The human pseudokinase SgK269 and its structurally related homologue SgK223, are oncogenic interacting scaffolds that promote the assembly of specific tyrosine kinase signaling pathways. Previous studies have shown that SgK223 and SgK269 homo-and hetero- associate  and recently the structures of SgK223 and SgK269 were solved [2, 3]. These structures revealed a dimerisation interface composed of the helices that flank the pseudokinase domain (PK domain). Additionally, the SgK223 structure revealed an oligomerisation interface formed by the interaction of the aG helix of the PK domain with the N-lobe of the PK domain. This study identified energetic hotspots that are important for dimerisation of SgK223. In addition, mutagenesis within the aG helix of SgK223 and SgK269 disrupted the ability of these proteins to oligomerise.
In this study, we carried out single site alanine mutagenesis to determine the energetic hotspots at the dimerisation interface of SgK269. Furthermore, we carried out mutagenesis within the N-lobe of SgK223 and SgK269, to investigate the role of this interface in homo- and hetero-oligomerisation. Single alanine mutants at the dimerisation interface of SgK269 failed to disrupt dimerisation. Future studies will involve using double/triple mutants or replacing hydrophobic residues to charged residues at the dimerisation interface. Single mutations within the N-lobe of SgK223 disrupted homo- and hetero-oligomerisation. However, single mutations within the N-lobe of SgK269 failed to disrupt oligomerisation, hence it is likely oligomerisation of SgK269 may occur through a different interface that does not involve the N-lobe of the PK domain.
The future directions on this project will be to functionally characterise the interactions of SgK223 and SgK269 in a cellular setting. Microscopy techniques such as super-resolution and lattice light sheet microscopy will be used to identify the localisation of these proteins, and how they alter cell morphology and migration.