Biological roles and mechanisms of nitric oxide reactions with iron-sulfur cluster transcriptional regulators

Lead Research Organisation: University of East Anglia
Department Name: Chemistry

Abstract

Nitric oxide is a poisonous 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 nitric oxide exerts its toxic effects is through reaction with a widespread group of proteins that contain a type of cofactor made from both iron and sulfur (called an iron-sulfur cluster). Members of this group play crucial roles in a very wide range of cellular processes. To avoid nitric oxide toxicity, disease-causing (as well as benign) bacteria have evolved protective systems that function to detoxify nitric oxide by removing it through chemical reaction. The fact that iron-sulfur cofactors are particularly sensitive to nitric oxide 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 nitric oxide detoxification response in the presence of nitric oxide. Despite the importance and widespread nature of the reaction of iron-sulfur clusters with NO, very little is known about this reaction process. This application is focussed on understanding how NO-responsive iron-sulfur cluster-containing regulators function. Here, we propose to investigate two such regulators (called WhiD and NsrR). One (WhiD) is a member of a family of proteins that are found only in a small number of bacteria (including Mycobacteria tuberculosis, the causative agent of tuberculosis, one of the world's major killers, and Streptomyces coelicolor, the source of many of the antibiotics currently in use in the clinic). Members of this protein family are known to play key roles in these bacteria in cell developmental processes associated with stress response, and are crucial for the ability of M. tuberculosis to survive in the inhospitable environment of a human host for years, in a dormant state that is highly resistant to antibiotics. The other (NsrR), is a member of a widely distributed but largely unstudied family of regulators. It functions as a primary NO sensor by controlling the cellular response to NO toxicity. Recent work in our laboratories has revealed important new insight into the nature of these regulatory proteins, including, for the first time, detailed mechanistic information about the reaction of a protein-bound iron-sulfur cluster with nitric oxide, leading to the formation of previously unreported products. We now propose to exploit these recent advances to explore, using a wide range of methods, the biochemistry of the reaction of NO with these proteins. This will reveal unprecedented mechanistic insight into how NO-sensing regulatory proteins function, and provide information that will be of general importance for all iron-sulfur protein NO reactions.

Technical Summary

The ability to sense and respond to NO is important for the survival and adaptability of many bacteria. One of the major ways by which nitric oxide exerts its toxic effects is through reaction with iron-sulfur cluster-containing proteins, which are found in all cell types, playing crucial roles in a very wide range of processes, including respiration and protein synthesis. The particular sensitivity of iron-sulfur clusters to NO has been exploited in Nature: several NO-responsive regulators are themselves iron-sulfur cluster proteins. This application is focussed on understanding how the iron-sulfur clusters of regulatory proteins sense NO, and we propose to achieve this by elucidating the mechanisms by which clusters react with NO, what the iron-nitrosyl products are and determining the effect of this is on the DNA-binding characteristics of the regulator. We propose to study two NO-responsive regulators from S. coelicolor with which we have made important recent progress: NsrR, 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. Our recent work on these two regulators has established them as ideal model systems, and we are now in a position to make important advances. We propose to use spectroscopic, kinetic and bioanalytical methods to reveal details about the cluster environments, the mechanism of reaction with NO and the products of cluster nitrosylation. For WhiD, we have shown that these are not the widely reported DNIC species. We will also investigate how reaction with NO affects DNA-binding. These studies will provide novel insight that will be important for understanding NO toxicity in general.

