Crystal Structure of the C-type Lectin-like Domain from the Human Hematopoietic Cell Receptor CD69
2001; Elsevier BV; Volume: 276; Issue: 10 Linguagem: Inglês
10.1074/jbc.m008573200
ISSN1083-351X
AutoresAndrea S. Llera, Fernando Viedma, Francisco Sánchez‐Madrid, José R. Tormo,
Tópico(s)Immunotherapy and Immune Responses
ResumoCD69, one of the earliest specific antigens acquired during lymphoid activation, acts as a signal-transducing receptor involved in cellular activation events, including proliferation and induction of specific genes. CD69 belongs to a family of receptors that modulate the immune response and whose genes are clustered in the natural killer (NK) gene complex. The extracellular portion of these receptors represent a subfamily of C-type lectin-like domains (CTLDs), which are divergent from true C-type lectins and are referred to as NK-cell domains (NKDs). We have determined the three-dimensional structure of human CD69 NKD in two different crystal forms. CD69 NKD adopts the canonical CTLD fold but lacks the features involved in Ca2+ and carbohydrate binding by C-type lectins. CD69 NKD dimerizes noncovalently, both in solution and in crystalline state. The dimer interface consists of a hydrophobic, loosely packed core, surrounded by polar interactions, including an interdomain β sheet. The intersubunit core shows certain structural plasticity that may facilitate conformational rearrangements for binding to ligands. The surface equivalent to the binding site of other members of the CTLD superfamily reveals a hydrophobic patch surrounded by conserved charged residues that probably constitutes the CD69 ligand-binding site.1E871E8I CD69, one of the earliest specific antigens acquired during lymphoid activation, acts as a signal-transducing receptor involved in cellular activation events, including proliferation and induction of specific genes. CD69 belongs to a family of receptors that modulate the immune response and whose genes are clustered in the natural killer (NK) gene complex. The extracellular portion of these receptors represent a subfamily of C-type lectin-like domains (CTLDs), which are divergent from true C-type lectins and are referred to as NK-cell domains (NKDs). We have determined the three-dimensional structure of human CD69 NKD in two different crystal forms. CD69 NKD adopts the canonical CTLD fold but lacks the features involved in Ca2+ and carbohydrate binding by C-type lectins. CD69 NKD dimerizes noncovalently, both in solution and in crystalline state. The dimer interface consists of a hydrophobic, loosely packed core, surrounded by polar interactions, including an interdomain β sheet. The intersubunit core shows certain structural plasticity that may facilitate conformational rearrangements for binding to ligands. The surface equivalent to the binding site of other members of the CTLD superfamily reveals a hydrophobic patch surrounded by conserved charged residues that probably constitutes the CD69 ligand-binding site.1E871E8I C-type lectin-like domain(s) natural killer NK-cell receptor domain(s) NK gene cluster carbohydrate-recognition domain(s) major histocompatibility complex mannose-binding protein root mean square dithiothreitol 4-morpholinoethanesulfonic acid The C-type lectin-like domain (CTLD)1 is a conserved protein module that has been found in numerous proteins with a wide range of functions (1Drickamer K. Curr. Opin. Struct. Biol. 1999; 9: 585-590Crossref PubMed Scopus (535) Google Scholar). This fold was initially identified in a group of C-type (Ca2+-dependent) animal lectins (sugar-binding proteins) that mediate both pathogen recognition and cell-cell communication by means of protein-carbohydrate interactions (2Drickamer K. Taylor M.E. Annu. Rev. Cell Biol. 1993; 9: 237-264Crossref PubMed Scopus (723) Google Scholar). Based on amino acid sequence comparisons, multidomain arrangement, and functional criteria the extensive superfamily of CTLDs has been subdivided in a variety of groups (3Drickamer K. Curr. Opin. Struct. Biol. 1993; 3: 393-400Crossref Scopus (212) Google Scholar, 4Day A.J. Biochem. Soc. Trans. 