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The Journal of Immunology, 2006, 176: 6873-6878.
Copyright © 2006 by The American Association of Immunologists

Evidence for a Domain-Swapped CD4 Dimer as the Coreceptor for Binding to Class II MHC1

Akiko Maekawa*, Bryan Schmidt*, Barbara Fazekas de St. Groth{dagger}, Yves-Henri Sanejouand{ddagger} and Philip J. Hogg2,*,§

* Centre for Vascular Research, University of New South Wales, Sydney, Australia; {dagger} Centenary Institute of Cancer Medicine and Cell Biology, Sydney, Australia; {ddagger} Laboratoire de Physique, Ecole Normale Superieure, Lyon, Cedex, France; and § Children’s Cancer Institute Australia, Randwick, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD4 is a coreceptor for binding of T cells to APC and the primary receptor for HIV. The disulfide bond in the second extracellular domain (D2) of CD4 is reduced on the cell surface, which leads to formation of disulfide-linked homodimers. A large conformational change must take place in D2 to allow for formation of the disulfide-linked dimer. Domain swapping of D2 is the most likely candidate for the conformational change leading to formation of two disulfide-bonds between Cys130 in one monomer and Cys159 in the other one. Mild reduction of the extracellular part of CD4 resulted in formation of disulfide-linked dimers, which supports the domain-swapped model. The functional significance of dimer formation for coreceptor function was tested using cells expressing wild-type or disulfide-bond mutant CD4. Eliminating the D2 disulfide bond markedly impaired CD4’s coreceptor function. Modeling of the complex of the TCR and domain-swapped CD4 dimer bound to class II MHC and Ag supports the domain-swapped dimer as the immune coreceptor. The known involvement of D4 residues Lys318 and Gln344 in dimer formation is also accommodated by this model. These findings imply that disulfide-linked dimeric CD4 is the preferred coreceptor for binding to APC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD4 is a type I integral membrane glycoprotein that is expressed on most thymocytes and on the subset of peripheral T lymphocytes that includes helper T cells. It plays a central role in T cell biology by functioning as a coreceptor for binding of TCR to class II MHC (MHCII)3 and Ag on APC (1). It also contributes directly to signal transduction through the noncovalent association of its cytoplasmic tail with the p56lck protein tyrosine kinase of the Src family (2, 3). CD4 is required to shape the T cell repertoire during thymic development and to permit appropriate peripheral activation of mature T cells.

The extracellular part of CD4 consists of four Ig-like domains, D1–D4. The backbone of Ig domains are defined by seven beta strands which form two beta sheets. The D1 and D4 disulfides are conventional cross-sheet Ig disulfides, while the D2 disulfide is atypical in that it links adjacent strands in the same beta sheet and is highly strained, or is what we have called a cross-strand bond (4). D2 is a truncated beta barrel containing 75 residues instead of the typical ~100 residues. The D2 cross-strand bond is cleaved on the cell surface apparently by thioredoxin (5), which is a thiol-disulfide oxidoreductase secreted by CD4+ T cells (6). Cleavage of the D2 bond leads to the formation of covalent dimers of CD4 on the cell surface linked intermolecularly through the D2 cysteines.

Disulfide-linked CD4 dimers have been observed on the surface of lymphoid, monocytoid, and dendritic cells (5, 7, 8, 9, 10). The ratio of CD4 dimer to monomer varies with cell type. The monomer predominates on blood lymphocytes and monocytes (8, 10) but the dimer is the major form of CD4 on epidermal Langerhans cells (9).

Because the D2 disulfide bond (Cys130–Cys159) is buried in the monomer, a large conformational change must take place to allow for formation of the disulfide-linked dimer. Using molecular modeling techniques, we showed that domain swapping of D2 is a good candidate for the conformational change, the hinge loop (or linker) being the loop between the E and F strands (11). The sequence of events, therefore, is reduction of the D2 disulfide followed by D2 domain swapping and finally formation of two disulfide bonds between Cys130 in one monomer and Cys159 in the other one. Indeed, no other conformational change in CD4 that we considered could accommodate formation of intermolecular disulfide bonds between the D2 cysteines. Two recent descriptions of a domain-swapped Ig-fold (12, 13) supports the feasibility of a domain-swapped D2.

