Crystal structure of HLA-A2 bound to LIR-1, a host and viral major histocompatibility complex receptor

Leukocyte immunoglobulin-like receptor 1 (LIR-1), an inhibitory receptor expressed on monocytes, dendritic cells and lymphocytes, regulates cellular function by binding a broad range of classical and nonclassical major histocompatibility complex (MHC) class I molecules, and the human cytomegalovirus MHC class I homolog UL18. Here we describe the 3.4-Å crystal structure of a complex between the LIR-1 D1D2 domains and the MHC class I molecule HLA-A2. LIR-1 contacts the mostly conserved ₂-microglobulin and 3 domains of HLA-A2. The LIR-1 binding site comprises residues at the interdomain hinge, and a patch at the D1 tip. The structure shows how LIR-1 recognizes UL18 and diverse MHC class I molecules, and indicates that a similar mode of MHC class I recognition is used by other LIR family members.

The LIR family (also known as the immunoglobulin-like transcripts (ILTs), monocyte/macrophage immunoglobulin-like receptors and CD85) comprises a set of immunoreceptors expressed on the surface of lymphoid and myeloid cells^{1, 2}. The LIRs, which are related to natural killer cell killer immunoglobulin-like receptors (KIRs) and the immunoglobulin A (IgA) receptor FcRI, are highly similar to one another, sharing 63−84% amino acid identity in their extracellular regions. All except LIR-4 are type 1 transmembrane proteins, containing either two or four immunoglobulin superfamily domains in their extracellular regions. One subset of cell surface LIR molecules (LIR-1, LIR-2, LIR-3, LIR-5 and LIR-8) transmits inhibitory signals through intracellular tyrosine-based inhibitory motifs, whereas another group (LIR-6, LIR-7, ILT7, ILT8, ILT10 and ILT11) transmits activatory signals by associating with signaling adaptor molecules^{1, 2}.

After being characterized as ILT2 (ref. 3), LIR-1 (the most broadly expressed LIR family member) was identified as the receptor for UL18 (ref. 4), a MHC class I homolog encoded by human cytomegalovirus⁵. UL18 shares 25% amino acid sequence identity with classical MHC class I molecules in the extracellular region, and associates with host-derived ₂-microglobulin (₂M)⁶ and with short peptides that show characteristics of those presented by classical MHC class I proteins⁷. LIR-1 and LIR-2 are also receptors for a broad range of MHC class I molecules, including classical (HLA-A, HLA-B and HLA-C) and nonclassical (HLA-E, HLA-F and HLA-G) molecules, which they bind with similar affinities (in the micromolar range, like several other low affinity cell-surface interactions) and kinetics^{8, 9}. In contrast, individual KIRs show allele-specific recognition of MHC class I molecules¹⁰. The structural nature of host MHC class I ligand recognition by LIR-1 and LIR-2 and the molecular basis of LIR-1 targeting by the human cytomegalovirus UL18 protein remain to be clarified. To address these issues and to gain information about ligand recognition by the LIR family of immunoreceptors, we solved the structure of the ligand-binding D1D2 fragment of LIR-1 in complex with a classical MHC class I ligand, HLA-A^*0201.

We determined the crystal structure of LIR-1 D1D2 bound to HLA-A^*0201 presenting a peptide derived from human immunodeficiency virus 1 Pol (residues 309−317; ILKEPVHGV) in space group P3₁21 to 3.4-Å resolution (R_cryst = 22.2%, R_free = 31.0%; Table 1 and Fig. 1a). We determined a 2F_o − F_c annealed omit electron density map relating to a portion of the LIR-1−HLA-A2 molecular interface (Fig. 1b). The D1D2 region of LIR-1 binds to MHC class I and UL18 with the same binding affinity as the full-length extracellular region (D1−D4)⁸. The structures of the proteins in the LIR-1−HLA-A2 complex resemble previously reported structures determined individually^{11, 12}: LIR-1 D1D2 contains two immunoglobulin superfamily domains arranged at a nearly perpendicular angle, and HLA-A2 includes a heavy chain with an 1-2 domain peptide-binding platform and a membrane-proximal 3 domain, which associates noncovalently with the ₂M light chain and bound peptide. Unlike T cell receptors (TCRs) and KIRs, which interact with the polymorphic peptide-binding platforms of MHC molecules^{10, 13}, LIR-1 D1D2 recognizes the side of the HLA-A2 molecule, forming two contact surfaces that encompass residues from the HLA-A2 3 domain, which is mainly nonpolymorphic, and ₂M, which is conserved (Fig. 1a and Table 2). The interaction is therefore consistent with recognition by LIR-1 of a broad range of MHC class I molecules in a peptide-independent way^{1, 2}.

