PA63 channel of anthrax toxin: an extended beta-barrel.
Anthrax toxin consists of three protein components: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA(63), generated by protease "nicking" of whole PA, is responsible for delivering the toxin's catalytic fragments (LF and EF) to the target cell's cytosol. In planar bilayer membranes, trypsin-nicked PA makes cation-selective voltage-gated channels with a pore diameter of > or =12 A. The channels are presumed to be heptameric "mushrooms", with an extracellular "cap" region and a membrane-inserted, beta-barrel "stem". Although the crystal structure of the water-soluble monomeric form has been resolved to 2.1 A and that of the heptameric "prepore"" to 4.5 A, the structure for the membrane-bound channel (pore) has not been determined. We have engineered mutant channels that are cysteine-substituted in residues in the putative beta-barrel, and identified the residues lining the channel lumen by their accessibility to a water-soluble sulfhydryl-specific reagent. The reaction with lumen-exposed cysteinyl side chains causes a drop in channel conductance, which we used to map the residues that line the pore. Our results indicate that the beta-barrel structure extends beyond the bilayer and involves residues that are buried in the monomer. The implication is that major rearrangement of domains in the prepore cap region is required for membrane insertion of the beta-barrel stem.
Nassi
Shilla
S
Department of Neuroscience and Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Collier
R John
RJ
Finkelstein
Alan
A
D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics; D001408:Bacillus anthracis / Q000737:chemistry* / Q000235:genetics; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D008051:Lipid Bilayers / Q000737:chemistry; D008698:Mesylates / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary / Q000235:genetics; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D007473:Ion Channels; D008051:Lipid Bilayers; D008698:Mesylates; D000644:Quaternary Ammonium Compounds; C030325:anthrax toxin; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; C009405:tetrabutylammonium; D003545:Cysteine