Structure-function analysis of the endoplasmic reticulum oxidoreductase TMX3 reveals interdomain stabilization of the n-terminal redox-active domain
Structure-function analysis of the endoplasmic reticulum oxidoreductase TMX3 reveals interdomain stabilization of the n-terminal redox-active domain
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DOI：
10.1074/jbc.m706442200
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发表时间：
2007-11-16
影响因子：
4.8
通讯作者：
Ellgaard, Lars
中科院分区：
文献类型：
--
作者：
Haugstetter, Johannes;Maurer, Michael Andreas;Ellgaard, Lars
内质网中的二硫键形成由具有一个或多个硫氧还蛋白样结构域的蛋白质二硫键异构酶家族的酶催化。我们最近发现了跨膜蛋白TMX 3，蛋白质二硫键异构酶家族的硫醇二硫键氧化还原酶。在这里，我们发现TMX 3的内质网腔区域包含三个硫氧还蛋白样结构域，一个N-末端氧化还原活性结构域（命名为a），随后是两个酶失活结构域（B和b '）。使用重组表达的TMX 3结构域构建体a、ab和ab '，我们比较了结构稳定性和酶性质。通过结构和生物物理方法，我们证明了减少的结构域具有典型的球状折叠结构域的特征，然而，氧化后大大不稳定。重要的是，通过B结构域的结构域间稳定化使得a结构域对氧化和还原形式的化学变性和蛋白水解更具抗性。结合TMX 3 abb '的分子模拟研究，实验结果为TMX 3的多结构域结构与其作为氧化还原酶的功能之间的关系提供了新的认识。总体而言，数据表明，除了它们作为底物和辅因子结合结构域的作用之外，氧化还原非活性硫氧还蛋白样结构域还在稳定相邻的氧化还原活性结构域中起作用。
Disulfide bond formation in the endoplasmic reticulum is catalyzed by enzymes of the protein disulfide-isomerase family that harbor one or more thioredoxin-like domains. We recently discovered the transmembrane protein TMX3, a thiol-disulfide oxidoreductase of the protein disulfide-isomerase family. Here, we show that the endoplasmic reticulum-luminal region of TMX3 contains three thioredoxin-like domains, an N-terminal redox-active domain ( named a) followed by two enzymatically inactive domains (b and b'). Using the recombinantly expressed TMX3 domain constructs a, ab, and abb', we compared structural stability and enzymatic properties. By structural and biophysical methods, we demonstrate that the reduced a domain has features typical of a globular folded domain that is, however, greatly destabilized upon oxidization. Importantly, interdomain stabilization by the b domain renders the a domain more resistant toward chemical denaturation and proteolysis in both the oxidized and reduced form. In combination with molecular modeling studies of TMX3 abb', the experimental results provide a new understanding of the relationship between the multidomain structure of TMX3 and its function as a redox enzyme. Overall, the data indicate that in addition to their role as substrate and co-factor binding domains, redox-inactive thioredoxin-like domains also function in stabilizing neighboring redox-active domains.