Clostridial neurotoxins comprising the seven serotypes of botulinum neurotoxins
and tetanus neurotoxin are the most potent toxins known to humans. Their potency
coupled with their specificity and selectivity underscores the importance in
understanding their mechanism of action in order to develop a strategy for
designing counter measures against them. To develop an effective vaccine against
the toxin, it is imperative to achieve an inactive form of the protein which
preserves the overall conformation and immunogenicity. Inactive mutants can be
achieved either by targeting active site residues or by modifying the surface
charges farther away from the active site. The latter affects the long-range
forces such as electrostatic potentials in a subtle way without disturbing the
structural integrity of the toxin causing some drastic changes in the
activity/environment. Here we report structural and biochemical analysis on
several mutations on Clostridium botulinum neurotoxin type E light chain with at
least two producing dramatic effects: Glu335Gln causes the toxin to transform
into a persistent apoenzyme devoid of zinc, and Tyr350Ala has no hydrolytic
activity. The structural analysis of several mutants has led to a better
understanding of the catalytic mechanism of this family of proteins. The
residues forming the S1' subsite have been identified by comparing this
structure with a thermolysin-inhibitor complex structure.
