STRUCTURAL DETERMINANTS OF THE SPECIFICITY FOR SYNAPTIC VESICLE-ASSOCIATED MEMBRANE PROTEIN SYNAPTOBREVIN OF TETANUS AND BOTULINUM TYPE-B AND TYPE-G NEUROTOXINS/

Tetanus and botulinum neurotoxins type B and G are zinc-endopeptidases of remarkable specificity. They recognize and cleave a synaptic vesic le-associated membrane protein (VAMP)/synaptobrevin, an essential prot ein component of the vesicle docking and fusion apparatus. VAMP contai ns two copies of a nine-residue motif, also present in SNAP-25 (synapt osomal-associated protein of 25 kDa) and syntaxin, the two other subst rates of clostridial neurotoxins. This motif was suggested to be a det erminant of the target specificity of neurotoxins. Antibodies raised a gainst this motif cross-react among VAMP, SNAP-25, and syntaxin and in hibit the proteolytic activity of the neurotoxins. Moreover; the vario us neurotoxins cross-inhibit each other's proteolytic action. The role of the three negatively charged residues of the motif in neurotoxin r ecognition was probed by site-directed mutagenesis. Substitution of ac idic residues in both copies of the VAMP motif indicate that the first one is involved in tetanus neurotoxin recognition, whereas the second one is implicated in binding botulinum B and G neurotoxins. These res ults suggest that the two copies of the motif have a tandem associatio n in the VAMP molecule.