Structure of the glycosylated adhesion domain of human T lymphocyte glycoprotein CD2

Structure of the glycosylated adhesion domain of human T lymphocyte glycoprotein CD2 Jane M Withkalt, Daniel F Wysslt, Gerhard Wagner TM, Antonio RN Arulanandam2, a, Ellis L Reinherz2, 4 and Michael A Recny 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA, 2Laboratory of Immunobiology, Dana Farber Cancer Institute, and 3 Department of Pathology and 4Department of Medicine, Harvard Medical School, Boston, MA 02115, USA and 5PROCEPT,Inc., 840 Memorial Drive, Cambridge, MA 02139, USA

Background: CD2, a T-cell specific surface glycopro-

tein, is critically important for mediating adherence of T cells to antigen-presenting cells or target cells. Domain 1 of human CD2 is responsible for cell adhesion, binding to CD58 (LFA-3) expressed on the cell to which the T cell binds. Human CD2 domain 1 requires N-llnked carbohydrate to maintain its native conformation and ability to bind CD58. In contrast, rat CD2 does not require N-linked carbohydrate, and binds to a different ligand, CD48. Results: The three-dimensional structure of the glyco sylated form of domain 1 of human CD2 has been de termined by NMR spectroscopy. The overall structure

resembles the typical 13-barrel of an immunoglobulin variable domain. Nuclear Overhauser enhancement contacts between the protein and the N-linked glycan have been tentatively identified. Conclusion: Based on our results, we propose a model showing how the N-linked glycan might be positioned in the human CD2 domain i structure. The model provides an explanation for the observed instability of deglycosylated human CD2, and allows residues that are important for CD58 binding to be differentiated from those affecting conformational stability via interactions with the glycan.

Structure 15 September 1993, 1:69-81 Key words: glycoprotein, immunoglobulin, NMR, receptor on T cell

Introduction

a 117 residue cytoplasmic tail, which is rich in proline and basic residues [11-13] and is required for CD2-mediated signal transduction [14]. All adhesion functions have been mapped to domain 1 of the CD2 extracellular region [15] which contains a single high mannose N-linked glycan that is required for maintaining the structural stability and functional CD58-binding prop erties of the CD2 receptor in humans [16] .

T lymphocytes display a unique array of cell-surface adhesion receptors which regulate the ability of the immune system to detect, and respond in a coordinated fashion, to the presentation of foreign antigens. Human CD2 is a transmembrane glycoprotein receptor (M r ~-,50-55 kDa) expressed on the surface of T lympho cytes and natural Miler cells which regulates adhesion between T cells and antigen-presenting cells (APCs), including epithelial cells and endothelial cells, or between cytolytic T cells and target cells [1,2]. Human CD2 binds specifically to leukocyte function-associated antigen-3 (LFA-3, CD58) present on APes [3,4]. The CD2-CD58 adhesion pair plays a key role in facilitating the specific recognition by the T-cell receptor (TCR) of peptide antigens presented by the major histocompatibility complex (MHC), leading to initiation of cellular immune responses [5-7]. CD2 has also been shown to be able to transduce activation signals. For example, perturbation of the CD2 extracellular domains with a combination of monoclonal antibodies which bind specific epitopes in both domains 1 and 2 can lead to activation of T cells independent of antigen recognition by the TCR [8,9].

In rodents, the only known ligand for CD2 is CD48, termed BCM1 in mice [17] and OX 45 in rats [18]. No gene for CD58 has been identified in these species. Human CD2 also binds CD48 but with a 100-fold lower affinity and is unable to support cell--cell conjugates [19]. Comparisons of chromosomal location, genomic organization and primary sequence homology between CD2, CD48 and CD58 have led to the hypothesis that all three receptors arose from duplication of a primordial IgSF domain; however, through evolutionary divergence rodent CD2 and human CD2 have come to recognize distinct functional ligands [19]. Data have been reported indicating that CD59 is an alternative ligand for human CD2, and that the binding site of CD59 overlaps that of CD58 [20,21], but other obser vations contradict this notion [19].

CD2 is a member of the immunoglobulin gene superfamily (tgSF) [10] and consists of a two-domain 185 amino acid residue extracellular portion, a 25 residue hydrophobic transmembrane segment and

The NMR structure of the unglycosylated adhesion domain of rat CD2 (r-sCD299) expressed in Escherichia coli [22] and the crystal structure of the entire extra cellular portion of rat CD2 (r-sCD2177) expressed in

*Corresponding author, tIM Withka and DF Wyss contributed equally to this work. (~ Current Biology Lid ISSN 0969-2126

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Structure 1993, Vol 1 No 1

mammalian cells [23] were reported recently. Both of these structures have characteristic immunoglobu lin (Ig) folding patterns, consisting of two J3-sheets composed of either nine (domain 1) or seven (domain 2) antiparallel [3-strands. The ollgosaccharides attached to each of four N-glycosylation sites were enzymatically trimmed back to single N-acetylglucosamine (GlcNAc) monosaccharide units to facilitate crystallization of rsCD2177. From these studies, it appears that the presence of these N-linked glycans is not required for rat CD2 to form a stable three-dimensional (3D) Ig fold. However, analysis of a deglycosylated soluble recombinant human CD2 protein, or a mutant CD2 transmembrane receptor lacking the N-glycosylation site within the adhesion domain, showed that neither would bind to CD58 or to monoclonal antibodies that define native adhesion domain epitopes [16]. These studies clearly demonstrate that there are structural and functional requirements for N-glycosylation in the human CD2 receptor. However, it is not yet clear whether the failure to bind CD58 after deglycosylation is due to disruption of CD2 structure, loss of contacts between the N-linked carbohydrate and CD58, or both. Sequence alignment of homologous regions in the amino-terminal domains of human and rat CD2 revealed that the position of the single N-linked glycan in the human CD2 adhesion domain is not conserved between these two species. Therefore, to examine the unique importance of the N-linked glycan in maintaining the conformational stability of human CD2, and to better understand whether the N-linked glycan might directly interact with CD58, we have determined the 3D solution structure of the fully glycosylated adhesion domain of human CD2 (husCD2105; Mr ,-~13.6 kDa) by NMR spectroscopy. To our knowledge, this is the first NMR structure reported for a glycoprotein containing a fully intact N-linked carbohydrate that is required for its functional activity. In a separate study [24] we have described the 1H resonance assignments and secondary structure analysis for hu-sCD2105 which provide the basis for the de tailed analysis of the tertiary structure reported here. The NMR structures have been determined by distance geometry (DG) methods using interproton and hydrogen bonding distance restraints and dihedral angle restraints from the 1H NMR data. While human CD2 domain i exhibits a typical Ig fold consisting of two [3sheets comprising antiparallel 13-strands packed tightly about a hydrophobic core, there are a number of interesting differences between the human and rat struc tures which may reflect the fact that rodent and human CD2 recognize distinct, albeit structurally homologous, ligands. Complete assignment of the high mannose N-linked glycan projecting from Asn65 has not been made as yet. However, we have tentatively identified two nuclear Overhauser enhancement (NOE) cross peaks defining contacts between the high mannose oligosaccharide and one amino acid residue. We also have indirect evidence which suggests a possible orientation for the functionally important glycan within

the 3D human CD2 structure. These data, together with mutational studies, provide substantial insight into the nature of the CD58 binding site.

