Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone
Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone
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DOI：
10.1111/febs.12592
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发表时间：
2014-01-01
期刊：
影响因子：
5.4
通讯作者：
Sargent, Frank
中科院分区：
文献类型：
--
作者：
Dow, Jennifer M.;Grahl, Sabine;Sargent, Frank
大肠杆菌是一种革兰氏阴性细菌，可以在无氧呼吸过程中使用硝酸盐。周质硝酸还原酶 NapA 的催化亚基含有两种类型的氧化还原辅因子，并通过双精氨酸蛋白转运途径跨细胞质膜输出。 NapD 是一种小细胞质蛋白，对于周质硝酸还原酶的活性至关重要，并与 NapA 的双精氨酸信号肽紧密结合。在这里，我们使用自旋标记和 EPR 证明，分离的 NapA 双精氨酸信号肽以其未结合的形式构建，并在与 NapD 相互作用时经历微小但显着的构象变化。此外，可以通过仅将亲和标签工程化到NapD上来分离包含全长NapA蛋白和NapD的复合物。分析超速离心表明 NapDA 复合物中的两种蛋白质以 1:1 摩尔比存在，复合物的小角 X 射线散射分析表明 NapA 在与其 NapD 伴侣结合时至少部分折叠。如果没有 NapA Tat 信号肽，则无法分离 NapDA 复合物。总而言之，这项工作表明 NapD 分子伴侣主要与该系统中的 NapA 信号肽结合，并指出 NapD 在钼辅因子插入中的作用。
Escherichiacoli is a Gram-negative bacterium that can use nitrate during anaerobic respiration. The catalytic subunit of the periplasmic nitrate reductase NapA contains two types of redox cofactor and is exported across the cytoplasmic membrane by the twin-arginine protein transport pathway. NapD is a small cytoplasmic protein that is essential for the activity of the periplasmic nitrate reductase and binds tightly to the twin-arginine signal peptide of NapA. Here we show, using spin labelling and EPR, that the isolated twin-arginine signal peptide of NapA is structured in its unbound form and undergoes a small but significant conformational change upon interaction with NapD. In addition, a complex comprising the full-length NapA protein and NapD could be isolated by engineering an affinity tag onto NapD only. Analytical ultracentrifugation demonstrated that the two proteins in the NapDA complex were present in a 1:1 molar ratio, and small angle X-ray scattering analysis of the complex indicated that NapA was at least partially folded when bound by its NapD partner. A NapDA complex could not be isolated in the absence of the NapA Tat signal peptide. Taken together, this work indicates that the NapD chaperone binds primarily at the NapA signal peptide in this system and points towards a role for NapD in the insertion of the molybdenum cofactor.