NK Cell Inhibitory Receptor Ly-49C Residues Involved in MHC Class I Binding

Jonas Sundbäck, Adnane Achour, Jakob Michaëlsson, Hannah Lindström and Klas Kärre
J Immunol January 15, 2002, 168 (2) 793-800; DOI: https://doi.org/10.4049/jimmunol.168.2.793

Abstract

Mouse NK cells express Ly-49 receptors specific for classical MHC class I molecules. Several of the Ly-49 receptors have been characterized in terms of function and ligand specificity. However, the only Ly-49 receptor-ligand interaction previously described in detail is that between Ly-49A and H-2Dd, as studied by point mutations in the ligand and the crystal structure of the co-complex of these molecules. It is not known whether other Ly-49 receptors bind MHC class I in a similar manner as Ly-49A. Here we have studied the effect of mutations in Ly-49C on binding to the MHC class I molecules H-2Kb, H-2Db, and H-2Dd. The MHC class I molecules were used as soluble tetramers to stain transiently transfected 293T cells expressing the mutated Ly-49C receptors. Three of nine mutations in Ly-49C led to loss of MHC class I binding. The three Ly-49C mutations that affected MHC binding correspond to Ly-49A residues that are in contact or close to H-2Dd in the co-crystal, demonstrating that MHC class I binding by Ly-49C is dependent on residues in the same area as that used by Ly-49A for ligand contacts.

Natural killer cells express inhibitory receptors specific for MHC class I (1). Target cells lacking expression of a cognate MHC class I are killed by NK cells, in accordance with the missing self hypothesis (2). In the mouse, the major family of NK cell receptors specific for classical MHC class I molecules is the Ly-49 receptors (3). They belong to the C-type lectin-like family and are type II proteins. Even though the Ly-49 receptors are homologous to C-type lectins, they do not bind Ca2+ and lack the carbohydrate-binding motif present in other C-type lectins (4).

More than 23 genes of the Ly-49 family have been cloned, termed Ly-49a–w (5, 6, 7, 8, 9, 10, 11), with splicing variants and allelic variation among some of the family members. All Ly-49 family members are not present in a given mouse strain; C57BL/6 is the best-characterized mouse strain, where 14 Ly-49 genes, Ly49a–n, have been mapped to the NK gene complex on mouse chromosome 6 (9, 12, 13). Of these 14 C57BL/6 Ly-49 genes, only Ly-49A–J are expressed as full-length gene products. The Ly-49 family of molecules consists of both inhibitory and activating receptors, where the former have an immunoreceptor tyrosine-based inhibitory motif in their cytoplasmatic domains and the latter associate with the immunoreceptor tyrosine-based activation motif containing adaptor molecule DAP12 (14, 15). The Ly-49 receptors are expressed on overlapping subpopulations of NK cells and some T cells (16).

The function and ligand specificity has been determined for several of the Ly-49 receptors (17). Many of the receptors show broad specificity in that they bind several alleles of MHC class I. Ly-49A+ NK cells bind to and are functionally inhibited by cells expressing H-2Dd, H-2Dp, and H-2k (18, 19, 20, 21, 22), but expressed as a transgene on T cells, Ly-49A mediated binding and inhibition by all MHC haplotypes tested except H-2b (23). Ly-49C is functionally inhibited by H-2Kb but do bind the majority of H-2 class I alleles tested (7, 23, 24). Ly-49C and I are peptide-selective receptors (23, 25, 26), while Ly-49A is peptide dependent but not selective (27, 28, 29).

The aim of this study was to identify residues in Ly-49C potentially important for interaction with MHC class I molecules. A model of Ly-49C, based on the crystal structure of rat mannose binding protein A (rMBP-A)4 (30), was used to select residues to mutate. We introduced mutations in Ly-49C by substituting residues with the corresponding residues in Ly-49A. The mutated receptors were tested for binding to soluble MHC class I tetramers.

