An ion pair in class II major histocompatibility complex heterodimers critical for surface expression and peptide presentation.

In this report we demonstrate that the ion pair Arg-80α and Asp-57β, located in the peptide-binding site of nearly all class II major histocompatibility complex (MHC) proteins, is important for surface expression and function of the murine class II heterodimer I-Ad. Charge reversal at either of these two residues by site-directed mutagenesis generated mutant class II molecules that failed to appear at the cell surface. This defect in surface expression was partially reversed when the invariant chain was present or when the mutants were paired with the corresponding charge-reversed variant of the opposite chain. Surprisingly, surface expression was restored when cells expressing the single-site mutants were cultured at reduced temperature. In addition, the substitution of Asp-57β with residues found in alleles of class II molecules associated with diabetes resulted in heterodimers that were inefficiently expressed at the cell surface and presented foreign peptide poorly. Together, these results demonstrate that the formation of a salt-bridge between Arg-80α and Asp-57β is required for efficient surface expression of class II MHC molecules, therefore representing an important step in the assembly and transport of functional class II heterodimers to the cell surface.

In this report we demonstrate that the ion pair Arg-80␣ and Asp-57␤, located in the peptide-binding site of nearly all class II major histocompatibility complex (MHC) proteins, is important for surface expression and function of the murine class II heterodimer I-A d . Charge reversal at either of these two residues by site-directed mutagenesis generated mutant class II molecules that failed to appear at the cell surface. This defect in surface expression was partially reversed when the invariant chain was present or when the mutants were paired with the corresponding charge-reversed variant of the opposite chain. Surprisingly, surface expression was restored when cells expressing the single-site mutants were cultured at reduced temperature. In addition, the substitution of Asp-57␤ with residues found in alleles of class II molecules associated with diabetes resulted in heterodimers that were inefficiently expressed at the cell surface and presented foreign peptide poorly. Together, these results demonstrate that the formation of a salt-bridge between Arg-80␣ and Asp-57␤ is required for efficient surface expression of class II MHC molecules, therefore representing an important step in the assembly and transport of functional class II heterodimers to the cell surface.
T lymphocytes respond to foreign antigens by detecting peptide fragments of those antigens bound to products of the major histocompatibility complex (MHC) 1 and displayed on the surface of antigen presenting cells (reviewed in Ref. 1). The threedimensional structures of both class I and II MHC molecules complexed with self-or foreign peptide antigens have recently been solved, revealing much about the molecular basis of peptide binding to MHC molecules (2)(3)(4)(5)(6)(7)(8). Structurally, both classes of MHC molecules are similar, as predicted by sequence comparison (9); however, they differ in fine detail, especially in the peptide-binding site (7,8). These differences partly account for the distinct manner in which each class of molecule binds peptide. In class I molecules, residues forming the "ends" of the peptide-binding site bury the termini of bound peptides (3)(4)(5)(6). In contrast, in class II molecules, both ends of the peptidebinding site are "open," allowing the termini of bound peptides to extend out of the site (7,8). Consequently, peptides bound to class I molecules are predominantly 8 -10 residues in length (10 -14), whereas peptides bound to class II molecules tend to be 13-25 residues in length (15)(16)(17).
In both classes of MHC molecules, the presence of bound peptides influences their tertiary structure and efficiency of appearance on the cell surface. The binding of self-and foreign peptides to the class I heavy chain promotes the folding, assembly, and surface expression of class I molecules by stabilizing the class I complex (18,19). Similarly, peptide binding to class II molecules influences the conformation, stability, and surface appearance of class II molecules (20 -24). Thus, detailed functional analysis of the peptide-binding sites of both classes of molecules will be necessary to understand the role of peptide binding in determining the tertiary structure, and hence, surface expression of MHC molecules and subsequent display of foreign antigens to the immune system.
