Structural Analysis of Botulinum Neurotoxins Type B and E by Cryo-EM

Abstract

Botulinum neurotoxins (BoNTs) are the causative agents of a potentially lethal paralytic disease targeting cholinergic nerve terminals. Multiple BoNT serotypes exist, with types A, B and E being the main cause of human botulism. Their extreme toxicity has been exploited for cosmetic and therapeutic uses to treat a wide range of neuromuscular disorders. Although naturally occurring BoNT types share a common end effect, their activity varies significantly based on the neuronal cell-surface receptors and intracellular SNARE substrates they target. These properties are the result of structural variations that have traditionally been studied using biophysical methods such as X-ray crystallography. Here, we determined the first structures of botulinum neurotoxins using single-particle cryogenic electron microscopy. The maps obtained at 3.6 and 3.7 Å for BoNT/B and /E, respectively, highlight the subtle structural dynamism between domains, and of the binding domain in particular. This study demonstrates how the recent advances made in the field of single-particle electron microscopy can be applied to bacterial toxins of clinical relevance and the botulinum neurotoxin family in particular.

Keywords: BoNT/B; BoNT/E; Clostridium botulinum; botulinum neurotoxin; botulism; cryo-EM.

Figure 1

(a) Phylogenetic tree of clostridial neurotoxins prepared with MEGAX [33] from protein sequences aligned with CLUSTALO [34]. (b) Schematic representation of the botulinum neurotoxin domain structure. The toxins are expressed as single-chain proteins but are later proteolytically converted to their active di-chain form, where LC is linked by a single disulphide bridge to HC. The LH_N fragment has been defined previously as the combined LC + H_N domains [35].

Figure 2

(a,b) Cryo-EM maps of BoNT/B and BoNT/E at an estimated resolution of 3.6 and 3.7 Å, respectively. The maps, as well as comparative 2D class averages of the toxins in (c,d), reveal clear secondary elements and domain arrangements illustrated by a schematic diagram (LC in green, H_N in blue, H_C in grey).

Figure 3

(a,b) 3D reconstruction of BoNT/B (teal) and BoNT/E (red), respectively. (c,d) Close-up map of secondary features from each domain, including the mutated catalytic site (mutations marked by *, E231Q/H234Y and E213Q/H216Y for BoNT/B and BoNT/E, respectively); α-helix of H_N, β-sheet of H_CN, and the ganglioside-binding site of H_CC.

Figure 4

(a) Superposition of the cryo-EM (teal) and crystal (PDB 1EPW, light grey) coordinates of BoNT/B. (b,c) B factor-scaled ribbon representations of the crystal and cryo-EM structures of BoNT/B, respectively. (d) Superposition of the cryo-EM (red) and crystal (PDB 3FFZ, light pink) coordinates of BoNT/E. (e,f) B factor-scaled ribbon representations of the crystal and cryo-EM structures of BoNT/E, respectively.