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The Journal of Immunology, 2005, 175: 977-984.
Copyright © 2005 by The American Association of Immunologists

Crystal Structure of Mouse CD1d Bound to the Self Ligand Phosphatidylcholine: A Molecular Basis for NKT Cell Activation1,2

Barbara Giabbai*, Stèphane Sidobre{dagger}, M. D. Max Crispin{ddagger}, Yovan Sanchez-Ruìz§, Angela Bachi§, Mitchell Kronenberg{dagger}, Ian A. Wilson{ddagger} and Massimo Degano3,*

* Biocrystallography Unit and {dagger} Mass Spectrometry Unit, DIBIT San Raffaele Scientific Institute, Milan, Italy; {ddagger} Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92121; and § Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
NKT cells are immunoregulatory lymphocytes whose activation is triggered by the recognition of lipid Ags in the context of the CD1d molecules by the TCR. In this study we present the crystal structure to 2.8 Å of mouse CD1d bound to phosphatidylcholine. The interactions between the ligand acyl chains and the CD1d molecule define the structural and chemical requirements for the binding of lipid Ags to CD1d. The orientation of the polar headgroup toward the C terminus of the {alpha}1 helix provides a rationale for the structural basis for the observed V{alpha} chain bias in invariant NKT cells. The contribution of the ligand to the protein surface suggests a likely mode of recognition of lipid Ags by the NKT cell TCR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Athird lineage of Ag-presenting molecules, CD1 proteins are able to bind lipids or glycolipids on the surface of professional APCs. CD1-bound lipid Ags are presented for recognition by T cells via a specific TCR-mediated interaction (1). CD1 molecules share a highly similar tertiary fold with class I MHC molecules, but the CD1 binding cavity is modified to create a deeper, narrower cleft lined by hydrophobic residues, which reflects the unique Ag binding specificity of these molecules (2).

CD1 molecules can be classified into two groups based on sequence similarity. Human CD1a, -b, -c, and -e as well as orthologous molecules in other species can be included in group I (3), and human and murine CD1d and their orthologs belong to group II (4). This distinction is paralleled by a clearly different functional role in vivo. All group I CD1 proteins present microbial lipids and lipoglycans (5, 6, 7) as well as self glycosphingolipids to T lymphocytes (8). Group I CD1-restricted T cells include double-negative (9), CD8+, and CD4+ phenotypes (10) and are involved in the acquired immune response or may participate in pathogenic autoimmune reactions, such as the response to brain-derived lipids in multiple sclerosis. Group II CD1 molecules restrict the response of NKT cells, a subset of T lymphocytes expressing NK receptors (11). Most NKT cells in mice are activated through the engagement of their TCR with a CD1d-ligand complex (12). The precise nature of this ligand is still unknown, but NKT cells are considered to be self-reactive, and therefore, this ligand is thought to be an autologous lipid Ag. The glycosphingolipid {alpha}-galactosyl ceramide ({alpha}GalCer),4 when bound to CD1d, strongly activates NKT cells. Because this compound was derived from a marine sponge, and because it has an {alpha} anomeric linkage of the hexose sugar to the ceramide lipid, which is rare in nature, {alpha}GalCer is not considered to be the natural ligand for NKT cells. This compound is a potent TCR agonist (13), however, and it has been instrumental in defining the characteristics and physiological role of these cells.

The great majority of {alpha}GalCer-reactive NKT (iNKT) cells bear a semi-invariant TCR (invTCR) (11) with a conserved {alpha}-chain rearrangement (V{alpha}24J{alpha}18 in humans; the homologous V{alpha}14J{alpha}18 in mice) and a limited use of V{beta} gene segments (V{beta}11 in humans; largely V{beta}8.2 in mice) (14). Other ligands that can be bound to CD1d include cellular phospholipids, such as phosphatidylinositol (PI) that act as chaperones in stabilizing the hydrophobic CD1d cavity (15). These compounds could represent an inactive form of a ligand that becomes highly antigenic during inflammation and could then trigger NKT cell activation, but to date it has not been possible to activate most NKT cells with phospholipids (15, 16, 17). A notable exception is represented by the 24.8.A iNKT cell hybridoma that is activated by the self phospholipid phosphatidylethanolamine (PE) containing a polyunsaturated acyl chain (18).

NKT cells are a paradigm of T regulatory lymphocytes, able to secrete large amounts of cytokines, such as IL-4, IL-10, or IFN-{gamma}, within minutes of TCR ligation (19). IFN-{gamma} secretion by NKT cells can dramatically potentiate the rejection of tumors (20). Administration of {alpha}GalCer to diabetes-prone NOD mice blocked the onset of autoimmunity, either by stimulating the synthesis of Th2 cytokines such as IL-4 or by causing hyporesponsiveness of autoreactive T cells (21). Hence, NKT cells can act as rheostats of the immune response.