We have been interested in the biochemical basis of recognition of peptide antigens bound to MHC molecules by T cells. We have characterized the T cell receptor (TCR) expressed by the murine T cell clone D5 (25), which recognizes arsonate (Ars)conjugated peptides presented by the class II molecule I-A d (26). We have engineered single-site substitutions in the putative MHC/peptide antigen-binding site of the D5 TCR and have identified several residues that are important in recognition of the MHC⅐peptide complex (27,28). Subsequently, we sought to compensate these single-site TCR mutations with complementary mutations in the I-A d molecule. In the course of these studies, we identified an ion pair, Asp-57 of the ␤-chain and Arg-80 of the ␣-chain of I-A d , that plays an essential role in surface expression of I-A d as well as peptide presentation.

MATERIALS AND METHODS
Cell Lines-The following T hybridomas expressing the TCR of the T cell clone D5 (25) (33) were used to transiently express I-A d ␣and ␤-chains, described below. All cells were grown in complete medium containing Dulbecco's modified Eagle medium (high glucose), 10 mM HEPES, 100 units/ml penicillin, 100 g/ml streptomycin, 50 M ␤-mercaptoethanol, 2 mM glutamine, and 10% fetal calf serum (Hyclone Laboratories, Logan, UT) in 10% CO 2 incubators at 37°C unless indicated. CTLL-20 cells (34) used to assay IL-2 were maintained in complete medium supplemented with rat IL-2 (Collabo-rative Research, Bedford, MA). RT2 cells were maintained in medium supplemented with HAT (Life Technologies, Inc.).
DNA Constructions-Plasmid constructions were carried out by standard techniques (35). Plasmids containing A␣ d cDNA (A␣ d -Sal-EXV, [36]), A␤ d cDNA (A␤ d -EXV (37)), and murine invariant chain cDNA (mIi-EXV (37)) were kindly provided by Dr. N. Braunstein. Control plasmid encoding chimeric molecule 2B4␤-, which consists of the extracellular domains of the 2B4 TCR ␤-chain fused to the transmembrane and cytoplasmic domains of the TCR -chain (␤-in pCDL-SR␣ (38)) was kindly provided by Dr. I. Engel (National Institutes of Health, Bethesda, MD). EcoRI fragments containing A␣ d and A␤ d cDNA were subcloned into the polylinker EcoRI site of the expression plasmid pLGP-3, kindly provided by Dr. P. Hogan (Harvard Medical School, Boston, MA), which is derived from the plasmid pCD and contains an SV40 promotor for transient expression of cDNA in COS cells and fd to generate single-stranded templates for mutagenesis (39). Site-directed mutagenesis was carried out on uracil-labeled, single-stranded templates, as described previously (27), using oligonucleotides listed in Table I to generate variants. Variants were initially screened by analyzing restriction enzyme sites introduced or destroyed by the mutagenic oligonucleotides ( Table I). Sequences of variants were confirmed by dideoxynucleotide sequencing the entire ␣1or ␤1-domain of each plasmid using Sequenase (U. S. Biochemical Corp.).
Transfection of COS Cells-COS cells were transfected essentially as described (40). A total of 2 ϫ 10 6 COS cells was plated in complete medium in 75-cm 2 flasks 24 h prior to transfection. Cells were washed twice with Dulbecco's modified Eagle's medium/10 mM HEPES and cultured with a total of 10 g each of ␣and ␤-chain plasmid DNA in 6 ml Dulbecco's modified Eagle's medium, 10 mM HEPES, 100 M chloroquine (Sigma), 250 g/ml DEAE-dextran (Pharmacia) for 4 h at 37°C. The DNA solution was removed, and cells were left in 6 ml of shock medium containing Dulbecco's modified Eagle's medium, 10 mM HEPES, 10% dimethyl sulfoxide for 3 min at room temperature. Shock medium was removed and replaced with 20 ml of complete medium. Cells were cultured for 2 days prior to analysis. As a control, in each experiment COS cells were mock-transfected with medium lacking I-A d plasmids or were transfected with I-A d ␣-chain plasmid alone. When indicated, 20 g of plasmid DNA encoding the invariant chain or the co-transfection control molecule 2B4␤-was co-transfected along with I-A d plasmid DNA.