Results and discussion

We report the 3D structure of the N glycosylated 105 residue adhesion domain of human CD2 (hu-sCD2105). Because human CD2 requires N-glycosylation to be stable and functional, we could not prepare 13C/15Nisotopically labeled protein in E. coli in order to facilitate assignments of 1H resonances from heteronuclear edited multi dimensional NMR experiments. Hence, we were required to use the CD2 glycoprotein adhesion domain derived from two-domain hu-sCD2182. This fragment contains the first 105 amino acids of the extracellular segment and has a single N-glycosylation site at Asn65. This carbohydrate binding site contains a single high mannose N-glycomer [(Man)n(GlcNAc)2, where n = 5-9] when the recombinant protein is expressed in Chinese hamster ovary (CliO) cells [16]. Consequently the 1H assignments for hu-sCD2m5 were achieved fol~ lowing standard 2D NMR techniques [25,26]. The evaluation of 2D homonuclear NMR data to obtain 1H assignments as well as interproton distance and dihedral angle information is hampered by resonance overlap for proteins of this size. These difficulties were reduced by recording spectra at different temperatures as well as by performing heteronuclear 2D NMR experiments on a hu-sCD2m5 sample selectively labeled with 15N-lysine. A complete analysis of the secondary structure and 1H resonance assignments is reported elsewhere [24]. Interproton distances, dihedral angles and hydrogen bonding information used in structure calculations as conformational restraints were obtained using 2D homonuclear nuclear Overhauser enhancement spectroscopy (NOESY) [27,28], total correlation spectroscopy (TOCSY) [29] and double quantum filtered correlated spectroscopy (2QF-COSY) [30-32] techniques. Evaluation of calculated model structures

Solution structures of hu-sCD2105 were determined by DG methods [33] using DGII in the INSIGHT II package (BIOSYM Technologies, San Diego) and incorporating interproton and hydrogen bonding distance restraints as well as dihedral a @ e restraints. These calculations included 740 NOE restraints consisting of 8 intra-residue, 328 sequential, 37 (i+ 2) NOEs, 12 (i+ 3) NOEs, 14 (i + 4) NOEs and 341 long range NOEs. Additionally, 50 hydrogen bonding distances as well as dihedral angle restraints for 79 qbangles and 19 Z1 angles were included. A total of 115 DG structures were calculated and 59 structures were accepted on the basis of interproton distance and angle violations as compared with the experimental data as well as the non-bonded contact violations. Of these 59 structures, 18 of the best distance geometry structures were energy min-

Structure of the adhesion domain of human CD2 W i t h k a et al.

imized to reduce repulsive non-bonded contacts and the backbone atoms are shown superimposed in Fig. la. Fig. l b displays all backbone heavy atoms for the DG structure with the lowest error function.

lular domains by ab initio methods [13]. In retrospect, the contributions to the circular dichroism signals that led to the prediction of some helical structure may be due to turns connecting the [3-sheets.

The framework of hu-sCD2105 is composed of antiparallel [3-strands [strands A (residues 7-12), B (residues 16-20), C (residues 32-37), C' (residues 45-47), C" (residues 53-55), D (residues 60-63), E (residues 67-71), F (residues 80-86) and G (residues 94-103) (see Fig. 2)] and is relatively well defined with an average root mean square deviation (rmsd) from the mean structure of 0.641 ~ for backbone atoms and 1.094 & for all heavy atoms. The corresponding average rmsd for both loops and 13-strands (excluding terminal residues 1% and 104-105) is 1.111 ~ for backbone atoms and 1.800& for all heavy atoms, indicating significant flexibility in some of the longer loop regions as shown in Table 1. The three-residue C" strand is positioned between the C'C" and C"D flexible loop regions and is the least well defined [3-strand, showing only two typical antiparallel NOE contacts to the adjacent C' strand. The structural elements defined in the human CD2 structure by NMR differ from the 0t/13 structure previously proposed on the basis of circular dichroism measurements [15 ] and computer modeling studies of the amino acid sequence profiles of murine and human CD2 extracel-

The definition of dihedral angles in the ensemble of model :structures can be evaluated by the calculation of order parameters as described previously by Hyberts et al. [34]. The angular order parameters (sang le) can identify the degree of fluctuation in a dihedral angle where the two limits are defined for an exact angle (sangle= 1) and a randomly distributed angle (sangle= 1/~/N, where N is equal to the number of structures considered). Order parameters for the backbone dihedral angles, qb and q,, have been calculated for all residues using the 18 model structures and are shown in Fig. 3. For the majority of residues, the backbone dihedral angles are well defined, with the exception of some residues in the loop regions. The most significant fluctuations occur for residues in the BC, C'C" and C"D loops. Evaluation of backbone dihedral angles (qb, 4 ) for all 18 structures was performed by analysis of Ramachandran maps as shown in Fig. 4a. As a comparison, (qb, 4 ) values for all residues in all model structures with order parameters greater than 0.9 are plotted in Fig. 4b. The majority of these values are consistent with the allowed regions

Fig. 1. (a) Superposition of backbone atoms of residues 7-103 for 18 model structures of hu-sCD2105 with the [3sheet containing strands B, D and E oriented in front. The N-linked g]ycosylation site at Asn65 in the DE loop is highlighted in yellow. (b) DG-II structure of hu-sCD2105 with the lowest error function. All heavy atoms of the backbone are shown. Sequence numbers are given for every fifth residue.

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Structure 1993, V o l 1 N o 1

violations which were all within 5.64 ° of the upper and lower limits were observed per model structure. No hydrogen bonding distance violations occurred in any model structure.

