Structural basis for recognition of the nonclassicalMHC molecule HLA-G by the leukocyte Ig-likereceptor B2 (LILRB2�LIR2�ILT4�CD85d)Mitsunori Shiroishi*, Kimiko Kuroki*, Linda Rasubala*, Kouhei Tsumoto†, Izumi Kumagai‡, Eiji Kurimoto§, Koichi Kato§,Daisuke Kohda*, and Katsumi Maenaka*¶

*Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; †Departmentof Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwanoha, Kashiwa, Chiba 277-8562, Japan; ‡Department ofBiomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 6-6-11-606, Sendai 980-8579, Japan; and §Graduate Schoolof Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan

Edited by Jack L. Strominger, Harvard University, Cambridge, MA, and approved August 29, 2006 (received for review June 22, 2006)

HLA-G is a nonclassical MHC class I (MHCI) molecule that cansuppress a wide range of immune responses in the maternal–fetalinterface. The human inhibitory immune receptors leukocyte Ig-like receptor (LILR) B1 [also called LIR1, Ig-like transcript 2 (ILT2), orCD85j] and LILRB2 (LIR2�ILT4�CD85d) preferentially recognizeHLA-G. HLA-G inherently exhibits various forms, including �2-microglobulin (�2m)-free and disulfide-linked dimer forms. Nota-bly, LILRB1 cannot recognize the �2m-free form of HLA-G orHLA-B27, but LILRB2 can recognize the �2m-free form of HLA-B27.To date, the structural basis for HLA-G�LILR recognition remains tobe examined. Here, we report the 2.5-Å resolution crystal structureof the LILRB2�HLA-G complex. LILRB2 exhibits an overlapping butdistinct MHCI recognition mode compared with LILRB1 and dom-inantly recognizes the hydrophobic site of the HLA-G �3 domain.NMR binding studies also confirmed these LILR recognition differ-ences on both conformed (heavy chain�peptide��2m) and freeforms of �2m. Binding studies using �2m-free MHCIs revealeddifferential �2m-dependent LILR-binding specificities. These resultssuggest that subtle structural differences between LILRB familymembers cause the distinct binding specificities to various forms ofHLA-G and other MHCIs, which may in turn regulate immunesuppression.

crystal structure � immune suppression � MHC class I � �2m-free MHC �maternal–fetal interface

In the maternal–fetal interface, special immune suppression sys-tems exist that allow the fetus to evade certain maternal immune

responses (1). HLA-G is a nonclassical MHC class I (MHCI)molecule that is expressed only on placenta trophoblasts and thymicepithelial cells. Trophoblast cells do not express major classicalMHCIs (HLA-A and -B) to avoid induction of T cell responses.Instead, these cells express HLA-G, -C, and -E to suppress thematernal immune responses by binding to inhibitory immunereceptors. The reported receptors for HLA-G are limited butinclude leukocyte Ig-like receptors (LILRs, also called LIR, Ig-liketranscript (ILT), or CD85), LILRB1 (LIR1�ILT2�CD85j), andLILRB2 (LIR2�ILT4�CD85d), as well as KIR2DL4 (killer cellIg-like receptor 2DL4), expressed on natural killer cells and someT cells. Because the LILRBs are expressed on a wide range ofleukocytes and mediate inhibitory signals, HLA-G is believed tohave a pivotal role in a broad range of immune suppressionfunctions in the placenta. Like other classical MHCIs, HLA-Gcomprises a heavy chain, �2-microglobulin (�2m), and an 8- to 10-aapeptide derived from proteolytically degraded proteins inside cells.In addition to the tissue expression pattern, HLA-G has a numberof unique characteristics. HLA-G shows very limited polymorphismand exists in several forms of spliced variants, including soluble anddomain-deleted forms. In addition, HLA-G forms a disulfide-linked dimer in solution and at the cell surface (2, 3). This dimermediates much more efficient inhibitory signals than HLA-G

monomers by binding to LILRB1 and LILRB2, as shown by cellularand biochemical studies (2–4). The recent crystal structures of theHLA-G monomer (5) and dimer (4) provided the structural basisfor efficient signaling. The HLA-G dimer exposes LILR-bindingsites that are easily accessible for receptor binding, enabling oneHLA-G dimer to bind two receptors simultaneously, which posi-tions the intracellular domains of the receptors close to each other,supporting high efficiency of signaling.

Interestingly, �2m-free heavy chains (fHCs) of HLA-G are foundin the placenta (6). fHCs of MHCIs are also expressed in someother tissues, including activated T cells and tumor cells. In addition,the pathological allele HLA-B27 exists as an unusual fHC ho-modimer form (7). Thus, fHCs are believed to have physiologicalroles in regulating immune responses. To date, only two receptorsfor fHC, have been shown to be members of the LILR familyLILRB2 and LILRA1 (LIR6�CD85i), by elucidating the binding ofHLA-B27 fHC tetramer to LILRB2- and LILRA1-transfected cells(8). Gonen-Gross et al. (6) showed that the fHC of HLA-G cannotbind to LILRB1, a close relative of LILRB2, but did not examinewhether the fHC of HLA-G can bind to LILRB2.

