Crystal structure of a soluble CD28-Fab complex

Crystal structure of a soluble CD28-Fab complex

Edward J Evans1, Robert M Esnouf2, Raquel Manso-Sancho1, Robert J C Gilbert2, John R James1, Chao Yu1,Janet A Fennelly1, Cheryl Vowles1, Thomas Hanke3, Bjorn Walse4, Thomas Hunig5, Poul Sørensen4,6,David I Stuart2 & Simon J Davis1

Naive T cell activation requires signaling by the T cell receptor and by nonclonotypic cell surface receptors. The most

important costimulatory protein is the monovalent homodimer CD28, which interacts with CD80 and CD86 expressed on

antigen-presenting cells. Here we present the crystal structure of a soluble form of CD28 in complex with the Fab fragment

of a mitogenic antibody. Structural comparisons redefine the evolutionary relationships of CD28-related proteins, antigen

receptors and adhesion molecules and account for the distinct ligand-binding and stoichiometric properties of CD28 and the

related, inhibitory homodimer CTLA-4. Cryo-electron microscopy–based comparisons of complexes of CD28 with mitogenic

and nonmitogenic antibodies place new constraints on models of antibody-induced receptor triggering. This work completes

the initial structural characterization of the CD28–CTLA-4–CD80–CD86 signaling system.

Nonclonotypic costimulatory proteins expressed on the surfaces ofleukocytes profoundly influence the course of immune responses.CD28, the first of these molecules to be discovered, was initiallythought to satisfy the predictions of a ‘two-signal’ model of lympho-cyte activation1 by providing a qualitatively distinct second signal, butit is now clear that CD28 amplifies the transcriptional effects of T cellreceptor (TCR) triggering rather than initiating a distinct geneexpression program2,3. The molecular basis of this effect is yet to bedetermined. One proposal4 is that, after ligand binding and receptortriggering, CD28 is recruited to and alters the stoichiometry of amembrane-proximal signaling complex formed around a scaffoldconsisting of the adaptors LAT and SLP76 that is assembled inresponse to TCR ligation. Costimulatory CD28 signaling sustains Tcell activation by consolidating immunological synapse formation5,6

and by enhancing both cell cycle progression through upregulated D-cyclin expression7,8 and T cell survival by inducing the expression ofthe antiapoptotic protein Bcl-XL (refs. 9,10). CD28 is constitutivelyexpressed on almost all mouse T cells and human CD4+ T cells and on50% of human CD8+ T cells. CD28 deficiency impairs T cellproliferation11–13, immunoglobulin class switching11, germinal centerformation14 and T helper type 2 responses15. The CD28 orthologsICOS16 and CTLA-4 (ref. 17) provide additional costimulatory andinhibitory signals, respectively.

CD28, ICOS and CTLA-4 are covalent homodimers consisting ofpaired V-set (variable domain–like) immunoglobulin superfamily(IgSF) domains attached to single transmembrane domains andcytoplasmic domains bearing tyrosine-dependent signaling motifs18.

These motifs are phosphorylated by Src tyrosine kinase(s), but exactlyhow this is triggered by ligand binding is unknown. CD28 and CTLA-4 bind the shared, related ligands CD80 and CD86, which each consistof tandem V-set and C1-set IgSF domains; ICOS binds a distinct,CD80-related ligand known as ICOSL or LICOS19,20. CD28 has littleadhesive capacity21 even though the strengths of its ligand interactionsare comparable to those of conventional adhesion molecules22, sug-gesting that costimulatory interactions may require synapse forma-tion. Whereas the interactions of CD80 and CTLA-4 are each bivalentand therefore are likely to be avidity driven23–25, interactions involvingCD28 and CD86 are monovalent26. In the case of CD28 and CTLA-4homodimers, distinct quaternary structural arrangements are theproposed source of these stoichiometric differences. Along withaffinity differences measured in surface plasmon resonance (SPR)–based assays, the stoichiometric differences result in the formation ofsignaling complexes whose stabilities are likely to vary by more thanfour orders of magnitude26. The affinity of CD86 for CTLA-4 is about8% of that of CD80, whereas its affinity for CD28 is 20% of that ofCD80 (ref. 10). Relative to their competing CTLA-4 binding affinities,therefore, CD86 binds CD28 two- to threefold more effectively thanCD80. For this reason it has been argued that CD86 is likely to be themore effective costimulatory ligand in vivo and that CD80 is probablyinhibitory26. Antibodies are potent ligands of CD28, and a correlationexists between epitope location and the extent of autonomousmitogenic signaling induced by the antibodies27. Whereas nonmito-genic antibodies bind the ligand-binding region of the V-set domainof CD28, all of the mitogenic (or ‘superagonistic’27) antibodies to

Published online 6 February 2005; doi:10.1038/ni1170

1Nuffield Department of Clinical Medicine, The University of Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK. 2Division of Structural Biology,The University of Oxford, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford, OX3 7BN, UK. 3TeGenero AG, D-97076 Wurzburg, Germany.4Active Biotech Research AB, PO Box 724, Lund, SE-220 07, Sweden. 5Institut fur Virologie und Immunbiologie, University of Wurzburg, D-97078 Wurzburg, Germany.6Present address: LEO Pharma A/S, Industriparken 55, DK-2750 Ballerup, Denmark. Correspondence should be addressed to D.I.S. ([email protected])or S.J.D. ([email protected]).

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human CD28 and to rat CD28 identified thus far bind epitopessensitive to mutations creating alterations in the C¢¢D loop.

Structures of the ligand-binding domains of human CD80, CD86and CTLA-4 have yielded important insights into the nature of thesignaling complexes formed by these molecules23–25,28. We presenthere the crystal structure of a monomeric form of the extracellularregion of the fourth and final element of this signaling system, CD28,in a complex with the Fab fragment of a mitogenic antibody. We usecryo-electron microscopy (cryo-EM) to confirm that homodimericcontacts observed in the crystal lattice are comparable to thosemediating native CD28 homodimerization and to compare thestructures of CD28 in complexes with mitogenic and nonmitogenicwhole antibodies. Our data account for the distinct binding specifi-cities and stoichiometric properties of CD28 and CTLA-4, redefine theevolutionary relationships of CD28-related proteins, antigen receptorsand adhesion molecules, and place new constraints on models ofantibody-induced receptor triggering.

