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• W2011265839 abstract "Natural cytotoxicity receptors (NCR) mediate lysis of a variety of tumor and virus-infected cells by natural killer (NK) cells. Upon engagement, NCR trigger the cytolytic activity and cytokine release of NK cells through association with ITAM-containing signaling molecules. To further understand the function of these receptors in activation of natural cytotoxicity, we determined the crystal structure of the extracellular ligand binding domain of human NKp46, one of three known NCR, at 2.2-Å resolution. The overall fold and disposition of the two C2-set immunoglobulin domains are similar to the D1D2 domains of inhibitory killer cell Ig-like receptor (KIR) and Ig-like transcript (ILT) receptors. As the cellular ligands of NKp46 have not yet been defined, the known ligand binding sites of KIR and ILT were compared with the corresponding structural regions of NKp46 in an effort to identify potential areas suitable for molecular recognition. A potential binding site for influenza hemagglutinin is located near the interdomain hinge, a region that mediates ligand binding in KIR. The structural similarity of NKp46 to inhibitory KIR receptors may have implications for how NK cells balance activating and inhibitory signals. Natural cytotoxicity receptors (NCR) mediate lysis of a variety of tumor and virus-infected cells by natural killer (NK) cells. Upon engagement, NCR trigger the cytolytic activity and cytokine release of NK cells through association with ITAM-containing signaling molecules. To further understand the function of these receptors in activation of natural cytotoxicity, we determined the crystal structure of the extracellular ligand binding domain of human NKp46, one of three known NCR, at 2.2-Å resolution. The overall fold and disposition of the two C2-set immunoglobulin domains are similar to the D1D2 domains of inhibitory killer cell Ig-like receptor (KIR) and Ig-like transcript (ILT) receptors. As the cellular ligands of NKp46 have not yet been defined, the known ligand binding sites of KIR and ILT were compared with the corresponding structural regions of NKp46 in an effort to identify potential areas suitable for molecular recognition. A potential binding site for influenza hemagglutinin is located near the interdomain hinge, a region that mediates ligand binding in KIR. The structural similarity of NKp46 to inhibitory KIR receptors may have implications for how NK cells balance activating and inhibitory signals. NK 1The abbreviations used are: NK, natural killer; NCR, natural cytotoxicity receptor; MHC, major histocompatibility complex; ITIM, immunoreceptor tyrosine-based inhibitory motif; ITAM, immunoreceptor tyrosine-based activating motif; ILT, Ig-like transcript; KIR, killer cell Ig-like receptor; MIRAS, multiple isomorphous replacement with anomalous signaling; SMAC, supramolecular activation cluster; CTLR, C-type lectin-like receptor; PEG, polyethylene glycol; MOPS, 4-morpholinepropanesulfonic acid; HLA, human leukocyte antigen; TCR, T cell receptor. cells contribute to the innate immune response by directly lysing certain autologous cells that have undergone tumor transformation or infection. NK cell-mediated cytotoxicity is modulated through the participation of two classes of cell surface receptors: activating receptors, which trigger the cytotoxic lysis of target cells, and inhibitory receptors, which transmit signals to block natural cytotoxicity. Inhibitory receptors, such as members of the KIR and murine Ly49 receptor families, primarily recognize class I major histocompatibility complex (MHC) molecules on target cells. Cells that have diminished or altered expression of class I MHC molecules, as may occur following viral infection or transformation, are unable to engage inhibitory receptors and become susceptible to lysis by NK cells (the “missing self” hypothesis) (1Karre K. Ljunggren H.G. Piontek G. Kiessling R. Nature. 