Biochemical and structural characterisation of human phosphatidylinositol transfer protein Nir2 and American hookworm lipid binding protein Na-FAR-1

This study focused on biochemical and structural characterisation of two lipid binding proteins: human phosphatidylinositol transfer protein (PITP) Nir2 and American hookworm fatty acid and retinol binding protein (FAR) Na-FAR-1. Nir2 is a large multi-domain PITP that has recently been implicated in phosphoinositide signalling, where it was demonstrated to regulate phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] homeostasis. Nir2 acts by reciprocally transporting phosphatidylinositol (PI) and phosphatidic acid (PA) between the plasma membrane (PM) and the endoplasmic reticulum (ER), which allows PI(4,5)P2 to be re-synthesised at the PM. Upon cell stimulation, Nir2 translocates to the ER-PM contact sites and is believed to associate with the PM by binding to PA via its C-terminal LNS2 domain. Due to the proposed role of LNS2-PA binding in Nir2 targeting, a detailed investigation of the binding mechanism is desirable, which could help to reveal more details about Nir2 function in the cell. Expression screening of the difficult-to-express Nir2 LNS2 domain yielded a highly-expressed construct that was employed for characterisation of LNS2-PA binding. The data suggested that Nir2 LNS2 binds PA in a specific manner, interacts with both the polar and apolar regions of PA and might associate with the membrane via both hydrophobic and polar interactions. Although the structure of the LNS2 domain could not be determined, several assays are proposed for the identification of LNS2-PA interaction inhibitors that could be used as tool compounds in the investigation of Nir2 and its homologs. Na-FAR-1 is a small lipid binding protein secreted by the human hookworm Necator americanus that infects hundreds of millions of people globally. Na-FAR-1 is known to bind a range of lipid ligands including fatty acids, retinoids and phospholipids, and was proposed to play a role in parasite-host interactions by facilitating nutrient uptake or sequestering lipid signalling molecules in the host tissues. The structure of Na-FAR-1 has been determined previously, but the molecular details of ligand binding by Na-FAR-1 remained unclear. In this study, the high-resolution structure of Na-FAR-1 in complex with its natural ligand oleic acid was determined, and the ligand binding sites were mapped. Furthermore, phospholipid binding by Na-FAR-1 was investigated, and resonance assignment of Na-FAR-1 in complex with PA was carried out, which can be used to obtain the structure of the complex. In addition, Na-FAR-1’s interaction with lysophosphatidic acid was demonstrated in vitro. As lysophosphatidic acid is a mediator of inflammation, the interaction might have important biological implications if it also occurs in vivo.