LILRs are encoded within the human leukocyte receptor com-plex 19q13.4 and are closely related to the killer cell Ig-likereceptors (KIR�CD158) (9). Although LILRB1 is widely expressedon monocytes, dendritic cells, B cells, and subsets of natural killerand T cells, LILRB2 is almost exclusively expressed on the my-elomonocytic lineage. Both LILRB1 and LILRB2 have immuno-receptor tyrosine-based inhibitory motifs in their cytoplasmic tailsto recruit the protein tyrosine phosphatase SHP-1, resulting in theinhibitory signaling. In addition to the maternal–fetus tolerance(10), LILRB2 is thought to be involved also in regulating thetolerogenic antigen-presenting cells (APCs) induced by regulatoryT cells (11) and CD68� macrophages and neutrophils in synoviumfrom rheumatoid arthritis patients (12).

Both LILRB1 and LILRB2 have four tandem Ig-like domains inthe extracellular region, and the N-terminal two Ig-like domains areresponsible for MHCI recognition. Killer cell Ig-like receptorfamily members recognize the peptide-binding region of MHCIsand thus exhibit some peptide selectivity (13). By contrast, ligands

Author contributions: M.S. and K.M. designed research; M.S., K. Kuroki, L.R., E.K., and K.M.performed research; K.T. and I.K. contributed new reagents�analytic tools; M.S., E.K.,K. Kato, D.K., and K.M. analyzed data; and M.S., K. Kato, and K.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Abbreviations: MHCI, MHC class I; LILR, leukocyte Ig-like receptor; SPR, surface plasmonresonance; �2m, �2-microglobulin; fHC, �2m-free heavy chain; APC, antigen-presentingcell; PIR, paired Ig-like receptor.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID code 2DYP).

¶To whom correspondence should be addressed. E-mail: kmaenaka@bioreg.kyushu-u.ac.jp.

© 2006 by The National Academy of Sciences of the USA

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for most LILR family members are yet to be identified, but LILRB1and LILRB2 have been shown to bind to a broad range of MHCIsby recognizing the �3 domain and �2m of MHCIs, both of which areconserved among classical and nonclassical MHCIs. As mentionedearlier, however, the ligand recognition of LILRB1 is different fromLILRB2 in terms of �2m dependency, even though the tworeceptors show high sequence identity (81%). On the other hand,our previous report showed that LILRB1 and LILRB2 show someallele-dependent recognition, such as preferential binding toHLA-G in comparison with other MHCIs (14).

The crystal structure of the LILRB1�HLA-A2 complex clearlyshowed that LILRB1 binds two sites, the �3 domain and �2m ofHLA-A2, simultaneously (15). As mentioned above, HLA-G has anumber of unique characteristics compared with other classicalMHCIs. In addition, LILRB2 exhibits different MHCI-bindingcharacteristics than LILRB1. However, the structural characteris-tics of any MHCI complex with LILRB2 and the structural char-acteristics of any receptor complex of HLA-G have yet to beelucidated. Here, we report the crystal structure of the LILRB2�HLA-G complex. The structure demonstrated that the bindinginterface of the LILRB2�HLA-G complex shows some overlapwith that of the LILRB1�HLA-A2 complex. However, the bindinginterface also shows some significant differences, which are ob-servable in both the crystal structure and NMR binding studiesusing 15N-labeled �2m. LILRB2 adopts a rather distinct bindingmode by using a small number of different amino acids fromLILRB1, resulting in predominant recognition of the �3 domainand providing the �2m-independent MHCI recognition that isdemonstrated by surface plasmon resonance (SPR) studies onLILRB2 binding to various forms of HLA-G and MHCIs. Morehydrophobic interactions were observed between LILRB2 and the�3 domain of HLA-G compared with the LILRB1�HLA-A2complex. It is reasonable to suggest that these interactions confera higher affinity for HLA-G. Based on these structural and func-tional results, the physiological roles for LILR recognition ofvarious forms of HLA-G as well as other MHCIs are discussed.

ResultsOverall Structure of the LILRB2 Complex with HLA-G. The crystalstructure of the complex of HLA-G and LILRB2 was solved bymolecular replacement [the program used was Amore; the searchprobes were the structures of HLA-G. Crystallographic data andrefinement statistics are summarized in Table 1, which is publishedas supporting information on the PNAS web site, and the 2Fo � Fcmap is shown in Fig. 1B. The overall complex structure demon-strated that LILRB2 recognizes the �3 and �2m domains of HLA-Gby using the N-terminal Ig-like domain (site 2) and the interdomainregion (site 1), respectively (Fig. 1A). At first glance, this complexstructure looks similar to the LILRB1�HLA-A2 complex reportedby Willcox et al. (15). Unexpectedly, however, Fig. 2 shows that therecognition modes of LILRB1 and LILRB2 are not the same, whichplausibly contributes to different ligand specificities as described inthe next paragraph.

