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ANTHRAX TOXIN DECIPHERED
Structure, mechanism of third toxin component determined
STU BORMAN, C&EN WASHINGTON
Three proteins from the bacterium Bacillus anthracis, known collectively as "anthrax toxin," are responsible for the microorganism's toxic and potentially lethal effects. Two--protective antigen and lethal factor--had been structurally analyzed previously. Now, after a three-year effort, scientists have determined the crystal structure and mechanism of action of the catalytic portion of the third component, edema factor.
The study was carried out by associate professor Wei-Jen Tang of the Ben May Institute for Cancer Research at the University of Chicago; crystallographer Andrew Bohm of Boston Biomedical Research Institute, Watertown, Mass., and Tufts University School of Medicine, Boston; and coworkers [Nature, 415, 396 (2002)]. A key element in being able to determine the structure of the protein, Bohm tells C&EN, was the realization that its catalytic portion could be crystallized, even though the entire protein was extremely resistant to crystallization.
Anthrax researcher Robert C. Liddington of the Burnham Institute, La Jolla, Calif., explains in a Nature commentary that inhalation anthrax, the most serious type of anthrax infection, begins when inhaled B. anthracis spores are ingested by immune-system macrophages in the lungs. Macrophages normally destroy such invaders, but in this case the spores kill the macrophages instead. The bacterium then finds its way into the bloodstream, where it secretes the three-component anthrax toxin.
Protective antigen forms a membrane-inserting heptamer that makes it possible for the other two proteins to enter target cells. Lethal factor then disrupts a key cell-signaling pathway and kills immune-system cells. Liddington and coworkers determined the crystal structure of protective antigen about five years ago [Nature, 385, 833 (1997)] and that of lethal factor last year [Nature, 414, 229 (2001); C&EN, Oct. 29, 2001, page 9].
Edema factor's mechanism of action involves recruitment of the endogenous regulatory protein calmodulin. Edema factor not only binds calmodulin, sequestering it and preventing it from carrying out its signal transduction functions and other normal roles, but binding of calmodulin also converts it into an active conspirator in edema factor's toxic mission.
Tang, Bohm, and coworkers have now discovered exactly how this occurs by analyzing crystal structures they obtained of edema factor's catalytic region with and without calmodulin. Their study shows that binding calmodulin induces a major conformational rearrangement of edema factor's catalytic domain. The rearrangment makes the enzyme highly efficient at catalyzing the conversion of adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cyclic AMP). The resulting overproduction of cyclic AMP causes cell death and fluid accumulation (edema) that contribute to anthrax infection.
The structure of edema factor is sufficiently different from the stuctures of human and animal enzymes of the same class--the adenylyl cyclases--that edema factor might be easy to block selectively with a small-molecule drug, the researchers note.
"Three years ago, when we started this project, B. anthracis was an obscure agricultural pathogen with interesting biological properties," Tang says. "Now anthrax is front and center in every clinician's mind, and within months of the first bioterrorism case we have the structures for all three toxins. We hope this work will quickly lead to new therapies."
ANTHRAX IN ACTION Crystal structures of edema factor's catalytic portion in its inactive (left) and calmodulin-bound states. Edema factor's enzymatic core (green) binds a single metal ion (purple) at the active site in both structures. Calmodulin (red) activates edema factor by inserting itself between the enzyme's turquoise and yellow segments, causing a complete rearrangement of the purple loop. In its active-state conformation, the purple loop stabilizes the orange loop, which in turn positions the substrate for catalysis.
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Copyright © 2002 American Chemical Society