The functional significance of dimer formation for coreceptor function was tested using cells expressing wild-type or disulfide-bond mutant CD4 that does not form dimers. Eliminating the D2 disulfide bond markedly impaired CD4’s coreceptor function. Moreover, modeling of the complex of the TCR and domain-swapped disulfide-linked CD4 dimer bound to MHCII and Ag supports this dimer as the immune coreceptor. These findings imply that domain-swapped dimeric CD4 is the preferred coreceptor for binding to MHCII.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reduction of soluble CD4

Recombinant soluble human CD4 (residues 1–369) was provided by the National Institutes of Health AIDS Research and Reference Reagent Program. Human thioredoxin was a gift from A. Holmgren, (Karolinska Institute, Stockholm, Sweden) and was stored in HEPES-buffered saline containing 10 mM EDTA and 100 µM DTT. A sample of the thioredoxin was inactivated by alkylating the active site cysteines with 10 mM N-ethylmaleimde# for 10 min at 37°C. The unreacted maleimide was removed by dialysis.

Detection of murine CD4 dimers

TCR+, murine CD4+ Jurkat T cells (1 x 107) were washed twice with PBS and labeled with 100 µM sulfosuccinimidobiotin (Pierce) for 30 min at room temperature, and unreacted sulfosuccinimidobiotin was quenched with 200 µM glycine for 30 min at room temperature. The cells were incubated with L3T4 mAb for 30 min at 37°C, washed five times with PBS, and lysed in 0.5 ml of 50 mM Tris-HCl (pH 8.0) buffer containing 0.15 M NaCl, 0.5% Triton X-100, 0.05% Tween 20, and a protease inhibitor mixture (Roche) for 30 min at 4°C. The lysate was centrifuged at 14,000 x g at 4°C, and the supernatant was incubated with goat anti-rat IgG-Sepharose for 90 min at 4°C. The pellet was washed five times with PBS and the CD4 was released from the beads by boiling the pellet in 30 µl of Laemmli’s SDS-PAGE sample buffer for 2 min. The CD4 was resolved by SDS-PAGE on a NuPAGE Novex 4–12% Bis-Tris Gel (Invitrogen Life Technologies) with MOPS running buffer under nonreducing conditions and transferred to polyvinylidene difluoride membranes. The membrane was incubated with a 1/2000 dilution of streptavidin-peroxidase (Amersham Biosciences) and developed by chemiluminescence.

Cloning and mutagenesis of murine CD4 cDNA

A cDNA library was generated by reverse transcription of the poly(A)+ RNA isolated from C57BL/6 mouse thymus with oligo(dT) primer (Invitrogen Life Technologies), followed by T4 DNA polymerase to obtain the double-stranded cDNA. The coding region of the mouse CD4 cDNA was amplified by PCR with a sense primer, 5'-CCTGTGCAAGAAGCAGAGTGAAGG-3' and an antisense primer, 5'-GCAAGCTCTGCAGACAGACAGGCT-3', and the amplified fragment was subcloned into the pCR-Script vector (Stratagene). The integrity of the cDNA was confirmed by sequencing and then subcloned into the mammalian expression vector pCXN2, which is under the control of the chicken beta-actin promoter and the CMV immediate early enhancer (14). Cys135, Cys165, or both Cys135 and Cys165 were replaced with alanine by site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis kit (Integrated Sciences).

