Interactions of High-Affinity Cationic Blockers with the Translocation Pores of B. anthracis, C. botulinum, and C. perfringens Binary Toxins
Interactions of High-Affinity Cationic Blockers with the Translocation Pores of B. anthracis, C. botulinum, and C. perfringens Binary Toxins
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DOI：
10.1016/j.bpj.2012.07.050
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发表时间：
2012-09-19
影响因子：
3.4
通讯作者：
Nestorovich, Ekaterina M.
中科院分区：
文献类型：
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作者：
Bezrukov, Sergey M.;Liu, Xian;Nestorovich, Ekaterina M.
最近推出了阳离子β-环糊精衍生物，作为三种二元细菌毒素的高效、潜在通用阻断剂：炭疽杆菌的炭疽毒素、肉毒杆菌的C2毒素和产气荚膜梭菌的iota毒素。二元毒素由两个独立的组分组成：作用于某些细胞内靶标的酶促 A 组分，以及在靶细胞膜上形成寡聚通道的结合/易位 B 组分。在这里，我们研究了 PA(63)、C2IIa 和 Ib 通道（分别为炭疽、C2 和 iota 毒素的 B 成分）中两种结构不同的 β-环糊精（AmPr beta CD 和 AMBnT beta CD）的结合和解离反应速率常数的电压和盐依赖性。对于所有三个通道，在带正电荷的氨基的硫代烷基连接体上携带额外疏水芳香基团的阻断剂 AMBnT beta CD 与 AmPr beta CD 相比，表现出明显更强的结合。这种效应被视为阻断剂在通道中的停留时间增加，而表征结合反应速率的阻断之间的时间实际上保持不变。令人惊讶的是，电压灵敏度（以阻滞剂停留时间的对数斜率表示为电压的函数）在所有六个研究案例中实际上是相同的，表明三个通道之间的结构相似性。此外，与 AmPr β CD 相比，更有效的 AMBnT β CD 阻滞剂显示出结合和解离速率常数的盐依赖性更弱。通过估计所施加的跨膜场、长程库仑和与盐浓度无关的短程力的相对贡献，我们发现后者代表了主要的相互作用，这解释了高阻塞效率。在寻找通道腔中负责短程力的推定基团时，我们使用 PA(63) 的 F427A 突变体进行了测量，该突变体缺乏功能上重要的苯丙氨酸钳。我们发现阻断剂的结合率实际上是保守的，但停留时间和相应的结合常数下降了一个数量级以上，这也减少了两种阻断剂的效率之间的差异。
Cationic beta-cyclodextrin derivatives were recently introduced as highly effective, potentially universal blockers of three 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, which acts on certain intracellular targets, 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 beta-cyclodextrins (AmPr beta CD and AMBnT beta CD) in the PA(63), C2IIa, and Ib channels (B components of anthrax, C2, and iota toxins, respectively). With all three channels, the blocker carrying extra hydrophobic aromatic groups on the thio-alkyl linkers of positively charged amino groups, AMBnT beta CD, demonstrated significantly stronger binding compared with AmPr beta CD. This effect is seen as an increased residence time of the blocker in the channels, whereas the time between blockages characterizing the binding reaction on-rate stays practically unchanged. Surprisingly, the voltage sensitivity, expressed as a slope of the logarithm of the blocker residence time as a function of voltage, turned out to be practically the same for all six cases studied, suggesting structural similarities among the three channels. Also, the more-effective AMBnT beta CD blocker shows weaker salt dependence of the binding and dissociation rate constants compared with AmPr beta CD. 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 PA(63), which lacks the functionally important phenylalanine clamp. We found that the on-rates of the blockage were virtually conserved, but the residence times and, correspondingly, the binding constants dropped by more than an order of magnitude, which also reduced the difference between the efficiencies of the two blockers.