HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 28, 19536-19544, July 14, 2006

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 281/28/19536    most recent M603076200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shiroishi, M. Articles by Maenaka, K. Search for Related Content PubMed PubMed Citation Articles by Shiroishi, M. Articles by Maenaka, K. Social Bookmarking                      What's this?

Crystal Structure of the Human Monocyte-activating Receptor, "Group 2" Leukocyte Ig-like Receptor A5 (LILRA5/LIR9/ILT11)^*

From the Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

Received for publication, March 31, 2006 , and in revised form, April 26, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Human leukocyte Ig-like receptor B1 (LILRB1) and B2 (LILRB2) belong to "Group 1" receptors and recognize a broad range of major histocompatibility complex class I molecules (MHCIs). In contrast, "Group 2" receptors show low similarity with LILRB1/B2, and their ligands remain to be identified. To date, the structural and functional characteristics of Group 2 LILRs are poorly understood. Here we report the crystal structure of the extracellular domain of LILRA5, which is an activating Group 2 LILR expressed on monocytes and neutrophils. Unexpectedly, the structure showed large changes in structural conformation and charge distribution in the region corresponding to the MHCI binding site of LILRB1/B2, which are also distinct from killer cell Ig-like receptors and Fc receptors. These changes probably confer the structural hindrance for the MHCI binding, and their key amino acid substitutions are well conserved in Group 2 LILRs. Consistently, the surface plasmon resonance and flow cytometric analyses demonstrated that LILRA5 exhibited no affinities to all tested MHCIs. These results raised the possibility that LILRA5 as well as Group 2 LILRs do not play a role in any MHCI recognition but could possibly bind to non-MHCI ligand(s) on the target cells to provide a novel immune regulation mechanism.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Leukocytes are regulated by opposing signaling pathways triggered by interactions of cell surface receptors with their ligands. Leukocyte Ig-like receptors (LILRs³ or LIRs, also called Ig-like transcripts (ILTs) and CD85) are encoded in human chromosome 19q13.4, along with the other Ig-like receptors, including the killer cell Ig-like receptor (KIR) family, FCAR and NKp46 (1). The LILR family has two or four tandem Ig-like extracellular domains and is divided into three functional categories: inhibitory, activating, and secreted receptors (2). The inhibitory LILR receptors have long cytoplasmic tails with two to four immunoreceptor tyrosine-based inhibitory motifs, in which the phosphorylation of tyrosine residues recruits the Src homology 2 domain-containing tyrosine phosphatase-1, transducing negative signals to inhibit activation signals. In contrast, the activating receptors have short cytoplasmic tails lacking immunoreceptor tyrosine-based inhibitory motifs and an arginine residue in the transmembrane region, which binds to negatively charged residues of FcR harboring the immunoreceptor tyrosine-based activation motif. The LILRs are mainly expressed on myelomonocytic cells, although some are also expressed on a wide range of leukocytes.

LILRA5 (LIR9/ILT11/CD85f) is expressed as both a membrane-bound activating and a secreted form (3). The membrane-bound form of LILRA5 has two Ig-like domains in the extracellular region with typical features of activating receptors (an arginine in the transmenbrane region and a short cytoplasmic domain), whereas the secreted form only has the extracellular domain. In contrast with LILRB1, which is expressed on a range of leukocytes, the expression of the activating form of LILRA5 has been detected on CD14⁺ monocytes (3). The expression of LILRA5 has also been detected in neutrophils and monocytes but only at the mRNA level (3). Cross-linking of LILRA5 molecules on monocytes using anti-LILRA5 antibody induced the mobilization of calcium and the secretion of cytokines released in the early stages of inflammatory responses, such as interleukin-1, tumor necrosis factor-, and interleukin-6 (3). Thus, LILRA5 may modulate CD14⁺ monocyte function under inflammatory conditions.

