Crystal Structure of the A Domain from the a Subunit ... - Science Direct

Cell, Vol. 80, 631-638, February24, 1995, Copyright© 1995 by Cell Press

Crystal Structure of the A Domain from the a Subunit of Integrin CR3 (CD11b/CD18) Jie-Oh Lee,* Philippe Rieu,t M. Amin Arnaout,t and Robert Liddington* * Laboratory of X-Ray Crystallography Dana Farber Cancer Institute and Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School Boston, Massachusetts 02115 1Leukocyte Biology and Inflammation Program Nephrology Division Department of Medicine Massachusetts General Hospital and Harvard Medical School Charlestown, Massachusetts 02129

Summary We have determined the high resolution crystal structure of the A domain from the (~ chain of integrin CR3. The domain adopts a classic a/I] "Rossmann" fold and contains an unusual Mg =+ coordination site at its surface. One of the coordinating ligands is the glutamate side chain from another A domain molecule. We suggest that this site represents a general metal ion-dependent adhesion site (MIDAS) for binding protein ligands. We further propose that the J~ subunits of integrins contain a MIDAS motif within a modified A domain. Our crystal structure will allow reliable models to be built for other members of the A domain superfamily and should facilitate development of novel adhesion modulatory drugs. Introduction

The A domain is a region of approximately 200 amino acids that is present in one or more copies in many proteins involved in cell-cell, cell-matrix, and matrix-matrix interactions (Figure 1). Many members of this superfamily mediate, through their A domain(s), adhesion functions that underlie such important physiologic processes as morphogenesis, cell migration, hemostasis, inflammation, immunity, and wound healing. The genomic organization, functional diversity, and ancient lineage of the A domain suggest that it originated from a primordial gene that subserved a morphoregulatory role, still retained in some matrix proteins, and was later incorporated into other proteins and modified to mediate specific adhesion events critical for host survival (Colombatti et al., 1993). The leukocyte integrin complement receptor type 3 (CR3, also known as CD11b/CD18, ~MI32, Mol, OKM-1, and Mac-l) is the major integrin of phagocytic cells. It is a member of the 132integrin family, which consists of four glycoprotein heterodimers, each with a distinct a subunit (CD11 a, CD11 b, CD11 c, and CD1 ld) noncovalently associated with a common 13subunit (CD18) (Arnaout, 1990b; Cobbold et al., 1994; Rieu and Arnaout, 1995). J32integrins

mediate the stable adhesion of leukocytes to endothelium and the subsequent transendothelial migration into inflamed organs (Arnaout, 1993; Hynes, 1992). CR3 also mediates aggregation of phagocytes (Arnaout et al., 1985), ingestion of opsonized particles, and the generation of oxygen free radicals and release of hydrolytic enzymes =n response to particulate stimuli (Arnaout et al., 1983). Inherited deficiency of 132integrins (called leukocyte adhes=on molecule deficiency or LAD) results in life-threatening pyogenic infections and poor wound healing due to the inability of circulating phagocytes to extravasate into infected tissues and to clear pathogens through phagocytosis and cell-mediated killing (Arnaout, 1990a). While essential for host survival, 132 integrin-mediated influx and inflammatory functions in phagocytes often exacerbate the local pathologic lesions and tissue injury in many noninfectious disease states including hemorrhagic shock, burns, atherosclerosis, and hyperacute rejection (Albelda et al., 1994; Arnaout, 1990b; Ross, 1993). In several animal models of inflammation (Carlos and Harlan, 1990), monoclonal antibodies to CR3 and other 132integrins markedly reduce the influx and inflammatory functions of leukocytes, thus preserving tissue integrity and host survival. Antagonists that target CR3 and other [32 integrins may thus be useful in therapeutic interventions of inflammatory diseases. The functions of CR3 in leukocyte extravasation and inflammation are mediated through its binding to several physiologic ligands, including iC3b, the major complement C3 opsonin (Arnaout et al., 1983; Wright et al., 1983), CD54 (intercellular adhesion molecule-I, ICAM-1 [Diamond et al., 1991 ; Simmons et al., 1988]), and the coagulation factors fibrinogen and factor X (Altieri et al., 1988). Recent studies have shown that the CR3 A domain (also called I domain) is an independent structural and functional unit, having many of the binding functions of the intact receptor: a recombinant CR3 A domain binds divalent cations (Michishita et al., 1993), iC3b (Ueda et al., 1994), CD54, fibrinogen (Zhou et al., 1994), and is the binding site for NIF, a hookworm-derived neutrophil adhesion inhibitor (Moyle et al., 1994; Rieu et al., 1994). We therefore undertook a crystallographic analysis of the CR3 A domain in an attempt to understand the molecular basis for its specific adhesive interactions and to gain insight into interactions mediated by other members of the A domain superfamily. Results and Discussion Crystal Structure of the OR3 A Domain We crystallized a recombinant A domain from the CD1 lb subunit of CR3 in the presence of Mg2+ and solved its structure at 1.7 ,~, resolution using the technique of multiwavelength anomalous dispersion (MAD) with a selenomethionine derivative and synchrotron radiation. The electron density map calculated from the experimental data was immediately interpretable (Figure 2), and a complete

