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ARTICLE |
CORRESPONDENCE Ian A. Wilson: wilson{at}scripps.edu OR Vipin Kumar: vkumar{at}tpims.org
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Innate pathways of immunity are important not only in providing a rapid response against infectious agents, but also in the effective induction and/or modulation of adaptive responses. Natural killer T (NKT) cells that share the cell-surface receptors of NK cells and, in addition, express an antigen receptor (TCR) are generally stimulated by lipid antigens and rapidly secrete large amounts of IFN-
NKT cells recognize lipid antigens when bound by nonpolymorphic, MHC class Ilike CD1 molecules (4). APCs, such as dendritic cells, macrophages, and subsets of B cells, express CD1 molecules. The CD1 family consists of five distinct genes (CD1ae) that can be categorized into two groups. In humans, group 1 consists of CD1a, CD1b, CD1c, and CD1e, whereas CD1d is the only member of group two and is also expressed in mice.
The first crystal structure of mouse CD1d revealed a deep hydrophobic ligand binding groove, which seems to be well-suited for the binding of long chain hydrophobic compounds, such as lipids, where electron density for two long aliphatic chains was observed, but not fully interpreted, in the binding groove (5). Recently, this bound self-ligand was identified as phosphatidyl choline (PC), and the CD1d crystal structure confirmed that the two lipid tails are inserted into the A' and F' pockets, with the headgroup extending out of the groove for T cell recognition (6).
Other structural determinations of a variety of CD1ligand complex structures, such as CD1asulfatide (7), CD1alipopeptide (8), CD1bphosphatidyl inositol, and CD1bGM2 (9), CD1bglucose monomycolate (10), mouse CD1d-galactosyl ceramide (
-GalCer; short-chain, C8; reference 11), and human CD1d
-GalCer (long-chain, C26; reference 12), have provided fascinating insights into the general mode of ligand binding and elucidated the basis for CD1 isotype-specific variation. Each lipid ligand inserts its aliphatic moieties, either two alkyl chains for glycolipids or one alkyl chain for lipopeptides, into the deeply buried hydrophobic cavities of CD1, thereby positioning the diverse T cell epitopes, carbohydrate moieties for glycolipids and peptidic moieties for lipopeptides, above the hydrophobic binding groove on the CD1 surface for inspection by TCRs specific for either the ligand and/or the CD1 isoform (13).
Because different lipids are either present in or traffic to distinct endocytic compartments, it has been suggested that the different CD1 isotypes have evolved to target these various intracellular locations. CD1b and CD1d are localized mainly in the late endosomes or lysosomes where microbial lipids accumulate after infection, whereas CD1a recycles back to the cell surface via passage through early or recycling endosomes (14, 15). Recently, a family of endosomal lipid transfer proteins, the saposins, has been shown to be necessary for the presentation of certain lipids by CD1. It has been proposed that these saposins have the ability to extract and bind monomeric lipids from the membrane, which could then be transferred through a yet unknown mechanism to CD1 in the endosomal compartments (1618).
Characterization of lipid-reactive NKT cells and their function is important for understanding the role of this NKT cell subset in the spectrum of immune responses. NKT cells have regulatory effects in both experimental models and in human autoimmune diseases, such as diabetes and multiple sclerosis (2, 3). These cells are autoreactive and express a memory/activation phenotype. NKT cells are heterogeneous, but most of their phenotypic and functional characteristics have been derived from studies of the major mouse NKT cell population that expresses TCRs with an invariant V14+
-chain (V
24+ in human). A nonmammalian glycolipid,
-GalCer, has been invaluable as a highly potent, stimulatory ligand for these NKT cells in the context of CD1d.
-GalCer, unlike most mammalian glycolipids, has its carbohydrate moiety attached via an
rather than a ß linkage. Thus, very little is known about the phenotype or physiological relevance of self-glycolipidreactive NKT cells compared with the
-GalCerreactive invariant NKT cells.