RESULTS

Structure determination

CD28 was expressed as a cleavable IgG1 Fc chimera (CD28Fc) thatbound antibodies and soluble CD80 (sCD80; ref. 26). Cleaved, fullyglycosylated or deglycosylated CD28 homodimers failed to crystallize.Complexes formed with the Fab fragments of two mitogenic anti-bodies to CD28 (9D7 and 5.11A1)27 and a third, nonmitogenic

antibody (7.3B6)27 also failed to crystallize. However, gentle reductionand alkylation generated a stable CD28 monomer that bound sCD80with wild-type affinity (Supplementary Fig. 1 online; ref. 26) andproduced usable crystals with the mitogenic 5.11A1 Fab. Data col-lected at the European Synchrotron Radiation Facility microfocusbeamline (ID13), although weak, extended to a resolution of 2.7 A.The data were phased by molecular replacement; representative finalelectron density is in Supplementary Figure 2 online, and datacollection and refinement statistics are in Table 1.

Structure of the monomer

The asymmetric unit of the crystal consists of monomeric CD28bound to the 5.11A1 Fab fragment (Fig. 1a). The extracellular domainof CD28 consists of a single antiparallel b-barrel of 20 � 25 � 40 A3

Table 1 Data collection and refinement statistics

Data collection statistics

Beamline ESRF ID13

Wavelength (A) 1.008

Resolution limits (A) 25–2.7

Space group C2

Unit cell dimensions (A) 191.2 � 47.4 � 71.8

b-angle (1) 94.4

Number of observations 103,158

Unique reflections 17,413

Completeness (%)a 96.8 (97.4)

I /s(I)a 9.5 (1.3)

Rmerge (%)b 15.0

Structure refinement statistics

Resolution limits (A) 25–2.7

Number of reflections in working set 16,560

Number of reflections in (5%) test set 842

R factor (%)c 23.9

Rfree (%)c,d 28.2

Number of atoms:

Protein; sugar; water 4,250; 42; 203

Geometry r.m.s. deviations:

Bond lengths (A); bond angles (1) 0.009; 1.5

Average B factors (A2):

Main chain; side chain; sugars; water 53.6; 54.7; 73.2; 62.1

CD28; Fab H chain; Fab L chain 53.5; 56.1; 52.5

B-factor r.m.s. deviations (A2):

Main chain bonds; side chain bonds 1.8; 2.9

Main chain angles; side chain angles 2.9; 4.1

aNumbers in parentheses refer to the shell of highest resolution (2.80�2.70 A).bRmerge ¼ S|Ij � oI4| / SIj, where Ij is the intensity of an individual observation of a reflectionand oI4 is the average intensity of that reflection. cR factor ¼ S ||Fo| � |Fc|| / S|Fo|, where|Fo| is the observed structure factor amplitude and |Fc| is the calculated structure factoramplitude. dRfree is the R factor calculated against the test data excluded from refinement.

CD28

*CD80

TCRδ

CD28

CTLA-4

PD-1

a

c

d

b

Figure 1 Structure of the sCD28 monomer. (a) Two orthogonal views of the

asymmetric unit of the crystal, with sCD28 in red and the 5.11A1 Fab in

green. (b) Secondary structure of sCD28. (c) a-carbon representations of

sCD28 (PDB accession number 1yjd; red), CD80 domain 1 (PDB accession

number 1dr9; light blue) and TCRd V domain (PDB accession number

1tvd; gray) in two orthogonal views, after superposition with SHP51.

*, The conserved G-strand b-bulge. (d) SHP-superimposed a-carbon

representations of the V-set domains of sCD28 (red), human CTLA-4

(PDB accession number 1i8l; blue) and mouse PD-1 (PDB accessionnumber 1npu; purple). The diagrams in c,d, left, are shown in the same

orientation as in b.

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(Fig. 1b). Electron density was continuous for CD28 from Asn1 toLys118, although the BC loop from Leu28 to Ser30 was particularlypoorly ordered. We found no density that could be interpreted for the16 residues linking the extracellular domain to the first residue of thetransmembrane domain (Phe135). A synthetic, disulfide-bondedpeptide corresponding to this sequence was also disordered in solutionaccording to 1H–nuclear magnetic resonance spectroscopy (C.Y., S.J.D.and A.K. Downing, data not shown).

The extracellular domain of CD28 has the same topology as thevariable (V) domains of antigen receptors; that is, the b-sheetsare formed by the DEBA and A¢GFCC¢C¢¢ b-strands (Fig. 1b). Auto-mated structure comparisons using the DALI29 server (EuropeanBioinformatics Institute) indicated that, excluding CTLA-4 (discussedbelow), nine of the ten most CD28–like protein domains present in theProtein Data Bank are V domains, followed by the V-set IgSF domainsof adhesion molecules. We did superpositioning of CD28 with a TCRdV domain and an adhesion molecule–type V-set domain from CD80(ref. 23; Fig. 1c; r. m. s. differences between CD28 and the TCRd andCD80 V-set domains are both 1.6 A but for 84 and 75 equivalentresidues, respectively). CD28 showed all the hallmarks of antigenreceptor V domains: the substantial b-strand A and B interactionstypical of antigen receptors rather than the limited A¢-G contacts ofadhesion molecule V-set domains; C¢- and G-strand b-bulges char-acteristic of antigen receptor domains, centered in this case on Glu46and Asn111, rather than the b-bulges located immediately after thesestrands found in adhesion molecule V-set domains; and the substan-tially greater overall twist of the A¢GFCC¢C¢¢ sheet that distinguishesantigen receptor V domains from adhesion receptor V-set domains(Fig. 1c). For CD28, however, the disruptive effect of the G-strandb-bulge is exaggerated by a side chain–main chain hydrogen bondinvolving Asn107 of b-strand G and Lys95 of b-strand F. The extendedC¢C¢¢ loop also differentiates CD28 (and CTLA-4) from antigen recep-tor V domains. N-glycosylation sites are located on the DEBA sheet(Asn19 and Asn74), on the b-bulge in strand G (Asn111) and at the‘top’ (Asn53) and ‘bottom’ (Asn87) of the domain. Distinct electrondensity was present at three sites (Asn19, Asn53 and Asn87), withsome density at a fourth site (Asn111). At full occupancy, the largestcontiguous, unglycosylated surface consists of the entire A¢GFCC¢C¢¢face, whereas the DEBA face is likely to be completely obscured.

