Structure-function relationships of the Mycobacterium tuberculosis transcription factor WhiB1.
Structure-function relationships of the Mycobacterium tuberculosis transcription factor WhiB1.
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DOI：
10.1371/journal.pone.0040407
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Green J
中科院分区：
文献类型：
--
作者：
Smith LJ;Stapleton MR;Buxton RS;Green J
类WhiB蛋白家族成员具有铁硫簇，参与放线菌发育过程的调控。结核分枝杆菌具有七种Wbl蛋白。M.结核病WhiB 1对O2相对不敏感，但对一氧化氮（NO）非常敏感。一氧化氮亚硝基化WhiB 1铁硫簇并促进DNA结合; WhiB 1的脱辅基形式也结合DNA。然而，铁硫簇收购和DNA结合的WhiB 1的分子要求的特点很差。通过定点突变产生了WhiB 1变体，并评估了相应蛋白质获得铁硫簇和/或与WhiB 1启动子DNA结合的能力。WhiB 1 N-末端区域的所有四个Cys残基（Cys 9、37、40和46）都是掺入[4Fe-4S]簇所必需的，而可能的替代簇配体Asp 13（通过与M. smegalopathy WhiB 2）不是。预测WhiB 1的C-末端区域包含蛋白质的DNA结合结构域，该结构域由预测的β-转角（58 GVWGG 62）和随后的两个氨基酸基序（72 KRRN 75和78 TKAR 81）组成，这两个基序在WhiB 1蛋白中是保守的。β-转角的Gly残基（Gly 58、61和62）和下游保守区的正电荷残基（Lys 72、Arg 73、Arg 74、Lys 79和Arg 81）是WhiB 1 DNA结合所必需的。对M.结核病whiB 1和表征相应的蛋白质已被用来探讨NO反应性转录因子WhiB 1的结构与功能的关系。这表明N-末端区域中的所有四个保守的Cys残基对于铁-硫簇的掺入是必需的，但对于DNA结合不是必需的。在C-末端区域的氨基酸取代的变体的分析揭示了预测的β-转角和两个保守的带正电荷的基序在促进DNA结合中发挥的关键作用，但不是铁-硫簇收购，由WhiB 1。
Members of the WhiB-like (Wbl) protein family possess iron-sulfur clusters and are implicated in the regulation of developmental processes in Actinomycetes. Mycobacterium tuberculosis possesses seven Wbl proteins. The [4Fe-4S] cluster of M. tuberculosis WhiB1 is relatively insensitive to O2 but very sensitive to nitric oxide (NO). Nitric oxide nitrosylates the WhiB1 iron-sulfur cluster and promotes DNA-binding; the apo-forms of WhiB1 also bind DNA. However, the molecular requirements for iron-sulfur cluster acquisition and for DNA-binding by WhiB1 are poorly characterized. WhiB1 variants were created by site-directed mutagenesis and the abilities of the corresponding proteins to acquire an iron-sulfur cluster and/or bind to whiB1 promoter DNA were assessed. All four Cys residues (Cys9, 37, 40, and 46) in the N-terminal region of WhiB1 were required for incorporation of a [4Fe-4S] cluster, whereas a possible alternative cluster ligand Asp13 (by analogy with M. smegmatis WhiB2) was not. The C-terminal region of WhiB1 is predicted to house the DNA-binding domain of the protein consisting of a predicted β-turn (58GVWGG62) followed by two amino acid motifs (72KRRN75 and 78TKAR81) that are conserved in WhiB1 proteins. Gly residues (Gly58, 61 and 62) in the β-turn and positively-charged residues (Lys72, Arg73, Arg74, Lys79 and Arg81) in the downstream conserved regions were required for binding of WhiB1 DNA. Site-directed mutagenesis of M. tuberculosis whiB1 and characterization of the corresponding proteins has been used to explore structure-function relationships of the NO-responsive transcription factor WhiB1. This showed that all four conserved Cys residues in the N-terminal region are required for incorporation of iron-sulfur clusters but not for DNA-binding. Analysis of variants with amino acid substitutions in the C-terminal region revealed the crucial roles played by a predicted β-turn and two conserved positively-charged motifs in facilitating DNA-binding, but not iron-sulfur cluster acquisition, by WhiB1.
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DOI：
10.1073/pnas.0700490104
发表时间：
2007-07-10
影响因子：
11.1
作者：
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通讯作者：
Steyn, Adrie J. C.
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作者：
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DOI：
10.1042/bj20101440
发表时间：
2010-12-15
期刊：
The Biochemical journal
影响因子：
--
作者：
Smith LJ;Stapleton MR;Fullstone GJ;Crack JC;Thomson AJ;Le Brun NE;Hunt DM;Harvey E;Adinolfi S;Buxton RS;Green J
通讯作者：
Green J