Recently, self-lipids have been identified as ligands for both invariant (i) and variant CD1d-restricted NKT cells (1921). Although iNKT cells recognize isoglobotrihexosyl ceramide (iGB3), a distinct CD1d-restricted population recognizes sulfatide when presented by CD1d (19, 20). Staining of cells with sulfatideCD1dtetrameric complexes ex vivo demonstrated that sulfatide-reactive NKT cells are present in the liver, thymus, and spleen. Interestingly, during the normal course of experimental autoimmune encephalomyelitis (EAE), a prototype model for T cellmediated autoimmune disease that is characterized by inflammation and demyelination in the central nervous system (CNS), NKT cells reactive against sulfatideCD1d tetramers, but not -GalCerCD1d tetramers, are increased severalfold within the CNS (19). Furthermore, the treatment of mice with 20 mg sulfatide prevents antigen (MOG35-55 peptide)-induced EAE in CD1d+/+ mice, but not in CD1d/ C57BL/6 mice (19). Thus, a sulfatide-reactive subset of NKT cells can be targeted for manipulation of autoimmune responses in experimental autoimmunity.
In an attempt to characterize self-glycolipid recognition by NKT cells and, more precisely, to elucidate the structural differences in ligand binding and NKT cell stimulation between -anomeric (
-GalCer) versus ß-anomeric (sulfatide) glycolipids by mouse CD1d, we crystallized CD1d in complex with a synthetic sulfatide self-antigen and determined its three-dimensional structure at 1.9 Å resolution.
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Results |
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To analyze the fine specificity of sulfatide-reactive T cells, we have used synthetic homologues or analogs of sulfatide comprised of different fatty acid chain lengths (C16C24) to characterize the immunodominant sulfatide species. In vitro proliferation or cytokine secretion assays were used to determined the most active species as cis-tetracosenoyl (C24:1, mono-unsaturated fatty acid) sulfatide (Fig. 1 A). Other sulfatides, including tetracosanoyl- (C24, saturated fatty acid), palmitoyl- (C16, saturated fatty acid), and lyso-sulfatides (lacking the fatty acid chain) did not induce a significant immune response (Fig. 1 A). Furthermore, adjuvant-free administration of cis-tetracosenoyl sulfatide in three different mouse strains resulted in significant amelioration of a chronic and relapsing form of EAE (unpublished data). Sulfatides, either naturally derived from bovine brain or as synthetic cis-tetracosenoyl species, can be efficiently loaded onto purified mCD1d, as judged by isoelectric focusing gel electrophoresis (Fig. 1 B). The negative charge on the sulfatide promotes a shift in the migration of exogenously loaded mCD1d compared with endogenous CD1d, which is likely loaded with a neutral self-antigen, such as PC (6).
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The sulfatide negative charge arises from the 3' sulfate group. Because mono-GM1 that contains sialic acid, which is also negatively charged, does not induce proliferative/cytokine responses, and because GM1CD1d-tetramers do not stain NKT cells (unpublished data), it seems unlikely that the negative charge alone is responsible for specific recognition by NKT cells. In addition, the 3' sulfate group is required for the activation of sulfatide-specific NKT cells because ß-GalCer, which is identical to the sulfatide but lacks the 3' sulfate group, does not stimulate this NKT cell pool. As 3'-sulfated -GalCer is also not able to activate sulfatide-specific NKT cells (unpublished data), it is suggested that these sulfatide-reactive NKT cells are specific for the configuration of the sulfated galactose moiety (ß form active,
form inactive) at the CD1d surface.
Structure determination of the CD1dsulfatide complex
Soluble and fully glycosylated, heterodimeric mouse CD1d-ß2M protein (residues 1279 heavy chain and 199 ß2M) was secreted by Spodoptera frugiperda (SF9) cells upon infection with recombinant baculovirus and purified to homogeneity by affinity and size-exclusion chromatography, as described below in Materials and methods. After partial deglycosylation of the protein using Endoglycosidase H (Endo H), synthetic cis-tetracosenoyl sulfatide was loaded by incubating CD1d with a 10-fold molar excess of sulfatide without any detergents. The CD1dsulfatide complex was further purified by size-exclusion chromatography. More than 95% of the CD1d protein appeared to be loaded with sulfatide, as judged by native IEF gel electrophoresis (unpublished data). Using the sitting drop vapor diffusion method, we crystallized the CD1dsulfatide complex and determined its three-dimensional structure by molecular replacement using the protein coordinates of the CD1d short-chain -GalCer complex (PDB code 1Z5L) as the search model (Table I and Fig. 2; reference 11). The crystal structure was refined to a final Rcryst and Rfree of 18.6 and 24.8%, respectively. The asymmetric unit of the crystal contains two CD1dsulfatide complexes (A and B), which are very similar (root-mean-square deviation of 0.55 Å for all C
atoms). Thus, we describe here only the crystal structure for complex A, except where stated otherwise.
