Cell, Vol. 68, 1145-1162,

March 20, 1992, Copyright

0 1992 by Cell Press

Crystal Structure of a Soluble Form of the Human T Cell Coreceptor CD8 at 2.6 A Resolution Daniel J. Leahy,’ Richard Axel,*t and Wayne A. Hendrickson’+ *Department of Biochemistry and Molecular Biophysics THoward Hughes Medical Institute Columbia University New York, New York 10032

Summary A secreted fragment of the extracellular portion of human CD8a has been expressed in CHO cells, and a deglycosylated and proteolyzed form of this fragment has been crystallized. We report here the crystal structure of this fragment as refined at 2.6 A resolution. The structure was solved by molecular replacement using a superposition of ten variable domains from immunoglobulin light chains as the search model. Only the N-terminal 114 amino acids of CD8a are visible in the electron density maps. The domain formed by these residues possesses a fold typical of immunoglobulin variable domains and associates to form F,-like homodimers. Introduction T lymphocytes recognize peptide antigen bound to major histocompatibility complex (MHC) molecules on the surface of target cells. Recognition of the peptide-MHC molecule complex is mediated by the T cell antigen receptor (TCR) and either CD4 or CD8. The variable regions of the TCR are thought to contact the antigenic peptide and the surrounding polymorphic regions of MHC, whereas CD8 and CD4 contact nonpolymorphic regions of class I and class II MHC molecules, respectively. This coordinate engagement of the TCR and either CD4 or CD8 with an MHC molecule on target cells is required to shape the T cell repertoire during thymic development and permits activation of mature T cells. During thymic development, only those T cells that recognize self-MHC are selected to mature, a process known as positive selection. Positive selection is thought to result from interaction of TCR’ CD4+ CD8’ thymocytes with MHC, such that thymocytes whose TCRs recognize class I MHC turn off CD4 expression, whereas thymocytes recognizing class II MHC extinguish CD8 expression @obey et al., 1991). This results in the selection of ciass l-reactive cells that are committed to a CD8 lineage. The involvement of CD8 in thymic selection has been demonstrated by antibody-blocking studies (Ramsdell and Fowlkes, 1989; Zuniga-Pflucker et al., 1990) and more recent genetic experiments. For example, in homozygous mice with mutations in the CD8 gene, mature class l-reactive cells fail to develop (Fung-Leung et al., 1991). These selection events in the thymus generate mature T cells that express either CD4 or CD8. Mature T cells that recognize antigen associated with class I MHC (largely

killer cells) express CD8, whereas T cells that recognize class II-associated antigens (largely helper cells) express CD4 (Swain, 1983). Antibody-blocking studies, along with gene transfer experiments (Dembic et al., 1987; Gabert et al., 1987) have demonstrated that CD4 and CD8 are essential for efficient T cell activation in the periphery. The function of CD8 is likely to be mediated by association with nonpolymorphic regions of class I MHC molecules on target cells (Norment et al., 1988; Rosenstein et al., 1989). Mutations in the extracellular region of CD8 can interfere with the ability of CD8’ cells to adhere to class I MHC-bearing cells (Sanders et al., 1991). Moreover, mutational analyses have identified a seven residue loop on the a3 domain of class I MHC that is required for CD8 binding (Connolly et al., 1988; Potter et al., 1989; Salter et al., 1990). These experiments suggest that CD8 and TCR recognize different domains of the class I MHC molecule. T cell activation may therefore involve the formation of a ternary complex, in which CD8 and TCR on the T cell surface associate with the same MHC molecule on the target cell. The engagement of CD8 with class I MHC molecules is likely to trigger an intracellular signal through p56rck,a src-related tyrosine kinase physically associated with CD4 and CD8 in lymphoid cells (Veillette et al., 1988). In this manner, the CD8-~56’“” complex may function in much the same way as growth factor receptors in which binding to an extracellular domain activates an intracellular tyrosine kinase. CD8 is found on the surface of T cells both as a disulfidelinked a2 homodimer and a disulfide-linked a8 heterodimer. Both CD8a and 8 contain an N-terminal extracellular domain that shares sequence and structural features with immunoglobulin-variable domains (Littman et al., 1985; Sukhatme et al., 1985; Norment and Littman, 1988). The single immunoglobulin domain of CD8a is followed by a 48 residue linker that precedes the putative transmembrane domain. In CDBa, a highly charged amino acid cytoplasmic tail contains the site of association of p56rCk(Turner et al., 1990). To provide a structural basis for the understanding of CD8 function in T cell recognition and T cell signaling, we generated secreted forms of CD8 for crystallographic analysis. One of these, after glycolytic and proteolytic processing, produced crystals suitable for diffraction analysis. We describe here the 2.6 A crystal structure of this soluble CD8a (sCD8) homodimer. The structure of the N-terminal domain of CD8 provides insight into how the immunoglobulin domain of CD8 may interact with class I MHC molecules. Results Expression and Crystallization of sCD8 The extracellular region of CD8 consists of 162 amino acids: an N-terminal immunoglobulin domain is followed by a 48 residue linker preceding the putative transmembrane segment. Termination codons were introduced into the linker region of the CD8a cDNA in order to generate

Cd 1146

truncated, secreted forms of the CD8 protein. The nonsense mutations were introduced into the cDNA following residues 114, 146, and 162 (CD8-114, -146, and -162, see Figure 1) by in vitro mutagenesis, and the cDNAs were transfected into CHO cells in an amplifiable expression cassette. Figure 2A shows the results of an immunoprecipitation of CD8 from the supernatants of these transfectants. CDB-114, which encodes the N-terminal immunoglobulin-like domain alone, produced no detectable secreted protein in CHO cells. CD8-146, which encompasses the immunoglobulin-like domain and 32 linker residues (containing a single cysteine), directed the synthesis of a secreted disulfide-linked homodimer of CD8. CDB-162, which includes the entire extracellular domain, generated a secreted protein that behaved as a monomer on nonreducing SDS gels. Cys-143 forms an interchain disulfide in wild-type CD8 as well as in CD8-146 (Snow et al., 1985a; Kirszbaum et al., 1989; see also protease treatment section in Experimental Procedures). We speculated that the lack of an interchain disulfide bond in CD8162 could reflect the formation of an intrachain disulfide between Cys-143 and Cys-160. This aberrant intrachain bond may reflect a high degree of flexibility in the regions of CD8a immediately adjacent to the cell membrane. To avoid potential structural flexibility and to ensure a dimeric state, we pursued crystallization trials with the 146 amino acid product. A population of CHO cells expressing CD8-146 (sCD8) was treated with increasing concentrations of methotrexate (MTX) to amplify the CDB-146 sequences. Individual colonies were obtained expressing l-2 mglliter of tissue culture supernatant, but continued rounds of amplification failed to increase the amount of sCD8 beyond this level. Extensive crystallization trials with sCD8 purified from these tissue culture supernatants failed to yield crystals. Flexibility of the 32 amino acids C-terminal of the immunoglobulin-like domain, as well as structural heterogeneity due to glycosylation, were suspected as potential barriers to crystallization. Native CD8 exhibits O-linked glycosylation (Snow et al., 1984). The disparity between the expected protein molecular mass (17,500 kd) of CD8-146 and that observed on SDS-polyacrylamide gels (w29,OOO kd per monomer) suggested that the CD8 expressed by CHO cells was also 0-glycosylated. Native polyacrylamide gels of sCD8 revealed at least four differently charged species (see Figure 2C), suggesting sialic acid heterogeneity, a feature consistent with O-linked glycosylation (Thomas and Winzler, 1969). Treatment of sCD8 with neuraminidase reduced the size of sCD8 monomers, as judged by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), increased the pl of sCD8 above pH 8.8, and reduced the charge heterogeneity to a single major species (see Figure 2). Crystallization trials with neuraminidase-treated sCD8, however, also failed to produce crystals. Subsequent treatment with 0-glycosidase produced material that crystallized readily, but the crystals were long (>l mm), thin (), and F& (Xl)F.l - IF,f)/XIF,I) are shown. Values are for all data unless two values are given, in which case the value in parentheses is for all data, and the value outside the parentheses is for all data having IFI > 20. Root mean square deviations in bond lengths and angles for the final model are 0.011 A and 3.1°, respectively. a Number of unique reflections. b Total number of measured reflections. ’ Number of reflections for which multiple measurements exist. ’ Fraction of theoretically possible data measured. e R,,,g, (XII - lZ).

