Bound Bacterial Glycolipid the Crystal Structure of Human Cd1b with A

The Crystal Structure of Human CD1b with a Bound Bacterial Glycolipid Thil Batuwangala, Dawn Shepherd, Stephan D. Gadola, Kevin J. C. Gibson, Nathan R. Zaccai, Alan R. Fersht, This information is current as Gurdyal S. Besra, Vincenzo Cerundolo and E. Yvonne Jones of September 26, 2021. J Immunol 2004; 172:2382-2388; ; doi: 10.4049/jimmunol.172.4.2382 http://www.jimmunol.org/content/172/4/2382 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Crystal Structure of Human CD1b with a Bound Bacterial Glycolipid1,2

Thil Batuwangala,3* Dawn Shepherd,3† Stephan D. Gadola,‡ Kevin J. C. Gibson,§ Nathan R. Zaccai,* Alan R. Fersht,¶ Gurdyal S. Besra,§ Vincenzo Cerundolo,4† and E. Yvonne Jones4*

The human MHC class I-like molecule CD1b is distinctive among CD1 alleles in that it is capable of presenting a set of glycolipid species that show a very broad range of variation in the lengths of their acyl chains. A structure of CD1b complexed with relatively short acyl chain glycolipids plus detergent suggested how an interlinked network of channels within the Ag-binding groove could accommodate acyl chain lengths of up to 80 carbons. The structure of CD1b complexed with glucose monomycolate, reported in this study, conﬁrms this hypothesis and illustrates how the distinctive substituents of intracellular bacterial glycolipids can be

accommodated. The Ag-binding groove of CD1b is, uniquely among CD1 alleles, partitioned into channels suitable for the compact Downloaded from accommodation of lengthy acyl chains. The current crystal structure illustrates for the ﬁrst time the binding of a natural bacterial lipid Ag to CD1b and shows how its novel structural features ﬁt this molecule for its role in the immune response to intracellular bacteria. The Journal of Immunology, 2004, 172: 2382–2388.

he CD1 proteins are a family of Ag-presenting molecules nation of these structural and functional studies suggested that the

that present lipid Ags to T lymphocytes (1). Human CD1 alkyl components of the Ags bind within a hydrophobic groove in http://www.jimmunol.org/ T molecules segregate into two groups according to se- the CD1 protein, the hydrophilic glycan moiety protrudes from the quence homology: group 1 contains CD1a, CD1b, CD1c, and binding groove, making contacts with the TCR (5). CD1e molecules, whereas group 2 contains CD1d molecules. This model for lipid Ag presentation has been recently confirmed Binding studies and structural studies have led to the definition by the crystal structures of human CD1b (6) and CD1a (7) in complex of a general molecular mechanism for lipid Ag presentation by with specific glycolipids. In addition, the structure of CD1b suggested CD1 molecules. The crystal structure of mouse (m)CD1d1 sug- that, unlike other CD1 molecules, this molecule has a unique binding gested that CD1 molecules accommodate hydrophobic acyl chains groove architecture characterized by a series of channels capable of in a hydrophobic binding groove (2). Functional studies supporting accommodating a very broad range of acyl chain length. this view were conducted using a series of analogues of glucose Refolding of denatured CD1b molecules in the presence of gan- by guest on September 26, 2021 monomycolate (GMM)5 presented by CD1b molecules varying in glioside GM2 and phosphatidylinositol (PI) was obtained using a glycosylation, hydroxylation, and lipid length (3, 4). The combi- protocol based on the inclusion of single acyl chain detergents in the refolding to prevent CD1b precipitation (6). The three-dimensional structure of CD1b molecules refolded using this protocol *Cancer Research UK Receptor Structure Group, The Division of Structural Biology, and †Cancer Research UK Tumor Immunology Group, The Weatherall Institute of showed the presence of detergent moieties occupying the channels Molecular Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, that were not filled by the lipid ligands. To test the hypothesis that ‡ United Kingdom; Department of Rheumatology and Clinical Immunology, University of longer acyl chain lipids (such as mycolates and GMM) occupy the full Berne, Berne, Switzerland; ¤School of Biosciences, The University of Birmingham, Bir- mingham, United Kingdom; and ¶Medical Research Council Laboratory of Molecular set of interlinked channels, the crystal structure of CD1b-GMM com- Biology, Medical Research Council Centre, Cambridge, United Kingdom plex, refolded in the absence of detergent, was determined. Received for publication September 5, 2003. Accepted for publication November 25, 2003. Materials and Methods The costs of publication of this article were defrayed in part by the payment of page Protein expression, refolding, and crystallization charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ␣ ␣ The extracellular 1- 3 domains of human CD1b (residues 1Ð283) and 1 ␤ ␤ This work was funded by United Kingdom Medical Research Council (Grants 2-microglobulin ( 2m) were synthesized separately using a prokaryotic G9900061 (to E.Y.J.) and G0000895 (to G.S.B.)), the European Commission Inte- expression system (pET; Novagen, Madison, WI) (6). Both recombinant grated Programme (Structural Proteomics in Europe, Grant QLRT-2001-00988), Can- proteins were purified from Escherichia coli inclusion bodies and solubi- cer Research UK (Grants C375/A2321 (to E.Y.J.) and C399/A2291 (to V.C.)), and the lized in 6 M guanidine-HCl solution containing 10 mM DTT. GMM was Cancer Research Institute. E.Y.J. is a Cancer Research UK Principal Research Fellow. purified from Nocardia farcinica as previously described (8), solubilized at G.S.B. is a Lister Jenner Research Fellow. 200 ␮g/ml in 150 mM NaCl and 0.5% Tween 20 solution, and sonicated. 2 ␤ The atomic coordinates of the CD1b-GMM structure have been deposited in the CD1b and 2m proteins were refolded with the GMM by oxidative refold- Protein Data Bank (http://www.rcsb.org; PDB ID code 1UQS). ing chromatography using a protocol previously described (9, 10). The 3 T.B. and D.S. contributed equally to this work. concentrated refolding mix was purified by size-exclusion chromatography using a HiLoad 26/60 Superdex-75 (Amersham Pharmacia, Piscataway, 4 Address correspondence and reprint requests to Dr. Vincenzo Cerundolo, Cancer Research UK Tumor Immunology Group, The Weatherall Institute of Molecular NJ). Crystallization conditions were screened using purified monomeric Medicine, Nuffield Department of Medicine, University of Oxford, Oxford OX3 9DS, CD1b-GMM glycolipid complexes (7.5 mg/ml in 20 mM Tris-Cl (pH 6.5), U.K. E-mail address: [email protected]; or Dr. E. Yvonne Jones, 25 mM NaCl, and 2 mM EDTA). Nanoliter-scale crystallization experi- Cancer Research UK Receptor Structure Group, The Division of Structural Biology, ments were set up using a Cartesian Technologies Microsys MIC4000 The Henry Wellcome Building for Genomic Medicine, Roosevelt Drive, Headington, (Genomic Technologies, Huntingdon, U.K.) (11, 12). Conditions yielding Oxford OX3 7BN, U.K. E-mail address: [email protected] crystals in nanoliter-scale sitting drops (100 nl of protein plus 100 nl of 5 Abbreviations used in this paper: GMM, glucose monomycolate; PI, phosphatidyl- reservoir solution) were subsequently scaled up and optimized by hand ␤ ␤ ␮ inositol; 2m, 2-microglobulin; RMSD, root-mean-square deviation; m, mouse. pipetting. Optimally diffracting crystals were grown at 20¡C from 2- l