Figure 1. Overall structure of the LIR-1−HLA-A2 complex. (a) Ribbon diagram of the LIR1 D1D2−HLA-A2 structure. Cysteines are shown in ball-and-stick form with disulfide bonds in yellow. C, C termini of HLA-A2 heavy chain and LIR-1 D1D2. Dashed lines, disordered regions (see Methods). (b) The D1D2−HLA-A2 model in the region of the 2M (cyan)−D1D2 (yellow) interface hinge contact area superimposed on a 3.4-Å SIGMAA-weighted 2Fo − Fc annealed omit electron density map contoured at 1.0 . (c) Conformational change in LIR-1 and KIR2DL1 after MHC class I binding. Comparison of the KIR2DL1−HLA-C14 and LIR-1−HLA-A2 complex structures. The D1 domains of KIR2DL1 (ref. 14) and LIR-1 D1D2 (green C representations) not in complexes are superimposed on the D1 domains of their bound counterparts (red C representations). In both complexes, the D1D2 interdomain angle is increased in the bound D1D2 structure compared with that of the free D1D2 structure. Full Figure and legend (44K)

Table 1. Data collection and refinement statistics for the LIR-1−HLA-A2 complex Full Table

Table 2. Amino acid contacts at the LIR-1−MHC class I interface Full Table

Although the structures of free HLA-A2 and HLA-A2 in a complex are not very different (r.m.s. deviation on all C atoms of 0.91 Å), comparison of the structures of free and bound LIR-1 D1D2 showed a change in the D1D2 interdomain angle (Fig. 1c). The angles between the long axes of the LIR-1 D1 and D2 domains were calculated as 84°, 85° and 90° in crystal structures of free LIR-1 D1D2 (ref. 11). When in a complex with HLA-A2, the LIR-1 D1D2 angle increases to 100°, indicating substantial flexibility of the D1D2 angle in the unbound state. Although crystal contacts could be involved in this interdomain shift, the interdomain angle in the co-crystal structure allows optimal contacts with both the 3 and ₂M domains of HLA-A2, and is therefore likely to reflect stabilization of a particular LIR-1 hinge region conformation that facilitates binding. A structurally analogous increase of similar size (10°) in the interdomain angle of KIR2DL1 noted after binding to HLA-Cw4 (Fig. 1c) was also attributed to optimization of domain orientation for ligand binding, and was not considered relevant to inhibitory signal initiation¹⁴.

There are two distinct contact areas in the LIR-1−HLA-A2 interface (Fig. 2): the A'CC'FG face at the tip of LIR-1 D1 contacts the HLA-A2 3 domain (with the exception of D1 residue Lys42, which contacts ₂M residue Asp96), and the LIR-1 D1-D2 interdomain hinge region contacts the ₂M domain. The contacting residues on the 3 domain are located at the end of strand A and in the A-B loop (residues 193−200) and the E strand (residue 248; Fig. 3a). Contact regions on ₂M are located toward the N-terminus (residues 1−4), in the F-G strand loop (residues 86−89) and in the G strand itself (residues 91−94, 96 and 99). The size of the LIR-1−HLA-A2 interface (1,700 Ų total buried solvent-accessible surface area) is marginally larger than those of KIR-MHC interfaces (1,500Ų; ref. 10) and similar to those of TCR-MHC complexes (1,700−1,900 Ų; ref. 13). Of the LIR-1 binding surface that is buried after complex formation (835 Ų), about 70% is involved in contacts with 14 ₂M residues, with the remainder contacting 6 3 domain residues (Table 2 and Fig. 2). This relative dominance of ₂M is unprecedented among HLA-binding immunoreceptors, including KIRs¹⁰, TCRs¹³ and CD8 (ref. 15), and is partly responsible for the broad recognition properties of LIR-1.