Flow Cytometry-Culture supernatants from B cell hybridomas used as control and I-A d -reactive mAb for flow cytometry are listed in Table  II. All B cells hybridomas were purchased from American Type Culture Collection (Rockville, MD), except K24 -199 cells, kindly provided by Dr. D. McKean (Mayo Clinic, Rochester, MN). Cell staining was carried out as described (27) using saturating concentrations of mAb supernatants determined by staining I-A d on transfected RT2 L cells (data not shown). Cells were analyzed on a FACScan flow cytometer (Becton Dickinson) using the FACScan software program. Dead cells were excluded from analysis by staining with propidium iodide. Results of flow cytometry of transfected COS cells are displayed as histograms of cell number versus fluorescence intensity or are expressed as "fluorescence units" (40). A total of 5000 cells was analyzed for each sample. Fluorescence units were calculated as the product of the number of "positive" cells exhibiting fluorescence above a reference point and the mean fluorescence of those cells. The reference point was set such that mocktransfected COS cell populations contained fewer than 1% of total cells with fluorescence above this level when the I-A d test mAb was used (see Fig. 1). In general, fewer than 1% of total COS cells transfected with I-A d plasmids displayed fluorescence above this reference point when stained with control mAb. The mean fluorescence of the positive cells was calculated from mean channel fluorescence provided by the FACScan program using the formula: Mean fluorescence ϭ 10 (mean channel fluorescence/256) .
Peptide Presentation Assay-A total of 10 5 T cell hybridomas was cultured in triplicate with 2 ϫ 10 4 transfected COS cells and serial 3-fold dilutions of peptide antigen in 200 l in 96-well microtiter plates. After 20 h, supernatants were removed, frozen, and tested for the ability to promote the growth of the IL-2-dependent cell line CTLL-20. Viability of the CTLL-20 cells was measured by their reduction of 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolum bromide (Sigma) using a V max microplate reader (Molecular Devices, Menlo Park, CA) by recording OD 570 nm after subtracting OD 650 nm . Arsonate-conjugated Ova peptides spanning residues 33-49 and 33-50, Ars-Ova (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49) and Ars-Ova(33-50), were prepared as described previously (26). The Ova(323-339) peptide was synthesized by M. Berne (Tufts University, Boston, MA) and purified by high performance liquid chromatography (HPLC). HPLC-purified peptide cI(12-26) was kindly provided by Dr.  RESULTS We individually substituted Lys for several negatively charged residues in the ␣-helical regions of the ␣1and ␤1domains of the I-A d heterodimer by site-directed mutagenesis (Table I and Fig. 9). These helices form the "walls" of the peptide-binding site of MHC molecules and, when bound with foreign peptide, are recognized by the TCR. We chose to express these variant I-A d molecules in COS cells because COS cells can transiently express murine class II molecules even though they fail to express the invariant chain (37). We analyzed expression of these variant I-A d molecules by flow cytometry using a variety of mAb recognizing different epitopes on I-A d (Table II). We then tested the ability of the variant class II molecules expressed on COS cells to present foreign peptides to I-A d -restricted, antigen-specific T cell hybridomas.

Role of Negatively Charged Residues in Surface Expression of I-A d and Presentation of Foreign Peptide-Between 10 and 50%
of COS cells transiently transfected with wild-type (WT) ␣and ␤-chains of I-A d displayed significant surface staining with I-A d -reactive mAb MK-D6 above staining with control mAb (Fig. 1). In contrast, mock-transfected COS cells failed to stain specifically with mAb MK-D6 (Fig. 1). COS cells transfected with I-A d ␣or ␤-chain DNA alone failed to stain with mAb recognizing either I-A d ␣or ␤-chains (see below and data not shown), demonstrating the strict requirement for both ␣and ␤-chains of I-A d for surface expression of I-A d heterodimers as previously reported (37).
