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.
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. Consequently, they are usually transported within proteins or protein-mediated lipid aggregates. Examples of vertebrate 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/CRBP/CRABP) family (∼14 kDa) that are confined to the cytoplasm, where they may bind FAs, retinol or retinoic acid, depending on the isoform. These proteins are variously involved in transport and storage of lipids and also in the delivery of small signalling lipids to their destinations .
Nematodes, both free-living and parasitic to plants and animals, exhibit several additional types of lipid-binding proteins that are not found in other phyla. Prominent examples include the nematode polyprotein antigens (NPAs), such as ABA-1, whose structure has previously been solved . These are synthesized as large polypeptide precursors that are post-translationally processed down to multiple copies of small lipid-binding proteins of ∼14 kDa [3–7]. Other examples include the nemFABPs (nematode FABPs), which are similar to the intracellular FABP/CRBP/CRABP family of proteins, but in nematodes have structural modifications found in no other group of animals and are not confined to the cytosol [8,9]. The subject of the present paper is an unusual class of lipid-binding protein, the FA and retinol-binding proteins (FARs). These occur in several isoforms of ∼20 kDa, eight having been found encoded within the genome of the free-living species Caenorhabditis elegans, each of which binds FAs and retinol to varying extents . There are as yet uncharted numbers of FAR isoforms in parasitic nematodes, but FARs have drawn attention because they are secreted by both plant- and animal-parasitic species and their encoding transcripts are relatively abundant [11–18]. They have also proven useful for serodiagnosis, have shown promise in experimental vaccines [19,20] and have been proposed to facilitate infection by manipulating host lipid-mediated defences [21,22]. At least one FAR has been shown to bind an anthelmintic drug, these drugs being typically hydrophobic and so may require carrier proteins to conduct them to their site of action within parasites . The definitive role of FARs in parasitism is not known, but their presence in secretions of the worms, coupled with their ligand-binding propensities, suggests roles for them either in acquiring lipids from the hosts, or in delivering or in sequestering signalling lipids and so modifying the tissues the parasites occupy or the immune responses against them.
The structure of one FAR protein, C. elegans FAR-7 (Ce-FAR-7), has been solved by X-ray crystallography, revealing a helix-rich structure that is unlike any type of lipid-binding protein previously described . The mRNA for Ce-FAR-7 does not encode a secretory signal peptide and its amino acid sequence indicates that it is in a different subfamily of FARs from those that are secreted from the synthesizing cell. We set out to confirm the expression pattern of a secreted FAR protein from a parasite and characterize its structure and ligand-binding characteristics. The protein Na-FAR-1 derives from the blood feeding intestinal hookworm of humans, N. americanus. This parasite and the other hookworm of humans, Ancylostoma duodenale, together infect over 300 million people worldwide [25,26], causing considerable morbidity, together with adverse social and economic consequences. We have determined the structure of Na-FAR-1 by both X-ray crystallography and NMR spectroscopy in solution. We find that it has a similar overall fold to Ce-FAR-7, indicating that FARs are structurally conserved despite considerable sequence diversity. But, in addition to the structural differences between Ce-FAR-7 and Na-FAR-1, we find that their ligand-binding sites differ significantly in position and form.
Protein expression and purification
Recombinant Na-FAR-1 (rNa-FAR-1) was expressed in BL21 (λDE3) Escherichia coli cells as described . For native crystallographic studies, Na-FAR-1 was purified to homogeneity, as previously described , from cells grown in LB media. Selenomethionine-labelled protein was purified from B834 cells grown in M9 minimal medium supplemented with a cocktail of free amino acids (each 0.5 g·l−1) and selenomethionine (50 mg·l−1; Generon).
For NMR studies, samples of unlabelled, 15N-labelled and 13C15N-labelled protein were purified by nickel-affinity, size exclusion and reverse-phase chromatographies, as described , from cells grown in M9 minimal medium containing 15NH4Cl, [13C6]-glucose or their unlabelled equivalents.
