Probing the structure of the diphtheria toxin channel. Reactivity in planar lipid bilayer membranes of cysteine-substituted mutant channels with methanethiosulfonate derivatives.
Probing the structure of the diphtheria toxin channel. Reactivity in planar lipid bilayer membranes of cysteine-substituted mutant channels with methanethiosulfonate derivatives.
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DOI：
10.1085/jgp.110.3.229
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发表时间：
1997-09
影响因子：
3.8
通讯作者：
Finkelstein, A
中科院分区：
文献类型：
--
作者：
Huynh, P D;Cui, C;Zhan, H;Oh, K J;Collier, R J;Finkelstein, A
先前的工作已经确定，白喉毒素T结构域中从残基322至382的61个氨基酸延伸形成的通道在离子传导性质上与由整个T结构域形成的通道没有区别。在毒素的水溶性形式的晶体结构中，这一段的大部分是α-螺旋发夹，命名为TH 8 -9。本研究旨在确定TH 8 -9中的哪些残基排列通道的离子传导途径;即，其内腔或入口。为此，我们将TH 8 -9的51个残基（328-376）中的49个突变为半胱氨酸，与平面脂质双层中的突变T结构域蛋白形成通道，然后确定它们是否与添加到浴溶液中的小的、带电荷的、脂质不溶性的、巯基特异性甲硫基磺酸盐（MTS）衍生物反应。反应的指示以及残留物衬在离子传导途径上的指示是单通道电导和/或闪烁行为的突然变化。这项研究的结果在两个方面令人惊讶。首先，在测试的TH 8 -9中的49个半胱氨酸取代的残基中，23个以最不寻常的模式与MTS衍生物反应，该模式由两个区段组成：一个从329延伸至341（13个中的11个反应），另一个从347延伸至359（13个中的12个反应）;这两个区段之外的残基似乎都没有反应。第二，在每个半胱氨酸突变体通道表现出MTS效应，只有一个过渡的单通道电导（或闪烁行为）发生，而不是几个预期的多聚体通道。我们的结果与通道的α-螺旋或β-链模型不一致，而是提出了一种开放的柔性结构。此外，与常识相反，它们表明通道不是多聚体的，而是仅由T结构域的一个TH 8 -9单元形成。
Previous work has established that the 61 amino acid stretch from residue 322 to 382 in the T-domain of diphtheria toxin forms channels indistinguishable in ion-conducting properties from those formed by the entire T-domain. In the crystal structure of the toxin's water-soluble form, the bulk of this stretch is an α-helical hairpin, designated TH8-9. The present study was directed at determining which residues in TH8-9 line the ion-conducting pathway of the channel; i.e., its lumen or entrances. To this end, we singly mutated 49 of TH8-9's 51 residues (328–376) to cysteines, formed channels with the mutant T-domain proteins in planar lipid bilayers, and then determined whether they reacted with small, charged, lipid-insoluble, sulfhydryl-specific methanethiosulfonate (MTS) derivatives added to the bathing solutions. The indication of a reaction, and that the residue lined the ion-conducting pathway, was a sudden change in single-channel conductance and/or flickering behavior. The results of this study were surprising in two respects. First, of the 49 cysteine-substituted residues in TH8-9 tested, 23 reacted with MTS derivatives in a most unusual pattern consisting of two segments: one extending from 329 to 341 (11 of 13 reacted), and the other from 347 to 359 (12 of 13 reacted); none of the residues outside of these two segments appeared to react. Second, in every cysteine mutant channel manifesting an MTS effect, only one transition in single-channel conductance (or flickering behavior) occurred, not the several expected for a multimeric channel. Our results are not consistent with an α-helical or β-strand model for the channel, but instead suggest an open, flexible structure. Moreover, contrary to common sense, they indicate that the channel is not multimeric but is formed from only one TH8-9 unit of the T-domain.