Ni(II) and Co(II) sensing by Escherichia coli RcnR
Ni(II) and Co(II) sensing by Escherichia coli RcnR
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DOI：
10.1021/ja710067d
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发表时间：
2008-06-18
影响因子：
15
通讯作者：
Chivers, Peter T.
中科院分区：
文献类型：
--
作者：
Iwig, Jeffrey S.;Leitch, Sharon;Chivers, Peter T.
大肠杆菌RcnR和结核分枝杆菌CsoR是最近鉴定的细菌金属响应DNA结合蛋白大家族的创始成员。RcnR控制的金属外排蛋白RcnA的表达只在响应Ni（II）和Co（II）离子。在这里，镍（II）和钴（II）与野生型和突变型RcnR蛋白的相互作用进行检查，以了解这些金属作为变构效应器的功能。两种金属都以纳摩尔亲和力与RcnR结合，并稳定蛋白质以使其变性。X射线吸收和电子顺磁共振光谱揭示了六个协调的高自旋网站为每个金属，含有硫醇配体。实验数据支持三重N-末端配位基序（NH 2-Xaa-NH-His），这是常见的两种金属。然而，Ni（II）-和Co（II）-RcnR络合物显示出不同的剩余的协调环境。每个金属配位保守的Cys配体，但具有不同的M-S距离。Co（II）-硫醇盐配位以前在Ni（II）-/Co（II）-响应性金属调节剂中没有观察到。RcnR从蛋白质的N-末端区域募集配体的能力将其与CsoR区分开来，CsoR使用较低的配位几何结构来结合Cu（I）。这些研究促进了Ni（II）-RcnR和NikR（大肠杆菌中的另一种Ni（II）响应性转录调节因子）之间的比较，以更好地了解大肠杆菌中不同的镍水平是如何被感知的。RcnR中Ni（II）-和Co（II）-结合位点的表征，结合所有RcnR/CsoR家族成员的生物信息学分析，确定了可能定义配体结合特异性的四个氨基酸指纹，导致不同类别的RcnR/CsoR蛋白质之间的相似性和差异的新图像。
Escherichia coli RcnR and Mycobacterium tuberculosis CsoR are the founding members of a recently identified, large family of bacterial metal-responsive DNA-binding proteins. RcnR controls the expression of the metal efflux protein RcnA only in response to Ni(II) and Co(II) ions. Here, the interaction of Ni(II) and Co(II) with wild-type and mutant RcnR proteins is examined to understand how these metals function as allosteric effectors. Both metals bind to RcnR with nanomolar affinity and stabilize the protein to denaturation. X-ray absorption and electron paramagnetic resonance spectroscopies reveal six-coordinate high-spin sites for each metal that contains a thiolate ligand. Experimental data support a tripartite N-terminal coordination motif (NH2-Xaa-NH-His) that is common for both metals. However, the Ni(II)- and Co(II)-RcnR complexes are shown to differ in the remaining coordination environment. Each metal coordinates a conserved Cys ligand but with distinct M-S distances. Co(II)-thiolate coordination has not been observed previously in Ni(II)-/Co(II)-responsive metalloregulators. The ability of RcnR to recruit ligands from the N-terminal region of the protein distinguishes it from CsoR, which uses a lower coordination geometry to bind Cu(I). These studies facilitate comparisons between Ni(II)-RcnR and NikR, the other Ni(II)-responsive transcriptional regulator in E coli, to provide a better understanding how different nickel levels are sensed in E coli. The characterization of the Ni(II)- and Co(II)-binding sites in RcnR, in combination with bioinformatics analysis of all RcnR/CsoR family members, identified a four amino acid fingerprint that likely defines ligand-binding specificity, leading to an emerging picture of the similarities and differences between different classes of RcnR/CsoR proteins.