The LILR family members can be divided into two groups: "Group 1" (LILRA1/A2/A3/B1/B2) and "Group 2" (LILRA4/A5/A6/B3/B4/B5) types (4). Group 1 types have over 70% sequence similarity to LILRB1 and LILRB2, which bind to a broad range of human major histocompatibility complex (MHC) class I molecules (MHCIs: HLA-A, -B, -C, -E, -F, and -G) (5-7). Since the crystal structure of the LILRB1-HLA-A2 complex showed that the residues on the MHCI binding site of the Group 1 receptors were well conserved, it is likely that all of the Group 1 receptors have the potential to bind MHCIs. In contrast, Group 2 receptors show less than 60% sequence similarity to LILRB1 and LILRB2 with amino acid substitutions on the MHCI binding site. Preliminary cellular studies (8-11) have shown that Group 2 LILRs do not bind some MHCI molecules, although ligands for the Group 2 receptors remain to be identified. As a result, it has proved difficult to investigate the Group 2 type LILRs.

Here we report the crystal structure of the whole extracellular domain of LILRA5 at 1.85 Å resolution. This is the first example of a three-dimensional Group 2 LILR structure. The structure showed that the overall structure of LILRA5 resembled the N-terminal two Ig-like domains of LILRB1/B2 previously shown to be responsible for MHCI recognition in these receptors (4). However, there were large differences in the local structure and surface electrostatic potential of the region corresponding to the MHCI recognition site of LILRB1/B2. The amino acid substitutions responsible for these changes are well conserved in Group 2 LILRs. In addition, surface plasmon resonance and flow cytometric analyses demonstrated that LILRA5 did not recognize all tested MHCIs. Taken together, these results clearly suggested that LILRA5 is not a MHCI-recognizing receptor but binds to unknown ligands to modulate functions of monocytes and neutrophils. By analogy, it seems likely that the other Group 2 LILR members also recognize as yet unidentified ligand(s). The identification of ligands for the Group 2 receptors may provide insight into the mechanisms regulating immune responses.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Cloning, Expression, and Purification of LILRA5—A fragment of the gene encoding the LILRA5 extracellular domain was produced by two steps of PCR amplification, using a cDNA library of human peripheral blood mononuclear cells. The forward and reverse primers used to clone the cDNA of LILRA5 were 5'-CACCATGGTCTCATCCGTCTG-3' and 5'-GACTTGTTTTGTGACGGACTGAG-3', respectively. For introduction of the restriction enzyme sites (NdeI and HindIII), 5'-GGAATTCCATATGGGGAACCTCTCCAAAGCCAC-3' (forward) and 5'-TAGGCAAGCTTATGAGACCGGAATCTCCAGGAG-3' (reverse) primers were used. The resultant PCR fragment including the whole extracellular domain (residues 1-196) with additional N-terminal methionine was digested with the restriction enzymes NdeI and HindIII and ligated into the pGMT7 expression vector (12). Inclusion bodies of LILRA5 were expressed in Escherichia coli strain BL21(DE3)pLysS and prepared as previously reported for LILRB1 and LILRB2 (7). These were isolated from cell pellets by sonication and were washed with detergent solution containing 0.5% Triton X-100. The purified LILRA5 inclusion bodies were then solubilized with denaturant solution (6 M guanidine hydrochloride, 50 mM MES, pH 6.5, 10 mM EDTA). 1 h prior to refolding, dithiothreitol (2 mM final concentration) was added into the protein solution to reduce disulfide bonds. The solubilized protein solution was diluted slowly into the refolding buffer (0.1 M Tris-HCl, pH 8.0, 0.4 ML-arginine, 2 mM EDTA, 3.73 mM cystamine, 6.37 mM cysteamine) to the final protein concentration of 2 µM and stirred for 48 h at 4 °C. Then the refolding solution was concentrated with a VIVAFLOW50 system (Sartorius) and an Amicon Ultra concentrator (Millipore). The concentrated LILRA5 solution was loaded on gel filtration column, Superdex 75. Further purification was performed by anion exchange chromatography with ResourceQ (Amersham Biosciences).

Production of Selenomethionyl (SeMet) LILRA5—The inclusion bodies of SeMet derivative LILRA5 were obtained from E. coli strain BL21(DE3)pLysS grown in M9 minimal medium supplemented with 1 mM MgSO₄, 0.1 mM CaCl₂, 0.4% glucose, all L-amino acids except methionine, and 25 mg/liter SeMet. The SeMet LILRA5 was refolded and purified in the same way as the native protein described above.

