The Structure of Bradyrhizobium japonicum Transcription Factor FixK2 Unveils Sites of DNA Binding and Oxidation
The Structure of Bradyrhizobium japonicum Transcription Factor FixK2 Unveils Sites of DNA Binding and Oxidation
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DOI：
10.1074/jbc.m113.465484
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发表时间：
2013-05-17
影响因子：
4.8
通讯作者：
Gruetter, Markus G.
中科院分区：
文献类型：
--
作者：
Bonnet, Mariette;Kurz, Mareike;Gruetter, Markus G.
FixK（2）是一种调节蛋白，其激活α-变形杆菌慢生型大豆根瘤菌的缺氧和微氧、内共生和固氮生活方式的大量基因。FixK（2）属于cAMP受体蛋白（CRP）超家族。尽管大多数CRP家族成员受效应分子的共同调节，但FixK（2）的活性受其位于DNA结合结构域附近的单个半胱氨酸（Cys-183）的氧化以及可能的蛋白水解的负向控制。在这里，我们报告了FixK（2）的三维X射线结构，它是CRP超家族FixK亚组的代表。只有当（i）使用氧化和蛋白酶不敏感的蛋白变体（FixK（2）（C183 S）-His（6）），其中Cys-183被丝氨酸取代，C末端与六组氨酸标签融合，以及（ii）允许该蛋白与30-mer双链靶DNA形成复合物时，结晶才成功。FixK（2）-DNA复合物的结构在1.77埃的分辨率下解析，在该分辨率下蛋白质形成同源二聚体。精确的蛋白质-DNA接触被确定，这导致了对典型靶序列的确认，即所谓的FixK（2）盒。C末端是表面暴露的，这可以解释其对特异性切割和降解的敏感性。氧化敏感的Cys-183也是表面暴露的，并且与DNA非常接近。因此，我们提出了一种机制，即半胱氨酸硫醇氧化后产生的含氧酸引起静电排斥，从而防止特异性DNA结合。
FixK(2) is a regulatory protein that activates a large number of genes for the anoxic and microoxic, endosymbiotic, and nitrogen-fixing life styles of the alpha-proteobacterium Bradyrhizobium japonicum. FixK(2) belongs to the cAMP receptor protein (CRP) superfamily. Although most CRP family members are coregulated by effector molecules, the activity of FixK(2) is negatively controlled by oxidation of its single cysteine (Cys-183) located next to the DNA-binding domain and possibly also by proteolysis. Here, we report the three-dimensional x-ray structure of FixK(2), a representative of the FixK subgroup of the CRP superfamily. Crystallization succeeded only when (i) an oxidation- and protease-insensitive protein variant (FixK(2)(C183S)-His(6)) was used in which Cys-183 was replaced with serine and the C terminus was fused with a hexahistidine tag and (ii) this protein was allowed to form a complex with a 30-mer double-stranded target DNA. The structure of the FixK(2)-DNA complex was solved at a resolution of 1.77 angstrom, at which the protein formed a homodimer. The precise protein-DNA contacts were identified, which led to an affirmation of the canonical target sequence, the so-called FixK(2) box. The C terminus is surface-exposed, which might explain its sensitivity to specific cleavage and degradation. The oxidation-sensitive Cys-183 is also surface-exposed and in close proximity to DNA. Therefore, we propose a mechanism whereby the oxo acids generated after oxidation of the cysteine thiol cause an electrostatic repulsion, thus preventing specific DNA binding.