Figure 2. Interaction surfaces used by LIR-1 and HLA-A2. Grasp38 representations of the D1D2−HLA-A2 structure (center), with D1D2 contact sites (yellow) on HLA-A2 (left), and HLA-A2 contact sites (yellow) on D1D2 (right). Full Figure and legend (19K)

Figure 3. Amino acid contacts at the LIR-1−MHC class I interface. (a) Comparison of the LIR-1 binding epitope (residues in color) on the HLA 3 domain across classical and nonclassical MHC class I, UL18 and the MHC-like molecules FcRn, HFE and ZAG. Only MHC class I homolog proteins that have been tested for binding to LIR-1 are included. Red, blue and gold indicate nonconservative substitution, conservative substitution and conservation of the consensus sequence (purple), respectively. (b) Conservation of 3- and 2M-contacting residues across the LIR family. Receptor function: i, inhibitory; +, activatory. s, soluble receptor. Colors of amino acid alterations from the LIR-1 reference sequence are as in a, with deletions marked in red. Full Figure and legend (56K)

Previous studies have suggested that both 3 and ₂M interaction surfaces in the crystal structure contribute energetically to MHC class I and UL18 recognition. Domain-swapping experiments have shown that LIR-1 D1-D4 binding was abolished when the 3 domain of HFE was incorporated into UL18 and MHC class I proteins⁸, consistent with the idea that specific LIR-1 D1 contacts with 3 domain residues in the crystal structure are energetically important. Moreover, a proteolytic fragment of LIR-1 D1D2, (residues 1−99, referred to as D1, but containing residues 97−99 of the interdomain hinge region) bound UL18 and HLA-Cw0702 with only one-quarter to one-third the affinity that of D1D2 (ref. 8). The binding of the D1 fragment alone can now be understood as an interaction with the MHC class I 3 and ₂M domains, with the reduction in affinity being caused by the absence of one or more of the D2 residues in the interdomain hinge region (residues 100, 127, 184 and 187), which make contacts to ₂M in the case of D1D2.

To further rationalize the broad recognition of MHC class I by LIR-1, we examined the sequences of classical and nonclassical MHC class I molecules, as well as MHC class I−like molecules that do not bind LIR-1 (FcRn, HFE and ZAG)⁸ in the 3 domain regions identified from the LIR-1−HLA-A2 structure as making contacts with LIR-1 (3 residues 193−196, 198 and 248). With the exception of Ala193, these all involve amino acid side-chain-specific interactions. In all classical and nonclassical MHC class I molecules examined, residues Glu198 and Val248 are conserved, residue 193 is either a proline or an alanine, and residue 194 is an uncharged hydrophobic amino acid (Fig. 3a). In addition, residues 195−197 are conserved in HLA-A, HLA-B, HLA-C, HLA-E and HLA-F. The most divergent sequence is that of HLA-G, which contains two amino acid changes, at positions 195 (Ser to Phe) and 197 (His to Tyr). The positions and nonconservative nature of these changes indicate that they could affect interaction with HLA-G, consistent with the slightly higher (three- to fourfold) affinities of LIR-1 and LIR-2 for HLA-G relative to other MHC class I molecules⁹. In contrast to most MHC class I molecules, FcRn, HFE and ZAG each contain four to six amino acid changes, of which three to four are nonconservative, from the MHC class I consensus sequence in the contact residues. The comparison described above indicates that in addition to residues on ₂M, LIR-1 recognizes a sequence motif on the 3 domain that is essentially restricted to classical and nonclassical MHC class I molecules, and that 3 domain contacts provide essential energetic contributions to binding energy. Subtle differences in the ₂M and 3 domain orientation could also affect LIR-1 binding, although the relative positions of the ₂M and 3 domains are mostly conserved in available structures of MHC class I proteins, and of the MHC homologs FcRn and HFE.

