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

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.

Keywords: Necator americanus; X-ray; fatty acid-binding protein; nematode; nuclear magnetic resonance (NMR); parasite; protein structure; retinol-binding protein.

Figure 1. FAR protein relationships and Na-FAR-1 expression

(a) Western blot with anti-Na-FAR-1 serum that specifically recognizes native Na-FAR-1 in N. americanus extracts (lane 3, 0.5 μg) and excreted/secreted (ES) products (lane 4, 0.5 μg) at an approximate M_r of 14 kDa, but not in L3 extracts (lane 1) and L3 ES products (lane 2) at the same loading, indicating the specific expression of Na-FAR-1 in adult stage as a secreted protein. The antiserum also recognized the recombinant Na-FAR-1 at 16 kDa (with His-tag, lane 5, 20 ng). There was no cross-reaction with FAR homologues from dog hookworm A. caninum (Ac-FAR-1, lane 6); B. malayi (Bm-FAR-1/lane 7, Bm-FAR-2/lane 8) and non-relevant recombinant protein Ac-SPI (lane 9) loaded at the same amount (20 ng). (b). Neighbour joining tree of Na-FAR-1 and other nematode FAR proteins. The tree was generated in jalview 2.8 [69] using the BLOSUM 62 matrix from a T-coffee WS sequence alignment [70] with FAR protein amino acid sequences of: N. americanus recently identified FAR proteins (NECAME_09996, NECAME_04475, NECAME_04474, NECAME_14206, NECAME_14205 and NECAME_14203), the free living nematode C. elegans (Ce-FAR-1 to Ce-FAR-8), the human parasitic nematodes O. volvulus (Ov-FAR-1), B. malayi (Bm-FAR-1), animal parasitic nematodes A. caninum (Ac-FAR-1 and Ac-FAR-2), A. ceylanicum (Ace-FAR-1), O. ostertagi (Oo-FAR-1) and H. polygyrus (Hp-FAR-1) and the plant parasitic nematodes G. pallida (Gp-FAR-1) and M. javanica (Mj-FAR-1). All the parasite proteins are coloured blue. See Supplementary Figure S1 for the multiple sequence alignment from which the tree was constructed. (c–g) Localization of Na-FAR-1 within adult male (c and d) and female (e–g) worms. Indirect immunofluorescence localization with rabbit anti-Na-FAR-1 serum stains the intestinal cells of adult N. americanus worms. Na-FAR-1 was also detected on the copulatory bursa and cloacal aperture of male worms. (h) Control carried out using pre-immune serum. Scale bars represent 100 μm.

Figure 2. The structure of Na-FAR-1

(a) Stereoscopic view (wall-eyed) of the superimposed apo- and holo-Na-FAR-1 structures shown in cartoon form. The apo structure determined by NMR is coloured from blue at the N-terminus through to red at the C-terminus. The holo form determined by X-ray crystallography is shown in grey. (b) The ensemble of the 20 lowest energy structures of apo-Na-FAR-1 shown in two orientations related by a 90° rotation. (c) The holo-Na-FAR-1 structure shown in b-factor putty representation with the palmitate shown as sticks. (d) Expanded view of the bound palmitate and its environment with 2Fo-Fc electron density within 4 Å of the ligand displayed as a mesh at 1σ and shaded yellow and Fo-Fc difference density displayed at ±3σ (green and red).

Figure 3. Na-FAR-1 cavities and comparison with Ce-FAR-7

(a) Sequence alignment of Na-FAR-1 and Ce-FAR-7. Secondary structure elements are indicated in boxes (α-helix) and lines (loops) coloured grey for Na-FAR-1 and yellow Ce-FAR-7. Comparison of Na-FAR-1 apo (b and e) and holo (c and f) forms with Ce-FAR-7 (d and g) shown in cartoon representation and coloured from blue (N-terminus) to red (C-terminus) viewed from two orientations related by a 90° rotation about the horizontal axis. Internal cavities accessible to a probe of 1.925 Å, equivalent to a methylene group are shown as transparent surfaces with the surrounding sidechains shown as sticks. (PDB accession codes Na-FAR-1 apo 4UET and holo 4XCP; Ce-FAR-7, 29WY)

Figure 4. Lipid binding by recombinant Na-FAR-1

(a) Lipid fractions bound to bacterially-expressed Na-FAR-1 detected by TLC. Lipids were extracted from the protein and fractions were analysed by TLC in conditions for resolving separately neutral (left panel) and polar (right panel) lipid classes. Standards and samples applied to TLC plates were: Hol, Na-FAR-1 purified without an HPLC step; Ec, extract from whole E. coli cells; LIV, standard mix of lipids from rat liver homogenate; STD, E. coli whole extract. CHO, cholesterol; TG, triglycerides; CHOe, cholesterol esters; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol; CL, cardiolipin. (b) GC–MS analysis of non-esterified FAs isolated from lipids associated with Na-FAR-1 purified from E. coli (dark grey) or found in E. coli extracts (light grey). FAs detected were: myristic (14:0), palmitic (16:0), palmitoleic (16:1), margarinic (17:0), heptadecenoic (17:1), stearic (18:0), oleic (18:1Δ9), vaccenic (18:1Δ11) and nonadecenoic acid (19:1). (c) FAs found in PLs isolated from purified Na-FAR-1 (dark grey) or found in E. coli extracts (light grey). (d and e) Fluorescent ligand binding by Na-FAR-1. (d) Fluorescence emission spectra of 1 μM DAUDA excited at 345 nm in buffer, on the addition of 1 μM Na-FAR-1 and after successive additions of oleic acid (81 nM, 810 nM and 8.1 μM). (e) Fluorescence emission spectra of 5 μl of 0.15 mM retinol in ethanol added to the fluorescence cuvette and excited at 350 nm and on the addition of 1.5 μM Na-FAR-1, compared with 1.5 μM protein in buffer and buffer alone.

Figure 5. Titration of Na-FAR-1 with sodium oleate followed by NMR spectroscopy

¹⁵N HSQC spectra of 0.4 mM Na-FAR-1 with successive additions of sodium oleate. The spectrum of the apo protein is shown in dark blue, with spectra coloured through purple to red at the final protein–ligand ratio of 1:10. A subset of the backbone and side chain (lettered) assignments are shown and the trajectories of selected residues indicated with arrows. The inset shows an expanded view of the cross-peaks from Gly⁷⁸ backbone amide with our interpretation of the protein–ligand stoichiometry responsible for each distinct cross-peak position.