Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom.
PLoS One. 2012;7(7):e40407. doi: 10.1371/journal.pone.0040407. Epub 2012 Jul 5.
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 O(2) 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 ((58)GVWGG(62)) followed by two amino acid motifs ((72)KRRN(75) and (78)TKAR(81)) 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.
WhiB 样（Wbl）蛋白家族的成员拥有铁硫簇，并且与放线菌发育过程的调控有关。结核分枝杆菌拥有 7 种 Wbl 蛋白。结核分枝杆菌 WhiB1 的 [4Fe-4S] 簇对 O(2)相对不敏感，但对一氧化氮（NO）非常敏感。一氧化氮亚硝化为 WhiB1 的铁硫簇并促进 DNA 结合；WhiB1 的脱辅基形式也能与 DNA 结合。然而，铁硫簇获取和 WhiB1 与 DNA 结合的分子要求尚未得到很好的描述。
通过定点突变创建 WhiB1 变体，并评估相应蛋白获取铁硫簇和/或与 whiB1 启动子 DNA 结合的能力。WhiB1 N 端区域的四个 Cys 残基（Cys9、37、40 和 46）都需要用于掺入 [4Fe-4S] 簇，而可能的替代簇配体 Asp13（与 M. smegmatis WhiB2 类似）则不需要。WhiB1 的 C 端区域预计包含该蛋白的 DNA 结合域，由一个预测的 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，这些基序在 WhiB1 蛋白中是保守的。β 转角中的 Gly 残基（Gly58、61 和 62）和下游保守区域中的带正电荷残基（Lys72、Arg73、Arg74、Lys79 和 Arg81）对于 WhiB1 DNA 的结合是必需的。
通过对结核分枝杆菌 whiB1 的定点突变和相应蛋白的特性分析，探索了对 NO 反应性转录因子 WhiB1 的结构-功能关系。结果表明，N 端区域的四个保守 Cys 残基都需要用于铁硫簇的掺入，但不需要用于 DNA 结合。对 C 端区域具有氨基酸取代的变体的分析表明，预测的 β 转角和两个保守的带正电荷的基序在促进 WhiB1 的 DNA 结合方面发挥了至关重要的作用，而不是铁硫簇的获取。