Several previous studies have addressed the Ly-49A-H-2Dd interaction either by mutagenesis of the ligand or by co-crystallization. The crystal structure of Ly-49A in complex with its ligand H-2Dd, published during this work (31), demonstrated two distinct sites of interaction between Ly-49A and H-2Dd termed site 1 and site 2. Site 1 had a relatively small binding area covering the glycosylation site at residue N176, while site 2 had a large binding area spanning the α1, α2, α3, and β2-microglobulin (β2m) domains of H-2Dd. Mutational studies of H-2Dd have suggested that site 2 is the predominant site of the Ly-49A-H-2Dd interaction (22, 32, 33, 34). The Ly-49C-MHC class I interaction has not been studied as extensively. Lian et al. (35) mutated Ly-49C substituting with corresponding Ly-49I residues and tested for binding to MHC class I transfected cells, where the mutation I226T had the most striking effect in that it did not bind any MHC class I molecule tested. This was also the only mutation that corresponded to a residue mutated in this study (K225N/I226T).

Three of the nine Ly-49C mutations made in this study, N160D/T162K, I195P/P196S, and K225N/I226T, severely decreased MHC class I binding. Our data demonstrate that Ly-49C binding to MHC class I involves residues that correspond to Ly-49A residues in contact with H-2Dd in the co-crystal.

Materials and Methods

Modeling

Models for the Ly-49A and Ly-49C NK inhibitory receptors were created on the basis of their 40 (Ly-49A) and 37% (Ly-49C) homology in the C-type lectin-like domain (CTLD) to the amino acid sequence of the carbohydrate recognition domain of rMBP-A (entry number 1 MSB in the Brookhaven Protein Data Bank (pdb)). The models will be described in detail elsewhere. Briefly, the residues Ly-49A148–256 and Ly-49C152–260 were aligned onto rMBP-A using disulfide constraints, and secondary structure prediction was used to confirm this alignment. The models were created with COMPOSER, part of the SYBYL program package (Tripos Associates, St. Louis, MO), and subsequently geometry-optimized using the following procedure. The backbone was fixed, and geometry optimization using the Powell algorithm was performed to a convergence point of 0.05 root mean square deviations in the total energy term. The dielectric model consisted of a distance-dependent dielectric cut-off of 9 Å and an ε value of 4 to simulate solvent effects with charges taken from the internal dictionary. All manipulations of molecules and energy minimizations were performed using the molecular modeling program SYBYL version 6.3. Atomic coordinates for the models of Ly-49A and Ly-49C will be made available on request.

Ly-49C mutations

The cDNA of Ly-49A (C57BL/6 allele) (36) and Ly-49C (A/Sn allele, identical with the BALB/c allele) (37) was subcloned into the eukaryotic expression vector pcDNA3 (Clontech Laboratories, Palo Alto, CA). Mutations N160D/T162K, K225N/I226T, and R234G in Ly-49C were created using the Transformer site-directed mutagenesis kit (Clontech Laboratories) with overlapping primers encoding the mutation. The other mutations in Ly-49C were created using the QuickChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) using overlapping primers encoding the mutation in Ly-49C-pcDNA3. All mutations were verified by sequencing.

Cells and transfections

293T, a human embryonal kidney cell line, was transiently transfected using either Lipofectamine Plus (Life Technologies, Gaithersburg, MD) or GenePorter (Gene Therapy Systems, San Diego, CA) according to accompanied protocol. Cells were stained with Abs and tetramers 2 days after transfection.

MHC class I tetramers

The production of soluble MHC class I H chain fused with a BirA substrate peptide (H-2KbBsp, H-2DbBsp, H-2DdBsp) and murine (m)β2m and the in vitro refolding of the MHC-Bsp/mβ2m/peptide complex have been described previously (38, 39). The generation of the H-2DdBsp construct has been previously reported (26). Synthetic peptides (HIV-1-derived P18-I10 (RGPGRAFVTI), OVA257–264 (SIINFEKL), Sendai virus (SEV) N324–332 (FAPGNYPAL), and lymphocytic choriomeningitis virus gp33 (KAVYNFATM)) were purchased from Research Genetics (Huntsville, AL). Briefly, MHCBsp, mβ2m, and peptide were refolded by dilution in the presence of leupeptin (2 μg/ml), pepstatin A (2 μg/ml), and PMSF (0.2 mM) for 48 h. The refolded complexes were purified by size exclusion chromatography on a Superose 12 10/30 column (Amersham Pharmacia Biotech, Piscataway, NJ), enzymatically biotinylated by incubation with BirA enzyme and biotin (Avidity, Denver, CO) according to instructions of the manufacturer. Free biotin was removed by gel filtration using NAP-5 desalting columns (Amersham Pharmacia Biotech). The MHC complexes were then quickly frozen and stored at −70°C. Tetramers were produced by mixing biotinylated MHC/mβ2m/peptide complexes with streptavidin-PE (Molecular Probes, Eugene, OR) at a 4:1 molar ratio. The percentage of biotinylated MHC molecules in each preparation was assessed by a gel shift assay and was >80%.