1986; 319: 675-678Crossref PubMed Scopus (1708) Google Scholar, 2Karre K. Scand. J. Immunol. 2002; 55: 221-228Crossref PubMed Scopus (245) Google Scholar, 3Ljunggren H.G. Karre K. J. Exp. Med. 1985; 162: 1745-1759Crossref PubMed Scopus (647) Google Scholar). The ligands of activation receptors are less well defined but also encompass class I MHC molecules and their homologs. For example, the stress-inducible MHC class I-related chains A and B and UL16 binding proteins are ligands of the activation receptor NKG2D (4Bauer S. Groh V. Wu J. Steinle A. Phillips J.H. Lanier L.L. Spies T. Science. 1999; 285: 727-729Crossref PubMed Scopus (2062) Google Scholar, 5Cosman D. Mullberg J. Sutherland C.L. Chin W. Armitage R. Fanslow W. Kubin M. Chalupny N.J. Immunity. 2001; 14: 123-133Abstract Full Text Full Text PDF PubMed Scopus (1045) Google Scholar). NK receptors that regulate natural cytotoxic functions belong to two structural classes, the Ig-like and the C-type lectin-like. Both classes have activating and inhibitory members. Despite this structural diversity, certain features unite receptors with common functions; inhibitory receptors contain cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs), through which they recruit specific Src homology 2-containing phosphatases following ligand engagement (6Lanier L.L. Annu. Rev. Immunol. 1998; 16: 359-393Crossref PubMed Scopus (1486) Google Scholar). Activating receptors, in contrast, have short cytoplasmic tails lacking tyrosine-based signaling motifs but contain a charged residue in their transmembrane domains through which they associate with activating motif-containing adapter proteins. The natural cytotoxicity receptors (NCR) are a recently characterized family of Ig-like activation receptors that appear to be major triggering receptors in tumor cell recognition, although their ligands are not yet identified (7Moretta A. Biassoni R. Bottino C. Mingari M.C. Moretta L. Immunol. Today. 2000; 21: 228-234Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 8Moretta A. Bottino C. Vitale M. Pende D. Cantoni C. Mingari M.C. Biassoni R. Moretta L. Annu. Rev. Immunol. 2001; 19: 197-223Crossref PubMed Scopus (1500) Google Scholar, 9Pessino A. Sivori S. Bottino C. Malaspina A. Morelli L. Moretta L. Biassoni R. Moretta A. J. Exp. Med. 1998; 188: 953-960Crossref PubMed Scopus (477) Google Scholar, 10Sivori S. Pende D. Bottino C. Marcenaro E. Pessino A. Biassoni R. Moretta L. Moretta A. Eur. J. Immunol. 1999; 29: 1656-1666Crossref PubMed Scopus (356) Google Scholar, 11Pende D. Parolini S. Pessino A. Sivori S. Augugliaro R. Morelli L. Marcenaro E. Accame L. Malaspina A. Biassoni R. Bottino C. Moretta L. Moretta A. J. Exp. Med. 1999; 190: 1505-1516Crossref PubMed Scopus (628) Google Scholar, 12Vitale M. Bottino C. Sivori S. Sanseverino L. Castriconi R. Marcenaro E. Augugliaro R. Moretta L. Moretta A. J. Exp. Med. 1998; 187: 2065-2072Crossref PubMed Scopus (611) Google Scholar, 13Sivori S. Vitale M. Morelli L. Sanseverino L. Augugliaro R. Bottino C. Moretta L. Moretta A. J. Exp. Med. 1997; 186: 1129-1136Crossref PubMed Scopus (434) Google Scholar). To date, the three known NCR are NKp46 and NKp30, which are expressed by circulating NK cells, and NKp44, which is expressed only by activated NK cells (9Pessino A. Sivori S. Bottino C. Malaspina A. Morelli