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model was built starting at residue D132 and finishing at residue K315 (numbered as in Arnaout et al., 1988) (Figure 3). The A domain comprises alternating amphipathic c~ helices and hydrophobic [3 strands that conform to the classic a/l~ open sheet (also called "Rossmann," dinucleotide-binding, or "doubly wound") fold. Five parallel and one short antiparaliel 15 strands form a central sheet that is surrounded by seven ct helices. The primary sequence with secondary structure assignments is shown in Figure 4A. A secondary structure prediction, based on an alignment of known A domain sequences and FT-IR spectroscopy, shows remarkably good agreement with our crystal structure (Perkins et al., 1994). The CR3 A domain contains a single cysteine at the beginning of the domain, which is disordered in our crystal structure. VLA-1 (CD49a/ CD29, a1~1) and VLA-2 (CD49b/CD29, ~t2131)contain a second cysteine within the ct/~ fold: modeling studies indicate that it is buried in the interior of the domain and cannot participate in disulfide bond formation (data not shown). Certain other A domain proteins, including von Willebrand

Figure 2. A Portionof the ElectronDensityMapSuperimposedon the Final RefinedCoordinates (a) In!tialmapcalculateddirectlyfrom the experimentalmeasurements at 2 A resolution.(b) Final2Fo-Fc map calculatedfrom experimental data and calculatedphasesat 1.7/~ resolution.

factor (vWF) and collagens, have a cysteine at both the N- and C-termini of the domain that form a disulfide bridge; this is entirely consistent with our three-dimensional structure, since the termini are close together in space. A single metal-binding site is located on the surface of the A domain, at the top of the 13sheet (Figures 3 and 5). We ascribe the electron density peak to a Mg 2+ion, because it is the only metal present in the crystallization buffer and the short bond distances (2.0 _+ 0.1 ,A) and octahedral coordination distinguish it from water. The Mg2+ ion has coordination sites for six ligands: three ligands are the hydroxyl oxygen atoms of $142, $144, and T209, and two are water molecules ((01 and 0)2). 0)2 also makes a hydrogen bond to the main chain carbonyl oxygen of E244. The two aspartates, D140 and D242, which have previously been implicated in metal binding (Michishita et al., 1993), do not coordinate the metal directly, but occupy a secondary coordination sphere: each carboxylate group makes hydrogen bonds to water molecule (ol and one hydroxyl side chain (D140 to $142 and D242 to $144) (Figure 5). In solution, we expect the sixth Mg 2÷ coordination site to be occupied by a water molecule, but in our crystal structure, it is occupied by a carboxylate oxygen atom from residue E314 of a neighboring A domain (bond distance 2.0 ,,~). Comparison

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The A domain adopts the classic dinucleotide-binding fold, a very common topology that is present in a wide variety of intracellular enzymes. The position of the ligand-binding site can be predicted in this class of structures from topology diagrams (reviewed by Branden and Tooze, 1991). A crevice is formed at the top of the 13 sheet, where the connecting loops from two 13strands run in opposite directions. This crevice forms a ligand-binding site in all known

Crystal Structure of CD1 l b A Domain 633

Figure 3. Schematic Stereo Diagram of the A Domain Structure Strand and helix assignments are as in Figure 4A. The Mg~+ ion is shown as a blue ball.