1994; 22: 83-88Crossref PubMed Scopus (153) Google Scholar). Among them, those that are predicted to bind sugars through coordination to a conserved Ca2+ ion are also known as carbohydrate-recognition domains (CRDs). The structure of several CRDs has been solved, and their binding to sugars has been thoroughly characterized (1Drickamer K. Curr. Opin. Struct. Biol. 1999; 9: 585-590Crossref PubMed Scopus (535) Google Scholar, 5Weis W.I. Drickamer K. Annu. Rev. Biochem. 1996; 65: 441-473Crossref PubMed Scopus (1022) Google Scholar). Increasing evidence shows, however, that many of the modules that adopt a CTLD fold lack the Ca2+-coordinating residues that mediate the classical C-type lectin sugar binding properties, suggesting that they may serve functions other than saccharide recognition (1Drickamer K. Curr. Opin. Struct. Biol. 1999; 9: 585-590Crossref PubMed Scopus (535) Google Scholar). The CTLD fold is widely represented among proteins that mediate the innate immune response (6Weis W.I. Taylor M.E. Drickamer K. Immunol. Rev. 1998; 163: 19-34Crossref PubMed Scopus (913) Google Scholar). In particular, a conserved genomic region known as natural killer (NK) gene cluster (NKC) encodes for a group of receptors with CTLD-containing sequences that are involved in modulation of NK-cell activity and natural host defense (6Weis W.I. Taylor M.E. Drickamer K. Immunol. Rev. 1998; 163: 19-34Crossref PubMed Scopus (913) Google Scholar, 7Brown M.G. Scalzo A.A. Matsumoto K. Yokoyama W.M. Immunol. Rev. 1997; 155: 53-65Crossref PubMed Scopus (147) Google Scholar). Most of these proteins are type II transmembrane receptors, usually expressed as disulfide-linked homo- or heterodimers. Each subunit comprises a single extracellular CTLD, specifically named NK domain (NKD), connected by a neck region of variable length to a single membrane-spanning region and a short intracellular N-terminal portion (reviewed in Refs. 8Lanier L.L. Annu. Rev. Immunol. 1998; 16: 359-393Crossref PubMed Scopus (1496) Google Scholar and 9López-Botet M. Bellón T. Curr. Opin. Immunol. 1999; 11: 301-307Crossref PubMed Scopus (146) Google Scholar). The cytoplasmic regions are often involved in signaling by recruitment, through specific sequence motifs, of kinases and phosphatases. Alternatively, some activating receptors, bearing short cytoplasmic tails devoid of any specific signaling sequence, associate noncovalently with membrane-anchored signaling molecules (10Bléry M. Olcese L. Vivier E. Hum. Immunol. 2000; 61: 51-64Crossref PubMed Scopus (98) Google Scholar). Some receptors encoded in the NKC, such as the CD94/NKG2 heterodimers and the mouse Ly49 family, have proved to bind molecules of the major histocompatibility complex (MHC) (11Ugolini S. Vivier E. Curr. Opin. Immunol. 2000; 12: 295-300Crossref PubMed Scopus (67) Google Scholar, 12López-Botet M. Llano M. Navarro F. Bellón T. Semin. Immunol. 2000; 12: 109-119Crossref PubMed Scopus (155) Google Scholar), but for many other receptors ligands have not yet been identified. CD69, one of the first described members of the NKC family of receptors (13Hamann J. Fiebig H. Strauss M. J. Immunol. 1993; 150: 4920-4927PubMed Google Scholar, 14López-Cabrera M. Santis A.G. Fernández-Ruiz E. Blacher R. Esch F. Sánchez-Mateos P. Sánchez-Madrid F. J. Exp. Med. 1993; 178: 537-547Crossref PubMed Scopus (265) Google Scholar, 15Ziegler S.F. Levin S.D. Johnson L. Copeland N.G. Gilbert D.J. Jenkins N.A. Baker E. Sutherland G.R. Feldhaus A.L. Ramsdell F. J. Immunol. 1994; 152: 1228-1236PubMed Google Scholar), is present at the cell surface as a disulfide-linked homodimer, with subunits of 28 and 32 kDa resulting from the differential glycosylation at a single extracellularN-linked glycosylation site (see Ref. 16Sánchez-Mateos P. Sánchez-Madrid F. Eur. J. Immunol. 1991; 21: 2317-2325Crossref PubMed Scopus (46) Google Scholar and reviewed in Ref. 17Testi R. D'Ambrosio D. De Maria R. Santoni A. Immunol. Today. 