L. Moretta L. Biassoni R. Moretta A. J. Exp. Med. 1998; 188: 953-960Crossref PubMed Scopus (477) Google Scholar, 11Pende D. Parolini S. Pessino A. Sivori S. Augugliaro R. Morelli L. Marcenaro E. Accame L. Malaspina A. Biassoni R. Bottino C. Moretta L. Moretta A. J. Exp. Med. 1999; 190: 1505-1516Crossref PubMed Scopus (628) Google Scholar, 12Vitale M. Bottino C. Sivori S. Sanseverino L. Castriconi R. Marcenaro E. Augugliaro R. Moretta L. Moretta A. J. Exp. Med. 1998; 187: 2065-2072Crossref PubMed Scopus (611) Google Scholar, 13Sivori S. Vitale M. Morelli L. Sanseverino L. Augugliaro R. Bottino C. Moretta L. Moretta A. J. Exp. Med. 1997; 186: 1129-1136Crossref PubMed Scopus (434) Google Scholar, 14Cantoni C. Bottino C. Vitale M. Pessino A. Augugliaro R. Malaspina A. Parolini S. Moretta L. Moretta A. Biassoni R. J. Exp. Med. 1999; 189: 787-796Crossref PubMed Scopus (382) Google Scholar). NKp46 has been implicated in NK cell-mediated lysis of several autologous tumor and pathogen-infected cell lines, including melanoma, erythroleukemia, Epstein-Barr virus-transformed B cells, and M. tuberculosis-infected monocytes (9Pessino A. Sivori S. Bottino C. Malaspina A. Morelli L. Moretta L. Biassoni R. Moretta A. J. Exp. Med. 1998; 188: 953-960Crossref PubMed Scopus (477) Google Scholar, 10Sivori S. Pende D. Bottino C. Marcenaro E. Pessino A. Biassoni R. Moretta L. Moretta A. Eur. J. Immunol. 1999; 29: 1656-1666Crossref PubMed Scopus (356) Google Scholar, 13Sivori S. Vitale M. Morelli L. Sanseverino L. Augugliaro R. Bottino C. Moretta L. Moretta A. J. Exp. Med. 1997; 186: 1129-1136Crossref PubMed Scopus (434) Google Scholar, 15Vankayalapati R. Wizel B. Weis S.E. Safi H. Lakey D.L. Mandelboim O. Samten B. Porgador A. Barnes P.F. J. Immunol. 2002; 168: 3451-3457Crossref PubMed Scopus (124) Google Scholar). In antibody-mediated redirected lysis studies, NKp46 was shown to strongly activate NK cell functions including cytokine production and cytolytic activity, and blocking of NKp46 inhibited NK cell lysis of a series of class I MHC-negative target cells (9Pessino A. Sivori S. Bottino C. Malaspina A. Morelli L. Moretta L. Biassoni R. Moretta A. J. Exp. Med. 1998; 188: 953-960Crossref PubMed Scopus (477) Google Scholar, 10Sivori S. Pende D. Bottino C. Marcenaro E. Pessino A. Biassoni R. Moretta L. Moretta A. Eur. J. Immunol. 1999; 29: 1656-1666Crossref PubMed Scopus (356) Google Scholar, 13Sivori S. Vitale M. Morelli L. Sanseverino L. Augugliaro R. Bottino C. Moretta L. Moretta A. J. Exp. Med. 1997; 186: 1129-1136Crossref PubMed Scopus (434) Google Scholar). NKp46 has two Ig-like extracellular domains followed by a ∼40-residue stalk region, a type I transmembrane domain, and a short cytoplasmic tail. Through a positively charged residue in the transmembrane region, the receptor associates with the ITAM-containing FcϵRIγ and CD3ζ (12Vitale M. Bottino C. Sivori S. Sanseverino L. Castriconi R. Marcenaro E. Augugliaro R. Moretta L. Moretta A. J. Exp. Med. 1998; 187: 2065-2072Crossref PubMed Scopus (611) Google Scholar, 16Moretta A. Bottino C. Millo R. Biassoni R. Curr. Top. Microbiol. Immunol. 1999; 244: 69-84PubMed Google Scholar). The gene encoding NKp46 is localized on the leukocyte Ig-like receptor complex on human chromosome 19, which contains a group of homologous immunoreceptors including the Ig-like transcripts (ILT), KIR, leukocyte-associated Ig-like receptors (LAIR), IgA Fc receptor (FcαRI), and platelet collagen receptor glycoprotein VI (17Martin A.M. Kulski J.K. Witt C. Pontarotti P. Christiansen F.T. Trends Immunol. 