Rc,, PllFol - IFcIII~IFol).

1983) alone (both with and without loop regions and with and without side chains) as the search model resulted in the correct solution somewhere in the top ten peaks, but not usually in the top three. The Patterson correlation-refined rotation function solution with the “composite” VL domain as the search model was used as the model for a translation function and produced a single clear solution in space group P6422. The translation function value for this solution was 7.1 o (correlation coefficient of ,308) while the next highest peak in this space group was at 4.20 (.212). The highest peak in the enantiomorphic space group P6222 was at 4.30 (.210). The initial electron density map, calculated with (21F,I /Fcj)elQcalccoefficients using the rigid-body refined “composite” model molecular replacement solution, revealed 70 of 113 modeled CD8a residues to be in reasonable electron density. The regions with unclear density principally comprised loop regions and strands forming the dimer interface. The task of rebuilding and refinement fell mostly to placing poorly defined loop residues and determining the register of a 6 strand that contained a six residue insertion relative to most antibodies. In general, while the electron density maps in certain regions could be very well defined and convincing, these maps were only marginally instructive in rebuilding poorly modeled regions. The strategy adopted for rebuilding was to focus on a particular area and rebuild it several ways, each time maintaining good stereochemistry, using only energetically favorable values for all torsion angles, and fitting the available electron density as well as possible. In most cases a solution would present itself that produced much improved electron density for that region. Improvements

in the electron density maps were invariably accompanied by improvements in stereochemistry and torsion angles following restrained refinement. Several attempts were made to extend residues off the current C-terminus, but at no time during the course of the refinement was convincing protein-like electron density observed in this region beyond Ala-l 14. There is sufficient volume to accommodate the additional 27 C-terminal residues in this region of the crystal lattice without disrupting crystal packing. These results, in addition to the relatively low R value achieved with the current model (see Table 2) and the similar crystals obtained when approximately ten residues have been removed by carboxypeptidase Y or when residues 142-l 46 have not been removed by proteolysis, have led us to conclude that residues 115-141 are disordered in this crystal. We will refer to the N-terminal immunoglobulin-like domain of CD8a, for which electron density is visible as CD8aDl. Refinement and stereochemical statistics for the current model of CD8a are shown in Table 2. Representative views of the electron density map are shown in Figure 3. The quality of the electron density maps is in general quite high. Carbonyl density is seen for most residues and provides assurance that the Ramachandran angles are accurate. A plot of the Ramachandran angles is shown in Figure 4. The single nonglycine outlier in this plot is serine 100 at cp = 66O, w = 2O. This residue occurs in a tight turn, however, where such values are not uncommon (Richardson, 1981). Three short loop regions have poor electron density and average 6 factors above 50 AZ: Ser-27 to Ser31, Gln-54 to Lys-56, and Arg40 to Ala-43. In these regions sufficient main chain density is present to provide confi-

CD8 Crystal Structure 1149

Figure 3. Representative

Electron Density

(A) A representative view of the electron density calculated with 21F., - IF,/ coefficientsand displayed with the current CD8a model. The densityiscontoured at 1.20. Visiblesidechains include Trp-35 in the lower right foreground and Phe-107, Phe-93, and Leu-36 from top to bottom in the left foreground. (B) The electron density in the vicinity of cysteine residues 22,33, and 94 associated with the current model. The sulfur atoms are labeled SG for each residue. The electron density is from a 21F,I - /F,I map contoured at 1.20.

dence that the course of the main chain is generally correct, but the exact main chain torsion angles and side chain positions are not well defined. The electron density in these regions deteriorates if the data below 5.0 A spacings are excluded from the map calculations.

Structure Description The overall fold of the N-terminal domain of CD8a is identical to that found in immunoglobulin variable (Ig-V) domains with two antiparallel 5 sheets of five and four strands connetted by a conserved topology of loops. These domains

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60

Figure 4. A Ramachandran

(Q,I+I) Plot for CD8a

Energy contours of 2,4,6, and 8 kcallmol as calculated by the method of Peters and Peters (1981) are plotted. Glycine residues are represented by boxes, proline residues are represented by triangles, and all other residues are represented by crosses.

associate to form immunoglobulin F,-like homodimers with an exact (in this case crystallographic) 2-fold axis of symmetry. Figure 5 shows both a ribbon drawing and a stereoplot of the a carbon backbone of the CD8aDl dimer viewed perpendicular to the molecular dyad axis with the CDR-like loops oriented upward. Owing to the high degree of structural relatedness, the strand nomenclature of Ig-V domains (A, B, C, C’, C”, D, E, F, and G) has been adopted as well as the terms CDRl -like for the B-C loop, CDRBlike for the C-c” loop, and CDR9like for the F-G loop. Figure 6 displays an alignment of the amino acid sequence of CD8a with the sequences of immunoglobulin REI, the N-terminal domain of human CD4, rat CD8a (0X8), and mouse CD8a (Lyt-2). The positions of the 8 strands in human CD8a are labeled in Figure 6, and the residues in CD8a and CD4, whose positions and side chain orientations are conserved following superposition with REI, are stippled. Figure 7 is a diagram of the main chain hydrogen-bonding pattern found in CD8a, as determined by the program DSSP of Kabsch and Sander (1983). The structure of CD8aDl is perhaps best understood by contrast with the well-known structures of immunoglobulin Vk domains (Amzel and Poljak, 1979; Alzari et al., 1988) and the more recently determined structure of the N-terminal domain of CD4 (CD4Dl) (Ryu et al., 1990; Wang et al., 1990). For simplicity, immunoglobulin REI (Epp et al., 1975) has been selected as a prototypical VL domain for comparison with CD8aDl. Plots of a carbon chains of REI and CD4Dl following superposition with CD8aDl are shown in Figure 8 and demonstrate the highly conserved 8 framework structure of these molecules. Least-squares superposition of the main chain atoms from the framework regions of CD8aD1, CD4D1, and REI (the stippled regions