sitting drops, using a 1:1 protein to precipitant (100 mM Na citrate (pH Atomic interactions between glycolipid and protein were calculated us-

5.6), 0.20Ð1.25 M ammonium sulfate, and 0.20Ð0.75 M Li2SO4) ratio and ing LIGPLOT (19). Volume calculations for cavities within the protein 5Ð15 mM urea as an additive in the drops. were calculated using VOLUMES (R. M. Esnouf, unpublished program). Variant glycolipids were modeled into the CD1b binding groove and en- Structure determination and analysis ergy minimized in CNS. The CD1b-GMM speciﬁc TCR, LDN5 (3), was built using sequence information from the IMGT database (20) and modeled Crystals were ﬂash frozen at 100 K in mother liquor containing 20Ð25% using SWISS-MODEL (21). Superimposition of coordinates for comparative glycerol. X-ray diffraction data were collected at beam lines 14.2 (Syn- analyses was conducted using SHP (22). Sequence alignments were performed chrotron Radiation Source (SRS), Daresbury, U.K.) and ID-14 EH4 (Eu- using the MULTALIN web interface (23), and the output was formatted using ropean Synchrotron Radiation Facility (ESRF), Grenoble, France) with ESPRIPT (24). Hydrophobicity calculations were performed via the 0.98- and 1.01-Å radiation, respectively, recorded on Area Detector Sys- PROTSCALE web interface (25) using the Eisenberg method (26). Figures tems Corporation (Poway, CA) Quantum 4 charge-coupled device detec- were produced using BOBSCRIPT (27) and RASTER3D (28). tors. The data were processed using DENZO, SCALEPACK (13), and TRUNCATE (CCP4 suite). Processing statistics are given in Table I. The

crystals belong to spacegroup P3121 with diffraction to 3.8 Å at SRS 14.2 Mass-spectrometric analysis of GMM (with unit cell dimensions, a ϭ b ϭ 97.5 Å, c ϭ 115.2 Å) and to 3.1 Å at ID-14 EH4 (with unit cell dimensions, a ϭ b ϭ 97.0 Å, c ϭ 114.8 Å). The GMM extracted from N. farcinica was analyzed by mass spectrometry as crystallographic asymmetric unit contains one molecule of CD1b-GMM previously described (29). The mass of the GMM bound in the CD1b- with a solvent content of 67%. GMM crystal structure was analyzed using the following protocols. Drops The CD1b-GMM structure was initially solved from data collected at containing crystals were removed by pipette from crystallization trays and SRS 14.2 by molecular replacement with the program EPMR (14) using dissolved in 6 M guanidium-HCl to denature the protein and dislodge the coordinates for unliganded CD1b derived from the CD1b-PI structure (Pro- GMM from the CD1b binding groove. This treatment also cleaved off the sugar group in the GMM to afford free mycolic acids. Separation of my-

