INFLUENZA VIRUS-M2 INTEGRAL MEMBRANE-PROTEIN IS A HOMOTETRAMER STABILIZED BY FORMATION OF DISULFIDE BONDS
INFLUENZA VIRUS-M2 INTEGRAL MEMBRANE-PROTEIN IS A HOMOTETRAMER STABILIZED BY FORMATION OF DISULFIDE BONDS
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DOI：
10.1016/0042-6822(91)90115-r
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发表时间：
1991-07-01
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：
HOLSINGER, LJ;LAMB, RA
测定了甲型流感病毒M2膜整合蛋白的寡聚体结构。在非还原条件下的SDS-聚丙烯酰胺凝胶上，流感A/Udorn/72病毒M2形成二硫键连接的二聚体（30 kDa）和四聚体（60 kDa）。蔗糖梯度分析和化学交联分析表明，M2的低聚体形式是由一对二硫键连接的二聚体或二硫键连接的四聚体组成的四聚体。此外，观察到少量150- 180，000 kDa的交联物质，现有数据表明其仅含有M2多肽。通过将两个细胞外和单个细胞质半胱氨酸残基中的每一个转化为丝氨酸残基并在真核细胞中表达改变的M2蛋白，来检查M2半胱氨酸残基在二硫键形成中的作用及其在形成寡聚体中的作用。在残基17或19处去除N-末端半胱氨酸中的任一个表明形成了由一对非共价缔合的二硫键连接的二聚体组成的四聚体，这表明每个半胱氨酸残基同样能够形成二硫键。当两个半胱氨酸残基从M2 N-末端结构域中去除时，未观察到二硫键连接的形式。当溶解在洗涤剂中时，这种双半胱氨酸突变体失去了与M2特异性mAb的反应性，并在蔗糖梯度上表现出改变的沉降模式。然而，这种双半胱氨酸突变体在膜中的化学交联表明它可以形成四聚体。总之，这些数据表明，二硫键的形成，虽然不是必要的寡聚体组装，稳定的M2四聚体从破坏去污剂溶解。
The oligomeric structure of the influenza A virus M2integral membrane protein was determined. On SDS-polyacrylamide gels under nonreducing conditions, the influenza A/Udorn/72 virus M2 forms disulfide-linked dimers (30 kDa) and tetramers (60 kDa). Sucrose gradient analysis and chemical cross-linking analysis indicated that the oligomeric form of M2is a tetramer consisting of either a pair of disulfide-linked dimers or disulfide-linked tetramers. In addition, a small amount of a cross-linked species of 150–180,000 kDa, which the available data suggest contains only M2 polypeptides, was observed. The role of M2 cysteine residues in disulfide bond formation and their role in forming oligomers were examined by converting each of the two extracellular and single cytoplasmic cysteine residues to serine residues and expressing the altered M2 proteins in eukaryotic cells. Removal of either one of the N-terminal cysteines at residues 17 or 19 indicated that tetramers formed that consisted of a pair of noncovalently associated disulfide-linked dimers, suggesting that each of the cysteine residues is equally competent for forming disulfide bonds. When both cysteine residues were removed from the M2 N-terminal domain, no disulfide-linked forms were observed. When solubilized in detergent this double-cysteine mutant lost reactivity with a M2 specific mAb and exhibited an altered sedimentation pattern on sucrose gradients. However, chemical cross-linking of this double-cysteine mutant in membranes indicated that it can form tetramers. Taken together, these data suggest that disulfide bond formation, although not essential for oligomeric assembly, stabilizes the M2 tetramer from disruption by detergent solubilization.