Abs and flow cytometry

A1 mAb (anti-Ly-49A) (40, 41) and SW5E6 mAb (anti-Ly-49C/I) (42, 43) were purified from hybridoma culture supernatants on a protein G column (Amersham Pharmacia Biotech). The mAb 4LO3311 (anti-Ly-49C) (43, 44) and the mAbs YE1/32 and YE1/48 (7) have been described previously. For staining, 106 cells were washed and resuspended in PBS with 1% FCS. All Abs were diluted in PBS with 1% FCS and used at concentrations of 2–20 μg/ml. Tetramers were used at a monomeric protein concentration of 20–30 μg/ml in PBS with 2% FCS. All incubations were performed for 60 min on ice. After the final washing, labeled cells were analyzed by flow cytometry in a FACScan (BD Biosciences, Mountain View, CA).

Results

Molecular models of Ly-49 receptors used for selection of residues possibly interacting with MHC class I

Ly-49A and Ly-49C show differences in ligand specificity. We compared the amino acid sequences of these receptors to identify residues in Ly-49 possibly involved in the interaction with MHC class I molecules. The carbohydrate recognition domain harbors the function and specificity of C-type lectins (reviewed in Ref. 45). Therefore, we speculated that residues within the corresponding CTLD of Ly-49 should affect MHC class I binding. There are 51 residues differing between Ly-49A and Ly-49C within the 125 amino acids encoding the CTLD. To predict residues pointing out toward the solution with the potential of direct contact with residues in MHC class I, we created molecular models of Ly-49A and -C based on the crystal structure of rMBP-A (30). We focused the analysis on residues differing in charge, size, or hydrophobicity between Ly-49A and Ly-49C. The residues were selected with the ambition to cover the whole CTLD. In total, three single and six double mutations were made, changing residues in Ly-49C to the corresponding residues in Ly-49A (Fig. 1⇓). The mutations were verified by sequencing in both directions and were cloned into the mammalian expression vector pcDNA3.

FIGURE 1.
FIGURE 1.

Ly-49C mutations. Residues that were mutated are indicated in a monomer of Ly-49C, modeled based on the crystal structure of rMBP-A (pdb entry 1 MSB; see Materials and Methods). The image was prepared with the Swiss PDB Viewer (59 ) and the Persistence of Vision Ray Tracer (www.povray.org).

Mutated Ly-49C receptors bind equally well to the 4LO331 mAb but differ in SW5E6 mAb binding

Our experimental strategy relied on the possibility of comparing the effect of the mutations to that of wild-type (wt) Ly-49C, and it was therefore necessary to obtain similar expression levels of the different molecules. Our initial attempts to establish stable transfectants yielded large variations in expression levels of Ly-49C, and we therefore switched to a transient expression strategy. Ly-49A and -C and the mutated Ly-49C genes were transiently transfected into the 293T cell line. This expression strategy yielded comparable levels of expression of the molecules, as determined by Ab staining in flow cytometry.

Because there was a possibility that the mutations in Ly-49C could affect not only binding to MHC class I molecules but also that to the Ab used to monitor receptor expression, two different anti-Ly-49C mAbs were used. Staining with the Ly-49C-specific mAb 4LO3311 demonstrated similar levels of expression of the mutated and wt Ly-49C receptors (Fig. 2⇓A). The 4LO3311 epitope has been mapped to position 129 in the stalk of Ly-49C (35), and the staining by this mAb is therefore unlikely to be affected by mutations in the CTLD. However, SW5E6 staining of the transfected 293T cells revealed a variable expression pattern of the mutated Ly-49C receptors (Fig. 2⇓B). One of the mutations in Ly-49C, I195P/P196S, resulted in the total loss of staining with the SW5E6 mAb, while staining of the N160D/T162K and K225N/I226T mutated receptors was low. We concluded that the transiently transfected mutated Ly-49C receptors were expressed at similar levels and that the residues I195/P196 are critically involved in SW5E6 mAb binding.

