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

点击翻译按钮获取中文摘要

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.