COS cells expressing WT I-A d and the 11 variant Lys-substituted heterodimers listed in Table I were analyzed by flow cytometry using several mAb reactive against either the ␣1or ␤1-domains of I-A d (Table II). The relative efficiency of surface expression of WT and variant I-A d molecules was estimated by calculating fluorescence units for each combination of ␣and ␤-chains (Fig. 2). As shown in Fig. 2, of the 11 Lys substitutions, only one, D57K␤, prevented surface expression of I-A d , TTGACTAAGGATTCAAATTTC Destroy DrdI a Variant polypeptides are designated by the amino acid substitution they carry; for example, D57K␤ represents the substitution of Asp-57 in the I-A d ␤-chain with Lys, using the single-letter code for amino acid residues and the I-A b numbering system (9). b (ϩ)-sense oligonucleotides used in mutagenesis are shown in 5Ј to 3Ј direction. Bases in bold are those that differ from the wild-type sequence. Only base changes in the variant codon, shown underlined, change the amino acid sequence; other changes are silent and were chosen to introduce or destroy a restriction site, shown in italic.
since neither the ␣-chain-reactive mAb K24 -199 nor any of the ␤-chain-reactive mAb recognized COS cells transfected with this variant. The remaining 10 Lys substitutions did not affect surface expression of I-A d since they all reacted with ␣-chain mAb K24 -199 and at least one ␤-chain mAb. Of these, seven (E51K␣, E59K␣, E70K␣, E74K␤, D76K␤, E84K␤, and E87K␤) had no effect on mAb reactivity: COS cells expressing these variant heterodimers stained with all mAb tested at intensities within a factor of three compared to COS cells expressing WT I-A d . In contrast, the remaining three substitutions (E59K␤, E66K␤, and E69K␤) eliminated recognition by one or more of the ␤-chain mAb. The E59K␤ heterodimer failed to stain with mAb BP107 and N22, the E66K␤ heterodimer failed to stain with mAb 34 -5-3S, and the E69K␤ heterodimer failed to stain with mAb MK-D6, 34 -5-3S, BP107, 28 -16-8S, and M5/114. Therefore, Glu-59␤, Glu-66␤, and Glu-69␤ are likely to be solvent accessible in I-A d , in agreement with the orientation of the corresponding residues in the human class II molecule HLA-DR1 (7,8). The loss of reactivity of mAb with these variant I-A d molecules agrees with previous reports demonstrating that the N-terminal half of the ␣-helix of the ␤1-domain of I-A molecules contributes to the epitopes of several mAb (for example, see Refs. 36,52). In addition, our results agree with previous epitope mapping and provide new or more detailed information regarding the reactivity of several mAb (see Table II).
We determined whether any of the 10 substitutions that were tolerated with respect to surface expression of I-A d affected the presentation of foreign peptides to an I-A d -restricted T cell hybridoma. Fig. 3A shows representative experiments indicating that COS cells expressing each of these variant heterodimers were able to present Ars-Ova(33-50) to D5h cells. Fig. 3B summarizes the results of several such experiments. When the slight differences in surface expression between the variant and WT I-A d heterodimers are taken into account (see Fig. 2), each of these 10 variant heterodimers were within 10-fold as efficient at peptide presentation as WT I-A d (Fig. 3B). These results demonstrate that although three of the 10 substitutions, including E59K␤, E66K␤, and E69K␤, completely eliminated recognition by at least one mAb, none profoundly affected peptide presentation.