Of the six LIR-1 amino acids that contact the HLA 3 domain, four (Arg36, Tyr38, Arg39 and Lys41) are conserved in LIR-2 (Fig. 3b), whereas alternative uncharged polar amino acids are substituted at positions 43 (Thr to Ser) and 76 (Tyr to Gln). Superposition of the D1 domain of each receptor indicates these residues occupy similar positions in LIR-1 and LIR-2. Conservation of interactions involving these residues is consistent with the similar affinities and broad specificity of both receptors for MHC class I molecules.

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The LIR-1−HLA-A2 structure can help in the interpretation of previous studies suggesting that LIR-1 uses a common binding interaction to recognize UL18 and MHC class I molecules⁸. Domain-swapping experiments have indicated that LIR-1 D1 and the MHC class I and UL18 3 domains are interaction sites⁸, as now verified for LIR-1 recognition of HLA-A2. Mutagenesis of LIR-1 identified four D1 residues (Tyr38 and one or more of Tyr76, Asp80 and Arg84) that affected binding to UL18 when substituted¹¹. Tyr38 and Tyr76 are involved in specific contacts with HLA-A2 3 domain residues in the region of positions 193−196 (Table 2a). Together with the fact that contacts between LIR-1 and ₂M are likely to be conserved in the viral homolog, and the finding that the presence or absence of bound peptide in UL18 does not affect binding to LIR-1 (ref. 8), these results indicate that the LIR-1−MHC class I and LIR-1−UL18 interaction interfaces are similar. If so, we can use the LIR-1 D1D2−HLA-A2 crystal structure as a first-order model for a LIR-1−UL18 complex. Whereas both cis and trans interactions with LIR-1 have been postulated as modes of UL18 action¹⁶, a similar binding mode would favor recognition in trans, such as UL18 on an human cytomegalovirus−infected cell engaging LIR-1 on an effector cell, or alternatively UL18−LIR-1 interaction between opposing membranes in intracellular compartments.

Despite the similarities in the binding of LIR-1 to UL18 and to MHC class I molecules, UL18 achieves an affinity in the nanomolar range, over 1,000-fold higher than that of LIR-1−MHC class I interactions⁸. When the 13 potential N-linked glycosylation sites of UL18 (ref. 5) are mapped onto the D1D2−HLA-A2 structure, the predicted LIR-1 binding site is one of the few contiguous surfaces that does not contain a potential glycosylation site (Fig. 4a), consistent with the observation that the nature of the carbohydrate attached to UL18 (complex or high-mannose carbohydrates) does not affect binding to LIR-1 (ref. 8). A more likely explanation for increased affinity for the viral homolog is a more favorable interaction of LIR-1 with amino acids on the 3 domain of UL18 than the comparable region of MHC class I proteins. Comparison of the UL18 sequence in the region analogous to the LIR-1-contacting residues in HLA-A2 (Fig. 3a) shows conservation of Asp196, a residue also conserved throughout classical and nonclassical MHC class I molecules, but substituted nonconservatively in FcRn, HFE and ZAG (Fig. 3a). In HLA-A2, this residue makes contacts with LIR-1 Tyr76, a residue that has been linked to the interaction with UL18 (ref. 11). The remaining five residues within the UL18 counterpart of the LIR-1 contact region on the HLA-A2 3 domain (residues 193−196, 198 and 248) differ between the UL18 and MHC class I sequences. Two substitutions are conservative (Ser to Asn at 195; Val to Ala at 248), whereas three are nonconservative (Pro to Asn at 193; Ile to Gln at 194; Glu to Arg at 198). These differences, perhaps together with an optimal orientation of the UL18 3 and ₂M domains, may contribute to the increased affinity of UL18 for LIR-1.