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proteins of this class and often includes a metal ion. Most metal ions play a catalytic role and are frequently found in phosphoryl transfer enzymes. In the bacterial chemotaxis protein CheY, a Mg 2+ ion has also been shown to play an important structural role (Bellsolell et al., 1994). In the A domain, the Mg 2+ ion is found in an analogous location, suggesting that it may form part of a ligand-binding site (see below). There are now several atomic resolution descriptions of Mg 2+ coordination by proteins (Bellsolell et al., 1994; Pal et al., 1990; Shirakihara and Evans, 1988). The common features of Mg 2÷coordination are six ligands arranged with octahedral geometry, bond distances of - 2.0 ,~.,coordination by oxygen atoms, and at least two ligands with formal negative charges. The A domain is highly unusual in having no negatively charged coordinating residue, except for that provided by the glutamate side chain from the next molecule. The most similar coordination is found in the GTPase p21 ras in complex with a GTP analog (Pal et al., 1990): a serine hydroxyl and a threonine hydroxyl are provided by the protein; the other coordinating ligands are two water molecules and two negatively charged phosphate oxygens from GTP; an aspartate side chain occu-

Figure 4. Primary Sequence of CD1 lb A Domain with the Secondary Structure, and Conservation of MIDAS in Other Domains (A) Sequence and secondary structure assignment of the CD11b A domain. MIDAS amino acid residues are indicated in bold letters. Numbers refer to amino acids in the intact CD1 lb subunit (Arnaout et al.,

1988). (B) Alignment of the MIDAS motif in the A domain superfamily. The names of various proteins is preceded by h, r, ra, m, and ch to reflect human, rabbit, rat, mouse, or chicken origin, respectively. Other abbreviations are described in the legend to Figure 1. Numbers indicate intervening amino acids separating the three components of MIDAS (DXSXS, T, and D, outlined) in each domain.

Cell 634

Figure 5. Stereo Diagram of the MIDAS Site Coordinating oxygen atoms are shown in red, and hydrogen bonds are shown by dashed red lines. E314 from another A domain molecule is shown in gold. The protein backboneis shown schematicallyas a gray ribbon.

pies a secondary coordination sphere, hydrogen bonding to a water molecule and the serine hydroxyl, in a similar fashion to that in the A domain. These observations suggest that stable Mg 2÷ion coordination requires a negatively charged group such as the carboxylate side chain of aspartic or glutamic acid.

The "MIDAS" Touch Our results show that the Mg 2+ ion binds to a site on the surface of the CR3 A domain and is further coordinated by the glutamate side chain of another A domain molecule (Figure 5). Recombinant A domain has been shown to bind Mg 2÷ in solution and to bind certain protein ligands in a metal-dependent fashion: alanine mutagenesis of the Mg~÷-coordinating residues D140GS (to A140GA) and D242 abolished metal binding to the recombinant protein and its ability to bind iC3b and NIF (Michishita et al., 1993; Ueda et al., 1994; Rieu et al., 1994). Similar mutations in VLA-2 (CD49b/CD29) have been shown to reduce collagen binding (Kamata et al., 1994). We extended these observations to the interaction of CD54 and fibrinogen with the CR3 A domain. As can be seen in Figure 6, binding of CD54 and, to a lesser degree, fibrinogen, was metal dependent, as binding was reduced in the presence of EDTA or to the mutant D242A. These studies indicate that metal coordination within the A domain plays an important role in protein ligand binding. Our crystal structure, revealing a Mg2+-binding site with an extra "available" coordination site, suggests a direct role for this site in protein ligand binding. We call this the metal ion-dependent adhesion site (MIDAS) motif. The

conserved features of this motif are a DXSXS sequence (residues 140-144, where X represent any amino acid), and a threonine (T209) and aspartate (D242) from other parts of the chain (Figure 4B). Although the X residues in the DXSXS sequence of CR3 are glycines, these are not essential, since the main chain has no unusual torsion angles. Inspection of the sequences of known A domains suggests that the majority possess a MIDAS motif, and many others appear to have a modified motif (Figure 4B). We note that the MIDAS motif consists of noncontiguous amino acids, as predicted (Michishita et al., 1993), unlike the EF-hand Ca2÷-binding motif. An analogous metal-

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Figure 6. Effectof MIDAS on Ligand Bindingto the CD11b A Domain Histograms (mean _+ SD) from four independentexperimentseach done in triplicate,showingbindingof fibrinogenand sCD54to immobilized wild-typeand D242A mutant CD1 lb A domain in the presence of divalent cations (Mg2÷/Ca2÷),and the effect of EDTA on binding of these proteinsto wild-typeA domain. Binding of iC3b, NIF, and the MAb 44 are shownfor comparisons.Bindingto the wild-typeA domain in the presenceof cationswas assigneda valueof 100 for each ligand.