Establishment of T cell lines expressing murine wild-type or disulfide-bond mutant CD4

Jurkat 8.2 T cells stably expressing the 5.CC {alpha}- and beta-chains of TCR specific for resides 86–103 of moth cytochrome c (15) (MCC) were cultured in RPMI 1640 medium containing 10% FCS, 100 U · ml–1 penicillin, 100 µg · ml–1 streptomycin, 2 mM L-glutamine, 0.1 mM 2-ME, 250 µg · ml–1 xanthine, 15 µg · ml–1 hypoxanthine, and 1.5 µg · ml–1 mycophenolic acid at 37°C and 5% CO2. Murine wild-type or disulfide-bond mutant CD4 was transfected into human CD4– Jurkat 8.2 T cells by electroporation with GenePulsar (Bio-Rad) at 400 V and 960 µF and selected in medium containing 400 µg · ml–1 G418 (Invitrogen Life Technologies). More than 40 clones were isolated and expression levels of TCR and CD4 were assessed by flow cytometry. One million T cells were incubated with either a control FITC- or PE-conjugated mAb or a FITC-conjugated L3T4 mAb, a PE-conjugated V{alpha} 11.1, 11.2b,d TCR mAb, or a FITC-conjugated Vbeta3 TCR mAb for 20 min at room temperature in PBS. The cells were washed with PBS containing 1% BSA, fixed with 1% formaldehyde, and analyzed by flow cytometry using a FACScan and CellQuest software. All Abs were purchased from BD Pharmingen. Clones expressing similar levels of TCR and wild-type or mutant CD4 were selected for assay.

T cell activation assay

T cells (2 x 105) were incubated with the B cell lymphoma cell line CH27 and MCC in 500 µl of RPMI 1640 medium containing 10% FCS, 100 U · ml–1 penicillin, 100 µg · ml–1 streptomycin, 2 mM L-glutamine, 0.1 mM 2-ME, and 5 ng · ml–1 PMA acetate at 37°C and 5% CO2. Supernatants were collected at discrete times and assayed for IL-2 by enzyme immunoassay (eBioscience). The capacity of the different clones to produce IL-2 was verified by activating with the anti-CD3{epsilon} mAb, OKT3, and was found to be comparable in all clones tested. T cells (2 x 105) were incubated in wells coated with OKT3 for 24 h at 37°C and 5% CO2.

Structural figures

A low-energy conformation of the D1-D4 dimer was sought using the following assumptions. First, the pair of D1-D2 domains is in the domain-swapped disulfide-linked configuration (11). Second, the pair of D3-D4 domains is in the configuration found in the crystal structure of the whole D1-D4 extracellular part (16). In practice, harmonic restraints were set on backbone atoms, except for those belonging to the D2-D3 interface (residues 103–114, 132–135, 146–157, 171–182, 199–208, 232–235, 251–254, and 275–282), with a 100-kcal · mol–1 · Å–1 force constant. Specifically, at each step of the optimization process, restrained atoms belonging to the pair of D1-D2 domains, on the one hand, to the pair of D3-D4 domains, on the other hand, were (implicitly) rotated and translated, a best fit being performed so as to minimize restraint energy. The optimization was performed with a simulated annealing protocol. A 1-nsec molecular dynamics simulation at 800 K followed by 100 K temperature jumps and 100-psec molecular dynamics simulations, the average conformation obtained during the last, 300 K, simulation being next energy minimized. Because of the harmonic restraints, the 800 K temperature does not yield denaturation (17). Instead, it speeds up the sampling of the relative positioning of the D1-D2 pair of domains with respect to the D3-D4 pair, both pairs behaving as nearly rigid bodies, as far as their backbone atoms are concerned. Calculations were performed using the CHARMM program (18) and EEF1 (19). Note that EEF1 refers not only to the implicit solvent model but also to specific electrostatics options (19).

