Title: Efficient Leukocyte Ig-like Receptor Signaling and Crystal Structure of Disulfide-linked HLA-G Dimer
Abstract: HLA-G is a nonclassical major histocompatibility complex class I (MHCI) molecule, which is expressed in trophoblasts and confers immunological tolerance in the maternal-fetal interface by binding to leukocyte Ig-like receptors (LILRs, also called as LIR/ILT/CD85) and CD8. HLA-G is expressed in disulfide-linked dimer form both in solution and at the cell surface. Interestingly, MHCI dimer formations have been involved in pathogenesis and T cell activation. The structure and receptor binding characteristics of MHCI dimers have never been evaluated. Here we performed binding studies showing that the HLA-G dimer exhibited higher overall affinity to LILRB1/2 than the monomer by significant avidity effects. Furthermore, the cell reporter assay demonstrated that the dimer formation remarkably enhanced the LILRB1-mediated signaling at the cellular level. We further determined the crystal structure of the wild-type dimer of HLA-G with the intermolecular Cys42-Cys42 disulfide bond. This dimer structure showed the oblique configuration to expose two LILR/CD8-binding sites upward from the membrane easily accessible for receptors, providing plausible 1:2 (HLA-G dimer:receptors) complex models. These results indicated that the HLA-G dimer conferred increased avidity in a proper structural orientation to induce efficient LILR signaling, resulting in the dominant immunosuppressive effects. Moreover, structural and functional implications for other MHCI dimers observed in activated T cells and the pathogenic allele, HLA-B27, are discussed. HLA-G is a nonclassical major histocompatibility complex class I (MHCI) molecule, which is expressed in trophoblasts and confers immunological tolerance in the maternal-fetal interface by binding to leukocyte Ig-like receptors (LILRs, also called as LIR/ILT/CD85) and CD8. HLA-G is expressed in disulfide-linked dimer form both in solution and at the cell surface. Interestingly, MHCI dimer formations have been involved in pathogenesis and T cell activation. The structure and receptor binding characteristics of MHCI dimers have never been evaluated. Here we performed binding studies showing that the HLA-G dimer exhibited higher overall affinity to LILRB1/2 than the monomer by significant avidity effects. Furthermore, the cell reporter assay demonstrated that the dimer formation remarkably enhanced the LILRB1-mediated signaling at the cellular level. We further determined the crystal structure of the wild-type dimer of HLA-G with the intermolecular Cys42-Cys42 disulfide bond. This dimer structure showed the oblique configuration to expose two LILR/CD8-binding sites upward from the membrane easily accessible for receptors, providing plausible 1:2 (HLA-G dimer:receptors) complex models. These results indicated that the HLA-G dimer conferred increased avidity in a proper structural orientation to induce efficient LILR signaling, resulting in the dominant immunosuppressive effects. Moreover, structural and functional implications for other MHCI dimers observed in activated T cells and the pathogenic allele, HLA-B27, are discussed. During pregnancy, the fetus can be the allogenic object for the maternal immune system, and thus a special system of immune tolerance is necessary for escaping from maternal immune surveillance to achieve a successful pregnancy. However, knowledge of the molecular mechanism of the maternal-fetal immune tolerance is still limited. In the maternal-fetal interface, the fetal extravillous cytotrophoblasts do not express major histocompatibility complex class I molecules (MHCIs), 5The abbreviations used are: MHCI, major histocompatibility complex class I molecules; MHC, major histocompatibility complex; LILR, leukocyte Ig-like receptor; β2m, β2-microglobulin; GFP, green fluorescent protein; MFI, mean fluorescence intensity; r.m.s.d., root mean square deviation; NK, natural killer; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. HLA-A or -B, on the cell surface but do express minor classical MHCI, HLA-C, and nonclassical MHCIs, HLA-E and -G (1LeMaoult J. Le Discorde M. Rouas-Freiss N. Moreau P. Menier C. McCluskey J. Carosella E.D. Tissue Antigens. 