Crystal Structure of CD1lb A Domain 635

dependent protein-protein interaction has been seen recently in the complex between gelsolin and actin, in which a Ca2÷ ion coordinates side chains from both proteins, providing a bridge between them (McLaughlin et al., 1993). In principle, MIDAS can contribute to protein ligand binding in two ways: directly through coordination of an acidic residue and/or indirectly through allosteric effects induced by Mg 2+ binding. Our crystallographic studies and recent mutagenesis data provide support for both of these mechanisms, operating to different extents with different ligands. Alanine mutagenesis of DE752 and EE758 in iC3b (Taniguchi-Sidle and Isenman, 1994) and E34 in CD54 (Staunton et al., 1990) greatly reduced interaction of these ligands with CD11b/CD18 and CD1 la/CD18, respectively, suggesting that an acidic residue from these ligands may directly coordinate the Mg 2+ cation. Indirect effects may explain MIDAS-dependent binding of NIF, since this interaction appears to require A domain residues (~413D loop) distant from the MIDAS motif (Rieu et al., 1994). Additional ligand binding and mutagenesis studies will be required to evaluate these possibilities and extend them to other members of the A domain superfamily. A MIDAS Motif in Integrin p Subunits? Ligand recognition by integrins is not limited to their subunits. In integrins a,b~3 (CD41/CD61 ) and ~v[33(CD51/ CD61), whose a chains lack an A domain, the metal-dependent binding of several protein ligands also involves a defined region in their 13subunits (D'Souza et al., 1988; Smith and Cheresh, 1988). We note that all integrin 13subunits (Argraves et al., 1987b; Fitzgerald et al., 1987; Law et al., 1987; MacKrell et al., 1988; Hogervorst et al., 1990; Ramaswamy and Hemler, 1990; Sheppard et al., 1990; Erie et al., 1991; Moyle et al., 1991) contain an absolutely conserved DXSXS sequence (residues 119-123 in [33), and point mutations replacing D119, $121, and $123 with alanine in 133abolish fibrinogen and RGD-peptide binding to m,b~3 (CD41/CD61) (without affecting heterodimer formation or cell surface expression) (Bajt and Loftus, 1994). Similarly, a mutation in [31 at D130 (equivalent to D119 in [33) abolishes ligand binding to ~5131 (Takada et al., 1992). Most significantly, RGD-containing peptides cross-link to a specific site on the [3 chain (K125 in [33) next to the DXSXS sequence. To explain these data, it was proposed that integrin subunits contain an EF-hand metal-binding motif that includes the DXSXS sequence (Loftus et al., 1990). However, while mutations of the DXSXS sequence abolish ligand binding, mutations of aspartate residues immediately downstream, which would form part of the putative EFhand, have no effect on ligand binding (Bajt and Loftus, 1994). We suggest that these data are more consistent with a MIDAS motif in the 13subunits, perhaps embedded in an A-like domain. No sequence homology has been detected between the ~ subunit A domain and the 13subunits using standard computer algorithms. However, the three-dimensional fold of the A domain, in common with other a/[3 open sheet structures, has a striking alternation of buried hydrophobic 13 strands and hydrophilic helices that surround the 13sheet. This alternating pattern is re-

