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

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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 (including training sessions) to identify any commercial opportunities arising from research activities and mechanisms to ensure that potential beneficiaries and investors are informed. The applicants are keen to exploit any commercial opportunities, although it is recognised that 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.