2003; 62: 273-284Crossref PubMed Scopus (143) Google Scholar). Toward T cells, the classical MHCIs present 8-10 amino acid peptides processed inside cells (e.g. proteasome) to induce peptide-specific T cell immune responses. Thus, the loss of HLA-A and -B expression significantly suppresses maternal T cell responses. Although HLA-C and -E are expressed in normal cells, the expression of HLA-G is restricted to a few tissues as follows: extravillous trophoblasts, thymus epithelial cells, and some tumors (1LeMaoult J. Le Discorde M. Rouas-Freiss N. Moreau P. Menier C. McCluskey J. Carosella E.D. Tissue Antigens. 2003; 62: 273-284Crossref PubMed Scopus (143) Google Scholar). In contrast with polymorphic classical MHCIs, HLA-G shows the limited polymorphism, suggesting that HLA-G may potentially have a role as a common ligand for generic immunosuppressive receptors in the protection of fetus cells from the maternal immune cells. Recently, several immunologically relevant cell-surface receptors were found to mediate the negative regulation of immune cells through binding to classical and nonclassical MHCIs. The receptors of HLA-G reported to date are CD8, leukocyte Ig-like receptor B1/B2 (LILRB1/LILRB2, also known as LIR1/LIR2, ILT2/ILT4, and Cd85j/Cd85d), and KIR2DL4. Although the molecular basis of the KIR2DL4-HLA-G interaction remains the subject of debate (1LeMaoult J. Le Discorde M. Rouas-Freiss N. Moreau P. Menier C. McCluskey J. Carosella E.D. Tissue Antigens. 2003; 62: 273-284Crossref PubMed Scopus (143) Google Scholar), LILRB1/2 and CD8-HLA-G recognitions have been studied (2Chapman T.L. Heikeman A.P. Bjorkman P.J. Immunity. 1999; 11: 603-613Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 3Shiroishi 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. Maenaka K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8856-8861Crossref PubMed Scopus (444) Google Scholar, 4Gao G.F. Willcox B.E. Wyer J.R. Boulter J.M. O'Callaghan C.A. Maenaka K. Stuart D.I. Jones E.Y. van Der Merwe P.A. Bell J.I. Jakobsen B.K. J. Biol. Chem. 2000; 275: 15232-15238Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). LILRB1 is expressed in a wide range of leukocytes, including natural killer (NK) cells and T cells, although LILRB2 is expressed in a restricted set of immune cells, including monocytes and dendritic cells (5Brown D. Trowsdale J. Allen R. Tissue Antigens. 2004; 64: 215-225Crossref PubMed Scopus (244) Google Scholar). Both LILRBs have four Ig-like domains in the extracellular region, and the N-terminal two Ig-like domains are responsible for MHCI recognitions. Upon MHCI binding, LILRBs mediate the negative signal by three or four immune receptor tyrosine-based inhibitory motifs in the intracellular domain. The HLA-G molecule in the maternal-fetal interface recognizes LILRBs to inhibit immune response of a wide range of maternal immune cells, including myelomonocytic cells, T cells, and NK cells. On the other hand, HLA-G has several soluble forms as splice variants (6Le Bouteiller P. Legrand-Abravanel F. Solier C. Placenta. 2003; 24 (Suppl. A): 10-15Crossref PubMed Scopus (51) Google Scholar, 7Fujii T. Ishitani A. Geraghty D.E. J. Immunol. 1994; 153: 5516-5524PubMed Google Scholar, 8Ishitani A. Geraghty D.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3947-3951Crossref PubMed Scopus (478) Google Scholar). The soluble forms of HLA-G induced the dysfunction of CD8+ T cells, which may promote semi-allogenic pregnancy (9Morales P.J. Pace J.L. Platt J.S. Phillips T.A. Morgan K. Fazleabas A.T. Hunt J.S. J. Immunol. 2003; 171: 6215-6224Crossref PubMed Scopus (111) Google Scholar, 10Fournel S. Aguerre-Girr M. Huc X. Lenfant F. Alam A. Toubert A. Bensussan A. Le Bouteiller P. J. Immunol. 2000; 164: 6100-6104Crossref PubMed Scopus (431) Google Scholar, 11Contini P. Ghio M. Poggi A. Filaci G. Indiveri F. Ferrone S. Puppo F. Eur. J. Immunol. 