Structural Comparison of the LILRB2�HLA-G and LILRB1�HLA-A2 Com-plexes. There are some major differences in the contact interfaceswith both the �3 domain and the �2m (details of the contactresidues are summarized in Table 2 and Fig. 5, which are publishedas supporting information on the PNAS web site). LILRB2 formsa more intimate contact interface with the �3 domain (red arrowsin Fig. 2 A and B). Fig. 2 C–F shows that LILRB2 interacts with amuch larger surface area of the �3 domain of HLA-G (site 2, 460Å2) and slightly larger surface area of �2m (site 1, 610 Å2) comparedwith the LILRB1�HLA-A2 complex (�3, 280 Å2; �2m, 570Å2). Thisfinding indicates that LILRB2 predominantly binds to the �3domain, whereas LILRB1 preferentially binds to �2m, which ex-plains why LILRB2 can bind to MHCIs with much less �2m

dependency than LILRB1 (as described under Different �2m De-pendency of MHCI Recognition of LILRBs).

In terms of specific amino acid interactions at the LILRB2–�2minterface (site 1), Val-183 of LILRB2 forms a hydrophobic inter-action with Trp-67, whose aromatic ring also forms a typical�–cation interaction with Lys-91 of �2m (Fig. 6A, which is pub-lished as supporting information on the PNAS web site). Bycontrast, Glu-184 of LILRB1 (the equivalent position of Val-183 ofLILRB2) forms a salt bridge with Lys-91 of �2m, causing thehydrophobic face of Trp-67 to rotate around the C�–C� bond to adifferent orientation to form the �–cation interaction with Lys-91of �2m as well as interactions with Ile-92 and Val-93 (Fig. 6B). Itcauses the distinct binding interfaces on �2m between LILRB1 andLILRB2: LILRB2 interacts with the region closer to the bottom ofthe �2 domain, whereas the LILRB1-binding interface is closer tothe membrane-proximal site (Fig. 2 C and E). This finding isconsistent with the NMR studies described in a later section (Fig.2 D and F). Notably, previous work has shown that LILRA1(LIR6�CD85i), like LILRB2, can bind to both conformed (heavychain�peptide��2m) and �2m-free HLA-B27 (8, 16) and also hasVal-183, suggesting that LILRA1 likely has a binding mode that issimilar to that of LILRB2. All other ‘‘group 1’’ LILRs except forLILRB1 contain Val-183 and are thus likely to adopt a LILRB2-type MHCI recognition mode if they can bind to any MHCIs.

Furthermore, the C�E loop (residues 153 and 154) of LILRB2makes contact with the N-terminal site of �2m, but that ofLILRB1 forms a 310 helix that is not involved in MHCI binding(Fig. 6 C and D). The FG loop (Asp-177 and Asn-179) ofLILRB2 can recognize the N-terminal site of �2m together withthe BC loop (residues 124–126). In contrast, LILRB1 uses onlythe BC loop for MHCI recognition (Fig. 6 C and D). Thus, thisinterface on �2m likely contributes to stability of the recognitionmode of LILRB2 in a manner distinct from that of LILRB1.

On the other hand, the �3 domain interacts with different facesof LILRB1 and LILRB2 (site 2, Figs. 1 and 2). LILRB1 mainly usesthe C strand for recognition of the �3 domain (e.g., Tyr-38, Arg-36,

Fig. 1. Overall structure of the LILRB2 complex of HLA-G. (A) Ribbon diagramof the LILRB2�HLA-G1 complex. Red, LILRB2; cyan, HLA-G heavy chain; green,�2m; blue, nonapeptide. LILRB2 uses two binding interfaces: D2-�2m (site 1) andD1-�3(site2)domain.Thestickmodelsof theaminoacids involved inthecomplexformation are shown as follows. Site 1: orange, D2 domain of LILRB2 (Trp-67,Asp-177, Asn-179, and Val-183); sky blue, �2m (Lys-6 and Lys-91). Site 2: magenta,D1 domain of LILRB2 (Arg-36, Tyr-38, Lys-42, Ile-47, and Thr-48); blue, �3 domain(Phe-195, Tyr-197, and Glu-229). The descriptions of site 1 and site 2 apply to Figs.1–4. (B) 2Fo � Fc map (green mesh) contoured at 1� onto the stick model of theLILRB2�HLA-G complex (around Phe-195 and Tyr-197 of HLA-G).