Mutations at Asp-57␤ Diminish Surface Expression of I-A d and Presentation of Foreign Peptides-As shown above (Fig. 2), the D57K␤ substitution impaired surface expression of I-A d heterodimers. We further investigated the role of Asp-57␤ in surface expression of I-A d by substituting Ser, Ala, or Arg at  (Table II). I-A d mAb K24 -199 is ␣-chain specific, whereas the remaining I-A d mAb are ␤-chain specific. Fluorescence units were calculated for staining of the COS cells and are representative of several independent experiments. this position (Table I). Ser and Ala were chosen because these residues are found at position ␤-57 in several natural variants of murine and human class II molecules (9).  (Fig. 4B). Co-transfection of the invariant chain reproducibly enhanced surface staining of variant heterodimers D57S␤ and D57A␤ by mAb K24 -199, MK-D6, and M5/114. Enhancement of staining by mAb K24 -199 was 14fold (Ϯ 7-fold, n ϭ 4) for D57S␤ and 13-fold (Ϯ 5-fold, n ϭ 4) for D57A␤; by MK-D6 was 3.2-fold (Ϯ 0.7-fold, n ϭ 4) for D57S␤ and 2.3-fold (Ϯ 0.5-fold, n ϭ 3) for D57A␤; and by M5/114 was 3.6-fold (Ϯ 1.0-fold, n ϭ 4) for D57S␤ and 3.8-fold (Ϯ 1.1-fold, n ϭ 4) for D57A␤. In fact, when the invariant chain was cotransfected, surface staining of these variant heterodimers was nearly as intense as observed with WT I-A d (Fig. 4B); for D57S␤ and D57A␤ staining was 89% (Ϯ 15%, n ϭ 4) and 78% (Ϯ 26%, n ϭ 4), respectively, of wild-type levels using mAb M5/114. Co-expression of the invariant chain reproducibly enhanced expression of variants D57K␤ and D57R␤ to levels slightly above background (Fig. 4B); however, this effect was not observed in experiments in which the efficiency of COS cell transfection was low. Co-transfection with plasmid DNA encoding the chimeric cell surface molecule 2B4␤- (38) in place of invariant chain DNA did not restore surface expression of I-A d variants substituted at Asp-57␤ (Fig. 4B).
The relative efficiency of transfection of WT or variant DNA in these experiments was monitored by co-transfection of DNA encoding 2B4␤-. Surface expression of the reporter molecule 2B4␤was approximately equivalent when it was co-transfected with either WT or variant I-A d DNA (Fig. 4C), demonstrating that the decreased expression of these variants was not due to nonspecific effects such as toxicity or inhibition of the expression of cell surface molecules.
We tested whether the D57S␤ or D57A␤ substitution had any effect on the presentation of foreign peptides to I-A d -restricted T hybridomas in experiments in which surface expression of these variants was made equivalent to WT I-A d by co-transfecting the invariant chain. Fig. 5 shows that COS cells expressing variant heterodimers D57S␤ or D57A␤ were at least 25-fold less efficient at presentation of Ars-Ova (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49) peptide to the D5h T hybridoma compared to WT I-A d . These substitutions also diminished presentation of Ova(323-339) peptide to T hybridoma 3DO-54.8 and cI(12-26) peptide to 9C127 (data not shown), both of which recognize peptides presented by I-A d . These results demonstrate that the identity of the side chain at position 57 in the I-A d ␤-chain not only plays a role in surface expression but also influences peptide presentation.

Charge Reversal at Arg-80␣ Diminishes Surface Expression of I-A d and Presentation of Foreign Peptide but Compensates
Charge Reversal at Asp-57␤-In HLA-DR1, Asp-57␤ forms an interchain salt-bridge with Arg-76␣ (8) the residue of human class II molecules corresponding to Arg-80␣ of murine class II molecules (9) (see Fig. 9). Therefore, we tested whether substitutions at Arg-80␣ would also affect surface expression of I-A d by substituting Asp or Glu for Arg-80␣. Fig. 6 shows that the variant heterodimer R80D␣ was not expressed at levels above background and that the variant R80E␣ was expressed at

FIG. 3. Response of D5h T cell hybridoma to antigenic peptide presented by COS cells expressing wild-type or variant I-A d heterodimers.
In A, COS cells expressing WT or the indicated ␣-(top) or ␤-chain (bottom) variant I-A d were tested for the ability to present the antigenic peptide Ars-Ova (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50) to the I-A drestricted T cell hybridoma D5h. The difference in the potency of peptide presented by WT I-A d in the two experiments is due to variation in transfection efficiency and the response of D5h and IL-2 indicator cells. In B, the relative ability of variant heterodimers to present antigenic peptide to the D5 TCR in several experiments was calculated as described under "Materials and Methods" and is represented by solid circles (WT ϭ 1.0). Bars represent the geometic mean of these independent experiments. levels slightly above background; in experiments in which COS cell transfection was more efficient, greater surface expression of the variant R80E was observed. When the invariant chain was co-transfected, surface expression of variant heterodimers R80E␣ or R80D␣ was almost as great as WT I-A d (Fig. 6). These results suggest that Arg-80 in the I-A d ␣-chain also plays an important role in surface expression of class II molecules.
