Structural basis of cholesterol binding by a novel clade of dendritic cell modulators from ticks

Two crystal structures of Japanin, an 18 kDa immune-modulatory lipocalin from the Brown Ear Tick (Rhipicephalus appendiculatus), have been determined at 2.2 and 2.4 Å resolution. In both crystal forms the protein is in complex with cholesterol, which sits in a closed pocket at the centre of the lipocalin barrel. Both crystal forms are dimers, which are also observed in solution. Molecular modelling suggests that previously-described members of a tick protein family bearing high sequence homology to Japanin are also likely to bind cholesterol or cholesterol derivatives.

molecule of cholesterol. It is noted that no cholesterol was added exogenously at any stage during purification or crystallisation. As was predicted from the sequence, the protein folds as a lipocalin, with an 8-stranded anti-parallel barrel at its centre. The three crystallographically-independent molecules superimpose with an overall C α rmsd of 1.3 Å across 152 residues (overlap computed with the program Theseus 10 ). The main sites of conformational mobility are the hairpin loop 48-58, the loop 105-112 and the C-terminus, residues 145-152. A search against the Protein Databank reveals that the closest structural homologues are the female-specific histamine-binding protein (FS-HBP2; PDB IDs 3g7x, 1qft) with a rmsd C α of 2.7 Å over 133 residues; and the OmCI complement inhibitor (PDB IDs 2cm4, 2cm9, 3zuo, 3zui, 5hcc, 5hcd, 5hce) with a rmsd C α of 2.5 Å over 123 residues 11 . Two disulphide bonds are observed (Japanin residues Cys28-Cys150 and Cys114-Cys138), the latter taking two alternative conformations. Residues Asn35 and Asn131 bear N-linked glycans, but they are not in close proximity, neither within the monomer nor in the context of the dimer 12 . Figure 1 shows two views of the protein.
Japanin dimer. Both crystal forms contain the same Japanin dimer, which has an interface area of about 1090 Å 2 , involving 32 residues and a calculated solvation free energy gain upon formation of the interface of −9.5 kcal/mole (as computed with the protein interfaces, surfaces and assemblies' service PISA at the European Bioinformatics Institute 13 ). In the orthorhombic form, the two molecules in the asymmetric unit form the dimer (see Fig. 2a). In the tetragonal crystals, the same dimer is formed by the asymmetric unit and a symmetry-related molecule across a twofold axis. The tetragonal crystal-form dimer and the orthorhombic crystal-form dimer superpose with an rmsd of 1.5 Å over 297 C α s.
The dimer interface involves the 67-75 and 105-112 loops, the 93-99 strand and the 129-141 helix. Figure 2b shows details of the dimer interface between molecules A and B in the lattice, centred around the Phe93A:Phe93B side chain stacking contact, with two additional pairs of hydrophobic residues forming contacts across the interface: Pro139A:Tyr70B and Tyr97A:Ile137B (and equivalent ones due to the twofold symmetry of the dimer, Pro139B:Tyr70A and Tyr97B:Ile137A). Two additional hydrophobic contacts are formed between CH2 moieties of Ser112 and Pro72 (again intermolecularly). Buried in the middle of this hydrophobic patch are the 2.0 Å hydrogen bonds between the side chain of Arg95 on one molecule and the main chain oxygen atom of Ile 94 on the neighbouring molecule. At the surface rim of the interface the Asp98A:Arg132B (and Arg132A:Asp98B) salt bridges are found. The dimer is likely to represent the solution species, as indicated by Multi-Angle Laser Light Scattering (MALLS) in solution (see Fig. 2c).
