Unravelling the structural biology of a hypoxia scaffold protein using novel NMR approaches

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Oxygen homeostasis is tightly regulated to ensure that cells and tissues are supplied with sufficient oxygen. Rapid reaction and adaptation to low oxygen concentrations ('hypoxia') can reduce damage and enable cells to remain viable. This process is known as the 'hypoxic response'. In low oxygen conditions, hypoxia-inducible transcription factors (HIFs) control expression of factors that facilitate the hypoxic response. However, under normoxic conditions, specific proline residues in one of the HIF subunits (HIFa) are hydroxylated by a prolyl hydroxylase (PHD). These hydroxyproline modifications lead to ubiquitination of HIF by the VHL E3 ubiquitin ligase complex and its eventual degradation.A scaffold protein called LIMD1 is critical to the hypoxic response signalling pathway. LIMD1 interacts with and collocates PHD and VHL. The objective of this iCASE PhD project is to characterize the structural biology of the protein/protein interactions mediated by LIMD1 in hypoxic signalling. The PhD student will use structural, biophysical and biochemical approaches to explore the structure/function relationship of LIMD1, including cutting-edge NMR methods for studying supramolecular protein complexes. LIMD1 has structured domains and intrinsically disordered regions, both of which contain important protein recognition sites. We have teamed up with NMR-Bio, a Grenoble-based company that develops novel reagents that facilitate NMR analyses of large protein complexes. The student will spend three months in Grenoble where they will receive training in bacterial and eukaryotic protein production techniques for NMR studies. The results of the structural and biophysical analyses of LIMD1 conducted in York will drive molecular cell biology studies performed in collaboration with Dr Tyson Sharp's group at the Barts Cancer Institute, London.The dysfunction of the hypoxic response is implicated in many human diseases, including the growth and spread of cancers. The structure/function analyses conducted in this iCASE PhD project will illuminate the role of LIMD1 in these processes.