Oh K J, Zhan H, Cui C, Altenbach C, Hubbell W L, Collier R J
Jules Stein Eye Institute, Department of Chemistry and Biochemistry, University of California Los Angeles 90095-7008, USA.
Biochemistry. 1999 Aug 10;38(32):10336-43. doi: 10.1021/bi990520a.
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.
通过半胱氨酸扫描诱变，对白喉毒素的分离T结构域在28个连续位点（残基328 - 355）进行了突变，这些位点构成了天然毒素中的TH8螺旋和TL5螺旋间环。在用巯基选择性氮氧化物试剂对突变蛋白进行衍生化后，我们在将其插入磷脂双层之前和之后，检测了每个氮氧化物的迁移率及其对极性和非极性顺磁试剂的可及性。在pH 8的溶液中用这些蛋白获得的数据通常与毒素晶体结构的预测一致。在pH 4.6时膜结合后，观察到该结构域发生了主要的结构重组，这显著降低了该区域大多数残基对极性试剂镍（II）-乙二胺二乙酸络合物（NiEDDA）的可及性。这些残基中的许多对非极性试剂O₂的可及性也降低了。沿序列的氮氧化物侧链对这些试剂的周期性可及性表明，TH8在膜结合状态下基本保持螺旋结构，一个表面与蛋白质相关，另一个表面面向双层的疏水内部。此外，TL5环在膜中似乎也变成了α螺旋，一个表面与蛋白质接触，另一个表面与双层内部接触。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酶部分跨膜转运提供了一个结构框架。
