Two Snapshots of Electron Transport across the Membrane INSIGHTS INTO THE STRUCTURE AND FUNCTION OF DsbD
Two Snapshots of Electron Transport across the Membrane INSIGHTS INTO THE STRUCTURE AND FUNCTION OF DsbD
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DOI：
10.1074/jbc.m900651200
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发表时间：
2009-04-24
影响因子：
4.8
通讯作者：
Beckwith, Jon
中科院分区：
文献类型：
--
作者：
Cho, Seung-Hyun;Beckwith, Jon
在大肠杆菌中，周质蛋白二硫键异构酶DsbC通过细胞质膜蛋白DsbD从细胞质硫氧还蛋白-1（Trx 1）转移电子而保持还原状态。DsbD的跨膜结构域（DsbD β）包含八个跨膜区段（TM），含有两个氧化还原活性半胱氨酸（Cys-163和Cys-285），每个半胱氨酸都暴露于膜的两侧。TM 1中的Cys-163和TM 4中的Cys-285可分别与DsbD的胞质Trx 1和周质Trx样结构域相互作用。当Cys-163和Cys-285为二硫键时，TM 1和TM 4的C-末端半部分暴露于水，而这些TM的N-末端半部分不暴露于水。为了评估DsbD β的两个半胱氨酸被减少时可能的构象变化，我们已经确定了TM 1和TM 4部分的可及性。我们用半胱氨酸取代这些TM片段中的氨基酸，并确定烷基化可及性。我们发现，在TM 1和TM 4中的单Cys置换的烷基化可及性在氧化和还原的DsbD β中是相同的，表明DsbD β在两种氧化还原状态之间的相对静态构象。我们还发现，当Cys-163和Cys-285被氧化或还原时，TM 2和TM 3的氨基酸可及性没有变化。总之，这些结果支持DsbD β在氧化和还原状态之间的转换中的相对静态结构，但提高了与Trx蛋白相互作用时构象变化的可能性。此外，我们还发现在TM 3的细胞质近端部分中存在暴露于水的残基，从而可以更详细地表征DsbD β中的空腔。
In Escherichia coli, the periplasmic protein disulfide isomerase, DsbC, is maintained reduced by transfer of electrons from cytoplasmic thioredoxin-1 (Trx1) via the cytoplasmic membrane protein, DsbD. The transmembrane domain of DsbD (DsbD beta), which comprises eight transmembrane segments (TMs), contains two redox-active cysteines (Cys-163 and Cys-285), each of which is water-exposed to both sides of the membrane. Cys-163 in TM1 and Cys-285 in TM4 can interact with cytoplasmic Trx1 and a periplasmic Trx-like domain of DsbD, respectively. When Cys-163 and Cys-285 are disulfide-bonded, the C-terminal halves of TM1 and TM4 are water-exposed, whereas the N-terminal halves of these TMs are not. To assess possible conformational changes of DsbD beta when its two cysteines are reduced, we have determined the accessibility of portions of TM1 and TM4. We substituted cysteines for amino acids in these TM segments and determined alkylation accessibility. We find that the alkylation accessibility of single Cys replacements in TM1 and TM4 is the same in oxidized and reduced DsbD beta, indicating a relatively static conformation of DsbD beta between the two redox states. We also find that the accessibility of amino acids of TM2 and TM3 when Cys-163 and Cys-285 are oxidized or reduced shows no change. Together, these results support a relatively static structure of DsbD beta in the switch between the oxidized and the reduced state but raise the possibility of conformational changes when interacting with Trx proteins. In addition, we also find water-exposed residues in the cytoplasmic proximal portion of TM3, allowing a more detailed characterization of the cavity in DsbD beta.