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Although the sulfated galactose headgroup makes an additional hydrogen bond with CD1d, as compared with the short-chain -GalCer variant PBS-25 (11), its electron density is less well-defined. The ß-linkage between the galactose and the ceramide backbone in the sulfatide results in a much higher degree of solvent exposure of the headgroup, whereas the two short hydrogen bonds (2.5Å and 2.6Å) between Asp153 and the
-linked galactose of PBS-25 pull the galactose toward the
2-helix in an orientation horizontal to the CD1 surface, which results in more intimate contacts with CD1d, as reflected by similar B-values of the PBS-25 galactose and the surrounding CD1 residues.
Comparison of - versus ß-anomeric ligands
A comparison of the orientation of the sulfatide and the PBS-25 ligand in the mCD1d binding groove (Fig. 5, A and B) illustrates that their lipid backbones are bound in a similar location, likely due to the specific hydrogen bonding between the ceramide moiety and the CD1 residues, whereas the different linkage between the galactose headgroups and the lipid ( vs. ß) results in conformational differences between each CD1dlipid complex. The ß-anomeric galactose (sulfatide) is highly exposed and projects out of the binding groove perpendicular to the
1- and
2-helices (Fig. 5 A), whereas the
-anomeric galactose (PBS-25) sits flat atop the binding groove between both helices, thereby burying the lipid backbone while simultaneously exposing less surface area for recognition by the semi-invariant V
14 NKT cell receptor (Fig. 5 A and B, bottom). However, the short-chain
-GalCer ligand PBS-25 induces the formation of a roof above the F' pocket (Fig. 5 B) from CD1d residues, which can now play a role in CD1dTCR interaction. Small chemical differences in the structure and binding of CD1d-specific ligands can then be directly transmitted to the CD1d surface and, therefore, can aid in ligand discrimination by NKT cells. In the case of the sulfatide ligand, where the galactosylsulfate moiety sticks out of the groove, interaction with the variant sulfatide-reactive NKT cells is likely to be different from iNKT cells. Here, the TCR has to accommodate a more protruding headgroup and a different conformation of residues on the CD1d surface. In addition, the sulfatide ligand exposes more of its lipid backbone, which could now play a role in the T cellrecognition process and help explain the biological differences among various sulfatide species that have different length fatty acid chains (Fig. 1 A).
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Discussion |
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All of the four lipid ligands that have been crystallized in complex with either human (12) or mouse (6, 11) CD1d so far show a common mode of lipid binding. The C18 sphingosine chain is always inserted in the F' pocket, and fatty acid (C8C26) is always inserted in the A' pocket. This orientation is similar for PC, where the shorter fatty acid chain (C12) is inserted in the A' pocket and the longer fatty acid (C24) in the F' pocket. Nevertheless, a sphingolipid with a C18 fatty acid, as found in sulfatides from natural sources, could insert its two alkyl chains in either pocket if the length of the alkyl chain is the only factor responsible for ligand binding. But the crystal structure of the CD1d-GalCer (PBS-25) complex revealed that the short fatty acid (C8) is surprisingly inserted only in the A' pocket. The orientation still needs to be determined for the recently identified family of
-anomeric microbial ligands, the glycuronosyl ceramides (C14; references 24 and 25). Thus, we propose that the N-amide linkage between the fatty acid and the sphingosine together with the hydroxyl groups of the sphingosine chain (Fig. 2 C, highlighted in yellow), which forms a precise hydrogen-bonding network with CD1, constrains the ceramide backbone that orients the respective tails into the A' and F' pockets.
It is not known whether the immunodominance of cis-tetracosenoyl sulfatide is due to a more efficient binding to CD1d or to the presence of specific TCR repertoires that are stimulated preferentially by individual sulfatide species. In this regard, it is interesting to note that the T cell hybridoma specific for lyso-sulfatide does not recognize any of the other sulfatides examined so far (unpublished data). Whether NKT cells specific for each individual sulfatide express a unique TCR or whether overlapping TCR repertoires exist is currently not clear and should be determined by generating T cell hybridomas reactive to each of the individual sulfatides. Of interest, however, is that although the hydrogen bonding network formed between the lipid backbone and CD1d is responsible for the orientation of the different lipid ligands in the binding groove, slight differences in the positioning of sulfatide and -GalCer can be observed when both structures are superimposed (not depicted). The same is true when the binding of
-GalCer to CD1d is compared in both mouse and human crystal structures. The exchange of one residue at the CD1d surface (Trp153 in human and Gly155 in mouse) results in a different positioning of the galactose, whereas the lipid backbone is bound in the same orientation (26). Therefore, it is possible that the biological differences between the different sulfatide species shown in Fig. 1 A are not only the result of differential loading characteristics of the ligands, but rather the consequence of subtle structural differences upon ligand binding due to either differences in fatty acid chain length and/or saturation (C16 vs. C24:1), or, in case of lyso-sulfatide, the complete lack of a fatty acid chain. However, it cannot be completely ruled out that the stronger NKT cell activation by cis-tetracosenoyl sulfatide is a result of the increased solubility of the mono-unsaturated and more polar nervonyl (C24:1) fatty acid versus the fully saturated tetracosanoyl (C24:0) fatty acid, which could lead to an increased efficiency of loading onto CD1.
