The C-terminus of botulinum neurotoxin type A light chain contributes to solubility, catalysis, and stability.
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 Zn2+-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 Km, but at a 10-fold slower kcat. Thus, regions within the C-terminus of LC/A contribute to solubility, stability, and catalysis.
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI, USA.
Michael R
MR
Baldwin
Marite
M
Bradshaw
Eric A
EA
Johnson
Joseph T
JT
Barbieri
D000595:Amino Acid Sequence; D000818:Animals; D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003014:Clostridium botulinum / Q000737:chemistry; D008969:Molecular Sequence Data; D009465:Neuromuscular Agents / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012995:Solubility
D009465:Neuromuscular Agents; D010455:Peptides; D011498:Protein Precursors; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D019274:Botulinum Toxins, Type A