in Figure 6) result in root mean square differences in atom positions of 0.95 A for CD8aDl and REI, 1.03 A for CD8aDl and CD4D1, and 1.07 A for CD4Dl and REI. The A strand of Ig-VL domains typically begins in an antiparallel 8 arrangement with strand B on one sheet, but crosses over to a parallel 8 arrangement with the latter part of strand G on the other sheet. The crossover occurs immediately following a proline that is conserved in the majority of VL region families (Kabat et al., 1987) and this proline follows a cis peptide bond in all known VL crystal structures. This cis proline is conserved in CD8aDl (proline 7) and, as in immunoglobulins, alters the course of strand A away from an antiparallel arrangement with strand B. Strand A does not then, however, proceed to a parallel 8 arrangement with strand G as in immunoglobulins. Instead, strand A veers away from contact with the body of the protein for three residues (Leu-8, Asp-g, and Arg-10). This region of strand A is involved in a crystal lattice contact that encompasses a salt bridge between Asp-9 and His-106 from a symmetry-related molecule. Whether this unusual main chain course also represents the structure of the A strand in solution and other noncrystalline states, or is instead more Ig-VL-like in solution and is disrupted by the lattice contact in the crystal, is not certain. Figure 38 shows a view of the three cysteines in CD8aDl and the electron density in this region. The electron density unambiguously demonstrates a disulfide bond between Cys-22 and Cys-94. This disulfide bond is homologous to the canonical immunoglobulin disulfide bridge between strand B of one sheet and strand F of the other sheet. The thiol group of Cys-33, however, is within van der Waals contact range of this disulfide bond, and a disulfide bond between Cys-22 and Cys-33 can be modeled with relatively minor structural perturbations. As can be seen in Figure 8c the disposition of the CDRBlike and CDRl-like loops relative to the framework strands differs between CD8aDl and REI. As in VL domains, the CD8aDl dimer interface is principally made through interactions between the G and C’ strands of different monomers. The c’ strand is longer in CD8aDl than in VL domains, and two additional G strand residues near the CDR3 loop interact with this longer C’strand, shifting the CDR3-like loop toward the dimer interface. The shift of the CDR3-like loop of CD8aDl toward the dimer interface makes room for a similar shift in the CDRl-like loop. The “hinge”pointatwhichthecourseoftheCD8aDl CDRl-like loop deviates from a more immunoglobulin-like CDRl is at Gly-32. Gly-32 has main chain torsion angles of cp = -91° and QI = -157O and is conserved between rat, mouse, and human CD8a. The importance of the G and C’ strands (and adjacent loops) for dimerization can be seen by comparison of CD4Dl with CD8aDl and REI. Figure 58 shows the dimer interaction in CD8aDl mediated by the CDRS-like and C-C’ loops (and the following G and C’ strands, respectively), while Figures 8a and 8c show how these structural elements are foreshortened by several residues in CD4Dl relative to CD8aDl and REI. CD4Dl does not form dimers. Another instance in which

(CD8 Crystal Structure 1151

Figure 5. Ribbon Diagram and Ca Plot of CD8 Dimer (A) An artist’s ribbon drawing of the CDBaDl homodimer oriented with the molecular dyad axis is situated vertically in the plane of th ie page. The CDR-like loops form the top surface of the molecule as shown, and the CDRl-like, CDRP-like, and CDRS-like loops are labeled 1, 2, and 3, respectively, for one subunit and l’, 2, and 3’ for the other subunit. The C-termini extend from the bottom of the molecule. The loops forming the dimer interface are the CDR9like loops (top) and C-C’ loops (bottom). (8) A stereodiagram of the a carbon backbone of the CDBaDl homodimer with one monomer in solid lines and the other monomer in dlashed lines. The orientation is as in (A). The N- and C-termini are labeled, and every tenth residue is marked with a circle.

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OX8 Lyt-2 CD&x

40 WLFRNSSSELLQPTF:~YVSssR KPQAPELRIFPKKMDAELGQKVDLV VLGS-VSQG WLFQNSSSKLPQPTFWYMASSH SQFRVSPLDRTWI?J&E?%%K YLLSNPTS 8 WLFQPRG-AAASPTFLiLXLSQNK DIQ~~sP$BLSngVgnaI~, SQD-IIKUM&~QTPG---KA@&~Xl%---A KKVVLGKKEDl'W%'Pk ASQK-KSI@&S&PS---N----QX&,GNQGSF -c'-B-A-c-

QLQLSPK~~DAEIGQEVET “LRDAQ

Figure 6. Sequence

Alignments

Alignments of amino acid sequences of the extracellular portions of rat CD8a (0X8), mouse CD&r (Lyt-2) human CD8a (CDBa), and the f%: N-terminal domains of human CD4 (CD4) and immunoglobulin REI (REI). The numbering is 70 60 80 OX8 SKL--NDILDPN-LFSARKE---NNKYILTLSKFSTKN~~YYF~IT~a~VMYFSPL:~~ seauential for human CD8a. Shown below the Lyt-2 NKITWDEKLNSSKLFSAMRDT--NNKYVLTLNKFSKENEGYYl sequences are the extents of each of the 6 CD&x strands in human CDBa. Cysteines conserved . ..m----m.LQ 2;: LTK-GPSKLN--DRADSRRSLWDQG%Z?PLU3?3LKIEbS )QKE---EYQLL in all sequences or in the CD8a sequences are -C”-D-E-F-Genclosed in boxes. The arrowheads mark resi0 * 15; dues that are the final amino acids encoded by OX8 Q&SIIT'%ITRAPTP~~~PTGTPRP~i@ F!‘ PGASGSVEGMGLG; * an exon in human CDBa. The diamond marks QKVNSTTTKPVLRTPSPVH-PTGTSQPQRPE RGSV--KGTGLDF IYIWAPL Lyt-2 wea LPAKPTTT-PAPRPPTPAPTIASQPLSLRPE if!+ PAAGGAVHTRGLDF the last residue of human CD8a seen in the IYIWAPL REI ITR electron density maps. The asterisks mark the CD4 VFG last residues encoded by the expression constructs described in the text. The scissors marks the sate at whrch Staphylococcal V8 protease cleaves human CD8a. The beginning of the putative transmembrane region is marked by a line and the notation TM. The sequences of human CDEa, CD4, and REI were aligned by examination of the crystal structures following least-squares superposition of conserved framework residues. Rodent CD8a sequences were aligned with human CD8a by first aligning conserved residues and then assigning insertions or deletions to loop regions. To identify objectively residues similar in structure, a metric, dCa/cos2(8/2), where dCa is the distance between the a carbons and 6 is the angle made by vectors defining the a to 6 carbon direction in compared structures, was defined. This metric simply assesses the distance between a carbons and the direction of the side chains in superimposed structures. Residues in CD4 and CDBa for which the value of this metric is less than 2 A when compared with REI are stippled in this figure along with their counterparts in REI.

CD4Dl differs from CD8aDl and VL domains occurs in the loop connecting the D and E strands. CD4Dl has a turn of helix in this loop, while CD8aDl and VL domains do not. The dimer interface and the relationship of monomers within a dimer are very similar for CD8aDl and REI. Both the G and C’ strands of immunoglobulins contain highly conserved f3 bulges that are found at the dimer interface and are believed to play an important role in facilitating dimerization (Chothia et al., 1985; Colman, 1988). Both of these bulges are present in CD8aDl. The conserved sequence motif for the C’strand bulge in immunoglobulins

is P-X1-X2-L-X2, where Xl is hydrophilic and X2 is hydrophobic, and this motif is reproduced in CD8aDl (residues 46-50, see Figure 6). The conserved sequence motif found in the G strand 6 bulges of immunoglobulins is F-GX-G. While the Ca positions of this bulge are virtually reproduced between Ig-V domains and CD8aD1, the CD8aDl sequence through this bulge is F-S-H-F (residues 104107). The conserved glycine in the fourth position of the Ig-V bulge motif typically has (cp,~) values energetically unfavorable for nonglycine residues (e.g., rp = 108’ and IJJ = 136O in REI). A flip of the peptide bond between

Figure 7. The Main Chain Hydrogen-Bondmg Pattern of Human CD8uDl Arrows represent the hydrogen bonds. Hydrogen bonds were identified with the program DSSP of Kabsch and Sander (1983)

~l~D3Crystal Structure

Figure 8. Cu Superpositionsof REI

CD8, CD4, and

Stereodiagrams of (1 carbon traces of the CD&D1 monomer superimposed with the N-terminal domam of CD4 (Ryu et al., 1990; Wang et al., 1990) and the V, monomer from immunoglobulin REI (Epp et al., 1975). In each case, CD8aDl is shown in solid lines with the N- and C-termini labeled. Every tenth residue of CD8aDi is marked with a circle, and the molecules are oriented with the CDR loops pointed upward. (a) and (b) show different views of a superposition of the CD8aDl monomer with CD4Dl. (c)and (d) show similar views, as in (a) and(b), respectively, of a superposition of CD&D1 with the V, domain of REI. (a) and (c) show the truncation of the CDR3 (top left) and C-C’ (bottom left) loops rn CD4 relative to CDBaDl and REI. (b) and (d) show the remarkably conserved B sheet framework of CD4D1, CDBaDl, and REI. The extension of the CDRZlike loop (top right) in CDBaDl and CD4DI relative to REI is also apparent by comparison of (b) and (d).