Western blotting and immunolocalization of Na-FAR-1
Antiserum prepared against recombinant Na-FAR-1 was raised in three rabbits by subcutaneous injection with 0.7 mg of purified recombinant Na-FAR-1 in Freund's complete adjuvant. Antiserum was tested by ELISA and Western blot analysis against the recombinant protein. To analyse the expression of Na-FAR-1 in the worm, soluble extracts of adult N. americanus, as well as recombinant proteins (each 100 ng) of homologues from Ancylostoma caninum FAR-1 (Ac-FAR-1); Brugia malayi FARs (Bm-FAR-1 and Bm-FAR-2) and the unrelated protein, recombinant Ac-SPI (serine protease inhibitor from A. caninum) were separated on a 4%–20% gradient SDS precast polyacrylamide gel (Invitrogen) and subsequently electrotransferred on to a PVDF membrane (Millipore). Rabbit anti-Na-FAR-1 serum was diluted 1:5000 into PBS (phosphate buffered saline), pH 7.4, with 0.05% Tween-20 and incubated with transferred membrane for 2 h. Horseradish peroxidase-conjugated goat anti rabbit-IgG was used as secondary antibody and ECL was used to develop the reaction (GE).
To determine the tissue-specific localization of Na-FAR-1, adult N. americanus worms were prepared as previously described . Briefly, adult worms were collected from the intestines of hamsters infected with N. americanus L3 [third (infective) larval stage of a nematode] for 45 days and fixed with 10% formalin. The fixed worms were sectioned and mounted on glass slides. The non-specific binding sites on worm sections were blocked with 5% FBS in PBS for 1 h. The rabbit anti-Na-FAR-1 serum was applied (1:500 dilution) to each tissue section and incubated for 2 h at room temperature in a humidified chamber. Pre-immune rabbit serum at the same dilution was used as a negative control. Sections were washed six times for 5 min each in PBS and probed with anti-rabbit Cy3-conjugated IgG (Rockland). Sections were viewed under a Nikon TE-2000 Inverted fluorescence microscope using a 550 nm excitation filter block and emission at 565 nm.
Crystallization, data collection, processing and structure solution
We have shown previously that Na-FAR-1 crystallizes in two crystal forms, one of which (form 2) shows significant twinning . Here, in order to obtain phasing information, selenomethionine-substituted protein was purified and crystallized, selecting only the cubic crystal form 1. Crystals were frozen in a stream of cool nitrogen gas (100 K) and brought to the Diamond Light Source, station I04 (DLS) for X-ray diffraction data collection.
Data were collected at 0.7° increments per image, for a total of 200 images [wavelength 0.9793 Å (1 Å=0.1 nm)] and processed by the automatic processing routines fast_dp, which utilized XDS , POINTLESS and SCALA . The structure was solved using the SAS protocol of Auto-Rickshaw . The input diffraction data were prepared and converted for use in Auto-Rickshaw, using programs of the CCP4 suite . FA values were calculated using the program SHELXC . Based on an initial analysis of the data, the maximum resolution for sub-structure determination and initial phase calculation was set to 2.14 Å based on the scaling statistics and the increase in Rmeas in the highest resolution bin. All the four heavy atoms requested were found using the program SHELXD . The correct handedness for the substructure was determined using the programs ABS  and SHELXE . Initial phases were calculated after density modification using the program SHELXE. 83.9% of the model was built using the program ARP/wARP . Despite a solvent content above 70%, only one copy of the protein was observed in the asymmetric unit. The model was completed by hand using COOT  and iterative rounds of BUSTER . Ligands and water were added using COOT. Based on its prevalence in the lipids co-purifying with recombinant Na-FAR-1, palmitate was fitted into unoccupied electron density within the binding pocket. Further electron density that may represent additional ligand molecules was observed, but as the density was incomplete and not at full occupancy, it was left unmodelled. The geometry of the finished models was validated using Molprobity .