Crystallization and Data Collection—Purified SeMet LILRA5 (20 mM Tris, pH 9.0, 50 mM NaCl) was concentrated to 10 mg/ml. An initial crystallization trial was performed using the crystallization screening kits, Crystal Screen I&II (Hampton Research) and Wizard I&II (Emerald Biostructures). 0.2 µl of protein solution was mixed in a 1:1 ratio with the crystallization reservoir solution on the Intelli-Plate (Art Robbins Instruments) using an autocrystallization setup system for sitting drop vapor diffusion, HYDRA-Plus1 (Apogent Discoveries). Square rod-type crystals were obtained in the condition of Crystal Screen II-48 (0.1 M Bicine, pH 9.0, 10% polyethylene glycol 20000, and 2% dioxane) at 20 °C. Crystals suitable for data collection were grown from 2-µl hanging drops containing the same protein/condition mixture. Multiwavelength anomalous dispersion data were collected with an ADSC Quantum 315 CCD detector at the beamline BL41XU of SPring-8 (Harima, Japan). Data collection was carried out at 100 K with crystals soaked in the reservoir solution containing 20% ethylene glycol. The diffraction data were processed and scaled with the HKL2000 program package (Table 1).

Surface Plasmon Resonance Analyses—Surface plasmon resonance experiments were performed using a BIAcore2000^TM (BIAcore AB, St. Albance, UK). The biotinylated human MHC class I molecules were prepared as described previously (7) and immobilized on the research grade sensor chip CM5 (BIAcore AB) on which streptavidin was covalently immobilized using the amine coupling method. The LILRA5 and LILRB1 solution was buffer-exchanged to HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20), and LILRA5 (or LILRB1) solution was flowed over at a rate of 10 µl/min.

Flow Cytometric Analysis for Binding of LILRA5 to HLA-Cw4-transfected 721.221 Cells—We constructed a vector containing LILRA5 fused to a C-terminal biotin ligase (BirA) recognition tag (GSLHHILDAQKMVWNHR). Tagged LILRA5 was refolded and purified as the wild type protein. Biotinylated LILRA5 was prepared as described previously (7). The LILRA5 tetramer was produced by incubation of biotinylated LILRA5 with phycoerythrin-conjugated streptavidin (Molecular Probes) for 15 min on ice. LILRB1 tetramer was prepared in the same manner. HLA-Cw4-transfected 721.221 cells (kindly provided by Dr. P. Parham) were also produced. Cells were incubated with either LILRB1 or LILRA5 tetramer for 1 h on ice. Untreated control cells were incubated with phycoerythrin-conjugated streptavidin. Subsequently, stained cells were analyzed by EPICS ALTRA (Beckman Coulter). Cell surface expression of HLA-Cw4 was monitored by 0.01 mg of fluorescein-labeled W6/32 anti-HLA monoclonal antibody (H-199; Leinco Technologies) or fluorescein-labeled mouse IgG2a (I-106; Leinco Technologies).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Crystallization and Structural Determination of LILRA5 Extracellular Domain—The whole extracellular domain of LILRA5 (residues 1-196) was overexpressed in E. coli as inclusion bodies and renatured by dilution refolding (7). One major peak was obtained from gel filtration and further purified by ion exchange chromatography. Crystals of the LILRA5 protein (space group was primitive orthorhombic (P2₁2₁2₁), and unit cell parameters were a = 28.0 Å, b = 58.5 Å, c = 131.2 Å) were obtained in 0.1 M HEPES, pH 7.5, 20% polyethylene glycol 4000, and 10% isopropyl alcohol, and the data set was collected to the resolution limit of 2.2 Å at SPring-8. It proved impossible to solve the structure of LILRA5 by molecular replacement using the structure of either LILRB1 or LILRB2 as a search probe. Therefore, we produced SeMet derivative protein in order to perform multiwavelength anomalous dispersion analysis. The SeMet derivative of LILRA5 was refolded, purified, and concentrated using a protocol similar to that described for the native protein. It was not possible to crystallize the SeMet protein in similar conditions to the native protein, but crystals were obtained in 0.1 M Bicine, pH 9.0, 10% polyethylene glycol 20000, and 2% dioxane. The crystal structure of the extracellular domain of LILRA5 was determined in the space group of P2₁2₁2₁ to 1.85 Å resolution by the multiwavelength anomalous dispersion method (details given under "Experimental Procedures" and in Table 1). The refined structure of the LILRA5 extracellular domain included two Ig-like domains (domain 1 (D1; residues 3-96) and domain 2 (D2; residues 100-195)), with a short linker connecting the two domains (residues 97-99) (Fig. 1, A and B). The AB (residues 106-116) and CC' (residues 136-138) loops of D2 were disordered (Fig. 1, A and C). The average B-factor of D2 (46.0 Ų) was higher than that of domain 1 (D1) (24.3 Ų), which may be due to the relatively loose packing of D2 in the crystal.