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Table 3. Fractional Sl Q2 F3 R4 v5 S6 P7 L6 D9 RlO Tll w12 N13 L14 G15 El6 T17 V16 El9

.39 .26 .02 .66 .02 .30 .25 .47 .95 .42 .35 .oo .52 .43 .54 .32 .53 .oo .34

Solvent Accessibility L20 .oo K21 .36 c22 .oo Q23 .30 V24 .05 L25 .45 L26 .07 527 .77 N26 .66 P29 .43 T30 .60 531 .20 G32 .06 c33 .oo s34 .oo w35 .oo L36 .OO F37 .lO 036 .OO

in the CD6 dimer P39 R40 G41 A42 A43 A44 s45 P46 T47 F46 L49 L50 Y51 L52 553 Q54 N55 K56 P57

.33 55 .OO .21 .73 .34 .09 .04 .45 .02 .02 .oo .lO .Ol .23 .72 .65 .73 .49

K56 A59 A60 E61 G62 L63 D64 T65 Q66 R67 F66 S69 G70 K71 R72 L73 G74 D75 T76

26 .14 .17 .66 .97 .I4 .31 .52 .79 .06 .06 .39 .14 .51 .23 .54 .63 .37 .13

F77 V76 L79 T60 L61 562 D63 F64 R65 R66 E67 N66 E69 G90 Y91 Y92 F93 c94 s95

.02 .OO .oo .I5 .OO .30 .34 .42 .OO .61 .61 .03 .20 .OO .lO .oo .OO .oo .oo

A96 L97 S96 N99 SlOO 1101 Ml02 Y103 F104 s105 H106 F107 V106 P109 vi10 Flll L112 P113 All4

.OO .06 .06 .63 .24 .04 .Ol .23 .05 .oo .53 .04 .oo .26 .oo .27 .14 .47 .96

The fractional solvent accessibility for each residue in the crystal structure of the human CD6a dimer is given. Fractional solvent accessibility calculated as described in Experimental Procedures.

His-106 and Phe-107 in the CD8aDl bulge, however, generates the energetically favorable (cp,~) values of (-74, 149) for Phe-107. This peptide bond flip results in (cp,~) values of (-72O, 164O) for His-l 06 as compared with (-72O, 11”) for the similar residue in REI. The side chain hydroxyl group of Ser-105 in CD8aDl (Ser-105 is comparable with the glycine in the second position of the Ig-V bulge motif) makes a hydrogen bond to the carbonyl oxygen atom of His-106, presumably stabilizing the “flipped” orientation of the peptide bond between His-106 and Phe-107. Both rat and mouse CD8a are similar to human CDBa, in that the second position of this bulge motif is a serine, and the fourth position is not a glycine. Human CD88 has the immunoglobulin-like F-G-X-G sequence in its putative G strand, but does not have the conserved C’ bulge sequence (Norment and Littman, 1988). If isolated protomers from CD8aDl and REI dimers are superimposed by least-squares superposition, a rotation of 12O is needed to align the other two protomers of the dimers. This value compares with values to 4O-6O for pairwise comparisons of immunoglobulins (Colman, 1988), not including RHE and LOC, whose dimer relationship differs dramatically from that found in all other immunoglobulins (Furey et al., 1983; Chang et al., 1985). A difference from VL domains that is shared by CD8aDl and CD4Dl is the CDRP-like loop, which is in both cases extended by several residues relative to VL domains. This feature is best seen by comparison of Figures 8b and 8d. Despite similar lengths, the CDR2-like loops of CD8aDl and CD4Dl do not follow a similar course. The C-terminal end of the C’ strand in CD8aDl continues its antiparallel 6 interaction with the C strand for approximately two residues beyond the point at which the CD4Dl C’ strand has diverged from its interaction with the C strand. This divergence of the C’ strand from the rest of CD4Dl gives the CD4Dl CDR2-like loop the appearance of having been “peeled” away from the body of the molecule. The CD4Dl CDRP-like loop is not involved in any lattice interactions that could potentially give rise to this “peeling.”

was

Table 3 lists the fractional solvent accessibility for each residue in CD8aDl. This table provides a measure with which to distinguish surface versus buried residues and is included to aid the design and interpretation of experiments with mutants or other CD8aDl sequence variants. Lattice Contacts Excluding dimer interface interactions, three regions of CD8aDl are involved in crystal lattice contacts. The A and G strands form a symmetrical contact with the A and G strands of another molecule. As noted above, Asp-9 makes a salt bridge to His-106 from a different dimer pair and may contribute to the unusual courseof the main chain of the A strand in this region. A sulfate ion appears to stabilize this region of lattice contact and forms a salt link between Arg-10 and Arg4 from a different molecule. In addition, the charged atoms of Lys-21 and Glu-19 from a third molecule also approach within 3 A of this sulfate site. The side chain of Glu-16 also makes a salt bridge to Lys-71 from this third molecule. The presence of a sulfate ion at this lattice contact may explain the requirement for sulfate in the crystallization conditions. The guanidinium group of Arg-85, which makes a salt bridge to Asp-83, is stacked on the guanidinium group of Arg-85 from a symmetry-related molecule. Since the guanidinium group of Arg-85 also stacks (on its other side) with the guanidinium group of Arg-67, this lattice contact involves the parallel stacking of four arginine side chains. Arg-67 is neutralized through a salt link with Asp-64, which is unusual by contrast with other immunoglobulin family members. In immunoglobulins and CD4 asalt link between Arg-67 and an Asp at position 88 is highly conserved. CD8aDl has an Asn at position 88. The side chain positions of Arg-67 and Asn-88 in CD8a are virtually identical to the corresponding Arg-Asp pair in REI. A third lattice interaction involves symmetry-related aspartic acids at position 75. The interaction of these residues occurs around a P-fold symmetry axis of the crystal (different from that coinciding with the molecular dyad) and

(CD9 Crystal Structure 1155

Figure 9. Space-Filling

Models of CDBaDI

and HLA-A2 Shown Adjacent to One Another

HLA-A2 model is based on that from Bjorkman et al. (1987). The loop in the a3 domain of HLA-A2, which has been associated with CD8 binding (residues 223-229) has been colored red, and the CDRl-like and CDRP-like loops of CD&D1 have been colored blue for one monomer and yellow for the other monomer. The more central colored loops of CD8aDi are the CDRl-like loops, while the lateral colored loops are the CDRP-like loops. The HLA-A2 molecule is oriented so that the a3 and p2 microglobulin domains are on the bottom, and the small notch visible on the top of the molecule corresponds to the peptide-binding cleft formed by the al and a2 domains. The models were displayed with the program QUANTA (Polygen Company, Waltham, MA).

