The Structure of the Bacterial Oxidoreductase Enzyme DsbA in Complex with a Peptide Reveals a Basis for Substrate Specificity in the Catalytic Cycle of DsbA Enzymes
The Structure of the Bacterial Oxidoreductase Enzyme DsbA in Complex with a Peptide Reveals a Basis for Substrate Specificity in the Catalytic Cycle of DsbA Enzymes
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DOI：
10.1074/jbc.m109.011502
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发表时间：
2009-06-26
影响因子：
4.8
通讯作者：
Scanlon, Martin J.
中科院分区：
文献类型：
--
作者：
Paxman, Jason J.;Borg, Natalie A.;Scanlon, Martin J.
革兰氏阴性菌中的氧化蛋白质折叠导致半胱氨酸残基对之间形成二硫键。这是一个多步骤的过程，其中二硫醇-二硫化物氧化还原酶DsbA起着核心作用。DsbA的结构包括未知功能的全螺旋结构域和硫氧还蛋白结构域，其中活性位点半胱氨酸在氧化、底物结合、还原形式和DsbB结合形式之间穿梭，其中DsbB是再氧化DsbA的膜蛋白。大多数DsbA酶与各种各样的还原底物相互作用，并且几乎没有特异性。然而，一些DsbA酶现在已经被确定，具有狭窄的底物库，并出现与较少数量的底物特异性相互作用。DsbA-底物复合物的瞬时性质阻碍了我们对DsbA酶与其底物相互作用的因素的理解。在这里，我们报告的晶体结构之间的复合物大肠杆菌DsbA和一个肽与来自基板的序列。在DsbA-肽复合物中鉴定的结合位点与在DsbA-DsbB复合物中观察到的DsbB的结合位点不同。该结构揭示了DsbA-肽相互作用的细节，并提出了一种机制，通过该机制，DsbA可以同时显示出对底物的广泛特异性，但对DsbB表现出特异性。这种结合模式得到了溶液核磁共振数据以及功能数据的支持，这表明DsbA的底物特异性可以通过在复合物结构中识别的结合界面处的变化进行修改。
Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.