tein Data Bank (PDB) entry 1GZQ, (6)). The solution was unambiguous Downloaded from with a correlation coefficient of 67% for data from 20.0- to 3.8-Å resolu- colic acids from the protein was conducted by organic extraction using tion. The structure was refined against the higher resolution data collected chloroform/methanol. The organic phase containing the liberated mycolic acid at ID-14 EH4 using rigid-body, positional, and group B-factor refinement was dried under a nitrogen stream, and the resulting pellet was dissolved in in CNS (15) (Table I). chloroform/methanol. This sample was analyzed by electrospray mass spec- troscopy. Measurements were conducted in positive-ion mode and performed An initial Fo-Fc map was calculated using the molecular replacement solution (unliganded) after rigid-body, positional, and B-factor refinement. on a triple quadrupole LCT instrument (Micromass, Altrincham, U.K.) fitted At a contour level of 2.0 ␴, tubular electron density was clearly visible in with an atmospheric pressure electrospray source. Samples were directly in- ␮ the AЈ,TЈ,FЈ, and CЈ channels. The density spanning AЈ-TЈ-FЈ was con- jected (10 l) using a Rheodyne injector (Rheodyne, Bensheim, Germany) http://www.jimmunol.org/ tinuous with the exception of missing density at three points in the TЈ with methanol as the mobile phase. The machine was run at a flow rate of 200 ␮ channel giving gaps of ϳ0.5, 2.3, and 2.5 Å. Density was also visible for l/min with the cone voltage at 35 V and the spraying needle voltage set at 3 some hydroxyl groups of the sugar head group. kV. The scan rate was 1 s for the mass range from 200 to 2000 Da. Between each further refinement round, the model was manually checked and glycolipid atoms built in using O (16), with reference to Results 2Fo-Fc and Fo-Fc electron density maps generated using phase information calculated from the models. Glycolipid parameter and topology files for Overall structure of CD1b-GMM CNS were generated using PRODRG (17). Water molecules were auto- matically picked using CNS and manually verified. Protein stereochemistry The structure of CD1b-GMM was determined by molecular re- was validated using PROCHECK (18). Statistics for the refined structure placement (see Materials and Methods) and refined at 3.1-Å res- by guest on September 26, 2021 are shown in Table I. olution (Table I). The final model contains CD1b residues 3Ð282

Table I. Crystallographic statistics

ESRF ID-14 EH4 SRS 14.2

Data collection

Spacegroup P3121 P3121 Unit cell (Å) a ϭ b ϭ 97.0, c ϭ 114.8 a ϭ b ϭ 97.5, c ϭ 115.2 ␣ ϭ ␤ ϭ 90¡, ␥ ϭ 120¡ ␣ ϭ ␤ ϭ 90¡, ␥ ϭ 120¡ Resolution range (Å)20Ð3.1 20Ð3.8 Completeness (outer) (%) 99.9 (100.0)a 100.0 (100.0)a Total observations 150,075 134,566 Unique reflections 11,697 6,512 Average I/␴(I) (outer)a 34.9 (5.7)a 19.4 (4.8)a a a Rmerge (outer) (%) 7.7 (60.4) 22.6 (93.1) Model refinement Maximum resolution (Å) 3.10 Reflections (working set/test set)c 10,887/800 d Rwork/Rfree (%) 23.3/29.1 RMSD from standard stereochemistry Bonds (Å) 0.009 Angles (¡) 2.11 Number of atoms Protein/lipid/water 3,011/76/109 Mean B-factors Protein/lipid/water 84.6/76.8/86.0 Ramachandran plot Most favored/additional (%) 69.9/29.8 Generous/disallowed (%) 0/0.3

a Values in parentheses refer to the highest resolution shells (3.21Ð3.10 Å for ID-14 EH4 and 3.93Ð3.80 Å for SRS 14.2 datasets). b ϭ⌺ Ϫ͗͘ ⌺͗ ͘ Rmerge (Iobs I )/ I . c All reflections with F Ͼ 0 were included. d ϭ⌺͉ Ϫ ͉ ⌺ R Fobs Fcalc / Fobs, where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively. Rfree is as for Rwork but calculated for a test set comprising reflections not used in refinement. 2384 CD1b-GMM CRYSTAL STRUCTURE

␣ ␣ ␤ (extracellular 1- 3 domains), 2m residues 1Ð100, and one GMM molecule (Fig. 1, A and B). To determine any signiﬁcant differences in conformation or relative domain positioning between different CD1b-ligand structures, the CD1b-GMM structure was superimposed with the CD1b-PI and CD1b-GM2 structures (PDB entries 1GZQ and 1GZP, respectively). The CD1b-GMM crystals are of a different

space group to those of CD1b-PI/GM2 (P3121 rather than C2221), and some conformational changes in peripheral regions of the protein are observed due to differences in crystal packing. There are no significant differences in main-chain conformations apart from ␣ those regions. The relative position of the 3 domain with respect ␣ ␣ to the 1 2 domain differs by 5.5¡ between the CD1b-GMM and CD1b-PI structures. This hinge-like effect results from a confor- ␣ ␣ mational flexibility of the linker region (between the 2 and 3 domains) centered on residues 175Ð184 (Fig. 1A). A similar point ␣ ␣ ␣ of flexion has been noted between the 1 2 and 3 domains of classical MHC class I molecules (30). Downloaded from The electron density for the glycolipid The Ag-binding groove of CD1b is a network of hydrophobic ␣ ␣ channels in the core of the 1 2 domain (Fig. 1C). Three channels, denoted as AЈ,CЈ, and FЈ, connect directly to the surface, and a Ј ␣ fourth channel, the tunnel T , runs through the core below the 1

␣ Ј Ј Ј http://www.jimmunol.org/ and 2 helices. A ,T, and F are sequentially connected, whereas CЈ also connects to TЈ and leads from the TCR recognition surface to ␣ a portal in the side of the molecule beneath the 2 helix (Fig. 1A) (6). Electron density maps calculated using phase information from the ﬁnal model show clear density for GMM. At a level of 2.5 ␴

on a simulated annealing Fo-Fc omit map calculated after omitting the GMM, the density is continuous for the sugar head group, ␣-chain, and meromycolate chain (Fig. 1B). The meromycolate chain completely occupies the AЈ,TЈ, and FЈ channels, and protrudes out of the FЈ channel into the TCR recognition surface. by guest on September 26, 2021 Additional, weaker electron density is visible extending beyond the TCR binding surface to the same height as the sugar head group of the GMM. This suggests the presence of a longer meromycolate chain bound at a partial occupancy; however, this minor species is not included in the ﬁnal model (see next section). The ␣-chain occupies the CЈ channel and stops well short of the portal. There was no electron density visible at any stage of model building beyond what is seen in the ﬁnal model. The electron density for the protein shows the CЈ channel portal in an open state.

