Generating a Metal-responsive Transcriptional Regulator to Test What Confers Metal Sensing in Cells.
Generating a Metal-responsive Transcriptional Regulator to Test What Confers Metal Sensing in Cells.
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DOI：
10.1074/jbc.m115.663427
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发表时间：
2015-08-07
期刊：
影响因子：
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通讯作者：
Robinson NJ
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文献类型：
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作者：
Osman D;Piergentili C;Chen J;Chakrabarti B;Foster AW;Lurie-Luke E;Huggins TG;Robinson NJ
背景：金属特异性转录与细胞的一组金属传感器的相关性质有关。结果：一种残基取代法使DNA结合甲醛传感器能够检测锌和钴。结论：较弱的DNA亲和力和较强的锌(II)亲和力使锌(II)传感具有较小的偶联自由能。意义：相对亲和力决定了锌(II)的最佳传感器，而不是钴。来自鼠伤寒沙门氏菌(一种CSOR/RcnR样转录抑制因子)的frmR被证明抑制了frmRA操纵子-启动子，甲醛但不能减轻细胞内的锰、铁、钴、镍、铜或锌(II)的抑制作用。相比之下，突变的FrmRE64H(获得了RcnR金属配体)的抑制作用可被钴和锌(II)缓解。出乎意料的是，FRMR已经被发现与Co(II)、Zn(II)和Cu(I)结合，而且金属和甲醛都会触发变构反应，从而削弱DNA亲和力。然而，细胞内源金属传感器(RcnR、ZntR、Zur和CueR)的感觉金属位置对于它们的同源金属来说都比FrmR更紧密。此外，内源金属传感器在竞争中胜过FRMR。金属敏感型FrmRE64H突变体与金属的亲和力比frmR高约1个数量级。由于FrmRE64H的钴亲和力明显弱于内源性钴传感器，因此FrmRE64H的钴传感增益仍然是个谜。钴传感需要谷胱甘肽，谷胱甘肽可能有助于钴的获取，从而赋予动力学优势。对于锌(II)，FrmRE64H的金属亲和力接近同源锌(II)传感器的金属亲和力。与直觉相反的是，在金属敏感的FrmRE64H中，锌(II)的变构耦合自由能比非金属敏感的FrmRE64H要小。通过测定每个细胞的frmR和FrmRE64H四聚体的拷贝数，然后估计启动子的占有率作为细胞内锌(II)浓度的函数，我们展示了锌(II)亲和力的适度收紧，加上载脂蛋白的DNA亲和力减弱，如何共同作用使FrmRE64H的相对性质(与ZntR和Zur相比)足以在细胞内检测锌(II)。
Background: Metal-specific transcription has been correlated with the relative properties of a cells' set of metal sensors. Results: A one-residue substitution enabled a DNA-binding formaldehyde sensor to detect Zn(II) and cobalt. Conclusion: Weaker DNA affinity combined with tighter Zn(II) affinity enabled Zn(II) sensing with a smaller coupling free energy. Significance: Relative affinity determined the best sensor in the set for Zn(II) but not for cobalt. FrmR from Salmonella enterica serovar typhimurium (a CsoR/RcnR-like transcriptional de-repressor) is shown to repress the frmRA operator-promoter, and repression is alleviated by formaldehyde but not manganese, iron, cobalt, nickel, copper, or Zn(II) within cells. In contrast, repression by a mutant FrmRE64H (which gains an RcnR metal ligand) is alleviated by cobalt and Zn(II). Unexpectedly, FrmR was found to already bind Co(II), Zn(II), and Cu(I), and moreover metals, as well as formaldehyde, trigger an allosteric response that weakens DNA affinity. However, the sensory metal sites of the cells' endogenous metal sensors (RcnR, ZntR, Zur, and CueR) are all tighter than FrmR for their cognate metals. Furthermore, the endogenous metal sensors are shown to out-compete FrmR. The metal-sensing FrmRE64H mutant has tighter metal affinities than FrmR by approximately 1 order of magnitude. Gain of cobalt sensing by FrmRE64H remains enigmatic because the cobalt affinity of FrmRE64H is substantially weaker than that of the endogenous cobalt sensor. Cobalt sensing requires glutathione, which may assist cobalt access, conferring a kinetic advantage. For Zn(II), the metal affinity of FrmRE64H approaches the metal affinities of cognate Zn(II) sensors. Counter-intuitively, the allosteric coupling free energy for Zn(II) is smaller in metal-sensing FrmRE64H compared with nonsensing FrmR. By determining the copies of FrmR and FrmRE64H tetramers per cell, then estimating promoter occupancy as a function of intracellular Zn(II) concentration, we show how a modest tightening of Zn(II) affinity, plus weakened DNA affinity of the apoprotein, conspires to make the relative properties of FrmRE64H (compared with ZntR and Zur) sufficient to sense Zn(II) inside cells.