Overall Structure—The amino acid sequence identities among the other Ig-like receptors encoded within the LRC region (hereafter, designated as the LRC receptors), such as LILRA5, KIR, NKp46, and FcR, are 35-41%, lower than those among LILR family receptors (Fig. 2). However, the overall structure of LILRA5 closely resembled that of the LRC receptors: KIRs (for KIR2DL3, root mean square deviations of 1.24 Å for 153 C atoms) (21-23), NKp46 (1.22 Å for 151 C atoms) (24), FcR (1.20 Å for 115 C atoms) (25), LILRB1 (0.92 Å for 143 C atoms) (26), and LILRB2 (1.09 Å for 147 C atoms) (27) (Figs. 1A and 2) (see supplemental Table S1). The acute D1-D2 hinge angle of LILRA5 was also similar to related receptors (LILRB1, KIR2DL2, KIR2DL3, LILRB2, and NKp46). The total buried area at the interdomain interface was 867 Ų, relatively small compared with the other LRC receptors (LILRB1 (957 Ų), KIR2DL1 (1071 Ų), KIR2DL2 (949 Ų), KIR2DL3 (933 Ų), FcR (1139 Ų), and NKp46 (891 Ų)) but larger than that of LILRB2 (779 Ų).

View larger version (36K): [in this window] [in a new window]   FIGURE 1. Crystal structure of the LILRA5 extracellular domain. A, ribbon drawing of the structure of LILRA5 in a rainbow color scheme from blue (N-terminal) to red (C-terminal). The dotted lines represent the disordered residues of loop regions (residues 106-116 and 136-138). The star indicates the predicted N-linked glycosylation site. B, topological diagram of LILRA5 structure. The arrows show the direction of the -strands. Cylinders labeled PP show polyproline II-type helices. C, left, overall view of domain 2 of LILRA5 (magenta) superimposed on LILRB1 (yellow). The region between 106 and 116 is disordered. Right, close-up view of the box in the left panel. LILRA5 and LILRB1 are shown in magenta and yellow stick models, respectively. Hydrogen bonds made between A' and G strands are shown in green dashed lines. The green dotted circle indicates the residue 105 following the disordered loop of LILRA5. The black dotted circle indicates the C-terminal residue of LILRA5 unable to form the A'GFCC' -sheet due to the distortion caused by Pro194 (see text).

View larger version (54K): [in this window] [in a new window]   FIGURE 2. Alignment of LILRA5 amino acid sequences and other related receptors encoded in the LRC region. Secondary structure elements of LILRA5 are shown above the sequences. The arrows indicate -strands, gray cylinders indicate 310 helices, and PP indicates polyproline II-type helices. The dashed lines indicate the disordered regions. The residues conserved in all receptors are shaded in black, and the residues conserved in LILRs are surrounded by squares. The asterisks indicate the residues involved in the MHCI binding of LILRB1.

Second, LILRA5 has the D strand interacting with a portion of the E strand in the D2 domain, not observed in LILRB1 and LILRB2 but found in KIRs (Fig. 3A, right; see supplemental Fig. S1). The D and E strands open up toward the outside and render Phe¹⁵⁴ able to make hydrophobic interactions with Leu¹⁴⁸ and Arg¹²⁶ of the BC loop. Similar interactions are also observed in KIRs, which have Phe at the equivalent position of Phe¹⁵⁴ of LILRA5 (Fig. 2). These strands do not directly interact with the MHCIs. As yet, it is uncertain whether this structural difference has an effect on ligand recognition. However, the neighboring region, including the BC and FG loops, is positively charged in LILRA5 (Arg¹²⁴, Arg¹²⁶, Arg¹⁷⁷, and His¹⁷⁹) with some hydrophobic residues (Leu¹²⁵, Leu¹⁴⁸, Phe¹⁵⁴), in contrast with LILRB1/B2 and KIRs, where the equivalent region is negatively charged (Fig. 4, also discussed below). This region is responsible for the receptor recognition toward the peptide-binding groove (KIRs) or 2m domain (LILRB1/B2) of MHCIs. This surface composition of LILRA5 is significantly distinct from KIRs and LILRB1/B2 and thus again probably inhibits the MHCI binding.