2002; 23: 81-88Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 18Wagtmann N. Rojo S. Eichler E. Mohrenweiser H. Long E.O. Curr. Biol. 1997; 7: 615-618Abstract Full Text Full Text PDF PubMed Google Scholar). NKp46 genes have also been identified in other mammals, including cow, rat, and mouse (19Storset A.K. Slettedal I.O. Williams J.L. Law A. Dissen E. Eur. J. Immunol. 2003; 33: 980-990Crossref PubMed Scopus (87) Google Scholar, 20Biassoni R. Pessino A. Bottino C. Pende D. Moretta L. Moretta A. Eur. J. Immunol. 1999; 29: 1014-1020Crossref PubMed Scopus (135) Google Scholar, 21Falco M. Cantoni C. Bottino C. Moretta A. Biassoni R. Immunol. Lett. 1999; 68: 411-414Crossref PubMed Scopus (42) Google Scholar). Despite common function, NKp46 has no significant homology to NKp30 and NKp44, which are encoded elsewhere in the genome. In efforts to understand the molecular basis of NCR-mediated NK cell activation, we determined the crystal structure of the extracellular ligand binding domains of human NKp46. Protein Expression, Refolding, and Purification—The ectodomain of NKp46 (residues 1-235, excluding the signal sequence) was expressed in pET-22b with a C-terminal tag containing Leu-Glu followed by a His6 purification tag. The receptor was expressed as inclusion bodies in BL21(DE3) cells and reconstituted by a dilution refolding procedure. In brief, inclusion bodies were redissolved in 8 m urea and rapidly diluted in a solution containing 0.1 m Tris-HCl, pH 8.0, 0.4 m arginine, 5 mm cysteamine, and 1 mm cystamine. The refolded protein was purified by immobilized metal affinity chromatography using nickel-nitrilotriacetic acid-agarose (Qiagen) followed by size exclusion chromatography on a Superdex 200 column (Amersham Biosciences). Crystallization and Preparation of Heavy Atom Derivatives—NKp46 (9 mg/ml) was crystallized at 18 °C by hanging drop vapor diffusion over solutions containing 8-11% polyethylene glycol (PEG) 2000, 0.1 m MOPS at pH 7.2. Initial crystallization conditions were identified using microbatch procedures with an Oryx 6 robot crystallization station (Douglas Instruments). Crystals (0.07 × 0.07 × 0.3 mm) belong to space group P61 and contain one molecule per asymmetric unit. Heavy atom reagents were screened for reaction with NKp46 in solution by mass spectrometry (22Sun P.D. Hammer C.H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 161-168Crossref PubMed Scopus (18) Google Scholar). Mercury derivatives were generated using relatively high heavy atom reagent concentrations and shorter soak times (23Sun P.D. Radaev S. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1099-1103Crossref PubMed Scopus (20) Google Scholar, 24Sun P.D. Radaev S. Kattah M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1092-1098Crossref PubMed Scopus (47) Google Scholar). Crystals Hg1 and Hg2 (Table I) were prepared by quick-soaking crystals in a solution containing 10 mm HgCl2, 10-14% PEG 2000, 50 mm HEPES at pH 7.0 for 75-110 min. An additional mercury derivative (crystal Hg3) was prepared by soaking in a solution containing 10 mm Hg(CH3COO)2, 10% PEG 2000, 50 mm HEPES at pH 7.0 for 1 h. Lead-soaked crystals were prepared with longer soak times (4 h) in 16.7 mm (CH3)3Pb(CH3COO)2 (trimethyllead acetate), 10% PEG 4000, 50 mm MOPS at pH 7.2.Table ICrystallographic data collection and refinement statisticsNativeHg1aThe Hg1 and Hg2 crystals were soaked in HgCl2 for 75 and 110 min, respectively; the Hg3 crystal was soaked in Hg(CH3COO)2 for 1 h, and the lead crystal was soaked in trimethyl lead acetate for 4 h.Hg2Hg3PbWavelength (Å)1.00930.99311.00930.99310.9392Unit cell parameters (Å)a = b = 85.73, c = 59.52a = b = 86.18, c = 59.34a = b = 86.27, c = 59.03a = b = 86.04, c = 59.22a = b = 85.02, c = 59.21Resolution