Figures of structural superimpositions were generated using the crystallographic structures of the complex between human {alpha}beta TCR and human HLA-DR4 MHCII bound to peptide Ag (PDB code 1J8H) and the complex between D1 and D2 of human CD4 and murine I-Ak MHCII (PDB code 1JL4). These composite structures were further superimposed with either the dimeric form of the full extracellular fragment of CD4 (PDB code 1WIQ) or with CD4 in a D2 domain-swapped dimer form (11). Pairwise structural comparison of structures was performed using the DaliLite server (European Bioinformatics Institute, Cambridge, U.K.) (20) and figures were generated using PyMol (Delano Scientific) (21).

Statistical analysis

Data are presented as means and SDs. All variables were examined for normality and homogeneity of variance. Normality of the distribution was assessed using the Shapiro-Wilk test. One-way ANOVA and post hoc tests, parametric or nonparametric as appropriate, were used to compare mean values. All analyses were performed using the statistical package SPSS version 13, and the statistical significance was defined as p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Soluble CD4 formed disulfide-linked dimers under mild reducing conditions

Experimental evidence for formation of domain-swapped disulfide-linked CD4 dimers was sought by incubating the extracellular part of CD4 under mild reducing conditions. We hypothesized that selective reduction of the D2 disulfide would trigger D2 domain swapping and formation of intermolecular disulfide bonds between the D2 cysteine.

Incubation of a domain 1-4 fragment of CD4 under mild reducing conditions (1 mM DTT for 5 min at 37°C) resulted in formation of disulphide-linked dimers (Fig. 1A). For a protein-protein complex to remain associated under the conditions of SDS-PAGE, it must be covalently linked or be of such high affinity (pM Kd or better) that it survives SDS denaturation and dilution. As expected, there was no dimer formation under conditions where all three CD4 disulfides as well as any interchain disulfides will be reduced (20 mM DTT for 5 min at 100°C).


Figure 1
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  		FIGURE 1. Soluble CD4 formed disulfide-linked dimers under mild reducing conditions and murine CD4 formed dimers on the T cell surface. A, Soluble CD4 was incubated in HEPES-buffered saline without (lane 1) or with 20 mM DTT at 100°C (lane 2) or 1 mM DTT at 37°C (lane 3) for 5 min. Five micrograms of protein was resolved on nonreducing SDS-PAGE and stained with Coomassie brilliant blue. The sizes of Mr markers are indicated at the left. B, Jurkat T cells expressing murine CD4 were labeled with sulfosuccinimidobiotin and the CD4 was immunoprecipitated with L3T4 mAb, resolved on SDS-PAGE, and blotted with streptavidin-peroxidase to detect the biotin label. The blot was of CD4 from 5 x 106 cells.

 
Murine CD4 formed dimers on the cell surface

Human CD4 forms dimers on the cell surface linked covalently through the D2 cysteines (5), and we sought to confirm that murine CD4 undergoes the same processing. Murine CD4+ Jurkat T cells were labeled with the amine-reactive biotin-linked probe sulfosuccinimidobiotin. The CD4 was immunoprecipitated with L3T4 mAb, resolved on SDS-PAGE, and the CD4 was detected by blotting with streptavidin-peroxidase. Both monomeric and dimeric CD4 was detected (Fig. 1B), confirming that murine CD4 forms covalent dimers like the human protein. Dimers of murine CD4 have also been demonstrated on the surface of KR3 T cell hybridoma cells (10).

Elimination of the CD4 D2 disulfide bond severely impaired coreceptor activity

The coreceptor function of CD4 was evaluated in culture using engineered Jurkat T cells (15). The functions of wild-type or a D2 disulfide-bond mutant (either or both D2 cysteines were mutated to alanine) CD4 was compared. This mutant is predicted to be a mimic of the reduced CD4 (5).

