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

Sara Košenina,Geoffrey Masuyer,Markel Martínez-Carranza,Pål Stenmark,Jonathan R Davies

doi:10.3390/toxins14010014

Sara Košenina, Geoffrey Masuyer + Show 3 more

Open Access

https://doi.org/10.3390/toxins14010014

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Journal: Toxins	Publication Date: Dec 23, 2021
Citations: 5	License type: CC BY 4.0

Affiliation: Stockholm University, University of Bath, Lund University

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.

Highlights

The botulinum neurotoxins (BoNT) are potent bacterial toxins produced mainly by toxigenic Clostridium botulinum species [1]
The holotoxins are expressed as single-chain proteins that need to be proteolytically activated into a functional di-chain form, which consists of a ~50 kDa light chain (LC) linked by a single disulphide bond to the ~100 kDa heavy chain (HC) [3]
The map for BoNT/B appeared overall more precise than that of BoNT/E (Figure 2), even though image processing showed a slight preferential orientation for BoNT/B, which may partially explain the variation in local resolution

Summary

Introduction

The botulinum neurotoxins (BoNT) are potent bacterial toxins produced mainly by toxigenic Clostridium botulinum species [1]. The map for BoNT/B appeared overall more precise than that of BoNT/E (Figure 2), even though image processing showed a slight preferential orientation for BoNT/B, which may partially explain the variation in local resolution For both toxins, particle sizes were consistent with monomers, and secondary structure could clearly be distinguished in the 2D average classification, so that typical features of the individual domains were identifiable. For BoNT/B, the lack of density in some areas, the HCC subdomain, suggests that the preferred particle orientation observed in the angular distribution map (Supplementary Figure S1) is the main reason affecting map quality and resolution, local dynamism of the domain cannot be excluded Such effect might be compensated by collection of larger datasets with altered parameters or grid preparation. Observations from the cryo-EM map suggest HC/E is inherently dynamic thanks to the flexible linker with HN, which would affect the local resolution even though it does not imply a significant conformational change like the one observed in the progenitor complex

Comparison with X-ray Crystal Structures

Constructs

Protein Expression and Purification

Cryo-EM Imaging

Cryo-EM Data Processing

Model Building and Validation