Recognition of sulfatide by CD1d-restricted NKT cells has important implications in autoimmune diseases of the CNS, as sulfatides are one of the major glycolipid components of the myelin membranes that are targeted in such diseases as multiple sclerosis (MS). MS is a demyelinating disease mediated by a T cellguided immune response that is either initiated from antigen-presenting events in the CNS or induced after the peripheral activation by a systemic molecular mimicry response (27, 28). Indeed, in MS patients, increased serum levels of glycolipids (29, 30) and antibodies directed against them have been reported (3134). T cells specific for glycolipids have been isolated from MS patients. Their frequency in five active MS patients was three times higher compared with five normal individuals (35). Recently, it has been demonstrated that sulfatide binds promiscuously to all of the CD1a, CD1b, CD1c, and CD1d isoforms (36). Because CD1 molecules are up-regulated on macrophages in areas of demyelination in chronic-active MS lesions but not in silent lesions in the brain (37), it is possible that self-glycolipids from myelin could be presented during local inflammation to T cells. Microglia, as well as infiltrating macrophages, could either engulf myelin components or internalize them by Fc receptors or by complement receptormediated phagocytosis after binding to myelin-specific antibodies. Thus, activated APCs in the CNS could not only present peptides (MHC) to T cells, but also glycolipids (CD1). Thus, myelin glycolipidreactive T cells could potentially influence the inflammation and demyelination in the CNS. It is clear from our data (19 and unpublished data) that the peripheral activation of sulfatide-reactive T cells after adjuvant-free administration of sulfatide ameliorates experimental autoimmune disease of the CNS. Because CD1 molecules, unlike the classical MHC molecules, are nonpolymorphic, insight into the molecular recognition of sulfatide by the CD1d molecules and their interaction with a unique CD1d-restricted NKT cell population will be extremely valuable in the potential development of non-HLAdependent therapeutic approaches for autoimmune demyelinating diseases in humans.
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Materials and Methods |
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Reagents.
Purified sulfatides from bovine myelin and other semi-synthetic sulfatides, including lyso-sulfatide, palmitoyl-sulfatide, and tetracosanoyl-sulfatide, were purchased from Matreya Inc. Synthetic cis-tetracosenoyl sulfatide was synthesized as reported previously (38). For biological experiments, lipids were dissolved in vehicle (0.5% Tween 20 and 0.9% NaCl solution) and diluted in PBS. For structural studies, cis-teracosenoyl sulfatide was dissolved in DMSO. All monoclonal antibodies were purchased from BD Biosciences.
Cell preparation.
Hepatic lymphocytes were isolated using established Percoll gradient methods. In brief, animals were killed by CO2 inhalation and perfused with chilled PBS. To obtain lymphocytes, the liver was removed, cut into small pieces, passed through a 70-µm nylon cell strainer (Falcon; Becton Dickinson), and suspended in DMEM supplemented with 2% heat-inactivated FBS. After centrifugation (1,500 g for 10 min), the cell pellet was washed and resuspended in the same medium. Lymphocytes were isolated from parenchymal hepatocytes, nuclei, and Kupffer cells by Percoll (35% Percoll containing 100 U/ml heparin) gradient separation. Splenocytes were obtained by passing the spleen through a 70-µm nylon strainer and suspension in DMEM with 2% heat-inactivated FBS. Erythrocytes were lysed with RBC lysis buffer (BD Biosciences).
Monoclonal antibodies and flow cytometry.
