Structural requirements in the membrane-spanning domain of the paramyxovirus HN protein for the formation of a stable tetramer.
Structural requirements in the membrane-spanning domain of the paramyxovirus HN protein for the formation of a stable tetramer.
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副粘病毒 HN 蛋白跨膜结构域形成稳定四聚体的结构要求。
DOI：
10.1006/viro.1995.1569
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Pohlmann,S
中科院分区：
文献类型：
--
作者：
Parks,GD;Pohlmann,S
副粘病毒血凝素神经氨酸酶 (HN) 是一种 II 型同源四聚体整合膜糖蛋白，由一对通过非共价键连接在一起的二硫键连接的二聚体组成。为了确定内部未切割的信号锚定 (S/A) 结构域在稳定四聚体形成中的作用，在 HeLa 细胞中表达了含有 S/A 取代的 cDNA 衍生的 HN 突变体。通过蔗糖梯度沉降和糖苷内切酶处理来检查蛋白质的四聚体组装和内质网至高尔基体的转运。对 19 个残基 S/A 的亮氨酸扫描取代分析鉴定出 S/A C 末端的 2 个极性残基（Ser 31 和 Tyr 36），它们对于稳定四聚体的形成非常重要。虽然 Ala、Cys 和 Gly 可以在功能上取代 Set 31，形成稳定的四聚体，但用 Leu 或 Phe 取代会导致突变体被检测为二硫键连接的二聚体。这些结果表明，31 位的小氨基酸，而不是特定的残基本身，是 S/A 中重要的组装要求。与位置 31 的大小要求相反，Tyr 36 用 Phe 的保守取代产生了 HN 突变体，该突变体以二聚体、四聚体和更高阶寡聚体的混合物形式沉积，表明正确的组装需要在该位置有 Tyr。被检测为二硫键连接二聚体的 S/A 突变体仅表现出 ER 至高尔基体转运的轻微减少（WT 的约 50%），这与 S/A 取代影响四聚体稳定性而不是具有转运能力的寡聚体形成的观点一致。这些数据表明，HNS/A C 端区域的两个位置对于稳定四聚体的组装有不同的结构要求。
The paramyxovirus hemagglutinin-neuraminidase (HN) is a type II homotetrameric integral membrane glycoprotein composed of a pair of disulfide-linked dimers that are held together by noncovalent bonds. To determine the role of the internal uncleaved signal-anchor (S/A) domain in stable tetramer formation, cDNA-derived HN mutants containing S/A substitutions were expressed in HeLa cells. The assembly into tetramers and ER-to-Golgi transport of the proteins were examined by sucrose gradient sedimentation and by endoglycosidase treatment. A leucine-scanning substitution analysis of the 19 residue S/A identified 2 polar residues (Ser 31 and Tyr 36) in the C-terminal end of the S/A that were important for the formation of a stable tetramer. While Ala, Cys, and Gly could functionally replace Set 31 in the formation of a stable tetramer, substitution with Leu or Phe resulted in mutants that were detected as disulfide-linked dimers. These results indicate that a small amino acid in position 31, rather than a specific residue per se, is an important assembly requirement in the S/A. In contrast to the size requirement for position 31, the conservative substitution of Tyr 36 with Phe produced an HN mutant that sedimented as a mixture of dimers, tetramers, and higher order oligomers, suggesting that proper assembly requires a Tyr in this position. The S/A mutants that were detected as disulfide-linked dimers showed only a slight reduction in ERto-Golgi transport (∼50% of WT), consistent with the proposal that the S/A substitutions had affected tetramer stability and not the formation of a transport-competent oligomer. These data indicate that there are different structural requirements for two positions in the C-terminal region of the HN S/A for the assembly of a stable tetramer.