may involve the sharing of a proton situated on this axis. A shared proton in this instance would be an example of a type A monobasic acid salt, as described by Speakman (1972). As these crystals were obtained at pH 5.6, this scenario would require an anomalously high pK, for these aspartyl groups in the crystallization conditions. While rare, carboxyl-carboxylate interactions and anomalously high pK,s for acidic groups in proteins are not unprecedented (Sawyer and James, 1982). The main chain density around Asp-75 (located in a tight turn connecting the D and E strands) is unambiguous, but the side chain density is weak for the 6 carbon and diffuse for the carboxyl groups, precluding a precise characterization of this interaction. Discussion We have solved the crystal structure of a fragment of the extracellular portion of human CD8a at 2.6 A resolution. The crystals used in this study were obtained only after

enzymatic removal of O-linked glycosylation. The crystal structure reveals an N-terminal domain with the fold characteristic of immunoglobulin variable domains. This domain forms an F,-like homodimer. As in the N-terminal domain of CD4, the CDR2-like loop of CD8a is extended relative to the CDRP loops of immunoglobulins. Electron density is not seen for amino acids beyond the immunoglobulin-like domain of CD8a (residues 115-141) despite their presence in the crystallized fragment. The interaction of CD8 and the TCR with a class I MHCpeptide complex generates an intracellular signaling event in both developing and mature T cells. The determination of the structure of CD8 permits a more detailed consideration of the CD8 class I MHC interaction. Figure 9 shows space-filling models of the class I MHC molecule HLA-A2 (Bjorkman et al., 1987) and the CD8a homodimer placed adjacent to one another. The loop on the HLA-A2 a3 domain that has been proposed as the principal CD&binding site (Salter et al., 1990) has been colored red, and the peptide-binding cleft in HLA-A2 is visible as a slight notch

Cell 1156

Figure 10. Electrostatic

Potential Surfaces of CD8a and HLA-A2

Molecular surfaces of CD8a (A) and HLA-A2 (6) (Bjorkman et al., 1987) are shown with the electrostatic surface potential represented by color. Regions of positive potential are shown in blue, and regions of negative potential are shown in red. The surfaces were calculated and displayed using the program GRASP (Nicholls et al., 1991). CD8 is shown looking down the molecular dyad axis toward the CD&like loops. HLA-A2 is shown oriented as in Figure 9. and the proposed CDB-binding site can be identified by comparison with this figure.

on the top of the HLA-A2 structure. Assuming that the TCR must contact HLA around the peptide-binding groove, these structures show that it is entirely plausible that CD8 and the TCR simultaneously bind to the same HLA molecule, as suggested by the experiments of Salter et al. (1990). It is also apparent from Figure 9 that, depending on the mode of binding, one CD8 dimer could simultaneously bind to two different HLA molecules. The loop on the a3 domain of class I MHC molecules that has been proposed as the CD&binding site (residues 222-229) contains four highly conserved acidic residues. This high concentration of negatively charged residues indicates that electrostatic interactions are likely to be an important feature of CD8 binding to class I MHC. Figure 10 shows representations of the molecular surfaces of HLA-A2 and the CD8a homodimer with the local electrostatic potential depicted by color. As is seen in this figure and by examination of amino acid sequences, the surface of CD8 bearing the CDR-like loops is predominantly positively charged, while the proposed CDB-binding site on HLA molecules is predominantly negatively charged. Other surfaces of CD8 display roughly balanced mixtures of positive and negative charge. The surface charge distribution of CD8 thus seems most consistent with the surface containing the CDR-like loops interacting with the proposed CD8-binding site on class I MHC molecules.

A study of the ability of CD8 mutants to bind to the class I MHC is also consistent with involvement of the CDR-like loops of CD8 in class I MHC binding (Sanders et al., 1991). Mutations located in the CDRl-like and CDR2-like loops of CD8 interfered most strongly with class I MHC binding in a cell adhesion assay. If the interaction between CD8 and class I MHC is confined to the CDR-like loops on CD8 and the proposed site on the a3 domain of class I MHC, then the simultaneous binding of two HLA molecules by one CD8 dimer would require involvement of the more distally located CDR2-like loops, while involvement of the more midline CDR3-like loops would be consistent with the binding of one CD8 dimer to a single HLA molecule. Despite the presence of more or less discrete surfaces in CD8 (the overall shape of CD8 is more like a box with rounded edges than, for example, a sphere), it is uncertain whether surfaces in addition to, or other than, the surface composed of the CDR-like loops will be involved in interactions with class I MHC molecules. CD4 and CD8 interact with class II and class I MHC molecules, respectively. Class I and class II MHC molecules are thought to adopt similar structures (Brown et al., 1988). Through these interactions, CD4 and CD8 appear to mediate analogous functions in T cell development and activation. CD4 and CD8, however, exhibit significantly different structural features, the most striking of which are

CD8 Crystal Structure 1157

the following: first, the existence of the N-terminal immunoglobulin-like domain of CD4 as a monomer, while the N-terminal immunoglobulin-like domain of CD8 forms an F,-like dimer; and, second, the presence of four immunoglobulin-like domains in the extracellular region of CD4, while the extracellular region of CD8 consists of a single immunoglobulin-like domain connected to a transrnembrane segment by a “stalk” region of ~48 amino acids (see Figure 1). The single structural feature shared by CD4 and CD8 and not present in immunoglobulin variable domains is an insertion of six residues in the CDRBlike loop. The functional significance of these structural features remains unclear, but Fleuryet al. (1991) haveshown that the extended CDR2-like loop in CD4 is unlikely to be iInvolved in interactions with class II MHC molecules, as deletion of this loop fails to impair CD4-class II MHC cell adhesion. The experiments of Fleury and coworkers have also led them to propose that CD4 does not contact class II MHC molecules along the surface encompassing the CDR-like loops, but rather along a surface including one side of the CDRl-like and CDR3-like loops of the N-terminal domain and a part of the second immunoglobulin-like domain. The surface in CD8 analogous to this proposed surface in CD4 would correspond to the left “half” of the CD8 dimer when viewed perpendicular to the dimer axis, as in Figure 5. This surface in CD8 is only marginally obscured by dimer hormation. If CD8 contacts class I MHC principally along tlhis surface, then two MHC-binding sites would be availatble on the CD8 dimer. Structure of the “Stalk” Region If CD8 binds to the membrane-proximal a3 domain of class I MHC as proposed, then the ~48 amino acids spanning tile N-terminal immunoglobulin domain and the transmembrane segment must traverse the length of a TCR, at least 510A. It is unlikely that a globular structure of this dimension could be formed with the number of amino acids available, implying that this region of CD8a adopts an extended structure. The absence of electron density for the 27 C-terminal amino acids in the V8sCD8 crystal structure indicates disorder in this region, but does not distinguish between a rigid structure whose orientation relative to the crystal lattice is variable and a “floppy” or overall indeterminate structure. The C-termini of dimer-related CD8a immunoglobulin domains are 34 A apart, indicating that at least a portion of the residues spanning the immunoglobulin and transmembrane regions are not interacting with their dimer counterparts. O-linked glycosylation is an important feature of the 48 amino acid membrane-proximal “stalk” region. Biochemical studies (Snow et al., 1985a) and our crystallographic results indicate that the N-terminal immunoglobulin-like domain of CD8a contains no glycosylation. The results of a carbohydrate analysis of sCD8 are thus consistent with virtually all of the seven serines and threonines in the “stalk” region being glycosylated (see Table 1). The presence of many prolines around these serines and threonines is also consistent with the majority of these sites being glycosylated (Wilson et al., 1991). We have shown