range (Å)50-2.250-3.050-3.450-3.150-2.7Total observations347,92261,64227,44946,32185,580Unique reflections12,7855,1413,4594,1456,818Completeness (%)97.8 (83.3)bValues in parentheses are given for the highest resolution shell.99.8 (100.0)90.9 (16.5)95.0 (97.8)99.5 (99.4)〈I/σ(I)〉21.2 (2.2)13.2 (2.2)17.5 (4.8)7.9 (1.7)17.5 (3.0)R sym (%)cRsym=Σhkl|Ihkl-〈Ihkl〉|/ΣhklIhkl.7.1 (30.8)13.8 (75.9)5.5 (18.1)10.5 (53.0)6.2 (39.4)Phasing statisticsR iso (%)dRiso=Σ||FPH|-|FP||/Σ|FP|, where F ph is the structure factor amplitude of the derivative crystal and F p is that of the native crystal.0.2810.2680.2740.117Number of sites3333Phasing power (acentric/centric)ePhasing power = (heavy atom amplitude)/(lack of closure error).2.5/1.52.3/1.32.6/1.50.60/0.52R cullis (acentric/centric)fR cullis = (lack of closure error)/(isomorphous difference).0.56/0.690.60/0.740.56/0.650.91/0.87Overall figure of merit (15-3 Å)0.59Refinement statisticsResolution range (Å)20-2.2R cryst (%)gR=Σ|Fobs-Fcalc|/Σ|Fobs|. R cryst and R free were calculated from working and test sets of reflections, respectively; the test set was composed of 1,284 (10%) reflections that were excluded from refinement.20.6R free (%)25.0Mean B-factor (Å2)41.9Number of atomsProtein1,500Water87r.m.s. deviations from idealityBond lengths (Å)0.008Angles (°)1.47Ramachandran plot, residues in:Most favored regions (%)94.6Allowed regions (%)100a The Hg1 and Hg2 crystals were soaked in HgCl2 for 75 and 110 min, respectively; the Hg3 crystal was soaked in Hg(CH3COO)2 for 1 h, and the lead crystal was soaked in trimethyl lead acetate for 4 h.b Values in parentheses are given for the highest resolution shell.c Rsym=Σhkl|Ihkl-〈Ihkl〉|/ΣhklIhkl.d Riso=Σ||FPH|-|FP||/Σ|FP|, where F ph is the structure factor amplitude of the derivative crystal and F p is that of the native crystal.e Phasing power = (heavy atom amplitude)/(lack of closure error).f R cullis = (lack of closure error)/(isomorphous difference).g R=Σ|Fobs-Fcalc|/Σ|Fobs|. R cryst and R free were calculated from working and test sets of reflections, respectively; the test set was composed of 1,284 (10%) reflections that were excluded from refinement. Open table in a new tab Data Collection and Processing—Prior to data collection, crystals were soaked in solutions containing glycerol (20-25%) for ∼1 min and flash-frozen directly in a stream of liquid nitrogen. Native diffraction data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at www.ser.anl.gov/new/members.html. Derivative data were measured at beamline X9b at the National Synchrotron Light Source, Brookhaven National Laboratory. All data were integrated and scaled with HKL2000 (25Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). Structure Solution and Refinement—Heavy atom sites were initially interpreted by SOLVE (26Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar), and additional sites were located by difference Patterson techniques and refined using MLPHARE (27Collaborative Computational Project Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The space group was confirmed by refinement of anomalous occupancies. MIRAS phases were initially calculated to 3.0 Å using data from three mercury- and one lead-derivatized crystals. Although the lead-soaked crystal was only weakly derivatized, its inclusion with the mercury derivatives appeared to improve the quality of the phases. Following solvent flattening and phase extension to 2.2 Å, the map was readily interpretable. Refinement of the structure against native data was carried out with CNS version 1.0 (28Brunger 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 model building was performed using the program “O” (29Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). Residues in the CC′ loop of domain 2 were refined with lower occupancies due to weak electron density. Geometry of the refined structure was validated according to Ramachandran plot criteria of Lovell et al. (30Lovell S.C. Davis I.W. Arendall III, W.B. de Bakker P.I. Word J.M. Prisant M.G. Richardson J.S. Richardson D.C. Proteins. 