Mass-spectrometric analysis of GMM GMM puriﬁed from the bacterium N. farcinica comprises a range of species with molecular mass spanning from 930.0 to 1040.1 Da

(C50-GMM-C58-GMM; Fig. 1E). Recovery of GMM from CD1b- GMM crystals using organic solvents proved unsuccessful. How- ever, under basic denaturation conditions as revealed by mass spectrometry, four peaks could be accounted for as free mycolic acids liberated from bound GMM species in the CD1b-GMM crys- FIGURE 1. Structure of the CD1b-GMM complex and ligand-binding tals (Fig. 1F). These correspond to mycolic acids containing a total groove of CD1b. A, Overall structure of CD1b-GMM complex. The main of 51, 53, 55, or 57 carbon atoms in the meromycolate chain plus ␣ ␣ ␤ chain for the 1- 3 domains of CD1b plus 2m is depicted schematically in ribbon representation (in blue and gray, respectively), and the nonhy- drogen atoms of the glycolipid GMM are drawn as van der Waals spheres (carbon in gray; oxygen in red). Residues responsible for ﬂexibility be- as van der Waals spheres. Atoms occupying each channel and the channel ␣ ␣ ␣ tween the 1 2 and 3 domain are colored in gold. B, Electron density for label are in matched colors. D, The chemical structure of the C60 species the glycolipid in the CD1b-GMM crystal structure. GMM is drawn in of GMM. In A–C, the structure is viewed from a common orientation. E, ball-and-stick (carbon in black; oxygen in red). The electron density is Mass spectrum of the GMM sample after extraction from N. farcinica. F, from a Fo-Fc omit map calculated after simulated annealing with the gly- Mass spectrum of mycolic acids recovered from CD1b-GMM crystalliza- colipid omitted, contoured at 2.5 ␴, and represented as a red mesh. C, The tion drops. The total number of carbons in the meromycolate and ␣-chains ligand-binding groove of CD1b. The hydrophobic binding surface is drawn in each species is indicated above each peak. Values on the mass spectra as a gray mesh with carbon from the acyl tails occupying the groove drawn are given in daltons. The Journal of Immunology 2385

␣ the -chain (denoted as C54,C56,C58, and C60, respectively) counting the total number of carbons up to the ester group bridging the glucose moiety, i.e., including the ␣, ␤, and carboxylate atoms. The reﬁned structure for CD1b-GMM contains the largest of the GMM species detected, with 8 carbon atoms in the ␣-chain and 49 carbons atoms in the meromycolate chain (i.e., the C60 species; Fig. 1D). As described above, weaker electron density seen protruding further out of the FЈ channel could possibly be due to an even larger species of GMM bound at low occupancy. Atoms were not modeled into this density because the presence of such species was not conﬁrmed by mass spectrometry and the x-ray data were of limited resolution. The mass spectrum of the lipid sample directly after extraction from N. farcinica shows the predominant species to be a C54-GMM (Fig. 1E), whereas the C58-GMM is the predominant species in the crystallization drops (F). This would suggest that the process of refolding has enriched the fraction of CD1b-GMM complexes containing larger-species GMM.

Comparison with CD1b-PI/GM2 structures Downloaded from The CD1b-GMM structure was compared with the CD1b-PI/GM2 structures to analyze binding groove architectures, glycolipid positions, and protein-glycolipid interactions. In both the CD1b-PI and GM2 structures, the relatively short lipid acyl chains occupy the CЈ and AЈ channels, whereas the FЈ and TЈ channels are oc-