FIGURE 2.
FIGURE 2.

Cell surface expression of the mutated Ly-49C receptors. A, 293T cells were transiently transfected with the indicated Ly-49 cDNAs (filled histogram) or a negative control (expression vector only, open histogram). After 2 days cells were stained with the indicated mAbs. One representative experiment is shown. B, Summary of anti-Ly-49C mAbs staining of the mutated receptors. Data shown are the average of 5–13 independent experiments ± SE. ∗, p < 0.05; ∗∗, p < 0.01 (by paired t test). Staple shades mark wt Ly-49C and mutations that affect MHC binding.

Three of the mutated Ly-49C receptors show decreased binding to H-2Kb

To study the effect of the Ly-49C mutations in terms of H-2 specificity, we used soluble MHC class I tetramers to stain the transiently transfected Ly-49C receptors in flow cytometry. Four different MHC class I tetramers, H-2Kb-OVA, H-2Kb-SEV, H-2Db-gp33, and H-2Dd-P18-I10 were prepared.

Ly-49A does not bind to H-2Kb (23, 26). Ly-49C to Ly-49A mutations at residues involved in shaping the allele specificity of Ly-49C would therefore be expected to lead to the loss of H-2Kb binding. In a previous study we have demonstrated that function and binding of Ly-49C to H-2Kb is peptide selective; the H-2Kb molecules on RMA-S cells stabilized with the OVA peptide are more potent in inhibiting Ly-49C+ NK cells than RMA-S cells with H-2Kb-SEV peptide complexes (25). This finding was confirmed using H-2Kb tetramers refolded with each of these two peptides, showing that they differ in binding to Ly-49C-transfected cells (26). By using H-2Kb refolded with the two different peptides, it might be possible to identify Ly-49C mutations affecting the peptide-selective recognition (i.e., recognition of H-2Kb refolded with one peptide, but not the other).

The mutations N160D/T162K, I195P/P196S, and K225N/I226T in Ly-49C resulted in a profound decrease in binding of both the H-2Kb-OVA and -SEV tetramers (Fig. 3⇓). When comparing the relative staining pattern of the wt and mutated Ly-49C receptors with each H-2Kb-peptide tetramer, as expected, the H-2Kb-SEV tetramers stained the transfected cells to a lesser extent than the H-2Kb-OVA tetramers; binding of the H-2Kb-SEV tetramer to wt Ly-49C was 43% of the H-2Kb-OVA tetramer binding. However, the relative staining pattern with the different mutated Ly-49C receptors was the same regardless of the peptide used to refold H-2Kb. This showed that none of the mutations affected peptide selectivity in the recognition of H-2Kb by Ly-49C.

FIGURE 3.
FIGURE 3.

H-2Kb tetramer staining of the mutated Ly-49C receptors. H-2Kb tetramers refolded with either OVA (SIINFEKL) or SEV (FAPGNYPAL) peptides were used to stain the Ly-49 transiently transfected cells. The data shown are the average of 3–10 independent experiments ± SE. ∗, p < 0.05; ∗∗, p < 0.01 (by paired t test). Staple shades mark wt Ly-49C and mutations that affect MHC binding.

H-2Db tetramers bind the mutated Ly-49C receptors with a similar pattern as the H-2Kb tetramers

Ly-49C has been shown to bind both H-2Kb- and H-2Db-soluble tetramers (23, 26). If Ly-49C binds these two H-2 class I molecules in the same manner, the Ly-49C mutated receptors that affected H-2Kb tetramer binding should also influence H-2Db tetramer binding.

Staining of the Ly-49C-transfected cells with H-2Db-gp33 tetramers was comparable to the H-2Kb-OVA tetramer staining, but here the consequence was even more profound for two of the three mutations that affected H-2Kb binding (Fig. 4⇓). H-2Db tetramer staining of the I195P/P196S mutated receptor was completely lost, and staining of the K225N/I226T mutant was very low. H-2Db staining of the N160D/T162K mutated receptor was equivalent to that of H-2Kb-OVA (51 and 43%, respectively, of wt Ly-49C) but was significantly higher than the staining of the two mutated receptors mentioned above. Ly-49A-transfected 293T cells were stained with the H-2Db-gp33 tetramer to the same levels as wt Ly-49C, as previously reported (26).

