COOPERATIVE EXOSITE-DEPENDENT CLEAVAGE OF SYNAPTOBREVIN BY TETANUS TOXIN LIGHT-CHAIN

The light chain (L chain) of tetanus neurotoxin (TeNT) has been shown to have been endowed with zinc endopeptidase activity, selectively dir ected toward the Gln(76)-Phe(77) bond of synaptobrevin, a vesicle-asso ciated membrane protein (VAMP) critically involved in neuroexocytosis. In previous reports, truncations at the NH2 and COOH terminus of syna ptobrevin have shown that the sequence 39-88 of synaptobrevin is the m inimum substrate of TeNT, suggesting either the requirement of a well defined three-dimensional structure of synaptobrevin or a role in the mechanism of substrate hydrolysis for residues distal from the cleavag e site. In this study, the addition of NH2- and COOH-terminal peptides of synaptobrevin, S 27-55 (S-1) and S 82-93 (S-2), to the synaptobrev in fragment S 56-81 allowed the cleavage of this latter peptide by TeN T to occur. This appears to result from an activation process mediated by the simultaneous binding of S-1 and S-2 with complementary sites p resent on TeNT as shown by surface plasmon resonance experiments and t he determination of kinetic constants. All these results favor an exos ite-controlled hydrolysis of synaptobrevin by TeNT, probably involving a conformational change of the toxin. This could account for the high degree of substrate specificity of TeNT and, probably, botulinum neur otoxins.