Molecular basis of the endopeptidase activity of type A botulinum neurotoxin

Clostridial botulinum neurotoxins (BoNTs) cause neuroparalysis by blocking neurotransmitter release. The toxin\u27s light chain (LC) acts as a Zn2+-endopeptidase. Type A botulinum nuerotoxin light chain (BoNT/A LC) was expressed in E. coli to high yield level. The recombinant BoNT/A LC, which retained the proteolytic activity and secondary structure, was used in this study. During the translocation of the LC to the cytosol, it is exposed to the endosomal low pH. Low pH showed a dramatic change in the BoNT/A LC polypeptide folding as indicated by differential heat denaturation. Furthermore, binding of 1-anilinonaphthalenesulfonate revealed exposure of hydrophobic domains of BoNT/A LC at low pH. Low pH induced changes were completely reversible. Fluorescence measurements suggested that the two Trp residues are buried and constrained in a hydrophobic environment. The reversibility rules out a possible prerequisite of acid treatment of LC for an ultimate functional conformation. BoNT/A is a zinc-endopeptidase that contains the consensus sequence HEXXH in LC. Substitution of Glu-224 in the motif with Gln (E224Q) resulted in a total loss of the endopeptidase activity, whereas substitution with Asp (E224D) retained ∼1% of the enzymatic activity. However, k m values for wild type and E224D LC were similar. Global structure, Zn2+ content, and substrate binding ability are retained in the mutants. Titration of Zn2+ to EDTA-treated wild-type and mutant proteins indicated similar Zn2+ binding affinity constants. These results suggest an essential and direct role of the carboxyl group of Glu-224 in the hydrolysis of the substrate. The location of the carboxyl group at a precise position is critical for the enzymatic activity. BoNT contains one Zn2+/molecule. The active-site Zn 2+, which was easily displaced from the active site by ethylenediaminetetraacetate, reversibly binds to the LC in a stoichiometric manner. Enzymatic activity was completely abolished in zinc-depleted LC (apo-LC). However, Zn 2+ replenishment partially restored the activity in reZn2+ -LC. Comparable Km values in holo- and reZn2+-LC were observed, indicating a similar substrate binding ability. Removal of the zinc causes irreversible tertiary structural change while the secondary structure remains unchanged. Zinc-binding leads to enhanced thermal stability of LC, which is not identical in native holo-LC and reZn 2+-LC