Understanding the molecular mechanism of iron-sulfur cluster biogenesis

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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, and future employment of the state-of-the-art training in biochemistry, spectroscopy, structural biology and mass spectrometry provided to the PDRAs and to 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.
- policy makers and commercial stakeholders, who are likely to be interested in the anticipated advances in understanding of how FeS clusters are assembled on scaffold proteins and subsequently transferred. We are focussed here on bacterial proteins, but analogous systems are found in lower and higher eukaryotes, enabling comparison to be made between the assembly machineries of the different kingdoms. In the longer term, the detailed knowledge about the FeS biogenesis systems gained as a result of this work may be exploited via a better understanding of a fundamental and essential cellular process, and of the myriad of disease states that are associated with errors in FeS cluster biogenesis. 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.