Table 1. Average root mean square deviations (rmsd) from the mean structure.

Backbone atoms rmsd (A) Residues 7-103 [~-sheet residues

1.111 0.641

All heavy atoms

Range (A)

rmsd (A)

0.965 1.322 0.479--0.864

Range (A)

1.800 1.094

Structure of the adhesion d o m a i n of h u m a n C D 2

The human CD2 adhesion domain has a characteristic Ig folding pattern. It has two 13-sheets, one composed of three antiparalle113 strands (strands D, E and B), the other of five (strands C", C', C, F and G). In addition, 13-strand A makes parallel NOE contacts to strand G, forming a structural motif characteristic of a 13-barrel. Residues 1-5 at the amino terminus are unstructured, showing only sequential HN(i) I-I0t(i- 1) NOE connectivities. However, a strong HN(i)-HN(i + 1) NOE contact between Asn5 and Ala6 has been observed indicating a change of chain direction. Thus, the unstructured amino-terminal pentapeptide points away from the 13barrel in all calculated model structures.

1.551 2.021 0.953-1.401

Average rmsd for secondary structural elements from the mean structure

Backbone atoms A strand AB loop

rmsd (A) 0.697 0.738

Range (a) 0.465 1.209 0.501-1.213

g strand

0.714

0.378-1.157

BC loop C strand CC' loop C' strand C'C" loop C" strand C'D loop D strand DE loop E strand EF loop F strand FG loop G strand

1.623 0.562 1.254 0.543 2.120 1.159 1.502 0.931 1.129 0.626 0.975 0.449 0.818 0.605

1.094-2.258 0.379~).820 0.623 2.089 0.305q1923 1.211-3.497 0.7691.980 0.872-2,662 0,592-1,542 0.545 1.884 0.322 1.100 0.716-1.361 0.323~).707 0.470 1.326 0.372 1.479

The average rmsds (,a)from the mean structure and the range of these values are shown for the 18 model structures of human CD2. Average rmsds for individual secondary structural dements are also shown. Rmsds were calculated based on the superposition of the backbone atoms for residues 7 103.

in the Ramachandran diagram. Residues indicated by an (x) in the forbidden regions originate from glycine residues. The final 18 minimized model structures agree well with the experimental NMR data, with an average of 2.1 interproton distance violations greater than 0.2A per model structure and no individual NOE violation greater than 0.37A. An average of 3.4 dihedral angle

The 3D fold of human CD2 domain i (with the 13-sheet containing the putative CD58 binding site oriented in front) is illustrated in Fig. 5a. In Fig. 5b, the opposite face of CD2, which contains the single N-glycosylation site at Asn65, is shown. The relative orientation of the two 13-sheets results from side chain contacts between hydrophobic residues in the core. These contacts principally inw)lve residues teu19, Tyr60, Leu62, Leu68, and Ile70 on strands B, D and E and Trp35, Ala45, Phe47, Tyr81 and Phe98 on strands C, C', F and G as shown in Fig. 6. When all the model structures are considered as a group, the loops connecting the various strands of the 13-sheets appear somewhat disordered (see Figs. la and 3 and Table 1), particularly the BC loop (residues 21-31), the C'C" loop (residues 48-52) and the C"D loop (residues 56-59). In the initial portion of the long BC loop, residues Ile21 and Pro22 make NOE contacts with Phe98 on the G strand and the terminal residues of this loop, Ile30 and Asp31, show NOE contacts to

Human CD2 A

B

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75

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99

Fig. 2. A m i n o acid sequence and 13strand assignments for human CD2 (husCD2105) and rat CD2 (dashed line - rsCD299 [22], solid line r-sCD2177 [23]). Residue Asn65, the site of N-linked glycosylation in human CD2 is boxed.

Structure of the a d h e s i o n d o m a i n of h u m a n CD2 W i t h k a e t al.

++llllilflbll!M!!+Iv,l!J!l!i!!dlililil!O! ° llll[llll l il ll tUil [lill ll l ill ll

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Fig. 3. The angular order parameter (Sang le) calculated [34] for all 18 model structures for the backbone dihedral angles, ~(top) and q; (bottom), as a function of residue number•

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Fig. 4. (a) Ramachandran plot for all residues in the 18 model structures. (b) Ramachandran plot only for residues with angular order parameters greater than 0.9. The (d?,~) values indicated by an (x) are for glycine residues. Based upon the hard sphere model, accessible regions for the upper limit and the lower limit in the Ramachandran plots are indicated by solid and dashed lines [35], respectively.

Asp87 and Thr88 in the FG loop. However, residues 23-29 show no medium or long range NOE contacts to any other region of the protein and these residues are largely disordered in the calculated structures. The C'C" and C"D loops are interrupted by the short three residue C"strand. Both loops and the C" strand are poorly ordered and show very few NOE contacts to other regions of the protein• However, in most model structures, the C'C" loop and the C" strand appear relatively flat and maintain the surface of the ]3-sheet containing the C',C, F and G strands (see Fig. la). The AB loop (residues 13-15), CC' loop (residues 38-44), DE loop (residues 6446), EF loop (residues 72~9) and FG loop (residues 87@3) are significantly more defined in the structures as a result of observed NOE contacts with portions of surrounding 13-strands and loop structures (see Figs. la and 3 and Table 1). The CC' loop is projected away from the surface of t h e well defined 13sheet containing the C", C', C, F

and G strands in all structures. Although the orientation of the CC' loop is fairly well defined, the amplitude of its projection away from the face of the [3-sheet varies between structures. Role of the N-linked o l i g o s a c c h a r i d e

The single N-linked glycosylation site in the adhesion domain of human CD2 at Asn65 is located at t h e top of the DE loop in the face of the [£sheet containing strands B, D and E (see Figs. la and 5b). This site is occupied with only high mannose oligosaccharides when recombinant hu-sCD2182 is expressed in CHO cells• While the exact carbohydrate glycotype of CD2 on human T cells is not known, the carbohydrate class is unlikely to switch from high mannose to either a complex or hybrid glycotype in different mammalian cell types. CliO derived hu-sCD2105 binds CD58, as does human CD2 expressed in monkey kidney (COS1) cells [16] or murine L cells [7]. Recombinant human

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Structure 1993, Vol 1 No 1

(c)

(d)

Fig. 5. Ribbon diagrams illustrating the overall folds for human and rat CD2s. In (a) the average structure of hu-sCD2105 is shown with the p-sheet implicated in CD58 binding (G, F, C, C'and C") in the foreground. (b) The opposite face of the average structure of hu-sCD2105 is shown with the glycosylation site at Asn65 indicated by a triangle. (e) NMR structure of domain 1 of rat CD2 (r-sCD299) in a similar orientation as in (b). (d) Crystal structure of domain 1 of rat CD2 (r-sCD2177) in a similar orientation as in (b). (Figures prepared using the MOLSCRIPT program [36]).