Although the central role of CD1d/NKT cell pathway in the modulation of several types of immune responses has been established, the molecular and structural details of lipid Ag binding to CD1d and the activation of the TCR of iNKT cells remain elusive. Crystal structures of human CD1b (hCD1b) bound to PI or the GM2 ganglioside showed a complex series of tunnels able to bind the hydrophobic acyl chains of ligands (22) and solved the conundrum of how long chains, such as those present in mycolic acids, could be accommodated in the CD1 binding cavity (23). The structure of an hCD1a/sulfatide complex showed how the CD1 binding groove is tailored in different isoforms to bind specific lipidic ligands, and how the hydrophobic acyl chains could also be part of the TCR recognition surface (24). These structural differences between CD1 molecules are clearly encoded by the primary structure that delineates various frameworks for the selection of Ags. Thus, a detailed knowledge of the atomic interactions involved in ligand binding to CD1d proteins provides further insight into the structural basis for lipidic Ag recognition by NKT cells. Thus, we determined the crystal structure of murine CD1d (mCD1d) bound to insect phosphatidylcholine (PC) to 2.8 Å resolution. The CD1d/ligand complex shows a different arrangement of hydrophobic tunnels compared with both hCD1a and hCD1b, and a distinct orientation of the Ag polar headgroup. The contribution of the Ag to the receptor recognition surface underlines how CD1d-restricted TCRs must sample highly localized structural changes to activate NKT cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Protein production and purification

Murine CD1d was produced in stably transfected Drosophila melanogaster S2 cells following previously described protocols (25). The protein was purified using a combination of immobilized metal affinity, size exclusion, and anion exchange chromatography. Purity levels were judged to be >95% from Coomassie Blue-stained denaturing SDS-PAGE gels. The protein concentration was estimated using the bicinchoninic acid assay.

High performance TLC (HPTLC) and mass spectrometric analysis

One hundred micrograms of pure recombinant mCD1d was extracted twice with a 1/1 mixture of methanol and chloroform in Eppendorf tubes. Organic phases were concentrated to 10 µl under a flow of nitrogen gas and spotted on HPTLC silica plates. Chromatograms were developed in glass tanks with a 25/25/25/10/0.02 solvent mixture of ethyl acetate, 1-propanol, chloroform, methanol, and sodium chloride. All plates were prerun in the same solvent mixture. Plates were dried, dipped in a 1% (v/v) solution of phosphomolybdic acid in ethanol, and heated for 10 min at 100°C. The standards used were a mixture of total myelin lipids from bovine cauda equina and commercially available PC or phosphatidylserine. For mass spectrometric analysis, developed plates were reversibly stained using iodide vapors. The ligand band was scraped off with a scalpel and extracted overnight in chloroform-methanol (3/1, v/v). The organic phase was injected in a QSTAR Pulsar quadrupole time-of-flight mass spectrometer (MDS Sciex) equipped with a nano-electrospray source for precursor ion scanning (PIS) experiments. PIS of the 184.075 m/z characteristic for the phosphocholine headgroup can identify PC species with enhanced sensitivity. Individual PC-containing molecules were monitored in negative mode on a Bruker Esquire 3000+ Ion Trap (Bruker Daltonics) to assess the fatty acid composition of PC after addition of 5 mM ammonium chloride.

Crystallization

Murine CD1d was concentrated to 6 mg/ml using ultrafiltration devices. Orthorhombic crystals (a = 41.80 Å, b = 107.01 Å, c = 110.63 Å, {alpha} = {beta} = {gamma} = 90°) in space group P212121 were reproducibly obtained with the hanging drop vapor diffusion method using 100 mM Tris (pH 7.4), 200 mM (NH4)2SO4, 25% polyethylene glycol 4000 as precipitant by mixing equal volumes of protein and precipitant solution. Typically, 100 x 50 x 50-µm crystals appeared after 3 wk at 25°C. Crystals were transferred to a cryoprotectant solution containing 100 mM Tris (pH 7.4), 200 mM (NH4)2SO4, 30% polyethylene glycol 4000, and 15% glycerol and plunged in liquid N2 for long-term storage at 100 K.

Data collection

Diffraction data were collected at beamline 7.1 of the Stanford synchrotron radiation laboratory using the oscillation method at a wavelength of 1.0 Å in 1° scans on a MAR345 imaging plate detector. Crystals were kept at 100 K during data collection. Data were indexed, integrated, and reduced using MOSFLM and SCALA. Intensities were converted to structure factors using the French and Wilson algorithm in the program TRUNCATE (26).