Predictably, CD28 most resembles CTLA-4 (r.m.s. difference of1.1 A for 96 equivalent residues; Figs. 1d and 2), but there areimportant differences. The b-strand G-disrupting side chain–mainchain interaction involving Asn107 is absent from CTLA-4. Moreover,CTLA-4 lacks the single-residue insertion (Glu108) that furtherdisrupts b-strand G in CD28. Also, the CC¢ loop is two residueslonger in CTLA-4, extending it beyond the plane formed by the smallb-sheet consisting of b-strands A¢, G¢ and F, preventing CD28-likehomodimerization (discussed below). Finally, whereas the C¢¢ strandforms the first strand in the second b-sheet of CTLA-4, it extends theA¢GFCC¢ face of CD28, as in most V-set domains. Although distantlyrelated in sequence, programmed death 1 (PD-1) is structurally verysimilar to CD28 (r.m.s. deviation of 1.4 A for 97 equivalent residues)and CTLA-4 (Fig. 1d; ref. 30). PD-1 shares b-strand C¢¢ topology withCD28 and its b-strand G configuration with CTLA-4. Together, CD28,CTLA-4 and PD-1 form a previously unknown subset of the IgSFconsisting of nonclonotypic receptors with considerable structuralsimilarity to antigen receptors.

Ligand-binding face

Mutations creating alterations of the MYPPPY sequence (amino acids99–104) conserved in CD28 and CTLA-4 result in substantial losses

(490%) in ligand binding, identifying this as the core of the ligand-binding site in each case28. The structure of the FG loop MYPPPY(amino acids 99–104) motif of CD28, including the cis-trans-cis main-chain conformation of the three proline residues that directs the sidechains of Tyr100, Pro102, Pro103 and Tyr104 of CTLA-4 toward CD80and CD86 (ref. 25), is almost identical to the equivalent region ofCTLA-4 (r.m.s. difference for the six a-carbons of less than 0.51 A;Fig. 3a). In CD28, the loop is stabilized by a water molecule that formshydrogen bonds with the carbonyl oxygens of the cis prolines (Pro101and Pro103; Supplementary Fig. 2 online).

Loop-grafting experiments31 and chemical footprinting analysis32

indicate that CD80 forms contacts outside the FG loop of CD28.Structures of CTLA-4–ligand complexes24,25 have shown that bothCD80 and CD86 bind the conserved residues Glu33, Arg35 and Glu97.The contiguous surface buried by both ligands in CTLA-4 has almostthe same contours (Fig. 3b) and electrostatic potential (Fig. 3c, left) asthe equivalent region of CD28. These surfaces also share a high degreeof electrostatic complementarity with the CTLA-4-binding surfaces ofCD80 and CD86 (Fig. 3c, right). Beyond this region, however, theputative ligand-binding faces of CD28 and CTLA-4 are different. Thefour independent views of CTLA-4–ligand binding offered bythe complex structures show considerable binding-mode plasticity.At the extremes, the position of CD80 differs from that of CD86 by arotation of 25–301 about the long axis of CD80 centered onb-strand F, generating ligand-specific contacts with peripheral, non-conserved residues in CTLA-4: CD80 contacts Leu106 of b-strand G,whereas CD86 contacts Thr53 in b-strand C¢. These residues aresubstituted nonconservatively in CD28, with aspartic acid and ty-rosine, respectively (Fig. 2). Along with Tyr105, which contacts bothligands and is substituted by Leu105 in CD28, these seem to be thelikely sources of differential ligand specificity and binding strength forCD28 and CTLA-4. Adjacent to the ligand-binding region, the break

A A′ B

B

C′′C′C

C C′ C′′

FED

D E

G

G

G′

F

CD28CTLA-4

A′A

Figure 2 Structure-based alignment of the CD28 and CTLA-4 extracellular

domain sequences. Structures were aligned with SHP as shown in

Figure 1d. The following features are highlighted: red, structurally equivalent

residues conserved in all mammalian CD28 and CTLA-4 sequences; green

and yellow, conserved CD28-specific (green) and CTLA-4-specific (yellow)

sequences; gray, b-strands; pale blue and orange, residues forminghomodimeric contacts (pale blue) and 5.11A1 Fab-contacts (orange);

maroon and dark blue, CTLA-4 residues that contact CD80 (maroon) and

CD86 (dark blue).


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in b-strand G creates a CD28-specific ‘pocket’ surrounded by His38,Phe93, Lys95, Asp106 and Lys109, and with Val5, Asn107 and Ser110at its base (Fig. 3d).

Molecular interfaces

The 5.11A1 Fab combining site contacts residues in the C, C¢, C¢¢ and Fstrands, and C¢¢D and FG loops of CD28, consistent with publishedwork showing that 5.11A1 binding is disrupted by C¢¢ strand and C¢¢Dloop alterations27. The interface is unremarkable as an example ofantibody-antigen recognition according to comparisons with surveyedantibody-antigen complexes33: it is comparable in size (1,249 A2 ofsurface is buried versus 1,175–1,755 A2 for other complexes) and hassimilar numbers of interacting residues (29 versus 27–39) and equiva-lent composition (24% of the CD28-contacting Fab residues arecharged, 21% are polar, 55% are hydrophobic and 40% are aromatic).Similarly, the Fab has neither unusual elbow angles nor unconventionalcomplementarity-determining region loop lengths or conformations.