cupied by detergent molecules (6). An analysis of the CD1b-PI and http://www.jimmunol.org/ GM2 structures when combined with the mutagenesis analysis by FIGURE 2. Comparison of the binding grooves of CD1b-GMM and Niazi et al. (31) on residues affecting glycolipid Ag presentation CD1b-PI. The van der Waals surface of the CD1b binding groove is de- led to the hypothesis that the meromycolate chain of a GMM li- picted in gray/green and gray/yellow for the CD1b-GMM and CD1b-PI gand would occupy the network of AЈ,TЈ, and FЈ channels (6). The structures, respectively. GMM and PI are drawn in ball-and-stick (carbon in gray; oxygen in red). In A and C, the view is rotated by 90¡ about the current structural analysis confirms that the meromycolate chain of Ј Ј Ј Ј horizontal from that of Fig. 1A, and the A -T junction is indicated by an GMM is bound in the CD1b groove traversing from the A (via T ) arrow. In B and D, the view is as in Fig. 1A, and the FЈ-TЈ junction is indicated Ј ␣ Ј to the F channel, whereas the -chain occupies the C channel. by an arrow. In E and F, superimpositions of the two surfaces are viewed in Comparison of side-chain conformations between the CD1b- close-up to highlight differences in the AЈ-TЈ and FЈ-TЈ junctions. The orien- GMM and CD1b-PI structures generally reveals relatively minor tation is as in A and B, respectively, and the GMM is drawn in ball-and-stick. by guest on September 26, 2021 conformational changes for residues lining the channels (root- mean-square deviation (RMSD) values of Ͻ0.50 Å between equiv- tal structure was used as a template for the modeling of these lipids alent side-chain atoms). However, side-chain conformations for into the binding groove. Lipids were inserted into the binding the residues lining the connecting regions between the FЈ-TЈ chan- groove, and the complexes were energy minimized with the pro- nels and for residues at the bottom of the AЈ channel are significantly tein backbone harmonically restrained. The substituent groups in different in the two structures (RMSD values of 0.50Ð1.50 Å). the resultant models were positioned at a wide range of points in The volume in the channel around each acyl carbon atom was the AЈ channel, AЈ-TЈ junction, or TЈ tunnel. Two representative calculated for the CD1b-glycolipid structures (effectively section- examples are illustrated in Fig. 3. Analysis of the model structures ing the channel using atom coordinates as grid points). In terms of revealed no stereochemical clashes. The CD1b binding groove is volume (Fig. 2), the most significant differences between CD1b- capable of accommodating these groups with only minor changes GMM and CD1b-PI/GM2 are in the connecting regions between to side-chain conformations (for example, Fig. 3). Volume calcu- the AЈ-TЈ and TЈ-FЈ channels (i.e., corresponding to the regions lations on the channels reveal that there is no significant change in with high RMSD for the comparison of side chains) (Fig. 2, A and the total volume of the binding groove; however, volume calcula- B). The connecting regions between these channels are curved or tions along sections of the binding groove show that there are local bent, with AЈ-TЈ taking a hairpin shape. The differences in channel changes in volume. The substituent groups are accommodated by volume can be attributed to the fact that, in the CD1b-GMM struc- slight increases (typically 5Ð10%) in the channel volume at these ture, the acyl chain is continuous and tracks smoothly around the points. Because these increases are achieved by changes in side- curve, whereas in CD1b-PI/GM2, the acyl chain termini and de- chain conformations, they tend to be counterbalanced by subtle tergent termini occupy these regions and point into the outer wall decreases in volume extending through neighboring regions. These of the bend. Residues forming the wall of the channels have altered model-based results imply a degree of flexibility in the CD1b bind- conformations to accommodate these termini. The key role of the ing groove, consistent with this molecule’s ability to bind lipids residues at the AЈ-TЈ junction in presenting long acyl chain ligands containing substituent groups at a wide range of positions. The has been previously highlighted by mutagenesis data (31). current structural data provide no evidence of any mechanism whereby differences in such substituent groups could influence Modeling of other lipids into the binding groove TCR recognition as reported by Grant et al. (33). Mycobacterial mycolic acids that bear substitutions along the meromycolate chain have been identified (Fig. 3) (32). These sub- Comparison of binding grooves between different human CD1 stitutions can be cyclopropyl, methyl, methoxy, or keto groups, molecules and occur at variable positions on the chain. To investigate how In an attempt to generate additional insights into the similarities CD1b could accommodate such structures, the CD1b-GMM crys- and differences in the binding groove architectures of the CD1 2386 CD1b-GMM CRYSTAL STRUCTURE

related to the conservation of hydrophobic patches 1, 4, 13, and 14 (Fig. 4). Because CD1c exhibits a similar pattern in its primary structure, it is likely that it too has a CD1b-like AЈ channel. This conservation of AЈ structure is consistent with the shared ability of CD1a, CD1b, and CD1c to bind sulfatide (34); however, Zajonc et al. (7) note that variations in the detailed architecture in this region are important in modulating the ligand-binding characteristics of CD1a vs the other CD1s. The remainder of the binding groove of CD1b is a distinctive network of channels rather than the single FЈ pocket reported for CD1a (7), because it is partitioned by bulky side chains pointing into the center of internal cavities (e.g., F77 and F114). These features map to hydrophobic patches 5 and 9 (Fig. 4). Their effect is an increase in the area of the hydrophobic surface and the formation of a tubular channel (Fig. 4B). The hydrophobicity analysis indicates that CD1c has some similarity to CD1b in these regions and thus may also differ from the single FЈ cavity or pocket-type structure seen in mCD1d1 (2) and CD1a (7). In CD1b, the combination of an interconnecting TЈ tun-

nel with a partitioned binding groove confer the unique features Downloaded from that allow it to bind lipids with longer acyl chains.

Lipid head group positions and TCR recognition To gain insight into how TCRs bind to the CD1b-GMM complex, CD1b-GMM and a model of a cognate TCR, LDN5 (Ref. 3 and