FIGURE 4.
FIGURE 4.

H-2Db tetramer staining of the mutated Ly-49C receptors mirrored H-2Kb staining. H-2Db tetramers refolded with the lymphocytic choriomeningitis virus peptide gp33 (KAVYNFATM) were used to stain the transiently transfected cells. The data shown are average of three to nine independent experiments ± SE. ∗, p < 0.05; ∗∗, p < 0.01 (by paired t test). Staple shades mark wt Ly-49C and mutations that affect MHC binding.

None of the introduced Ly-49A residues is sufficient to allow H-2Dd binding by Ly-49C

Finally, we used H-2Dd-P18-I10-soluble tetramers to stain the mutated Ly-49C receptors. In our hands, H-2Dd binds to Ly-49A, but not C, as assessed by both tetramer binding and a cell-cell conjugation assay (26) (J. Sundbäck, unpublished observations). Because Ly-49C was mutated to the corresponding residues in Ly-49A, there was a possibility that the mutations would lead to a gain of H-2Dd binding.

The transiently transfected cells were stained with the Ly-49A-specific mAb A1 and H-2Dd-P18-I10 tetramers. As expected, Ly-49A-expressing cells were stained brightly by both, while neither bound to cells expressing wt Ly-49C. None of the transfected cells expressing mutated Ly-49C receptors were stained by the mAb A1 or the H-2Dd tetramers (data not shown), demonstrating that it was not sufficient to introduce these Ly-49A residues in Ly-49C to gain H-2Dd binding. To investigate whether the mutated Ly-49C receptors would be detected by other Ly-49A-specific mAbs, the transiently transfected cells were also stained with the mAbs YE1/32 and YE1/48. None of these mAbs stained the mutated receptors, while wt Ly-49A-transfected cells were stained brightly (data not shown).

Discussion

We have identified Ly-49C residues in three areas of the CTLD as important for the interaction with H-2Kb and H-2Db: N160/T162 located in the strand between the first β strand and the α1 helix, I195/P196 in the strand between the α2 helix and the β2 strand, and K225/I226 in the loop between the β2′ and β3 strands (secondary structure designation according to Ref. 31). The two latter mutations had an even more profound effect on Ly-49C binding to H-2Db. The reduced H-2Kb binding by the mutated Ly-49C receptors occurred regardless of the peptide used to refold this molecule. We also mapped the epitope of the anti-Ly-49C/I-specific Ab SW5E6 to residues I195/P196. In general, the H-2 tetramer binding pattern of the different mutated Ly-49C receptors mirrored the staining pattern with the SW5E6 mAb; SW5E6 blocks the Ly-49C-H-2 interaction, linking the mutations with the functional outcome of Ab blocking and H-2 class I binding. None of the Ly-49C mutations that severely reduced H-2 tetramer binding showed a significant difference in staining with the Ly-49C-specific mAb 4LO3311 compared with wt Ly-49C. It cannot be excluded that the correlation between loss of SW5E6 staining and tetramer binding to the mutated Ly-49C molecules was due to incorrect folding of the mutated molecules. However, according to the Ly-49C model all mutated residues projected out toward the solvent, and it is therefore unlikely that the mutations would affect refolding of the molecules. In a previous study it was shown that the stalk region in Ly-49C was necessary to gain receptor specificity (20). We observed decreased ligand binding by mutations in the CTLD of Ly-49C. It is possible that the correct stalk region is needed for proper dimerization of the Ly-49C receptor to gain full binding specificity. Nevertheless, Ly-49C molecules with the stalk from Ly-49A (which is shorter) still bound several MHC class I alleles (20). We observed reduction of binding to all MHC class I alleles tested by some of the Ly-49C molecules mutated in the CTLD, indicating that these mutated residues were directly involved in the interaction with the ligands.

Because a molecular model of Ly-49C was used to select the mutations, it may first be pertinent to compare the model with the crystal structure of Ly-49A, which was published during the course of this study (31). The general structure of the Ly-49C model was very similar to that of Ly-49A in the crystal, with a close match in the α1 helixes and the β strand backbone atoms (data not shown). The greatest divergences between main chain atoms were located in the loop between the β2′ and β3 strands and a shift in the α2 helix. The high homology between the Ly-49C model and the Ly-49A crystal shows that the model was a good foundation for selection of residues to mutate, as confirmed in the functional tests of the mutations.

