Analyzing topography of membrane-inserted diphtheria toxin T domain using BODIPY-Streptavidin: At low pH, helices 8 and 9 form a transmembrane hairpin but helices 5-7 form stable nonclassical inserted segments on the cis side of the bilayer
Analyzing topography of membrane-inserted diphtheria toxin T domain using BODIPY-Streptavidin: At low pH, helices 8 and 9 form a transmembrane hairpin but helices 5-7 form stable nonclassical inserted segments on the cis side of the bilayer
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DOI：
10.1021/bi049354j
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发表时间：
2004-07-20
期刊：
影响因子：
2.9
通讯作者：
London, E
中科院分区：
文献类型：
--
作者：
Rosconi, MP;Zhao, G;London, E
白喉毒素T结构域在低pH条件下介导的膜插入是A链向细胞质转位的关键。为了定义T结构域的膜形貌，使用含有深度插入的T结构域的模型膜囊泡检查生物素化的Cys残基暴露于顺式和反式双层表面。为此，测量生物素与外部和囊泡包埋的BODIPY标记的链霉亲和素的反应性。发现T结构域以生理相关方向插入大约70-80%的分子。在该取向中，位于疏水螺旋8和9之间的环中的残基349暴露于双层的反式侧，而沿着T结构域的疏水螺旋5-9区域的其他溶液暴露的残基沿着位于顺式表面附近。一个协议开发，以检测残留物的移动来回跨膜表明，T结构域序列之间的顺式和反式两侧的双层没有迅速平衡。在膜插入之前将链霉亲和素结合到生物素化的残基仅抑制螺旋8和9之间的环中的残基的T结构域孔形成。孔形成实验使用避免在蛋白质初始插入时可能发生的瞬时膜缺陷/渗漏的干扰的方法。结合起来，这些结果表明，在低pH值下，疏水螺旋8和9形成跨膜发夹，而疏水螺旋5-7形成非经典的深度插入的非跨膜状态。我们建议，这代表了一种新的前易位状态，是不同于先前定义的后易位状态。
Low pH-induced membrane insertion by diphtheria toxin T domain is crucial for A chain translocation into the cytoplasm. To define the membrane topography of the T domain, the exposure of biotinylated Cys residues to the cis and trans bilayer surfaces was examined using model membrane vesicles containing a deeply inserted T domain. To do this, the reactivity of biotin with external and vesicle-entrapped BODIPY-labeled streptavidin was measured. The T domain was found to insert with roughly 70-80% of the molecules in the physiologically relevant orientation. In this orientation, residue 349, located in the loop between hydrophobic helices 8 and 9, was exposed to the trans side of the bilayer, while other solution-exposed residues along the hydrophobic helices 5-9 region of the T domain located near the cis surface. A protocol developed to detect the movement of residues back and forth across the membranes demonstrated that T domain sequences did not rapidly equilibrate between the cis and the trans sides of the bilayer. Binding streptavidin to biotinylated residues prior to membrane insertion only inhibited T domain pore formation for residues in the loop between helices 8 and 9. Pore formation experiments used an approach avoiding interference from transient membrane defects/leakage that may occur upon the initial insertion of protein. Combined, these results indicate that at low pH hydrophobic helices 8 and 9 form a transmembrane hairpin, while hydrophobic helices 5-7 form a nonclassical deeply inserted nontransmembraneous state. We propose that this represents a novel pre-translocation state that is distinct from a previously defined post-translocation state.