Cholesterol Exposure at the Membrane Surface Is Necessary and Sufficient to Trigger Perfringolysin O Binding
Cholesterol Exposure at the Membrane Surface Is Necessary and Sufficient to Trigger Perfringolysin O Binding
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DOI：
10.1021/bi9002309
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发表时间：
2009-05-12
期刊：
影响因子：
2.9
通讯作者：
Heuck, Alejandro P.
中科院分区：
文献类型：
--
作者：
Flanagan, John J.;Tweten, Rodney K.;Heuck, Alejandro P.
Perfringolysin O (PFO) 是胆固醇依赖性溶细胞素的原型，是一类作用于真核细胞膜的细菌成孔毒素。 PFO 的成孔机制表现出对膜胆固醇的绝对需求，但 PFO C 端结构域的结构排列与靶膜中胆固醇分布之间复杂的相互作用知之甚少。在此，我们表明，PFO 与双层的结合以及最终形成跨膜孔的一系列事件的启动取决于膜表面游离胆固醇的可用性，而膜核心中磷脂和胆固醇的酰基链堆积的变化，或去污剂抗性结构域的存在或不存在与 PFO 结合无关。此外，添加鞘磷脂（细胞膜中膜筏的典型成分）可抑制 PFO 与膜的结合。最后，添加不与 PFO 相互作用但插入膜中并取代与磷脂（例如表胆固醇）结合的胆固醇的分子，减少了触发 PFO 结合所需的胆固醇量。总而言之，我们的研究表明，当胆固醇浓度超过磷脂的结合能力时，就会触发 PFO 与膜的结合，然后这种过量的胆固醇就会自由地与毒素结合。
Perfringolysin O (PFO) is the prototype for the cholesterol-dependent cytolysins, a family of bacterial pore-forming toxins that act on eukaryotic membranes. The pore-forming mechanism of PFO exhibits an absolute requirement for membrane cholesterol, but the complex interplay between the structural arrangement of the PFO C-terminal domain and the distribution of cholesterol in the target membrane is poorly understood. Herein we show that PFO binding to the bilayer and the initiation of the sequence of events that culminate in the formation of a transmembrane pore depend on the availability of free cholesterol at the membrane surface, while changes in the acyl chain packing of the phospholipids and cholesterol in the membrane core, or the presence or absence of detergent-resistant domains do not correlate with PFO binding. Moreover, PFO association with the membrane was inhibited by the addition of sphingomyelin, a typical component of membrane rafts in cell membranes. Finally, addition Of Molecules that do not interact with PFO, but intercalate into the membrane and displace cholesterol from its association with phospholipids (e.g., epicholesterol), reduced the amount of cholesterol required to trigger PFO binding. Taken together, our studies reveal that PFO binding to membranes is triggered when the concentration of cholesterol exceeds the association capacity of the phospholipids, and this cholesterol excess is then free to associate with the toxin.