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etc.), with additional interactions formed by the F strand (Tyr-76)(Fig. 3 B and D; see also Fig. 7, which is published as supportinginformation on the PNAS web site). In contrast, LILRB2 accom-modates the membrane-proximal AB loop, including Phe-195 andTyr-197 of the HLA-G �3 domain, in a large cleft between the 310

helices and the C strand (Fig. 3 A and C), a space unoccupied in theLILRB1 complex (Fig. 3D).

Previous binding studies and structural reports of HLA-G pro-

posed that the hydrophobic amino acids Phe-195 and Tyr-197 formhydrophobic interactions with LILRB1 and LILRB2 (4, 5, 14). Thecurrent structure clearly showed that Phe-195 is located close to thehydrophobic faces of Thr-48 and Ile-47 of LILRB2 and that Tyr-197directly interacts with Arg-36 and Tyr-38 of LILRB2 (Fig. 3 A andC). In contrast, LILRB1 uses a different set of residues on the C andF strands, Tyr-76 and Tyr-38, to make contacts with the 195–197loop of HLA-A2 (Fig. 3 B and D). Assuming that HLA-G recog-

Fig. 2. Structural comparison of the LILRB2�HLA-Gand LILRB1�HLA-A2 complexes. (A) The LILRB2�HLA-Gcomplex is superimposed onto the LILRB1�HLA-A2complex around the MHCI regions. The molecular sur-face of HLA-G is shown. Cyan, HLA-G heavy chain;green, �2m; red, LILRB2; yellow, LILRB1. The D1 do-main of LILRB2 has more binding interface on the �3domain (site 2). The different interactions are ob-served in D2–�2m interfaces (site 1). (B) This compleximage is produced by rotating the image in A aroundthe horizontal axis �90°. (C and E) The buried surfaceareas of LILRB2�HLA-G (C) and LILRB1�HLA-A2 (E). Theburied surface areas were calculated by using the pro-gram SURFACE (CCP4 suite) with a 1.4-Å probe radius.Cyan, HLA heavy chain; green, �2m; red, LILRB1 orLILRB2. The buried surfaces are shown in yellow. (Dand F) NMR analysis of LILRB2–HLA-Cw7 and LILRB1–HLA-Cw7 interactions. The model orientation is similarto that of C and E. Mapping of amino acids whose1H-15N heteronuclear sequential quantum correlationpeaks were perturbed upon complex formation isshown (orange, �0.09 ppm chemical shift changed;magenta, �50% intensity reduced; red, disappeared;green, �0.09 ppm chemical shift changed; white, un-assigned). (D) Interaction of conformed (heavy chain�peptide��2m) (Left) and free (Right) forms of �2m withLILRB2. (F) The same binding analysis as in D withLILRB1.

Fig. 3. LILR binding interfaces (site 2) of the �3 domain of the LILRB2�HLA-G and LILRB1�HLA-A2 complexes. (A and C) LILRB2�HLA-G complex. (B and D)LILRB1�HLA-A2 complex. Cyan, HLA-G heavy chain; light blue, HLA-A2 heavy chain; green and light green, �2m; magenta, LILRB2; yellow, LILRB1. (A and B) Thebinding interface around the 195–197 loop of HLA-G. (C and D) The binding interface around the cleft between the first 310 helix and the C strand of LILRBs.

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nizes LILRB1 in a similar way to HLA-A2, Phe-195 is likely tointeract with the hydrophobic faces of Tyr-76 and Tyr-38, whereasTyr-197 may be too far from LILRB1 to be involved in directcontact. Therefore, Phe-195 and�or Tyr-197 induce the higherbinding affinity of HLA-G to LILRB2 and LILRB1, even thoughthe mechanisms may be different.

An additional HLA-G-binding site composed of Lys-42 onLILRB2 is observed at the interface with the �3 domain (Glu-229).In the LILRB1�HLA-A2 complex, there is no observable interac-tion between Lys-42 and the �3 domain at this location; instead,Lys-42 forms a salt bridge with Asp-96 of �2m (Table 2 and Figs.5 and 8, which are published as supporting information on thePNAS web site). Thus, this interaction could contribute to thedifferent recognition modes of LILRB2 and LILRB1.

