Abstract
Botulinum neurotoxins (BoNTs) can be used therapeutically to treat a wide range of neuromuscular and neurological conditions. A collection of natural BoNT variants exists which can be classified into serologically distinct serotypes (BoNT/B), and further divided into subtypes (BoNT/B1, B2, …). BoNT subtypes share a high degree of sequence identity within the same serotype yet can display large variation in toxicity. One such example is BoNT/B2, which was isolated from Clostridium botulinum strain 111 in a clinical case of botulism, and presents a 10-fold lower toxicity than BoNT/B1. In an effort to understand the molecular mechanisms behind this difference in potency, we here present the crystal structures of BoNT/B2 in complex with the ganglioside receptor GD1a, and with the human synaptotagmin I protein receptor. We show, using receptor-binding assays, that BoNT/B2 has a slightly higher affinity for GD1a than BoNT/B1, and confirm its considerably weaker affinity for its protein receptors. Although the overall receptor-binding mechanism is conserved for both receptors, structural analysis suggests the lower affinity of BoNT/B2 is the result of key substitutions, where hydrophobic interactions important for synaptotagmin-binding are replaced by polar residues. This study provides a template to drive the development of future BoNT therapeutic molecules centered on assessing the natural subtype variations in receptor-binding that appears to be one of the principal stages driving toxicity.
Highlights
Botulinum neurotoxins (BoNTs) are bacterial toxins produced predominantly by Clostridium botulinum. In their active form, BoNTs are composed of a di-chain polypeptide consisting of a ~100 kDa heavy chain and ~50 kDa light chain (LC) which remain linked by a single disulfide bond [1]
The heavy chain contains two domains: the receptor-binding domain (HC ) which is responsible for targeting neuronal cells at the presynaptic neuromuscular junction, and the translocation domain (HN ) which transfers the LC across the endosomal membrane within the cytosol
Separate crystallization experiments containing only GD1a and HC /B2 were successful in producing crystals in the space group C121, containing the ganglioside-bound complex which diffracted to a resolution of 1.8 Å (Table 1)
Summary
Botulinum neurotoxins (BoNTs) are bacterial toxins produced predominantly by Clostridium botulinum. BoNT/B, /G, and /DC instead bind to vesicle protein 2 (SV2) family as their protein receptor. BoNT/B, /G, and /DC instead bind to either either Synaptotagmin I or II (SytI or SytII) [4,5,6,7,8,9,10,11]. BoNTs can be further subcategorized into subtypes based on their amino. BoNTs can be further subcategorized into subtypes based on their amino acid sequences. BoNT/B1 studieshave haveconfirmed confirmed reduced toxicity and suggested it is due due to receptor-binding differences [21,22,23]. Identical residues are shown with a blue background, and similar residues with red Residues underlined in orange are involved in, or neighboring, the Synaptotagmin-binding site and characters. Residues underlined in orange are involved in, or neighboring, the Synaptotagminresidues underlined in blue are involved in, or neighboring, the ganglioside-binding site
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