Materials and Methods), were superimposed onto all available http://www.jimmunol.org/ crystal structures of MHC-TCR complexes. Due to the elevated position of the glycolipid head group of GMM and the structure of ␣ the 2 helix (which at its midpoint forms a more prominent apex, ␣ unlike that of the 2 helix in MHC class I or II molecules; Fig. 1A), FIGURE 3. Conformational changes of residue side chains to accom- there are steric clashes with the backbone of the TCR in all re- modate additional functional groups present on modeled lipids. Modeling sultant model complexes. Detailed conclusions must therefore into the CD1b binding groove of a type 3 ␣-mycolic acid (A) and a type 2 await the structure determination of a CD1-TCR complex. However, methoxymycolic acid (B). i, Chemical structures of modeled lipids. ii, Close-up views of the modeled CD1b-glycolipid structures centered on the these docking experiments imply that the complementarity-determin- by guest on September 26, 2021 methoxy group of the methoxymycolic acid and a cyclopropyl and alkene ing region 3 loops of a TCR would sample parts of the lipid. group of the ␣-mycolic acid. The positions of the substituent groups de- In addition to the structure of the glycolipid head group, its picted in the close-up view are circled in yellow on the chemical structures. position is likely to be an important determinant for recognition by For comparison, coordinates of the CD1b-GMM crystal structure are TCR. Site-directed mutagenesis experiments have identified sev- shown (dark-gray ball-and-stick). Modeled coordinates are depicted as eral residues in CD1b that appear to be required for TCR recog- ball-and-stick (carbon in black on protein side chains and in gray on lipid; nition (35). These residues fall into two groups in the context of the oxygen in red; nitrogen in blue). Bonds between substituent group atoms in ␣ structure, the first group being R79, E80, and D83 on the 1 helix, the modeled lipids are colored yellow. iii, Global view of the positions of and the second group being T165 and T157 on the ␣ helix (35) the substituent groups in the CD1b binding groove. The panels show the 2 (Fig. 5A). Both groups of residues may contribute to TCR recog- surface of the CD1b binding groove drawn as a gray mesh with the substituent group atoms drawn as yellow van der Waals spheres. The view in nition directly by making contacts with the complementarity-de- A is from beneath the binding groove (rather than from above as in Fig. termining region loops of the TCR; however, they may also con- ␣ 2A). The view in B is into the side of the binding groove through the 1 tribute by stabilizing a specific orientation of the glycolipid head ␣ helix (rather than through the 2 helix as in Fig. 2B). group by hydrogen bonding. Indeed the CD1b-GM2, PI, and GMM crystal structures show that residues from both clusters (specifically R79 and T157) can make hydrogen bonds to the gly- molecules, the sequences of CD1a, CD1b, CD1c, CD1d, and colipid headgroups (6) (Fig. 5, B–D). Mutagenesis of other CD1b mCD1d1 were analyzed in terms of hydrophobicity (Fig. 4A). The residues affects Ag-presenting function to a lesser degree (35), and analysis reveals a particularly complex pattern of hydrophobicity in the crystal structures, these do not make any direct interactions in CD1b, and in Fig. 4B, specific regions/patches of hydrophobic- with the glycolipid (Fig. 5A). In common with the CD1a structure ity are related to distinct structural features in the binding groove. (7), the CD1b-GMM structure reveals a portion of acyl tail pro- Of the other family members, CD1c shows the most similarity to truding from the FЈ pocket. This is an additional component of the CD1b in the hydrophobicity analysis. However, certain features glycolipid that may potentially contribute to TCR recognition. (e.g., patch 7; Fig. 4) that map to the TЈ channel are unique to CD1b. It has previously been predicted that the substitution of G98 Discussion by a valine in CD1a and CD1c would abolish the continuous TЈ Refolding of denatured CD1b molecules with different ligands, tunnel (Fig. 4E) (6). This has now been confirmed for CD1a by the either in the presence of single acyl chain detergents (6) or using determination of its crystal structure in complex with sulfatide (7). a protocol based on oxidative refolding chromatography (10) re- The CD1a structure also reinforces the conclusion that the AЈ results in an identical three-dimensional structure of CD1b. The ar- gion is the section of the binding groove that shows most similarity chitecture of the CD1b binding groove is therefore independent of between group 1 family members. The presence of CD1b-like AЈ refolding pathway or ligand. This structural stability is particularly channel and AЈ-TЈ junction in the CD1a binding groove can be important for the generation of CD1b tetramers for the monitoring The Journal of Immunology 2387 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Comparison of CD1 molecules. A, Comparison of hydrophobic patches between the different CD1 sequences. Hydrophobicity is colored as depicted in the scale bar. B—D, Three views of the CD1b binding groove with key residues drawn in ball-and-stick; the surface of the binding groove is drawn as a solid pale-blue surface, and GMM is drawn in gray van der Waals spheres. The colors of the residues in B–D correspond to the colored numbers of the hydrophobic patches in A.InB, the view is at ϳ45¡ to the upper surface of the binding groove. The view in C is that of Figs. 1A and 2B, whereas in D, it is rotated by 180¡ about the vertical (as in Fig. 3B). E, Sequence alignment between CD1b, CD1a, and CD1c. F, Sequence alignment between CD1d and mCD1d1.

of glycolipid-specific T cell responses. Structural and mass-spec- conformation changes in CD1b. The mechanisms controlling this trometric analysis reveals that longer GMM species are preferen- loading process require investigation. The current CD1b-GMM tially selected by CD1b. Given the potential contribution to TCR structure demonstrates how a meromycolate chain of 49 carbon recognition by acyl chains protruding out of the FЈ pocket, the atoms saturates the capacity of the AЈ-TЈ-FЈ superchannel. In con- preferential loading of longer GMMs may narrow the population trast, the ␣-chain of 8 carbons only partially occupies the CЈ chan- of T cells stained by CD1b-GMM tetramers. Such problems can be nel in CD1b-GMM. The previously reported structure for CD1b-PI circumvented in tetramer production by the use of purified samples indicates that a GMM with a longer ␣-chain will extend further of shorter chain GMMs that are known to bind to CD1b and be into this channel (6). The full capacity of this channel is 16 carbon recognized by CD1b-specific T cell clones (29). The ability to atoms; any additional ␣-chain carbon atoms may exit the binding ␣ refold incorporating long glycolipid species provides an invaluable groove via the portal under the 2 helix. tool for understanding the T cell response to bacterial infection. The crystal structure of CD1b-GMM provides the first detailed The complexity of the CD1b binding groove architecture implies information on the presentation of a natural bacterial glycolipid that in vivo loading of such long-chain meromycolates will require Ag. Recent functional studies have confirmed the ability of CD1b 2388 CD1b-GMM CRYSTAL STRUCTURE