Murine wild-type or D2 mutant CD4 was transfected into human CD4– Jurkat 8.2 T cells stably expressing both chains of the murine TCR that recognizes resides 86–103 of MCC (15). The CD4+TCR+ Jurkat cells were cocultured with the murine B cell lymphoma line CH27, which expresses MHCII that recognizes MCC peptide. Incubations were in the presence of PMA and different concentrations of MCC peptide and for different times. T cell activation was monitored by measuring IL-2 secreted into the medium. Cell surface expression of the wild-type or mutant CD4 on the T cell transfectants was confirmed by flow cytometry using Abs specific for murine CD4 and the {alpha}- and beta-chains of the TCR. Clones expressing similar levels of wild-type and mutant CD4 were selected for assay (data not shown).

Activation of T cell transfectants that express D2 mutant CD4 was markedly reduced as compared with cells expressing wild-type CD4. IL-2 production by CD4 mutant T cells was 3- to >55-fold lower than wild-type cells at all doses of Ag (Fig. 2A) and times of incubation (Fig. 2B).


Figure 2
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  		FIGURE 2. Elimination of the CD4 D2 disulfide bond severely impaired coreceptor activity. TCR+ Jurkat T cells expressing wild-type or D2 disulfide-bond mutant murine CD4 were cocultured with MHCII+ CH27 cells for 24 h in the presence of increasing concentrations of Ag (resides 86–103 of MMC; A) or for discreet times up to 24 h in the presence of 5 µg · ml–1 MCC (B). T cell activation was monitored by measuring IL-2 secreted into the medium. The bars and errors are the mean ± SD of four experiments using a single Jurkat cell clone. The results are representative of four different Jurkat cell clones. *, p < 0.05; **, p < 0.001.

 
The D2 disulfide/dithiol in human CD4 is required for dimer formation (5). As anticipated, dimers of murine CD4 were not detectable on the surface of the Jurkat T cells when the D2 disulfide was ablated (Fig. 3A). We cannot exclude the presence of a small amount of dimer, however, that is beyond the resolution of the experiment. The capacity of the different clones to produce IL-2 was verified by activating with an anti-CD3{epsilon} mAb and was found to be comparable in all clones tested (see e.g., Fig. 3B). In addition, the impaired activation of D2 mutant CD4 T cells was independent of the level of expression of CD4 in different clones (Fig. 3C).


Figure 3
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  		FIGURE 3. Controls for effects of ablation of the D2 disulfide on CD4 coreceptor activity. A, Jurkat T cells expressing wild-type (lane 1) or D2 disulfide-bond mutant (lane 2) murine CD4 were labeled with sulfosuccinimidobiotin, and the CD4 was immunoprecipitated with L3T4 mAb, resolved on SDS-PAGE, and blotted with streptavidin-peroxidase to detect the biotin label. The blot was of CD4 from 5 x 106 cells. The sizes of Mr markers are indicated at the left. B, TCR+ Jurkat cells expressing similar levels of wild-type or mutant CD4 were cultured for 24 h with MHCII+ CH27 cells and 50 µg · ml–1 MCC or in microtiter plate wells coated with the anti-CD3{epsilon} mAb OKT3. The bars and errors are the mean ± SD of four determinations of IL-2 concentration. **, p < 0.001. C, TCR+ Jurkat cells expressing different levels of wild-type or mutant CD4 were cocultured with MHCII+ CH27 cells for 24 h in the presence of 50 µg · ml–1 MCC. The bars and errors are the mean ± SD of four determinations of IL-2 concentration. D, TCR+ Jurkat cells expressing similar levels of wild-type, single D2 cysteine or double D2 cysteine CD4 mutants were cocultured with MHCII+ CH27 cells for 24 h in the presence of 50 µg · ml–1 MCC. The bars and errors are the mean ± SD of results with 3 (WT), 5 (C135A), 5 (C165A), or 12 (C153A,C165A) different Jurkat cell clones.

 
It was possible that replacing both D2 cysteine residues with alanine caused structural changes in addition to those resulting from ablation of the disulfide bond. This theory was tested by comparing the activation of T cells expressing either single D2 cysteine mutant (C135A or C165A) with the double cysteine (C135A,C165A) mutant. As anticipated, activation of the T cell transfectants expressing the single D2 cysteine mutants was reduced to the same extent as for the double cysteine mutant (Fig. 3D). Our conclusions regarding the consequence of ablating both D2 cysteines for coreceptor function would have required revision had the single mutants behaved differently than the double mutant in our assays.

