Kienker Paul K, Wu Zhengyan, Finkelstein Alan
Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA.
Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, Anhui, China.
J Membr Biol. 2016 Apr;249(1-2):181-96. doi: 10.1007/s00232-015-9859-9. Epub 2015 Dec 8.
The translocation domain (T-domain) of diphtheria toxin contains 10 α helices in the aqueous crystal structure. Upon exposure to a planar lipid bilayer under acidic conditions, it inserts to form a channel and transport the attached amino-terminal catalytic domain across the membrane. The TH5, TH8, and TH9 helices form transmembrane segments in the open-channel state, with TH1-TH4 translocated across the membrane. The TH6-TH7 segment also inserts to form a constriction that occupies only a small portion of the total channel length. Here, we have examined the TH5 segment in more detail, using the substituted-cysteine accessibility method. We constructed a series of 23 mutant T-domains with single cysteine residues at positions in and near TH5, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific reagents added to the solutions on either side of the membrane. For 15 of the mutants, the reagent caused a decrease in single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. We also found that reaction caused large changes in ionic selectivity for some mutant channels. We determined whether reaction occurred in the open state or in the brief flicker-closed state of the channel. Finally, we compared the reaction rates from either side of the membrane. Our experiments are consistent with the hypotheses that the TH5 helix has a transmembrane orientation and remains helical in the open-channel state; they also indicate that the middle of the helix is aligned with the constriction in the channel.
白喉毒素的转位结构域（T结构域）在水性晶体结构中含有10个α螺旋。在酸性条件下暴露于平面脂质双层时，它会插入形成通道，并将附着的氨基末端催化结构域转运穿过膜。TH5、TH8和TH9螺旋在开放通道状态下形成跨膜片段，TH1-TH4跨膜转运。TH6-TH7片段也会插入形成一个缩窄部分，该部分仅占据总通道长度的一小部分。在这里，我们使用半胱氨酸替代可及性方法更详细地研究了TH5片段。我们构建了一系列23个突变T结构域，在TH5及其附近位置带有单个半胱氨酸残基，监测它们在平面脂质双层中的通道形成，并探究添加到膜两侧溶液中的硫醇特异性试剂的影响。对于15个突变体，该试剂导致单通道电导降低，表明引入的半胱氨酸残基暴露在通道腔内。我们还发现该反应对一些突变通道的离子选择性产生了很大影响。我们确定反应是在通道的开放状态还是短暂闪烁关闭状态下发生的。最后，我们比较了膜两侧的反应速率。我们的实验与以下假设一致：TH5螺旋具有跨膜取向，并且在开放通道状态下保持螺旋结构；它们还表明螺旋的中部与通道中的缩窄部分对齐。
