Finkelstein A, Oh K J, Senzel L, Gordon M, Blaustein R O, Collier R J
Department of Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Int J Med Microbiol. 2000 Oct;290(4-5):435-40. doi: 10.1016/S1438-4221(00)80059-4.
The T-domain of diphtheria toxin, which extends from residue 202 to 378, causes the translocation of the catalytic A fragment (residues 1-201) across endosomal membranes and also forms ion-conducting channels in planar phospholipid bilayers. The carboxy-terminal 57-amino acid segment (residues 322-378) in the T-domain is all that is required to form these channels, but its ability to do so is greatly augmented by the portion of the T-domain upstream from this. Here we show that in association with channel formation by the T-domain, its hydrophilic 63-amino acid NH2-terminal region (residues 202-264) as well as the entire catalytic A fragment (residues 1-201) cross the lipid bilayer. The phenomenon that enabled us to demonstrate this was the rapid closure of channels at cis negative voltages when a histidine tag was placed at various positions in the NH2-terminal region of the T-domain or in the A fragment; the inhibition of this effect by trans nickel established that the histidine tag was present on the trans side of the membrane. Thus, all of the machinery necessary to translocate the A fragment across membranes is built into the 114 residues at the carboxy-terminal end of the T-domain (residues 265-378), without the requirement of any proteins in the plasma membrane (e.g., toxin receptor) or of any other cellular components.
白喉毒素的T结构域从第202位氨基酸延伸至第378位氨基酸，它可使催化性A片段（第1 - 201位氨基酸）穿过内体膜，并且还能在平面磷脂双分子层中形成离子传导通道。T结构域中羧基末端的57个氨基酸片段（第322 - 378位氨基酸）是形成这些通道所必需的全部，但T结构域中该片段上游部分能极大增强其形成通道的能力。在此我们表明，与T结构域形成通道相关的是，其亲水性的63个氨基酸的氨基末端区域（第202 - 264位氨基酸）以及整个催化性A片段（第1 - 201位氨基酸）穿过脂质双层。使我们能够证明这一点的现象是，当在T结构域的氨基末端区域或A片段的不同位置放置组氨酸标签时，通道在顺式负电压下会迅速关闭；反式镍对这种效应的抑制表明组氨酸标签位于膜的反式一侧。因此，将A片段转运穿过膜所需的所有机制都构建在T结构域羧基末端的114个氨基酸（第265 - 378位氨基酸）中，而无需质膜中的任何蛋白质（如毒素受体）或任何其他细胞成分。
