Topography of the hydrophilic helices of membrane-inserted diphtheria toxin T domain: TH1-TH3 as a hydrophilic tether.
Topography of the hydrophilic helices of membrane-inserted diphtheria toxin T domain: TH1-TH3 as a hydrophilic tether.
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插入膜的白喉毒素 T 结构域的亲水螺旋的形貌：TH1-TH3 作为亲水系链。
DOI：
10.1021/bi060587f
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发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
London,Erwin
中科院分区：
文献类型：
--
作者：
Wang,Jie;Rosconi,MichaelP;London,Erwin
在低pH触发的膜插入后，白喉毒素的T结构域帮助毒素的催化结构域跨膜转移。本研究对T区的亲水性N-末端螺旋(TH1、−、Th3)进行了研究。研究了低pH条件下的构象变化和膜插入后的形貌变化。这些实验包括在不同位置引入的单一半胱氨酸的双烷或BODIPY标记，随后测量双烷发射波长，双烷暴露于荧光猝灭剂，以及抗体与BODIPY基团的结合。将T结构域暴露在低pH溶液中，发现TH1的疏水表面在中性pH条件下处于天然状态，然后暴露在溶液中。当T结构域在低pH条件下被外加到脂泡中时，TH1的疏水面被埋在脂双层中。在低pH条件下，TH2和Th3螺旋也插入到双层中。然而，与螺旋Th5−Th9相反，当T结构域从浅插入(P)构象转换为深插入(TM)构象时，总的Th1−Th3插入较浅，并且Th1−Th3插入深度没有显著变化。通过链霉亲和素与生物素标记的半胱氨酸残基的结合来研究膜插入的T结构域的残基暴露在双层的外表面还是内表面。这些实验表明，当T结构域被外部添加到囊泡中时，整个Th1Th3片段保持在双层的顺式(外层)一侧。这项研究的结果表明，膜插入的Th1−Th3形成的自治片段既不深入双层，也不与疏水的Th5−Th9亚域形成的促进易位结构紧密相互作用。相反，Th1−Th3可能通过充当A链连接的柔性系绳来帮助易位。
After low pH-triggered membrane insertion, the T domain of diphtheria toxin helps translocate the catalytic domain of the toxin across membranes. In this study, the hydrophilic N-terminal helices of the T domain (TH1−TH3) were studied. The conformation triggered by exposure to low pH and changes in topography upon membrane insertion were studied. These experiments involved bimane or BODIPY labeling of single Cys introduced at various positions, followed by the measurement of bimane emission wavelength, bimane exposure to fluorescence quenchers, and antibody binding to BODIPY groups. Upon exposure of the T domain in solution to low pH, it was found that the hydrophobic face of TH1, which is buried in the native state at neutral pH, became exposed to solution. When the T domain was added externally to lipid vesicles at low pH, the hydrophobic face of TH1 became buried within the lipid bilayer. Helices TH2 and TH3 also inserted into the bilayer after exposure to low pH. However, in contrast to helices TH5−TH9, overall TH1−TH3 insertion was shallow and there was no significant change in TH1−TH3 insertion depth when the T domain switched from the shallowly inserting (P) to deeply inserting (TM) conformation. Binding of streptavidin to biotinylated Cys residues was used to investigate whether solution-exposed residues of membrane-inserted T domain were exposed on the external or internal surface of the bilayer. These experiments showed that when the T domain is externally added to vesicles, the entire TH1−TH3 segment remains on the cis (outer) side of the bilayer. The results of this study suggest that membrane-inserted TH1−TH3 form autonomous segments that neither deeply penetrate the bilayer nor interact tightly with the translocation-promoting structure formed by the hydrophobic TH5−TH9 subdomain. Instead, TH1−TH3 may aid translocation by acting as an A-chain-attached flexible tether.