Stanford, Calif. — Scientists here have begun unraveling the mechanism by which botulinum toxin attacks nerves, thereby causing paralysis.
Stanford, Calif. - Scientists here have begun unraveling the mechanism by which botulinum toxin attacks nerves, thereby causing paralysis.
Their work could help explain how botulinum toxin A functions in clinical and cosmetic applications. At the same time, the authors' findings one day could lead to treatments for botulism and similar illnesses.
"The most important finding of the paper is the structural basis for how the protease component of the botulinum toxin interacts with its target (Breidenbach MA, Brunger AT. Nature. 2004 Dec 16;432(7019):925-929. Epub. 2004 Dec 12.)," according to Axel T. Brunger, Ph.D., a Howard Hughes Medical Institute investigator and professor in Stanford University's departments of molecular and cellular physiology, neurology and neurological sciences, and the Stanford Synchrotron Radiation Laboratory (SSRL).
To elucidate this process, Dr. Brunger and graduate student Mark A. Breidenbach employed X-ray crystallography at the SSRL and at Advanced Light Source (ALS) at the Lawrence Berkeley National Laboratory. Their first challenge was to crystallize a SNARE/botulinum A complex in a fashion suitable for laboratory analysis.
Breidenbach's early efforts in this area tended to slice the SNARE target in two. As such, it could yield no meaningful insights.
"To crystallize this complex, we had to stabilize it. By introducing two mutations, we made it a dead enzyme, unable to function. We had to use two mutations rather than one in order to accomplish that," Dr. Brunger tells Dermatology Times.
In particular, to the single-site mutation E224Q (which substantially impaired substrate turnover; Li L et al. Biochemistry. 2000 Mar 7;39(9):2399-2405.), Mr. Breidenbach added a second mutation known to impair catalysis, Y366F. This mutation eliminated substrate turnover at the conditions necessary for crystallization (Binz T et al. Biochemistry. 2002 Feb 12;41(6):1717-1723.).
Dr. Brunger explains, "Both mutation sites were close to the so-called active site, which is the site of the protease that does the cutting. By changing those residues, it disrupted the ability of the enzyme to cut the protease."
Ultimately, Breidenbach and Dr. Brunger's research revealed that the SNARE protein literally attaches itself around the botulinum toxin A enzyme at more than two dozen sites. Thus bound, the protein is able to utilize a large portion of the enzyme's surface for specific interactions.
Extensive area of interaction
By having a large interaction area, botulinum toxin A achieves a high degree of specificity with just a single unit. In contrast, many enzymatic reactions achieve such specificity through large complexes of auxiliary proteins that work together.
"We found a truly extensive area of interaction between the protease and its target, which is quite unusual for proteases. That's quite interesting and also provides potentially the basis for novel types of vaccines or anti-toxins to treat botulism," Dr. Brunger says.
Dr. Brunger hopes to study the structures of other botulinum enzymes, as well as a closely related neurotoxin that causes tetanus. His work could provide a springboard for treatments that compete with specific binding sites on a neurotoxin's surface. Such compounds would produce an immediate effect.
"Knowledge of the structure should help in coming up with novel kinds of inhibitors that are very specific and produce few side effects," he says. "Based on our structure, one could perhaps design inhibitors targeted at sites that are very specific for a particular protease and that presumably don't interact with other proteases in the body. Whether that's possible remains to be seen. But our research provides a good starting point."