Iron-sulfur cluster-containing sensor regulators: mechanistic and structural studies of DNA-binding

为了生存，细菌必须能够感知并适应(不断变化的)环境。这包括试图在人类宿主中建立感染的病原体。调控蛋白通过与DNA特异结合来控制基因转录，在感知和响应环境变化方面发挥着关键作用。其中许多蛋白质含有一种特殊的辅因子，由铁和硫组成，称为铁-硫簇。在调节蛋白质中，这个簇起到传感器的作用，它通过经历导致蛋白质构象变化的反应来检测特定的信号。这些变化导致DNA结合的开启或关闭，从而传递原始信号以产生必要的细胞反应。Rrf2家族调控着细菌中广泛存在的一些最重要的细胞途径，包括铁代谢、铁-硫簇辅因子的生物合成以及对氧化和亚硝化胁迫的反应。许多RRF2家族调控因子结合一个铁-硫簇辅助因子，该簇的反应性支撑了调控因子的传感功能。结果表明，尽管Rrf2蛋白在序列和总体结构上看起来彼此相似，但它们结合的簇类型和结合方式因Rrf2蛋白而异。最近，我们在理解这些调控因子的机制和结构特征方面取得了很大进展，包括其中两个调控因子簇结合形式的第一结构。这揭示了该簇如何与以前在其他铁-硫簇蛋白中未观察到的蛋白质结合的特征。我们的工作也导致了对该簇如何与其特定的信号分子(例如细胞毒素一氧化氮)反应的详细功能理解，以及这种反应如何导致蛋白质形状的变化，从而可能影响蛋白质与DNA的结合能力。尽管有这些最近的进展，我们仍然对Rrf2家族蛋白质与DNA的相互作用以及这如何影响对信号分子的反应知之甚少。由于DNA溶液的粘度，这很难在溶液中研究，因此目前几乎所有可用的信息都与非DNA结合形式有关。我们已经开发了一种被称为质谱学的分析技术的应用，该技术为具有辅因子结合的蛋白质等非常大的分子提供准确的质量信息。这为铁硫簇协因子的反应提供了前所未有的洞察力。现在，我们已经成功地建立了可以检测到与DNA结合的RRF2蛋白的条件，在这种条件下，所需的低浓度意味着粘度不是问题。这为通过研究它们与DNA的结合以及当它们以DNA结合的形式时的反应性来获得对这些调节蛋白的基本了解打开了可能性。这将使我们能够解决目前无法通过其他方法解决的问题。例如，我们将能够确定关闭DNA结合的传感反应形状变化发生在哪个时间点。我们还成功地以DNA结合的形式确定了两个RRF2调节剂的3-4？分辨率结构，这开始揭示蛋白质的特殊形状和蛋白质与DNA之间的相互作用点的细节。这项工作虽然是基础性的，但将大大促进对细菌如何感知和克服不适宜居住条件的理解，包括它们在试图建立宿主感染时遇到的那些条件。

In order to survive, bacteria must be able to sense and adapt to their (changing) environment. This includes pathogens trying to establish infection in a human host. Regulatory proteins, which control gene transcription by specifically binding to DNA, play key roles in sensing and responding to environmental change. Many of these proteins contain a special cofactor, consisting of iron and sulfur, called an iron-sulfur cluster. In regulatory proteins, this cluster functions as the sensor, where it detects a particular signal by undergoing a reaction that leads to protein conformational changes. These changes cause DNA-binding to be turned on or off, and thus transduce the original signal to produce the necessary cellular response.The Rrf2 family of regulators is widespread amongst bacteria and controls some of the most important cellular pathways, including iron metabolism, the biosynthesis of iron-sulfur cluster cofactors, and responses to oxidative and nitrosative stresses. Many Rrf2 family regulators bind an iron-sulfur cluster cofactor, and the reactivity of this cluster underpins the sensing function of the regulator. It turns out that, although Rrf2 proteins appear to be similar to one another in terms of sequence and overall structure, the type of cluster they bind, and the way that they bind it, varies from one Rrf2 protein to another.Recently, we have made a lot of progress in understanding mechanistic and structural features of these regulators, including the first structures of cluster bound forms of two of them. This revealed features of how the cluster is bound to the protein not previously observed in other iron-sulfur cluster proteins. Our work has also led to detailed functional understanding of how the cluster reacts with its particular signal molecule, for example the cytotoxin nitric oxide, and how this reaction leads to changes in shape of the protein that are likely to affect the protein's ability to bind DNA.Despite this recent progress, we still know relatively little about the interaction of Rrf2 family proteins with DNA and how this affects the response to signaling molecules. This is difficult to study in solution because of the viscosity of DNA solutions and so nearly all information currently available relates to non-DNA-bound forms. We have developed the application of an analytical technique called mass spectrometry, which provides accurate mass information for very large molecules such as proteins with their cofactors bound. This has provided unprecedented insight into the reactions of iron-sulfur cluster cofactors. Now we have succeeded in establishing conditions under which Rrf2 proteins bound to DNA can be detected, where the low concentrations necessary mean that viscosity is not a problem. This opens up the possibility to gain fundamental insight into these regulatory proteins by studying their binding to DNA and reactivity when in their DNA-bound forms. This will enable us to address questions that cannot be tackled currently by other methods. For example, we will be able to determine at what point in the sensing reaction shape changes that turn off DNA binding occur. We have also succeeded in determining 3-4 Å resolution structures of two Rrf2 regulators in DNA-bound forms, and this is beginning to reveal details of the particular shape of the protein and the points of interaction between the protein the DNA. This work, although fundamental in nature, will significantly advance understanding of how bacteria sense and overcome inhospitable conditions, including those that they encounter when trying to establish infection in a host.