1994; 15: 479-483Abstract Full Text PDF PubMed Scopus (126) Google Scholar). Contrary to other NKC gene products, whose expression is restricted to NK cells, CD69 has been found in the surface of most hematopoietic lineages (reviewed in Ref. 18Marzio R. Mauel J. Betz-Corradin S. Immunopharmacol. Immunotoxicol. 1999; 21: 565-582Crossref PubMed Scopus (197) Google Scholar). It is one of the earliest markers induced upon activation in T and B lymphocytes, NK cells, macrophages, neutrophils, and eosinophils. In addition, it is constitutively expressed on monocytes, platelets, Langerhans cells, and a small percentage of resident lymphocytes in thymus and secondary lymphoid tissues (19Sánchez-Mateos P. Cebrián M. Acevedo A. López-Botet M. De Landázuri M.O. Sánchez-Madrid F. Immunology. 1989; 68: 72-79PubMed Google Scholar). CD69 is also present on B cell precursors in the bone-marrow, and recent studies with CD69-deficient mice revealed its modulatory role on B cell development and antibody synthesis (20Lauzurica P. Sancho D. Torres M. Albella B. Marazuela M. Merino T. Bueren J.A. Martı́nez,-A C. Sánchez-Madrid F. Blood. 2000; 95: 2312-2320Crossref PubMed Google Scholar). It has been demonstrated that CD69 acts as a signal-transmitting receptor. Its cytoplasmic portion is constitutively phosphorylated on serine residues (21Testi R. Phillips J.H. Lanier L.L. J. Immunol. 1988; 141: 2557-2563PubMed Google Scholar, 22Lanier L.L. Buck D.W. Rhodes L. Ding A. Evans E. Barney C. Phillips J.H. J. Exp. Med. 1988; 167: 1572-1585Crossref PubMed Scopus (232) Google Scholar). Even when the actual ligand that triggers this receptor is not known, cross-linking of CD69 by specific antibodies activates the extracellular signal-regulated kinase signaling pathway (23Zingoni A. Palmieri G. Morrone S. Carretero M. López-Botet M. Piccoli M. Frati L. Santoni A. Eur. J. Immunol. 2000; 30: 644-651Crossref PubMed Scopus (63) Google Scholar) and has been shown to induce rise in intracellular calcium concentration, synthesis of different cytokines and/or proliferation (24Cebrián M. Yague E. Rincón M. López-Botet M. De Landázuri M.O. Sánchez-Madrid F. J. Exp. Med. 1988; 168: 1621-1637Crossref PubMed Scopus (269) Google Scholar, 25Nakamura S. Sung S.S. Bjorndahl J.M. Fu S.M. J. Exp. Med. 1989; 169: 677-689Crossref PubMed Scopus (70) Google Scholar, 26Testi R. Phillips J.H. Lanier L.L. J. Immunol. 1989; 143: 1123-1128PubMed Google Scholar, 27Santis A.G. Campanero M.R. Alonso J.L. Tugores A. Alonso M.A. Yague E. Pivel J.P. Sánchez-Madrid F. Eur. J. Immunol. 1992; 22: 1253-1259Crossref PubMed Scopus (89) Google Scholar, 28De Maria R. Cifone M.G. Trotta R. Rippo M.R. Festuccia C. Santoni A. Testi R. J. Exp. Med. 1994; 180: 1999-2004Crossref PubMed Scopus (166) Google Scholar), and target lysis in interleukin-2-activated NK cells (22Lanier L.L. Buck D.W. Rhodes L. Ding A. Evans E. Barney C. Phillips J.H. J. Exp. Med. 1988; 167: 1572-1585Crossref PubMed Scopus (232) Google Scholar). In summary, CD69 wide distribution, along with its activating signal-transducing properties, suggest an important role of this receptor in the physiology of leukocyte activation. As a first step toward better understanding the structural basis for CD69 function, we have undertaken the production and analysis of soluble constructs of the CD69 extracellular region. We report here the three-dimensional structure of human CD69 NKD determined in two crystal forms. We compare it with other known CTLD structures, specifically those present in NKC receptors, describe its dimeric oligomerization, and suggest a putative ligand-binding site. The complete NKD of human CD69 (residues 82–199) was amplified from cDNA (14López-Cabrera M. Santis A.G. Fernández-Ruiz E. Blacher R. Esch F. Sánchez-Mateos P. Sánchez-Madrid F. J. Exp. Med. 1993; 178: 537-547Crossref PubMed Scopus (265) Google Scholar) by polymerase chain reaction and subcloned intoNdeI-BamHI restriction sites of pET 26b plasmid (Novagen) (29Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6225) Google Scholar) by double digestion and subsequent ligation. Restriction sites and a TAA termination codon were added to the insert using the following primers: CD695s, 