The most important lattice contact, located at the twofold axisbetween adjacent CD28 monomers, generates a plausible candidate forthe native CD28 homodimer. The contact buries a larger nonpolarinterface area consisting of residues with a higher propensity forforming dimer interfaces and containing a larger fraction of comple-tely buried atoms than the equivalent interface in CTLA-4 (ref. 34;

Supplementary Table 1 online). The observed ‘dimer’ has the overallshape and dimensions of the CTLA-4 homodimer (Fig. 4a), becauseresidues in b-strands A and G at the ‘edge’ of the domain mediatedimerization at least in part in each case. In other respects, thedimerization modes are fundamentally different. Whereas in CTLA-4 it involves residues adjacent to and including the stalk-like region, inthe CD28 crystals the contact is formed by the small, three-strandedA¢G¢F b-sheet present in all IgSF V-set domains. Superposition viaone of the monomers (Fig. 4b) indicated that the second monomerin CD28 is related to that in CTLA-4 by a 551 ‘rolling’ movementalong the domain ‘edge’ at b-strands A and G, followed by a 12-Atranslation parallel to the axis of rotation that aligns the twob-sheets of each monomer. In CTLA-4, only the DEBA sheets arealigned. At the core of the CD28 interface, His116 forms a main-chainhydrogen bond with Val86, and Ile91 and Ile114 form a centralhydrophobic core extended by Pro9, Met10 and Leu41 (Fig. 4c).Eight of the ten contacting residues at the interface are conserved in allmammalian CD28 sequences; five are conserved in CD28 but notCTLA-4 (Fig. 2). CD28-like homodimerization is prevented forCTLA-4 by the substitution of His116 by Tyr115, by the disruptionof the hydrophobic core by substitution of Ile114 by Gln113, andby the intrusion of the longer CC¢ loop into the plane formedby the small A¢G¢F b-sheet. Conversely, CTLA-4-like dimerization of

Figure 3 Properties of the ligand-binding faces

of CD28 and CTLA-4. (a) Superpositions, using

SHP, of the MYPPPY(99–104) sequences of

human CTLA-4 from the complexes with CD80

(dark blue; a-carbon r.m.s., 0.35 A and 0.37 A;

PDB accession number 1i8l) and CD86 (pale

blue; r.m.s., 0.43 A and 0.46 A; PDB accession

number 1i85), and from mouse CTLA-4not in complex (green; r.m.s., 0.45 A, 0.51 A,

0.48 A and 0.48 A; PDB accession number

1dqt) on CD28 (red). (b) CD28 residues and

GRASP surface (red) that are structurally

equivalent to the residues and surface of

CTLA-4 (blue) that are contacted by both CD80

and CD86. (c) The GRASP surfaces of CD28,

CTLA-4, CD80 and CD86 colored according to

their electrostatic potential, calculated at neutral

pH and contoured from �8.5 kT (where k is the Boltzman constant and T is the temperature in Kelvin; negative charge; red) through neutral (that is, 0 kT;

white) to +8.5 kT (positive charge; blue). CD80 and CD86 are in the configuration seen in complexes with CTLA-4, after a rotation of 1801 around the

indicated vertical axis, away from the binding interface. Lines of view in each panel are essentially perpendicular to the ligand-binding face of each

molecule. (d) Surface and underlying residues of a CD28-specific hydrophobic pocket close to the ligand-binding face.

P P

P

Y

Y

M

CD28

CD86

CD80

CTLA-4

180°

180°

a

b

c d

CD28

CTLA-4

V86

H116M10I114

I91

L41

a cb d

Figure 4 Structure of the crystallographic sCD28 homodimer. (a) GRASP surface representations of the putative sCD28 homodimer observed in the crystal

lattice (top) and the native CTLA-4 homodimer (PDB accession number 1i8l; bottom). The ligand-binding surface of CTLA-4 is in blue and the structurally

equivalent residues in CD28 are in red. Asparagine residues likely to be glycosylated in each protein are in green. The C termini (and the T cell surface) are

toward the top. (b) Two orthogonal views of the putative sCD28 (red) and native CTLA-4 homodimers after monomer superposition with SHP. The view of

sCD28 in the top diagram is identical to that in a. In the bottom diagram, the line of view is toward the T cell. (c) The putative sCD28 dimer interface with

key interacting residues. (d) The putative sCD28 homodimer (red), with oligomannose, bi- or tri-antennary N-glycans (green) modeled at each of the potential

glycosylation sites.


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CD28 is prevented by substitution of both Ile117 and Pro119by Lys. As noted for CTLA-4 (ref. 25), CD28 monomers do notre-dimerize at concentrations lower than 1 mg/ml in buffered saline(data not shown), indicating that the noncovalent interactionis weak. A model of the fully glycosylated homodimer is presentedin Figure 4d.

Docking CD80 on the putative CD28 homodimer (Fig. 5a), basedon superposition with the known CTLA-4–CD80 complex25, generatesan overall model very different from that of the CTLA-4 complex.Rather than parallel coligation around axes orthogonal to the mem-brane, the two CD80 molecules converge so that their membraneproximal domains ‘clash’ physically (Fig. 5b). In models based on theCD86–CTLA-4 complex24, the ‘clash’ is somewhat less striking, but forthe complex showing the least interference, based on CTLA-4 chain D,the shortest interatomic distance is still less than 2 A. In contrast to theligands, the putative CD28 dimer is ‘comfortably’ bivalent for 5.11A1.According to the lattice configuration, the two Fab fragments bind

almost directly opposite each other and parallel with the cell surface,forming a linear structure (Fig. 5c). Nonmitogenic antibody binding,in contrast, has been mapped to the ‘top’ of the molecule byalterations in the region of Val98 (ref. 27; Fig. 5c).

