Institute of Microbiology, Eidgenössiche Technische Hochschule Zurich, 8093 Zurich, Switzerland.
Proc Natl Acad Sci U S A. 2012 May 22;109(21):E1405-14. doi: 10.1073/pnas.1117003109. Epub 2012 May 1.
Reprogramming gene expression is an essential component of adaptation to changing environmental conditions. In bacteria, a widespread mechanism involves alternative sigma factors that redirect transcription toward specific regulons. The activity of sigma factors is often regulated through sequestration by cognate anti-sigma factors; however, for most systems, it is not known how the activity of the anti-sigma factor is controlled to release the sigma factor. Recently, the general stress response sigma factor in Alphaproteobacteria, σ(EcfG), was identified. σ(EcfG) is inactivated by the anti-sigma factor NepR, which is itself regulated by the response regulator PhyR. This key regulator sequesters NepR upon phosphorylation of its PhyR receiver domain via its σ(EcfG) sigma factor-like output domain (PhyR(SL)). To understand the molecular basis of the PhyR-mediated partner-switching mechanism, we solved the structure of the PhyR(SL)-NepR complex using NMR. The complex reveals an unprecedented anti-sigma factor binding mode: upon PhyR(SL) binding, NepR forms two helices that extend over the surface of the PhyR(SL) subdomains. Homology modeling and comparative analysis of NepR, PhyR(SL), and σ(EcfG) mutants indicate that NepR contacts both proteins with the same determinants, showing sigma factor mimicry at the atomic level. A lower density of hydrophobic interactions, together with the absence of specific polar contacts in the σ(EcfG)-NepR complex model, is consistent with the higher affinity of NepR for PhyR compared with σ(EcfG). Finally, by reconstituting the partner switch in vitro, we demonstrate that the difference in affinity of NepR for its partners is sufficient for the switch to occur.
重新编程基因表达是适应环境变化的重要组成部分。在细菌中，一种广泛存在的机制涉及到替代 sigma 因子，这些因子将转录重新定向到特定的调控子。sigma 因子的活性通常通过与其同源反 sigma 因子的隔离来调节；然而，对于大多数系统，尚不清楚如何控制反 sigma 因子的活性以释放 sigma 因子。最近，在α变形菌中的一般应激反应 sigma 因子σ(EcfG)被鉴定出来。σ(EcfG)被反 sigma 因子 NepR 失活，而 NepR 本身又受到响应调节剂 PhyR 的调节。这个关键调节剂通过其 σ(EcfG)sigma 因子样输出结构域 (PhyR(SL)) 对其 PhyR 接收结构域的磷酸化来隔离 NepR。为了了解 PhyR 介导的伙伴转换机制的分子基础，我们使用 NMR 解决了 PhyR(SL)-NepR 复合物的结构。该复合物揭示了一种前所未有的反 sigma 因子结合模式：PhyR(SL)结合后，NepR 形成两条延伸到 PhyR(SL)亚结构域表面的螺旋。对 NepR、PhyR(SL)和 σ(EcfG)突变体的同源建模和比较分析表明，NepR 与这两种蛋白质都具有相同的决定因素，在原子水平上表现出 sigma 因子模拟。疏水性相互作用的密度较低，以及 σ(EcfG)-NepR 复合物模型中缺乏特定的极性接触，与 NepR 与 PhyR 的亲和力高于 σ(EcfG)一致。最后，通过体外重建伙伴转换，我们证明了 NepR 与其伙伴的亲和力差异足以发生转换。