Preparation of NMR samples
Apo-Na-FAR-1 samples were delipidated by reverse phase-HPLC (high performance liquid chromatography) as previously described  using a C8-silica stationary phase and a water/acetonitrile gradient in the presence of 0.1% trifluoroacetic acid as the mobile phase. Samples were lyophilized, reconstituted and concentrated to approximately 0.6 mM in 20 mM sodium phosphate, pH 7.20. 2H2O was added to a final concentration of 5% (v/v). The sample used for residual dipolar coupling (RDC) measurements was prepared by partial alignment of the uniformly 13C15N-enriched Na-FAR-1 in a solution of magnetically aligned filamentous Pf1 bacteriophage  (ASLA biotech). The degree of alignment was evaluated by measuring the 2H quadrapolar splitting in the HDO resonance. After testing several conditions, a NaCl concentration of 300 mM with 9 mg·ml−1 of bacteriophage and 300 μM protein was selected.
NMR spectroscopy and analysis
All spectra were recorded at 311 K on a Bruker AVANCE 600 MHz spectrometer equipped with TCI cryoprobe. Resonances were assigned, as described previously . All spectra were processed in AZARA (Wayne Boucher, http://www.bio.cam.ac.uk/azara). Maximum entropy reconstruction  was used to enhance resolution of the indirect dimensions of 3D experiments. Spectra were analysed with CCPNmr analysis software . Frequency-based methods were employed to measure 1DNH  and 1DCαHα  couplings from in-phase/antiphase 15N-HSQC (heteronuclear single-quantum correlation spectroscopy) spectra. Distance restraints for structure calculations were derived from 3D 15N-NOESY-HSQC  and 3D 13C-NOESY-HSQC spectra, each recorded with 100 ms mixing time.
Structure calculations from NMR-based restraints
Distance restraints were derived from NOESY (nuclear Overhauser effect spectroscopy) cross-peaks with the initial mapping from normalized intensity to distance and grouped in distance bins. NOE distance restraints were incorporated in restrained MD calculations using the ambiguous distance restraints formalism  using ARIA 2.3  and CNS . Loose backbone dihedral restraints for regions of regular secondary structure predicted based on secondary chemical shifts by DANGLE  were incorporated during the high temperature phases of the simulations but omitted during the final cooling phase. RDC and hydrogen bond restraints were then introduced. The average RDC alignment tensor was estimated from the ensemble calculated using only NOEs with PALES  and used to incorporate the RDC restraints via the SANI potential  in square-well mode. The 20 structures that best satisfy the experimental restraints were chosen from 100 structures generated in the final iteration and refined in explicit water . The quality of these structures was analysed using PROCHECK_nmr  and their co-ordinates deposited in the Protein Data Bank under accession code 4UET. Structure figures were generated using PyMOL (http://www.PyMOL.org).
NMR relaxation measurements
15N-relaxation time constants, T1 and T2 were assessed using the method of Kay [55–57] at a field strength of 14.1 T. Relaxation delays for assessment of T1 were 101, 601, 1001 and 1401 ms whereas those for T2 were 17, 34, 68, 102 and 136 ms. Selected time points in each series were repeated in order to estimate the inherent error in calculation of cross-peak intensities. Relaxation times T1 and T2 were calculated using non-linear least squares fitting. Collection of 15N-HSQC-heteronuclear NOE experiments with and without saturation allowed extraction of 1H,15N NOE values. Both saturation and reference experiments were repeated for the purpose of error estimation.
Ligand-binding analysis by chemical shift perturbation
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. Double-labelled 13C-15N recombinant Na-FAR-1 (0.4 mM) was titrated by sequential addition of small volumes of unlabelled sodium oleate (Sigma–Aldrich) from a 125 mM stock solution in water (pH ∼9). Triple resonance experiments were recorded at selected points of the titration in order to assign the displaced cross-peaks. The titration started by addition of 0.5 molar equivalent of the ligand and was continued until turbidity due to the presence of insoluble oleic acid was observed.
Lipid extraction and analysis
Total lipids were extracted according to the methodology described by  and , with minor modifications. For unstripped Na-FAR-1 (no RP-HPLC purification), lipids were extracted from 15 mg of protein and compared with control apo-Na-FAR-1 (RP-HPLC purified protein). For comparison, approximately 3 ml of culture of E. coli BL21 (λDE3) cells were lysed by sonication. Each sample was mixed with 15 ml of CHCl3–CH3OH (2:1) and vigorously shaken for 15 min in an ice bath. The homogenate was washed with 250 μl of 2.9% (w/v) NaCl solution. After agitation, the phases were separated by centrifugation and the upper, aqueous phase discarded. The lower phase containing lipids was recovered and dried under a stream of N2 gas, re-dissolved in CHCl3 and stored at–20°C under N2 gas until analysis.
