Understanding the molecular mechanism of iron sulfur cluster biogenesis

Cells work thanks to 'fuel' which is absorbed from the environment. Small molecules made of iron and sulfur (called iron-sulfur clusters), which are attached to proteins, play key roles in the process by which energy stored in food (or light) is converted to a useable form in respiration (and photosynthesis). These iron-sulfur cluster molecules do not form spontaneously, however, and need to be assembled directly in the cell. This process poses an important problem: iron and sulfur are elements which are essential for life but, at the same time, also intrinsically toxic. Nature has thus engineered very complex and tightly regulated molecular machines, evolutionarily conserved across the kingdoms of life and all discovered within the past 15-20 years, to synthesise iron-sulfur cluster molecules and attach them to proteins in a very orderly and regulated way, making sure to minimise waste and the potential for harm to the cell. The importance of these machines for human life is illustrated by the number of diseases which increasingly appear to be linked to impairment of iron-sulfur cluster proteins and their formation. When any of the parts of these machines break down, disease occurs.
We have in the past focused a major part of our research efforts on understanding this important problem and on iron-sulfur cluster biochemistry in general, resulting in several seminal papers which have substantially advanced the field. An increasingly sophisticated understanding of the iron-sulfur cluster assembly machines of humans and bacteria is slowly emerging. However, we are still far from having the full picture, and understanding the steps involved in formation of the cluster is limited by the lack of detailed information on the precise sequence of events and nature of intermediates, which interactions are formed and how they regulate this process.
We propose a project aimed at understanding in unprecedented detail the mechanism by which the cluster is formed from iron and cysteine (the source of sulfur). Specifically, the experimental programme will address crucial outstanding questions including: the precise mechanism by which iron and sulfur are delivered, the steps that lead to cluster formation, the precise role of frataxin (a protein linked to the genetic disease Friedreich's ataxia in humans and know to be an important component of iron-sulfur cluster assembly), the process of cluster transfer to carrier and/or target proteins that require a cluster for function. We will use a powerful combination of different biophysical, structural and biochemical techniques which will allow us to reconstruct the whole mechanism. We have already developed all the necessary know-how and expertise in all the proposed techniques. Of particular novelty is the application of mass spectrometry to iron-sulfur cluster proteins under conditions in which the protein remains folded. This has the tremendous advantage that iron-sulfur clusters, and fragments thereof, remain bound to the folded protein so that by measuring accurately the mass of the protein with cofactors bound, the identity of the cofactor can be deduced. This has recently provided unprecedented insight into iron-sulfur cluster conversion and degradation in other systems, and is an extremely promising, novel methodology to elucidate the steps of de novo cluster assembly.
Overall, our research has important implications for our basic comprehension of these cellular processes in bacteria, which is to a large extent conserved in humans, with potential longer term medical benefits.

The aim of the project is to elucidate the mechanism of iron-sulfur (FeS) cluster biogenesis in unprecedented molecular detail by dissecting the fundamental steps that underpin their formation using non-denaturing mass spectrometry as a novel approach, in combination with established spectroscopic and structural techniques.
FeS clusters are essential prosthetic groups in proteins that play key roles in a wide range of cellular processes. They are assembled by specialised ancient machines that, when compromised by mutations or dysfunction, are linked to disease. The core components of the Isc FeS assembly machine are the desulfurase IscS, which converts cysteine into alanine and sulfur for use in cluster formation, the scaffold protein IscU, and their interacting partner proteins that include frataxin, ferredoxin, IscX, the chaperones HscA and B, and IscA.
This proposal stems from a recently begun collaboration between the two teams, which have different but complementary expertise in aspects of FeS cluster biochemistry. The proposed project will exploit preliminary studies demonstrating that Isc complexes and [2Fe-2S] IscU can be detected by non-denaturing mass spectrometry, where the protein remains folded, protein-protein interactions are maintained and non-covalently bound cofactors remain attached. This method will be used to elucidate steps in [2Fe-2S] cluster formation, coupling to form [4Fe-4S] clusters and transfer of [2Fe-2S] and [4Fe-4S] clusters to acceptor proteins. It will also exploit preliminary studies showing that Isc desulfurase activity can be detected in cell lysates by LC-MS. These, together with proven hybrid structural methods based on NMR and SAXS that can provide structural information for large complexes that are not easy to crystallise, and quantitative proteomics that will provide information on Isc protein levels under different conditions, will lead to a major breakthrough in defining the key processes of FeS cluster assembly.

This project involves a fundamental study of iron-sulfur (FeS) cluster assembly. 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:
- the biotechnology and pharmaceutical sectors and public sector laboratories, from the point of view of benefiting from the methodological advances in studies of complex cellular pathways involving cofactors. The project will also provide future state-of-the-art training in biochemistry, spectroscopy, structural biology and mass spectrometry to the PDRAs and PhD students and undergraduates working within the research groups who benefit from the expertise of the PDRAs;
- 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-sulphur 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.
- health professionals, policy makers and pharmaceutical stakeholders, who are interested in disease states that are associated with errors in FeS cluster biogenesis, which currently numbers approx. 10. Of particular interest is Friedreich Ataxia, an autosomal recessive neurodegenerative disease with an occurrence of 1 in 50000 and onset usually before 25 years of age. 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, Kings College London and UEA 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.