The C-terminus of botulinum neurotoxin type A light chain contributes to solubility, catalysis, and stability

Abstract

Botulinum neurotoxin type A (BoNT/A) is the etiological agent responsible for botulism, a disease characterized by peripheral neuromuscular blockade. BoNT/A is produced by Clostridium botulinum as a single chain protein that is activated by proteolytic cleavage to form a 50 kDa light chain (LC, 448 amino acids) and a disulfide bond-linked 100 kDa heavy chain (HC, 847 amino acids). Whilst HC comprises the receptor binding and translocation domains, LC is a Zn 2+-endopeptidase that cleaves at a single glutaminyl–arginine bond corresponding to residues 197 and 198 at the C-terminus of SNAP25. Cleavage of SNAP25 uncouples the neural exocytosis docking/fusion machinery. LC/A (LC 1–448) and several C-terminal deletion proteins of LC/A were engineered and expressed as His-tagged fusion proteins in Escherichia coli. LC 1–448 was purified, but precipitated upon storage. Approximately 40% of LC 1–448 was a covalent dimer due to the formation of inter-chain disulfide bond formation at Cys430. Conversion of Cys430 to Ser abolished dimer formation of LC 1–448, but did not improve solubility. Three C-terminal deletion peptides were engineered; LC 1–425 and LC 1–418 were expressed and could be purified as soluble and stable proteins, whilst LC 1–398 was soluble, but not stable to storage. Kinetic studies showed that LC 1–448 and LC 1–425 efficiently cleaved GST–SNAP25 and the fluorescent substrate SNAPtide, while LC 1–418 catalyzed the cleavage of both the SNAP25 and the fluorescent substrate SNAPtide with a similar K m, but at a 10-fold slower k cat. Thus, regions within the C-terminus of LC/A contribute to solubility, stability, and catalysis.