Among the LRC receptors, structures of LILRB1 (4), KIRs (32, 33), and FcR (25) have been determined in complex with their ligands. The comparison of the sequence of the ligand binding sites between these receptors and LILRA5 shows that the residues are not well conserved between these receptors and LILRA5 (Fig. 2). Electrostatic potential maps of the molecular surface clearly show the different charge distributions of LILRA5 compared with LILRB1, KIRs, and FcR, as partly described above (Fig. 4). LILRB1 has strongly negatively and positively charged regions, both of which are involved in the MHCI binding (2m and 3 domain). In addition, KIRs also contain a large negatively charged area crucial for MHCI binding (peptide-binding groove). However, in contrast with the above receptors, small negative and positive patches were dispersed on the surface of LILRA5, and a few hydrophobic areas were observed (Fig. 4). These features indicated that LILRA5 are likely to recognize as yet unidentified ligands. This suggestion is consistent with the biochemical study showing that LILRA5 does not interact with any of the tested MHCIs, as described below. Although surface charge distribution and less hydrophobic characteristics described above do not provide any potential candidate ligands, it is possible that LILRA5 forms a charge-complementary interface with unknown ligands, similarly to those of KIR-MHC, LILRB1-MHC, and FcR-Fc complexes, associated with low affinity and fast kinetics.

View larger version (53K): [in this window] [in a new window]   FIGURE 3. Structural comparison of MHCI binding sites between LILRBs and LILRA5. A, center, the LILRB1 (light pink) of the HLA-A2 complex model (4) (heavy chain; blue, 2-microglobulin; cyan) is superimposed onto LILRA5 (red). Left and right, large structural difference observed in D1 (right, orange dotted circle around the C' strand) and D2 (right, blue dotted circle around the DE loop). LILRB1 (light pink) and LILRB2 (light green) are superimposed onto LILRA5 (red). B, the ribbon model of CC' sheet of LILRA5 (red) is superimposed onto those (C strand and 310 helices) of LILRB1 (light pink). Conserved amino acids are shown in a ball model (left), and their colors are the same as those in amino acid comparison among LILR members (right). Key amino acids for structural changes are colored, and this coloring is also applied in the labels in C. The characters, h and s, on the sequence alignment indicate helix and sheet structures, respectively. C, details of local structures around the 310 helix of LILRB1 (left) and C' sheet of LILRA5 (right), highlighted by the orange dotted circle in A. The black dotted lines are shown in order for the main-chain structures to be easily recognized. The hydrophobic core of LILRB1 (left) and hydrophobic pocket of LILRA5 (right) are indicated by the red dotted circles.

View larger version (45K): [in this window] [in a new window]   FIGURE 4. Surface characteristics of LILRA5 and related LRC receptors. Transparent molecular surface of LILRA5, LILRB1, KIR2DL1, and FcR colored by electrostatic potential (positive, blue; negative, red) are drawn on their C traces. The residues involved in ligand binding are indicated by the green balls.

View larger version (33K): [in this window] [in a new window]   FIGURE 5. Binding analysis of LILRA5. A, surface plasmon resonance analysis of binding of human MHCI molecules to soluble LILRA5. Soluble LILRB1 (filled symbol) or LILRA5 (open symbol) was injected over the immobilized MHCI molecules (HLA-A2 (square), -B35 (circle), -Cw4 (triangle), -E (inverted triangle), and -G (diamond)). B, LILRA5 tetramer does not recognize HLA-Cw4 on the cell surface. To confirm the surface expression of HLA-Cw4 on 721.221 cells and HLA-Cw4-transfected 721.221 cells (721.221-Cw4), cells were stained with W6/32 anti-HLA class I antibody (left). Antibody-treated cells are shown in shaded histograms, and isotype control-treated cells are shown in open histograms. 721.221 cells and 721.221-Cw4 cells were treated with phycoerythrin-labeled LILR tetramers (LILRB1, middle panels; LILRA5, right panels). Negative controls are shown in open histograms, and tetramer-treated cells are shown in shaded histograms. 721.221-Cw4 cells were stained by LILRB1 tetramer but not LILRA5 tetramer.