Five of the nine mutations introduced in Ly-49C involved residues that, when compared with the corresponding residues of Ly-49A, form hydrogen bonds to H-2Dd in the Ly-49A-H-2Dd crystal. Ly-49A has two contact areas with H-2Dd in this co-crystal, site 1 and site 2. Among these five mutations, three are involved in hydrogen bonding at both sites (K161R, D248N/N250D, and I251Q), and two are involved in hydrogen bonding to site 2 exclusively (A169Q/N170T and I195P/P196S; see Table I⇓). The difference in residue numbers of the corresponding amino acids of Ly-49A and Ly-49C is due to a 4-aa-longer stalk region of Ly-49C.

View this table:
Table I.

Ly-49A residues interacting with H-2Dd in the co-crystal in comparison with the Ly-49C mutationsa

The Ly-49C mutations that led to reduced binding of MHC class I were all involved in or close to site 2 interactions; I195P/P196S corresponds to a site 2-only hydrogen bonding residue, K225N/I226T is close to the site 2 and distant to the site 1 binding site, and the mutation N160D/T162K is involved in both site 1 and site 2 interactions according to the Ly-49A-H-2Dd crystal (Fig. 5⇓). Therefore, it is most likely that Ly-49C is using the same receptor contact area as Ly-49A to bind H-2 class I, but the question remains of whether the two receptors are binding to H-2 at the same site(s). Further studies of mutations in the Ly-49C ligand(s) or crystallization of a co-complex between Ly-49C and, e.g., H-2Kb need to be performed to answer this question.

FIGURE 5.
FIGURE 5.

Model of Ly-49C and H-2Kb-OVA based on the crystal structure of Ly-49A-H-2Dd. Site 1 and site 2 interactions are shown. The H-2Kb H chain is yellow, β2m is gray, the peptide is orange, the Ly-49C-1 subunit is blue, and Ly-49C-2 is light blue. Ly-49C mutated residues N160D/T162K are shown in orange, I195/P196 are shown in green, and K225/I226 are shown in red. Mutated residues that do not affect the Ly-49C-H-2Kb interaction are shown as ball-and-stick molecules in backbone color. Ly-49C was threaded on the crystal structure of Ly-49A and was overlaid in the same orientation as Ly-49A in the Ly-49A-H-2Dd structure (pdb entry 1QO3). The crystal structure of H-2Kb-OVA (pdb entry 1VAC) was aligned with the α1α2 domains of H-2Dd in the crystal. The modeling and image were prepared using the Swiss PDB Viewer (59 ), and the image was refined with the Persistence of Vision Ray Tracer (www.povray.org).

The I195P/P196S mutation corresponds to the Ly-49A residue S192 that is hydrogen bonded to the amino-terminal part of the α2 domain of H-2Dd in site 2. H-2Db tetramer binding to this mutant was more radically affected than H-2Kb tetramer binding. This was also observed for the K225N/I226T mutated receptor. The Ly-49C-H-2Kb interaction leads to inhibition of effector cells (24), while the functional importance of the Ly-49C-H-2Db interaction is more controversial in the literature. The former interaction may thus be more robust and more difficult to completely disrupt by mutations of these residues. The I195P/P196S mutation also abolished SW5E6 binding, revealing the epitope for this mAb.

None of the K225 or I226 residues correspond to Ly-49A hydrogen bonding residues according to the crystal. However, K225 is in reach of the α2 domain of H-2 class I in site 2, where it builds up a shelf together with residues R227 and K228 under the H-2 α2 domain loop103–109. In another study of mutations in Ly-49C performed by Lian et al., (35) position I226 was mutated to a threonine. This mutation led to general loss of MHC class I binding in a cell-cell conjugation assay, suggesting that the K225N/I226T and I226T mutations disrupt the conformation of Ly-49C in the area built up by residues K225, R227, and K228, and that these residues are involved in the Ly-49C interaction with H-2 class I at a site 2 interaction.

