Structure and function at the lipid-protein interface of a pentameric ligand-gated ion channel.
Structure and function at the lipid-protein interface of a pentameric ligand-gated ion channel.
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五聚体配体门控离子通道的脂质-蛋白质界面的结构和功能。
DOI：
10.1073/pnas.2100164118
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发表时间：
2021
影响因子：
11.1
通讯作者：
Grosman,Claudio
中科院分区：
文献类型：
--
作者：
Kumar,Pramod;Cymes,GiselaD;Grosman,Claudio
虽然很早就有人提出膜蛋白可能含有紧密结合的脂类，但它们的特性、结合部位的结构以及它们的功能和结构相关性仍然难以捉摸。在某种程度上，这是因为紧密结合的脂类通常位于蛋白质的外围，密度图的质量通常较差，而且它们可能会被标准纯化程序中使用的洗涤剂分子所击败。作为表征五聚体配体门控离子通道(PLGICs)超家族天然结合脂类的一步，我们应用单粒子低温电子显微镜对完全没有洗涤剂溶解步骤的天然膜片段进行了研究。鉴于真核细胞分泌途径中膜脂组成的异质性，我们选择了在大肠杆菌内膜中表达的细菌pLGIC(Elic)进行研究。我们获得了未连接的ELIC的三维重建(2.5奥分辨率)，显示了在蛋白质-体-膜界面存在两种紧密结合的脂类的明显证据。其中一个与细胞质小叶中的“规则”双酰化磷脂一致，而另一个与细胞质小叶中的心磷脂的四酰化结构一致。在重建的时候。双极-脂质双层，ELIC保留了这个超家族成员的功能性质特征，因此，拟合的原子模型有望代表这个离子通道的(长期争论的)去连接-闭合的“静止”构象。值得注意的是，将心磷脂添加到磷脂酰胆碱膜上，恢复了只有磷脂酰胆碱的双层膜中大部分失去的离子通道活性。
Although it has long been proposed that membrane proteins may contain tightly bound lipids, their identity, the structure of their binding sites, and their functional and structural relevance have remained elusive. To some extent, this is because tightly bound lipids are often located at the periphery of proteins, where the quality of density maps is usually poorer, and because they may be outcompeted by detergent molecules used during standard purification procedures. As a step toward characterizing natively bound lipids in the superfamily of pentameric ligand-gated ion channels (pLGICs), we applied single-particle cryogenic electron microscopy to fragments of native membrane obtained in the complete absence of detergent-solubilization steps. Because of the heterogeneous lipid composition of membranes in the secretory pathway of eukaryotic cells, we chose to study a bacterial pLGIC (ELIC) expressed inEscherichia coli’s inner membrane. We obtained a three-dimensional reconstruction of unliganded ELIC (2.5-Å resolution) that shows clear evidence for two types of tightly bound lipid at the protein–bulk-membrane interface. One of them was consistent with a “regular” diacylated phospholipid, in the cytoplasmic leaflet, whereas the other one was consistent with the tetra-acylated structure of cardiolipin, in the periplasmic leaflet. Upon reconstitution inE. colipolar-lipid bilayers, ELIC retained the functional properties characteristic of members of this superfamily, and thus, the fitted atomic model is expected to represent the (long-debated) unliganded-closed, “resting” conformation of this ion channel. Notably, the addition of cardiolipin to phosphatidylcholine membranes restored the ion-channel activity that is largely lost in phosphatidylcholine-only bilayers.