Structural Changes Upon Complex Formation. Although LILRB1shows some interdomain angle changes (14–19°) upon complexformation, LILRB2 maintains the same interdomain angle (Figs. 9and 10, which are published as supporting information on the PNASweb site). Two 310 helices (residues 46–50 and 53–57) between theC and E strands of LILRB2 are formed upon HLA-G complexformation, whereas free LILRB2 shows only one 310 helix (residues52–55) (17) (Fig. 9 A and B). This conformation in the complexedstate of LILRB2 resembles that of LILRB1, even though theirMHCI-binding sites are not the same (Fig. 9C). In contrast, forbound HLA-G, the positional changes of only side chains and loopswithout significant conformational differences are observed incomparison with the free HLA-G structure (4, 5) (Fig. 11, which ispublished as supporting information on the PNAS web site). Ourpreliminary thermodynamic data showed that the LILRB2–HLA-G interaction tends to be entropic- and enthalpic-driven(K. Kuroki, M.S., L.R., K.T., I.K., D.K., and K.M., unpublisheddata), whereas the LILRB1–HLA-G interaction appears to beentropic-driven, as determined by van’t Hoff analysis and titrationcalorimetry (18). The LILRB1 free state already exhibits two 310helices and thus exhibits less conformational change in the localstructure being induced upon complex formation. Therefore, theconformational rearrangements of LILRB2 upon complex forma-tion may reflect some entropic loss in comparison with LILRB1.

NMR Analysis of LILRB1 and LILRB2 Binding to MHCI and �2m. Tofurther confirm the differences in MHCI binding of LILRB1 andLILRB2 directly in solution, NMR measurements were performed.We focused on a small subunit of MHCIs, �2m, because x-raycrystallographic data indicate that these two receptors embodydistinct modes of interactions with �2m in the MHCI complex. The�2m was expressed as a 15N-labeled protein and refolded with orwithout the nonlabeled heavy chain of MHCI and its cognatepeptide for identification of the LILR-binding interface. For NMRmeasurements, the HLA-Cw7 heterotrimer was used, because it ismuch more stable and less readily aggregated than HLA-G, andtherefore it is suitable for solution NMR analyses. 1H-15N hetero-nuclear single quantum coherence (HSQC) peaks originating from87 and 67 of 93 backbone amide groups of �2m were used asspectroscopic probes for the free and conformed (heavy chain�peptide��2m) forms, respectively. We successfully observed spec-

tral changes upon LILRB1 and LILRB2 complex formation. Fig. 2D and F Left show the amino acid residues of the conformed formof �2m that exhibited HSQC peaks significantly perturbed uponLILRB1 or LILRB2 complex formation. As expected from thecrystal structures of the LILR complexes, LILRB1 and LILRB2recognize �2m in solution, but their binding sites are different.The area of the LILRB1-binding site is widely distributed from the�2–�2m interface to the membrane-proximal site. In contrast, theLILRB2-binding site is located close to the �2 helix of MHCI,consistent with the crystal structure of the LILRB2 complexdescribed above. Furthermore, we also examined LILRB binding tothe free form of �2m. The results showed that the LILR-bindingsites of �2m are very similar to those of the conformed form (Fig.2 D and F Right), which indicates that LILRB1 and LILRB2 havethe ability to recognize �2m in a similar fashion either with orwithout heavy chain and peptide and suggests that the differentialLILR recognition modes are common to MHCIs and determined,at least partly, by LILR–�2m interactions.

Different �2m Dependency of MHCI Recognition of LILRBs. Ourprevious report of SPR analysis for LILRB–MHCI interactionsshowed that, unexpectedly, immobilization of MHCI (HLA-Cw7)by using a chemically biotinylated form of �2m inhibits binding toLILRB1 but not LILRB2 (14). Previous cellular-based assays haveshowed that the �2m-free homodimer form of recombinant HLA-B27 can bind to LILRB2-transfected but not to LILRB1-transfected cells (19). These results clearly indicated that LILRB1and LILRB2 have different ligand recognition modes in view of�2m dependency. Furthermore, the current crystallographic andNMR analyses clearly support this idea.

Here, detailed binding studies using a series of �2m-free MHCIforms were performed. The fHCs of HLA-G and HLA-Cw4 wereprepared following the standard refolding method for the �2m-associated MHCIs with the exception that no �2m was added. SPRanalysis showed that the immobilized fHCs of HLA-G1 and HLA-Cw4, which are not recognized by anti-�2m mAb BBM.1 or byanti-MHCI mAb w6�32, can bind to LILRB2 but not to LILRB1(Fig. 4 and Fig. 12, which is published as supporting information onthe PNAS web site). The �2m-associated MHCIs were also immo-bilized by means of the biotinylation tag of the heavy chain andacid-treated with a solution of 10 mM Gly�HCl (pH 2) to remove�2m. This method is common in the preparation of fHCs on the cellsurface. The acid-treated fHC was also able to bind to LILRB2 butnot to LILRB1 (data not shown). These results are consistent withthe cellular-based and SPR data described above.