Is the native CD28 homodimer recapitulated in the lattice?

Although the crystallographic CD28 dimer buries residues showing ahigher propensity for forming interfaces than those buried in nativeCTLA-4 homodimers, and a larger fraction of atoms are completelyburied, neither protein satisfies all the criteria34 required to distinguishphysiological, noncovalent homodimers from crystallographic arte-facts with any certainty, principally because the interfaces are too small(Supplementary Table 1 online). We therefore used cryo-EM andmutagenesis to compare the organization of the native and crystal-lographic CD28 homodimers.

Although CD28 is very small, we expected to be able to resolvecomplexes formed between whole 5.11A1 antibodies and uncleaved,

Membrane

a b c

Figure 5 Proposed ligand- and 5.11A1 Fab complexes formed by putative CD28 homodimers. (a) Orthogonal views of the extracellular region of modeled

CD80–CD28 homodimer complexes. With SHP, each of the CTLA-4 monomers from the CTLA-4–CD80 (PDB accession number 1i8l; dark blue) and CD86–

CTLA-4 (PDB accession number 1i85; pale blue) complexes was superimposed in turn on each of the monomers in the crystallographic CD28 homodimer(red). CD80 was then docked with CD28 according to the mode of ligand binding seen for the CTLA-4 monomer. (b) Superposition of CD28 and CTLA-4

homodimers as in Figure 4b, after the docking of CD80 to both molecules, as in a, based on the binding seen in PDB file 1i8l. (c) Two orthogonal views

of the complex formed by the crystallographic sCD28 homodimer (red) and bivalently bound 5.11A1 Fab (green). The position of the mutation disrupting

nonmitogenic antibody binding in rat CD28 is marked with light blue spheres. Top, horizontal line indicates approximate position of the membrane; the line

of view is parallel to the membrane. Bottom, the line of view is toward the T cell.

CD28Fc–5.11A1 mAba b

CD28Fc–7.3B6 mAb

1 2 3

4 5 6

71 60

68 55 59 70 70 21 49

98 44 67 94 66

Figure 6 Structures of CD28Fc complexes formed with mitogenic and nonmitogenic antibodies determined by cryo-EM. (a) Complexes were formed with

uncleaved, nonreduced CD28Fc and mitogenic (5.11A1; top) and nonmitogenic (7.3B6; bottom) whole antibodies. For each of the two sets of images, the

top row shows class-average images after classification of the cryo-EM images and the bottom row shows the corresponding, automatically identified, best-

matching reprojections of the crystal structure of the CD28-(5.11A1 Fab)2 dimer. Numbers in the images refer to the numbers of individual images used to

generate each class average. Far right, interpretations of the final cryo-EM image in each series. Boxed areas indicate regions of the CD28Fc chimera (light

gray) and antibodies (darker gray) expected to form the largest contiguous block of electron density. (b) Reconstruction of CD28Fc-5.11A1 complexes. In

diagrams 1–3, the CD28-(5.11A1 Fab)2 crystal structure was used as an alignment template; in diagrams 4–6, a chimeric model, based on the intersubunit

contacts of CTLA-4 homodimers and the CD28-5.11A1 Fab crystal structure, was used for alignment. Diagrams 1,4, alignment templates (left) and resulting

three-dimensional reconstructions (right). Diagrams 2,5, top and bottom reconstructions are presented in orthogonal views superimposed with the best fits of

the CD28-(5.11A1 Fab)2 crystal structure. Diagrams 3,6, top and bottom reconstructions are superimposed with the best fit of the chimeric model based onthe intersubunit contacts of CTLA-4 homodimers and the CD28-5.11A1 Fab crystal structure. Red, CD28; green or blue, Fab fragments.


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nonreduced CD28Fc chimeras using cryo-EM. Cryo-EM analysisshowed the formation of extended, tangled complexes when the twoproteins were mixed in equimolar amounts (data not shown). After areference-free alignment of the cryo-EM data to center the electrondensity (providing a common two-dimensional origin), we sortedsimilar images into classes using multivariate statistical analysis. Thisallowed the calculation of class averages, which we then used in multi-reference alignments to improve the class homogeneity (providing acommon three-dimensional phase origin). We iterated this processand then compared the final class averages with re-projections of thecrystal structure. The class-averaged 5.11A1 monoclonal antibody–CD28Fc electron density gave excellent matches to re-projections ofthe linear crystallographic complex consisting of the CD28 dimer andtwo 5.11A1 Fab fragments (Fig. 6a, top), suggesting that native CD28homodimers are similar to the crystallographic dimer and that thebasic unit of the extended complexes consists of bivalently bound andcrosslinked CD28Fc chimeras. As expected, the Fc portions of theantibody and CD28Fc chimera were mostly invisible after averagingbecause of flexibility in the hinge regions of each protein.

Simple modeling indicated that CD28-(5.11A1 Fab)2 complexesformed through CTLA-4-like dimerization would also be linear(Fig. 6b, diagram 4). To distinguish between CTLA-4-like andCD28-like complexes, we generated three-dimensional reconstructionsfrom the cryo-EM data. We obtained very similar reconstructions(Fig. 6b, diagrams 1 and 4) when we aligned the data with thecrystallographic CD28-(5.11A1 Fab)2 complex or a model of a CTLA-4-like CD28-(5.11A1 Fab)2 complex. A quantitative measure of thissimilarity is that the reconstructions could be aligned to one anotherwith a correlation coefficient of 99% and an R factor of 10%, valuesthat are as good as those obtained when the twofold symmetry of theindividual reconstructions was refined. We then automatically fittedthe two reconstructions with the atomic models used to align them,using the program URO35 (Fig. 6b, center and right). The crystal-lographic CD28-(5.11A1 Fab)2 complex gave very good fits to thereconstructions, regardless of the model used to align the data(Fig. 6b, diagrams 2 and 5). In contrast, the fit of the CTLA-4based model was poor in both cases (Fig. 6b, diagram 3 and 6),even though the reconstruction in Figure 6b, diagram 6, was gener-ated from alignments based on this model.