CD2 containing a mutation of Asn65 produced in COS cells is not functional [16]. Thus, it is reasonable to postulate that the glycosylation pattern for human CD2 expressed in either CHO cells, COS-1 cells or murine L cells is similar to that found in humans. Analysis of the glycoform heterogeneity of hu-sCD2105 by electrospray ionization mass spectrometry (ESI-MS) showed that the high mannose glycan is composed of heterogeneous isomeric structures (glycomers) with an iden-

tical [Man[31-4GlcNAc[31-4GlcNAc-] core trisaccharide, but having branched chains of varying lengths. The predominant glycoform species in our unlabeled sample, from which these structures were calculated, contains Man 5 ( ~ 20 %), Man 6 ( ,~ 34 %), Man 7 ( ,-~40 %) and Man 8 (,-~6%) glycomers (a detailed description of the glycan linkages and glycomer branching structures will be reported elsewhere: BB Reinhold, et at, & VN Reinhold, unpublished data). The Man 6 and Man 5

Structure of the adhesion domain of human CD2 Withka et a/.

Fig. 6. Hydrophobic core of hu-sCD2105 in stereo. Side chains (yellow) of residues Leu19, Tyr60, Leu62, Leu68 and lie70 from one [3-sheet (composed of [~strands B, D and E) and Trp35, Ala45, Phe47, Tyr81 and Phe98 on the other ~-sheet (composed of ~-strands C, C', F and G) are shown superimposed for 16 model structures. The backbone of the average structure (residues 7-103) is shown as a ribbon drawing with residues in r-strands shown in white and residues in loop regions shown in blue.

glycomers present in our sample arise from trimming the Man7 glycomer at different branch points. Fig. 7 illustrates the two three-branched Man7 isomeric structures which, based on ESI-MS and low energy collision analysis of the intact permethylated glycan, are the only Man7 glycomers present in our sample. Chemical shift assignments for the H1, H 2 and H 3 protons of at least seven mannosyl moieties have been made using natural abundance 13C-1H hetero single quantum correlation (HSQC) as well as homonuclear 2D NOESY and TOCSY techniques [37,38]. However, assignments for most of the H4, H 5 and H 6 protons have been hampered by the narrow chemical shift dispersion of glycomer proton resonances as well as by overlap with protein resonances in the aliphatic region. At this point, the iH assignments of the second GlcNAc monosaccharide has not been unambiguously identified due to resonance overlap. Mao Is, --2Man ,

I\ /3

6* Man~l ~

Manc~l

/ ~ Man~l - - 4GIcNAcffl--4GIcNAc

Man[~l--2Man~l ] Manc~l ~ 6 [/3 Man [cd--2Manal

Man~l N / 6 Man131__ 4GIcNAcffl--4GIcNAc

Man 1~l--2Man~l ] Fig. 7. The two possible isomeric structures for the predominant Man 7 glycomer present in our sample from which the NMR solution structures were calculated. These Man 7 glycomers have three branches that extend from the core [Man-(GIcNAc) 2] trisaccharide. The possible mannose residues involved in an ~1~2 linkage that could contact Gly90 in the FG loop are boxed. As discussed in the text, the mannose residue containing the H6 and H6, protons resonances which could conceivably contribute to the observed protein glycomer NOEs is indicated by an asterisk.

The fingerprint and aliphatic regions of 2D NOESY spectra at various mixing times have been analyzed extensively for possible protein-glycomer contacts. These NOE cross peaks can generally be differentiated from protein-protein NOEs due to the sharper line widths of

the glycomer resonances. Two protein-glycomer NOE contacts have been tentatively identified thus far (in addition to the sequential NOEs between all side chain protons of Asn65 and protons of the first, Ash-linked, GlcNAc) and occur between the H t of a mannose residue involved in an ~1--+2 linkage and the two alpha protons of Gly90 in the FG loop. Therefore, these NOEs must then define interactions between a mannosyl residue located on one of the two branches extending from the core [Man-(GlcNAc) 2] trisaccharide as shown in Fig. 7. In order to make contacts with Gly90, which is located in the middle of the FG loop, at least one of the mannosyl branches containing an ~1 --+2 linkage must extend upward from the top of the DE loop and (at least partially) fill the cleft between the BC loop and the DE loop. However, it cannot be totally excluded that these NOEs involve the unassigned H6 and H 6, proton resonances of the adjacent mannose residue involved in the cd-+6 linkage as indicated in Fig. 7. So far, only NOEs for the well resolved anomeric sugar protons have been analyzed since the other carbohydrate resonances appear in very crowded spectral regions and assignment of NOEs without isotope labeling is difficult. There is also indirect structural evidence which sup ports this proposed orientation of the N-linked glycan. In the rat CD2 structure, the BC loop shows a prominent kink as a result of NOEs between side chains of residues in this loop and the DE loop [22]. However, no such. NOEs are present for residues within the BC loop in human CD2. Thus, the human BC loop seems to be more disordered than the corresponding loop in the rat structure. This observation is quite surprising, given that the BC loops in human and rat CD2 exhibit > 90 % identity at the amino acid sequence level and represent the most homologous region of the proteins. Therefore, the absence of defined NOE contacts between the BC loop and the DE loop in the human structure further suggests that one or more of the glycomer branches is oriented towards the core of the protein, preventing residues in the BC loop from making contacts with residues on the [3-sheet that con tain strands B, D, E and their respective loops. There is additional, indirect evidence that the high mannose N-linked glycan might project towards residues at