Cholesterol binding. In each of the three crystallographically-independent molecules, fifteen residues lining the lipocalin pocket are involved in close intermolecular contacts with a cholesterol molecule, with a ligand:protein interaction surface of about 280 Å 2 , 3% of the surface of the molecule, see Fig. 3. Residues Ala6, Leu21, Val26, Val29, Thr31, Arg43, Leu45, Phe63, Leu86, Ala88, Leu104, Ser115 and Trp117 all make hydrophobic contacts shorter than 3.5 Å with the cholesterol molecule; the -OH group of cholesterol hydrogen bonds with the main chain NH of Glu23 (distance O-H: 2.0 Å) and to the Nε 2 atom of His17.
The presence of cholesterol in association with Japanin raises obvious questions as to the functional role of cholesterol binding to the protein: in particular, whether the presence of cholesterol in the lipocalin pocket is related to Japanin's immune-modulatory function. Cholesterol and a range of cholesterol derivatives have been shown to have immune-modulatory roles, either directly [14][15][16] or through interactions with the gut microbiota 17 .   Modelling of cholesterol derivatives in the Japanin ligand-binding pocket shows that epicholesterol, epicholestanol, epicoprostanol, and 7-dehydrocholesterol could fit in the pocket with minor rearrangements of the side chains. A 22(R)-hydroxy group would lead to a clash with Val76, making binding to derivatives carrying this group unlikely. Residues Thr31, Arg43, Ser115 and Trp117 could hydrogen bond to cholesterol derivatives bearing polar groups on the tail, such as 25-OH-cholesterol. Amino-acid side chains that carry a charge when on the surface of proteins at physiological pH are the exception rather than the rule within apolar protein pockets, and yet the Japanin pocket features Arg43 (completely conserved across members of the clade (Fig. 4)) and His17, whose Nε 2 atom is only 3.9 Å from the ligand's 3-hydroxy group. Thanks to the latter residue, cholesterol derivatives carrying a negative charge on the 3-hydroxy group (e.g. cholesterol sulfate) may therefore bind to the Japanin pocket more tightly than cholesterol. The Arg43 guanidinium side chain, on the other hand is buried at the base of the pocket and involved in a hydrogen bonding network with the main chain O atoms of Ala6, Asn8, Gln10, Thr31 and the hydroxyl group of the latter residue. It is possible that the Arg43 side chain could engage cholesterol derivatives carrying polar groups on the tail, such as 27-hydroxycholesterol 18 .

Japanin homologues are likely to bind cholesterol or cholesterol-derivatives. A number of tick
lipocalins have sequences that are highly similar to that of Japanin, forming a clade of tick dendritic cell modulators 4 . Figure 4 shows the sequence alignment of the Japanin protein to those homologues, annotated with the secondary structure elements and painted by conservation. The high degree of sequence conservation of the residues involved in the dimer interface suggests these proteins would also dimerise like Japanin. Based on sequence conservation and homology modelling, RA1, RA2 and RS are also all as likely to bind cholesterol as Japanin (no clashes in the pocket). Furthermore, the Dermacentor andersoni putative protein DA1244 would also be capable of binding cholesterol (most of the pocket is very conserved) and in fact could accommodate a larger ligand due to the substitution of Japanin F63 for DA1244 V66.

Discussion
Cholesterol and its derivatives, besides being major components of eukaryotic cellular membranes, play essential roles in cellular functions as important and as varied as intracellular transport; cell signalling; nerve conduction; regulation of lipid, glucose, and energy metabolism 19 ; drug metabolism and detoxification; and inflammation 20,21 . For example, cholesterol binding is crucial for human GPCR Smoothened to transmit native Hedgehog signalling 22,23 .
Tear lipocalins 24 and proteins belonging to different fold families have been reported to bind and either transport or chemically modify cholesterol [25][26][27] . Table 1 reports cholesterol-binding protein domains whose crystal structures were determined in their apo forms or in complex with either cholesterol or cholesterol derivatives. In the Japanin crystals, the cholesterol ligand is completely sequestered inside the protein, except In most cholesterol binding proteins of known structure, a conformational change would therefore be required for uptake and release of the ligand. Opening and closing of a protein "lid" to favour exchange of cholesterol has been invoked for example for the oomycete protein cryptogein 41 . The Japanin loop 18 SSKLFERNVG 27 is not kept in place by any strong bonds, and might therefore form an opening and closing lid, although its thermal motion in the crystals is not particularly higher than the rest of the structure.
