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

M Florencia Rey-Burusco,Kate Griffiths,Betina Córsico,Bin Zhan,Mads Gabrielsen,Andrew J Roe,Marina Ibáñez-Shimabukuro,Malcolm W Kennedy,Gisela R Franchini,Alan Cooper,Brian O Smith

doi:10.1042/bj20150068

Abstract

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.

Highlights

Lipids such as acids (FAs) and retinoids are relatively insoluble in water, can be susceptible to oxidation and are potentially damaging to membranes in their free form
Examples of vertebrate fatty acid (FA) and retinoid transporter proteins include serum albumins (∼64 kDa), which bind a range of compounds, lipocalins (∼20 kDa), which are found in many secretions and the proteins of the cytoplasmic FA-binding/cellular retinol-binding/cellular retinoic acid-binding protein (FABP/cellular retinol-binding protein (CRBP)/cellular retinoic acid-binding protein family (CRABP)) family (∼14 kDa) that are confined to the cytoplasm, where they may bind FAs, retinol or retinoic acid, depending on the isoform
Na-fatty acid- and retinolbinding protein (FAR)-1 had been identified in a gene survey of the N. americanus transcriptome under the name N. americanus LBP20 [62]

Summary

INTRODUCTION

Lipids such as acids (FAs) and retinoids are relatively insoluble in water, can be susceptible to oxidation and are potentially damaging to membranes in their free form. Apo-Na-FAR-1 samples were delipidated by reverse phaseHPLC (high performance liquid chromatography) as previously described [27] using a C8-silica stationary phase and a water/acetonitrile gradient in the presence of 0.1 % trifluoroacetic acid as the mobile phase. The 20 structures that best satisfy the experimental restraints were chosen from 100 structures generated in the final iteration and refined in explicit water [53] The quality of these structures was analysed using PROCHECK_nmr [54] and their co-ordinates deposited in the Protein Data Bank under accession code 4UET. In order to examine Na-FAR-1’s ligand binding properties by NMR, the chemical shift changes induced in 15N HSQC spectra of the protein by the presence of increasing amounts of ligand were analysed.

RESULTS AND DISCUSSION

DNH 1 DCαHα RDC Q factor Dihedral angles

CONCLUSION