DiscussionHLA-G is expressed at the maternal–fetal interface, where theexpression levels of classical MHCIs responsible for T cell responsesare reduced (20). There are several reports showing that solubleand membrane-bound forms of HLA-G induce a strong immuno-suppressive effect on a wide range of immune cells by binding toinhibitory immune cell receptors. To date, the reported receptorsfor HLA-G are LILRB1, LILRB2, KIR2DL4, and CD8. Ourpresent structural study provides a description of the complexedstates of both HLA-G and LILRB2. The structure showed that

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LILRB2‡ HLA-G1(red)‡ HLA-G1 fHC (green)

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Fig. 4. SPR analyses. Binding of LILRB2 (Left)and LILRB1 (Right) to HLA-G heterotrimer (redlines) and �2m-free HLA-G heavy chain (greenlines). Heterotrimers and �2m-free forms of MH-CIs were immobilized on the sensor chip at�2,000 response units (RU). Black lines show theresponses to the control protein (BSA).

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LILRB2 uses two sites, the tip of N-terminal domain and theinterdomain region, for binding to the �3 domain and �2m ofHLA-G, respectively. This binding mode is similar to that ofLILRB1 (Fig. 1); however, LILRB2 recognizes HLA-G in a distinctrecognition mode from that seen for the LILRB1�HLA-A2 com-plex (Fig. 2). The difference is derived from a surprisingly smallnumber of amino acid differences. LILRB2 uses the cleft betweenthe C strand and the 310 helices for binding to the 195–197 loop ofHLA-G, whereas LILRB1 uses the protruding site of the C strandwith a much smaller interface (Fig. 3). This structural characteristicexplains why LILRB2 binding to MHCI fHCs is of a sufficientaffinity to maintain the interaction between the two proteins,whereas the LILRB1 binding affinity is too low (Fig. 4). NMRbinding studies using 15N-labeled �2m in free and conformed(heavy chain�peptide��2m) forms demonstrated that the LILRB1-and LILRB2-binding sites on �2m are conserved in both forms,consistent with the complexed crystal structures (Fig. 2). This resultindicated that the LILR–�2m associations at least partly determineor support the variable recognition modes of the LILR�MHCIcomplex, achieved by the rearrangement of the interactions causedby only a few amino acid substitutions (e.g., Val-183) (Fig. 6).

HLA-G can bind to both LILRB1 and LILRB2 more stronglythan other classical and nonclassical MHCIs. Tyr-197 of HLA-Gdeeply intrudes into the hydrophobic pocket composed of Tyr-36and Arg-38 of LILRB2, forming typical �–� and �–cation inter-actions (Fig. 3) and providing a stronger and tighter associationthan His-197 of HLA-A2, which may form a somewhat weakerhydrophobic interaction with Tyr-36 (and Arg-38), explaining thehigher affinity of LILRB2 for HLA-G. Phe-195 of HLA-G faces thehydrophobic area comprising Ile-47 and Thr-48, whereas the equiv-alent Ser-195 of other classical MHCIs possibly excludes suchhydrophobic interactions at this location. However, the same sce-nario cannot be valid for the LILRB1�HLA-G interaction, becauseLILRB1 shows a different MHCI-binding interface compared withLILRB2. Based on the crystal structure of the LILRB1�HLA-A2complex (15), hydrophobic interactions can also be formed betweenTyr-38 and Tyr-76 of LILRB1 and Phe-195 of HLA-G, supportinga higher affinity for HLA-G. Our previous functional and structuralreport on the disulfide-bonded dimer form of conformed HLA-Gshowed that both LILRB1 and LILRB2 can provide significant highaffinity by avidity effects (4). Both the present LILRB2�HLA-Gcomplex structure and the previously reported LILRB1�HLA-A2complex structure ensure 1:2 (dimer:receptor) binding stoichiom-etry and structural orientation of LILRB�HLA-G dimer com-plexes, which facilitates the close location of its intracellular do-main, which is responsible for signal transduction, resulting inefficient signaling.

The human cytomegalovirus MHCI homologue UL18 can bindto LILRB1 with a remarkably high affinity (range of 3–20 nM) (21)that is �1,000 times higher than HLA-G�LILRB1-binding affinity.A mutational study introducing LILRB1 residues into LILRB2showed that the Gln-76-Tyr�Arg-80-Asp�Trp-83-Arg mutantLILRB2 binds UL18 with a Kd of 0.5–1 �M, 10–30 times higheraffinity than the wild-type LILRB2 (Kd � 12–14 �M) but muchlower than LILRB1 (22). These mutation sites are not directlyinvolved in the MHCI binding of LILRB2, as shown by the presentcomplex structure. Thus, they may have an indirect effect and�ordifferent binding mode that results in the preferable UL18 binding.In contrast, both the Tyr-76-Ala�Asp-80-Ala�Arg-80-Ala and Tyr-38-Ala mutant LILRB1s show lower affinity (Kd � 40 nM).However, the affinities are much higher than that describing theLILRB2�UL18 interaction, even though Tyr-38 is directly involvedin the MHCI binding. Thus, the other LILR-binding sites of UL18,such as the �2m interface, may dominantly contribute to highaffinity to the LILRB1 template structure. Moreover, the relativeorientation between �3 and �2m sites should fit the bindinginterface of LILRs to show high affinity. Therefore, UL18 can

provide more appropriate positioning and surface area of the �3and �2m sites for LILRB1 than for LILRB2.