Exogenous thioredoxin inhibited CD4 coreceptor function

CD4+TCR+ Jurkat cells were cocultured with thioredoxin or redox-inactivated thioredoxin and CH27 cells for 24 h in the presence of MCC peptide. IL-2 production by CD4 wild-type T cells was reduced ~40% by incubation with thioredoxin (Fig. 4). Thioredoxin did not effect activation of CD4 mutant T cells. This is as expected because the D2 disulfide, which is thioredoxin’s target, is missing in the mutant. Redox-inactive thioredoxin had no effect on activation of either wild-type or mutant T cells, which indicates that thioredoxin’s effect in this system was a consequence of its oxidoreductase activity. This is consistent with our earlier observations (5).


Figure 4
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  		FIGURE 4. Exogenous thioredoxin inhibited CD4 coreceptor function. TCR+ Jurkat cells expressing wild-type or D2 disulfide-bond mutant murine CD4 were cocultured without (Nil) or with 10 µM thioredoxin (Trx) or redox-inactive thioredoxin (NEM-Trx) and MHCII+ CH27 cells for 24 h in the presence of 5 µg · ml–1 MCC. T cell activation was monitored by measuring IL-2 secreted into the medium. The bars and errors are the means ± SD of four experiments using a single Jurkat cell clone. The results are representative of two different Jurkat cell clones. **, p < 0.001.

 
Modeling of the complex of the TCR and domain-swapped disulfide-linked CD4 dimer bound to MHCII and Ag supported the domain-swapped dimer as the immune coreceptor

There is currently no report of a ternary complex consisting of a TCR, a MHCII, and a CD4. Binary complexes of TCR ({alpha} and beta subunits) and peptide-bound MHCII (HLA-DR4) (22) (PDB code 1J8H) and D1D2 of CD4 and MHCII (I-Ak) (23) (PDB code 1JL4) have been solved. The structure of the entire extracellular region (D1-D4) of CD4 is also available (16). This structure is of a CD4 dimer linked through D4.

Using molecular modeling techniques, a D1-D4 domain-swapped dimer was built, assuming domain swapping for the pair of D1-D2 domains (11) and that the pair of D3-D4 domains interact in the same way as in the D1-D4 crystal structure (16) (Fig. 5). To fulfill these two requirements, the interface between the D2-D3 domain has to reorganize. Residues of interest in the D2 domain-swapped dimer are Gln114, Phe182, and Phe201 (Fig. 5A), which have been shown to abrogate CD4 coreceptor function (24), and D4 residues Lys318 and Gln344 (Fig. 5B), which are known to be involved in CD4 dimerization (10).


Figure 5
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  		FIGURE 5. Model of a D2 domain-swapped CD4 dimer. (The domain-swapped CD4 dimer and TCR-MHCII-CD4 complex models are available at http://yh.sanejouand.com/Articles.html.) A, One molecule of the dimer is shown in red and the other in blue. The D2 disulfide bonds covalently linking the dimer are shown in space-filling representation. The C termini carboxyl groups are shown in yellow. Gln114, Phe182, and Phe201 have been shown to abrogate CD4 coreceptor function (24 ). B, Position of D4 residues Lys318 and Gln344, which are known to be involved in CD4 dimerization (10 ). The Gln344 residues are interacting in the dimer and the Lys318 side chains are within 10 Å of each other. The C termini carboxyl groups are shown in yellow.

 
Complex models of two TCRs, two MHCIIs, and either a D2 domain-swapped or D4-linked CD4 dimer were also constructed (Fig. 6). Considering that only 9, 19, and 13 aa connect the most C-terminal residue of the structures and the transmembrane-spanning segments of CD4, TCR{alpha}, and TCRbeta, respectively, the models present a view of how TCR and CD4 on a Th cell might interact with MHCII on an APC. Lys279 in the D3 domain of the CD4 dimer is predicted to interact in the vicinity of Glu59 in the beta subunit of MHCII (Fig. 5A).