We tested whether charge reversal of Arg-80␣ could compensate the defect in surface expression of the charge-reversed variants of Asp-57␤. Fig. 6 shows that surface expression of the variant D57R␤ was partly restored when it was paired with the variant R80E␣, even in the absence of the invariant chain.
Surface expression of the variant D57R␤ paired with R80E␣ was further enhanced in the presence of the invariant chain. Similar results were obtained when R80E␣ was transfected along with D57K␤ or when R80D␣ was transfected with D57K␤ or D57R␤ (data not shown). Thus, the defect in surface expression of I-A d chains containing individual charge reversals at Asp-57␤ or Arg-80␣ can be partially reversed by expression of the partner charge-reversed chain, suggesting direct interaction between Arg-80␣ and Asp-57␤.

Surface Expression of Mutant I-A d Heterodimers Is
Restored at Reduced Temperature-Thus far we have identified two residues in the peptide-binding site of I-A d , the substitution of which results in poor surface expression of class II heterodimers. We suspected that the single-and double-site variants we had constructed were poorly expressed on the cell surface because they were defective in an important step in the transport of class II heterodimers to the cell surface. Because exogenously added foreign antigen has been demonstrated to enhance surface expression of normal class II molecules (23), we attempted, with limited success, to enhance the surface expression of these class II variants by culturing transfected COS cells in the presence of high concentrations of Ova(323-339) peptide antigen (data not shown). Subsequently, we tested whether culturing transfected COS cells at reduced temperature could enhance surface expression of variant heterodimers, since normal class I molecules (55) and class II heterodimers (22) that are apparently devoid of endogenous peptides can be expressed on the surface of insect cells, which are grown at 25°C. Also, at reduced temperature class I molecules appear on the surface of RMA-S cells (56), which do not supply foreign peptides to newly synthesized class I molecules and consequently do not express class I molecules on the cell surface at 37°C (18). Fig. 8 shows that singly substituted variant class II heterodimers containing charge reversal at Asp-57␤ or Arg-80␣, which were expressed poorly at 37°C in the absence of the invariant chain, appeared on the surface of COS cells cultured at room temperature. After room temperature incubation, the level of surface expression of singly substituted charge-re-versed variants R80E␣ or D57R␤ was identical to expression of the doubly substituted charge-swapped variant R80E␣/D57R␤ and WT I-A d heterodimers, as determined by staining with mAb MK-D6 (Fig. 8A) and ␣and ␤-chain reactive mAb (Fig.  8B). The increase in fluorescence of singly substituted chargereversed variants at room temperature was due to an increase in both the number of cells expressing I-A d as well as the intensity of those cells (Fig. 8A). On the other hand, the decrease in fluorescence of R80E␣/D57R␤ and WT heterodimers (Fig. 8B) was primarily due to the reduction of cells expressing I-A d at room temperature, rather than a decrease in the intensity of staining (Fig. 8A). It is important to note that the pattern of staining of heterodimers containing WT or variant ␣and ␤-chains expressed in COS cells at room temperature and at 37°C was similar when tested with the ␣and ␤-chainspecific mAb (Fig. 8B), demonstrating that the differences in temperature did not cause selective changes in the epitopes or conformations of I-A d molecules detectable by these mAb. These results demonstrate that surface expression of mutant class II molecules expressed poorly at 37°C in the absence of the invariant chain can be enhanced at reduced temperature. DISCUSSION We have conducted extensive mutational analysis of negatively charged residues in the peptide/TCR-binding domain of the class II molecule I-A d , which has been modeled onto the structure of HLA-DR1 (Fig. 9). We have demonstrated the importance of the ion pair Arg-80␣ and Asp-57␤ in both surface expression of and peptide presentation by class II heterodimers. Our results suggest that interactions in the peptidebinding site, including the formation of the salt-bridge between Arg-80␣ and Asp-57␤ and possibly peptide binding, are important for folding and surface expression of class II molecules at 37°C. These results agree with previous studies demonstrating that residues pointing into the peptide-binding site within the ␣1and ␤1-domains influence surface expression of class II molecules (32,36,57,58). The mutant class II molecules described here resemble haplotype-mismatched class II ␣and ␤-chains, which are partially blocked in egress to the medial Golgi compartment (40), and mutant heterodimers that are blocked in a relatively late stage of transport to the cell surface (57). We hypothesize that an important step in the transport of class II molecules to the cell surface can be blocked by disrupting the salt-bridge between Arg-80␣ and Asp-57␤ or the hydrogen bonds between these residues and bound peptides.