Structure solution and refinement

The structure was solved using the molecular replacement technique as implemented in the program MOLREP (27) against data from 25 to 3.0 Å. Coordinates of one monomer of the monoclinic crystal form of mouse CD1 (2) (PDB code 1CD1) were used as the search model after resetting the B values to 38 Å2. After rigid body refinement, the R value was 0.40 with a correlation coefficient of 0.53. The structure was rebuilt by extensive manual adjustment using the program O (28) in a prime-and-switch likelihood-modified electron density map calculated with RESOLVE (29). Difference electron density maps showed unambiguously the presence of carbohydrates at three N-linked glycosylation sites. A frame shift affecting the terminal residues (273–279) of the heavy chain was apparent in the electron density maps and was correctly modeled in the mCD1d/PC complex. The structure was subjected to cycles of maximum likelihood positional and isotropic temperature factor refinement, as implemented in REFMAC5 (30), followed by manual model rebuilding. Sigmaa-weighted (31) and shake-omit maps (32) calculated with (2Fo-Fc, {phi}c) and (Fo-Fc, {phi}c) coefficients were simultaneously inspected to avoid model bias. Shake-omit maps were calculated after omitting stretches of 10 residues and perturbing the model coordinates to a final root mean square deviation (rmsd) of 0.5 Å, occasionally followed by five cycles of refinement. Only adjustments that resulted in decrease in Rfree and did not lead to divergence between Rcryst and Rfree were considered successful. The initial electron density maps exhibited weak positive difference density inside the mCD1d cavities that allowed the inclusion of short acyl chains to the model. The residual density improved in quality after careful refinement of the protein structure. Inclusion of C24/C12 PC in the model improved the refinement statistics by 4% (both Rcryst and Rfree) and accounted for all the residual electron density in the binding cavity. Modeling of a shorter acyl chain in the A' pocket (for instance, with a C18/C12 PC molecule) did not satisfy all residual density and resulted in higher R values after refinement. At later stages of refinement, one glycerol molecule was included in the model as well as 26 water molecules that displayed density >4.0{varsigma} in residual Fo-Fc maps and resulted in 2Fo-Fc density >1.2{varsigma} after refinement using ARP/wARP (33).

Model analysis

Model quality was judged using the programs O and OOPS2 (34). Protein-ligand contacts for the mCD1d-PC, hCD1b-PI (PDB code 1GZP), and hCD1b-GM2 (1GZQ) complex structures were analyzed using the program CONTACSYM (26). The rmsd values were calculated using the program LSQMAN. Molecular and solvent-accessible surfaces were calculated with the program MSMS (35). Surface clefts and binding site volume calculations were evaluated using the program SURFNET (36), defined as volumes accessible to 1.6, but not to 6.0, Å radius spheres.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PC is bound to recombinant mCD1d

Recombinant class I MHC molecules produced in insect cells are not devoid of Ag in the binding groove; rather, they bind a mixture of peptides from the growth medium (37). Furthermore, we previously characterized murine NKT cell hybridomas that reacted to soluble mCD1d without addition of exogenous Ag (38). To determine whether a specific ligand was bound to the recombinant protein, we performed an organic extraction of purified mCD1d, followed by HPTLC analysis. The major component of the organic phase had retention factors substantially identical with PC standards, both from a commercial source and in purified myelin lipids. Minor amounts of PE and sphingomyelin were observed (Fig. 1). The ligand was also not detected in the insect cell growth medium or in class I MHC molecules produced in the same expression system (not shown). To further characterize the nature of the ligand bound to mCD1d, we performed mass spectrometric analysis on the HPTLC-purified material (39, 40). We identified seven major molecular species of PC with total fatty acid compositions consistent with C36:2, C34:1, and C34:2 bound to mCD1d. Thus, PCs with different acyl chain compositions are the main molecular species bound to insect cell-produced mCD1d. PC shares a common structural framework with the epitome of group II CD1 ligands, {alpha}GalCer (Fig. 1). PE is also bound to recombinant mCD1d, consistent with previous studies showing its weak agonist activity toward iNKT cell hybridomas (18).



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  		FIGURE 1. Identification of the ligand bound to recombinant mCD1d produced in Drosophila melanogaster cells. A, HPTLC analysis of chloroform/methanol extraction of purified mCD1d. The major band from CD1d-extracted material corresponds to PC. Two low intensity bands with retention factors corresponding to PE and sphingomyelin were also observed in the experiments. Lane 1, Myelin lipids (ML) standards purified from bovine cauda equina; lane 2, organic phase from mCD1 extraction; lane 3, phosphatidylserine (PS); lane 4, PC. B, Mass spectrometric analysis of HPTLC-purified lipid from insect cell-produced mCD1d. Identification of CD1d-bound PC species by PIS on lipid extracts redissolved in 20 µl of MeOH/CHCl3 (3/1, v/v) and 5 mM ammonium acetate. At least seven PC molecules differing in fatty acid composition were identified. Based on the molecular masses observed, the peak at 788.03 Da is consistent with C36:2 PC. It should be noted that the intensity units reported on the y-axis do not correlate with the relative abundance of the different species. C, Comparison of the chemical structures of PC, as modeled in the mCD1d cavity of the crystal structure, with {alpha}GalCer.