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Structure 1993, Vol 1 No 1

the top of the C'C" loop and potentially stabilize this region. We have observed that the Arg48-Lys49 peptide bond of hu-sCD2182 is stable to digestion by clostripain at 25°C, whereas the Argl05-Vall06 and Arg146-Va1147 peptide bonds are rapidly cleaved (within minutes). Additionally, many of the side chain and backbone assignments for Arg48 were not obtained and very few NOEs were observed to the backbone atoms. As a result, this residue is relatively unrestrained in the calculations, and it is one of only three residues for which backbone assignments are incomplete [24]. This suggests that Arg48 is both highly mobile and solvent exposed, and therefore one might expect it to be readily accessible to proteolytic attack. However, if one branch of the N-linked glycan projecting from Asn65 fills the cleft between the DE and BC loops, a different branch of the high mannose oligosaccharide might project towards the C'C" loop and shield Arg48 from proteolytic attack by clostripain. This hypothesis is further supported by the observation that when the high mannose N-linked glycan is completely removed from hu-sCD2105 by digestion with the endoglycosidase pep tide N-glycosidase F, the Arg48-Lys49 peptide bond is rapidly cleaved (data not shown). However, additional structural information for the N-linked glycan is necessary to verify its proposed orientation and functional role. Proposed binding site of the natural ligand CD58 The 3D structure of human CD2 now provides new possibilities for interpreting whether specific residues formerly identified as being important for CD58 binding are indeed CD2-CD58 contact residues, or are perhaps involved in protein-glycomer interactions which stabilize native CD2 structure. Previous mutational anal-

yses on human CD2 identified six solvent-exposed residues as being important for CD58 binding: Lys43 (CC' loop), Gln46 (C' strand), Lys82 (F strand), Tyr86 (F strand), Asp87 (FG loop) and Gly90 (FG loop) [39,40]. These residues are highlighted in Fig. 8. The side chains of residues Gln46, Lys82 and Tyr86 are oriented towards the solvent exposed surface of the [3-sheet containing the C, C' and F strands. The side chain of Lys43 is also clearly oriented towards the center of the binding site which appears to be bracketed by the CC' and FG loops. However, in contrast to these four residues, the first residue of the FG loop, Asp87, has its side chain pointing away from the face of the [3-sheet implicated in CD58 binding and towards the high mannose oligosaccharide which is proposed to fill the cavity between the BC and DE loops. A cluster of charged and polar residues in the BC, C'C" and FG loops with solventexposed side chains are also found in this region (Fig. 9). These residues could conceivably participate in a network of hydrogen bonding interactions with various saccharide residues within the N-linked glycomer, which may be mediated by bridging water molecules. The potential NOE contacts observed between Gly90 and a mannose residue involved in an ~1--+2 linkage, taken together with the indirect evidence discussed above, would clearly bring the glycan into close proximity with this region of the polypeptide surface. These potential hydrogen-bonding interactions might also explain the appearance of disordered loops, particularly the BC loop, in the model structures. If the BC, FG and C'C" loops make hydrogen-bonded contacts with glycomer residues and do not interact with other regions of the protein, these structural constraints are not included in the calculations and therefore these

Fig. 8. Ribbon drawing of one model structure of hu-sCD2105 (residues 7103). Residues Lys43 (CC' loop), Gin46 (C'strand), Lys82 (F strand), Tyr86 (F strand), Asp87 (FG loop) and Gly90 (FG loop) which are implicated in the binding of CD58 are shown in yellow. The proposed binding site of CD58 involves the solvent-exposed surface of the ~sheet containing the C', C and F strands and is bracketed by CC', C'C" and FG loops. The N-linked glycosy[ation site at Ash65 (DE loop) is displayed in red.

Structure of the adhesion domain of human CD2 Withka et al.

loops would appear more disordered in the ensemble of model structures than they would actually be in the native structure. Such hydrogen-bonded contacts might also account, in part, for the stabilizing effect that the N-linked glycan has on the overall CD2 conformational structure. Removal of the high mannose glycomer would eliminate this putative network of interactions between various polypeptide side chains, water molecules and individual saccharide residues. This would presumably lead to an increase in the conformational flexibility of the BC, FG and C'C" loops which frame the CD58 binding site, thus destabilizing the native 3D structure of the adhesion domain. Multiple roles for residue Glyg0 in the FG loop are implicated in the binding of CD58. In addition to the potential NOE contacts of the alpha protons of Gly90 with an al--+2 mannosyl residue, the carbonyl of Gly90, which is directed towards the CD58 binding site in all 18 model structures, may be involved in hydrogenbonding interactions with CD58 residues. Furthermore, evaluation of the model structures indicate that the qb and ~ angles for Gly90 are well defined with an average value of 0 = 97 ° and ~ = 61°. Analysis of Ramachandran maps indicates that these values lie outside the regions allowed for residues with C6 atoms (see Figs. 4a and 4b). Therefore, substitution of a glutamic acid residue at this position, which was previously shown to inhibit CD58 binding [39], would clearly disrupt the structure of the FG loop. On the basis of detailed analysis of the human CD2 structures, we have recently made additional alanine

substitution mutations in human CD2 to further probe the apparent binding, site for CD58 (ARN Arulanandam, et al., & EL Reinherz, unpublished data). Of these residues, Asp32 (C strand), Lys34 (C strand), Arg48 (C'C" loop), Lys51 (C'C" loop) and Lys91 (FG loop) were found to disrupt binding of CD58. These results imply that the C strand and the C'C" loop are also in volved in the binding of CD58. All of the side chains for these residues are solvent exposed in the model structures. The side chains for these residues are relatively well defined and oriented into the proposed binding region, with the exception of Arg48 and Lys51 in the flexible C'C" loop. In addition, alanine-substitution mutations of Lys41 (CC' loop), Asn92 (FG loop) and Va193 (FG loop) were found to partially disrupt the binding of CD58. A summary of -all mutations known to "affect the binding of CD58 is shown schematically in Fig. 10. The CD58 binding surface thus appears to be concentrated about the solvent exposed surface of the C, C' and F strands. This surface is fairly flat, but contains a shallow depression centered about the C strand. The CD58 binding site for CD2 proposed by Driscoll et al. [22] and Jones et al. [23], and discussed by Springer [41], based on a prediction of human CD2 j3-strand assignments by alignment of homologous regions with the rat structure, is in line with the current findings. The surface of the 13-sheet is extended by the C'C" loop and bracketed by the CC' and FG loops (see Fig. 8). Electrostatic interactions appear to play a major role in the binding of CD58 since the majority of the residues implicated in binding are surface exposed and charged moieties. Therefore, mutations in the face of

Fig. 9. Space-filling model of hu-sCD2105 showing the putative glycan contact site. Residues implicated by direct and indirect evidence which suggest a possible orientation for the oligosaccharides are shown. In this figure, the model of CD2 is rotated to view the top of the BC, C'C" and FG loops. Potential NOE contacts are observed for the glycomer which is N-linked at Ash65 (purple) in the DE loop and for Gly90 (white) in the FG loop. The cluster of charged and polar residues in this region, including Asp28, Asp29 and Asp31 (red)in the BC loop, Arg48 and Lys49 (green'~in the C'C" loop and Asp87 (red), Thr88 (yellow), Lys89 and Lys91 (green) in the FG loop are shown.