Our previous association study between polymorphisms of Group 1 LILR, LILRB1, and rheumatoid arthritis in Japanese subjects demonstrated that LILRB1 is highly polymorphic, and one of those haplotypes is associated with the susceptibility to rheumatoid arthritis in HLA-DRB1 shared epitope-negative subjects (39). Interestingly, as pointed out by the review of Brown et al. (2), the cross-linking of LILRA5⁺ on the surface of monocytes confers calcium flux and secretion of proinflammatory cytokines interleukin-1 and tumor necrosis factor-, which are mainly produced by macrophages in the rheumatoid arthritis synovial fluid (40). Because LILRA5 is expressed not only as a membrane-bound form but as a secreted one, LILRA5 signaling is expected to be modulated by the ratio of these isoforms. Therefore, the elucidation of the LILRA5 signaling pathway, especially the identification of LILRA5 ligand(s), will give new insights to the molecular mechanism of the immunological tolerance and the pathogenesis of inflammatory diseases.

Conclusion—Here we report the first structure of a Group 2 LILR family member, LILRA5. The crystal structure of LILRA5 exhibits distinct features of both structural conformations and surface electrostatic potential from related LRC-encoded receptors, such as LILRB1/B2, KIRs, FcR, and NKp46. The key amino acids responsible for these characteristics are well conserved in Group 2 LILRs. The surface plasmon resonance binding and cell staining studies demonstrate that LILRA5 does not bind to any of the tested classical and nonclassical MHCIs. Taken together, these data suggest that LILRA5 as well as Group 2 LILRs could play a novel role in immune regulation by recognizing unknown ligand(s) other than MHCIs.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 2D3V) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by research fellowships and a research grant of the Japan Society for the Promotion of Science for young scientists (to M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Table S1.

¹ Supported in part by the Ministry of Education, Science, Sports, Culture and Technology of Japan and the Protein 3000 project.

² Supported in part by the Ministry of Education, Science, Sports, Culture and Technology of Japan, the Protein 3000 project, and the Kanae foundation. To whom correspondence should be addressed: Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Tel.: 81-92-642-6969; Fax: 81-92-642-6764; E-mail: kmaenaka{at}bioreg.kyushu-u.ac.jp.

³ The abbreviations used are: LILR, leukocyte Ig-like receptor; KIR, killer cell Ig-like receptor; MHC, major histocompatibility complex; MHCI, MHC class I; MES, 4-morpholineethanesulfonic acid; SeMet, selenomethionyl; D1, domain 1; D2, domain 2; Bicine, N,N-bis(2-hydroxyethyl)glycine.

⁵ M. Shiroishi, K. Kuroki, L. Rasubala, D. Kohda, and K. Maenaka, unpublished result.

	ACKNOWLEDGMENTS

 
We thank Drs. H. Arase, M. Yawata, and P. Parham for materials and helpful discussion. We are grateful to Drs. M. Kawamoto and N. Shimizu (SPring-8) and Drs. K. Sasaki and T. Obita (Kyushu University) for kind help with data collection.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