Lipid classes bound to Na-FAR-1were analysed by TLC (thin layer chromatography) on silica gel plates Si250 (J.T.Baker) with the methodology and solvent systems described by . Lipid samples obtained from holo-Na-FAR-1 and E. Coli lysates controls and standards were spotted on TLC plates (20×20 cm) previously activated at 100°C for 30 min and developed with methyl acetate–isopropanol–chloroform–methanol–0.25% KCl (25:25:25:10:9, by volume) for polar lipids and hexane/diethyl-ether/acetic acid (80:20:1, by volume) for neutral lipids.
Non-esterified FA and phospholipid (PL) TLC spots were scraped and extracted from the silica with pure chloroform for FAs and chloroform–methanol–water for PLs respectively. FA composition was analysed by GC (gas chromatography)–MS of their methyl ester derivatives, prepared with BF3-methanol according to the method of Morrison and Smith  as described previously . The individual FA methyl ester peaks were identified by comparison of their retention times with those of standards and by their mass spectra.
Fluorescence experiments were performed with a Fluorolog-3 Spectrofluorometer (Horiba-Jobin Yvon). Buffer alone was used to correct for Raman and background scattering. RP-HPLC delipidated Na-FAR-1 was employed in all ligand-binding experiments.
The ligand-binding capacity of Na-FAR-1 was investigated with the fluorescent ligands 11-(dansylamino)undecanoic acid (DAUDA) and retinol. Stock solutions (10 mM) were prepared in ethanol and then diluted in PBS for use in the assays. Retinol solutions were diluted in ethanol and added directly to the cuvette to minimize degradation.
The FA chain length preference of rNa-FAR-1 was tested by displacement of the fluorescent ligand DAUDA as described . Binding of non-fluorescent ligands was detected by a reversal of the wavelength shift and a decrease in fluorescence emission intensity on equal additions of test ligands to a DAUDA–rNa-FAR-1 complex recorded at the peak fluorescence emission wavelength of DAUDA in the protein (470 nm). The concentration of Na-FAR-1 in the cuvette was 1.5 μM. DAUDA ethanol stock solution was diluted 1:10000 in PBS for use in the assays at 1 μM. Stock solutions of all the non-fluorescent competitors were made to approximately 10 mM in ethanol, then diluted in PBS for use in the assays.
RESULTS AND DISCUSSION
Na-FAR-1 had been identified in a gene survey of the N. americanus transcriptome under the name N. americanus LBP-20 (sequence ID NAC00128) . LBP-20 proteins are currently named FAR due to their capacity for binding FAs and retinol and Na-FAR-1 was renamed accordingly . More recently, the genome of N. americanus has been published and at least six FAR proteins have been recognized (Figure 1; Supplementary Figure S1), including Na-FAR-1, under the sequence name NECAME_14208 . Na-FAR-1 cDNA predicts a 19364.57 Da protein with a 14 amino acid secretion signal peptide predicted by SignalP . The post-translational removal of the leader peptide would yield a mature protein of 155 amino acids with a molecular mass of 17082.49 Da.
(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 Mr 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  using the BLOSUM 62 matrix from a T-coffee WS sequence alignment  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.
A phylogenetic tree was constructed to show the relationship between Na-FAR-1 and FARs from other species (Figure 1b). The tree was constructed with amino acid sequences omitting any leader peptides identified by SignalP. The sequences included those of C. elegans (Ce-FAR-1–8), the human parasitic nematodes Onchocerca volvulus FAR-1 (Ov-FAR-1), B. malayi (Bm-FAR-1), animal parasitic nematodes A. caninum (Ac-FAR-1 and Ac-FAR-2), Ancylostoma ceylanicum FAR-1 (Ace-FAR-1), Ostertagia ostertagi FAR-1 (Oo-FAR-1) and Heligmosomoides polygyrus FAR-1 (Hp-FAR-1) and the plant parasitic nematodes Globodera pallida FAR-1 (Gp-FAR-1) and Meloidogyne javanica FAR-1 (Mj-FAR-1). FAR transcript levels in some of these parasites are notably high, particularly in the parasitic stages of life cycles [11–18,62,66]. Figure 1 shows that most FAR proteins known from parasites group together, including those from N. americanus, and a subset of FARs from C elegans (Ce-FAR-1, 2 and 6). As noted above, Ce-FAR-7, the only FAR protein whose structure was previously known, falls in a subfamily of FARs distant from all of the FAR proteins from parasites  (Figure 1b).
