Enhancement of the endopeptidase activity of botulinum neurotoxin by its associated proteins and dithiothreitol

Botulinum neurotoxins type A (BoNT/A), the most toxic substance known to ma n, is produced by Clostridium botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs), possibly through a polycistronic ex pression of a clustered group of genes. The botulinum neurotoxin complex is the only known example of a protein complex where a group of proteins (NAP s) protect another protein (BoNT) against acidity and proteases of the GI t ract. We now report that NAPs also potentiate the Zn2+ endopeptidase activi ty of BoNT/A in both in vitro and in vivo assays against its known intracel lular target protein, 25 kDa synaptosomal associated protein (SNAP-25). Whi le BoNT/A exhibited no protease activity prior to reduction with dithiothre itol (DTT), the BoNT/A complex exhibited a high protease activity even in i ts nonreduced form. Our results suggest that the bacterial production of NA Ps along with BoNT is designed for the NAPs to play an accessory role in th e neurotoxin function, in contrast to their previously known limited role i n protecting the neurotoxin in the GI tract and in the external environment . Structural features of BoNT/A change considerably upon disulfide reductio n, as revealed by near-UV circular dichroism spectroscopy. BoNT/A in the re duced form adopts a more flexible structure than in the unreduced form, as also indicated by large differences in Delta H values (155 vs 248 kJ mol(-1 )) of temperature-induced unfolding of BoNT/A.