Interactions of High-Affinity Cationic Blockers with the Translocation Pores of B. Anthracis, C. Botulinum, and C. Perfringens Binary Toxins

Abstract

Cationic β-cyclodextrin derivatives were recently introduced as highly effective, potentially universal blockers of binary bacterial toxins: anthrax toxin of Bacillus anthracis, C2 toxin of Clostridium botulinum, and iota toxin of Clostridium perfringens. The binary toxins are made of two separate components: the enzymatic A component and the binding/translocation B component, which forms oligomeric channels in the target cell membrane. Here we studied the voltage and salt dependence of the rate constants of binding and dissociation reactions of two structurally different β-cyclodextrins (AmPrβCD and AMBnTβCD) in the PA63, C2IIa, and Ib channels. With all three channels, the blocker carrying extra hydrophobic aromatic groups, AMBnTβCD, demonstrated stronger binding compared with AmPrβCD. This effect is seen as an increased residence time of the blocker in the channels, whereas the time between blockages stays practically unchanged. Surprisingly, the voltage sensitivity, expressed as a slope of the logarithm of the blocker residence time as a function of voltage, was practically the same for all six cases studied, suggesting structural similarities among the three channels. Also, the more-effective AMBnTβCD blocker shows weaker salt dependence of the binding and dissociation rate constants compared with AmPrβCD. By estimating the relative contributions of the applied transmembrane field, long-range Coulomb, and salt-concentration-independent, short-range forces, we found that the latter represent the leading interaction, which accounts for the high efficiency of blockage. In a search for the putative groups in the channel lumen that are responsible for the short-range forces, we performed measurements with the F427A mutant of PA63. We found that the on-rates of the blockage were virtually conserved, but the residence times dropped by more than an order of magnitude, reducing the difference between the efficiencies of the two blockers.