Mechanistic and Structural Insights into NO sensing by Iron-Sulfur Cluster Regulators

铁硫簇调节器对 NO 传感的机理和结构见解

基本信息

批准号：
BB/P006140/1
负责人：
Nicolas Le Brun
金额：
$ 51.41万
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：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FP006140%2F1
关键词：
Mechanistic Structural Insights into NO

项目摘要

Nitric oxide (NO) is a toxic molecule that is generated by soil bacteria and in our bodies as a defence against pathogenic organisms trying to establish infection. One of the major ways by which NO exerts its toxic effects is through reaction with a widespread group of proteins that bind a type of cofactor containing both iron and sulfur arranged as a cluster. Members of this group play crucial roles in a very wide range of processes, including respiration and protein synthesis. To avoid NO toxicity, pathogenic (as well as harmless) organisms have evolved protective systems that detoxify NO by removing it through chemical reaction. The fact that iron-sulfur clusters are particularly sensitive to NO (and their modification is a major route by which NO exerts its toxic effects) has been exploited in nature, through the evolution of a number of regulatory proteins that themselves contain an iron-sulfur cluster and which function as biological switches, turning on the cellular detoxification response in the presence of NO. Despite the importance and widespread nature of the reaction of iron-sulfur clusters with NO, we still know relatively little about this process. Some important progress has been made in recent years, but the difficulties associated with working with iron-sulfur proteins, which are fragile and must be handled in O2-free environments, and with detecting and unambiguously identifying intermediates and products of the cluster reaction with NO have, up to now, been major obstacles.The project described in this proposal will lead to a major advance in our understanding of how NO-responsive iron-sulfur cluster-containing regulators function. The major subject of our proposed study is an iron-sulfur cluster regulator that is a member of a large and not well understood family of regulators found in a wide range of pathogenic and non-pathogenic bacteria, in which it functions as a primary NO sensor by controlling the cellular response to NO toxicity. We will also study a second regulatory protein that belongs to a family found only in a small number of bacteria, but which includes the pathogen that causes tuberculosis, one of the world's major killers, and the bacterium that is the source of many of the antibiotics currently in use in the clinic. Members of this family play key roles in cell developmental processes associated with stress response, including sporulation and dormancy, which is important for the ability of the tuberculosis pathogen to survive in the inhospitable environment of a human host for years, in a state that is highly resistant to antibiotics.The project will build on three important recent breakthroughs. Firstly, we have established novel mass spectrometry methodologies that enable us to detect iron-sulfur cluster regulators with their clusters intact. This now provides the opportunity to follow by mass spectrometry the reaction of the cluster with NO by detecting and identifying intermediates and products formed. Secondly, we have developed novel ways of studying the same proteins using vibrational spectroscopy, providing characteristic signatures according to the iron-NO complexes formed. Finally, working with a group in France, we have determined the high resolution structure of one of the regulators with its iron-sulfur cluster bound. This is a first for this family of iron-sulfur cluster regulators and provides the ideal basis on which to understand how the cluster promotes DNA binding and how it reacts with NO. We will exploit these recent advances to explore using a range of approaches the biochemistry of the reaction of NO with these proteins, revealing unprecedented mechanistic insight into how NO-sensing regulatory proteins function, and providing clues about how NO sensing, and therefore survival, of pathogens could be disrupted/prevented.

一氧化氮 (NO) 是一种有毒分子，由土壤细菌产生，在我们体内用于防御试图建立感染的病原生物。 NO 发挥毒性作用的主要方式之一是与一组广泛存在的蛋白质发生反应，这些蛋白质结合一种含有铁和硫的辅助因子，排列成簇。该组的成员在非常广泛的过程中发挥着至关重要的作用，包括呼吸和蛋白质合成。为了避免 NO 毒性，致病（以及无害）生物体已经进化出保护系统，通过化学反应去除 NO 来解毒。事实上，铁硫簇对 NO 特别敏感（它们的修饰是 NO 发挥毒性作用的主要途径），这一事实已在自然界中得到利用，通过许多本身含有铁硫簇的调节蛋白的进化簇并起到生物开关的作用，在 NO 存在的情况下开启细胞解毒反应。尽管铁硫簇与 NO 的反应非常重要且广泛，但我们对这一过程仍然知之甚少。近年来取得了一些重要进展，但与铁硫蛋白的研究相关的困难，铁硫蛋白很脆弱，必须在无氧环境中处理，以及检测和明确识别与 NO 的簇反应的中间体和产物到目前为止，这一直是主要障碍。该提案中描述的项目将导致我们对 NO 响应性铁硫簇调节剂如何发挥作用的理解取得重大进展。我们提出的研究的主要主题是铁硫簇调节剂，它是在多种致病性和非致病性细菌中发现的一个大的、尚未被充分了解的调节剂家族的成员，其中它充当主要的一氧化氮传感器通过控制细胞对 NO 毒性的反应。我们还将研究第二种调节蛋白，该蛋白属于仅在少数细菌中发现的家族，但该家族包括导致结核病（世界主要杀手之一）的病原体，以及作为许多抗生素来源的细菌目前在诊所使用。该家族的成员在与应激反应相关的细胞发育过程中发挥着关键作用，包括孢子形成和休眠，这对于结核病病原体在人类宿主的恶劣环境中生存多年的能力非常重要。该项目将建立在最近的三个重要突破的基础上。首先，我们建立了新颖的质谱方法，使我们能够检测簇完整的铁硫簇调节剂。现在，这提供了通过检测和识别形成的中间体和产物，通过质谱法跟踪簇与 NO 的反应的机会。其次，我们开发了使用振动光谱研究相同蛋白质的新方法，根据形成的铁-NO 复合物提供特征信号。最后，我们与法国的一个小组合作，确定了其中一个调节剂及其铁硫簇结合的高分辨率结构。这是铁硫簇调节剂家族中的首例，为了解簇如何促进 DNA 结合以及它如何与 NO 反应提供了理想的基础。我们将利用这些最新进展，使用一系列方法探索 NO 与这些蛋白质反应的生物化学，揭示对 NO 传感调节蛋白如何发挥作用的前所未有的机制见解，并提供有关 NO 如何传感以及因此生存的线索。病原体可以被破坏/预防。