The role of zinc binding in the biological activity of botulinum toxin

Botulinum toxin is a zinc-dependent endoprotease that acts on vulnerable ce lls to cleave polypeptides that are essential for exocytosis. To exert this poisoning effect, the toxin must proceed through a complex sequence of eve nts that involves binding, productive internalization, and intracellular ex pression of catalytic activity. Results presented in this study show that s oluble chelators rapidly strip Zn2+ from its binding site in botulinum toxi n, and this stripping of cation results in the loss of catalytic activity i n cell-free or broken cell preparations. Stripped toxin is still active aga inst intact neuromuscular junctions, presumably because internalized toxin binds cytosolic Zn2+. In contrast to soluble chelators, immobilized chelato rs have no effect on bound Zn2+, nor do they alter toxin activity. The latt er finding is because of the fact that the spontaneous loss of Zn2+ from it s coordination site in botulinum toxin is relatively slow .When exogenous Z n2+ is added to toxin that has been stripped by soluble chelators, the mole cule rebinds cation and regains catalytic and neuromuscular blocking activi ty. Exogenous Zn2+ can restore toxin activity either when the toxin is free in solution on the cell exterior or when it has been internalized and is i n the cytosol. The fact that stripped toxin can reach the cytosol means tha t the loss of bound Zn2+ does not produce conformational changes that block internalization. Similarly, the fact that stripped toxin in the cytosol ca n be reactivated by ambient Zn2+ or exogenous Zn2+ means that productive in ternalization does not produce conformational changes that block rebinding of cation.