Identification of shallow and deep membrane-penetrating forms of diphtheria toxin T domain that are regulated by protein concentration and bilayer width
Identification of shallow and deep membrane-penetrating forms of diphtheria toxin T domain that are regulated by protein concentration and bilayer width
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DOI：
10.1074/jbc.272.40.25091
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发表时间：
1997-10-03
影响因子：
4.8
通讯作者：
London, E
中科院分区：
文献类型：
--
作者：
Wang, Y;Malenbaum, SE;London, E
白喉毒素富含α-螺旋的疏水性跨膜（T）结构域被认为在毒素的膜插入和其催化结构域跨膜易位中起核心作用。选择的剩余物，322（靠近螺旋TH 8的氨基末端），333（在螺旋TH 8内），和356（在螺旋TH 9内）被Cys取代并用荧光探针bimane标记，（残基333和356应位于跨膜状态的双层内，并且残基322不应穿透双层，将T结构域插入模型膜囊泡后，通过其荧光发射和其与氮氧自由基标记的磷脂的猝灭来表征bimane标记相对于脂质双层的位置。发现当将T结构域添加到含有二油酰磷脂酰胆碱的囊泡中时，所有三个残基都位于靠近外表面的位置。然而，在高T结构域浓度下或在较薄的含有二肉豆蔻酰磷脂酰胆碱的囊泡中，大部分残基333和356深深地渗透到膜中。相反，残基322在这些条件下保持暴露于水溶液，这些结论通过新的抗体结合方法得到证实。使用淬灭4，4-二氟-5，7-二甲基-4-硼杂-3 α，4 α-二氮杂-3-引达省（BODIPY）基团的荧光的抗体来评估BODIPY标记的322、333和356的暴露，残基333和356对外部添加的抗体的最大暴露仅在双甲烷荧光显示这些残基不穿透双层的条件下观察到。相反，残基322在所有条件下保持暴露。我们认为，深度穿透T结构域的构象代表了一种跨膜或近跨膜状态，其跨膜/非跨膜平衡的调控是理解白喉毒素膜插入和转运的关键。我们的研究结果表明，毒素-毒素相互作用可能在调节这种行为中发挥重要作用。
The alpha-helix-rich, hydrophobic transmembrane (T) domain of diphtheria toxin is believed to play a central role in membrane insertion by the toxin and in the translocation of its catalytic domain across membranes, In this report, T domain structure was studied using site-directed single-Cys mutants. The residues chosen, 322 (near the amino-terminal end of helix TH8), 333 (within helix TH8), and 356 (within helix TH9) were substituted with Cys and labeled with the fluorescent probe bimane, (Residues 333 and 356 should be located within the bilayer in the transmembrane state, and residue 322 should not penetrate the bilayer,) After insertion of T domain into model membrane vesicles, the location of bimane label relative to the lipid bilayer was characterized by its fluorescence emission and by its quenching with nitroxide-labeled phospholipids. It was found that when the T domain is added to dioleoylphosphatidylcholine-containing vesicles, all three residues reside close to the outer surface, However, at high T domain concentration or in thinner dimyristoleoylphosphatidylcholine-containing vesicles, a large fraction of residues 333 and 356 penetrate deeply into the membrane, In contrast, residue 322 remains exposed to aqueous solution under these conditions, These conclusions were confirmed by a novel antibody binding method, Antibodies that quench the fluorescence of 4,4-difluoro-5,7-dimethyl-4-bora-3 a,4a-diaza-3-indacene (BODIPY) groups were used to evaluate the exposure of BODIPY-labeled 322, 333, and 356, Maximum exposure of residues 333 and 356 to externally added antibody was only observed under conditions in which bimane fluorescence showed that these residues do not penetrate the bilayer, In contrast, residue 322 remained exposed under all conditions, We propose that the deeply penetrating T domain conformation represents a transmembrane or near-transmembrane state, The regulation of the transmembrane/nontransmembrane equilibrium should be a key to understanding diphtheria toxin membrane insertion and translocation. Our results suggest that toxin-toxin interactions may play an important role in regulating this behavior.