The ion pair Asp-57␤ and Arg-76␣, which corresponds to Arg-80␣ in murine class II ␣-chains, is present in nearly all class II molecules (9) and is likely to represent an important universal structural feature of class II molecules (8). Based on the structure of HLA-DR1 (8), the formation of this salt-bridge replaces the hydrogen-bonding network found at the end of the peptide-binding site of class I molecules in which the C termini of bound peptides are buried. Asp-57␤, present in the ␤-chain of most class II molecules, occupies a position analogous to Thr-143 conserved in all class I ␣2-domains; similarly, Arg-80␣, conserved in all ␣-chains of class II molecules, occupies a position analogous to Tyr-84, conserved throughout all class I ␣1domains (9). Together Tyr-84 and Thr-143 in class I heavy chains form hydrogen bonds with C termini of foreign and self-peptides (3)(4)(5)(6). The side chains of Asp-57␤ and Arg-80␣ in HLA-DR1 occupy roughly the same position as the side chains of Tyr-84 and Thr-143 in class I molecules, yet are located about 2 Å lower in the class II molecule due to slight changes in the ␣-helices (7). This lowering "opens" this end of the peptidebinding site of class II molecules, allowing longer peptides to bind and extend out of the site (7,8). In addition, Asp-57␤ and Arg-80␣ form hydrogen bonds with amide hydrogen and carbonyl oxygen atoms of bound peptides.
The interchain salt-bridge formed between Asp-57␤ and Arg-80␣ and the hydrogen bonds they form with the backbone of peptides bound to class II heterodimers might contribute a great deal to the stability of the class II heterodimer and its ability to bind peptides. The reversal of charge in singly substituted mutants D57R␤, D57K␤, R80E␣, and R80D␣ would bring together in close proximity similarly charged residues in the peptide-binding domains of assembling class II heterodimers, potentially destabilizing a portion of the interface between the ␣and ␤-chains and preventing these mutant class II heterodimers from adopting a conformation such that they can bind peptides efficiently. Similarly, the substitution of Ser or Ala for Asp-57␤ disrupts the salt-bridge, but unlike charge reversal at Asp-57␤, this substitution might be less disruptive because only one charged residue, Arg-80␣, remains unpaired. However, since the remaining unmutated charged residue is still available to hydrogen bond to the main chain of a bound peptide, the singly substituted mutant class II molecules might be stabilized to some degree when provided with peptide. The pairing of charge-swapped ␣-chains R80E␣ or R80D␣ with ␤-chains D57R␤ or D57K␤ restores a salt-bridge between ␣and ␤-chains; however, these variants might not bind peptides efficiently because hydrogen bonding between residues at these positions and carbonyl oxygen and amide hydrogen atoms of bound peptides is disrupted. Alternatively, the new chargeswapped salt-bridge might not be as strong as the original WT salt-bridge because the I-A d microenvironment that stabilizes the WT salt-bridge might destabilize the charge-swapped saltbridge, as predicted on theoretical grounds (59).
