Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state.
Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state.
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DOI：
10.1021/bi400249f
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发表时间：
2013-05-21
期刊：
影响因子：
2.9
通讯作者：
Ladokhin, Alexey S.
中科院分区：
文献类型：
--
作者：
Vargas-Uribe, Mauricio;Rodnin, Mykola V.;Kienker, Paul;Finkelstein, Alan;Ladokhin, Alexey S.
易位（T）结构域在白喉毒素进入细胞中起关键作用。内体酸化后，T结构域经历一系列构象变化，导致其插入膜并形成通道。最近，我们报道了C-末端组氨酸H322，H323和H372与谷氨酰胺的三重取代阻止了平面脂质双层中开放通道的形成。在这里，我们报告说，这种影响主要是由于H322的突变。我们进一步研究的功能和膜折叠的损失之间的关系，在一系列的突变体与C-末端组氨酸取代使用光谱测定。突变体的膜插入途径不同于野生型，如在pH 6.0-6.5下膜诱导的色氨酸荧光红移所揭示的。在H323和H372处替换但在H322处未替换的T结构域突变体在进一步酸化后重新获得野生型样光谱特征。圆二色性测量证实，受影响的突变体错误折叠插入囊泡。电导测量表明，取代H322显着减少了正确折叠的通道在平面双层的数量，但活性通道的属性似乎是不变的。我们认为H322在开放通道的形成中起着重要的作用，并参与引导T结构域的N-末端区域正确插入膜中。
The translocation (T) domain plays a key role in the entry of diphtheria toxin into the cell. Upon endosomal acidification, the T-domain undergoes a series of conformational changes that lead to its membrane insertion and formation of a channel. Recently, we have reported that the triple replacement of the C-terminal histidines H322, H323 and H372 with glutamines prevents the formation of open channels in planar lipid bilayers. Here, we report that this effect is primarily due to the mutation of H322. We further examine the relationship between the loss of functionality and membrane folding in a series of mutants with C-terminal histidine substitutions using spectroscopic assays. The membrane insertion pathway for the mutants differs from that of the wild type as revealed by membrane-induced red-shift of tryptophan fluorescence at pH 6.0–6.5. T-domain mutants with replacements at H323 and H372, but not at H322, regain wild type-like spectroscopic signature upon further acidification. Circular dichroism measurements confirm that affected mutants misfold during insertion into vesicles. Conductance measurements reveal that substituting H322 dramatically reduces the numbers of properly folded channels in a planar bilayer, but the properties of the active channels appear to be unaltered. We propose that H322 plays an important role in the formation of open channels and is involved in guiding the proper insertion of the N-terminal region of the T-domain into the membrane.
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