Structural characterisation of the catalytic domain of botulinum neurotoxin X - high activity and unique substrate specificity

Abstract

Botulinum neurotoxins (BoNTs) are among the most potent toxins known and are also used to treat an increasing number of medical disorders. There are seven well-established serotypes (BoNT/A-G), which all act as zinc-dependent endopeptidases targeting specific members of the SNARE proteins required for synaptic vesicle exocytosis in neurons. A new toxin serotype, BoNT/X, was recently identified. It cleaves not only the canonical targets, vesicle associated membrane proteins (VAMP) 1/2/3 at a unique site, but also has the unique ability to cleave VAMP4/5 and Ykt6. Here we report the 1.35 Å X-ray crystal structure of the light chain of BoNT/X (LC/X). LC/X shares the core fold common to all other BoNTs, demonstrating that LC/X is a bona fide member of BoNT-LCs. We found that access to the catalytic pocket of LC/X is more restricted, and the regions lining the catalytic pocket are not conserved compared to other BoNTs. Kinetic studies revealed that LC/X cleaves VAMP1 with a ten times higher efficiency than BoNT/B and the tetanus neurotoxin. The structural information provides a molecular basis to understand the convergence/divergence between BoNT/X and other BoNTs, to develop effective LC inhibitors, and to engineer new scientific tools and therapeutic toxins targeting distinct SNARE proteins in cells.

Crystal structure of the catalytic domain of the Weissella oryzae botulinum‐like toxin

“…The comparison also revealed a significant difference at the loop defining exosites 1 and 2 in LC/F (residues 172-182). In LC/Wo, these sites correspond to a much shorter linker (206-211), similar to the one observed in LC/X [21]. Interestingly, the Ca 2+ ion observed in LC/ Wo is situated within bonding distance (~3.5 A) of the superposed VAMP peptide (Fig.…”

“…LC/X, the closest structural homolog of LC/Wo, presented restricted access to its active site . LC/Wo on the contrary seems to have an open, negatively charged active site, a property shared with LC/B, which consistently has the highest sequence identity (~22%) to LC/Wo (Fig.…”

“…3A) and includes Arg409 and Tyr412, which are likely also important for substrate recognition, in addition to their role in catalysis [32]. LC/X, the closest structural homolog of LC/Wo, presented restricted access to its active site [21]. LC/ Wo on the contrary seems to have an open, negatively charged active site, a property shared with LC/B, which consistently has the highest sequence identity (~22%) to LC/Wo (Fig.…”

Variations in the Botulinum Neurotoxin Binding Domain and the Potential for Novel Therapeutics

“…This suggests that this toxin significantly diverged from other serotypes during its evolution. Despite this, recent structural characterisation of the LC has revealed a core fold common to all BoNTs [ 126 ]. Little is known about the BoNT/X H N and H C domains, and considering the novel characteristics of LC, attempts are underway to determine the specific receptor(s) that it targets and how it functions in vivo.…”

Novel Botulinum Neurotoxins: Exploring Underneath the Iceberg Tip

“…Given that the SNARE-selectivity of BoNTs relies on multiple, extended and very specific interactions between the L chain and the substrate [ 1 , 106 , 107 , 108 , 109 ], the ability of BoNT/X to cleave such a copious number of VAMP isoforms is very surprising. Even more surprising is the fact that, notwithstanding such a broad specificity, LC/X chain cleaves VAMP-2 with a 10-fold higher efficiency than LC/B [ 110 ]. Moreover, a BoNT/X derivative consisting of two nontoxic fragments enzymatically ligated (LC-HN and HC) can enter neurons and cause flaccid paralysis in vivo, thus suggesting that it can, in principle, cause botulism.…”