Structure-function relationships in diphtheria toxin channels: I. Determining a minimal channel-forming domain.
Structure-function relationships in diphtheria toxin channels: I. Determining a minimal channel-forming domain.
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白喉毒素通道中的结构-功能关系：I.确定最小通道形成域。
DOI：
10.1007/bf00234995
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Collier,RJ
中科院分区：
文献类型：
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作者：
Silverman,JA;Mindell,JA;Zhan,H;Finkelstein,A;Collier,RJ
白喉毒素(DT)是一种氨基酸外毒素，其活性形式是由两条多肽链通过链间二硫键连接而成。DT的N端A片段通过酶促灭活细胞的蛋白质合成机制来杀死细胞；它的C端B链是毒素与敏感细胞结合和A片段移位到胞浆中所必需的。该B片段由N端的T区(191-386位氨基酸)和C端的R区(387位氨基酸)组成，负责DT在脂质双层和细胞膜上形成的离子传导通道。为了进一步描述DT的通道形成区域，我们研究了DT的缺失突变体在几种pH条件下在脂质双层膜上形成的通道。由只含有T结构域(即，缺少A片段和/或R结构域)的突变体形成的通道，以及由用白介素2(IL-2)取代R结构域的突变体形成的通道，具有与野生型DT形成的通道基本相同的单通道电导和选择性。此外，删除T结构域的N-末端118个氨基酸对突变通道的单通道电导和选择性的影响也很小。总之，这些数据确定T结构域的61个氨基酸延伸，对应于DT晶体结构中包括α-螺旋TH8和TH9的区域，是毒素的通道形成区域。
Diphtheria Toxin (DT) is a 535 amino acid exotoxin, whose active form consists of two polypeptide chains linked by an interchain disulphide bond. DT's N-terminal A fragment kills cells by enzymatically inactivating their protein synthetic machinery; its C terminal B chain is required for the binding of toxin to sensitive cells and for the translocation of the A fragment into the cytosol. This B fragment, consisting of its N-terminal T domain (amino acids 191–386) and its C-terminal R domain (amino acids 387–535) is responsible for the ion-conducting channels formed by DT in lipid bilayers and cellular plasma membranes. To further delineate the channel-forming region of DT, we studied channels formed by deletion mutants of DT in lipid bilayer membranes under several pH conditions. Channels formed by mutants containing only the T domain (i.e., lacking the A fragment and/or the R domain), as well as those formed by mutants replacing the R domain with Interleukin-2 (Il–2), have single channel conductances and selectivities essentially identical to those of channels formed by wild-type DT. Furthermore, deleting the N-terminal 118 amino acids of the T domain also has minimal effect on the single channel conductance and selectivity of the mutant channels. Together, these data identify a 61 amino acid stretch of the T domain, corresponding to the region which includes α-helices TH8 and TH9 in the crystal structure of DT, as the channel-forming region of the toxin.