We obtained additional evidence that native CD28 forms dimerscomparable to those seen in the crystal lattice rather than CTLA-4 likehomodimers by mutagenesis, using two approaches. The main contactmediating dimerization in the lattice (Fig. 7a) only partially over-lapped with the region that would form CTLA-4 like homodimers(Fig. 7a), indicating that the two regions could be altered by mutation

separately (Fig. 7b). Early attempts to produce secreted CD28 mono-mers by simply altering the interchain disulfide bond yielded unde-tectable expression (P.A. van der Merwe, personal communication),suggesting that CD28 folding is homodimerization dependent andthat CD28 expression could be used to assay for successful homo-dimerization. Mutations producing alterations in the residues formingcontacts in the crystallographic dimer (Ile91 and Ile114) completelyabrogated surface expression of CD28, whereas similar alterations ofresidues that would be expected to form the interface of a CTLA-4 likedimer (Asp15 and Lys118) were well tolerated (Fig. 7c), indicatingthat the former residues constitute the true dimerization interface. In asecond approach, we tested the prediction, based on the structure ofthe crystallographic dimer, that a steric ‘clash’ involving theirmembrane-proximal domains prevents the simultaneous binding ofligands to native CD28 homodimers: in SPR-based binding assays,native CD28 bound bivalently to bacterially expressed CD86 lackingthe membrane-proximal domain (Supplementary Fig. 3 online).Overall, the cryo-EM data and mutation experiments suggest thatnative CD28 forms homodimers that resemble the crystallographicdimer, rather than a CTLA-4-like homodimer.

Cryo-EM analysis of CD28Fc–7.3B6 antibody complexes

Cryo-EM analysis also showed that like 5.11A1, the nonmitogenicantibody 7.3B6 (ref. 27) formed large, tangled complexes when mixedat a ratio of 1:1 with CD28Fc (data not shown). Three-dimensionalreconstructions of these complexes could not be generated without anappropriate model on which to base the alignments. We relied insteadon the calculation of class averages using multivariate statisticalanalysis and automated classification of the images based on refer-ence-free and multi-reference alignments. The antibody 7.3B6 formedbivalent, V-shaped complexes with CD28Fc (Fig. 6a, bottom), directlyconfirming that this antibody is also capable of crosslinking CD28.Antibody 7.3B6 blocked CD80 and CD86 binding in SPR-based assays(Supplementary Fig. 4 online), indicating that the V-shaped com-plexes resulted from interactions at the ‘top’ of the protein. Theepitopes of other nonmitogenic antibodies examined thus far havebeen similarly localized to the ligand-binding region of CD28 (ref. 27).

DISCUSSION

The structure we have presented here provides explanations for severalimportant functional similarities and differences between CD28 andCTLA-4. Differences in loop and strand configuration outside theligand-binding regions of each molecule constitute evidence of anancient duplication of the CD28 and CTLA-4 precursor gene. Thecore of each ligand-binding site and, in particular, the conformation of

Figure 7 Mutational analysis of the native CD28

homodimer. (a) GRASP surface of the CD28

monomer, with the set of residues forming

contacts in the crystallographic dimer in pale

red and the set of residues equivalent to those

forming contacts in the CTLA-4 homodimer in

pale blue; residues common to both sets are

purple. (b) Positions of residues altered in full-length, surface-expressed CD28: I91 and I114

(both mutated to R), which form contacts in the

crystallographic dimer, are red, and D15 and

K118 (mutated to R and E, respectively), which

are equivalent to residues forming contacts in the

CTLA-4 homodimer, are blue. (c) Relative expression of wild-type (WT) and mutated CD28 on the surface of transiently transfected human HEK 293T cells.

Expression was measured as the mean fluorescence of cells labeled with antibody to CD28 (7.3B6) and Alexa 488–conjugated mouse IgG secondary

antibody. Data are representative of two experiments. Only mutations disrupting contacts in the crystallographic dimer abrogate expression.

I91

I114

D15

K118

a cb Mutations:

100%

50%

Rel

ativ

e flu

ores

cenc

e

0%

Moc

k

D15R

I91R

I114

R

K118E W

T

"CD28" interface

"CTLA-4" interface


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the MYPPPY motifs are nonetheless so highly conserved that it is notsurprising that interactions between CD28 and B7 family proteins aredetectable across species after 300 million years of divergent evolu-tion36. The high degree of conservation of the ligand binding surfacesand extreme complementarity of the CTLA-4–CD80 interface25 sug-gest that subtle conformational differences contribute to the 8- to40-fold variation in the affinities of CD28 and CTLA-4 for CD80 andCD86 (ref. 26). The substitution of Leu106, Thr53 and Tyr105adjacent to the binding site in CTLA-4 may also contribute to thesebinding differences. The G-strand ‘pocket’ unique to CD28 seems tobe worth investigating as a target for small, potentially therapeuticcompounds, given its proximity to the ligand-binding site.

The key functional distinction between CD28 and CTLA-4, whichhas been established in SPR-based binding assays26, is that CD28 ismonovalent rather than bivalent. This stoichiometric difference aloneis estimated to reduce the stability of signaling complexes formed byCD28 by about 99%. For CTLA-4, binding is also likely to bestabilized by the formation of linear arrays with bivalent CD80homodimers23. As a result, the shortest-lived activating complexformed by CD28 (that is, with CD86) is expected to have only0.01–0.001% of the stability of the most potent inhibitory complexformed by CTLA-4 (that is, with CD80; ref. 26). Simple modelingbased on the assumptions that CD28 forms homodimers comparableto those seen in the crystals of the sCD28-Fab complex and that theligands interact with CD28 in the way that they bind CTLA-4 suggeststhat the observed functional monovalence26 results simply from stericinterference with binding. Solution kinetic analyses26 indicate that,even for the parallel arrangement of ligands in CTLA-4–CD80 com-plexes25, occupancy at one site reduces the ‘on rate’ for binding at thesecond site by about 86%. Therefore, even if direct physical ‘clashes’ donot completely prevent bivalent binding, the ‘on rate’ for binding of asecond ligand to CD28 might nevertheless be so low as to beundetectable. Such effects are likely to be compounded in vivo byconstraints in flexibility imposed by the attachment of the moleculesto the cell surface.

