NsrR from Streptomyces coelicolor is a nitric oxide-sensing [4Fe-4S] cluster protein with a specialized regulatory function.
NsrR from Streptomyces coelicolor is a nitric oxide-sensing [4Fe-4S] cluster protein with a specialized regulatory function.
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DOI：
10.1074/jbc.m115.643072
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发表时间：
2015-05-15
期刊：
影响因子：
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通讯作者：
Le Brun NE
中科院分区：
文献类型：
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作者：
Crack JC;Munnoch J;Dodd EL;Knowles F;Al Bassam MM;Kamali S;Holland AA;Cramer SP;Hamilton CJ;Johnson MK;Thomson AJ;Hutchings MI;Le Brun NE
背景：NsrR家族蛋白是一类含[2Fe-2S]或[4Fe-4S]簇的全局调节蛋白。结果如下：天蓝色链霉菌NsrR仅调节三个基因，并且它是与NsrR调节的启动子紧密结合的蛋白质的[4Fe-4S]形式。结论：[4Fe-4S] NsrR具有只与一氧化氮应激反应相关的特异性功能。重要性：NsrR家族的成员很可能都是[4Fe-4S]蛋白。Rrf 2家族转录因子NsrR控制多种细菌中响应一氧化氮（NO）的基因表达。NsrR的NO敏感模块的精确形式是争议的主题，因为先前已经观察到含有[2Fe-2S]或[4Fe-4S]簇的NsrR蛋白。光学，穆斯堡尔，共振拉曼光谱和自然质谱表明，天蓝色链霉菌NsrR（ScNsrR），以前报道含有一个[2Fe-2S]簇，可以分离含有一个[4Fe-4S]簇。ChIP-seq实验表明ScNsrR调节子很小，仅由hmpA 1、hmpA 2和ScNsrR本身组成。hmpA基因编码NO解毒黄血红蛋白，表明ScNsrR具有专注于NO解毒的专门调节功能，并且不像一些NsrR直系同源物那样是全局调节剂。EMSA和DNase I足迹法表明，[4Fe-4S]形式的ScNsrR特异性地和紧密地结合到所鉴定的靶基因的启动子区域中的11-bp反向重复序列，并且DNA结合在与NO反应后被消除。共振拉曼数据与三个Cys残基和一个含氧残基的簇配位一致，对ScNsrR变异体的分析表明，高度保守的Glu-85可能是第四个配体。最后，我们证明，一些低分子量的硫醇，但重要的是没有生理相关的硫醇，如半胱氨酸和类似物的mycothiol，结合弱的[4Fe-4S]簇，并暴露这种结合形式的O2导致簇转换为[2Fe-2S]形式，它不结合到DNA。这些数据有助于解释观察到的[2Fe-2S]形式的NsrR。
Background: NsrR family proteins are [2Fe-2S] or [4Fe-4S] cluster-containing global regulators. Results: Streptomyces coelicolor NsrR regulates only three genes, and it is the [4Fe-4S] form of the protein that binds tightly to NsrR-regulated promoters. Conclusion: [4Fe-4S] NsrR has a specialized function associated only with nitric oxide stress response. Significance: Members of the NsrR family are most likely all [4Fe-4S] proteins. The Rrf2 family transcription factor NsrR controls expression of genes in a wide range of bacteria in response to nitric oxide (NO). The precise form of the NO-sensing module of NsrR is the subject of controversy because NsrR proteins containing either [2Fe-2S] or [4Fe-4S] clusters have been observed previously. Optical, Mössbauer, resonance Raman spectroscopies and native mass spectrometry demonstrate that Streptomyces coelicolor NsrR (ScNsrR), previously reported to contain a [2Fe-2S] cluster, can be isolated containing a [4Fe-4S] cluster. ChIP-seq experiments indicated that the ScNsrR regulon is small, consisting of only hmpA1, hmpA2, and nsrR itself. The hmpA genes encode NO-detoxifying flavohemoglobins, indicating that ScNsrR has a specialized regulatory function focused on NO detoxification and is not a global regulator like some NsrR orthologues. EMSAs and DNase I footprinting showed that the [4Fe-4S] form of ScNsrR binds specifically and tightly to an 11-bp inverted repeat sequence in the promoter regions of the identified target genes and that DNA binding is abolished following reaction with NO. Resonance Raman data were consistent with cluster coordination by three Cys residues and one oxygen-containing residue, and analysis of ScNsrR variants suggested that highly conserved Glu-85 may be the fourth ligand. Finally, we demonstrate that some low molecular weight thiols, but importantly not physiologically relevant thiols, such as cysteine and an analogue of mycothiol, bind weakly to the [4Fe-4S] cluster, and exposure of this bound form to O2 results in cluster conversion to the [2Fe-2S] form, which does not bind to DNA. These data help to account for the observation of [2Fe-2S] forms of NsrR.