2003; 33: 125-134Crossref PubMed Scopus (320) Google Scholar). Therefore, the HLA-G-LILRB and HLA-G-CD8 interactions may induce a wide range of the immunological tolerance. Sequencing studies of both overall eluted and purified peptides have shown that HLA-G can display limited but still diverse sets of peptides (12Lee N. Malacko A.R. Ishitani A. Chen M.C. Bajorath J. Marquardt H. Geraghty D.E. Immunity. 1995; 3: 591-600Abstract Full Text PDF PubMed Scopus (298) Google Scholar, 13Diehl M. Munz C. Keilholz W. Stevanovic S. Holmes N. Loke Y.W. Rammensee H.G. Curr. Biol. 1996; 6: 305-314Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). This peptide presentation is more similar to that of classical MHCIs than that of nonclassical MHCI, HLA-E, which can present a very limited repertoire of peptides, including the MHCI signal sequence and heat shock and viral proteins. HLA-G shows the slow transport to the cell surface and the long half-life on the cell surface because of its truncated intracytoplasmic domain lacking an endocytosis motif (14Park B. Lee S. Kim E. Chang S. Jin M. Ahn K. Immunity. 2001; 15: 213-224Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 15Park B. Ahn K. J. Biol. Chem. 2003; 278: 14337-14345Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The result is that HLA-G selects a limited set of high affinity peptides for presentation, even though it has the peptide presentation mechanism of the classical MHCIs. The recently reported crystal structure of the HLA-G C42S mutant monomer (16Clements C.S. Kjer-Nielsen L. Kostenko L. Hoare H.L. Dunstone M.A. Moses E. Freed K. Brooks A.G. Rossjohn J. McCluskey J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3360-3365Crossref PubMed Scopus (121) Google Scholar) also showed that the peptide recognition of HLA-G included an extensive network of contacts, supporting the constrained mode of the peptide binding. HLA-G has two free cysteine residues (Cys42 and Cys147) unlike most of other MHCIs. Boyson et al. (17Boyson J.E. Erskine R. Whitman M.C. Chiu M. Lau J.M. Koopman L.A. Valter M.M. Angelisova P. Horejsi V. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16180-16185Crossref PubMed Scopus (192) Google Scholar) reported that the bacterial recombinant soluble form of HLA-G can form a disulfide-linked dimer with the intermolecular Cys42-Cys42 disulfide bond. Moreover, the soluble HLA-G1 expressed by human embryonic kidney 293 cells also showed the mixture of monomer, disulfide-linked dimer, and oligomer forms, which could reduce the CD8 expression level on cytotoxic T lymphocyte (9Morales P.J. Pace J.L. Platt J.S. Phillips T.A. Morgan K. Fazleabas A.T. Hunt J.S. J. Immunol. 2003; 171: 6215-6224Crossref PubMed Scopus (111) Google Scholar). However, it is uncertain how much effect each form has. On the other hand, the membrane-bound form of HLA-G can also form a disulfide-linked dimer on the cell surface of the Jeg3 cell line, which endogenously expresses HLA-G (18Gonen-Gross T. Achdout H. Arnon T.I. Gazit R. Stern N. Horejsi V. GoldmanWohl D. Yagel S. Mandelboim O. J. Immunol. 2005; 175: 4866-4874Crossref PubMed Scopus (99) Google Scholar) and also HLA-G transfectants (17Boyson J.E. Erskine R. Whitman M.C. Chiu M. Lau J.M. Koopman L.A. Valter M.M. Angelisova P. Horejsi V. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16180-16185Crossref PubMed Scopus (192) Google Scholar, 19Gonen-Gross T. Gazit R. Achdout H. Hanna J. Mizrahi S. Markel G. Horejsi V. Mandelboim O. Hum. Immunol. 2003; 64: 1011-1016Crossref PubMed Scopus (27) Google Scholar, 20Gonen-Gross T. Achdout H. Gazit R. Hanna J. Mizrahi S. Markel G. GoldmanWohl D. Yagel S. Horejsi V. Levy O. Baniyash M. Mandelboim O. J. Immunol. 2003; 171: 1343-1351Crossref PubMed Scopus (124) Google Scholar). The mutagenesis studies suggested that the HLA-G dimer was responsible for efficient LILRB1-mediated inhibition of the killing activity of NK cells (19Gonen-Gross T. Gazit R. Achdout H. Hanna J. Mizrahi S. Markel G. Horejsi V. Mandelboim O. Hum. Immunol. 2003; 64: 1011-1016Crossref PubMed Scopus (27) Google Scholar, 20Gonen-Gross T. Achdout H. Gazit R. Hanna J. Mizrahi S. Markel G. GoldmanWohl D. Yagel S. Horejsi V. Levy O. Baniyash M. Mandelboim O. J. Immunol. 