Biophysical simulations using the Protein Energy Landscape Exploration (PELE) algorithm revealed low binding energies for cholesterol, typical of a ligand 42 . However, in silico docking suggests that the Japanin pocket can accommodate a variety of cholesterol derivatives. Japanin's natural ligand(s) may therefore be cholesterol derivatives which -unlike cholesterol -were not available during recombinant protein production. It is also possible that a ligand present when Japanin is produced by the tick may be exchanged for a cholesterol derivative following secretion into the host. Indeed, arthropods are unable to synthesize cholesterol de novo 43 , so it is likely that a cholesterol ligand would be bloodmeal-derived. Moreover, uptake of Japanin by the host endocytic pathway would result in a low pH environment following endosomal acidification, perhaps promoting intracellular ligand release in favour of a cholesterol derivative with a negative charge on a moiety bound to the 3-hydroxy group. Alternatively, going from alkaline tick saliva into serum might be enough to trigger an exchange, especially at inflammatory sites, where pH is known to drop 44 . The presence of Arg43 and His17 in the binding pocket may point to pH changes as critical to ligand selectivity and to the ligand binding/ releasing process. Inside the pocket, without neighbouring acidic amino-acids side chains, the pK A of the guanidinium side chain of Arg43 and His17 may differ from the ones for the same amino acids in isolation. Their side chains may be uncharged at physiological pH, when the molecule is empty or cholesterol is in the pocket. Alternatively, a polar (charged) cholesterol derivative may be the physiological ligand, and a hydrogen bond (salt bridge) between a Arg43 and/or His17 and a polar (charged) cholesterol derivative would ensure permanent trapping of the ligand after sequestration. Last but not least, Japanin residues Thr31, Arg43, Ser115 and Trp117 could hydrogen bond to cholesterol derivatives bearing polar groups on the tail: oxidized derivatives of cholesterol are potent immune-suppressors, with 25-hydroxycholesterol for example acting as an inhibitor of humoral and cellular responses 45 . The most relevant of these compounds (given the role of Japanin in dendritic cell biology) is 7-alpha, 25-dihydroxycholesterol (7,25,DHC), which functions as a guidance cue for EBI2-expressing dendritic cells, positioning them in a location where they encounter blood-borne particulate antigens 46 . Overlaying 7,25,DHC onto the cholesterol molecule in the crystal structure places the 7-hydroxyl group within hydrogen bonding distance of Serine 115, a residue in the completely conserved 114 CSLWV 118 motif in the Japanin clade (see the alignment in Fig. 4).
Whatever the physiological ligand(s), lipids play important roles in the metabolism and activation of immune cells 47 , and it is plausible that Japanin's function is mediated by ligand sequestration. For example, a recent study discovered that cholesterol crystals activate Syk and PI3 kinases in human macrophages and dendritic cells, driving IL-1 production in a Syk-and PI3K-dependent manner, and activating the downstream MAP kinases; in these human innate immunity cells, cholesterol induces S100 and MMP1 expression via tyrosine kinases 48 . Olesoxime, a cholesterol-like compound, has been shown to favor oligodendrocyte maturation in culture and promote myelin regeneration in rodents 49 . The effects of cholesterol-deprivation on immunity are suggested by the phenotype of Staggerer mice which are defective in the cholesterol-binding ROR nuclear receptor and exhibit defects in the immune and inflammatory response 50 . Collectively these observations suggest that cholesterol sequestration by Japanin, perhaps localised at the bite site, might be directly responsible for at least some of Japanin's immune-modulatory properties.  Table 1. Cholesterol-binding proteins of known structure.
In summary, the Japanin's crystal structures have revealed it to be the prototypic cholesterol-binding lipocalin, a group which is very likely to include other members of the Japanin-like clade of hard tick lipocalins, while also hinting at directions for future research into the mechanism of action of this unusual immune-modulatory molecule.