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flected in hydropathy plots: for the c~ subunit A domain, the plot is a good predictor of secondary structure, with 13strands at the peaks and (x helices in the valleys (Figure 7). For five other crystal structures in this class that we tested, similar results were obtained (data not shown). So we searched for parallels in hydropathic properties in the integrin 13subunits. We averaged the hydropathic profiles for the six integrin A domains (those of CD11a-CD11c, CD49a, CD49b, and ctE) and all eight human integrin [3 subunits and aligned the sequences to the DXSXS motif (Figure 7). The plot for the [3subunits is remarkably similar, especially in the first half of the domain (e.g., residues 110-210 in [33) (Figure 7). We therefore propose that the integrin 13subunits contain a MIDAS motif within a threedimensional fold similar to the (~ subunit A domain. Interestingly, this putative 13A domain is also the site of several naturally occurring mutations that impair heterodimer formation in 132 integrins (reviewed by Arnaout and Michishita, 1993), suggesting a broader function of the A domain not only in ligand recognition but also in formation of the integrin heterodimeric structure. Concluding Remarks The present work elucidates the structure of an integrin domain and suggests a general mechanism for metalmediated protein-protein interactions. The structure should be valuable in elucidating the adhesion functions of the A domain in other members of the superfamily and may lead to the design of novel therapeutics. Experimental Procedures Purification and Crystallization Wild-type A domain was expressed as a glutathioneS-transferase fusion proteinin Escherichiacoti, cleaved,and purifiedas described previously(Michishitaet al., 1993).It wasthen digestedovernightwith 0.5% immobilizedtrypsin(Pierce)at 4°C in phosphate-bufferedsaline (PBS) (pH 7.4), and the trypsinwas removedby filtration.The protein was next loadedonto a Ni-NTA (Qiagen)column and eluted with a linear gradientof 0-200 mM imidazole.Fractionswere pooled,dialyzed against20 rnM Tris (pH 8.2), 10 mM EDTAbuffer, and loaded ontoa S-Sepharosecationexchangecolumn(Pharmacia).The protein was elutedwith a 0-500 mM NaCIgradientand further purifiedon a Superdex-75gelfiltrationcolumn(Pharmacia)equilibratedwith 10 mM imidazole(pH 6.5), 60 mM KCI,and 10 mM EDTA.Finally,the protein was passedthrough a G-25 desaltingcolumnto removeEDTAand concentratedfor crystallizationusinga centriconunitwitha 10Kmolecular mass cutoff (Amicon). The selenomethionine(SeMet)derivative of the A domainwas expressedin E. coli as described(Harrisonet al., 1994)and purified exactlyas for wild type.

Cell 636

Crystals were grown using the sitting drop vapor diffusion method by mixing equal volumes (50 Id) of protein (12 mg/ml) and reservoir solution (12% polyethylene glycol [PEG] 8K, 0.1 M Tris [pH 8.5], 1 mM MgCI2, 10 mM 13-mercaptoethanol) at room temperature. Crystals grow to a typical size of 0.5 mm x 0.05 mm x 0.05 mm in 2-3 weeks and belong to the tetragonal space group P43 with unit cell dimensions a = 45.7 A, c = 94.3 A. The SeMet A domain crystals grow under identical conditions and are isomorphous with the wild type.