Planned Impact

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. These fundamental studies have already provided a major breakthrough in understanding iron-sulfur cluster sensitivity to nitric oxide, and the work proposed here will exploit this head start to maximum effect. In the longer term, the detailed knowledge about WhiD/NsrR and other Wbl/Rrf2 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. The work outlined in this proposal will lay the groundwork for future structural studies which would be the first step in developing inhibitors of these sensing pathways. We will evaluate the data that emerges from this work for potential commercial exploitation. The vital role that iron, including iron-sulfur clusters, and metal ions in general play in maintaining health (in all living organisms) is really not well appreciated by the general public and the major beneficiaries of the research, in terms of appreciating this important area, are likely to be school children and the general public. We will present our work, at the appropriate level, as we have done often in the recent past, at outreach events for both the general public and particularly high school students to encourage the next generation to study science and in particular chemistry and biology, and to encourage a better appreciation of research in general. In this way, we will ensure impact of this research beyond academia. UEA has a well established infrastructure for schools and public outreach projects. Together with partner organizations such as Norwich City Council, Norfolk Museums Service, Eastern Daily Press, the BBC, and the BBSRC Institute of Food Research and John Innes Centre, it won a Beacon of Public Engagement award 'CueEast' (Community University Engagement East) in 2007, making it one of a handful of national public engagement coordinating centres. This provides an ideal environment for increasing impact of the research conducted at the University.

Publications

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Crack JC (2014) Techniques for the production, isolation, and analysis of iron-sulfur proteins. in Methods in molecular biology (Clifton, N.J.)

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Crack JC (2012) Bacterial iron-sulfur regulatory proteins as biological sensor-switches. in Antioxidants & redox signaling

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Crack JC (2016) Biochemical properties of Paracoccus denitrificans FnrP: reactions with molecular oxygen and nitric oxide. in Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry

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Crack JC (2018) Redox-Sensing Iron-Sulfur Cluster Regulators. in Antioxidants & redox signaling

 
Description We have established the type of cluster that is bound by the NO-sensing regulator NsrR and discovered ways in which it can undergo cluster conversion. We have determined the genes that are regulated by NsrR in the medically important strain Streptomyces coelicolor and found that this regulator has a much more specialised function here compared to other organisms.
We have investigated in detail the mechanism of reaction of iron-sulfur cluster regulators (NsrR and WhiD) with NO using a range of novel techniques. We have applied kinetic and spectroscopic approaches available in our labs and through collaboration have applied advanced iron-specific spectroscopies including nuclear resonance vibrational spectroscopy (NRVS). Furthermore, we have developed a novel mass spectrometry approach to gaining high resolution structural and mechanistic information. Several papers have been published as a result of this award.
Exploitation Route Our work (when fully published) will provide unprecedented high resolution mechanistic data on the reactions of the cytotoxin and signalling molecule nitric oxide with iron-sulfur clusters. This is fundamental research and builds a detailed understanding of cellular processes that could be exploited in a number of ways. Principal amongst these will be further advances in fundamental understanding of iron-sulfur clusters and the reactions of NO. Additional eventual uses could also include applications in healthcare and biotechnology.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology,Other

 
Description Our findings have only recently been published and some are still being prepared for publication. Published articles are being used by the research community to gain further insight into fundamental cellular processes involving reactions of small molecules such as O2 and the cytotoxin and signalling molecule nitric oxide with iron-sulfur cofactors. It was recently demonstrated that the O2-sensor of many bacteria, FNR, is essential for the virulence of pathogenic E. coli. We are aware of current developments towards novel antimicrobials based on inhibition of transcription factors such as FNR. Impact from this has not yet arisen but it is a possibility.
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description BBSRC Responsive mode
Amount £764,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2017 
End 04/2020
 
Description Newton International Fellowship
Amount £127,000 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2015 
End 02/2017
 
Description Discover Chemistry and Pharmacy 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Schools
Results and Impact Individual questions were raised; contributed to an overall increased understanding of research.

Many of the attendees of the summer school choose to study Chemistry or pharmacy at University
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015
 
Description Public lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact The talk resulted in several questions and discussion continued into a post talk gathering.

No direct impacts that I am aware of.
Year(s) Of Engagement Activity 2012
 
Description School Visit (Lincolnshire) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact I visited a School in Lincolnshire to present an accessible but research led talk on the roles of metals in biology to a Year 12 class of approx 15 pupils.
Year(s) Of Engagement Activity 2015