Materials and Methods
Protein expression and purification. Protein expression and purification was as previously described 4 . In brief, polyhistidine-tagged recombinant protein was produced by Sf9 cells infected with recombinant baculovirus, then purified using Talon resin followed by gel filtration.
Crystallisation, X-ray diffraction and data processing. Crystals were grown in the course of several weeks by the vapour diffusion method in sitting drops at 21 °C, set up using an Oryx nano crystallisation robot (Douglas, UK).
The crystallisation drops were set up by mixing 0.12 μl of protein solution at OD 280 = 6.6 (approximately 5.2 mg/ml) in gel filtration buffer (50 mM Tris.HCl, pH 7.5, 150 mM NaCl) with 0.08 μl of the crystallisation screen, and were equilibrated against 70 μl of mother liquor.
Japanin-P 4 1 2 1 2: square-bipyramidal crystals initially grew from condition H9 of the Molecular Dimensions ProPlex screen: 0.1 M imidazole pH 7.0 and 50% v/v 1-methyl-pentan-(2,4)diol (MPD). The crystals could be reproduced by screening around the condition and grew in the pH interval 6.9-7.2 and MPD range 40-50%. A 2.2 A X-ray diffraction dataset was collected from one such crystal and indexed in a tetragonal primitive space group. Scaling and systematic extinctions suggested P4 1 2 1 2 or P4 3 2 1 2 (details in Table 2). Japanin-C222 1 : a prismatic crystal grew in condition 1 of the JCSG + screen 51 : 0.2 Li 2 SO 4 , 0.1 M CH 3 COON a pH 4.5 and 50% v/v PEG 400. A 2.4 Å X-ray diffraction dataset was collected and indexed in a C-centred orthorhombic space group. Systematic extinctions suggested C222 1 . The same C222 1 crystal form also grew in condition H5 of the Molecular Dimensions Structure screen 2-41: 0.01 M cetyltrimethylammonium bromide, 0.5 M NaCl and 0.1 M MgCl 2 . This crystal gave 2.5 Å diffraction data and a structure that is equivalent to the one described here.
Diffraction data were collected on beamline ID29 at the ESRF, Grenoble, France. X-ray data integration and scaling were done using the computer programs XDS 52 and Scala 53 , in the CCP4 suite, run from the data processing suites xia2 54 and autoPROC 55 . Table 2 shows the crystallographic data collection and processing statistics.
Phasing and refinement. The Japanin-P4 1 2 1 2 crystal form was phased by molecular replacement with the computer program Phaser 56 in the CCP4 suite, using a search model obtained from PDB ID 1qft, modified using Chainsaw 57 in the CCP4 suite and further manual trimming of loops. One copy in the asymmetric unit in P4 1 2 1 2 gave a good initial hit which was then improved by iterative automated model building in Buccaneer 58 in the CCP4 suite, using 2Fo-Fc maps computed in autoBUSTER 59 and alternating cycles of full B refinement and TLS refinement. The program Coot 60 was then used for manual rebuilding, again using autoBUSTER for refinement. The model for the bound cholesterol was built in the residual electron density (see Fig. 3) once the model for the protein had been completed, starting from the idealised coordinates and stereochemical dictionary downloaded from the Hic-UP server 61 .
The Japanin-C222 1 crystal form was phased by molecular replacement using the Japanin-P4 1 2 1 2 coordinates as a search model. The program Phaser 56 in the CCP4 suite placed two molecules in the asymmetric unit of C222 1 . Refinement in autoBUSTER and manual building in Coot followed. All stages of refinement implemented automated non-crystallographic symmetry restraints. Table 3 contains the final crystallographic refinement data and statistics.
The diffraction data and coordinates have been deposited as PDB IDs 4boe and 4bqu for the tetragonal and orthorhombic crystal forms respectively.