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

含铁硫簇的传感器调节器：DNA 结合的机制和结构研究

基本信息

批准号：
BB/V006851/1
负责人：
Nicolas Le Brun
金额：
$ 61.45万
依托单位：
University of East Anglia
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2022
资助国家：
英国
起止时间：
2022 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FV006851%2F1
关键词：
Iron sulfur cluster containing sensor

项目摘要

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.

为了生存，细菌必须能够感知并适应其（不断变化的）环境，其中包括试图在人类宿主中建立感染的调节蛋白，这些蛋白通过与 DNA 结合来控制基因转录，在感知中发挥着关键作用。许多这些蛋白质含有一种特殊的辅助因子，由铁和硫组成，称为铁硫簇，在调节蛋白中，该簇起到传感器的作用，通过发生反应来检测特定信号。导致蛋白质构象变化。引起 DNA 结合的变化打开或关闭，从而转导原始信号以产生必要的细胞反应。Rrf2 调节因子家族广泛存在于细菌中，控制着一些最重要的细胞途径，包括铁代谢、生物合成许多 Rrf2 家族调节因子结合铁硫簇辅助因子，并且对氧化和亚硝化应激做出反应，并且该簇的反应性支撑了铁硫簇辅助因子的传感功能。事实证明，尽管 Rrf2 蛋白在序列和整体结构方面似乎彼此相似，但它们结合的簇类型以及结合方式因一种 Rrf2 蛋白而异。我们在理解这些调节剂的机制和结构特征方面取得了很多进展，包括其中两个簇结合形式的第一个结构，这揭示了簇如何与以前在其他铁硫中观察到的蛋白质结合的特征。我们的工作。还导致人们对簇如何与其特定信号分子（例如细胞毒素一氧化氮）反应以及该反应如何导致蛋白质形状变化（可能影响蛋白质结合 DNA 的能力）的详细功能了解。尽管最近取得了进展，但我们对 Rrf2 家族蛋白与 DNA 的相互作用以及它如何影响对信号分子的反应仍然知之甚少，由于 DNA 溶液的粘度以及目前可用的几乎所有相关信息，这很难在溶液中进行研究。到我们开发了一种称为质谱分析技术的应用，它可以为非常大的分子（例如与其辅因子结合的蛋白质）提供准确的质量信息，这为铁硫簇的反应提供了前所未有的见解。现在我们已经成功地建立了可以检测 Rrf2 蛋白与 DNA 结合的条件，其中所需的低浓度意味着粘度不是问题，这为通过研究它们的结合来获得对这些调节蛋白的基本了解提供了可能性。 DNA 和这将使我们能够解决目前其他方法无法解决的问题，例如，我们将能够确定在传感反应中什么时候发生形状变化，从而关闭 DNA 结合。我们还成功确定了 DNA 结合形式的两个 Rrf2 调节剂的 3-4 Å 分辨率结构，这开始揭示蛋白质的特定形状以及蛋白质与 DNA 之间相互作用点的细节。尽管本质上是基础性的，但将显着增进对如何感知细菌并克服恶劣条件，包括它们在试图在宿主体内建立感染时遇到的条件。