Preventing voltage-dependent gating of anthrax toxin channels using engineered disulfides.
The channel-forming component of anthrax toxin, (PA(63))(7), is a heptameric water-soluble protein at neutral pH, but under acidic conditions it spontaneously inserts into lipid bilayers to form a 14-stranded beta-barrel ion-conducting channel. This channel plays a vital role in anthrax pathogenesis because it serves as a conduit for the membrane translocation of the two enzymatic components of anthrax toxin, lethal factor and edema factor. Anthrax channels open and close in response to changes in transmembrane voltage, a property shared by several other pore-forming toxins. We have discovered an unexpected phenomenon in cysteine-substituted channels that provides a window into this gating process: their normal voltage-dependent gating can be abolished by reaction with methanethiosulfonate (MTS) reagents or exposure to oxidizing conditions. Remarkably, this perturbation is seen with cysteines substituted at sites all along the approximately 100 A length of the channel's beta-barrel. In contrast, reaction with N-ethylmaleimide, a thiol-reactive compound that does not form a mixed disulfide, does not affect gating at any of the sites tested. These findings, coupled with our biochemical detection of dimers, have led us to conclude that MTS reagents are catalyzing the formation of intersubunit disulfide bonds that lock channels in a conducting state, and that voltage gating requires a conformational change that involves the entire beta-barrel.
Anderson
Damon S
DS
Molecular Cardiology Research Institute, Tufts Medical Center, Boston, MA 02111, USA.
Blaustein
Robert O
RO
D000942:Antigens, Bacterial / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004574:Electromagnetic Fields; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D018000:Receptors, Peptide / Q000737:chemistry*
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D004220:Disulfides; D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D018000:Receptors, Peptide; C030325:anthrax toxin; C070309:anthrax toxin receptors