Figure 6
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  		FIGURE 6. Models of the TCR-MHCII-CD4 complex in which CD4 is linked either through domain swapping of D2 or through D4 only. A, TCR-MHCII-CD4 dimeric complex with CD4 as the domain-swapped disulfide-linked dimer. CD4 dimer is shown in red and blue, with the D2 disulfide bonds covalently linking the dimer shown in space-filling representation. The {alpha}- and beta-chains of MHCII are shown in green and the {alpha}- and beta-chains of the TCR in purple. The assembly is oriented with the APC membrane on top and the T cell membrane on the bottom. The C termini carboxyl groups are shown by space filling. Cellular anchor points for both CD4 and the TCR are approaching coplanar, supporting the functional feasibility of this complex. B, TCR-MHCII-CD4 dimeric complex with CD4 linked through the D4 domain, as crystallographically determined. CD4 monomers are shown in red and blue, MHCII is shown in green, the TCR is shown in purple, and the peptide Ag in black. Cellular anchor points for the CD4 molecules are clearly nonplanar with the anchor points of the TCR, implying that this complex in unlikely to be feasible on the cell surface.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A number of different receptors have been shown to form disulfide-linked dimers on the cell surface, including immune system receptors. The dimers form either during maturation of the protein in the endoplasmic reticulum and Golgi (e.g., TCR (25) and CD8 (26)) or after arriving at the cell surface (e.g., m3 muscarinic (27) and IL-3 (28) receptors). In most cases, dimer formation has been demonstrated to be important for function (29). CD4 is an example of a receptor that dimerizes after arriving at the cell surface.

The very low affinity of binding of monomeric CD4 to MHCII, Kd > 100 µM (30), implies that a CD4 dimer or multimer is the productive receptor for MHCII. Indeed, self-association of CD4 has been suggested to be important for productive CD4-MHCII binding (3, 31). Dimers of CD4 rather than higher order oligomers, however, have been the focus of most investigations (10, 16). The nature of the dimerization is controversial. Both the D1 (32, 33, 34) and D4 (3, 31, 35, 36) domains have been implicated in dimerization. The observation that soluble CD4 crystallizes as a dimer linked through D4 (16) focused attention on this domain.

D4 residues Lys318 and Gln344 have been shown to be important for CD4 dimerization on the surface of T cells (10). The relevance of a solely D4-linked dimer is questionable, however, considering that the Kd for dimerization of the soluble protein is ~1 mM (16, 37). Assuming a concentration of CD4 on the cell surface of ~10 µM (16), only ~1% of the total CD4 is predicted to be dimeric based on a Kd of 1 mM. However, in epidermal Langerhans cells, for instance, most of the CD4 is dimeric (9). A weak interaction via D4 is also inconsistent with the SDS- and PAGE-stable CD4 dimers observed on primary and transfected cells (5, 8, 9, 10).

A domain-swapped dimer, on the other hand, will persist under SDS-PAGE conditions because of the covalent linking of the D2 cysteine residues. To accommodate the pair of D3-D4 domains in the domain-swapped dimer, a conformational change has to occur at the D2-D3 interface. Indeed, because the E-F loop of D2 involved in domain swapping belongs to this interface and because domain swapping implies a large motion of this loop (11), it is likely that the D2-D3 interface is destabilized as a consequence of domain swapping. As a matter of fact, the linker region between D2 and D3 is expected to be flexible (38), and mutations of residues Gln114, Phe182, and Phe201 (Gln114, Phe182, and Phe201 in the murine protein), which are partially buried and belong to the D2-D3 interface, have been shown to completely abrogate CD4 coreceptor function (24).

