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

一氧化氮(NO)是一种有毒分子，由土壤细菌产生，在我们的体内用来防御试图确定感染的病原体。NO发挥毒性作用的主要方式之一是通过与一组广泛存在的蛋白质反应，这些蛋白质结合了一种同时含有铁和硫的辅因子，并以簇的形式排列。这个组的成员在非常广泛的过程中发挥着关键作用，包括呼吸和蛋白质合成。为了避免无毒，致病(以及无害)生物体进化出了保护系统，通过化学反应将其清除，从而为NO解毒。铁-硫簇对NO特别敏感(其修饰是NO发挥其毒性作用的主要途径)这一事实在自然界中已经被利用，通过一些调节蛋白的进化，这些蛋白本身包含一个铁-硫簇并发挥生物开关的作用，在NO存在时启动细胞解毒反应。尽管铁-硫团簇与NO反应的重要性和广泛性，我们对这一过程仍然知之甚少。近年来已经取得了一些重要的进展，但与铁硫蛋白相关的困难，以及检测和明确识别NO簇合反应的中间体和产物，到目前为止仍然是主要障碍。铁硫蛋白是脆弱的，必须在无氧环境中处理，到目前为止，这一建议中描述的项目将导致我们对含NO反应的铁硫簇调控因子如何发挥作用的理解取得重大进展。我们拟议的研究的主要对象是一个铁-硫簇调节因子，它是在广泛的致病和非致病细菌中发现的一个庞大的、尚未被很好理解的调节因子家族的成员，在这个家族中，它通过控制细胞对NO毒性的反应而作为主要的NO感受器发挥作用。我们还将研究第二种调节蛋白，它属于一个只在少数细菌中发现的家族，但包括导致结核病的病原体，世界上主要的杀手之一，以及目前临床上使用的许多抗生素的来源。这个家族的成员在与应激反应相关的细胞发育过程中扮演着关键角色，包括产孢子和休眠，这对结核病病原体在高度抵抗抗生素的不适宜人类宿主的环境中存活多年的能力非常重要。该项目将建立在最近的三项重要突破的基础上。首先，我们建立了新的质谱学方法，使我们能够检测铁-硫簇调节剂，而它们的簇是完整的。这就提供了通过检测和鉴定中间体和产物，通过质谱学来跟踪该簇与NO的反应的机会。其次，我们开发了利用振动光谱研究相同蛋白质的新方法，根据形成的铁-NO复合体提供特征特征。最后，我们与法国的一个小组合作，确定了其中一个调节子的高分辨结构，它的铁-硫团簇结合。这对这一铁硫簇调节剂家族来说是第一次，并为了解该簇如何促进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的进展，利用一系列方法来探索NO与这些蛋白质反应的生物化学，揭示NO感知调节蛋白如何发挥作用的前所未有的机制洞察，并提供关于NO感知以及病原体的生存如何被干扰/防止的线索。

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.