The N160D/T162K mutation does not correspond to residues contacting H-2 according to the Ly-49A-H-2Dd crystal. However, these two residues surround residue K161, which corresponds to the hydrogen-bonding residue R157 in Ly-49A. R157 is involved in both site 1 and site 2 interactions. MHC class I tetramer staining of the N160D/T162K mutated Ly-49C receptor is reduced to about half that of wt Ly-49C, but the reduction in MHC class I tetramer binding by the N160D/T162K mutant was not as great as that for the I195P/P196S and K225N/I226T mutated receptors; particularly, H-2Db-gp33 binding was significantly higher compared with that of the two other mutants. The less dramatic decrease in MHC class I binding by the N160D/T162K mutated Ly-49C receptors could be due to the fact that these residues are not involved in direct contact with H-2 class I. It should be mentioned in this context that when the residue K161 was mutated to an arginine, we saw no reduction in H-2 binding. The lack of effect of the K161R mutated receptor could be explained by the fact that the size difference between lysine and arginine (both are positively charged) may not be sufficient to disturb the interaction with H-2 class I.

Six of the nine Ly-49C mutations did not affect MHC class I tetramer binding, of which the K161R, A169Q/N170T, D248N/N250D, and I251Q mutations correspond to residues in Ly-49A that form hydrogen bonds to H-2Dd in the co-crystal (Table I⇑). The arginine at position 234 in Ly-49C, which was mutated to a glycine with no decrease in H-2 binding, also has potential to contact the H-2 class I complex. On the average, MHC tetramer binding was reduced to <25% compared with wt Ly-49C for these mutations.

The Ly-49C residues K161, R234, D248/N250, and I251 are all involved in forming the site 1 binding site when Ly-49C is modeled according to the Ly-49A-H-2Dd crystal. At site 2 residues K161 and R234 could interact with the β2m subunit, and residues A169/N170, D248/N250, and I251 interact with the α3 domain of the H-2 class I complex according to the crystal. The lack of effect of these mutated receptors raises the question of whether Ly-49C is binding MHC class I at a different site compared with Ly-49A. If Ly-49C is binding to MHC class I in a similar manner as Ly-49A, it must be concluded that each of these mutations alone is not sufficient to interfere with the binding. None of the mutated Ly-49C receptors presumably involved in site 1 interactions affected MHC class I tetramer binding, except one, N160D/T162K (also involved in site 2 interaction). If Ly-49C is binding H-2 class I at similar sites as Ly-49A, this implies that MHC tetramer binding can be accounted for primarily by site 2 interactions, as concluded from studies of Ly-49A binding to mutated H-2Dd molecules (32). At site 2, the mutated Ly-49C receptors that did not decrease MHC binding are presumably binding the α3 domain of the H-2 H chain, while the mutated receptors that reduced binding are presumably contacting α2 domain residues. This is consistent with the idea that it is sufficient to exchange the α2 domain of H-2Dd to lose interaction with Ly-49A (46). It should also be noted that the α3 domain is flexible, as demonstrated by several H-2 class I crystal structures (31, 47, 48, 49, 50), opening the possibility for an altered interaction with this domain of H-2 by Ly-49C compared with the Ly-49A-H-2Dd interaction.

The H chain and β2m components of MHC class I tetramers were produced as recombinant unglycosylated proteins in an Escherichia coli expression system. Binding of these soluble MHC class I molecules to Ly-49 receptors proves that carbohydrates are not necessary for binding, as previously concluded (23, 26, 29). However, this does not exclude a positive or negative influence of glycans. It appears that the oligosaccharide at position N176 in H-2Dd can influence at least binding to Ly-49A, while the oligosaccharide at position N86 does not interfere with the Ly-49A-H-2Dd interaction (33, 51, 52). An N-acetylglucosamine and a fucose residue bound to N176 have been modeled to fit well into the polar open cavity at site 1 of the co-crystal of Ly-49A and H-2Dd (31). Interestingly, the Ly-49A residue S192, corresponding to P196 in the I195P/P196S mutation, is located on the flat surface of Ly-49 where the rest of the oligosaccharide can establish further interactions. Oligosaccharides may be of importance for Ly-49 binding to H-2 class I molecules. For example, Hanke et al. (23) used glycosylated, insect cell-produced tetramers and demonstrated that H-2Dd bound Ly-49C, which we did not observe with our nonglycosylated H-2Dd tetramers.