9. Altamirano, M. M., R. Golbik, R. Zahn, A. M. Buckle, and A. R. Fersht. 1997. Refolding chromatography with immobilized mini-chaperones. Proc. Natl. Acad. Sci. USA 94:3576. 10. Karadimitris, A., S. Gadola, M. Altamirano, D. Brown, A. Woolfson, P. Klenerman, J. L. Chen, Y. Koezuka, I. A. Roberts, D. A. Price, et al. 2001. Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography. Proc. Natl. Acad. Sci. USA 98:3294. 11. Brown, J., T. S. Walter, L. Carter, N. G. A. Abrescia, A. R. Aricescu, T. D. Batuwangala, L. E. Bird, N. Brown, P. P. Chamberlain, S. J. Davis, et al. 2003. A procedure for setting up high-throughput nanolitre crystallization experiments. II. Crystallization results. J. Appl. Crystallogr. 36:315. 12. Walter, T. S., J. Diprose, J. Brown, M. Pickford, R. J. Owens, D. I. Stuart, and K. Harlos. 2003. A procedure for setting up high-throughput nanolitre crystallization experiments. I. Protocol design and validation. J. Appl. Crystallogr. 36:308. 13. Otwinowski, Z., and W. Minor. 1997. Processing of x-ray diffraction data collected in oscillation mode. In Methods in Enzymology: Macromolecular Crys- tallography, Vol. 276, Pt. A. C. W. Carter, Jr., and R. M. Sweet, eds. Academic, New York, p. 307. 14. Kissinger, C. R., D. K. Gehlhaar, and D. B. Fogel. 1999. Rapid automated molecular replacement by evolutionary search. Acta Crystallogr. D 55:484. FIGURE 5. Head group orientation of different lipids from crystal struc- 15. Brunger, A. T., P. D. Adams, G. M. Clore, W. L. DeLano, P. Gros, tures and positions of residues implicated in TCR recognition. A, View of R. W. Grosse-Kunstleve, J. S. Jiang, J. Kuszewski, M. Nilges, N. S. Pannu, et al. the TCR binding surface of the CD1b-GMM complex with residues im- 1998. Crystallography & NMR system: a new software suite for macromolecular plicated in functional studies as critical to TCR recognition indicated as structure determination. Acta Crystallogr. D 54:905. Downloaded from 16. Jones, T. A., J. Y. Zou, S. W. Cowan, and M. Kjeldgaard. 1991. Improved spheres at their C␣ positions. Those drawn in red mark residues that, when methods for building protein models in electron-density maps and the location of mutated, cause a Ͼ1-log reduction in CD1b Ag presentation, whereas those errors in these models. Acta Crystallogr. A 47:110. in purple show a Ͼ0.5-log reduction (mutagenesis data from Ref. 35). 17. van Aalten, D. M., R. Bywater, J. B. Findlay, M. Hendlich, R. W. Hooft, and Comparison of GMM (B), GM2 (C), and PI (D) structures in complex with G. Vriend. 1996. PRODRG, a program for generating molecular topologies and CD1b. The main chain for the ␣ and ␣ helices of CD1b is represented as unique molecular descriptors from coordinates of small molecules. J. Comput. 1 2 Aided Mol. Des. 10:255. coil (yellow). The glycolipid and key side chains from CD1b are shown in 18. Laskowski, R. A., D. S. Moss, and J. M. Thornton. 1993. Main-chain bond ball-and-stick (lipid carbon atoms in gray; protein carbon atoms in black; lengths and bond angles in protein structures. J. Mol. Biol. 231:1049. http://www.jimmunol.org/ oxygen in red; nitrogen in blue). Hydrogen bonds are depicted as dashed 19. Wallace, A. C., R. A. Laskowski, and J. M. Thornton. 1995. LIGPLOT: a pro- lines. The view in B–D is somewhat tipped about the horizontal from that gram to generate schematic diagrams of protein-ligand interactions. Protein Eng. 8:127. of Fig. 1A. 20. Lefranc, M. P. 2003. IMGT databases, web resources and tools for immunoglob- ulin and T cell receptor sequence analysis, http://imgt.cines.fr. Leukemia 17:260. 21. Guex, N., and M. C. Peitsch. 1997. SWISS-MODEL and the Swiss-PdbViewer: molecules to present full-length GMM to T cells (T.-Y. Cheng and an environment for comparative protein modeling. Electrophoresis 18:2714. B. Moody, unpublished data). Further insights into T cell recog- 22. Stuart, D. I., M. Levine, H. Muirhead, and D. K. Stammers. 1979. Crystal structure of cat muscle pyruvate kinase at a resolution of 2.6 Å. J. Mol. Biol. 134:109. nition of CD1b-glycolipid complexes await the determination of 23. Corpet, F. 1988. Multiple sequence alignment with hierarchical clustering. Nu-

