Redox-Dependent Domain Rearrangement of Protein Disulfide Isomerase Coupled with Exposure of Its Substrate-Binding Hydrophobic Surface
Redox-Dependent Domain Rearrangement of Protein Disulfide Isomerase Coupled with Exposure of Its Substrate-Binding Hydrophobic Surface
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DOI：
10.1016/j.jmb.2009.11.049
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发表时间：
2010-02-19
影响因子：
5.6
通讯作者：
Kato, Koichi
中科院分区：
文献类型：
--
作者：
Serve, Olivier;Kamiya, Yukiko;Kato, Koichi
蛋白质二硫键异构酶（PDI）是内质网中的主要蛋白质，通过催化二硫键的形成、重排和断裂而作为含二硫键蛋白质的必需折叠催化剂和分子伴侣起作用。该酶具有模块化结构，具有四个硫氧还蛋白样结构域a、B、b '和a'，沿着具有C-末端延伸。同源的a和a'结构域在其直接参与硫醇-二硫键交换反应的活性位点中含有一个半胱氨酸对，而B'结构域则为底物多肽的非结构化区域提供主要结合位点。在这里，我们报告了氧化还原依赖的分子内重排的B'和a'结构域的特异腐质霉，嗜热真菌，阐明了结合使用核磁共振（NMR）和小角X射线散射（SAXS）方法。我们的NMR数据表明，底物结合到跨越这两个结构域的疏水表面，其在α '结构域的活性位点氧化后变得更多地暴露于溶剂。氢-氘交换和弛豫数据表明，α '畴的氧化还原态影响W畴的动力学性质。此外，小角X射线散射图谱显示，α '活性位点的氧化导致两个结构域的分离。在这些数据的基础上，我们提出了一种PDI作用的机理模型; a'结构域将其自身的二硫键转移到底物结合区疏水表面上容纳的未折叠蛋白质中，从而变成释放氧化底物的“封闭”形式。(C)2009爱思唯尔有限公司保留所有权利。
Protein disulfide isomerase (PDI) is a major protein in the endoplasmic reticulum, operating as an essential folding catalyst and molecular chaperone for disulfide-containing proteins by catalyzing the formation, rearrangement, and breakage of their disulfide bridges. This enzyme has a modular structure with four thioredoxin-like domains, a, b, b', and a', along with a C-terminal extension. The homologous a and a' domains contain one cysteine pair in their active site directly involved in thiol-disulfide exchange reactions, while the b' domain putatively provides a primary binding site for unstructured regions of the substrate polypeptides. Here, we report a redox-dependent intramolecular rearrangement of the b' and a' domains of PDI from Humicola insolens, a thermophilic fungus, elucidated by combined use of nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS) methods. Our NMR data showed that the substrates bound to a hydrophobic surface spanning these two domains, which became more exposed to the solvent upon oxidation of the active site of the a' domain. The hydrogen-deuterium exchange and relaxation data indicated that the redox state of the a' domain influences the dynamic properties of the W domain. Moreover, the SAXS profiles revealed that oxidation of the a' active site causes segregation of the two domains. On the basis of these data, we propose a mechanistic model of PDI action; the a' domain transfers its own disulfide bond into the unfolded protein accommodated on the hydrophobic surface of the substrate-binding region, which consequently changes into a "closed" form releasing the oxidized substrate. (C) 2009 Elsevier Ltd. All rights reserved.