Figure 4. Implications of UL18−LIR-1 interaction. (a) View of D1D2−HLA-A2 structure with the approximate positions of potential UL18 glycosylation sites mapped onto the HLA-A2 structure. Potential N-linked glycosylation sites (pink spheres) are distant from the LIR-1 binding site. The closest predicted O-linked glycosylation site (gray sphere) is also distant from the binding site. Three additional O-linked glycosylation sites are predicted in UL18 in a region corresponding to sequence between the carboxyl terminus of the HLA-A2 ectodomain and the transmembrane region. (b) Closer view of the LIR-1−HLA-A2 interface in the region outlined by the dotted rectangle in a. The structures of free LIR-1 (ref. 11) and LIR-2 (ref. 17) are superimposed on the bound LIR-1 structure to show movement of the loop of residues 76−84 (mostly disordered in the bound LIR-1 structure (see Methods). The side chains of LIR-1 Asp80 (which have been linked to the binding of LIR-1 UL18; ref. 11) and its counterpart in LIR-2, Arg80, are shown as ball-and-stick representation. Full Figure and legend (32K)

UL18 is highly selective for LIR-1, binding over 3,000-fold more strongly then to LIR-2 (ref.11). The crystal structure of LIR-2 D1D2 identified conformational alterations relative to LIR-1 in both established elements of the UL18 binding site: an alternative rotamer conformation of Tyr38, and an 11-Å shift in the loop of residues 76−84 (ref. 17). When the structures of LIR-1 D1 and LIR-2 D1 not in complexes are superimposed onto LIR-1 D1 in the LIR-1−HLA-A2 complex structure (Fig. 4b and Methods), the LIR-1 loop of residues 76−84 (residues 78−83 are disordered in the LIR-1−HLA-A2 structure) is oriented toward the 3 domain. However, the main chain conformation of the LIR-2 loop (which contains a one-residue deletion and two glycine-to-nonglycine substitutions relative to LIR-1) is shifted 11 Å away from the contact interface, preventing interaction of residues 79−82 with the MHC class I (here representing UL18; Fig. 4b). In addition, the three residues in the D1 loop of residues 76−84 that have been linked to UL18−LIR-1 binding (Tyr76, Asp80 and Arg84) are substituted nonconservatively in LIR-2 (to Gln76, Arg80 and Trp83)¹¹. Consequently, even assuming considerable flexibility in this region of LIR-2, UL18 may interact preferentially with LIR-1 side chains at these positions, as indicated by the demonstration that a LIR-2 D1D2 protein containing LIR-1 residues at these positions bound UL18 10-fold more tightly than did wild-type LIR-2 D1D2 (ref. 11). Differences in the LIR-2 region of the loop of residues 76−84 may also provide an explanation for the slightly higher (three- to fourfold) affinities for LIR-1 binding to HLA-A, HLA-B, and HLA-C molecules compared with that for LIR-2 (ref. 9).

Comparison of the LIR-1 residues that interact with the HLA-A2 ₂M domain with other LIR receptors separates the LIR family members into two groups (Fig. 3b). Group 1 members, which include the inhibitory receptors LIR-1 and LIR-2, the soluble receptor LIR-4, and the activatory receptors LIR-6a, LIR-6b and LIR-7, show high conservation (10 of 13) of these residues. Group 2 members, which comprise LIR-3, LIR-5 and LIR-8 (all of which are likely to transmit inhibitory signals), and ILT7, ILT8 and ILT11 (not represented in the LIR family), show poor conservation (4 of 13) in which >85% of the changes are either nonconservative substitutions or deletions. Similar results are obtained by a comparison of the HLA-A2 3 domain−contacting residues conserved or substituted conservatively between LIR-1 and LIR-2 (residues 36, 38, 39, 41 and 43). Thus, the ligands of other group 1 members are likely to include MHC class I or MHC class I-like molecules noncovalently associated with ₂M, as already demonstrated for LIR-2 and LIR-6 (refs. 1,2,18). In contrast, group 2 members LIR-3, LIR-5 and LIR-8 seem unlikely to engage MHC class I proteins using a binding mode similar to that of LIR-1, and most probably engage a different set of ligands.