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Structure 1993, VoI 1 No 1

G

F

C

C'

C"

E E

the crystal structure, one might expect that human CD2 would also dimerize in solution. We find no evidence to support this from our NMR experiments (the line widths are relatively narrow and intermolecular NOEs are absent from the 1H-1H homonuclear NMR spectra). Molecular weight analysis of both hu-sCD2105 and husCD2182 by HPLC gel permeation chromatography [ 16] and equilibrium sedimentation analysis of two-domain hu-sCD2182 [42] are also consistent with CD2 adopting a monomeric form in solution. Structural information on the CD2-CD58 co-complex will be required to verify the proposed heterophilic interaction of CD2 and CD58 and to determine precisely which residues are involved in CD58 binding. Comparison of the adhesion domains of human and rat CD2

KIOI

Q1~031102

Fig. 10. A schematic of the [3-sheet containing strands G, F, C, C' and C" and the respective CC', C'C" and FG loops. Residues implicated in the total disruption of CD58 binding are shown in circles. Mutations to residues which result in partial disruption of CD58 binding are shown in rectangles.

this sheet, including the CC' loop, would be expected to inhibit CD2-CD58 interactions by disrupting key contact residues required for binding. On the basis of the structural data presented here, it also appears that Asp87 and Gly90 (in the FG loop) and Arg48 (in the C'C" loop) may be involved in protein-glycomer interactions which may stabilize these loop regions. Mutations which disrupt protein-glycomer interactions would likewise be expected to disrupt CD2-CD58 binding by destabilizing these loops, which frame the CD58 binding site. The proposed CD58 binding site is nearly identical to the site of homophilic CD2-CD2 adhesion at the amino-terminal domains of rat sCD2177, seen between two independent copies of the protein in the crystal structure [23]. Since CD2 and CD58 are structurally homologous, it is likely that the heterophilic interaction is quite similar to this homophilic interaction in which hydrogen bonding exists between residues on the tips of the FG loop of the amino-terminal domain of one rat CD2 and the CC' loop of another. Given that a homophilic interaction is observed for rat CD2 in

The solution structure of" rat CD2 domain 1 was solved by Driscoll et al. [22] for an unglycowlated form of CD2 expressed as a fusion protein in E. coli (Fig. 5c), and the crystal structure of two-domain rat CD2 expressed in mammalian cells was solved by Jones et aL [23] (Fig. 5d). In the later study, rat CD2 glycoprotein was expressed in CliO cells and only GlcNAc monosaccharide units were retained at each of the glycosylation sites following treatment with endoglycosidase H. The adhesion domain of rat CD2 contains three N-linked glycosylation sites located at the bottom of the EF loop (ASn67), the middle of the F strand (Asn77) and the top of the FG loop (Asn84) (see Fig. 2). None of these sites is conserved in the human structure, and it is clear that intact glycosylation is not required for rat CD2 to fold properly. Human and rat CD2 are ~ 41% identical with respect to their primary sequences, and even though they differ in their functional requirement for N-glycosylation and recognize different ligands, the respective adhesion domains adopt similar Ig folds in solution. However, there are a number of interesting differences between rat and human CD2 with respect to the lengths of the [3-strands and their inter-connecting loops. These variations in secondary structure have been described in detail elsewhere [24]. As discussed previously, the major structural difference between the human and rat CD2 structures is the conformation of the inter-sheet BC loop which provides indirect evidence for the orientation of the high mannose glycomer with respect to the human CD2 polypeptide conformation. Other structural differences include the length of the CC' loop, which contains a two residue insert in the human structure when compared with the rat structure. Although this loop is oriented similarly in human and rat CD2, the increased length of this loop in human CD2 creates a 'bracket' of charged residues projecting away from the flat surface of the [3-sheet (see Fig. 8). As described above, this CC' loop is clearly important for CD58 binding and contains a series of four charged residues (including Lys43) which are not found in the rat structure.

Structure of the adhesion domain of human CD2 Withka et a/.

In the crystal structure of rat CD2, [3-bulges have been identified in the C' strand at Leu38 and in the G strand at Arg87 [23]. Similar backbone distortions are found at homologous positions in human CD2 at Lys43 and Asn92. However, these residues are defined to be in the CC' and FG loops, respectively, and are not a part of the network of [3-strands. Thus, they lack some characteristics of classical 13-bulges. Comparison of human CD2 to other immunoglobulin structures

Many structural features of IgSF domains are conserved in the adhesion domain of human CD2 with the e x c e p tion of the conserved disulfide bond linking the two 13sheets through strands B and F. However, comparison of the adhesion domain of human CD2 with other immunoglobulin domains indicates the presence of several functionally important differences. The CC' and FG loops in CD2, which are implicated in both homophilic interactions (rat) and heterophilic adhesion (human) are significantly longer than the corresponding loops in human CD4 [43,44]. The C'C" loop, implicated in the binding of CD58, is relatively flat in human and rat CD2 and maintains the surface of the [3-sheet consisting of the C', C, F and G strands. This orientation is similar in CD8 [45]; however, the C'C" loop in CD4, which is crucial to the binding of the HIV coat glycoprotein, gp120, is oriented at an angle relative to the respective [3-sheet [43,44,46].

p e r i m e t e r of t h e CD58 b i n d i n g site via a n e t w o r k of h y d r o g e n bonds. This m a y explain the abolition of CD58 binding t h a t o c c u r s w h e n h u m a n CD2 is deglycosylated. Several a m i n o acid residues in d o m a i n 1 have b e e n previously identified b y mutational analyses as being i m p o r t a n t for CD58 binding. O n e of these, Asp87, appears instead to p o i n t into t h e cavity p r o p o s e d to c o n t a i n t h e N-linked c a r b o h y d r a t e , and m a y t h e r e f o r e affect CD58 binding by destabilizing p r o t e i n - c a r b o h y d r a t e int e r a c t i o n s instead o f b y m a k i n g direct c o n t a c t w i t h CD58. Our m o d e l also p r o p o s e s t h a t t h e N-linked carboh y d r a t e itself is n o t a ligand for CD58, and allows for differentiation b e t w e e n a m i n o acid residues involved in CD58 b i n d i n g a n d t h o s e that instead i n t e r a c t w i t h c a r b o h y d r a t e residues. This inform a t i o n s h o u l d b e useful for designing drugs that disrupt C D 2 - C D 5 8 binding, w h i c h c o u l d suppress T-cell activation and m i g h t t h e r e f o r e be beneficial in t h e t r e a t m e n t of a u t o i m m u n i t y and o t h e r T-cell m e d i a t e d diseases.

