Light Chain Separated from the Rest of the Type A Botulinum Neurotoxin Molecule Is the Most Catalytically Active Form

Abstract

Botulinum neurotoxins (BoNT) are the most potent of all toxins. The 50 kDa N-terminal endopeptidase catalytic light chain (LC) of BoNT is located next to its central, putative translocation domain. After binding to the peripheral neurons, the central domain of BoNT helps the LC translocate into cytosol where its proteolytic action on SNARE (soluble NSF attachment protein receptor) proteins blocks exocytosis of acetyl choline leading to muscle paralysis and eventual death. The translocation domain also contains 105 Å -long stretch of ∼100 residues, known as “belt,” that crosses over and wraps around the LC to shield the active site from solvent. It is not known if the LC gets dissociated from the rest of the molecule in the cytosol before catalysis. To investigate the structural identity of the protease, we prepared four variants of type A BoNT (BoNT/A) LC, and compared their catalytic parameters with those of BoNT/A whole toxin. The four variants were LC + translocation domain, a trypsin-nicked LC + translocation domain, LC + belt, and a free LC. Our results showed that Km for a 17-residue SNAP-25 (synaptosomal associated protein of 25 kDa) peptide for these constructs was not very different, but the turnover number (k cat) for the free LC was 6-100-fold higher than those of its four variants. Moreover, none of the four variants of the LC was prone to autocatalysis. Our results clearly demonstrated that in vitro, the LC minus the rest of the molecule is the most catalytically active form. The results may have implication as to the identity of the active, toxic moiety of BoNT/A in vivo.

Engineering botulinum neurotoxin domains for activation by toxin light chain

“…The greater potency of self‐activated LH N /A in eSCNs may be attributable to the dissociated LC/A becoming free of H N /A. This is in agreement with the higher specific activity of the LC/A moiety compared to LH N /A and BoNT/A [21].…”

Cleavage of SNAP25 and Its Shorter Versions by the Protease Domain of Serotype A Botulinum Neurotoxin

“…In light of the inhibitor development problems, we extended that study to include two C-terminally truncated LcA and demonstrated that a full length BoNT/A Lc containing 1–448 residues has the highest catalytic activity because its C-terminal appeared to play a product removal role from the active site of LcA [24]. There was little variation in the substrate K m catalyzed by these Lcs and by various BoNT/A forms [25].…”

Small Molecule Inhibitors as Countermeasures for Botulinum Neurotoxin Intoxication

“…Thus, the BoNT/A LC is catalytically active only after translocation into the neuronal cytosol and dissociation from the HC. Experimental evidence indicates that the BoNT/A LC, after separation from the rest of the holotoxin, is the actual active component [ 47 ]. In contrast, the orientation of the BoNT/B protective belt allows for active site access relative to the BoNT/A holotoxin [ 39 ], making the BoNT/B LC catalytically active prior to reduction of the disulfide bond.…”