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Critter Chemistry

January 10, 2000

Black Widow Toxin Reveals Its Structure

Mairin Brennan

The structure of -latrotoxin—the neurotoxic protein in the venom of the black widow spider—has been determined by researchers at Imperial College , London [Nat. Struct. Biol., 7, 48 (2000)]. Using cryogenic electron microscopy (cryo-EM), Yuri A. Ushkaryov, Marin van Heel, Elena V. Orlova, and coworkers showed that the soluble protein that forms pores in presynaptic membranes and wreaks havoc on the nervous system is a tetramer with a propeller-like structure.

Cryo-EM is a useful approach for studying proteins such as -latrotoxin that are difficult to crystallize. In this technique, a suspension of molecules is frozen rapidly at very low temperatures. Projections from thousands of randomly oriented individual molecules taken by an electron microscope can be exploited to generate a three-dimensional image of the molecule. Cryo-EM works well for larger molecules, which allow better alignment of the individual projections. And it works better for molecules that have symmetry, an attribute that enabled the team to determine the structure of the 520-kilodalton tetramer at a resolution of 14 , Ushkaryov says.

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This is "another example of the tremendous contributions that cryo-EM can make to resolving the structure of macromolecules and their interactions," says Joachim Frank, Howard Hughes Medical Institute investigator and laboratory chief at the New York State Department of Health in Albany. In the postgenomic era, visualizing macromolecular interactions in the cell will require an approach that goes a step beyond bioinformatics in making sense of genomic information, he says. Cryo-EM of single particles is particularly suited for this task, he suggests.

The goal of Ushkaryov's research is to understand exocytosis, the process by which a cell discharges particles too large to diffuse through the cell membrane. -Latrotoxin, which binds to nerve-cell terminals, stimulates a massive discharge of neurotransmitters into the synaptic cleft. Thus, the molecule has been used to study synaptic function, although its mode of action is complex: In addition to forming stable pores in the membrane, the toxin binds to two different receptors and may activate two different exocytosis pathways.

Cryo-EM revealed that -latrotoxin exists as a hydrophilic dimer in the absence of divalent cations. In the presence of either Ca2+ or Mg2+, however, it forms a tetramer in which the subunits rearrange to produce a hydrophobic region that enables the toxin to "flop" onto the lipid bilayer membrane. Once it has adhered, the tetramer makes channels in the membrane so that extracellular Ca2+ can enter the cell and stimulate exocytosis, Ushkaryov explains.

"But this is a very simple scenario," he says, "and I don't think people would be so interested in the toxin if this was the only thing it could do. -Latrotoxin also stimulates exocytosis in the absence of extracellular Ca2+, although Mg2+ must be present."

In fact, how Ca2+ stimulates exocytosis is an enigma that many people are trying to explain, Ushkaryov says. Although regulatory proteins that bind Ca2+ have been identified on synaptic vesicles, no one knows the mechanism by which vesicles fuse with the plasma membrane to disgorge their contents. Indeed, Ushkaryov questions whether Ca2+ influx through toxin-created membrane pores is enough to trigger the release of neurotransmitters from vesicles. He suggests that the surge might be due, in part, to neurotransmitters in the cytoplasm leaking out through the pores.

In a commentary accompanying the article, electron microscopist Helen R. Saibil at Birkbeck College, London, suggests that -latrotoxin's efficacy derives from synergism between the different pathways. The toxin could both stimulate exocytosis and activate signal pathways releasing intracellular Ca2+, she says, setting the stage for influx of extracellular Ca2+ that would propagate the exocytosis cycle.

Time will tell. "I have spent 18 years studying -latrotoxin and its receptors," Ushkaryov says. "Right now, we are trying to figure out how the toxin stimulates release of neurotransmitters in the absence of extracellular Ca2+."

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