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

• 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

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（变化）环境。这包括试图在人类宿主中建立感染的病原体。通过特异性结合DNA来控制基因转录的调节蛋白在灵敏度和对环境变化的反应中起关键作用。这些蛋白质中的许多含有特殊的辅助因子，由铁和硫组成，称为铁硫簇。在调节蛋白中，该簇作为传感器起作用，在该传感器中，它通过经历导致蛋白质构象变化的反应来检测特定信号。 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.许多RRF2家族调节剂结合了铁硫簇辅助因子，该簇的反应性支撑了调节剂的灵敏度函数。事实证明，尽管RRF2蛋白在序列和整体结构，它们绑定的群集的类型以及它们结合的方式方面似乎相似，并且从一种RRF2蛋白变化到另一种。这揭示了该簇如何与以前在其他铁硫簇蛋白中观察到的蛋白质结合的特征。我们的工作还导致了对簇如何与其特定信号分子反应的详细功能理解，例如细胞毒素一氧化氮，以及该反应如何导致蛋白质形状的变化，这些蛋白质形状会影响蛋白质结合DNA的能力。尽管如此，尽管最近的进展可能对rrf2家族蛋白的相互作用对rrf2家族的相互作用相关，但对rrf2家族蛋白的相互作用对DNA的反应对DNA的反应构成了对DNA的反应，而不是对DNA的反应效果。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称为质谱的分析技术的应用，该技术为非常大的分子（例如蛋白质及其辅助因子结合）提供了准确的质量信息。这为铁硫簇辅因子的反应提供了前所未有的见解。现在，我们成功地建立了可以检测到与DNA结合的RRF2蛋白的条件，而低浓度的必要浓度意味着粘度不是问题。这打开了通过研究与DNA结合形式时与DNA的结合和反应性的结合，从而为这些调节蛋白提供了基本见解的可能性。这将使我们能够解决其他方法当前无法解决的问题。例如，我们将能够确定在传感反应形状变化中关闭DNA结合的位置。我们还成功地确定了DNA结合形式的两个RRF2调节剂的3-4Å分辨率结构，这开始揭示蛋白质特定形状的细节以及DNA蛋白质之间的相互作用点。尽管本质上的基本基础，但会大大促进细菌的感知和克服荒凉的疾病，包括试图在宿主中建立感染时遇到的疾病。

项目成果

Probing the Reactivity of [4Fe-4S] Fumarate and Nitrate Reduction (FNR) Regulator with O2 and NO: Increased O2 Resistance and Relative Specificity for NO of the [4Fe-4S] L28H FNR Cluster

• DOI: 10.3390/inorganics11120450
• 发表时间: 2023-12-01
• 期刊: INORGANICS
• 影响因子: 2.9
• 作者: Crack，Jason C.;Amara，Patricia;Le Brun，Nick E.
• 通讯作者: Le Brun，Nick E.

Structural determinants of DNA recognition by the NO sensor NsrR and related Rrf2-type [FeS]-transcription factors.

• DOI: 10.1038/s42003-022-03745-7
• 发表时间: 2022-07-30
• 期刊: COMMUNICATIONS BIOLOGY
• 影响因子: 5.9
• 作者: Rohac, Roman;Crack, Jason C.;de Rosny, Eve;Gigarel, Oceane;Le Brun, Nick E.;Fontecilla-Camps, Juan C.;Volbeda, Anne
• 通讯作者: Volbeda, Anne