Mononuclear cells were isolated and suspended in FACS buffer (12 x 106/ml) containing 0.02% NaN3 in PBS (wt/vol) and 2% FBS (vol/vol) and treated with antibodies against FcR- (2.4G2) to block nonspecific binding and then labeled with the indicated monoclonal antibodies. For flow cytometry staining, 2.5 µg of tetramerized mCD1d was used. Flow cytometric analysis was performed on a FACSCalibur instrument using CELLQuest software (Becton Dickinson).
Tetrameric mCD1dlipid complexes.
Mouse CD1d protein was generated using the baculovirus expression system essentially as described previously (19). Unloaded mCD1d tetramers were prepared by preincubating biotinylated mCD1d protein with vehicle only. To produce glycolipidmCD1d tetramers, biotinylated mCD1d protein was incubated with sulfatide at a molar ratio of 1:6 for 1820 h at 22°C.
Measurement of proliferative and cytokine responses.
For proliferative response to the self-glycolipids, 8 x 105 splenocytes from naive C57BL/6 (CD1d+/+) or CD1d/ mice were cultured in vitro for 72 h in the presence of increasing concentrations of cis-tetracosenoyl sulfatide (550 µg/ml). [3H]thymidine incorporation was quantitated as described previously (19, 39). For cytokine secretion, supernatants from 48-h cultures were collected and IFN- and IL-4 levels were determined using typical sandwich ELISA assays using reagents from BD Biosciences as described previously (19, 39). IL-2 secretion by sulfatide-reactive NKT cell hybridomas was determined in culture supernatants by standard ELISA.
In vitro loading of sulfatides onto mouse CD1d.
Soluble mouse CD1d (9 µg/18 µl in PBS) was mixed with 4 µg (2.0 µl) of purified bovine brainderived sulfatide or the synthetic cis-tetracosenoyl sulfatide and incubated at 37°C for 6 h followed by washing with 500 µl of PBS and concentrated using 10 kD mol wt cut-off centrifugal concentrators (Millipore). After adjusting the remaining volume, equal amounts of protein were subjected to IEF gel electrophoresis to monitor the loading efficiency of sulfatide onto CD1d.
Protein expression, purification, and crystallization.
The baculovirus transfer vector pAcUW51 (BD Biosciences) containing the nucleic acid sequence for the extracellular domains of mCD1d and ß2M, including signal sequence, was provided by M. Kronenberg. To engineer a CD1d construct for crystallization purposes, the biotinylation domain, generally used for the preparation of CD1 tetramers followed by a hexahistidine tag, was removed by digestion with the restriction enzyme BamHI and replaced with a short hexahistidine tag that was introduced by ligation of two complementary BamHI-restricted oligonucleotides. A SalI site was incorporated in the oligonucleotide to verify the integration of the fragment by restriction digestion. Three colonies were analyzed by sequencing to find one clone with the right orientation of the hexahistidine tag. The constructed plasmid was cotransfected into SF9 cells with BaculoGold DNA using Cellfectin reagent (Invitrogen) according to the manufacturer's specifications. After several rounds of virus amplification, the protein was expressed for 3 d in shaking flasks (145 rpm) at 28°C using SF9 cells (2 x 106 cells/ml) with a multiplicity of infection of 3. The expression experiment was typically performed on a 58-liter scale. SF9 cells were removed from the cell culture media by centrifugation, and the media was further concentrated to 1 liter and washed several times with PBS buffer. The protein was purified from the concentrated media by ion metal affinity chromatography on a Ni-NTA resin (QIAGEN) in 50 mM Tris/HCl buffer, pH 8.0, followed by anion-exchange chromatography using MonoQ HR 10/10 (GE Healthcare) in 10 mM Tris/HCl buffer, pH 8.0, with a linear gradient of 0250 mM NaCl. Two of the four total N-linked carbohydrates were removed after the proximal N-acetyl glucosamine residue by Endo H cleavage, as judged by mass spectrometry analysis (unpublished data). In brief, 1 mg CD1d protein was incubated with 50 mU Endo H in 100 mM sodium acetate, pH 5.9, at 37°C for 2 h and purified by size-exclusion chromatography (Superdex S200 10/300 GL; GE Healthcare). The synthetic sulfatide ligand was then loaded onto the partially deglycosylated protein by incubating 3 mg CD1d with a 10-fold molar excess of synthetic sulfatide (0.12 ml of a 5 mg/ml solution in DMSO) in 100 mM sodium acetate buffer, pH 5.9, for 6 h at 37°C with intermittent agitation. The loaded CD1d protein was purified by ion-exchange chromatography on a MonoQ column using a shallow gradient of NaCl (0150 mM over 25 column volumes). Loading of sulfatide onto CD1d was observed by native IEF gels that monitored a shift toward the cathode due to the additional negative charge from the sulfate moiety. The CD1d fractions from the MonoQ run were pooled and concentrated to 7.5 mg/ml in 10 mM Hepes, pH 7.5, and 25 mM NaCl. The best crystals were obtained at 22°C by mixing 1 µl protein (7.5 mg/ml) with 1 µl precipitant solution (2 M ammonium sulfate, 0.1 M sodium cacodylate, pH 6.5, 0.2 M NaCl, and 10 mM manganese chloride) and grown for 2 wk for data collection.