2003; 171: 1343-1351Crossref PubMed Scopus (124) Google Scholar). Recently, the β2m-free form of HLA-G also forms disulfidelinked dimers and multimers on the cell surface mainly by Cys42-mediated disulfide bonds, similar to the dimer form of normal β2m-associated HLA-G protein described above (18Gonen-Gross T. Achdout H. Arnon T.I. Gazit R. Stern N. Horejsi V. GoldmanWohl D. Yagel S. Mandelboim O. J. Immunol. 2005; 175: 4866-4874Crossref PubMed Scopus (99) Google Scholar). The disulfide-linked homodimer was also observed in the β2m-free form of HLA-B27, which has a free cysteine (Cys67). Previous reports (21Kollnberger S. Bird L. Sun M.Y. Retiere C. Braud V.M. McMichael A. Bowness P. Arthritis Rheum. 2002; 46: 2972-2982Crossref PubMed Scopus (203) Google Scholar, 22Allen R.L. Raine T. Haude A. Trowsdale J. Wilson M.J. J. Immunol. 2001; 167: 5543-5547Crossref PubMed Scopus (141) Google Scholar) suggested that this homodimer would be directly associated with the development of ankylosing spondylitis, whereas others argued that the dimer formation may be part of multimers that exhibit significant function for signal transduction (23Colbert R.A. Curr. Mol. Med. 2004; 4: 21-30Crossref PubMed Scopus (66) Google Scholar, 24Damjanovich S. Matyus L. Damjanovich L. Bene L. Jenei A. Matko J. Gaspar R. Szollosi J. Immunol. Lett. 2002; 82: 93-99Crossref PubMed Scopus (11) Google Scholar). Furthermore, the significant level of the expression of the disulfide-linked β2m-free MHCI dimers was observed at the cell surface of activated but not resting normal T cells, suggesting that the MHCI dimers are important for regulating the activation of T cells (25Santos S.G. Powis S.J. Arosa F.A. J. Biol. Chem. 2004; 279: 53062-53070Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Our previous report (3Shiroishi 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. Maenaka K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8856-8861Crossref PubMed Scopus (444) Google Scholar) showed that LILRB1 and LILRB2 preferentially bound to the monomer form of HLA-G in comparison with the other classical MHCIs, and its binding site overlapped with that of CD8. Recently the crystal structure of the HLA-G C42S mutant monomer was reported (16Clements C.S. Kjer-Nielsen L. Kostenko L. Hoare H.L. Dunstone M.A. Moses E. Freed K. Brooks A.G. Rossjohn J. McCluskey J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3360-3365Crossref PubMed Scopus (121) Google Scholar), proposing the HLA-G dimer model. However, the three-dimensional structure and the receptor binding characteristics of the disulfide-linked wild-type HLA-G dimer have never been experimentally evaluated. Here we report the LILR binding and signaling studies, and we have determined the crystal structure of the HLA-G dimer. First, we performed LILRB1/2 binding studies of the monomer and dimer forms of HLA-G, clearly showing that both overall apparent affinities of the dimer are much higher than those of the monomer by the avidity effect. Next, the LILRB1 NFAT-GFP reporter cell assay (26Arase H. Mocarski E.S. Campbell A.E. Hill A.B. Lanier L.L. Science. 2002; 296: 1323-1326Crossref PubMed Scopus (981) Google Scholar, 27Ohtsuka M. Arase H. Takeuchi A. Yamasaki S. Shiina R. Suenaga T. Sakurai D. Yokosuka T. Arase N. Iwashima M. Kitamura T. Moriya H. Saito T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8126-8131Crossref PubMed Scopus (84) Google Scholar) clearly demonstrated that the dimer formation remarkably augmented the LILRB1-mediated signaling. Furthermore, in order to reveal the structural basis for the high LILR affinity and efficient LILR signaling of the HLA-G dimer, we determined the crystal structure of the wild-type HLA-G, which formed a disulfide-linked dimer via the Cys42-Cys42 disulfide bond in the crystals. The structural orientation of the dimer was additionally stabilized by the extended interface interactions of loops between β-strands below the α1-helix, in the center where Cys42 was located. The HLA-G dimer exhibited the oblique configuration to expose two LILRB1/2- and CD8-binding sites upward from the membrane