2003; 50: 437-450Crossref PubMed Scopus (3892) Google Scholar). Analysis of interdomain hinge angles was performed with the program HINGE (31Snyder G.A. Brooks A.G. Sun P.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3864-3869Crossref PubMed Scopus (104) Google Scholar). Surface area calculations were made with CONTACT and AREAIMOL (27Collaborative Computational Project Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). Molecular figures were prepared with Molscript (32Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), Raster3D (33Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3878) Google Scholar), GRASP (34Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5318) Google Scholar), and Pymol (35DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar). Potential glycosylation sites were identified by NetOGlyc and NetNGlyc (36Hansen J.E. Lund O. Tolstrup N. Gooley A.A. Williams K.L. Brunak S. Glycoconj. J. 1998; 15: 115-130Crossref PubMed Scopus (454) Google Scholar). Overview of the Structure of NKp46 D1D2—The crystal structure of the extracellular ligand binding domain of NKp46 was determined to 2.2 Å by MIRAS phasing. Data collection and phasing statistics are listed in Table I. The refined structure includes residues 4-191, corresponding to the two N-terminal Ig domains. No interpretable electron density was found for the 40-residue stalk region that connects the two Ig domains to the transmembrane region. This suggests that the stalk is highly flexible and does not adopt a defined secondary structure. The two Ig domains of NKp46 are arranged in a V-shaped conformation, with an interdomain hinge angle of 85° (Fig. 1). Each Ig domain resembles that of a C2-set Ig fold, with two antiparallel β-sheets formed by strands ABE and C′CFG. NKp46 deviates from the canonical C2 Ig fold in that the first β-strand in each domain is interrupted by a cis proline and is split into two shorter strands A and A′, which pair with strands B and G, respectively. Additional short strands are formed between strands F and G; in D1 this short strand pairs with strand F, while in D2 it pairs with strand G of D1. NKp46 contains short regions of helical conformation, including a 310 helix in the EF loop of each domain. Residues preceding strand G adopt a polyproline II-type helical conformation, as observed in ILT2 (37Chapman T.L. Heikema A.P. West Jr., A.P. Bjorkman P.J. Immunity. 2000; 13: 727-736Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). These residues overlap with sequence motifs similar to the WSXWS motifs first identified in hematopoietic receptors (37Chapman T.L. Heikema A.P. West Jr., A.P. Bjorkman P.J. Immunity. 2000; 13: 727-736Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar); in NKp46 these motifs are WSXXS and WSFPS in the N- and C-terminal domains, respectively. The side chains of the two serine residues within this motif are hydrogen bonded to main chain atoms in strand F, thus contributing to the stability of the core Ig fold. The observed hinge angle of NKp46 (85°) is very similar to unliganded ILT2 (86°) and KIR2DL2 (84°) but is slightly larger than HLA-Cw3-bound KIR2DL2 (75°). Acute hinge angles are also common to Fc receptors such as FcγRIII and FcϵRI although their relative domain orientation is different (38Garman S.C. Kinet J.P. Jardetzky T.S. Annu. Rev. Immunol. 