Materials and methods

Protein expression, purification and sample preparation

Biological implications

T h e role t h a t N-linked oligosaccharides play in g l y c o p r o t e i n s t r u c t u r e a n d f u n c t i o n is o f t e n q u i t e varied and n o t c o m p l e t e l y u n d e r s t o o d , b u t t h e r e are a n u m b e r of e x a m p l e s w h e r e N-glycosylation is i m p o r t a n t for t h e folding, transport, or biological activity of s e c r e t e d g l y c o p r o t e i n s or transm e m b r a n e g l y c o p r o t e i n r e c e p t o r s [47,48]. Hum a n CD2 requires N-linked glycosylation in dom a i n l to maintain its s t r u c t u r e and ability to b i n d its ligand, CD58. This i n t e r a c t i o n is critically imp o r t a n t for T cells to b i n d a n t i g e n - p r e s e n t i n g cells and target cells, and w i t h o u t C D 2 - C D 5 8 adhesion t h e r e c o g n i t i o n of M H C / a n t i g e n c o m p l e x e s b y the T-cell r e c e p t o r is n o t o p t i m a l for T-cell b i n d i n g and activation. In this w o r k , w e have b e g u n to u n d e r s t a n d h o w N-glycosylation m e d i a t e s t h e c o n f o r m a t i o n a l stability, and t h u s t h e a d h e s i o n properties, of hum a n CD2. In o u r model, t h e c a r b o h y d r a t e fills a cavity b e t w e e n t h e BC, C ' C " a n d FG loops o n t h e t o p of t h e Ig-likc ]3-barrel, p o t e n t i a l l y making c o n t a c t s w i t h a cluster o f c h a r g e d a n d polar residues w h i c h stabilize flexible loops o n t h e

Methods for the construction of mammalian cell expression vec tors coding for secretion of a soluble version of two-domain hu-sCD2182 receptor in CHO ceils, purification of hu-sCD2182 and production of one-domain hu-sCD2105 from clostripain di gestion of hu-sCD2182 have been described in detail elsewhere [16]. High producing hu-sCD2182CHO cell clones (> 8 mg 1-1) maintained in 1 I.tMmethotrexate were seeded in 8 liter spinner vessels at a density of 105 cells per ml with 8 gm 1-1 CYTODEX3 microcarriers (Sigma) and grown in alpha MEMcontaining 6 mM glutamine, 4gml -I glucose, 10% fetal calf serum, and l gM methotrexate. Cultures were maintained at 37°C with replacement of the medium every three days. One domain hu-sCD2105 was purified by a combination of immunoafilnityand size exclusion chromatography essentially as described previously [16]. A sample of hu-sCD2182 selectivelyenriched with 15N-lysinewas obtained by replacing the unlabeled lysine in the alpha MEM with pure, crystalline 15N-lysinepurchased from Cambridge Iso tope Research Laboratories (Wobum, MA). Two 8 liter suspension cultures were grown in the presence of 15N-lysinesupplemented alpha MEM for three days prior to harvesting,when the medium was replaced in one of the spinner cultures and second labeling was performed. During the production phase for 15N lysine labeled hu-sCD2182,the methotrexate was removed from the medium, the serum was increased to 15% and 0.1gm1-1 pluronic F68 was added. 15N lysine labeled hu-sCD2105was prepared from two-domain hu-sCD2182 by clostripain digestion as described [16], although in order to improve purity the adhesion domain was repurified by immunoaffinitychromatography prior to size exclusion chromatography. Protein samples (1.0-1.5 mM concentration) were prepared for homonuclear 2D NMR experiments by dialysis into 20mM deuterated acetate buffer at pH4.5 in H20 and D20.

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NMR spectroscopy and structure calculations Sequence specific resonance assignments were determined by standard 2D homonuclear NMR m e t h o d s and included a corn bination of 2Q-COSY, 2QF-COSY, TOCSY and NOESY techniques [25,26]. NMR experiments were carried out o n Bruker AMX500 or AMX600 spectrometers at temperatures of 25°C. TOCSY and NOESY experiments were run at two additional temperatures, 13°C and 37°C, to resolve degenerate resonances. Data were processed either o n a Sun Sparc station 2 or a Sill con Graphics 4D/35 Personal Iris using Felix software (BIOSYM Technologies, San Diego). Resonance assignments and a complete description of the experimental procedures will b e described elsewhere [241. Interproton distances used as restraints in the structure calculations were predominantly obtained from NOESY experiments at 25°C in D 2 0 and 90% H20:10%D20 with mixing times ('~m) of 76 ms and 100 ms. Contours were counted for each cross-peak with the most intense cross-peaks consisting of 14 levels. Representative volumes for well resolved cross-peaks at each contour level were determined by 2D vol ume integration using EASY [49] for the 7 6 m s NOESY data. Distances were calibrated by relating the average representative volume(-W6) for every two c o n t o u r levels to distances (A.) o n a linear scale where the most intense cross-peaks observed for sequential N H ( i ) - H a ( i - 1 ) NOEs in 13-strands corresponded to interproton distances of 2 . 4 ~ [34]. Therefore, u p p e r distance b o u n d s of 2.4A, 2.6&, 2 . 9 ~ 3.3A, 3.7A, 4.0A and 4.2& were assigned. Proton distances corresponding to very weak crosspeaks observed at Tm = 76 ms and cross-peaks only observed at ~m 100 ms were included in the calculations with an u p p e r distance restraint of 5.0~. Appropriate pseudo atom corrections were added to the u p p e r limits w h e r e necessary for prochiral ~-methylene and methyl groups [25]. Van der Waals' contact distances were used as lower distance bounds. A total of 740 NOE distance restraints were evaluated and used in the calculations. An additional 320 intra-residue restraints were obtained; however, they were found to b e unnecessary in the calculations due to covalent restraints. Hydrogen b o n d i n g restraints included in the calculation were determined based u p o n the presence of typical NOEs for antiparaltel ~-strands. The lower and u p p e r b o u n d s were 1.80~_ and 2.30A. for H N - O distances and 2.50A and 3.30~ for N--O distances. Dihedral angle restraints for qb were included in the calculations for 79 residues. The 3J(HN,H~) coupling constants were not rigorously obtained and estimates of large coupling constants were determined from 2QF-COSY cross-peaks as well as intense intra-residue H N - H ~ TOCSY and the corresponding weak intra residue HN H a NOESY cross peaks [25,26,50]. These restraints were included in the calculations with a lower b o u n d of - 1 8 0 ° a n d an u p p e r b o u n d of - 2 0 °. Stereospecific assignments for 13 methylene protons for six residues and the dihedral angle, Z1, for 19 residues were de termined using 3J(H~z,H13) coupling constants from 2QF-COSY data and TOCSY spectra at "gm 12, 22 and 32 ms as well as the relative intensitites of HN H~ NOEs at "~m = 76 ms and Hc~-H13 NOEs at ~m = 40 and 7 6 m s [25,26,51,52]. The dihedral restraints, Z1, were included in the calculations in one of the three staggered rotamer conformations ( - 60 °, 180 °, + 60 °) ± 60 °. =