 
Crystal structure of mCD1d bound to PC

We crystallized mCD1d in complex with a mixture of Drosophila-derived PCs and solved its structure to 2.8 Å resolution (Table I). The electron density maps improved during refinement and facilitated the interpretation of a PC molecule with fatty acid chains of C24 and C12 composition (Fig. 2). The inclusion of the long fatty acid chain was guided by the residual electron density observed in the binding cavity, which already was very strong within the A' and F' pockets and at the glycerol moiety from the initial stages of refinement. Modeling of a shorter fatty acid, such as C18, resulted in clear residual Fo-Fc density at the 3{varsigma} level inside the binding pocket and higher crystallographic R values. A C24 fatty acid is extremely rare in insect phospholipids, because it is largely found in sphingolipids (41). Nevertheless, the modeled Ag shows the possible binding mode for both short- and long-chain fatty acids in the binding pocket.


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  		Table I. Data collection and refinement statisticsa

 


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  		FIGURE 2. Electron density of the recombinant mCD1d ligand. The electron density was calculated using sigmaa-weighted (2Fo-Fc, {phi}c) coefficients after omitting the ligand atoms. The map is contoured at 1.0{varsigma}, colored in blue, and limited to 2.5 Å from the ligand atoms. The C24/C12 PC molecule fits the continuous density extending from the glycerol junction to the interior of both A' and F' binding cavities. The mCD1d backbone is depicted as a light blue tube, and the PC molecule as cylinders colored according to atom type (carbon in yellow, nitrogen in blue, oxygen in red, and phosphorus in green). This figure was generated with DINO (<www.dino3d.org>).

 
The overall structure of mCD1d bound to PC (Fig. 3) is substantially similar to the previously deposited structure (2) where no ligand was included (rmsd, 0.67 Å for all C{alpha}, 1.72 Å for all atoms in the Ag binding domain). The electron density clearly shows the presence of three N-linked glycans in the mCD1d/PC complex. Remarkably, the Asn165 site shows complete density for a hexasaccharide due to its involvement in lattice contacts with two symmetry-related mCD1d molecules in the crystal.



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  		FIGURE 3. Structure of the mCD1d/PC complex. A, Ribbon diagram of mCD1d/PC with helices in pink, {beta}-strands in blue, and {beta}2-microglobulin in cyan. The bound ligand is represented as a space-filling model, with carbon atoms in yellow, oxygens in red, nitrogen in blue, and phosphorus in magenta. B, The Ag-binding domain of mCD1d bound to PC viewed from the TCR perspective. The N-linked carbohydrates covalently attached to mCD1d are shown in a ball-and-stick representation. The figures were generated using MOLSCRIPT (53 ) and RASTER3D (54 ).

 
Interaction of lipid Ags with mCD1d

The PC molecule is bound to mCD1d with the acyl chains inserted in the A' and F' pockets, and the hydrophilic phosphocholine extending from the central opening of the cleft toward the protein surface. The A' pocket of mCD1d is occupied by the longer acyl chain of PC linked to position 2 of glycerol that is typically esterified by unsaturated chains. The chain adopts a curved conformation pivoting around a pole formed by residues Cys12 and Phe70 (Fig. 4). The conformation of the chain suggests that fatty acids bearing up to four double bonds, such as arachidonic acid, could optimally fit the C-shaped A' pocket. No unsaturation was modeled in the bound Ag due to the intermediate resolution of the crystallographic data. However, the conformation of the fatty acid suggests that double bonds present at position 9, 12, or 15 would impose the correct curvature to the alkyl chain to optimally fit the A' pocket. The acyl chain is bound to the protein exclusively via van der Waals’ interactions. However, the side chains of Gln14, Ser28, Thr37, and His38 extend in the A' pocket within 4.5 Å of the acyl chain and are poised for hydrogen bonding with donors or acceptors branching from the hydrocarbon chain. Hydroxyl groups at the C11, C12, or C17 position of the acyl chain could take advantage of these polar interactions and display higher binding affinities to mCD1d. The A' pocket also displays two small cavities branching from the main tunnel that could accommodate methyl substituents on the acyl chain. The volume of the A' cavity (530 Å3) of mCD1d can fit up to 28 methyl groups, and, similarly to other lipid-binding proteins, shorter acyl chains can be accommodated. The quality of the electron density, despite the heterogeneity of lipids bound to mCD1d, suggests that both long and short acyl chains bind to the A' pocket in the same conformation. These findings are consistent with biochemical data showing a broad specificity of group II CD1 proteins with respect to acyl chain length in modified {alpha}GalCer derivatives.