Na-FAR-1 localization within the parasite
Na-FAR-1-encoding mRNA had been detected in adult and larval L4 N. americanus life stages  and analysis of differences in gene expression between infective larvae (iL3) and adult parasitic stages shows that Na-FAR-1 is mainly expressed in the adult, blood-feeding stage . Immunohistochemistry revealed that Na-FAR-1 protein occurs in the cloacal aperture and copulatory bursa of male worms (Figures 1c and 1d) and in the intestine (Figures 1f and 1g). The presence of Na-FAR-1 in the parasite's intestinal cells may indicate involvement in transportation and storage of lipids derived from host blood and its location in copulatory bursa of male worms suggests its function may additionally be related to reproduction. Na-FAR-1 was detected in the intestinal cells but not the reproductive structures of females (Figure 1e).
3D structure of Na-FAR-1
The 3D structure of Na-FAR-1 was determined by both protein X-ray crystallography and solution state NMR spectroscopy. Na-FAR-1, which had not been subjected to reverse phase HPLC purification to strip out co-purifying ligands, crystallized as previously reported and diffracted to 2.14 Å . Molecular replacement using the Ce-FAR-7 structure proved unsuccessful and we therefore obtained the crystal structure of Na-FAR-1 in complex with co-purifying ligands, hereafter referred to as holo-Na-FAR-1, by anomalous dispersion from crystals of Se-Met-labelled protein (Table 1). In contrast, NMR spectra of unstripped Na-FAR-1 in solution, like those of other FAR proteins we have tested, were characterized by broad signal peaks indicative of multiple conformations and/or conformational exchange. However, stripped Na-FAR-1 gave good solution NMR spectra and the structure of apo-Na-FAR-1 was determined from a total of 7289 NOE-derived distance restraints, 316 dihedral angle restraints and restraints derived from 177 RDCs (101 1DNH and 76 1DCaHa) observed in a sample that had been partially aligned in Pf1 filamentous bacteriophage (⇓Table 2).
The overall folds of the Na-FAR-1 structures in complex with ligands (holo) and without ligand (apo) are similar (Figure 2a–c). Superposition of the holo and apo structures gives a 1.814 Å co-ordinate RMSD (root-mean-square deviation) for all heavy atoms (1.579 Å for main chain heavy atoms only). Na-FAR-1 presents a wedge-shaped structure with two larger faces each of approximately 40 by 30 Å in area and of ∼17 Å in width at the wide end of the wedge. The fold is organized into 11 helices of various lengths, defining an internal cavity (Figure 3). The N-terminal helices, 310 (residues 3–5), α2 (8–11) and α3 (16–24) are co-planar with the C-terminal helices α9 (126–135), α10 (137–144) and α11 (147–152) and together with one of the longest helices, α6 (57–75), form one large face of the wedge. The other two long helices, α7 (79–99) and α8 (107–122), are co-planar with α5 (45–55) and form the other large face. These two faces enclose an internal cavity that is closed at the thick end of the wedge by α4 (29–37), which is almost perpendicular to the long axis of the molecule. Hydrophobic residues are located almost exclusively on the inward-facing sides of the helices with their side chains pointing towards the internal cavity. Most of the polar and charged residues are on the external surface of the protein generating a predominately hydrophilic surface. The notable exceptions are Ser88, Lys96 and Tyr100 whose side chains are located within the cavity, whereas His67 and Arg93 stand on either sides of the largest opening to the cavity.
Numbers in brackets indicate values in the highest resolution bin (2.27–2.14 Å). Abbreviation: PDB, protein structure database.
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