truncated (beltless) botulinum neurotoxin B heavy chain and the preliminary expression of the N-terminally truncated botulinum neurotoxin B translocation domain, are described. This provides the foundation for future structural characterisation of BoNT translocation.
| Date of Award | 4 Dec 2023 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Ravi Acharya (Supervisor), Vasanta Subramanian (Supervisor) & Sai Man Liu (Supervisor) |