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  		FIGURE 4. Interaction of ligand with mCD1d. A, Stereo view of the interactions of the C24 acyl chain with residues in the A' pocket. The molecular surface of mCD1d within 4.5 Å of the ligand is represented as a semitransparent surface. The mCD1d chain is displayed as a violet C{alpha} trace. The side chains of hydrophobic residues are in green; potential hydrogen bonding donors or acceptors are in red. B, Interaction of the C12 acyl chain in the F' pocket. C, Stereo view of the hydrogen bonding interactions between the PC headgroup and mCD1d. Atoms are colored according to element type: carbon in yellow, nitrogen in blue, oxygen in red, and phosphorus in green. The molecular surface was calculated in MSMS (35 ), and the figure was generated with DINO (<www.dino3d.org>).

 
The F' pocket is a linear, hydrophobic groove extending from the central opening of mCD1d toward the C-terminal end of the {alpha}1 helix (Fig. 4). The residues lining this pocket are exclusively hydrophobic, with no possibilities for hydrogen bonding interactions between ligand and protein. The electron density for the hydrocarbon chain observed extending from the C1 atom of glycerol in the F' pocket is considerably shorter (C12) than that found in the A' pocket and adopts a fully extended conformation. Further extension of the acyl chain in the cavity by more than four methylene groups is prevented by a wall of hydrophobic residues formed by Phe81, Val85, Ile96, Ile98, and Phe120. Hence, the main restriction posed by the F' pocket to the ligand is apparently the maximum length of the hydrocarbon chain that can be accommodated, limited to 16 carbon atoms. The acyl chain in the F' pocket is partially accessible to solvent around its central carbon atoms (Fig. 4). This access suggests that acyl chains can enter the F' pocket of mCD1d without requiring conformational changes in the protein, hence explaining the differences in pH-dependent binding between hCD1b and CD1d molecules (42, 43). Moreover, the acyl chain in the F' pocket could potentially kink toward the protein surface through this opening, similar to that observed for CD1a- and CD1b-bound ligands (22, 24).

Polar headgroup of mCD1d ligands interacts with protein surface residues

Although the volume and shape of the A' and F' pockets pose constraints on the length, degree of unsaturation, and substitutions of the hydrocarbon chains of lipidic Ags, they do not provide sufficient specificity to orient ligands when both acyl chains are shorter than 18 carbon atoms. Thus, the hydrogen bonding interactions between glycerol (or sphingosine of {alpha}GalCer) and mCD1d contribute to the specific orientation of the hydrocarbon chains in the A' and F' pockets (Fig. 4). The glycerol moiety of PC is bound via hydrogen bonding interactions with the side chains of residue Ser76, Thr156, and an ordered water molecule (Fig. 4). A weak polar interaction is observed between the carbonyl oxygen of the C24 acyl chain and the main chain nitrogen of Tyr73 that probably contributes only marginally to the overall enthalpy of PC binding to mCD1d. The sphingosine backbone of glycosphingolipids could interact with the same protein residues through similar interactions. These hydrogen bonds are clearly important for the binding of Ags to mCD1d, because {alpha}GalCer derivatives without hydroxyl groups on the 3 and 4 carbons of the sphingosine or with altered stereochemistry at these positions are less efficient in stimulating iNKT cells, most likely because of either decreased binding affinity for CD1d or faster dissociation rates. Thus, the structural and biochemical data demonstrate how the hydrogen-bonding interactions between the polar moiety of the Ag and CD1d are crucial determinants for the biological activity of ligands, and how they enhance Ag selectivity.

The PC moiety of PC is oriented toward the C terminus of helix {alpha}1, parallel to the direction of the acyl chain in the F' pocket (Fig. 3). The phosphate group is bound via a direct hydrogen bond to the side chain of Ser76 and a salt bridge interaction to the charged guanidinium group of Arg79. A highly ordered water molecule bridges the phosphate group of the Ag to the side chain of Asp80. The quaternary amino group of choline extends away from the protein surface without making specific contact. The headgroup of PC occupies a volume that corresponds to the P6 position of peptide Ags bound to class I MHC proteins (44). These results are in contrast to those observed for both CD1 isoforms studies performed to date. In the hCD1b structures in complex with GM2 ganglioside or PI, the polar moiety of the ligand has an opposite orientation and makes no specific contacts with the protein (22). In the hCD1a/sufatide structure, the headgroup is inserted more deeply in the F' pocket and sandwiched between the {alpha}1 and {alpha}2 helices. The lipid Ag headgroup orientation with respect to the Ag-presenting molecule also differs from that observed for glycopeptides bound to class I MHC molecules. Nevertheless, the high B values observed in all CD1/ligand crystal structures suggest a certain flexibility of the hydrophilic moiety of these lipid Ags.

