Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
PLoS One. 2012;7(8):e43676. doi: 10.1371/journal.pone.0043676. Epub 2012 Aug 24.
The alternate sigma factor sigH of Mycobacterium tuberculosis is expressed under stress and acts as a major regulator of several genes, including some other sigma factors and redox systems. While it is auto-regulated by its own promoter at the transcriptional level, its regulation at the post-translational level is through its cognate protein, an anti-sigma factor, RshA. Hither before RshA was believed to be a zinc-associated anti-sigma factor (ZAS) and the binding of RshA to SigH is redox dependent. Here, we show that RshA coordinates a [2Fe-2S] cluster using cysteines as ligands and native RshA has more affinity to [2Fe-2S] cluster than to zinc. Furthermore, we used amide hydrogen deuterium exchange mass spectrometry (HDX-MS), followed by site-directed mutagenesis in SigH and RshA, to elucidate the interaction mechanism of RshA and SigH and the potential role of metal ion clustering in SigH regulation. Three regions in SigH, comprising of residues 1-25, 58-69, 90-111, 115-132 and 157-196 and residues 35-57 of RshA show decreased deuterium exchange and reflect decreased solvent accessibility upon complexation with SigH. Of the three RshA mutants, created based on the HDX results, the RsHA E37A mutant shows stronger interaction with SigH, relative to WT RshA, while the H49A mutant abolishes interactions and the C(53)XXC(56)AXXA mutant has no effect on complexation with SigH. The D22A, D160A and E162 SigH mutants show significantly decreased binding to RshA and the E168A mutant completely abolished interactions with RshA, indicating that the SigH-RshA interaction is mediated by salt bridges. In addition, SigH-RshA interaction does not require clustering of metal ions. Based on our results, we propose a molecular model of the SigH-RshA interaction.
结核分枝杆菌的替代σ因子 SigH 在应激下表达，作为几个基因的主要调节剂，包括其他一些σ因子和氧化还原系统。虽然它在转录水平上通过自身启动子进行自我调节，但在翻译后水平上的调节是通过其同源蛋白，一种反σ因子 RshA 进行的。在此之前，RshA 被认为是一种锌相关的反σ因子（ZAS），并且 RshA 与 SigH 的结合是依赖于氧化还原的。在这里，我们表明 RshA 使用半胱氨酸作为配体来协调 [2Fe-2S] 簇，并且天然 RshA 对 [2Fe-2S] 簇的亲和力比对锌的亲和力更强。此外，我们使用酰胺氢氘交换质谱（HDX-MS），然后对 SigH 和 RshA 进行定点突变，以阐明 RshA 和 SigH 的相互作用机制，以及金属离子簇在 SigH 调节中的潜在作用。SigH 中的三个区域，包括残基 1-25、58-69、90-111、115-132 和 157-196，以及 RshA 的残基 35-57，在与 SigH 复合时显示出较低的氘交换，反映出较低的溶剂可及性。在基于 HDX 结果创建的三个 RshA 突变体中，与 WT RshA 相比，RshA E37A 突变体与 SigH 的相互作用更强，而 H49A 突变体则消除了相互作用，C(53)XXC(56)AXXA 突变体对与 SigH 的复合没有影响。D22A、D160A 和 E162 SigH 突变体与 RshA 的结合显著降低，而 E168A 突变体完全消除了与 RshA 的相互作用，表明 SigH-RshA 相互作用是通过盐桥介导的。此外，SigH-RshA 相互作用不需要金属离子的聚类。基于我们的结果，我们提出了 SigH-RshA 相互作用的分子模型。
