Conformation of the diphtheria toxin T domain in membranes:: A site-directed spin-labeling study of the TH8 helix and TL5 loop
Conformation of the diphtheria toxin T domain in membranes:: A site-directed spin-labeling study of the TH8 helix and TL5 loop
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DOI：
10.1021/bi990520a
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发表时间：
1999-08-10
期刊：
影响因子：
2.9
通讯作者：
Collier, RJ
中科院分区：
文献类型：
--
作者：
Oh, KJ;Zhan, HJ;Collier, RJ
分离的白喉毒素T结构域通过半胱氨酸扫描突变，在天然毒素的TH8螺旋和TL5螺旋环的28个连续位置(残基328-355)发生突变。在用巯基选择性氮氧化物试剂衍生突变蛋白后，我们检查了每个氮氧化物的流动性以及它在插入磷脂双层之前和之后对极性和非极性顺磁性试剂的可及性。用pH为8的溶液中的蛋白质获得的数据与根据毒素晶体结构的预测大体一致。在pH为4.6的膜上结合后，结构域发生了重大的结构重组，大大降低了该区域大多数残基与极性试剂镍(II)-乙二胺二乙酸酯络合物(NiEDDA)的可及性。其中许多残留物对非极性试剂O-2的可及性也有所降低。沿着这些试剂序列的氮氧化物侧链的周期性可及性表明，TH8在膜结合状态下大部分仍然是螺旋的，一个表面与蛋白质结合，另一个面向双层的疏水内部。此外，TL5环在膜上似乎也变成了阿尔法螺旋，一个表面与蛋白质接触，另一个与双层内部接触。这些发现为理解T结构域如何形成跨膜通道并介导白喉毒素的酶部分跨膜转运提供了一个结构框架。
The isolated T domain of diphtheria toxin was mutated by cysteine-scanning mutagenesis at 28 consecutive sites (residues 328-355) that comprise the TH8 helix and the TL5 interhelical loop in the native toxin. After derivatizing the mutant proteins with a sulfhydryl-selective nitroxide reagent, we examined the mobility of each nitroxide and its accessibility to polar and nonpolar paramagnetic reagents, before and after insertion into phospholipid bilayers. The data obtained with the proteins in solution at pH 8 are generally consistent with predictions from the crystal structure of the toxin. Upon membrane binding at pH 4.6, a major structural reorganization of the domain was seen, which dramatically reduced the accessibility of most residues in this region to the polar reagent nickel(II)-ethylenediaminediacetate complex (NiEDDA). Many of these residues also showed reduced accessibility to the nonpolar reagent O-2. Periodic accessibility of the nitroxide side chains along the sequence to these reagents shows that TH8 remains largely helical in the membrane-bound state, with one surface associated with protein and the other facing the hydrophobic interior of the bilayer. In addition, the TL5 loop also appears to become alpha-helical in the membrane, with one surface in contact with protein and the other in contact with the bilayer interior. These findings provide a structural framework for understanding how the T domain forms a transmembrane channel and mediates translocation of diphtheria toxin's enzymic moiety across a membrane.