=

Structures were calculated b y distance geometry (DG) methods [33] using DGII in the NMR refine m o d u l e of the INSIGHT II package (BIOSYM Technologies, San Diego). This procedure included b o u n d s smoothing in which triangular inequality distance limits are determined. Random distance matrices that satisfy the triangular inequality limits were determined by r a n d o m ized metrization and coordinates with appropriate distances are obtained and optimized by angular e m b e d d i n g and majorization procedures in 4D. Structures are optimized in 3D by simulated annealing and conjugate gradient minimization procedures as described by Havel [33]. Using the final restraint list, 115 struc-

tures were calculated of which 59 structures had error functions less than 1.0. Eighteen of the best structures were chosen based u p o n the n u m b e r and size of NOE and dihedral angle violations as well as n o n - b o n d e d contact violations. These 18 structures were subjected to 50 iterations of steepest descent and 200 iterations of conjugate gradient minimization to reduce repulsive n o n - b o n d e d contacts. Minimization was carried out using INSIGHT II package with the CFF91 force field (BIOSYM Technologies, San Diego) and included potential energy terms for distance and dihedral angle restraints. Atomic coordinates have b e e n deposited in the Brookhaven protein data bank. Acknowledgements: The authors would like to thank A Krezel, T Ha-

vel and J Habazettl for many useful discussions regarding structure detern~nation by NMR, B Reinhold and V Reinhold for submitting oligosaccharide results prior to publication, M Concino for helpful advice on mammalian cell culture, A Kister and P PaHai for assistance in CD2 structural analysis for mutational studies, P Driscoll and I Campbell for providing coordinates for the NMR structure of rat CD2, D Stuart for providing coordinates for the crystal structure of the extracellular region of rat CD2, S Hyberts for supplying programs to calculate order parameters and J Godoy, K Gordon, M Knoppets, K Sterne, H Stump and C Tully for expert technical assistance. This work was supported in part by a NIH post doctoral fellowship (F32GM15646 01) to JMW, a grant from the Swiss National Science Foundation to DFW, a NIH grant (AI 21226) to ELR and by a grant to GW from PROCEPT, Inc.

References 1. Moingeon, P., el aL, & Reinherz, E.L. (1989). The structural biology of CD2. ImmunoL Rev. 111, 111-144. 2. Bierer, B.E., Sleckman, B.P., Ratnofsky, S.E. & Burakoff, S.J. (1989). The biologic roles of CD2, CD4, and CD8 in T-cell activation. Annu. Rev. Immuno/. 7, 579--599. 3. Shaw, S., Ginther Luce, G.E., Quinones, R., Gress, R.E., Springer, T.A. & Sanders, M.E. (1986). Two antigen-independent adhe sion pathways used by human cy*otoxic T-cell clones. Nature, 323, 262-264. 4. Selvaraj, P., Plunkett, M.L, Dustin, M., Sanders, M.E., Shaw, S. & Springer, T.A. (1987). The T lymphocyte glycoprotein CD2 binds the cell surface ligand LFA-3. Nature, 326, 400403. 5. Bierer, B.E., Peterson, A., Barbosa, J., Seed, B. & Burakoff, S.J. (1988). Expression of the T-cell surface molecule CD2 and an epitope-loss CD2 mutant to define the role of lymphocyte function-a~ssociated antigen 3 (LFA-3) in T-cell activation. Proc. Natl. Acad Sci. USA, 85, 1194-1198. 6. Moingeon, P., Chang, H.C., Wallner, B.P., Stebbins, C., Frey, A.Z. & Reinherz, E.L. (199Q). CD2-mediated adhesion facilitates T lymphocyte antigen recognition function. Nature, 339, 312-314. 7. Koyasu S., et aL, & Reinherz, E.L (1990). Role of interaction of CD2 molecules with lymphocyte function-associated antigen 3 in T-cell recognition of nominal antigen. Proc. Natl. Acad Sci. USA, 87, 2603 2607. 8. Meuer, S.C., et al., & Reinherz, E.L. (1984). An alternative pathway of T-cell activation: a functional role for the 50kd Tll sheep erythrocyte receptor protein. Cell, 36, 897-906. 9. H~inig,T., Tiefenthaler, G., Meyer zum B{ischenfelde, K-H. & Meuer, S.C. (1987). Alternative pathway for activation of T cells by binding of CD2 to its cell-surface ligand. Nature, 326, 29~:~301. 10. Williams, AF. & Barclw, A.N. (1988). The immunoglobulin su perfamily domains for cell surface recognition. Annu. Rev. ImmunoL 6, 381-406. 11. Sewell, W.A., Brown, M.H., Dunne, J., Owen, M.J. & Crampton, M.J. (1986). Molecular cloning of the human T-lymphocyte surface CD2 ( T l l ) antigen. Proc. NatL Acav~ ScL USA, 83, 8718--8722. 12. Sayre, P.H., et a~, & Reinherz, E.L. (1987). Molecular cloning and expression of T l l cDNAs reveal a receptor-like structure on human T lymphocytes. Proc. Natl. AcavL Sci. USA, 84, 2941 2945.

Structure of the a d h e s i o n d o m a i n of h u m a n CD2 Withka et a/. 13.

14. 15.

16. 17.

18. 19.

20.

21. 22. 23. 24.

25. 26. 27. 28.

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Received: 8 July 1993; revised: 4 August 1993. Accepted: 4 August 1993.

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