1. Martin, A. M., Kulski, J. K., Witt, C., Pontarotti, P., and Christiansen, F. T. (2002) Trends Immunol. 23, 81-88[CrossRef][Medline] [Order article via Infotrieve]
2. Brown, D., Trowsdale, J., and Allen, R. (2004) Tissue Antigens 64, 215-225[CrossRef][Medline] [Order article via Infotrieve]
3. Borges, L., Kubin, M., and Kuhlman, T. (2003) Blood 101, 1484-1486[Abstract/Free Full Text]
4. Willcox, B. E., Thomas, L. M., and Bjorkman, P. J. (2003) Nat. Immunol. 4, 913-919[CrossRef][Medline] [Order article via Infotrieve]
5. Chapman, T. L., Heikeman, A. P., and Bjorkman, P. J. (1999) Immunity 11, 603-613[CrossRef][Medline] [Order article via Infotrieve]
6. Lepin, E. J., Bastin, J. M., Allan, D. S., Roncador, G., Braud, V. M., Mason, D. Y., van der Merwe, P. A., McMichael, A. J., Bell, J. I., Powis, S. H., and O'Callaghan, C. A. (2000) Eur. J. Immunol. 30, 3552-3561[CrossRef][Medline] [Order article via Infotrieve]
7. Shiroishi, M., Tsumoto, K., Amano, K., Shirakihara, Y., Colonna, M., Braud, V. M., Allan, D. S., Makadzange, A., Rowland-Jones, S., Willcox, B., Jones, E. Y., Van Der Merwe, P. A., Kumagai, I., and Maenaka, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 8856-8861[Abstract/Free Full Text]
8. Allen, R. L., Raine, T., Haude, A., Trowsdale, J., and Wilson, M. J. (2001) J. Immunol. 167, 5543-5547[Abstract/Free Full Text]
9. Colonna, M., Samaridis, J., Cella, M., Angman, L., Allen, R. L., O'Callaghan, C. A., Dunbar, R., Ogg, G. S., Cerundolo, V., and Rolink, A. (1998) J. Immunol. 160, 3096-3100[Abstract/Free Full Text]
10. Borges, L., Hsu, M. L., Fanger, N., Kubin, M., and Cosman, D. (1997) J. Immunol. 159, 5192-5196[Abstract]
11. Cella, M., Dohring, C., Samaridis, J., Dessing, M., Brockhaus, M., Lanza-vecchia, A., and Colonna, M. (1997) J. Exp. Med. 185, 1743-1751[Abstract/Free Full Text]
12. Reid, S. W., Smith, K. J., Jakobsen, B. K., O'Callaghan, C. A., Reyburn, H., Harlos, K., Stuart, D. I., McMichael, A. J., Bell, J. I., and Jones, E. Y. (1996) FEBS Lett. 383, 119-123[CrossRef][Medline] [Order article via Infotrieve]
13. Terwilliger, T. C., and Berendzen, J. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 849-861[CrossRef][Medline] [Order article via Infotrieve]
14. Terwilliger, T. C. (2000) Acta Crystallogr. Sect. D Biol. Crystallogr. 56, 965-972[CrossRef][Medline] [Order article via Infotrieve]
15. Perrakis, A., Morris, R., and Lamzin, V. S. (1999) Nat. Struct. Biol. 6, 458-463[CrossRef][Medline] [Order article via Infotrieve]
16. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. A 47, 110-119[CrossRef]
17. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
18. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta Crystallogr. Sect. D Biol. Crystallogr. 53, 240-255[CrossRef][Medline] [Order article via Infotrieve]
19. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef]
20. Nicholls, A., Sharp, K. A., and Honig, B. (1991) Proteins 11, 281-296[CrossRef][Medline] [Order article via Infotrieve]
21. Fan, Q. R., Mosyak, L., Winter, C. C., Wagtmann, N., Long, E. O., and Wiley, D. C. (1997) Nature 389, 96-100[CrossRef][Medline] [Order article via Infotrieve]
22. Snyder, G. A., Brooks, A. G., and Sun, P. D. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 3864-3869[Abstract/Free Full Text]
23. Maenaka, K., Juji, T., Stuart, D. I., and Jones, E. Y. (1999) Struct. Fold Des. 7, 391-398[Medline] [Order article via Infotrieve]
24. Foster, C. E., Colonna, M., and Sun, P. D. (2003) J. Biol. Chem. 278, 46081-46086[Abstract/Free Full Text]