Comparison of ligand binding modes in CD1 isoforms

The binding grooves of the various CD1 isoforms have clearly evolved to efficiently and selectively bind different subsets of lipid Ags (Fig. 5). The A' pocket of mCD1d shares highest similarity with hCD1b, but lacks the T' tunnel that allows the accommodation of extremely long chains, such as those found in mycolic acids. The A' pocket of hCD1a is limited in size by residue Val28, resulting in a restricted A' pocket volume. Despite these significant differences, this portion of the CD1 binding groove is relatively conserved in shape and position. The second acyl chain of lipid Ags is bound in completely different fashions in the three CD1 isoforms. In hCD1b, a lateral pocket that is not found in the other isoforms accommodates the acyl chain bound at the C1 position of glycerol. In hCD1a, the acyl chain of the sulfatide Ag extends into the F' cavity with an S-shaped kink. The lipid chain notably rises toward the protein surface because of an underlying defined by residues Val98, Leu116, Phe126, Phe144, and Val147. The homologous residues of hCD1b and mCD1d are smaller in size and do not induce a similar protrusion in the molecular surface. A unique feature of mCD1d is the ridge caused by Leu100, which prevents long acyl chains extending directly from the A' to the F' pocket and forces the hydrophilic glycerol moiety of PC toward the protein surface.



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  		FIGURE 5. A comparison of the binding cavities of different CD1 isoforms. The molecular surfaces of the cavities in the different complex are shown as semitransparent surfaces. The two views in each panel are related by a 90° rotation about the vertical axis. The protein residues from each isoform that define the structure and properties of the binding cavities are depicted. The ligands are shown as stick models. A, The mCD1d/PC; B, hCD1a/sulfatide; C, CD1b/PI/detergent complexes. Protein residues are in green. Ligand atoms are colored as follows: carbon atoms in yellow, nitrogen in blue, oxygen in red, sulfur in green, and phosphorus in magenta. The figure was generated using MSMS (35 ) and DINO (<www.dino3d.org>).

 
Suggested modes for invTCR interaction with CD1d/ligand complexes

We analyzed the surface clefts of the murine class I MHC molecule Kb bound to the superagonist peptide SIYR (Fig. 6) (44). These pMHC clefts are completely occupied by the CDR1 and CDR3 loops of both the {alpha}- and {beta}-chains of the 2C TCR upon formation of the TCR/MHC/peptide complex. The P4 peptide residue of the SIYR Ag is pinched between the CDR3{alpha} and CDR3{beta} loops, whereas the P6 is accommodated between CDR3{beta} and CDR1{beta}. Similar results have been obtained with other class I MHCs bound to different peptides as well as class II MHC molecules (not shown). Thus, the spatial distribution of the clefts on the Ag-presenting molecules could provide hints about the positioning of the CD1d-reactive TCR. The phosphocholine headgroup in the mCD1d/PC complex is surrounded by two large clefts, similar to the Kb/SIYR complex. The Ag headgroup is positioned similarly to the P6 residue of SIYR and, hence, is likely to interact with the CDR3{beta} and CDR1{beta} loops of the invTCR. This model is supported from differences in reactivity toward mCD1d-restricted glycolipids and phospholipids by invTCRs bearing different CDR3 regions or V{beta} rearrangements (45). To fill the surface clefts of the mCD1d/ligand complex, the footprint of the invTCR could be shifted toward the N-terminal region of the {alpha}1 helix. This difference in orientation together with either an upward displacement or a difference in the tilt angle of the invTCR would allow tighter binding interactions. The limited contribution of the headgroup to the total CD1d/Ag surface (110 Å) (2) compared with the typical corresponding peptide/MHC surfaces (~250 Å2) further underscores how the TCR must be able to sample fine changes in the antigenic surface and appropriately transduce these into lower kinetic off rates and tighter binding interactions.