Structure determination.
Crystals were flash-cooled at a temperature of 100 K in mother liquor containing 25% glycerol. Diffraction data from a single crystal was collected at Beamline 8.2.2 of the Advanced Light Source, Berkeley, CA, and then later from another crystal at Beamline 11.1 of the Stanford Synchrotron Radiation Laboratory and processed to 2.7 Å and 1.9 Å, respectively, with the Denzo-Scalepack suite (40) in spacegroup P2 (unit cell dimensions: a = 58.86 Å, b = 74.84 Å, c = 101.56 Å, and ß = 102.1°). Two CD1lipid complexes occupy the asymmetric unit with an estimated solvent content of 53.8% based on a Matthews' coefficient (Vm) of 2.66 A3/Da. Molecular replacement in P21 was performed with the lower resolution dataset in CCP4 (41) using the program MOLREP (42) and the CD1d-PBS-25 structure (15ZL) as the search model, with the ligand removed, which resulted in a Rcryst of 48.2% and a correlation coefficient of 0.46. Subsequent rigid-body refinement in REFMAC 5.2 to a resolution of 3.5 Å resulted in an Rcryst of 41.4%. The initial refinement included several rounds of restrained refinement against the maximum likelihood target in REFMAC 5.2. Tight restraints were maintained between the two molecules in the asymmetric unit throughout the refinement of the lower resolution dataset. At a later stage of refinement, carbohydrates were built at 5 out of the 8 N-linked glycosylation sites in both CD1 molecules. When the 1.9 Å high resolution dataset became available, it was then used for subsequent refinement of the model derived from the 2.7 Å resolution data. Before model building and restrained refinement, one round of rigid-body refinement was performed to 3.5 Å resolution that resulted in an Rcryst of 28%. Tight noncrystallographic symmetry restraints were applied only during the first round of refinement and then not used further, as they resulted in an increase in the Rfree value. The refinement progress was judged by monitoring the Rfree for cross-validation (43). The model was rebuilt into A-weighted 2FoFc and FoFc difference electron density maps using the program O (44). Water molecules were assigned during refinement in REFMAC using the water ARP module for >3
peaks in an FoFc map and retained if they satisfied hydrogen-bonding criteria and returned 2FoFc density >1
after refinement. Starting coordinates for the sulfatide ligand were obtained from the CD1asulfatide structure (1ONQ) and modified accordingly using the molecular modeling system INSIGHT II (Accelrys, Inc.). The sulfatide library for REFMAC (45) was created using the Dundee PRODRG2 server (46). Final refinement steps were performed using the translation, libration, and screw-rotation displacement procedure in REFMAC (47) with a total of six anisotropic domains (three per complex:
1-
2 domain,
3-domain, and ß2M) and resulted in improved electron density maps for the glycolipid ligand and a further drop in Rfree. The CD1dsulfatide structure has a final Rcryst of 18.6% and an Rfree of 24.8%. The quality of the model (Table I) was assessed with the program Molprobity (48).
Structure presentation.
The program Pymol (49) was used to prepare Figs. 2, 3, and 4. The programs Molscript (50), GRASP (51), and Raster3D (52) were used to prepare Fig. 5.
Accession codes.
Coordinates and structure factors for the CD1dsulfatide complex have been deposited in the Protein Data Bank under accession code 2AKR.
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Acknowledgments |
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This study was supported by National Institutes of Health grants GM62116, CA58896 (both to I.A. Wilson), and CA10066 (to V. Kumar), and The Skaggs Institute for Chemical Biology (to I.A. Wilson, C.-H. Wong, and D.M. Zajonc). This is manuscript number 17631-MB of The Scripps Research Institute.
The authors have no conflicting financial interests.
Submitted: 11 August 2005
Accepted: 27 October 2005
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References |
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