these sugars to be heavily sialylated (see Figure 2 and Table 1). The presence of many negatively charged sialic acids (as well as the large number of proline residues) may serve to keep this region in an extended form and repel it from the membrane surface while also preventing proteolysis. If, as for other tyrosine kinase-linked receptors, the association state of CD8 is important for transmembrane signaling, then the extensive sialylation may prevent unwanted aggregation and reduce the likelihood of inappropriate signaling. The failure of the N-terminal immunoglobulin domain of CD8 to be secreted as an independent unit (see Figure 2) suggests that glycosylation may be involved in ensuring proper CD8 expression. Many other cell surface receptors contain similar “stalk” regions. The low density lipoprotein receptor (Yamamoto et al., 1984), nerve growth factor receptor (Johnson et al., 1986) tumor necrosis factor receptor II (Smith et al., 1990) and OX-40 (Mallett et al., 1990) all contain membraneproximal regions of similar length that are either known or suspected to contain O-linked glycosylation. The mannose receptor contains a similar region of O-linked glycosylation between domains 3 and 4 of the eight extracellular carbohydrate recognition domains (Taylor et al., 1990). All of these regions of O-linked glycosylation share a high incidence of threonines, serines, and prolines. lntrachain Disulfide lsomerization Immunoglobulin-variable domains contain two absolutely conserved cysteines that form a conserved disulfide bond between the Band F strands. CD8aDl contains these two conserved cysteines, but also contains a third cysteine on the C strand that is present in both rodent and human CD8a (see Figure 6). Kirszbaum et al. (1989) found by biochemical methods that the disulfide bond in the a subunit of murine CD8 a6 heterodimers was made between the cysteines on the B and C strands. The intrachain disulfide bond found in the crystal structure of CD8a, however, connects the B and F strands and is homologous to the intrachain disulfide found in immunoglobulin domains. The apparent ability of CD8a to form an intrachain disulfide between a cysteine from the B strand to a cysteine on either the C or the F strand indicates that two structures with potentially different properties are possible for the CD8a protomer. The disulfide bond isomers need not represent drastically different structures, however, as indicated by the proximity and disposition of the three cysteinyl sulfurs (see Figure 38). In both the a2 homodimer structure presented here and the up heterodimers analyzed biochemically, the disulfide bridges found must have been present in near quantitative amounts, suggesting that these disulfides are not formed randomly. The presence of one disulfide bridge in a2 homodimers and another in a8 heterodimers suggests the simple model that the dimerization partner influences which intrachain disulfide bond is formed. The maintenance of slightly different a chain structures in a2 homodimers and aj3 heterodimers may reflect different functional roles for these molecules and help to prevent otherwise overlapping functions or interactions. It seems unlikely that these disulfide bonds would isomerize once CD8 has reached the cell surface,

Cell 1158

Figure 11. Superposition of lmmunoglobuhn Domains Used As Molecular Replacement Search Model The a carbon backbones of ten Ig-V, domains following least-squares superposition are shown in green. The molecules depicted are listed in Experimental Procedures. The molecules are oriented so that the CDA loops are on the top of this picture. The conserved immunoglobulin disulfide bond is shown in yellow, a conserved tryptophan (corresponding to Trp35 in CD8a) is shown in red, and a conserved arginine(corresponding to position 67 in CD&) is shown in blue.

but rather that the relative population of CD8 disulfide isomers is set by cellular events. It has been shown that a T cell clone can alter the relative populations of homo- and heterodimers in response to external stimuli (Terry et al., 1990). The multiple quaternary forms of CD8 (a2 homodimers, afi heterodimers, and complexes with CD1 a [Snow et al., 1985b]) are also noteworthy by contrast with CD4. This diversity in CD8 hints that CD8 may have functions without counterpart in CD4, or that CD4 and CD8 may use different mechanisms to accomplish similar functions. Molecular Replacement Search Model The molecular replacement search model used to solve this structure consists of a superposition of ten different immunoglobulin light chain variable domains. This “composite” search model was used in the rotation and translation searches without truncation or deletion of any residues, as described in Experimental Procedures. Figure 11 shows the a carbon backbones of the superimposed immunoglobulin domains, along with the conserved disulfide bonds and two representative side chain positions. By giving greater weight to highly conserved features and downweighting variable regions (such as the CDR loops), this search model is most representative of the desired

conserved structure and permits inclusion of more information during molecular replacement. Moreover, the resulting electron density maps are less idiosyncratically biased toward a particular model. Use of this “composite” or “average” VL domain as the molecular replacement search model eased the solution of the CD8 crystal structure. As the number of structures of related proteins increases, this type of search model should prove more useful. Experimental

Procedures

Site-Directed Mutagenesis A cDNA encoding human CD8a (Littman et al., 1985) was subcloned into M13, and site-directed mutagenesis was performed utilizing the method of Kunkel et al. (1987). Three mutants, each containing a stop codon in the region of the gene encoding the extracellular portion of the molecule, were created by changing the codon for Lys-115 from AAG to TAG (CDBK115Z) the codon for Ala-147 from GCG to TAG (CD8A147Z)and thecodonforTyr-163fromTAC toTAG(CD6Y163Z). Each mutant CD8 was sequenced in its entirety by the dideoxy method of Sanger et al. (1977). Expression A version of the dihydrofolate reductase (DHFR)-containing exprossion plasmid psT4DHFR (Deen et al., 1988) was modified so that the SV40 promoterwas replaced with the human cytomegalovirus intermediate/early enhancer/promoter (Eioshart et al., 1985) followed by SV40

CD8 Crystal Structure 11’59

spllice donor and splice acceptor sites, and the CD4 gene was replaced with the polylinker 5’-Xbal-Notl-SaclI-Sfil-EcoRI-PstI-Smal-BamHl-3’. The BamHl and Sfil sites were not unique to the polylinker in the resulting plasmid, which was subsequently named PLAY. Native CD8a and the CD8K115Z, CD8A147Z. and CD8Y163Z mutants were subcloned into PLAY, and 1 pg of each plasmid in 19 Kg of salmon sperm DNA was transfected into the DHFR~ CHO cell line DG44 (Urlaub et al., 1983) by calcium phosphateprecipitation(Wigleret al., 1977) using a kit from Specialty Media (Lavallette, NJ). The DG44 cells had been platedatx 104cells/100mm tissuecultureplate(Nunc)3daysprior to the transfection. Following overnight incubation with the calcium phosphate precipitate, the DG44 cells were washed, incubated for 24 hr in normal medium (Ham’s F-12 with 10% fetal calf serum), and then placed in selection medium (hypoxanthine- and thymidine-free Ham’s F-12 with 10% dialyzed fetal calf serum [GIBCO]). Colonies surviving this selection were subcloned and tested by ELISA for production of sC:DB. The cell lines secreting the highest amounts of sCD8 were subjected to several rounds of amplification by the addition of increasIng amounts of MTX. Initial MTX concentrations found useful ranged from 20 to 50 nM. All cell lines were maintained in selection medium with MTX until the protein purification stage, at which time they were adapted to the serum-free medium Ex-Cell 301 (JRH Scientific) with MTX. Continued rounds of amplification failed to increase the amount of sCD8 beyond l-2 mg per liter of tissue culture supernatant. All colonies surviving the selections were carefully subcloned and screened for sCD8 production, as many lost the ability to produce sCD8 during the selection. ELISA The presence of sCD8 was assayed using a sandwich ELISA kit available from T Cell Sciences (Cambridge, MA). The kit was used as recommended, except that both the initial and second stage antibodies were diluted several fold immediately prior to the assay, and protein A--purified OKT8 antibody was occasionally substituted for the first stage antibody. lmmunoprecipitation Cells grown to confluence in 24-well tissue culture plates were incubated overnight in 1 ml of methionine-freeselection medium containing 75 FCi of [%]methionine. Supernatants from these wells were removed, precleared for 1 hr at room temperature with 30 ~1 of protein A--Sepharose (Pharmacia) saturated with an isotype-matched control anti-murine H-2Kh antibody. Following removal of the anti-H-2Kk-protein A-Sepharose, 0.4 ml of each supernatant was added to two tubes, each containing 20 ~1 of protein A-Sepharose saturated with the monocl13nal anti-CD8 antibody OKT8. The supernatants were left to incubate for 1 hr at room temperature with occasional mixing. The OKT8saturated protein A-Sepharose was then centrifuged out, washed four tirnes with 100 m M NaCI, 10 m M Tris-HCI (pH 7.5), 0.2% NP-40, and resuspended in either reducing or nonreducing SDS sample buffer and boiled. The samples were then centrifuged, loaded directly onto 12.5% SDS-polyacrylamide gels, and electrophoresed according to the method of Laemmli (1970). Plurification One liter of tissue culture supernatant from cell lines maintained in the serum-free medium Ex-Cell 301 was filtered and brought to a pH of approximately8.5 bytheadditionof 6.0gofTrisBase. Thesupernatant was also made 0.02% in NaN3. The supernatant was then run over a 100 ml Mono-Q Sepharose (Pharmacia) anion-exchange column Immediately followed by a 15 ml wheatgerm lectin-Sepharose (Pharmacia) column, as used by Snow et al. (1984). During the development of this procedure, the presence of sCD8 was monitored by ELISA and the conditions adjusted to optimize yield. An sCD8-containing protein plaak was eluted from the wheatgerm lectin column with 5% N-acetylgl#ucosamine in 150 m M NaCI, IO m M NaH2P0,, and 0.02% NaN3 (pH 7.2). The wheatgerm lectin eluent was then concentrated by vacuum dialysis, dialyzed into 20 m M NaCHC02 (pH 5.4). and loaded onto a 1 ml Mono-S Sepharose fast pressure liquid chromatography cation exchange column (Pharmacia) preequilibrated with 20 m M HEPES (pH 8.0) and 0.02% NaN3. sCD8 was the first peak eluted from the Mono-S column with a salt gradient from 20 m M HEPES (pH 8.0). 0.02% NaN3 to 500 m M NaCI, 20 m M HEPES (pH 8.0), 0.02% NaN3.

