Specific binding of adamantane drugs and direction of their polar amines in the pore of the influenza M2 transmembrane domain in lipid bilayers and dodecylphosphocholine micelles determined by NMR spectroscopy.
Specific binding of adamantane drugs and direction of their polar amines in the pore of the influenza M2 transmembrane domain in lipid bilayers and dodecylphosphocholine micelles determined by NMR spectroscopy.
复制标题
DOI：
10.1021/ja102581n
复制
发表时间：
2011-03-30
影响因子：
15
通讯作者：
Hong, Mei
中科院分区：
文献类型：
--
作者：
Cady, Sarah D.;Wang, Jun;Wu, Yibing;DeGrado, William F.;Hong, Mei
流感M2蛋白的跨膜结构域（M2 TM）形成对病毒生命周期重要的四聚体质子通道。质子通道活性被含胺的金刚烷基药物金刚烷胺和金刚乙胺抑制，它们已被证明特异性地结合到M2 TM的Ser 31附近的孔。然而，极性胺是否指向通道的N-或C-末端尚未确定。阐明极性基团的方向将阐明药物结合抑制该质子通道的机制，并将促进新抑制剂的合理设计。在这项研究中，我们确定了极性胺的方向，使用M2 TM重建的脂质双层以及DPC胶束。13 C-2 H旋转回波双共振NMR实验的13 C-标记的M2 TM和甲基氘代金刚乙胺在脂质双层显示，极性胺指向的C-末端的通道，与甲基接近Gly 34。十二烷基磷酸胆碱（DPC）胶束中M2 TM的溶液NMR实验表明，药物结合引起蛋白质的显着化学位移扰动，这与M2 TM和M2（18-60）结合到脂质双层中所观察到的非常相似。特定的2 H-标记的药物允许分配的药物-蛋白质交叉峰，这表明金刚烷胺和金刚乙胺结合的孔在相同的方式为双层结合的M2 TM。这些结果强烈表明，金刚烷基抑制M2 TM实现不仅通过直接物理闭塞的孔，但也通过扰动的质子敏感残基His 37的平衡常数。在DPC胶束中，这是没有观察到与不同的洗涤剂，DHPC，再生相关的特定孔结合位点，强调了洗涤剂环境对膜蛋白的功能结构的显着影响。
The transmembrane domain of the influenza M2 protein (M2TM) forms a tetrameric proton channel important for the virus lifecycle. The proton-channel activity is inhibited by amine-containing adamantyl drugs amantadine and rimantadine, which have been shown to bind specifically to the pore of M2TM near Ser31. However, whether the polar amine points to the N- or C-terminus of the channel has not yet been determined. Elucidating the polar group direction will shed light on the mechanism by which drug binding inhibits this proton channel and will facilitate rational design of new inhibitors. In this study, we determine the polar amine direction using M2TM reconstituted in lipid bilayers as well as DPC micelles. 13C-2H rotational-echo double-resonance NMR experiments of 13C-labeled M2TM and methyl-deuterated rimantadine in lipid bilayers showed that the polar amine pointed to the C-terminus of the channel, with the methyl group close to Gly34. Solution NMR experiments of M2TM in dodecylphosphocholine (DPC) micelles indicate that drug binding causes significant chemical shift perturbations of the protein that are very similar to those seen for M2TM and M2(18–60) bound to lipid bilayers. Specific 2H-labeling of the drugs permitted the assignment of drug-protein cross peaks, which indicate that amantadine and rimantadine bind to the pore in the same fashion as for bilayer-bound M2TM. These results strongly suggest that adamantyl inhibition of M2TM is achieved not only by direct physical occlusion of the pore but also by perturbing the equilibrium constant of the proton-sensing residue His37. The reproduction of the pharmacologically relevant specific pore-binding site in DPC micelles, which was not observed with a different detergent, DHPC, underscores the significant influence of the detergent environment on the functional structure of membrane proteins.
登录
查看更多内容
影响因子：
2.7
作者：
Cady SD;Hong M
通讯作者：
Hong M
影响因子：
3.4
作者：
Chen, Hanning;Wu, Yujie;Voth, Gregory A.
通讯作者：
Voth, Gregory A.
影响因子：
2.9
作者：
Intharathep, Pathumwadee;Laohpongspaisan, Chittima;Hannongbua, Supot
通讯作者：
Hannongbua, Supot
DOI：
10.1073/pnas.0804958105
发表时间：
2008-08-05
影响因子：
11.1
作者：
Jing, Xianghong;Ma, Chunlong;Lamb, Robert A.
通讯作者：
Lamb, Robert A.
DOI：
10.1073/pnas.0811720106
发表时间：
2009-01-27
影响因子：
11.1
作者：
Khurana, Ekta;Dal Peraro, Matteo;Klein, Michael L.
通讯作者：
Klein, Michael L.