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Molecular mechanisms of the activation of type A botulinum neurotoxin endopeptidase
Clostridial neurotoxins (BoNTs) are among the most toxic substances presently known. They are a unique group of metalloproteases which catalyze single site cleavage of specific proteins involved in the docking and fusion of synaptic vesicles with plasma membrane for neurotransmitter release. BoNTs are classified into seven distinct serotypes, designated as A to G. Botulinum neurotoxins type A (BoNT/A) is produced by Clostridium botulinum type A as a complex with a group of neurotoxin associated proteins (NAPs). Our results suggest that the bacterial production of NAPs along with BoNT is designed for the NAPs to play an accessory role in the neurotoxin function, in contrast to their previously known limited role in protecting the neurotoxin in the GI tract and in the external environment. We have examined the structure of BoNT/A in aqueous solution, and found the structure in aqueous solution differs dramatically from that resolved by x-ray crystallography, both at secondary and quaternary levels. In terms of secondary structure, BoNT/A in aqueous solution has about 47% β-sheet structure as revealed by infrared spectroscopy, while x-ray crystallography revealed only 17% β-sheet structure. In terms of quaternary structure, BoNT/A exists as a dimer in aqueous solution, which contrasts with the reported monomeric structure of BoNT/A based on the x-ray crystallography. The dimeric form of BoNT/A can self-dissociate into monomeric form at a concentration lower than 50 nM. This concentration-dependent structural change has a significant impact on the endopeptidase activity of BoNT/A: the catalytic efficiency of the monomeric BoNT/A is about 4-fold higher than that of its dimeric form. This difference implies a sterically restricted catalytic site of BoNT/A in the dimeric form of BoNT/A. Reduction of disulfide bond between heavy chain and light chain of BoNTs is required for their enzymatic activity. We are investigating structural differences of BoNT/A under reducing and non-reducing conditions. Reduction of disulfide bond between heavy and light chains of BoNT/A induced a molten globule conformation at physiological temperature (37°C). This molten globule conformation of BoNT/A has been proven to be the enzymatically active structure.
Cai, Shuowei, "Molecular mechanisms of the activation of type A botulinum neurotoxin endopeptidase" (2001). Doctoral Dissertations Available from Proquest. AAI3027183.