We performed detailed binding studies using fHCs of HLA-Gand HLA-Cw4 to reveal the importance of �2m and the �3 domainfor binding to LILRB1 and LILRB2. The result showed thatLILRB1 cannot bind to the fHCs but that LILB2 can, consistentwith several previous reports. The physiological roles of fHC are notyet fully understood. However, it is of note that fHCs are induciblyexpressed on activation of T cells. LILRB2 on APCs can recognizefHCs, thus raising the possibility that LILRB2 can inhibit the APCfunction by binding to fHCs on activated T cells. Furthermore,because the LILRB2 expression is up-regulated on tolerogenicAPCs by regulatory T cells (11), it may be interesting to examinetheir fHC expression. The fHC of HLA-G is observed in theplacenta (6) and exhibits the same biochemical and structuralcharacteristics as fHCs of other MHCIs demonstrated in thepresent study, and, thus, it likely regulates APC function at thematernal–fetal interface.

The fHC of HLA-B27 is proposed to be directly associated withspondyloarthropathies. HLA-B27 forms a disulfide-linked (Cys-67-Cys-67) homodimer of fHC, which can bind to LILRB2- andLILRA1-transfected cells but not to LILRB1-transfected cells (19).The fHC dimer of HLA-B27 is likely to have a similar recognitionmode to the HLA-G dimer, possibly eliciting high affinity toefficiently transduce signaling (4). Further investigation is required,but it is plausible that the fHC regulation system may havesignificant physiological relevance.

Paired Ig-like receptor (PIR) B, a mouse homologue of LILRBs,can bind �2m with remarkably high affinity (approximately nano-molar range) and �2m-associated MHCIs with low affinity (ap-proximately micromolar range) (23). PIR-B can bind to the �2m-free fHC homodimer form of HLA-B27 (24), suggesting that PIR-Blikely has a function similar to LILRB2, although it is currentlyunknown whether PIR-B can bind to �2m-free fHC of mouseMHCIs.

Group 1 LILRs can be further categorized into LILRB1 andLILRB2 types with regard to MHCI-binding modes. The cleftbetween the C strand and the 310 helices is used as the MHCI-binding site on LILRB2 (site 2) but not on LILRB1. As describedabove, LILRA1 has similar amino acid composition to LILRB2 inthe cleft area and Val-183 regions, which are supposed to determinethe binding modes. Thus, LILRA1 is likely to belong to the LILRB2type of receptors, which is supported by the result that, likeLILRB2, LILRA1 can bind to the �2m-free B27 homodimer (19).The other group 1 LILRs also have the LILRB2-like amino acidsin these regions, similar to LILRA1. However, our recent data forthe structure of one of the ‘‘group 2’’ LILRs, LILRA5 (LIR9�ILT11�CD85f), showed that this cleft is filled by a C� strand that ischanged from the 310 helices (LILRB1 and LILRB2) to form the�-sheet with the C strand (25). Consistently, LILRA5 cannot bindto classical or nonclassical MHCIs (25). Furthermore, previouspreliminary reports demonstrated that the other group 2 LILRs,LILRB3 (19, 26), LILRB4 (27), and LILRB5 (28), cannot bind toMHCIs. Our speculation about the evolutionary aspect of the LILRfamily is that, initially, the ancestor receptor of PIRs�LILRsacquired MHC-binding function and subsequently evolved to dis-criminate between �2m-free (LILRB2 and LILRA1) and �2m-associated (LILRB1) MHCIs and to recognize unknown non-MHCI ligands (group 2 LILRs).

In conclusion, the LILRB2�HLA-G complex structure revealsthat LILRB2 shows remarkably distinct MHCI-binding recog-nition from LILRB1, binding more to the �3 domain than �2m.This finding could explain the �2m-independent MHCI bindingof LILRB2 demonstrated by the SPR binding studies, whichcontrasts with LILRB1. LILRB2 uses the cleft of D1 to tightlyassociate with the membrane-proximal loop of the HLA-G �3domain, including the aromatic amino acids Phe-195 and Tyr-197, which likely explains the high affinity of LILRB2 binding to

16416 � www.pnas.org�cgi�doi�10.1073�pnas.0605228103 Shiroishi et al.

HLA-G. The slight structural differences between members ofthe LILRB family achieve distinct ligand-binding specificities,including recognition of �2m-free MHCIs, which might regulateimmunologically relevant events. Moreover, because the MHCI-binding interfaces of LILRB2 are different from those ofLILRB1, LILRB1- or LILRB2-specific inhibitors can be de-signed for blocking either interaction.

