Isotopic labelling tools to aid NMR studies of protein structure and interactions: applications to the LIMD1 scaffold protein

The scaffold protein LIM domain-containing protein 1 (LIMD1) has been identified as a tumour suppressing protein with roles in a range of cell signalling pathways. In microRNA (miRNA) mediated silencing, LIMD1 has been shown to be a regulator of the miRNA induced silencing complex (miRISC), able to bind to both Argonaute (AGO) and GW182 proteins and promote their co-localisation. In hypoxic signalling, LIMD1 is thought to promote degradation of hypoxia inducible factor 1α (HIF-1α) through scaffolding of prolyl hydroxylases (PHD) and von-Hippel Lindau (VHL) proteins. The regions of LIMD1 responsible for binding to Argonaute-2 (AGO2), Trinucleotide Repeat Containing Adaptor 6A (TNRC6A), PHD2 and VHL have been mapped though co-immunoprecipitation assays (Co-IPs), but precise binding sites and motifs have not been identified. This thesis investigates the structural biology and biophysics of the interactions of LIMD1 with its partner proteins in miRNA mediated silencing and hypoxic signalling pathways. For each system, this involved recapitulating the reported interactions of LIMD1 in vitro before more in depth analysis could be achieved. Investigations into the interactions of LIMD1 in the context of miRNA mediated silencing were largely unsuccessful but informed analysis of LIMD1 in hypoxic signalling. In that context, reconstitution of complexes of LIMD1 with PHD2 and VHL in vitro was demonstrated using NMR spectroscopy. Compelling evidence was obtained for identification of the binding interface between LIMD1 and PHD2 which was consistent with previously reported in vivo data. Finally, new methodologies were developed that combined specific isotopic unlabelling with filtered/edited NOESY experiments to aid in structural studies of challenging proteins by NMR spectroscopy. Overall, this thesis addresses the challenges of studying the structural biology and interaction mechanisms of multi-domain proteins with large unstructured regions, and presents case studies for the use of NMR spectroscopy together with new methodologies to support this important area of biology.