Treatment of Ly-49A- or Ly-49C-expressing cells with the sulfated polysaccharide fucoidan blocks Ab and MHC class I binding (51, 53, 54). The carbohydrates on H-2Dd are sulfated, and inhibiting metabolic sulfation reduced binding to immobilized Ly-49A (55). The Ly-49C mutations I195P/P196S in this study and N198S in a previous report (35) led to loss of SW5E6 binding, and the I195P/P196S mutation also led to loss of MHC class I binding, suggesting that this region overlaps with the sulfated carbohydrate binding region, which remains to be tested.

As mentioned above, one difference between Ly-49C and Ly-49A in binding MHC class I is the peptide selectivity of Ly-49C, which is not seen for Ly-49A. Three of the peptides used in this context (Refs. 25 and 26 and this study) have been crystallized in complex with H-2Kb by Wilson et al. (49, 56). When comparing the crystal structure of H-2Kb-pOVA (pdb code 1VAC; allowing efficient Ly-49C binding) with the H-2Kb-pSEV (pdb code 2VAB) and H-2Kb-pVSV (pdb code 2VAA) crystals (both less permissive for Ly-49C binding), the most striking difference is located in the α2 domain loop built up by residues 103–109. The backbone atoms of this loop103–109 have a 1.8-Å root mean square deviation (2.4 Å for residue D106) in the H-2Kb-OVA crystal compared with the two other crystal structures (which are alike in this loop), where the loop is shifted down toward the site 2 binding site of Ly-49, demonstrating the flexibility of this loop. Simultaneous mutation of residues 103, 104, and 107 in this loop of H-2Dd reduce binding to Ly-49A (57), whereas single mutations of residues 104 and 107 did not affect the interaction with Ly-49A (33, 58). The Ly-49C mutation K225N/I226T that showed reduced MHC class I binding is close to this loop when Ly-49C is modeled onto the Ly-49A-H-2Dd co-crystal.

Another explanation for the peptide selectivity of Ly-49C has been proposed by Matsumoto et al. (32); the peptide selectivity of Ly-49C has been mapped to position 7 in the peptide (25). Changes in this position may influence the amino-terminal end of the α2 helix of H-2 class I, where Ly-49A binds at site 2. S192 in Ly-49A binds H-2Dd at this site, which corresponds to the Ly-49C residue P196 present in the I195P/P196S mutation that led to reduced MHC class I tetramer binding in this study.

In summary, our data demonstrate that Ly-49C residues I195/P195, K225/I226, and N160/T162 affect binding to MHC class I, and that these residues correspond to Ly-49A residues that are either in direct contact or close to its ligand in the co-crystal between Ly-49A and H-2Dd. This suggests that Ly-49C binding to MHC class I is similar to the Ly-49A-H-2Dd interaction.

Acknowledgments

We thank Dr. S. Lemieux for the mAb 4LO3311, Drs. J. Brennan and F. Takei for the YE1/32 and YE1/48 hybridomas, Dr. O. Kanagawa for the A1 hybridoma, Drs. C. Sentman and V. Kumar for the SW5E6 hybridoma, Dr. J. D. Altman for the H-2Kb construct, and Dr. T. N. M. Schumacher for the H-2Db construct.

Footnotes

  • ↵1 This work was supported by the Swedish Cancer Society, Karolinska Institute, Swedish Foundation of Strategic Research, Swedish Medical Research Council, Swedish Society for Medical Research, Robert Lundberg Memorial Foundation, and Lars Hierta Memorial Foundation. J.M. and A.A. are supported by the Network for Inflammation Research funded by the Swedish Foundation for Strategic Research.

  • ↵2 Address correspondence and reprint requests to Dr. Jonas Sundbäck, Microbiology and Tumor Biology Center, Karolinska Institute, Nobels väg 16, S-171 77 Stockholm, Sweden. E-mail address: jonas.sundback{at}mtc.ki.se

  • ↵3 Current address: Center for Molecular Medicine, Karolinska Hospital, Stockholm, Sweden.

  • ↵4 Abbreviations used in this paper: rMBP-A, rat mannose binding protein A; CTLD, C-type lectin-like domain; β2m, β2-microglobulin; m, murine; pdb, Brookhaven Protein Data Bank; wt, wild type, SEV, Sendai virus.

  • Received October 22, 2001.
  • Accepted November 13, 2001.

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