MethodsPreparation of Recombinant Proteins. The refolded protein fragmentof the two N-terminal extracellular Ig-like domains (residues 1–196)of LILRB2 was prepared as described in ref. 14. Soluble HLA-GCys-42 Ser mutant was refolded from inclusion bodies of heavychain and �2m and the synthesized peptide (RIIPRHLQL) andpurified as described in ref. 14. The purified HLA-G and LILRB2were mixed at a 1:1 molar ratio at low concentrations (�1 mg�mlof each) and concentrated by using an Amicon Ultra concentrator(Millipore, Billerica, MA) to 7–10 mg�ml. The buffer was finallyexchanged to 20 mM Tris�HCl (pH 8.0)�100 mM NaCl for furtherexperiments.

Crystallization, Data Collection, and Structural Determination. Crys-tals suitable for data collection were obtained in the conditionof 0.1 M Tris�HCl (pH 8.0)�45–50% PEG 400 at 20°C by thehanging drop vapor-diffusion method. All of the data sets werecollected at 100 K. A 5-day soak in dehydration buffer (0.1 MTris�HCl, pH 8.0�60% PEG 400) yielded crystals that diffractedto 2.5 Å. The final data set was collected at beamline BL41XUat SPring-8. Data were processed and scaled with the HKL2000program package (29). The crystals belonged to the space groupP3121 (a � b � 81.4 Å, c � 186.7 Å, � � 120°). The structurewas determined by molecular replacement by using Amore in theCCP4 suite (30) with coordinates of HLA-G (PDB ID code2D31) and LILRB2 (PDB ID code 2GW5) as the search probes.One clear solution was found by using the reflections in the rangeof 20–3.5 Å. The structure was further refined with the individ-ual B factor and positional refinements of CNS (31) by using the50- to 2.5-Å reflections and alternating with manual rebuildingin the interactive graphics program O (32). The Rfree and Rworkof the final model were 27.9% and 23.5%, respectively. Inter-molecular contact atoms were identified by using the programCONTACT (CCP4 suite) (30). The distances of each pair of

atoms were determined by following the report of Sheriff et al.(33). Buried surface areas were calculated by using NACCESSwith a 1.4-Å probe radius. The figures of structural informationwere prepared by using PyMOL (35) and GRASP (36).

Binding Analysis Using NMR. Uniformly 15N-labeled �2m ([15N]�2m)was prepared from Escherichia coli BL21 cells carrying an expres-sion plasmid grown in M9 minimal medium containing 1 g�liter15NH4Cl and purified by the same method as the unlabeled protein.HLA-Cw7 associated with 15N-labeled �2m (HLA-Cw7�[15N]�2m)was prepared in the same way as normal MHCIs. [15N]�2m andHLA-Cw7�[15N]�2m dissolved in the buffer (10 mM sodium phos-phate, pH 6.5�10% 2H2O) were concentrated to 0.1 mM.

LILRB1 and LILRB2 were dissolved in the same buffer used for�2m. For the direct observation of LILR binding interfaces of �2mas either the free or conformed (heavy chain�peptide��2m) form,a series of 1H-15N heteronuclear sequential quantum correlationspectra at 2:1 (LILRB:�2m) or 1:1 (LILRB:HLA-Cw7) ratios weremeasured at 30°C with a DMX500 spectrometer (Bruker, Billerica,MA) equipped with a cryogenic probe.

Binding Analysis Using SPR. The soluble form of LILRB1 andC-terminal biotinylated MHCIs were prepared as described in refs.14 and 34. The soluble LILRB1 and LILRB2 were dissolved inHBS-EP buffer (10 mM Hepes, pH 7.4�150 mM NaCl�3.4 mMEDTA�0.005% Surfactant P20) (Biacore, Uppsala, Sweden). SPRexperiments were performed by using a BIAcore2000 system(Biacore AB). All of the biotinylated proteins were immobilized onthe CM5 sensor chip, on which streptavidin was covalently immo-bilized by amine coupling. Chemically biotinylated BSA was used asa control protein. The data were analyzed by using Biaevaluation4.1 (Biacore AB) and ORIGIN 5 (Microcal, Northampton, MA)software.

We thank A. Nakagawa, M. Kawamoto, H. Sakai, N. Shimizu, and K.Hasegawa for assistance in data collection at Spring-8. We also thank P.Bowness, P. J. Bjorkman, and B. E. Willcox for discussion. This work wassupported by a Japan Society for the Promotion of Science (JSPS) Post-doctoral Fellowship for Young Researchers (to M.S.); a JSPS PostdoctoralFellowship for Foreign Researchers (to L.R.); the Ministry of Education,Science, Sports, Culture, and Technology of Japan (K.M., D.K., K. Kato,and E.K.); and the Protein 3000 Project (K.M., D.K., and K. Kato).

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