25. Herr, A. B., Ballister, E. R., and Bjorkman, P. J. (2003) Nature 423, 614-620[CrossRef][Medline] [Order article via Infotrieve]
26. Chapman, T. L., Heikema, A. P., West, A. P., Jr., and Bjorkman, P. J. (2000) Immunity 13, 727-736[CrossRef][Medline] [Order article via Infotrieve]
27. Willcox, B. E., Thomas, L. M., Chapman, T. L., Heikema, A. P., West, A. P., Jr., and Bjorkman, P. J. (2002) BMC Struct. Biol. 2, 6[CrossRef][Medline] [Order article via Infotrieve]
28. Bravo, J., Staunton, D., Heath, J. K., and Jones, E. Y. (1998) EMBO J. 17, 1665-1674[CrossRef][Medline] [Order article via Infotrieve]
29. de Vos, A. M., Ultsch, M., and Kossiakoff, A. A. (1992) Science 255, 306-312[Abstract/Free Full Text]
30. Livnah, O., Stura, E. A., Johnson, D. L., Middleton, S. A., Mulcahy, L. S., Wrighton, N. C., Dower, W. J., Jolliffe, L. K., and Wilson, I. A. (1996) Science 273, 464-471[Abstract]
31. Somers, W., Ultsch, M., De Vos, A. M., and Kossiakoff, A. A. (1994) Nature 372, 478-481[CrossRef][Medline] [Order article via Infotrieve]
32. Boyington, J. C., Motyka, S. A., Schuck, P., Brooks, A. G., and Sun, P. D. (2000) Nature 405, 537-543[CrossRef][Medline] [Order article via Infotrieve]
33. Fan, Q. R., Long, E. O., and Wiley, D. C. (2001) Nat. Immunol. 2, 452-460[Medline] [Order article via Infotrieve]
34. Sloane, D. E., Tedla, N., Awoniyi, M., Macglashan, D. W., Jr., Borges, L., Austen, K. F., and Arm, J. P. (2004) Blood 104, 2832-2839[Abstract/Free Full Text]
35. Chang, C. C., Ciubotariu, R., Manavalan, J. S., Yuan, J., Colovai, A. I., Piazza, F., Lederman, S., Colonna, M., Cortesini, R., Dalla-Favera, R., and Suciu-Foca, N. (2002) Nat. Immunol. 3, 237-243[CrossRef][Medline] [Order article via Infotrieve]
36. Kim-Schulze, S., Scotto, L., Vlad, G., Piazza, F., Lin, H., Liu, Z., Cortesini, R., and Suciu-Foca, N. (2006) J. Immunol. 176, 2790-2798[Abstract/Free Full Text]
37. Bleharski, J. R., Li, H., Meinken, C., Graeber, T. G., Ochoa, M. T., Yamamura, M., Burdick, A., Sarno, E. N., Wagner, M., Rollinghoff, M., Rea, T. H., Colonna, M., Stenger, S., Bloom, B. R., Eisenberg, D., and Modlin, R. L. (2003) Science 301, 1527-1530[Abstract/Free Full Text]
38. Arase, H., Mocarski, E. S., Campbell, A. E., Hill, A. B., and Lanier, L. L. (2002) Science 296, 1323-1326[Abstract/Free Full Text]
39. Kuroki, K., Tsuchiya, N., Shiroishi, M., Rasubala, L., Yamashita, Y., Matsuta, K., Fukazawa, T., Kusaoi, M., Murakami, Y., Takiguchi, M., Juji, T., Hashimoto, H., Kohda, D., Maenaka, K., and Tokunaga, K. (2005) Hum. Mol. Genet. 14, 2469-2480[Abstract/Free Full Text]
40. Feldmann, M., and Maini, R. N. (2001) Annu. Rev. Immunol. 19, 163-196[CrossRef][Medline] [Order article via Infotrieve]
41. Collaborative Computational Project, Number 4. (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760-763[CrossRef][Medline] [Order article via Infotrieve]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				M. Shiroishi, K. Kuroki, L. Rasubala, K. Tsumoto, I. Kumagai, E. Kurimoto, K. Kato, D. Kohda, and K. Maenaka Structural basis for recognition of the nonclassical MHC molecule HLA-G by the leukocyte Ig-like receptor B2 (LILRB2/LIR2/ILT4/CD85d) PNAS, October 31, 2006; 103(44): 16412 - 16417. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 281/28/19536    most recent M603076200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shiroishi, M. Articles by Maenaka, K. Search for Related Content PubMed PubMed Citation Articles by Shiroishi, M. Articles by Maenaka, K. Social Bookmarking                      What's this?