Nickel binding to NikA: an additional binding site reconciles spectroscopy, calorimetry and crystallography
Nickel binding to NikA: an additional binding site reconciles spectroscopy, calorimetry and crystallography
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DOI：
10.1107/s0907444906048712
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发表时间：
2007-02-01
影响因子：
2.2
通讯作者：
Heddle, Jonathan G.
中科院分区：
文献类型：
--
作者：
Addy, Christine;Ohara, Masato;Heddle, Jonathan G.
大肠杆菌需要细胞内镍作为许多酶的辅助因子，并且是厌氧呼吸所必需的。然而，高浓度的镍是有毒的，因此进出口系统已经发展到控制金属的细胞水平。大肠杆菌中的nik操纵子编码一个镍摄取系统，其中包括质周镍结合蛋白NikA。野生型NikA的晶体结构既与镍结合，又以载子形式存在。配体结构似乎显示了镍和蛋白质之间不寻常的相互作用，其中没有形成直接的键。晶体结构所显示的极不寻常的镍配位与早期的x射线光谱研究形成强烈对比。通过广泛的诱变和等温滴定量热法探测了已知的镍结合位点，发现即使大量的破坏性突变对镍的亲和力也几乎没有影响。镍结合位点突变体的晶体结构已被解决，并发现镍在远离先前表征的结合位点的位置被结合到两个组氨酸残基上。这个新颖的位点立即解决了晶体结构和其他生物物理分析之间的冲突。讨论了这两个结合位点的生理相关性。
Intracellular nickel is required by Escherichia coli as a cofactor for a number of enzymes and is necessary for anaerobic respiration. However, high concentrations of nickel are toxic, so both import and export systems have evolved to control the cellular level of the metal. The nik operon in E. coli encodes a nickel-uptake system that includes the periplasmic nickel-binding protein NikA. The crystal structures of wildtype NikA both bound to nickel and in the apo form have been solved previously. The liganded structure appeared to show an unusual interaction between the nickel and the protein in which no direct bonds are formed. The highly unusual nickel coordination suggested by the crystal structure contrasted strongly with earlier X-ray spectroscopic studies. The known nickel-binding site has been probed by extensive mutagenesis and isothermal titration calorimetry and it has been found that even large numbers of disruptive mutations appear to have little effect on the nickel affinity. The crystal structure of a binding-site mutant with nickel bound has been solved and it is found that nickel is bound to two histidine residues at a position distant from the previously characterized binding site. This novel site immediately resolves the conflict between the crystal structures and other biophysical analyses. The physiological relevance of the two binding sites is discussed.