Figure 5. LIR-1−MHC class I interactions at the cell surface. Comparison of MHC class I complexes with KIR2DL1 (ref. 14), the B7 TCR40, LIR-1 (including homology models representing the structures of D3 and D4; ref. 8) and CD8 (ref. 15). Horizontal dashed lines indicate the approximate locations of cell surfaces to which the full-length forms of the proteins are attached. The LIR-1−MHC class I interaction is depicted in trans (as explained in text). Dotted lines, mucin-like regions of the CD8 ectodomains; solid horizontal lines, interchain disulfide bonds. The structure of a MHC class I complex with a KIR that contains three immunoglobulin-like domains has not been determined, but would be expected to result in a larger intermembrane distance than the KIR2DL1−HLA-C interaction indicated. Full Figure and legend (25K)

The LIR-1 D1D2−HLA-A2 structure shows no contacts with or conformational changes in the HLA-A2 1-2 peptide-binding platform; thus, simultaneous interaction of LIR-1 D1D2 and either a KIR or a TCR with a single MHC class I molecule should also be possible in principle. Either might be physiologically relevant, as LIR-1 is coexpressed at the cell surface with TCRs on MHC class I−restricted CD8⁺ T cells and with KIRs on subsets of natural killer cells²¹. In contrast, simultaneous binding of LIR-1 and the T-cell coreceptors CD8 or CD8 , which contact MHC class I 3 and ₂M domains¹⁵, would not be possible. Although the CD8 and LIR-1 binding sites on MHC class I are mainly nonoverlapping, steric effects would exclude binding of both LIR-1 and CD8 to the same MHC class I molecule (Fig. 5), as confirmed by binding studies⁹. Thus, LIR-1 may inhibit activation signals on MHC class I−restricted T cells by competing with CD8 for binding to MHC class I complexes engaged by the TCR, with the dual effect of preventing stimulatory signals transmitted by CD8 and of recruiting SHP-1 phosphatase to the vicinity of the TCR, thereby decreasing the half-lives of phosphorylated signaling components. In this way, LIR-1 could potentially act as a potent 'negative coreceptor' on MHC class I−restricted T cells or natural killer cells expressing LIR-1.

LIR proteins are encoded within the leukocyte receptor cluster, a region of human chromosome 19 that also includes the genes for KIRs and the IgA receptor FcRI (ref. 22). These genes have probably diverged from a common ancestor, resulting in receptor families that interact in different ways with structurally diverse ligands. LIR-1 and KIRs use the D1-D2 interdomain hinge to interact with MHC class I molecules, but only LIR-1 uses an additional binding surface located toward the membrane-distal tip of D1 (Fig. 6). FcRI also uses the tip of its D1 domain to bind to the Fc portion of IgA, but does not use the D1-D2 interdomain hinge region used by LIR-1, KIRs and other Fc receptors encoded outside of the leukocyte receptor cluster, such as the FcR and FcRI proteins²³ (Fig. 6).

Figure 6. Comparison of the ligand-binding sites on the structures of KIR2DL1 (ref. 14), LIR-1, and FcRI (ref. 23). The positions of residues within 4 Å of the binding partner (an MHC class I molecule for KIR2DL1 and LIR-1, and the Fc region of IgA for FcRI) are indicated as yellow (binding site within the D1D2 interdomain hinge) or blue (binding site within D1) spheres. The LIR-1 76-84 loop region is indicated in magenta; residues associated with binding to UL18 (Asp80 and Arg84) are also marked as blue spheres. Full Figure and legend (30K)