5′GCGCGCGCGCATATGGTTTCTTCATGCTCTG; CD693, 5′GCGCGCGGATCCTTATTTGTAAGGTTTG. Automatic DNA sequencing (ABI PRISM, PerkinElmer Life Sciences) of the cloned 1insert using T7 promoter primer rendered the correct sequence. The resulting plasmid was transformed into Escherichia colistrain BL21(DE3), and these cells were grown in LB medium at 37 °C until the A 600 reached 0.7 cm−1 and then induced by addition of isopropyl-1-thio-β-d-galactopyranoside to 0.5 mm. After 4 h, cells were harvested and resuspended in Tris-HCl, pH 8.0, 0.2 m NaCl, 5 mm EDTA, 5 mm DTT. They were lysed by adding lysozyme to a final concentration of 1 mg/ml, and the viscosity was reduced by sonication. The protein was obtained as insoluble aggregates forming inclusion bodies, which were extensively washed three times in 50 mmTris-HCl, pH 8.0, 0.1 m NaCl, 1 mm EDTA, 1 mm DTT, 0.5% (v/v) Triton X-100 and once in 50 mm Tris-HCl, pH 8.0, 2 m NaCl, 1 murea, 1 mm EDTA, 1 mm DTT. The protein was solubilized in 25 mm MES, pH 6.5, 8 m urea, 10 mm EDTA, 1 mm DTT, and insoluble material was discarded by centrifugation. The CD69 NKD was refolded by the method of dilution of denaturing conditions following a modification of the protocol originally described for MHC class I molecules (30Garboczi D.N. Hung D.T. Wiley D.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3429-3433Crossref PubMed Scopus (579) Google Scholar). Urea-solubilized CD69 NKD was added by slow dilution to 1 liter of 0.1m Tris-HCl, pH 8.5, 400 mml-arginine, 2 mm EDTA, 6.3 mmcysteamine, 3.7 mm cysteamine, 0.1 mmphenylmethylsulfonyl fluoride. Repeated pulses were added every 12 h. After 36 h, the refolding mixture was concentrated under nitrogen to a volume of 5 ml and purified by gel filtration chromatography in 25 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.1 mm EDTA on a Superdex 200 column (Amersham Pharmacia Biotech). In these conditions, CD69 NKD elutes as a noncovalent dimer, although the peak shows a slight asymmetry, consistent with an equilibrium between the monomeric and dimeric forms. Correctly sized fractions were further purified by cation exchange chromatography using a Mono S column (Amersham Pharmacia Biotech). Protein was loaded in 25 mm Tris-HCl, pH 7.0 and eluted with a linear gradient to 500 mm NaCl in the same buffer. For crystallization, the protein was buffer-exchanged into 15 mm Hepes, pH 7.2, 50 mm NaCl and concentrated to 5 mg/ml. Crystals were obtained by mixing aliquots of the protein solution with an equal volume of the reservoir solution containing 0.1 m sodium acetate buffer, pH 4.8, 150 mm zinc sulfate or sodium sulfate, and 15% polyethylene glycol 6000. Two different crystal forms grew in these conditions. Long and thin prisms of square section, which belong to the tetragonal system, predominated, whereas larger irregular crystals, occasionally with a triangular shape and that belong to the trigonal system, appeared infrequently. The tetragonal crystals belong to the space group P43212 with unit cell dimensions a = b = 69.6 Å and c = 118.6 Å and contain one dimer in the asymmetric unit. The trigonal crystals belong to the space group P3121 with unit cell dimensions a = b = 48.4 Å, c = 119.9 Å, a = β = 90°, and γ = 120° and contain a monomer in the asymmetric unit. For cryogenic data collection, crystals were harvested in a modified reservoir solution, transferred to harvest buffer containing 15% glycerol or ethylene glycol, and flash-cooled by plunging into liquid propane. The high resolution data sets used for the structure refinement of both crystal forms were collected at beam line ID14-3 of the European Synchrotron Radiation Facility in Grenoble, France, using a MarResearch charge-coupled device, whereas the initial data set in the tetragonal form used for the structure determination was collected at beam line ID14–2 using an ADSC Quantum4 charge-coupled device. Data were integrated, scaled, and merged with the HKL package (31Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38777) Google Scholar) (see Table I).Table IData collection and refinement statistics for human CD69Data