An important issue is whether native CD28 forms a homodimerresembling that seen in the lattice. This seems very likely to be the casefor the following reasons. First, the contact has more of the character-istics of physiological dimers (rather than crystal contacts) than CTLA-4, in that a greater proportion of atoms are completely buried and theresidues involved are more often found in such interfaces34. Second,80% of these residues are conserved in all mammalian CD28sequences, with only half being also present in CTLA-4. Third, theproposed dimer uses the small, relatively flat A¢G¢F b-sheet that formsinterfaces between V-set domains and adjacent IgSF domains in othermolecules, notably CD4 and antibody light chains. Fourth, the con-figuration is compatible with interchain disulfide bond formation atCys123 and glycosylation at every potential site. Fifth, the predictionbased on the structure of the proposed dimer, that CD28 is monovalentbecause a membrane-proximal domain ‘clash’ prevents the simulta-neous binding of two ligands, was confirmed by data showing thatCD86 molecules lacking this domain bound simultaneously to CD28in SPR-based assays. Sixth, the structure was verified independentlyusing cryo-EM. Native homodimers formed linear bivalent complexeswith the Fab portions of whole 5.11A1 antibodies that were indis-tinguishable from re-projections of the crystallographic complex at theresolution of this technique. Three-dimensional reconstructions alsoindicated that the linear complexes were best fit by models based on thelattice contact rather than CTLA-4. Finally, and perhaps most persua-sively, mutations resulting in alterations of contacting residues in thecrystallographic interface prevented expression of the protein, whereas

mutations outside this region, for example, the surface that would haveformed a CTLA-4-like homodimer, were well tolerated. Overall, thedata suggest that CD28 exists in the single homodimeric state seen inthe lattice, rather than ‘relaxed’ and ‘parallel’ conformations37.

Although broadly similar, the CD28 and CTLA-4 homodimersdiffer by a rotation and translation, making it unlikely that oneevolved from the other in a single step. One possibility is that thehomodimers derive from a bivalent ancestor that was reliant only onthe interchain disulfide bond for dimerization and that the distinctdomain-domain contacts and stoichiometries emerged separately,driving functional diversification. The use of the GF strand ‘edge’ ofthe domain may have been the only configuration allowing orthogonalligand binding, the probable legacy of primordial IgSF recognition25.

Global structural comparisons indicate that the extracellular domainof CD28 is most like the V-domains of antigen receptors. A C¢¢ strandswitch in CTLA-4 to a conformation seen in TCR Va domains hasbeen noted38, but in CD28 the C¢¢ strand occupies the position seen inalmost all other V-set domains. The exchange of the C¢¢ b-strandbetween the two sheets seen in two highly related molecules suggeststhat such exchange can provide structural diversity with little energeticpenalty. CD28 and CTLA-4 also have clearly defined A strands,b-bulges in the same position as antigen receptors and substantialamounts of overall twist in the A¢GFCC¢C¢¢ face, all of which arecharacteristic of antigen receptor V domains. We propose that V-setdomains be subdivided into V1- and V2-set domains to reflect thesestructural differences. Given the structural similarities of antigenreceptors and CD28, their related functions (coactivation of T cells)and shared signaling properties (their dependence on phosphorylationby extrinsic kinases after interactions with monovalent ligands), itseems likely that CD28-related proteins and antigen receptors evolvedfrom a shared ancestor distinct from conventional adhesion molecules.

Our analysis places new constraints on models of antibody-inducedreceptor triggering. In the context of the observed correlation betweenepitope location and mitogenicity for antibodies to CD28 (ref. 27),our most important finding is that CD28 is bivalent for and will becrosslinked by both mitogenic and nonmitogenic antibodies. Alongwith the observation that antibodies to CD28 are not mitogenic insolution27, where crosslinking is likely to be favored, this indicates thatreceptors are not triggered by crosslinking alone. Antibody-inducedconformational changes are also unlikely to be responsible for signal-ing by any given set of antibodies, because the structural rearrange-ments accompanying antibody binding to protein antigens aregenerally minor and restricted to side-chain movements33 and becauseantibodies rarely if ever bind shared epitopes in precisely the sameway39. A third potential mechanism is that antibodies alter thearrangement of signaling proteins either by physically interfering inthe assembly of multicomponent signaling complexes or by effectingthe size-dependent segregation of signaling molecules in a wayanalogous to that proposed for TCR triggering by endogenousligands40,41. These processes would be topology dependent, accountingfor the observed correlation between epitope location and mitogeni-city27 and would explain, in the latter case, the requirement forantibody immobilization.

METHODSProtein expression and crystallization. Chimeric cDNA encoding, in the

following order, the signal peptide sequence of mouse immunoglobulin heavy

chain, residues 1–134 of the extracellular domain of CD28, a thrombin cleavage

site and the heavy-chain constant region 2 and 3 domains of mouse IgG1

(residues 103–323 of the secreted protein) was expressed in Lec3.2.8.1 cells

as described for sCD80 (ref. 42). The purified, thrombin-released homodimer


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was reduced with 1.5 mM dithiothreitol and was alkylated with 3.3 mM

iodoacetamide. A 2:1 molar mixture of the CD28 monomer and 5.11A1 Fab

prepared by papain digestion (Pierce Biotechnology) was concentrated to

15.6 mg/ml in HBS. Crystals were grown at 21 1C at the Oxford Protein

Production Facility in 200-nl ‘sitting drops’ with 0.2 M magnesium formate,

20% PEG 3350, set up with a Cartesian Robot (APS Robotics & Integration)43.