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  		FIGURE 6. Surface clefts suggest a mode for TCR/mCD1d interaction. A, The molecular surface of H-2Kb in complex with the SIYR peptide (PDB code 1B6J), with the ligand depicted as a ball-and-stick model. The contribution to the surface of the MHC residues interacting with the 2C TCR CDR1 and CDR2 loops of the {alpha}- and {beta}-chains are highlighted in pink and light blue, respectively. The positions of the CDR3 loops are indicated by labels. The surface clefts are rendered as semitransparent orange surfaces. The 2C TCR CDR loops fill the clefts as indicated by the labels. B, Molecular surface of mCD1d with PC as a stick model. The clefts of the mCD1d/PC complex suggest that the CDR1 and three of the {beta}-chains of the invTCR could fill the rightmost cavities and discriminate the Ag. Residues previously mapped by mutagenesis as important for Ag binding or TCR recognition are highlighted in blue. This figure was generated using SURFNET (36 ) and DINO (<www.dino3d.org>).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have identified PC as the major glycolipid bound to mCD1d molecules produced in insect cells. The presence of this phospholipid indicates that loading of ligands into the CD1d groove can occur without any specialized proteins that are present only in APCs of vertebrate cells. Indeed, common self lipids may constitute the initial natural ligand repertoire for CD1 molecules. CD1d molecules can thus bind diverse phospholipids and glycolipids. The crystal structure of murine CD1d in complex with PC clarifies the chemical and structural requirements for ligand binding to group II CD1 molecules. The hydrophobic A' and F' pockets together with the absence of the long tunnel observed in the hCD1b structure pose a constraint on the length of both acyl chains of the bound Ags. Notably, the hydrocarbon chains of the PI and PC ligands in these two structures access the A' cavity from the opposite ends (Fig. 5). The interaction between the acyl chain of PC and the A' pocket residues in mCD1d results in tighter contacts at the entry point compared with hCD1b, thus accounting for the different conformations observed. The curved shape of the A' cavity allows the accommodation of fatty acids containing cis double bonds, such as those found in phospho- and glycolipids, but apparently not trans unsaturated bonds, present, for instance, in PGs. The F' pocket accommodates optimally a saturated 16-carbon chain in a fully extended conformation, although one cannot exclude the possibility that longer hydrocarbon chains could extend toward the surface if local conformational rearrangements occurred. Taken together, the hydrophobic pockets of mCD1d suggest a clear preference for phospho- or glycolipid Ags bearing two short acyl chains, such as found in mammalian cells. Hence, the structural differences among mCD1d, hCD1a, and hCD1b in the arrangement of their binding tunnels explain their different functional roles in the immune system. The binding mode of PC to mCD1d also strengthens the idea that autologous lipids are involved in the activation of iNKT cells in vivo.

A model of interaction of the strong agonist {alpha}GalCer and CD1d based on mutagenesis data suggested that the acyl chain of the glycosphingolipid fits the F' pocket, whereas the sphingosine moiety fills the A' pocket. In hCD1a, the sphingosine chain of sulfatide is also accommodated in the A' pocket (24), strengthening the proposed model for {alpha}GalCer/CD1d interactions. The structure of mCD1d with bound PC suggests a different mode of interaction between the {alpha}GalCer Ag and the protein, where the sphingosine chain fits the F' pocket and the C24 acyl chain fits the A' pocket. This orientation of the hydrophobic tails of Ag would better complement the volumes of the A' and F' pockets, thus resulting in high entropic stabilization. This model is apparently in contrast with the interpretation of previous data showing how the {alpha}GalCer analog AGL587 that bears a short acyl chain (four carbon atoms) was unable to stimulate iNKT cells when bound to the Phe10Ala A' pocket mutant of mCD1d (38), although it could stimulate iNKT cells when bound to wild-type CD1d. The Phe10 residue of mCD1d contributes to the bottom of the curved A' tunnel and interacts via van der Waals’ interactions with the bound hydrocarbon chain. The results from the mutagenesis studies could reflect a lower stability of the mutants compared with wild-type mCD1d. The long acyl chain of {alpha}GalCer could provide ligand-induced stabilization of the CD1d mutant and result in efficient presentation to the invTCR.

Phospholipids, glycolipids, diacylglycerides, and sphingolipids share a common chemical and structural framework that meets the binding requirements of mCD1d. Indeed, PI binds to mCD1d, possibly acting as a chaperone for trafficking of the protein to the cell surface (15). However, the exact nature of the physiological ligands bound to group II CD1 molecules during inflammation to promote NKT cell activity is still elusive. The finding that PC molecules differing in acyl chain composition can bind to the recombinant protein confirms that mCD1d molecules display a relatively broad specificity for hydrocarbon chain length, similarly to fatty acid-binding proteins and nonspecific lipid-binding proteins (46, 47). The absolute predominance of van der Waals’ contacts together with the paucity of hydrogen bonding partners within the binding cavities impose a restraint only on the length of the ligand acyl chain. Moreover, our results strongly suggest an entropy-driven binding and imply that loading of Ag requires displacement of highly homologous endogenous ligands. Hence, subtle modifications in the hydrogen bonding interactions between protein and ligand could be major factors in shifting the binding equilibrium toward a specific Ag. Improved binding could derive, for instance, from a single hydroxylation either in the headgroup or on the acyl chains.