1999; 17: 973-976Crossref PubMed Scopus (30) Google Scholar, 39Zhang Y. Boesen C.C. Radaev S. Brooks A.G. Fridman W.H. Sautes-Fridman C. Sun P.D. Immunity. 2000; 13: 387-395Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The hinge angle of NKp46 is stabilized by both interdomain hydrogen bonds and hydrophobic interactions. Specifically, hydrogen bond pairings between the additional short strand of D2 and the G strand of D1 and between Arg145 and Asn175 and the presence of a salt bridge between Asp93 and His176 contribute to the hinge conformation. The hydrophobic hinge core is formed between residues from strands A′ and G in D1 and residues from strands C and C′, as well as the WSFPS motif in D2. The interface buries 1,021 Å2 of accessible surface area, and comparison of NKp46 sequences across species reveals that 11 of 18 hinge core residues are conserved, indicating that the acute domain orientation is likely to be preserved. Structural Comparison with Ig-like Immunoreceptors—The crystal structure of human NKp46 reported here can be contrasted with the structure of the NKp44 ectodomain, which is a single V-type Ig domain (40Cantoni C. Ponassi M. Biassoni R. Conte R. Spallarossa A. Moretta A. Moretta L. Bolognesi M. Bordo D. Structure (Camb.). 2003; 11: 725-734Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In addition, a third disulfide bond observed in the structure of NKp44 is absent in NKp46. Furthermore, NKp30 is also predicted to contain a single V-type Ig domain. The two-domain structure of NKp46 does resemble a number of Ig-like cell surface receptors. The closest structural relatives are other leukocyte receptor complex immunoreceptors, including KIR, ILT, and FcαRI (CD89), which are 31-35% identical to NKp46 at the protein sequence level. NKp46 also has structural similarity to FcγRIIb and other Fc receptors of known structure, although sequence identity to these is less than 20%. Structures of the D1D2 domains of three KIR family proteins have been determined alone and in complex with class I MHC ligands (31Snyder G.A. Brooks A.G. Sun P.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3864-3869Crossref PubMed Scopus (104) Google Scholar, 41Boyington J.C. Motyka S.A. Schuck P. Brooks A.G. Sun P.D. Nature. 2000; 405: 537-543Crossref PubMed Scopus (347) Google Scholar, 42Fan Q.R. Mosyak L. Winter C.C. Wagtmann N. Long E.O. Wiley D.C. Nature. 1997; 389: 96-100Crossref PubMed Scopus (143) Google Scholar, 43Fan Q.R. Long E.O. Wiley D.C. Nat. Immunol. 2001; 2: 452-460Crossref PubMed Scopus (224) Google Scholar, 44Maenaka K. Juji T. Stuart D.I. Jones E.Y. Struct. Fold. Des. 1999; 7: 391-398Abstract Full Text Full Text PDF Scopus (84) Google Scholar). Superposition of NKp46 with KIR2DL2 (Fig. 2A) resulted in a root mean square (r.m.s.) deviation of 1.1 Å for 71 α-carbon atoms in D1 and 0.9 Å for 78 α-carbon atoms in D2. The D1D2 moieties of ILT2 (LIR-1) and ILT4 (LIR-2) (37Chapman T.L. Heikema A.P. West Jr., A.P. Bjorkman P.J. Immunity. 2000; 13: 727-736Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 45Willcox, B. E., Thomas, L. M., Chapman, T. L., Heikema, A. P., West, A. P., Jr., and Bjorkman, P. J. (2002) BMC Structure Biology http://biomedcentral.com/1472-6807/2/6Google Scholar) are also closely related in structure to NKp46. Superposition of ILT2 with NKp46 (Fig. 2B) resulted in r.m.s. deviations of 0.9 Å (66 α-carbon atoms) and 1.4 Å (80 α-carbon atoms) for D1 and D2, respectively. The recently reported structures of the FcαRI ectodomain (46Ding Y. Xu G. Yang M. Yao M. Gao G.F. Wang L. Zhang W. Rao Z. J. Biol. Chem. 2003; 278: 27966-27970Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 47Herr A.B. Ballister E.R. Bjorkman P.J. Nature. 