The influenza virus M2 ion channel protein:: Probing the structure of the transmembrane domain in intact cells by using engineered disulfide cross-linking
The influenza virus M2 ion channel protein:: Probing the structure of the transmembrane domain in intact cells by using engineered disulfide cross-linking
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DOI：
10.1006/viro.1998.9552
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发表时间：
1999-02-01
期刊：
影响因子：
3.7
通讯作者：
Lamb, RA
中科院分区：
文献类型：
--
作者：
Bauer, CM;Pinto, LH;Lamb, RA
甲型流感病毒M-2整体膜蛋白是一个离子通道，允许质子在内体病毒粒子脱壳过程中进入病毒颗粒，并调节病毒感染细胞中反式高尔基网络的pH值。M-2蛋白是一种由97个残基组成的同质寡聚物，具有一个跨膜结构域，其残基覆盖了通道的孔区域，通道的生物活性形式是同质四聚体。为了了解TM结构域的结构排列，我们将TM结构域的每个残基依次变为半胱氨酸，并使用氧化二硫交联来识别邻近的残基。用碘对膜中的M-2蛋白进行氧化处理，在TM结构域残基27、34和41处产生最大的交联。当反应在37℃下进行时，使用催化剂Cu(II)（1,10-菲罗啉）(3)氧化膜上的M-2蛋白导致许多TM结构域残基交联，这表明膜上的TM结构域可以发生旋转运动。然而，对二硫键二聚体形成动力学分析表明，TM结构域残基27、30、34、37和41的形成速度最快。此外，当在4℃下进行氧化时，最大的交联发生在TM结构域残基27、30、34、37和41。这些位置与七重体的a和d位置相对应，因此这些生化数据与M-2四聚体的TM结构域形成四螺旋束是一致的。对膜中M-2蛋白在pH值为5.2时氧化形成的二硫键的分析表明，与pH值为74时相比，TM结构域残基40、42和43的交联大大减少。这种向TM结构域细胞质侧的残基交联的ph依赖性变化与低ph下M-2离子通道的激活相似。(C) 1999年学术出版社。
The influenza A virus M-2 integral membrane protein is an ion channel that permits protons to enter virus particles during uncoating of virions in endosomes, and it also modulates the pH of the trans-Golgi network in virus-infected cells. M-2 protein is a homo-oligomer of 97 residues with a single transmembrane (TM) domain whose residues encompass the pore region of the channel and the biologically active form of the channel is a homotetramer. To understand the structural arrangement of the TM domains, each residue of the TM domain was changed in turn to cysteine, and oxidative disulfide cross-linking used to identify residues in close proximity. Oxidative treatment of M-2 protein in membranes using iodine resulted in maximum cross-linking at TM domain residues 27, 34, and 41. Oxidation of M-2 protein in membranes using the catalyst Cu(II)(1,10-phenanthroline)(3) resulted in cross-linking of many TM domain residues when the reaction was allowed to proceed at 37 degrees C, suggesting that rotational movements of the TM domains in the membrane can occur. However, analysis of the kinetics of disulfide-linked dimer formation showed that TM domain residues 27 30, 34, 37 and 41 formed most rapidly. Furthermore, when oxidation was performed at 4 degrees C, maximum cross-linking occurred at TM domain residues 27 30, 34, 37 and 41. These positions correspond to the a and d positions of a heptad repeat Thus these biochemical data are consistent with the TM domain region of the M-2 tetramer forming a four-helix bundle. Analysis of the disulfide bonds that formed when oxidation of M-2 protein in membranes was performed at pH 5.2 showed greatly reduced cross-linking at TM domain residues 40, 42, and 43 than that found at pH 74. This pH-dependent change in cross-linking of residues toward the cytoplasmic side of the TM domain parallels with the activation of the M-2 ion channel at low pH. (C) 1999 Academic Press.