An amantadine-sensitive chimeric BM2 ion channel of influenza B virus has implications for the mechanism of drug inhibition
An amantadine-sensitive chimeric BM2 ion channel of influenza B virus has implications for the mechanism of drug inhibition
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DOI：
10.1073/pnas.0910584106
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发表时间：
2009-11-03
影响因子：
11.1
通讯作者：
Lamb, Robert A.
中科院分区：
文献类型：
--
作者：
Ohigashi, Yuki;Ma, Chunlong;Lamb, Robert A.
甲型流感病毒M2(A/M2)和乙型流感病毒BM2都是形成质子选择性离子通道的小分子膜蛋白。甲型流感病毒A/M2通道是抗病毒药物金刚烷胺(及其甲基衍生物金刚乙胺)的靶点，而BM2通道活性不受药物影响。孔-跨膜(TM)结构域肽的原子结构已通过X射线结晶学确定[Stouffer等人。(2008)自然451：596-599]和更大的M2多肽的核磁共振方法[Schnell和Chou(2008)自然451：591-595]。结晶学数据显示通道孔中的电子密度(分辨率为3.5埃)，与金刚烷胺堵塞通道孔一致。相反，核磁共振数据显示，4个金刚乙胺分子结合在螺旋的外侧，朝向细胞膜的细胞质一侧。药物结合包括与残基40-45的相互作用以及金刚乙胺和天冬氨酸残基44之间的极性氢键(D44)。这两个不同的药物结合部位导致了两种不相容的药物抑制机制。我们已经在金刚烷胺敏感的A/M2和金刚烷胺不敏感的BM2之间产生了嵌合通道，旨在定义药物结合部位。含有A/M2胞外区5个残基和A/M2TM区24-36个残基的两个嵌合体对金刚烷胺/金刚乙胺的敏感性为85%，比活性与WT BM2相当。这些功能数据表明，位于4个螺旋外侧的金刚烷胺/金刚乙胺结合部位不是与药物抑制A/M2离子通道有关的主要部位。
Influenza A virus M2 (A/M2) and the influenza B virus BM2 are both small integral membrane proteins that form proton-selective ion channels. Influenza A virus A/M2 channel is the target of the antiviral drug amantadine (and its methyl derivative rimantadine), whereas BM2 channel activity is not affected by the drug. The atomic structure of the pore-transmembrane (TM) domain peptide has been determined by x-ray crystallography [Stouffer et al. (2008) Nature 451: 596-599] and of a larger M2 peptide by NMR methods [Schnell and Chou (2008) Nature 451: 591-595]. The crystallographic data show electron density (at 3.5 angstrom resolution) in the channel pore, consistent with amantadine blocking the pore of the channel. In contrast, the NMR data show 4 rimantadine molecules bound on the outside of the helices toward the cytoplasmic side of the membrane. Drug binding includes interactions with residues 40-45 and a polar hydrogen bond between rimantadine and aspartic acid residue 44 (D44). These 2 distinct drug-binding sites led to 2 incompatible drug inhibition mechanisms. We have generated chimeric channels between amantadine-sensitive A/M2 and amantadine-insensitive BM2 designed to define the drug-binding site. Two chimeras containing 5 residues of the A/M2 ectodomain and residues 24-36 of the A/M2 TM domain show 85% amantadine/rimantadine sensitivity and specific activity comparable to that of WT BM2. These functional data suggest that the amantadine/rimantadine binding site identified on the outside of the 4 helices is not the primary site associated with the pharmacologic inhibition of the A/M2 ion channel.