The Effectors and Sensory Sites of Formaldehyde-responsive Regulator FrmR and Metal-sensing Variant.
The Effectors and Sensory Sites of Formaldehyde-responsive Regulator FrmR and Metal-sensing Variant.
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甲醛反应调节剂FRMR和金属感应变体的效应子和感觉位点。
DOI：
10.1074/jbc.m116.745174
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发表时间：
2016-09-09
期刊：
影响因子：
--
通讯作者：
Pohl E
中科院分区：
文献类型：
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作者：
Osman D;Piergentili C;Chen J;Sayer LN;Usón I;Huggins TG;Robinson NJ;Pohl E
DUF 156家族的DNA结合转录调节因子包括响应于钴和/或镍（RcnR，InrS）或铜（CsoR）的金属传感器加上响应于过硫化物的CstR和甲醛响应性FrmR。出乎意料的是，来自沙门氏菌鼠伤寒血清型的FrmR的变构机制在体外由金属触发，并且变体FrmRE 64 H在体内获得对Zn（II）和钴的响应性。在这里，我们建立了FrmR的变构机制是由甲醛在体外直接触发。对甲醛的敏感性需要在所有DUF 156蛋白中保守的半胱氨酸（FrmR中的Cys 35）。金属和甲醛去敏感FrmRE 64 H的晶体结构揭示了FrmR特异性氨基末端Pro 2邻近Cys 35，并且这些残基形成了推断的甲醛去敏感位点。有证据表明，空间上接近保守的半胱氨酸的残基调整DUF 156蛋白的敏感性高于或低于不同效应子的临界阈值，在细胞内产生特异性的信号。相对于FrmR，RcnR在体外对甲醛的反应性较低，并且RcnR在体内不感测甲醛，但是相互突变FrmRP 2S和RcnRS 2 P分别损害和增强甲醛在体外的反应性。甲醛解毒FrmA需要S-（羟甲基）谷胱甘肽，但谷胱甘肽抑制甲醛检测FrmR在体内和体外。定量每个细胞中FrmR分子的数量并将甲醛修饰建模为[甲醛]的函数，表明FrmR反应性得到优化，使得FrmR被修饰，并且frmRA在比生成S-（羟甲基）谷胱甘肽所需的更低的[甲醛]下被去阻遏。因此，FrmA的表达与其底物的积累相协调。
The DUF156 family of DNA-binding transcriptional regulators includes metal sensors that respond to cobalt and/or nickel (RcnR, InrS) or copper (CsoR) plus CstR, which responds to persulfide, and formaldehyde-responsive FrmR. Unexpectedly, the allosteric mechanism of FrmR from Salmonella enterica serovar Typhimurium is triggered by metals in vitro, and variant FrmRE64H gains responsiveness to Zn(II) and cobalt in vivo. Here we establish that the allosteric mechanism of FrmR is triggered directly by formaldehyde in vitro. Sensitivity to formaldehyde requires a cysteine (Cys35 in FrmR) conserved in all DUF156 proteins. A crystal structure of metal- and formaldehyde-sensing FrmRE64H reveals that an FrmR-specific amino-terminal Pro2 is proximal to Cys35, and these residues form the deduced formaldehyde-sensing site. Evidence is presented that implies that residues spatially close to the conserved cysteine tune the sensitivities of DUF156 proteins above or below critical thresholds for different effectors, generating the semblance of specificity within cells. Relative to FrmR, RcnR is less responsive to formaldehyde in vitro, and RcnR does not sense formaldehyde in vivo, but reciprocal mutations FrmRP2S and RcnRS2P, respectively, impair and enhance formaldehyde reactivity in vitro. Formaldehyde detoxification by FrmA requires S-(hydroxymethyl)glutathione, yet glutathione inhibits formaldehyde detection by FrmR in vivo and in vitro. Quantifying the number of FrmR molecules per cell and modeling formaldehyde modification as a function of [formaldehyde] demonstrates that FrmR reactivity is optimized such that FrmR is modified and frmRA is derepressed at lower [formaldehyde] than required to generate S-(hydroxymethyl)glutathione. Expression of FrmA is thereby coordinated with the accumulation of its substrate.