To decipher the toxin's molecular structure, the scientists bombard crystalline samples with high-intensity X-rays produced by the National Synchrotron Light Source (NSLS) at Brookhaven, one of the most widely used scientific facilities in the world. By studying how the X-rays are diffracted as they bounce off or pass through parts of the crystal, the scientists can work backward to reconstruct the shape and arrangement of the atoms in the molecule.

The scientists also did experiments to test the ability of type E neurotoxin and their lab-made mutant to cleave the target protein that prevents neurotransmitter release during a botulism infection. They found that the mutant, which differs from the wild type toxin by just one amino acid, binds specifically to the substrate protein, but does not perform the hydrolytic reaction to cleave it, as the wild type does.

The structural analyses reveal a likely explanation for the loss of toxicity: The substitution of the amino acid glutamine for glutamate in the catalytic domain of the toxin does not change the overall structure substantially. But it does increase the distance between a zinc atom and a water molecule crucial to the cleaving process by a mere 0.6 angstroms (an angstrom is one ten-billionth of a meter). That subtle change disturbs the electrostatic properties of the molecule at the active site, making it unable to cleave the target protein.

These details about the toxin ™s mechanism of action may suggest a variety of ways to inhibit that action and render the toxin harmless.

There are no Clostridium botulinum bacteria at Brookhaven Lab. For this work, the scientists obtained the gene for the part of the type E neurotoxin that actively cleaves proteins, the catalytic domain. They then expressed this gene to produce this portion of the neurotoxin protein, which by itself is not toxic. This work is done in strict compliance with Brookhaven ™s Institutional Biosafety committee regulations according to standards set by the U.S. Centers for Disease Control and Prevention in Atlanta. Only authorized scientists have access to the laboratory.

This research was supported by the U.S. Army Medical Research and Materiel Command.

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