Role of acidic residues in helices TH8-TH9 in membrane interactions of the diphtheria toxin T domain.
Role of acidic residues in helices TH8-TH9 in membrane interactions of the diphtheria toxin T domain.
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DOI：
10.3390/toxins7041303
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发表时间：
2015-04-14
期刊：
影响因子：
4.2
通讯作者：
Ladokhin AS
中科院分区：
文献类型：
--
作者：
Ghatak C;Rodnin MV;Vargas-Uribe M;McCluskey AJ;Flores-Canales JC;Kurnikova M;Ladokhin AS
白喉毒素易位结构域（T结构域）的pH触发的膜插入导致催化结构域转移到胞质溶胶中，这与作为货物递送系统的潜在生物医学应用相关。残基的质子化被认为在该过程中起关键作用，并且残基E349、D352和E362由于其在膜插入单元TH 8-TH 9内的位置而特别令人感兴趣。我们已经使用了各种光谱，计算和功能测定来表征携带双突变E349 Q/D352 N或单突变E362 Q的T结构域的性质。囊泡渗漏测量表明，这两种突变体与膜的酸性条件下比野生型。热展开和荧光测量，补充分子动力学模拟，表明突变体E362 Q是更容易酸不稳定，因为破坏本地分子内接触。荧光实验表明，E362 Q中而不是E349 Q/D352 N中电荷的去除对于TH 8-TH 9的插入是重要的。这两种突变体在进一步酸化时都采用最终的功能状态。我们的结论是，这些酸性残基参与的T结构域的pH依赖性的行动，和它们的替代品可用于微调膜相互作用的pH范围。
The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8–TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8–TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.
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影响因子：
4.2
作者：
Antignani A;Fitzgerald D
通讯作者：
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影响因子：
3.4
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