Crystals were cooled to 100K in the precipitating reagent containing 10%

glycerol, with a Cryostream liquid nitrogen system (Oxford Cryosystems). Data

were collected at the European Synchrotron Radiation Facility to a high-

resolution limit of 2.7 A on beamline ID13 with a 133-mm MAR charge-

coupled device camera. A total of 158 images of 11 oscillations were collected

with radiation of wavelength 1.008 A and a crystal to detector distance of

130 mm. Images were processed with HKL2000 software44. The crystals were of

space group C2 with unit cell dimensions of a ¼ 191.2 A, b ¼ 47.4 A, c ¼ 71.8 A

and b¼ 94.41. These data were 97% complete to 2.7 A but were extremely weak

in the highest resolution shells (I / s(I) ¼ 1.3, where I is intensity, for data

between a resolution of 2.8–2.7 A). Assuming one CD28 monomer and one Fab

molecule per asymmetric unit, the solvent content was estimated to be 57%.

Structure determination and refinement. The structure was solved by

molecular replacement in XPLOR v3.851 (ref. 45) using search models

generated from a single Fab (Protein Data Bank (PDB) accession number

15c8) by varying the elbow angle with an XPLOR script. A single unambiguous

solution was found with an elbow angle altered by �81 relative to that of the

Fab in PDB file 15c8. After rigid body refinement of each Fab domain using

Crystallography and Nuclear Magnetic Resonance System (CNS)46 software,

the R factor was 44.4%. Electron density maps phased from this model showed

limited density for CD28. The Fab model was modified to the sequence of

5.11A1 and, after two cycles of manual rebuilding using O software47 and

minimization in CNS, a molecular replacement solution for CD28 was then

found by using, as a search model, the regions of CTLA-4 (from PDB accession

number 1i8l) most conserved in CD28; that is, all the b-strands except C¢¢ and

the EF and FG loops. To reduce bias, we constrained the models for the Fab

constant domains to those from PDB file 15c8 during all but the final two

rounds of refinement. We refined the rest of the model by alternating positional

and restrained, individual B-factor refinement in X-PLOR 3.851 with manual

rebuilding in O. All refinement procedures used data from 25.0–2.7 A with a

random set of 5% of reflections excluded as a cross-validation set. The

data were corrected for anisotropy and were sharpened (applying a B-factor

of �30 A2) for all refinement in X-PLOR. For the penultimate refinement,

regions of the Fab constant region differing from that in PDB file 15c8 were

rebuilt. In the final round (done with CNS) single N-acetylglucosamine groups

were added to residues 19, 53 and 87 of CD28 and the water_pick script (CNS)

was used to add 203 water molecules. A final step of restrained, individual B-

factor refinement against the unsharpened data gave the current model, which

has an R factor of 23.9% and Rfree of 28.2% against all data to 2.7 A. The model

includes residues 1–118 of the CD28 monomer, although the region between

residues 28 and 30 has poor electron density, and has 81% of residues in the

most favored regions of the Ramachandran plot, with a further 17% in

additionally allowed regions. Full refinement statistics are in Table 1.

Cryo-EM. CD28Fc (25 pmol) was incubated on ice for 10 min with either

5.11A1 or 7.3B6 antibody at an equimolar ratio in 50 ml HBS. An aliquot was

applied to a glow-discharged Holey Carbon grid (Quantifoil), and was blotted

off before the grid was plunged into liquid ethane. Transmission electron

micrographs were recorded in low-dose conditions at liquid nitrogen tempera-

ture on a Philips CM200 FEG microscope at a magnification of 50,000�,

acquired over a large de-focus range. A selection of micrographs was then

digitized on a UMAX PowerLook 3000 scanner with a raster size of 8.322 mm,

resulting in a pixel size of 1.66 A. Images of the antibody complex (B530) were

selected from nine micrographs. Image processing was done with IMAGIC-5

(ref. 48) and SPIDER49 software. A multivariate statistical analysis and auto-

mated classification of the images (after an initial reference-free alignment and

multi-reference alignment) allowed class averages to be calculated with an

improved signal/noise ratio. The resulting class averages were used to align the

whole dataset and then the classification and multi-reference alignment

procedure was repeated. Classes showing a good signal/noise were selected

and optimally aligned in SPIDER to re-projections of the dimeric Fab-CD28

crystal structure.

Three-dimensional reconstruction of the CD28-5.11A1 image dataset was

done with FREALIGN50. The data were initially aligned with maps generated

either from the CD28(5.11A1)2 complex found in the crystal structure or from

a chimeric model in which the arrangement between the CD28 monomers was

modified to be equivalent to that in CTLA-4. The alignment of CD28

monomers with CTLA-4 monomers was done with the program SHP51, based

on the crystal structure of the CTLA-4 dimer25. Six iterations of FREALIGN

were done, after which the twofold symmetries of the maps were individually

refined using the program GAP52. For the map generated from the CD28

crystal structure alignment, the real-space correlation coefficient of this aver-

aging was 98% and the R factor was 11% (ref. 53). For the map generated from

a CTLA-4 type arrangement, the correlation coefficient was 98% and the R

factor was 10%. The CTLA-4 type reconstruction was then fitted to the CD28

type map, giving a correlation coefficient of 99% and an R factor of 10%. The

reconstructions were fitted with the atomic models used in the data alignment

with the program URO35.

PDB accession number. Coordinates of the sCD28 structure, 1yjd.

Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTSThe authors thank S. Ikemizu, E.Y. Jones, P.A. van der Merwe, K.M. Dennehy,S.H. Abidi and L. Hene for comments on the manuscript; and E. Mancini,J. Grimes and the staff of ID13 at ESRF for assistance with data collection.Supported by the Wellcome Trust, the Royal Society and the UK MedicalResearch Council through funding of the Oxford Protein Production Facility,and by Active Biotech Research AB.

COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Immunologywebsite for details).

Received 11 August 2004; accepted 19 January 2005

Published online at http://www.nature.com/natureimmunology/

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