collectionData setsP43212P43212P3121Resolution range aNumbers in parentheses correspond to the highest resolution shell. (Å)50.0–2.3050.0–1.9550.0–1.50(2.35–2.30)(2.00–1.95)(1.53–1.50)Measurements8059798431151066Unique reflections139292183626542Completeness (%)98.7 (98.4)99.1 (99.2)98.6 (100.0)I/ς(I)20.2 (4.0)27.5 (5.4)33.5 (15.6)R merge(%)6.8 (31.1)6.1 (20.2)6.2 (10.6)RefinementResolution range25.0–1.9520.0–1.50R cryst/R free24.8/27.022.9/24.4Reflectionsworking set19826 (90.0%)24561 (91.3%)test set2010 (9.1%)1961 (7.3%)Number of copies in asymmetric units21Number of nonhydrogen protein atoms bNumbers in brackets represent atoms in dual conformation.1926956 [54]Number of solvent atoms69115r.m.s. deviations from idealitybond length (Å)0.0070.009bond angle (°)1.411.57bonded B factors (Å2)2.271.82Ramachandran plot cAs calculated by PROCHECK (60).most favored (%)9092.4allowed (%)107.6generously allowed (%)00disallowed (%)00a Numbers in parentheses correspond to the highest resolution shell.b Numbers in brackets represent atoms in dual conformation.c As calculated by PROCHECK (60Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Open table in a new tab The CD69 structure was determined in the tetragonal crystal form, which became available first. The structure was solved by molecular replacement using the AMoRe package (32Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5038) Google Scholar) with truncated coordinates of the CD94 dimer (33Boyington J.C. Riaz A.N. Patamawenu A. Coligan J.E. Brooks A.G. Sun P.D. Immunity. 1999; 10: 75-82Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) as the search model. Structure factors from 15.0 to 5.0 Å were used for the rotation and translation functions. Model phases were improved and extended from 5.0 to 2.6 Å by iterative cycles of density modification in DM (34Cowtan K.D. Joint CCP4 ESF-EACMB Newslett. 1994; 31: 34Google Scholar), which consisted of solvent flattening and 2-fold averaging. The resulting electron density maps allowed unambiguous building of the molecule, including extensive portions, like helix α2, not present in the initial truncated model. Crystallographic refinement was carried out in CNS (35Brünger 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 (17024) Google Scholar) using standard procedures that included a bulk solvent correction and overall anisotropic scaling. Automatic refinement, employing the maximum likelihood amplitude target, was alternated with manual rebuilding in the graphics program O (36Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13036) Google Scholar) using both averaged and unaveraged ςA-weighted (37Read R.J. Acta Crystallogr. Sect. A. 1986; 42: 140-149Crossref Scopus (2053) Google Scholar) 2Fo −Fc, Fo −Fc, and omit electron density maps. Tight noncrystallographic symmetry restrains were initially applied to all regions except the flexible loops involved in lattice contacts. When the high resolution data set for this crystal form became available, the noncrystallographic symmetry restrains were gradually relaxed based on the behavior of the R free. All regions of CD69 NKD are well ordered, with the exception of the tip of the β2-β2′ hairpin, one residue at the N terminus, and the last two residues at the C terminus, which show poor density and high B factors. The model contains residues from 83 to 199 for both subunits, and 69 solvent atoms. The present R crysis 24.8, and R free is 27.0 for all data (F > 0) between 25.0 and 1.95 Å. The trigonal crystal form was readily solved using a partially refined model from the tetragonal crystal form and was similarly refined in CNS. Electron density maps clearly showed two possible conformations for residues at the carboxy end of helix α2. Therefore, residues 133 to 136 were modeled in two alternate conformations with half-occupancy. All residues, including the β2-β2′ hairpin, showed in the electron density maps. The model contains residues from 83 to 199 for both subunits, and 115 solvent atoms. The presentR crys is 22.9%, andR free is 24.4% for all data (F>0) between 20.0 and 1.50 Å. Refinement