Preventing voltage-dependent gating of anthrax toxin channels using engineered disulfides

Area of Science:

• Biophysics
• Structural Biology
• Toxinology

Background:

• The protective antigen (PA63) component of anthrax toxin forms a heptameric pore in lipid bilayers.
• This pore facilitates the entry of lethal factor and edema factor, crucial for anthrax pathogenesis.
• Anthrax toxin channels exhibit voltage-dependent gating, similar to other pore-forming toxins.

Purpose of the Study:

• To investigate the mechanism of voltage-dependent gating in anthrax toxin channels.
• To explore the role of specific channel regions and chemical modifications in gating.

Main Methods:

• Cysteine substitution within the PA63 channel beta-barrel.
• Chemical modification of substituted cysteines using MTS reagents and N-ethylmaleimide.
• Electrophysiological measurements to assess channel gating.
• Biochemical analysis to detect protein interactions.

Main Results:

• Reaction with MTS reagents or oxidizing agents abolished voltage-dependent gating in cysteine-substituted channels.
• This effect was observed irrespective of the cysteine substitution site along the beta-barrel.
• N-ethylmaleimide treatment did not alter channel gating.
• Biochemical assays detected dimer formation upon MTS treatment.

Conclusions:

• MTS reagents induce intersubunit disulfide bond formation, locking the channel in a conducting state.
• Voltage gating of anthrax toxin channels involves a conformational change across the entire beta-barrel.
• Disulfide bond formation inhibits this conformational change, thus abolishing gating.