In the model of the TCR-MHCII-CD4 complex in which CD4 is linked through D4 only, it is apparent that the cellular anchor points for the CD4 molecules and the TCR are not coincident with a planar T cell surface, irrespective of how the complex is viewed (23). In the complex with CD4 linked through swapping of D2, however, the cellular anchor points for the CD4 dimer and the TCR are approaching coincidence with a planar T cell surface. The membrane-proximal domains of each of the components are all roughly vertical and the Ag-binding groove of MHCII is at a ~45° angle with the membrane. Consistent with binding studies (39), there are no direct contacts between CD4 and the TCR in the model. The membrane-proximal ends of the TCR and the D2 domain-swapped CD4 are ~90Å apart, which should be ample space for the CD3{epsilon} and CD3{delta} subunits within or near this interval (40, 41). It is noteworthy that Lys279 in the D3 domain of CD4 interacts with MHCII in the vicinity of Glu59. The model raises questions about how the cytoplasmic portions of the TCR subunits and the CD4-associated p56lck coordinate in signaling and the positioning of the CD3 components within the TCR machinery.

The D2 domain-swapped model accommodates the known involvement of D4 residues Lys318 and Gln344 in CD4 dimerization (10). In the model, the Gln344 residues of the monomers interact in the dimer and the Lys318 residues are <10 Å apart (Fig. 6). It is conceivable that an interaction through D4 residues Lys318 and Gln344, although a weak interaction in itself, is important for aligning two CD4 monomers so that the D2 domains can swap efficiently to form the covalently linked dimer. Domain swapping involves partial denaturation of the protein. After the D2 disulfide has broken, strands F and G have to escape both monomers. Many interactions that stabilize the beta sheet are broken and the corresponding conformation is a high-energy one. As a consequence, the process is expected to be slow. In the prion protein, for instance, domain swapping and the corresponding disulfide exchange takes days (42). In CD4, however, the alignment provided by the interactions between the D4 domains may speed up the process. Also, an intermediary state with a transient disulfide bond established between the pair of Cys159 in both F strands is conceivable and would certainly help the system to progress toward the domain-swapped form.

Thioredoxin has been implicated in reduction of the D2 disulfide on the T cell surface (5). Exogenous thioredoxin inhibited the coreceptor activity of CD4 in our system, an effect that was dependent on the presence of the D2 disulfide and the redox activity of the oxidoreductase. The result implies that thioredoxin-mediated reduction of the D2 disulfide does not in itself lead to swapping of the domain and covalent dimer formation. This may relate to the mechanism by which thioredoxin cleaves disulfide bonds (43, 44). The Cys32 thiol of thioredoxin attacks the substrate disulfide bond, cleaving the bond. The mixed disulfide then decomposes via attack by the Cys35 thiol on the intermolecular disulfide, resulting in a stable disulfide between the active site cysteine residues and release of the reduced substrate. It is possible that the mixed disulfide between CD4 and thioredoxin is not being resolved or is resolved inefficiently, which would prevent swapping of D2 because of steric hindrance.

In conclusion, our observations imply that disulfide-linked dimeric CD4 is the preferred coreceptor for binding to MHCII. Strategies to promote dimerization of CD4 should, therefore, enhance the immune response, while inhibiting dimer formation is predicted to be immunosuppressive.


   Acknowledgments
 
We thank N. Tedla and Y. Kanaoka for valuable help and advice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Australian Research Council, the National Health and Medical Research Council of Australia, and the New South Wales Health Department. Back

2 Address correspondence and reprint requests to Dr. Philip J. Hogg, Center for Vascular Research, School of Medical Sciences, University of New South Wales, Sydney, New South Wales 2052, Australia. E-mail address: p.hogg{at}unsw.edu.au Back

3 Abbreviations used in this paper: MHCII, class II MHC; MCC, moth cytochrome c. Back

Received for publication December 2, 2005. Accepted for publication March 22, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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