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

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.

The ability to sense and respond to NO is important for the survival and adaptability of many bacteria. The particular sensitivity of iron-sulfur (FeS) clusters to NO has been exploited in nature: several NO-responsive regulators are themselves FeS cluster proteins. This application is focussed on understanding how NO sensing occurs in these proteins.
We propose to study two NO-responsive regulators, NsrR and WhiD, with which we have made important recent progress. NsrR is a member of the poorly studied Rrf2 family, regulates NO-detoxification systems that function to remove NO by redox reaction and is found in a wide range of pathogenic and non-pathogenic bacteria. WhiD is a member of the WhiB-like (Wbl) family of regulators (found only in the actinomycetes, which includes Mycobacteria and Streptomyces), which play key roles in cell developmental processes such as sporulation and the transition into dormancy. We have established the use of mass spectrometry under native conditions to detect the cluster bound form of iron-sulfur cluster regulators. By using isotopically substituted clusters, we will map the intermediates and products formed upon reaction with NO. We have also developed the use of ATR IR spectroscopy to study the iron-nitrosyl species formed during nitrosylation, and time-resolved stopped-flow experiments, as well as thermodynamic titrations, will be performed. Furthermore, we have very recently solved the structure of NsrR with its cluster bound, revealing important new insight into how the cluster modulates DNA-binding and how NO might disrupt it. We will exploit these recent breakthroughs to determine the mechanisms of the nitrosylation reaction in NsrR and WhiD, and FeS regulators in general, in unprecedented mechanistic and structural detail. The range of versatile techniques we develop here will open up possibilities for studies of other key NO/small molecule pathways.

This project involves a fundamental structure-function study of nitric oxide sensing regulatory proteins. The project will have diverse and far reaching impacts within the UK and internationally. The main beneficiaries of the proposed research will be the academic research community, but, as described in the beneficiaries section, this is potentially a broad group. Outside of academia, there are several groups of potential beneficiaries, including:
- policy makers and commercial stakeholders, who are likely to be interested in the anticipated advances in understanding how microorganisms, including pathogens, sense nitric oxide via iron-sulfur cluster regulatory proteins. In the longer term, the detailed knowledge about NsrR/WhiD and other Rrf2/Wbl family members gained as a result of this work may be exploited. Bacterial pathogens that cannot sense and respond to nitric oxide have decreased fitness or are unable to survive inside the host. Clearly, compounds that interfere with the NO sensing mechanisms of NsrR/WhiD could find widespread use as antibacterial drugs. Solving the structure of NsrR in its cluster-bound form is a major advance and this will be exploited in this application. The work outlined in this proposal will lay the groundwork for the future development of inhibitors of these sensing pathways. We will evaluate the data that emerges from this work for potential commercial exploitation.
These groups will benefit from the high quality publications arising from this work, which will be accessible to researchers working in private (pharmaceutical) and public sector laboratories (e.g. health agencies), and by advisors to policy makers. This will stimulate new research and inform decision making. Although the project involves basic research, UEA and Oxford have appropriate policies and support to identify any commercial opportunities arising from research activities and mechanisms to ensure that potential beneficiaries and investors are informed. In support of this statement, both PIs are independently currently developing technologies for commercial applications and their respective Universities are playing a key role in assisting with this. Thus, the applicants are keen to exploit any commercial opportunities, although it is recognised that, in this case, these are likely to arise in the longer term;
- the biotechnology and pharmaceutical sectors and public sector laboratories, from the point of view of benefiting from future employment of the state-of-the-art training in biochemistry, spectroscopy and X-ray crystallography provided to the PDRA and to PhD students and undergraduates working within the research groups who benefit from the expertise of the PDRA;
- schools and the general public, who benefit from engagement activities running parallel with the research effort, which seek to inspire the next generation of science undergraduates and scientists and to better inform the general public of key scientific concepts and issues over which society has an influence. The vital role that iron, including iron-sulfur clusters, and metal ions in general, play in maintaining health (of e.g. humans, molluscs, plants, yeast and bacteria) is really not well appreciated by the general public. Proteins that bind metal cofactors account for at least 30% of all proteins, and so this is a very important subgroup of proteins. The PIs have a lot of experience of delivering engaging presentations, in particular to A-level students.