PMID:18511418

Citation	Sikorra, S, Henke, T, Galli, T and Binz, T (2008) Substrate recognition mechanism of VAMP/synaptobrevin-cleaving clostridial neurotoxins. J. Biol. Chem. 283:21145-52
Abstract	Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) inhibit neurotransmitter release by proteolyzing a single peptide bond in one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors SNAP-25, syntaxin, and vesicle-associated membrane protein (VAMP)/synaptobrevin. TeNT and BoNT/B, D, F, and G of the seven known BoNTs cleave the synaptic vesicle protein VAMP/synaptobrevin. Except for BoNT/B and TeNT, they cleave unique peptide bonds, and prior work suggested that different substrate segments are required for the interaction of each toxin. Although the mode of SNAP-25 cleavage by BoNT/A and E has recently been studied in detail, the mechanism of VAMP/synaptobrevin proteolysis is fragmentary. Here, we report the determination of all substrate residues that are involved in the interaction with BoNT/B, D, and F and TeNT by means of systematic mutagenesis of VAMP/synaptobrevin. For each of the toxins, three or more residues clustered at an N-terminal site remote from the respective scissile bond are identified that affect solely substrate binding. These exosites exhibit different sizes and distances to the scissile peptide bonds for each neurotoxin. Substrate segments C-terminal of the cleavage site (P4-P4') do not play a role in the catalytic process. Mutation of residues in the proximity of the scissile bond exclusively affects the turnover number; however, the importance of individual positions at the cleavage sites varied for each toxin. The data show that, similar to the SNAP-25 proteolyzing BoNT/A and E, VAMP/synaptobrevin-specific clostridial neurotoxins also initiate substrate interaction, employing an exosite located N-terminal of the scissile peptide bond.
Links	PubMed PMC3258937 Online version:10.1074/jbc.M800610200
Keywords	Animals; Humans; Kinetics; Models, Biological; Mutagenesis; Peptides/chemistry; Protein Binding; Protein Structure, Tertiary; R-SNARE Proteins/chemistry; Rats; Recombinant Proteins/chemistry; Substrate Specificity; Tetanus Toxin/chemistry; Tetanus Toxin/metabolism; Transcription, Genetic; Vesicle-Associated Membrane Protein 2/chemistry
edit table

Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
CLOBO:BXA2		GO:0006508: proteolysis	ECO:0000269:		P		Figure 2: Botulinum toxin proteolytic activity on VAMP.	complete CACAO 3313

References

See Help:References for how to manage references in GONUTS.