The use of two binding surfaces on LIR-1 establishes a molecular link to the recognition surfaces used by KIRs and FcRI in ligand binding, and raises questions as to the nature of the evolutionary relationships between these immunoreceptors. Based on gene structure and the existence of orthologous murine receptors (the paired immunoglobulin-like receptors²⁴), it seems likely LIRs evolved before KIRs. The homology of chicken immunoglobulin-like receptors to LIRs and paired immunoglobulin-like receptors supports the idea of the existence of a common ancestor pre-dating the separation of bird and mammalian lineages²⁵. In contrast, several features of the KIRs, including the lack of rodent orthologs, high similarity between different KIR loci and differences between chimpanzee and human KIR sequences, indicate a more rapid and recent evolution, restricted to primates^{25, 26, 27}. In common with KIRs, FcRI lacks a murine ortholog, favoring the idea that it originated more recently than the LIRs. These arguments, in combination with the current structural data, are consistent with the recent proposal that KIR and FcRI genes evolved from recombinations, duplications and shuffling events involving ancestral LIR genes²⁵. The presence of two (rather than one) binding surfaces on LIR-1 may therefore be characteristic of a more ancient receptor, which subsequently diverged into KIRs, preserving the use of the D1-D2 interface region, FcRI, which uses a more extensive D1 membrane-distal binding site, and LIR-1, preserving both interaction sites.

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The HLA-A2 complex (comprising residues 1−276 of the mature A2 heavy chain, noncovalently associated with ₂M and a nonamer peptide (ILKEPVHGV) derived from human immunodeficiency virus 1 Pol) and LIR-1 D1D2 (residues 1−198 of the mature protein) were produced using existing methods involving expression in Escherichia coli and dilution refolding²⁸. Renatured LIR-1 and HLA-A2 complex were concentrated separately, and were purified by size-exclusion chromatography using a Superdex 75 column. The purified proteins were concentrated and quantified by absorption measurements at 280 nm. Extinction coefficients (280 nm) of 66,150 M^-1cm^-1 (HLA-A2 complex) and 48,275 M^-1cm^-1 (LIR-1) were calculated using amino acid analysis and as described²⁹.

The structure was determined by molecular replacement using the program AmoRe³¹ and the coordinates of LIR-1 D1D2 and HLA-A2. Unambiguous solutions were found in the cross-rotation and translation functions for HLA-A2 and LIR-1 (R_cryst = 41%; R_free = 43%) for data between 20 Å and 4.0 Å. After four-domain rigid-body refinement with REFMAC5, as implemented in the CCP4 program suite³², rebuilding was accomplished with the program O (ref. 33) using 2F_o − F_c annealed omit maps (Fig. 1b), alternating with reciprocal space refinement in the crystallography and nuclear magnetic resonance system (CNS)³⁴. Final rounds of simulated annealing refinement and subsequently B factor refinement using grouped temperature factors in CNS³⁴ resulted in a final R_cryst of 22.2% (R_free = 31.0%) for all data between 20 Å and 3.4 Å (Table 1 and Supplementary Fig. 1 online). The HLA-A2−LIR-1 complex model consists of residues 1−276 of the HLA-A2 heavy chain; peptide residues 1−9; ₂M residues 1−16, 21−73 and 76−99; and LIR-1 residues 4−27, 32−77, 84−138 and 141−198. The side chains of residues 17, 82 and 268 of HLA-A2; 41 and 94 of ₂M; and 33, 34, 52, 53, 56, 57, 84, 86 and 87 of LIR-1 were disordered and modeled as alanine residues. Disulfide bonds are found between LIR-1 residues 26 and 75, 122 and 174, and 134 and 144; HLA-A2 residues 101 and 164, and 203 and 259; and ₂M residues 25 and 80. For analysis of interdomain angles, contacts and buried surface areas, D1 is defined as residues 1−98 and D2 is defined as residues 99−198. Interdomain contact residues were identified using the program CONTACT³², and were defined as residues containing an atom of 4.0 Å of the partner domain. Buried surface areas were calculated using SURFACE³² with a 1.4-Å probe radius. Interdomain angles were calculated using the program Dom_angle³⁵, which determines the angle between the long axes of adjacent domains that are approximated by ellipsoids calculated from the coordinates. Molscript³⁶, Raster3D³⁷, Grasp³⁸ and PyMOL³⁹ were used to prepare Figures 1−5.

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