2003; 423: 614-620Crossref PubMed Scopus (225) Google Scholar) and its complex with Fcα (47Herr A.B. Ballister E.R. Bjorkman P.J. Nature. 2003; 423: 614-620Crossref PubMed Scopus (225) Google Scholar) also underscore the structural relationship between leukocyte Ig-like receptor complex members. The structure of free FcαRI (47Herr A.B. Ballister E.R. Bjorkman P.J. Nature. 2003; 423: 614-620Crossref PubMed Scopus (225) Google Scholar) was superimposed with NKp46 (Fig. 2C) with r.m.s. deviations of 2.1 Å (D1, 70 α-carbon atoms) and 1.9 Å (D2, 93 α-carbon atoms). NKp46 can be distinguished primarily from KIR, ILT, and FcαRI by the conformation and length of several loops. For example, the CC′ loop in D1 is shorter than in KIR, ILT2, and FcαRI. Like KIR and Fcα-complexed FcαRI, NKp46 contains a longer C′ strand in D1 as compared with ILT2 and free FcαRI, which are followed by a helical stretch and a D strand, respectively. The C′E loop in D1 has divergent conformations in the structures examined; in NKp46 this loop includes three proline residues and three basic residues and adopts an unusual meandering conformation. The nearby FG loop adopts similar conformations in NKp46 and FcαRI, while in ILT2 it is twisted relative to the plane of the FG strands and in KIR it is lengthened by three residues and forms a protruding flap (Fig. 2A). In D2, the C′E and FG loops vary in length among the receptors. The EF loop also varies, although the conformation in NKp46 resembles that in FcαRI, including a single turn of 310 helix. Implications for Ligand Binding—NKp46 has been reported to bind both influenza virus hemagglutinin and Sendai virus hemagglutinin-neuraminidase, and this interaction appears to involve terminal sialic residues on NKp46 (48Mandelboim O. Lieberman N. Lev M. Paul L. Arnon T.I. Bushkin Y. Davis D.M. Strominger J.L. Yewdell J.W. Porgador A. Nature. 2001; 409: 1055-1060Crossref PubMed Scopus (758) Google Scholar). Human NKp46 is predicted to contain a single N-linked (Asn195) and two O-linked (Thr104 and Thr204) glycosylation sites. Two of these sites (Asn195 and Thr204) are located in the membrane proximal stalk, which was disordered in the structure. Thr104 is located in D1D2 in a solvent-exposed position near the D1-D2 juncture. This location is close to the ligand binding site of KIR receptors (see below). However, none of the three putative glycosylation sites in human NKp46 are conserved in all species. To date, no cellular ligands of NKp46 have been identified, although NKp46 has been shown to mediate NK cell cytolysis of certain human tumor cells. A murine homolog of NKp46 has been identified (20Biassoni R. Pessino A. Bottino C. Pende D. Moretta L. Moretta A. Eur. J. Immunol. 1999; 29: 1014-1020Crossref PubMed Scopus (135) Google Scholar), which is 58% identical to the human receptor (Fig. 3). Intriguingly, it has been reported that murine tumor cells are susceptible to killing via human NKp46 (9Pessino A. Sivori S. Bottino C. Malaspina A. Morelli L. Moretta L. Biassoni R. Moretta A. J. Exp. Med. 1998; 188: 953-960Crossref PubMed Scopus (477) Google Scholar, 10Sivori S. Pende D. Bottino C. Marcenaro E. Pessino A. Bia" @default.
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• W2011265839 title "Crystal Structure of the Human Natural Killer (NK) Cell Activating Receptor NKp46 Reveals Structural Relationship to Other Leukocyte Receptor Complex Immunoreceptors" @default.
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