CD1b-glycolipid-TCR complex structures. cleic Acids Res. 16:10881. by guest on September 26, 2021 24. Gouet, P., E. Courcelle, D. I. Stuart, and F. Metoz. 1999. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15:305. Acknowledgments 25. Appel, R. D., A. Bairoch, and D. F. Hochstrasser. 1994. A new generation of We are grateful to the staff of the SRS (Daresbury, U.K.) and the ESRF and information retrieval tools for biologists: the example of the ExPASy WWW European Molecular Biology Laboratory outstation (Grenoble, France) for server. Trends Biochem. Sci. 19:258. 26. Eisenberg, D., E. Schwarz, M. Komaromy, and R. Wall. 1984. Analysis of mem- the assistance with x-ray data collection. We thank Jonathan Grimes, Erika brane and surface protein sequences with the hydrophobic moment plot. J. Mol. Mancini, and Geoff Sutton for help and advice during data collection; Rob- Biol. 179:125. ert Esnouf for providing advice and scripts for running VOLUMES; 27. Esnouf, R. M. 1999. Further additions to MolScript version 1.4, including reading Jonathan Diprose for writing the script for generating Fig. 4A; and Peter and contouring of electron-density maps. Acta Crystallogr. D 55:938. Ashton for carrying out the mass spectrometry measurements. 28. Merritt, E. A., and D. J. Bacon. 1997. Raster3D: photorealistic molecular graph- ics. In Macromolecular Crystallography, Part B, Vol. 277. Academic, San Di- ego, p. 505. References 29. Moody, D. B., V. Briken, T. Y. Cheng, C. Roura-Mir, M. R. Guy, D. H. Geho, 1. Porcelli, S., C. T. Morita, and M. B. Brenner. 1992. CD1b restricts the response M. L. Tykocinski, G. S. Besra, and S. A. Porcelli. 2002. Lipid length controls of human CD4Ϫ8Ϫ T lymphocytes to a microbial antigen. Nature 360:593. antigen entry into endosomal and nonendosomal pathways for CD1b presenta- 2. Zeng, Z., A. R. Castano, B. W. Segelke, E. A. Stura, P. A. Peterson, and tion. Nat. Immunol. 3:435. I. A. Wilson. 1997. Crystal structure of mouse CD1: an MHC-like fold with a 30. Madden, D. R. 1995. The three-dimensional structure of peptide-MHC com- large hydrophobic binding groove. Science 277:339. plexes. Annu. Rev. Immunol. 13:587. 3. Moody, D. B., B. B. Reinhold, M. R. Guy, E. M. Beckman, D. E. Frederique, 31. Niazi, K., M. Chiu, R. Mendoza, M. Degano, S. Khurana, D. Moody, A. Melian, Ј Ј S. T. Furlong, S. Ye, V. N. Reinhold, P. A. Sieling, R. L. Modlin, et al. 1997. I. Wilson, M. Kronenberg, S. Porcelli, and R. Modlin. 2001. The A and F Structural requirements for glycolipid antigen recognition by CD1b-restricted T pockets of human CD1b are both required for optimal presentation of lipid an- cells. Science 278:283. tigens to T cells. J. Immunol. 166:2562. 4. Ernst, W. A., J. Maher, S. Cho, K. R. Niazi, D. Chatterjee, D. B. Moody, 32. Watanabe, M., Y. Aoyagi, H. Mitome, T. Fujita, H. Naoki, M. Ridell, and G. S. Besra, Y. Watanabe, P. E. Jensen, S. A. Porcelli, et al. 1998. Molecular D. E. Minnikin. 2002. Location of functional groups in mycobacterial meromy- interaction of CD1b with lipoglycan antigens. Immunity 8:331. colate chains; the recognition of new structural principles in mycolic acids. Mi- 5. Porcelli, S. A., B. W. Segelke, M. Sugita, I. A. Wilson, and M. B. Brenner. 1998. crobiology 148:1881. The CD1 family of lipid antigen-presenting molecules. Immunol. Today 19:362. 33. Grant, E. P., E. M. Beckman, S. M. Behar, M. Degano, D. Frederique, 6. Gadola, S. D., N. R. Zaccai, K. Harlos, D. Shepherd, J. C. Castro-Palomino, G. S. Besra, I. A. Wilson, S. A. Porcelli, S. T. Furlong, and M. B. Brenner. 2002. G. Ritter, R. R. Schmidt, E. Y. Jones, and V. Cerundolo. 2002. Structure of Fine speciﬁcity of TCR complementarity-determining region residues and lipid human CD1b with bound ligands at 2.3 Å, a maze for alkyl chains. Nat. Immunol. antigen hydrophilic moieties in the recognition of a CD1-lipid complex. J. Im- 3:721. munol. 168:3933. 7. Zajonc, D. M., M. A. Elsliger, L. Teyton, and I. A. Wilson. 2003. Crystal struc- 34. Shamshiev, A., H. J. Gober, A. Donda, Z. Mazorra, L. Mori, and G. De Libero. ture of CD1a in complex with a sulfatide self antigen at a resolution of 2.15 Å. 2002. Presentation of the same glycolipid by different CD1 molecules. J. Exp. Nat Immunol. Med. 195:1013. 8. Moody, D. B., M. R. Guy, E. Grant, T. Y. Cheng, M. B. Brenner, G. S. Besra, 35. Melian, A., G. F. Watts, A. Shamshiev, G. De Libero, A. Clatworthy, M. Vincent, and S. A. Porcelli. 2000. CD1b-mediated T cell recognition of a glycolipid an- M. B. Brenner, S. Behar, K. Niazi, R. L. Modlin, et al. 2000. Molecular recog- tigen generated from mycobacterial lipid and host carbohydrate during infection. nition of human CD1b antigen complexes: evidence for a common pattern of J. Exp. Med. 192:965. interaction with ␣␤ TCRs. J. Immunol. 165:4494.