that is fully accessible for receptors, providing the plausible 1:2 (HLA-G dimer:receptors) complex model. These structural and functional results suggested that the HLA-G dimer conferred increased avidity in a proper structural orientation to show overall high affinity and efficient signaling to LILRB1/2 and probably CD8. We will further discuss the potential physiological roles of the HLA-G dimer in soluble and membrane-bound forms. Based on the present study of the HLA-G dimer, the structural and functional characteristics of the disulfidelinked β2m-free MHCI dimers, either of the pathogenic allele HLA-B27 or expressed on the activated T cells, will be also discussed. Production of Recombinant Proteins—Recombinant ectodomains of the HLA-G monomer, LILRB1, and LILRB2 were produced according to our previous report (3Shiroishi 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. Maenaka K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8856-8861Crossref PubMed Scopus (444) Google Scholar). The preparation of the biotinylated HLA-G monomer, LILRB1, and LILRB2 were essentially the same as for those without the tag. 6K. Kuroki, M. Shiroishi, D. Kohda, and K. Maenaka, unpublished data. Site-directed mutagenesis for the C42S mutant HLA-G monomer was performed by the two-step PCR method. The refolded monomer of wild-type HLA-G naturally formed the disulfidebonded dimer (10-30% of the total protein) for ∼20 days at 4 °C. For the binding studies, the HLA-G dimer was purified by gel filtration (Superdex 200 10/30, Amersham Biosciences). Native Gel Electrophoresis—The native-PAGE for characterizing the protein samples and analyzing the binding was performed by the Phast system (Amersham Biosciences). We used the commercially available native-PAGE buffer strip (0.25 m Tris, 0.88 m l-alanine, pH 8.8; Amersham Biosciences), and the experiment was performed at 15 °C. In the complex formation experiments, the sample mixture was incubated at 20 °C for 1 h before applying to the homogeneous 12.5% polyacrylamide gel (Amersham Biosciences). Less than 4 μl of samples on each lane was applied. Equilibrium Gel Filtration Studies—The equilibrium gel filtration was performed on a SMART system with Superdex-200 PC 3.2/30 column (Amersham Biosciences) at a flow rate of 60 μl/min. The column was equilibrated with 10 mm Hepes, pH 7.4, 150 mm NaCl, 3.4 mm EDTA, and 0.005% surfactant P20 (HBS-EP buffer) with 10 μm LILRB1 or 15 μm LILRB2. The mixtures of HLA-G dimer (10 μm for LILRB1 and 15 μm for LILRB2) and LILRB1/2 at molar ratios of 1:0 (dimer alone), 1:1, 1:2, and 1:3 were incubated at room temperature for 1 h before injections. Surface Plasmon Resonance Studies—Surface plasmon resonance experiments were performed using a BIAcore2000™ (BIAcore AB, St. Albans, UK) following the standard protocol of our previous report (3Shiroishi 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. Maenaka K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8856-8861Crossref PubMed Scopus (444) Google Scholar). Briefly, the biotinylated LILRBs were immobilized on the research grade Sensor Chip CM5 (BIAcore AB) on which streptavidin was covalently immobilized. The HLA-G dimer or C42S mutant monomer flowed over at 50 μl/min. Kinetic constants were derived using the curve fitting for the bivalent analyte model or the simple 1:1 binding model by the BIAevaluation version 3.2 (BIAcore). For equilibrium binding analyses, the equilibrating binding response at each concentration of analyte was calculated by subtracting the response measured in the control flow cell from the response in the sample flow cells. Affinity constants (Kd) were calculated by nonlinear curve fitting or by Scatchard analysis with the simple 1:1 Langmuir binding model using the program Origin version 5.0 (Microcal). In the experiments of reverse orientation, the biotinylated HLA-Gs were immobilized. LILRBs flowed over the immobilized HLA-Gs. LILRB1 NFAT-GFP Reporter Cell Assay—The chimera molecule that consisted of the extracellular domain of LILRB1 and the transmembrane and cytoplasmic domains of activating PILRβ (28Shiratori I. Ogasawara K. Saito T. Lanier L.L. Arase H. J. Exp. Med. 2004; 199: 525-533Crossref PubMed Scopus (105) Google Scholar) was transfected into a mouse T cell hybridoma carrying NFAT-green fluorescence protein (GFP) reporter gene and DAP12 by using retrovirus vector (26Arase H. Mocarski E.S. Campbell A.E. Hill A.B. Lanier L.L. Science. 