Structure and ligand binding of As-p18, an extracellular fatty acid binding protein from the eggs of a parasitic nematode.

Intracellular lipid-binding proteins (iLBPs) of the fatty acid-binding protein (FABP) family of animals transport, mainly fatty acids or retinoids, are confined to the cytosol and have highly similar 3D structures. In contrast, nematodes possess fatty acid-binding proteins (nemFABPs) that are secreted into the perivitelline fluid surrounding their developing embryos. We report structures of As-p18, a nemFABP of the large intestinal roundworm Ascaris suum, with ligand bound, determined using X-ray crystallography and nuclear magnetic resonance spectroscopy. In common with other FABPs, As-p18 comprises a ten ß-strand barrel capped by two short α-helices, with the carboxylate head group of oleate tethered in the interior of the protein. However, As-p18 exhibits two distinctive longer loops amongst ß-strands not previously seen in a FABP. One of these is adjacent to the presumed ligand entry portal, so it may help to target the protein for efficient loading or unloading of ligand. The second, larger loop is at the opposite end of the molecule and has no equivalent in any iLBP structure yet determined. As-p18 preferentially binds a single 18-carbon fatty acid ligand in its central cavity but in an orientation that differs from iLBPs. The unusual structural features of nemFABPs may relate to resourcing of developing embryos of nematodes.

Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus.

Fatty acid and retinol-binding proteins (FARs) comprise a family of unusual α-helix rich lipid-binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein [C. elegans FAR-7 (Ce-FAR-7)] is from a subfamily of FARs that does not appear to be important at the host/parasite interface. We have therefore examined [Necator americanus FAR-1 (Na-FAR-1)] from the blood-feeding intestinal parasite of humans, N. americanus. The 3D structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by NMR (nuclear magnetic resonance) spectroscopy and X-ray crystallography respectively, reveals an α-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand-binding cavity and an additional C-terminal α-helix. Titration of apo-Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein-ligand complexes can be formed. Na-FAR-1 and possibly other FARs may have a wider repertoire for hydrophobic ligand binding, as confirmed in the present study by our finding that a range of neutral and polar lipids co-purify with the bacterially expressed recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male.