Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Nature. 2013 Jun 27;498(7455):521-5. doi: 10.1038/nature12283. Epub 2013 Jun 5.
The hepatitis C virus (HCV) has developed a small membrane protein, p7, which remarkably can self-assemble into a large channel complex that selectively conducts cations. We wanted to examine the structural solution that the viroporin adopts in order to achieve selective cation conduction, because p7 has no homology with any of the known prokaryotic or eukaryotic channel proteins. The activity of p7 can be inhibited by amantadine and rimantadine, which are potent blockers of the influenza M2 channel and licensed drugs against influenza infections. The adamantane derivatives have been used in HCV clinical trials, but large variation in drug efficacy among the various HCV genotypes has been difficult to explain without detailed molecular structures. Here we determine the structures of this HCV viroporin as well as its drug-binding site using the latest nuclear magnetic resonance (NMR) technologies. The structure exhibits an unusual mode of hexameric assembly, where the individual p7 monomers, i, not only interact with their immediate neighbours, but also reach farther to associate with the i+2 and i+3 monomers, forming a sophisticated, funnel-like architecture. The structure also points to a mechanism of cation selection: an asparagine/histidine ring that constricts the narrow end of the funnel serves as a broad cation selectivity filter, whereas an arginine/lysine ring that defines the wide end of the funnel may selectively allow cation diffusion into the channel. Our functional investigation using whole-cell channel recording shows that these residues are critical for channel activity. NMR measurements of the channel-drug complex revealed six equivalent hydrophobic pockets between the peripheral and pore-forming helices to which amantadine or rimantadine binds, and compound binding specifically to this position may allosterically inhibit cation conduction by preventing the channel from opening. Our data provide a molecular explanation for p7-mediated cation conductance and its inhibition by adamantane derivatives.
丙型肝炎病毒 (HCV) 产生了一种小的膜蛋白 p7，它能够自我组装成一个选择性传导阳离子的大型通道复合物。我们希望研究一下该病毒蛋白孔道采用的结构解决方案，以实现阳离子的选择性传导，因为 p7 与任何已知的原核或真核通道蛋白都没有同源性。金刚烷衍生物可以抑制 p7 的活性，金刚烷衍生物是流感 M2 通道的有效抑制剂，也是抗流感感染的许可药物。这些金刚烷衍生物已被用于 HCV 的临床试验中，但由于各种 HCV 基因型之间药物疗效的巨大差异，在没有详细的分子结构的情况下，很难解释这一现象。在这里，我们使用最新的核磁共振 (NMR) 技术确定了这种 HCV 病毒蛋白孔道及其药物结合位点的结构。该结构表现出一种不寻常的六聚体组装方式，其中单个 p7 单体不仅与它们的直接相邻单体相互作用，而且还延伸更远与 i+2 和 i+3 单体相互作用，形成了一个复杂的、漏斗状的结构。该结构还指出了阳离子选择的机制：一个天冬酰胺/组氨酸环，在漏斗的狭窄端起到一个广泛的阳离子选择性过滤器的作用，而一个精氨酸/赖氨酸环，在漏斗的宽端，可能选择性地允许阳离子扩散进入通道。我们使用全细胞通道记录进行的功能研究表明，这些残基对于通道活性至关重要。对通道-药物复合物的 NMR 测量显示，在周边和形成孔的螺旋之间有六个等效的疏水性口袋，金刚烷或金刚乙胺结合在这些口袋中，化合物结合到这个位置可能会通过阻止通道打开来变构抑制阳离子传导。我们的数据为 p7 介导的阳离子传导及其被金刚烷衍生物抑制提供了分子解释。