sCD8 was the only protein visible on silver-stained amide gels of the Mono-S eluent.

SDS-polyacryl-

Gel Filtration Molecular weights were analyzed on a Superose-12 fast pressure liquid chromatography column (Pharmacia) using blue dextran 2,000,000, bovine serum albumin, ovalbumln. and cytochrome c as size standards. Neuraminidase Reaction Purified sCD8 was transferred into 100 m M NaCI, 10 m M NaCH&Oz, 10 m M EDTA (pH 5.2) buffer and concentrated to l-2 mglml by repeated centrifugation in Centricon-lo filters (Amicon). One unit of type Xneuraminidase(Sigma)wasaddedper 1 mgofsCD8,and thesample was left to incubate overnight at 37%. Progress of the reaction was monitored on native polyacrylamide gels, and additional neuraminidase was added until the reaction was complete. 0-Glycosidase Reaction Neuraminidase-treated sCD8 was transferred into 20 m M cacodylate (pH 6.6) buffer and concentrated to -2 mglml by repeated centrifugation in Centriconfilters. Thirty milliunits of endo-a-N-acetylgalactosaminadase (0-glycosidase, Boehringer Mannheim) was added per milligram of CD8 in three 10 mU aliquots over 24 hr, while the sample was incubating at 37%. Progress of the reaction was monitored as a size decrease in sCD8 on 12.5% SDS-polyacrylamide gels. Protease Treatment For treatment with Staphylococcal V8 protease, 0-glycosidase-treated sCD8 was transferred into 50 m M NaCI, 20 m M HEPES (pH 8.0), 0.02% NaN3 by repeated centrifugation in Centriconfilters. For every 2 mg of CD8,O.i mg of Staphylococcal V8 protease (Boehringer Mannheim) was added, and the sample was incubated at 37% for 30 min. The sample was then diluted 2-fold with deionized Hz0 (dHnO), loaded onto a Mono-S fast pressure liquid chromatography column, and eluted as described above. In general, the neuraminidase, O-glycosidase, and V8 protease reactions were carried out sequentially in the same Centriconfilter unit. The sCD8 peak was then concentrated and transferred into dHnO for crystallization trials. The SDSpolyacrylamidegel in Figure2Bshows that V8 treatment resulted in the loss of the interchain disulfide bond but with negligible size difference between V8- and non-VStreated monomers. N-terminal sequencing of the V8-treated sCD8 revealed only the appropriate N-terminus, and the only possible C-terminal proteolytic site for V8 consistent with these results is at Glu-141 (see Figure 6), indicating that the interchain disulfide bond was formed by Cys-143. 0-glycosidase-treated sCD8 was also treated with trypsin. chymotrypsin. elastase, subtilisin A, clostripain, and papain, but noneofthese treatments produced a stable fragment suitable for crystallization trials. Carboxypeptidase Y Reaction V8 protease-treated sCD8 was transferred into 50 m M sodium citrate (pH 5.6) buffer, and 0.1 mg of carboxypeptidase Y (Worthington) was added per 2 mg of sCD8. The sample was then incubated at 37% for 3 hr. The progress of the reaction was evaluated by noting the size of the sCD8 on 8%-25% SDS-polyacrylamide gradient gels. The reaction seemed to stop after removal of approximately ten amino acids, as judged by SDS-PAGE (data not shown). The carboxypeptidase Y-treated sCD8 produced crystals of a similar morphology to V8treated sCD8 under similar conditions, but these crystals were not pursued, as they were generally smaller than the crystals from the VB-treated material. Carboxypeptidase P Carboxypeptidase Y-treated sCD8 was transferred Into 50 m M sodium chloride, 20 m M sodium citrate(pH 5.2) buffer, and 1 U of carboxypeptidase P (Sigma) was added per 20 pg of sCD8. The sample was then incubated overnight at 37%, and the progress of the reaction was monitored by noting the size of the sCD8 on 8%-25% SDS-polyacrylamide gradient gels. Carboxypeptidase P treatment produced several fragments of sCD8 of the approximate size that would be expected for the N-terminal immunoglobulin-like domain alone (as judged by SDS-