Data Collection and Structure Determination Crystals were transferred briefly into a solution containing 12% PEG 8K, 0.1 M Tris (pH 8.5), 30% glycerol, and frozen in a stream of boiledoff nitrogen at 100 K prior to data collection. A low resolution data set was collected on a MarResearch Imaging plate using a Rigaku rotating anode source. All other data, including a high resolution native set, were collected on beam line X12C of the National Synchrotron Light Source at the Brookhaven National Laboratory using a MarResearch Imaging plate. The X-ray absorption edge of Se was determined by measuring the X-ray fluorescence spectrum from the crystal in situ; data were collected at three wavelengths: the inflection point (X1 = 0.9797 ,&,), peak (~.2 = 0.9793/k), and plateau (X3 = 0.9300 A) of the Se absorption edge (Hendrickson et al., 1985). All data were reduced with DENZO and scaled with SCALEPACK (Otwinowski, 1991) to Rsymmetry = 3%-4% and data completeness = 99.5%. Three Se positions were determined by inspection of Difference Pattersone, refinement with the program HEAVY (Terwilliger and Eisenberg, 1983), and inspection of Difference Fouriers. MAD phases were determined to 2.0 ,~, resolution using the program MLPHARE (Otwinowski, 1991) and combined with the SIR phases calculated from the Z3 and native data sets. The figure of merit was 0.65 overall to 2.0 ,~ resolution and varied between 0.91 at 5 ,~ and 0.46 at 2 ,~. The initial electron density map was immediately interpretable, and a complete atomic model was built, including most side chains, using the program FRODO (Jones, 1985). The initial model was refined at 2.0 A to an R factor of 25% using PROLSQ (Konnert and Hendrickson, 1980) and XPLOR (Br(Jnger, 1992). This refined SeMet A domain structure was used as the initial model for the native structure. Two rounds of XPLOR refinement and manual rebuilding, and the addition of water molecules, produced a final model with an R factor of 21% at 1.7 ~, resolution (f > 2c 7-1.7 ,~,)with good geometry (rms deviation on bond length = 0.017 A, bond angle = 3.0°). The final model contains all nonhydrogen atoms of residues D132 to K315 and 150 water molecules. The electron density for the first 5 and last 9 residues of the construct are not visible and are presumed to be disordered or cleaved by trypsin. Searches, Sequence Alignments, and Hydropathy Plots Homology searches were made using the BLAST program from Genetics Computer Group (GCG) Software package (Madison, Wl), sequence alignments were done using GCG's pileup (gap weight 3.0 and a gap length weight of 0.1), and the secondary structure predictions of the A domain family (Perkins et al., 1994) hydropathy was calculated from Eieenberg'e consensus hydrophobicity index (Eisenberg, 1984) and averaged over a 6-residue window. The secondary structure predictive value was assessed by analysis of the ~/13structures of carboxypeptidase, subtilisin, dihydropterine reductase, CheY, and flavodoxin. Coordinates were taken from the Brookhaven Protein Data Base. Functional Assays The recombinant (r) wild-type and m utani (D242A) forms of the CD1 l b A domain were adsorbed onto Immulon-2 96-well microtiters plates as described (Ueda et al., 1994). rN IF (a gift from Dr. M. Moyle [Corvas International Inc.]; Moyle et al., 1994), fibrinogen (Sigma), and a recombinant soluble (s) form of human CD54 (sCD54, containing all five immunoglobulin domains but lacking the intramembranous and cytoplasmic regions; a gift from Dr. J. Greve [Miles Research Center, West Haven, CT]; Greve et al., 1989) were labeled with sulfo-NHS-Biotin as described by the manufacturer (Pierce). 360 ~g of biotinylated sCD54 were deglycosylated (to enhance binding; Diamond et al., 1991), using 2.4 U of N-Glycanase F (Boehringer) in presence of protease inhibitors (2 p.g/ml each of leupeptine, chymostatin, antipain, pepstatin, and 2 mM phenylmethylsulfonylfluoride) for 18 hr at 37°C, and deglycosylation confirmed by electrophoresis of a sample on a polyacrylamide gel (Laemmli, 1970) followed by Coomassie staining. To

measure binding of biotinylated proteins to immobilized wild-type and mutant C D l l b A domain, 100 ng/ml of biotinylated NIF, 8 I~g/ml of deglycoeylated biotinylated sCD54, and 125 p.g/ml of biotinylated fibrinogen, in VBSG buffer (veronal-buffered saline (pH 7.4) with 0.10/o gelatin) containing 1 mM CaCI2 and I mM MgCI2 or 1 mM EDTA, were added to A domain-coated microtiter wells in triplicates and incubated at room temperature for 60 min. Wells were washed, incubated with alkaline phosphatase-coupled avidin, washed again, developed with substrate, and quantified colormetrically using a microplate reader. In parallel, binding of the murine anti-human CD11 b MAb 44 (Arnaout et al., 1983) to immobilized A domain was measured using alkaline phosphatase-coupled goat anti-mouse IgG. Binding of fluoresceinated sheep erythrocytes coated with iC3b (EAiC3b) to immobilized A domain was performed as previously described (Ueda et al., 1994).

Acknowledgments The corresponding author is M. A. A. We would especially like to thank Bob Sweet and his staff at the Brookhaven National Laboratory for assistance in data collection, and Laurie Bankston for critical reading of the manuscript and useful discussions. The coordinates of CD11 b A domain will be submitted to the Brookhaven Protein Data Bank. We thank Ms. Linda Costa for secretarial assistance. M. A. A., J. L., and P. R. were supported by National Institutes of Health grants RO1 DK48549 and PO1 AI-28465 to M. A. A.; P. R. was also supported by a fellowship grant from the American Heart Association, Massachusetts affiliate. R. L. was supported by NIH5P30CA0651. The equipment for this work was made possible by the Kresge and Rippel Foundations and by the National Institutes of Health. Received November 14, 1994; revised December 9, 1994.

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Crystal Structure of CD1 l b A Domain 637

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