Clever machinery
There are seven forms of botulinum neurotoxin produced by seven different strains of the Clostridium botulinum bacterium, explained Brunger, a Howard Hughes Medical Institute investigator who holds professorships in three Stanford departments-molecular and cellular physiology, neurology and neurological sciences, and the Stanford Synchrotron Radiation Laboratory (SSRL).
"The seven botulinum neurotoxins cut SNARE proteins at different sites along the surface," he said. "Why that is, we really don't know exactly."
For the Nature study, the researchers focused on one of the seven forms, botulinum serotype A, which is the active ingredient in Botox. Breidenbach, lead author of the study, spent months in Brunger's lab trying to crystallize a SNARE/botulinum A complex for laboratory analysis. Unfortunately, the botulinum A samples usually ended up slicing the SNARE target in two, rendering it useless.
"The trick that Mark found was to introduce two specific mutations in the botulinum enzyme that would inhibit its function, but not to the degree that it would affect its structure," Brunger said. "These two mutations prevented it from cutting, so we could observe how it interacted with an intact SNARE."
The SNARE/botulinum A crystals were then taken to SSRL and the Lawrence Berkeley National Laboratories, where their structures were determined using a technique called x-ray crystallography. The results, published in Nature, reveal a complicated, three-dimensional maze of twisted proteins that look like gift-wrapping ribbons gone awry.
"What we've shown is that part of the targeted SNARE protein literally wraps itself around the botulinum A enzyme, using a large portion of the enzyme's surface for specific interactions," Brunger noted. "That's the novel finding in our study."
It turned out that the SNARE p
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Contact: Mark Shwartz
mshwartz@stanford.edu
650-723-9296
Stanford University
12-Dec-2004