The structure of mCD1d bound to PC further defines the regions of the {alpha}1{alpha}2 domains responsible for TCR recognition. The hydrophilic portion of the Ag can interact with the surface residues of CD1d, effectively modifying the character of the surface presented for recognition to the TCR. Structure-based mutagenesis studies identified several residues of mCD1d that affect recognition of CD1/{alpha}GalCer complexes by iNKT cells (38, 48). Together with the present structure, we can finely dissect the contributions of these mutated residues to interaction with Ag or the TCR. Residues Arg79, Asp80, and Asp153, shown to be important for the stimulation of iNKT cells, all contact the hydrophilic headgroup of the PC ligand. They, therefore, also could contribute to both the binding and orientation of {alpha}GalCer and other Ags for TCR recognition. Residue Ser76 affected the reactivity of only some NKT cell hybridomas and is likely to contribute less to the interactions with the galactose moiety of {alpha}GalCer. Compared with MHC-glycopeptide complexes, the PC headgroup points further away from the Ag-binding domain and is oriented toward the highpoint of helix {alpha}2. Thus, the structure of CD1d in complex with PC suggests a modified TCR recognition of Ag compared with both hCD1b and class I MHC-glycopeptide complexes.

The orientation of the PC polar headgroup toward the C terminus of the {alpha}1 helix also supports the sampling of the Ag by the footprint of the TCR {beta}-chain. Hence, all available data suggest that the main role of the {alpha}-chain of the invTCR is in interacting largely, if not exclusively, with the mCD1d protein, and that of CDR1{beta} and CDR3{beta} is in encoding the fine Ag specificity. However, the highly diverse repertoire of CDR3{beta} sequences in the {alpha}GalCer-reactive NKT cells in vivo supports a germline-mediated recognition of the endogenous Ag(s) driving the expansion of these cells via the CDR1{beta} region (49). The exact definition of the TCR/CD1 interaction must await high resolution structural studies.

The structural aspects of lipid ligand binding to mCD1d derived from the present structure can now be exploited in the search for novel synthetic glycolipid ligands to modulate iNKT cell function in experimental disease models (21, 50, 51). A glycosphinglipid termed OCH, for example, bears only a C4 sphingosine chain compared with {alpha}GalCer and can induce selective secretion of IL-4 by iNKT cells. This ligand has a potentially high relevance for the down-modulation of autoimmunity. and indeed, in one study OCH suppressed the onset of the experimental model of multiple sclerosis in mice more effectively than {alpha}GalCer (50). OCH is clearly unable to fill the A' pocket of mCD1d, and indeed, it displays a reduced binding affinity and faster off rates (52). The lack of A' pocket interactions could force the headgroup of the ligand further away from the surface, modifying the interactions with the invTCR. The qualitatively and quantitatively different structural epitope exposed for TCR recognition would modify the affinity and kinetics of ternary complex formation, and that could result in differential signaling cascades in the T cells and ultimately in selective cytokine secretion. Although this hypothesis remains to be fully tested, it has tremendous potential for the development of a general, haplotype-independent, immunotherapeutic intervention in autoimmune disease.


   Acknowledgments
 
We kindly acknowledge support by the staff of SSRL beamline 7-1. We thank Drs. Giulia Casorati, Paolo Dellabona, and Marika Falcone for critical reading of the manuscript and helpful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Human Frontier Science Program Grant RG0168/2000-M (to M.K. and M.D.); National Institutes of Health Grants AI45053 (to M.K.), CA58896 (to I.A.W.), and GM62116 (to M.K. and I.A.W.); and grants from the Italian Multiple Sclerosis Foundation (to M.D.) and the Italian Foundation for Cancer Research (to M.D.). Back

2 Coordinates and structure factors of the CD1d/PC complex have been deposited in the Protein Data Bank (<www.rcsb.org>), accession code 1ZHN. Back

3 Address correspondence and reprint requests to Dr. Massimo Degano, Biocrystallography Unit, DIBIT Scientific Institute San Raffaele, via Olgettina 58, 20132 Milan, Italy. E-mail address: degano.massimo{at}hsr.it Back

4 Abbreviations used in this paper: {alpha}GalCer, {alpha}-galactosyl ceramide; h, human; HPTLC, high performance TLC; iNKT, {alpha}GalCer-reactive NKT; invTCR, semi-invariant TCR; m, murine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIS, precursor ion scanning; rmsd, root mean square deviation. Back

Received for publication March 21, 2005. Accepted for publication May 2, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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