Cell 1160

PAGE; data not shown), but with poor yield and loss of much protein as a precipitate of smaller molecular weights. The carboxypeptidase P-treated sCD8 produced crystals of a different morphology than V8treated sCD8 and in different conditions, but these crystals did not diffract to high angles. Carbohydrate Analysis Carbohydrate analyses were performed using the Dionex system of high pH anion-exchange chromatography, followed by pulsed amperometric detection (Dionex, Sunnyvale, CA). Analysis of neutral and amino sugars was done according to Hardy et al. (1988) using some of the modifications employed by Blithe et al. (1989). Sialic acid content was analyzed as described by Blithe et al. (1989). Crystallization Crystals were obtained by the “hanging drop” vapor diffusion method (MacPherson, 1982). Typically, 1 ul of sCD8 at -5-10 mglml in dH,O was mixed 1 :I with a 1 :l dilution of crystallization reservoir buffer with dH,O. The best crystals of V8 proteaselo-glycosidaselneuraminidase-treated sCD8 were obtained with a reservoir buffer of 250/n-30% (w/v) PEG 3350, 3.75% saturated ammonium sulfate, and 10 m M sodium citrate (pH 5.6). The drops were incubated at 20°C, and crystals wereusuallyobserved in I-2days. Thesecrystalsgrew to their full size in l-2 weeks and were initially characterized using a Huber precession camera mounted on a Rigaku RU-200 rotating anode (Molecular Structure Corporation, Woodland, TX). Polyacrylamide Gel Electrophoresis Both native and SDS denaturing polyacrylamide gels were run utilizing a Phast Gel system (Pharmacia). All samples were run according to the manufacturer’s instructions, except that the polarity of the electrodes was reversed for native gels of sCD8 after neuraminidase treatment. This change was necessary, owing to the increase of the pl of sCD8 following neuraminidase treatment above the pH of 8.8 used in the polyacrylamide gel buffer. Diffraction Measurements Data were collected at the CHESS using an oscrllation camera mounted on the Fl beam line. Diffraction images were recorded on Fuji HR-III imaging plates and digitized with the imaging plate reader available at CHESS (Bilderback et al., 1988). Data were collected from four crystals that were cooled to 4°C. A 0.1 m m collimator was used with 0.979 A radiation. A typical exposure time was 96 s for a 2.0° oscillation. Three crystals were mounted with the c’ axis (coincident with the long axis of the crystal) parallel to the capillary axis. These crystals were aligned to collect the beginning, middle, and end of the 30° necessary for a complete set of unique data in the P8222 (P6422) space group. Prior to the data collection, the c” axisof each crystal was offset -lo0 relative to the oscillation axis to achieve a more efficient coverage of measured reflections. The fourth crystal was aligned with C* perpendicular to the oscillation axis to measure the”missrng cone” of data. The crystal to imaging plate distance was 240 mm. Data Processing and Reduction Raw data images were indexed, integrated, and corrected for Lorentz and polarization effects with the program DENZO, written and kindly provided by 2. Otwinoski. Profile fit intensities for 20,488 fully recorded reflections were scaled and merged into 4,675 unique reflections with the ROTAVATA and AGROVATA programs from the CCP4 program package (The SERC [UK] Collaborative Computing Project No. 4, A Suite of Programs for Protein Crystallography, Daresbury Laboratory, Warrington, England, 1979). The merging R factor for all data 302.6 A was 0.105 (see Table 2). Molecular Replacement A composite molecular replacement search model was constructed from all of the immunoglobulin light chain variable (V,) domains with coordinates available in the Brookhaven Protein Data Bank. The coordinates for REI (Epp et al., 1975) RHE (Furey et al., 1983) AN02 (Brunger et al., 1991) 4-4-20 (Herron et al., 1989) HyHel-5 (Sheriff et al., 1987) J539 (Suh et al., 1986) KOL (Marquart et al., 1980) NEW (Poljak et al., 1974) MCG (Schiffer et al., 1973) and McPc603 (Satow et al., 1986) were aligned by least-squares superposition of the main

chain atoms of homologous framework residues. The aligned coordinate sets were then concatenated and used together without truncation or alteration to produce Patterson maps for the rotation and translation searches. Individual B factors were used as reported in the data bank, with the exception of REI and NEW, for which B factors were unavailable. An overall B factor of 14.0 A* was used for these two structures. The rotation function, Patterson correlation refinement, and translation function were all carried out using the program X-PLOR (Brunger, 1990b). For the rotation function, the aligned coordinate sets were centered in a Pi box of dimensions 60 by 60 by 70 A. The Patterson vector length cut off was 30 A, and data in the resolution range 15.0 to 4.0 A were used with a 20 cut off on IF,/. The asymmetric unit for the rotation function was found in Rao et al. (1980). The top peak in the rotation function also produced the top peak following Patterson correlation refinement (Brunger. 1990a), and this solution was used in a translation search in the two possible spacegroups P6222and P6422. The translation search was carried out with data (F > 20) between 10.0 and 4.0 A on a 1.33 A grid. Refinement The R value, Zi jF,( - /F,j l/ZjOF,j, for the translation function solution was ,502 for all data 8.0-3.0 A. This value compared with R values of ,550 to ,555 for the next highest translation function peaks in P6,22 and the highest translation function peaks in P6222. Rigid body refinement in X-PLOR of the translation function solution reduced the R value from ,502 to ,499 (all data 8.0-3.0 A) with a rotation of 0.11 o and a translation of 0.36 A. A 2jF,j - jF,j electron densrty map (coefficients of (21F,j - 1F,j)e’*“““) was synthesized with calculated values obtained from the rigid body-refined search model. This map was displayed with coordinates of the REI VL monomer extracted from the search model, and the interactive computer graphics program FRODO (Jones, 1985) was used to make insertions, deletions, and side chain substitutions in the REI model to construct a model for residues l-l 13 of CDBa. Cysteine residues 22, 33, and 94 were initially modeled as alanine, owing to uncertainty as to which residues participated in the intrachain disulfide bond. Side chains were rotated to eliminate contacts and provide the best fit to the electron density. The main chain region of two non-CDR-like loops was also rebuilt to provide a better fit to the density. The overall B factor for this model refined to 12.5 i\2, and this value was used in subsequent refinements. The CD8 model was then subjected to 200 cycles of conjugate gradient minimization in X-PLOR with a low weighting on X-ray terms to maintain good stereochemistry. This~minimization reduced the R value to ,366 (all data IF,/ > 20, 8.0-2.8 A), with root mean square deviations from ideality of 0.020 A for bond lengths and 4.5O for angles. Throughout refinement, maps calculated with (3jF,j - 2jF,j)e’@ ‘, (2jF,I - jF&e’*ca’c, and (IF,1 I F,j)e’vcdc coefficients were all examined. After several cycles of manual rebuilding with FRODO followed by minimization, an X-PLORsimulated annealing refinement was run (Brunger et al., 1990). After 60 cycles of conjugate gradient minimization, the model was “slow cooled” from 4000 K to 300 K in 25O decrements with 25 fs (50 steps at 0.5 fslstep) of dynamics at each temperature stage. The simulated annealing was followed by 120 cycles of minimization. The R value at this point was ,306 with root mean square deviation of 0.016 A in bond lengths and 4.2O in angles. Following several more rounds of model building and conjugate gradient refinement, the electron density began to show an unambiguous bridge between cysteines 22 and 94. This feature was modeled as a disulfide, and simulated annealing refinement was run again as before. This refinement reduced the R value to .272 with root mean square deviations of 0.014 A in bond lengths and 3.E” in angles. Continued cycles of model building and minimization coupled with refinement of individual B factors (targets of 1 50 and 2.00 for bond- and angle-related main chain atoms, respectively, and 2.00 and 2.50 for bond- and angle-related side chain atoms, respectively) and the addition of 23 water and 1 sulfate molecules have produced the current model, whose agreement statistics and stereochemical parameters are shown in Table 2. Calculation of Fractional Surface Accessibility Surface accessibility was calculated in X-PLOR, which uses the method of Lee and Richards (1971). The probe size was set to 1.4A. The X-PLOR script file was written by C. M. Ogata. To calculate frac-

t;F,

Crystal Structure

tional surface accessibility, the value for the accessibility for each residue was normalized by the value obtained for the same accessibility calculation performed on that residue as a tripeptide with adjacent residues truncated to glycines. Acknowledgments We thank Dan Denney for the gift of a plasmid containing the cytomegaltovirus intermediate/early promoter/enhancer and SV40 splice site sequences, Adelaide Caruthers for the DG44 cell line, Tom Livelli for advice and assistance with transfections, Lillian Eoyang and Leigh Zawel for technical assistance, Mary Ann Gawinowicz for N-terminal sequence analysis, Andrew Pound for amino acid analyses, Susan Pollak for carbohydrate analyses, Rachel Yarmolinsky for drawing the ribbon diagram of CD8 found in Figure 5, Kurt Drickamer and Ellen Robey for helpful discussions, and Tom Peat, Bill Weis. and Wei Yang for assistance with X-ray data collection. We gratefully acknowledge use of the CHESS facilihes. This work was supported in part by National Institutes of Health grant 5-31294-3600 to R. A. and by National Science Foundation grant DMB-89-17570 to W. A. H.; D. J. L. is a fellow of the Helen Hay Whitney Foundation. Atomic coordinates and structure factor amplitudes have been deposited with the Protein Data Bank, Chemistry Department, Brookhaven National Laboratory, Upton, NY 11973, from which copies are available. The costs of publication of this article were defrayed in part by thle payment of page charges. Thus article must therefore be hereby marked “advertisemenl’ in accordance with 18 USC Section 1734 solely to indicate this fact.

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impli-

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