2002; 296: 1323-1326Crossref PubMed Scopus (981) Google Scholar, 27Ohtsuka M. Arase H. Takeuchi A. Yamasaki S. Shiina R. Suenaga T. Sakurai D. Yokosuka T. Arase N. Iwashima M. Kitamura T. Moriya H. Saito T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8126-8131Crossref PubMed Scopus (84) Google Scholar). The various concentrations (0-160 ng/ml) of HLA-G dimer and monomer were immobilized on the 48-well tissue culture plate (BD Falcon) at 37 °C for 2 h. The reporter cells expressing the LILRB1-PILR chimera molecule (5 × 104/well) were stimulated by immobilized HLA-G monomer or dimer for 12 h, and the expression of GFP was analyzed by FACSCalibur. Crystallization, Data Collection, Structure Determination, and Refinement—The detailed description for crystallization of the disulfidelinked HLA-G dimer was published (51Shiroishi M. Kohda D. Maenaka K. Biochem. Biophys. Acta. 2005; 10.1016/j.bbapap.2005.10.006PubMed Google Scholar). Briefly, 0.2 μl of protein solution was mixed in a 1:1 ratio with the crystallization reservoir solution. The crystals were obtained in the condition of Wizard II-39 (100 mm CHAPS, pH 10.5, 20% (w/v) PEG8000, 200 mm NaCl) at 20 °C. A 3.2-Å diffraction data set was collected at 100 K, at beamline BL38B1 of Spring8 (Harima, Japan) (l = 1.0000 Å). The diffraction data were processed and scaled with HKL2000 program package. The detail crystallographic statistics are shown in Table 1. The HLA-G structure was phased by the molecular replacement procedure using Molrep in CCP4 package (29Number Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) with the search probe, the crystal structure of the HLA-E-peptide complex (Protein Data Bank code 1MHE) (30O'Callaghan C.A. Tormo J. Willcox B.E. Braud V.M. Jakobsen B.K. Stuart D.I. McMichael A.J. Bell J.I. Jones E.Y. Mol. Cell. 1998; 1: 531-541Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Two HLA-G monomers (chains A and B) were found by using the HLA-E model in the range 20 to 3.5 Å. After the rigid-body adjustment, one cycle of positional and overall B-factor refinement with the program Refmac5 (29Number Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar), the Fo-Fc map clearly showed electron density for the disulfide bond between Cys42 of the crystallographic 2-fold monomer (Fig. 4B), indicating that two disulfide-bonded dimers existed in this crystal, and each monomer in each dimer complex was placed along the crystallographic 2-fold axis. Further refinement with the grouped (2 groups per residue) B-factor refinement was carried out with Refmac5 and CNS (31Brunger 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. Warren G.L. Acta Crystallogr. Sect. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) and alternated with manual rebuilding in the interactive graphics program O (32Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). The final models include two HLA-G molecules (heavy chain, β2m and peptide) and show Rcryst 23.5% (Rfree = 29.8%) between 50 and 3.2 Å. Detailed crystallographic statistics are shown in Table 1. A Ramachandran plot of the backbone angles gave 85.3, 13.4, and 0.9% in most favored, additionally, and generously allowed regions, respectively, and 0.3% in the disallowed region. Ramachandran plot was calculated by PROCHECK (33Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Figures were generated using PyMOL, BOBSCRIPT (34Esnouf R.M. J. Mol. Graph. Model. 