Because the substitutions for Arg-80␣ and Asp-57␤ affect both suface expression of I-A d and presentation of peptides to the TCR, we propose that the defect in efficient surface expression of the variant heterodimers results from intracellular editing of the heterodimers as incorrectly folded proteins. Class II ␣and ␤-chains are synthesized initially in the endoplasmic reticulum as heterodimers complexed with the invariant chain and devoid of tightly bound peptides (21). The invariant chain forms multimers with class II heterodimers (60), precludes the binding of endogenous peptides (61,62) and may, like low affinity peptide binding, prevent the aggregation and denaturation of peptide-free class II heterodimers at physiological temperature (24). The invariant chain directs the intracellular transport of class II heterodimers (63), which includes passage through the Golgi complex and subsequent entry into the endocytic pathway. In specialized endocytic compartments (64 -66), the invariant chain is proteolytically processed and dissociates from class II heterodimers, resulting in the stable binding of peptides to class II molecules and their transport to the cell surface. Class II heterodimers that fail to acquire peptide undergo aggregation and denaturation, are presumably recognized as mis-folded proteins, and are blocked from transport to the cell surface (23). The class II mutants described in this report that are defective in surface expression might be inactivated by aggregation, denaturation, or degradation more rapidly than WT I-A d heterodimers. In the presence of the invariant chain, surface expression of these variants is partly restored, perhaps because the invariant chain stabilizes the association of the nascent polypeptides, provides a peptide to the peptide-binding site, or enhances the transport of the variant heterodimers to a subcellular compartment where peptides can be supplied.
In addition, the mutant class II molecules described here resemble class I molecules that fail to appear on the surface of RMA-S cells at physiological temperature but reappear at the cell surface at reduced temperature (56). It is likely that at reduced temperature, both class I and II MHC molecules do not require bound peptide for surface expression because, when expressed in insect cells cultured at reduced temperature, they appear on the cell surface apparently devoid of bound peptide (22,55). Perhaps at reduced temperature, rates of inactivation of peptide-deficient MHC molecules and the mutant class II molecules described here are decreased. Our results support the hypothesis that both class I and II molecules might be best suited for surface expression at physiological temperature only when bound with peptide.
Finally, the importance of Arg-80␣ and Asp-57␤ in surface expression and the function of I-A d might explain why these residues are conserved throughout nearly all class II molecules. Several naturally occurring class II molecules have been identified that contain substitutions at position ␤-57, which are analogous to the variants D57S␤ and D57A␤ described in this report. It is noteworthy that these alleles have been implicated in autoimmune disease. Deviations from Asp-57 in human class II molecule ␤-chains have been observed in alleles associated with insulin-dependent diabetes mellitus, including DQw3.2, DQw2, DQw1.1, and DQw1.AZH, in which Ser, Val, or Ala has been substituted for Asp-57␤ (67). In the I-A ␤-chain of non-obese diabetic mice, Ser is substituted for Asp-57␤ (68). Although such alleles have other substitutions in addition to the change for Asp-57␤ that might account for the association of these alleles with diabetes, the identity of the side chain at ␤-chain position 57 appears to determine susceptibility and resistance to diabetes: the presence of Asp appears to be protective against diabetes (67). Expression of these variant forms of class II molecules might result in the presentation of an abnormal array of self-or foreign peptides or an altered conformation of class II molecule on the cell surface. Indeed, selfpeptides eluted from I-A g7 molecules of non-obese diabetic mice are reportedly distinct from other peptides that bind class II molecules in that they possess acidic C-terminal residues (69). Thus, antagonists directed at the peptide-binding site of the variant class II molecule might be designed that selectively inhibit presentation of these abnormal polypeptides. FIG. 9. Location of tested residues on a predicted map of I-A d . Shown is a ribbon diagram of the top view of the peptide-binding site of HLA-DR1 bound with an influenza virus peptide (8) onto which the residues of I-A d tested in this report were modeled, based on the sequence similarity shared among class II molecules (9). Substitution of Asp-57␤ with Lys or Arg impaired surface expression of I-A d heterodimers, whereas substitution of 10 other negatively charged residues with Lys had little or no effect on surface expression or presentation of foreign peptide to an I-A d -restricted TCR. The substitution with Glu or Asp for Arg-80␣, predicted to form a salt-bridge with Asp-57␤ in I-A d , also impaired surface expression of I-A d and its ability to present foreign peptides.