1997; 15 (112-133): 132-134Crossref PubMed Scopus (1795) Google Scholar), and Raster 3D (35Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3878) Google Scholar).TABLE 1Data collection and refinement statisticsHLA-G dimerData collectionSpring8 (BL38B1)Space groupP21212Cell dimensions a, b, c (Å)94.70, 127.85, 72.60Resolution (Å)50-3.20 (3.31-3.20)Rsym or Rmerge0.090 (0.325)I/sI6.2 (2.1)Completeness (%)83.8 (77.7)Redundancy4.9 (4.0)RefinementResolution (Å)50-3.20 (3.31-3.20)No. reflections12,897 (1,162)Rwork/Rfree0.235 (0.272)/0.279 (0.376)No. protein atoms6,126B-factor47.3r.m.s.d.Bond lengths (Å)0.010Bond angles (°)1.363 Open table in a new tab 1:2 (HLA-G Dimer:LILRBs) Binding Stoichiometry—To characterize the biochemical properties of the HLA-G dimer, the dimer and C42S mutant monomer were prepared as described under “Experimental Procedures.” The intermolecular disulfide bond in the wild-type HLA-G dimer was confirmed by gel filtration, SDS-PAGE, and native-PAGE using the reducing agents (Fig. 1, A-C). As in the previous report by Boyson et al. (17Boyson J.E. Erskine R. Whitman M.C. Chiu M. Lau J.M. Koopman L.A. Valter M.M. Angelisova P. Horejsi V. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16180-16185Crossref PubMed Scopus (192) Google Scholar), the C42S mutation kept HLA-G as the monomer form (hereafter designated as the HLA-G monomer or the monomer), clearly indicating that the HLA-G dimer was formed dominantly via the Cys42-Cys42 disulfide bond. The LILRB1 binding to the HLA-G monomer was observed on native-PAGE, but not all of the monomer formed the LILRB1 complex because of the low affinities (Kd ∼2-5 μm) as described in our previous report (3Shiroishi 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. Maenaka K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8856-8861Crossref PubMed Scopus (444) Google Scholar) (data not shown). To determine the stoichiometry of the complex between LILRB1 and HLA-G dimer, a series of the mixture (1:1, 1:2, and 1:4 (HLA-G dimer:LILRB1)) was analyzed on native-PAGE (Fig. 1D). It showed that the full complex was composed of one HLA-G dimer and two LILRB1s (1:2 complex). On the other hand, the LILRB2 binding study by native-PAGE (Fig. 1F) demonstrated that the LILRB2 bound to the dimer but with very low affinity. To determine the binding stoichiometry unambiguously, equilibrium gel filtration was performed (Fig. 1, E and G). When protein concentrations in the running solution are above the dissociation constant, the elution profile will depend on the stoichiometry of the injected mixture and the true equilibrium stoichiometry of the complex. The stoichiometry of the HLA-G dimer-LILRB1 and -LILRB2 complexes was determined by applying samples, including either 1:0, 1:1, 1:2, or 1:3 (HLA-G dimer:LILRB1) molar ratios over a column equilibrated with 10 μm LILRB1 or 15 μm LILRB2. The injection of the 1:0 and 1:1 (HLA-G dimer:LILRB) showed a peak corresponding the dimer-LILRB complex and a trough where the free LILRBs migrated, indicating that the LILRBs of the running solution were consumed for the complex formation. On the other hand, the 1:3 molar ratio sample for both LILRBs generated two peaks corresponding to the HLA-G dimer-LILRB complex and the free LILRB, indicating the presence of excess free LILRBs. The 1:2 molar ratio sample showed only one complex peak and thus represented the true equilibrium complex stoichiometry. The 1:2 binding stoichiometry for LILRB1/2 reasonably conferred the avidity effect, which was observed in the later section of the surface plasmon resonance analysis and can be accounted for by the LILRB1 complex model structure of the HLA-G dimer (Fig. 6B). High Affinity LILRB Binding of HLA-G Dimer—The binding of the HLA-G dimer to LILRBs was further characterized by surface plasmon resonance. The HLA-G dimer and monomer were injected over sensor surfaces on which biotinylated LILRBs had been immobilized. Representative data for binding of HLA-G monomer and dimer to LILRBs were shown in Fig. 2. The monomer showed 1:1 binding with very fast dissociation rates (3.5-5 s-1) (Fig. 2, B and D). Affinity constants (Kd) of the monomer for LILRB1 and LILRB2 were 3.5 and 15 μm, respectively, derived from equilibrium analysis (Fig. 2E), which were similar to those from the opposite orientation studies of the HLA-G monomer (supplemental Fig. S1 and Table S1) (3Shiroishi M. Tsumoto K. Amano K. Shirakihara Y. Colonna M. Braud V.M. Allan D.S. Makadzange A. Rowland-Jones S. Willcox B.