statistics for both crystal forms are given in Table I. Structure superpositions were done with SHP (38Stuart D.I. Levine M. Muirhead H. Stammers D.K. J. Mol. Biol. 1979; 134: 109-142Crossref PubMed Scopus (385) Google Scholar). Solvent-accessible surface areas were determined with NACCESS (39Hubbard S.J. Thornton J.M. NACCESS. Department of Biochemistry and Molecular Biology, University College, London1993Google Scholar) using a probe radius of 1.4 Å and default atom radii (40Chothia C. J. Mol. Biol. 1976; 105: 1-12Crossref PubMed Scopus (1065) Google Scholar), and cavity volumes were determined with SURFNET (41Laskowski R.A. J. Mol. Graph. 1995; 13: 323-330Crossref PubMed Scopus (841) Google Scholar). Calculation of hydrogen bonds was carried out with HBPLUS (42McDonald I.K. Thornton J.M. J. Mol. Biol. 1994; 238: 777-793Crossref PubMed Scopus (1904) Google Scholar) using the program's default values. Figures were produced with GRASP (43Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5324) Google Scholar) and BOBSCRIPT (44Esnouf R.M. J. Mol. Graph. 1997; 15: 133-138Google Scholar) and rendered with RASTER3D (45Merrit E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2859) Google Scholar). The human CD69-NKD sequence was aligned to representatives of the NKD family using ClustalW at ExPASy on the Internet and were subsequently edited manually based on the known structures of rat mannose-binding protein (MBP)-A (PDB accession code 1ytt), mouse Ly49A (1qo3), and human CD94 (1b6e). Sequences were retrieved from GenBankTM (hCD69, NP_001772.1; hMAFA-L, AAC32200; hAICL, NP_005118; hLLT1, NP_037401; hNKRP1A, NP_002249; hKLRF1, AAF37804; hCLEC2, AAF36777.1; hCD94, NP_002253; hNKG2A, NP_002250.1; mLy49A, I49361) and Swiss-Prot (mCD69,P37217; rMBP-A, P19999). The alignment figure was drawn using ESPript (46Gouet P. Courcelle E. Stuart D.I. Metoz F. Bioinformatics ( Oxf. ). 1999; 15: 305-308Crossref PubMed Scopus (2560) Google Scholar). Human CD69 consists of a 40-residue intracellular domain, a 21-residue transmembrane region, and an extracellular portion that comprises a 20-residue neck and an NKD of 118 amino acids. Soluble forms of CD69 NKD, comprising residues 82 to 199, were prepared by in vitro refolding from material expressed in E. coli. Recombinant CD69 NKD retains binding to a panel of specific monoclonal antibodies recognizing four distinct epitopes (16Sánchez-Mateos P. Sánchez-Madrid F. Eur. J. Immunol. 1991; 21: 2317-2325Crossref PubMed Scopus (46) Google Scholar), and it behaves as a noncovalent dimer during gel filtration (results not shown). The structure of CD69 NKD was determined by molecular replacement in two different crystal forms, tetragonal and trigonal, and refined to 1.95 and 1.50 Å, respectively. The quality of the diffraction data and refinement statistics are given in TableI. The tetragonal crystal form contains two molecules in the asymmetric unit, related by a molecular 2-fold axis. The electron density is continuous in the final 2F o− Fc map from residues 83 to 199, except for the exposed β2-β2′ hairpin that shows weak density and appears to be disordered (see below for a description of secondary structure elements). In the trigonal crystal, the CD69 NKD dimer is crystallographic, and there is a single molecule in the asymmetric unit. In this crystal form, all residues (83 to 199), including the β2-β2′ hairpin, are in good, continuous density in the final electron density maps. The C-terminal end of helix α2 shows static disorder and has been modeled in two alternate conformations in the trigonal form. Pairwise superpositions of the three independent copies for CD69 NKD give r.m.s. deviations from 0.27 to 0.40 Å for main chain atoms between residues 89 and 198. The largest differences are focused in the domain N terminus, around the β turn (residues 86–89), which precedes strand β0, and the β2-β2′ hairpin. In the tetragonal form, this flexible hairpin is exposed to the solvent, whereas in the trigonal crystal it is better ordered because of crystal packing interactions. Description of the structure is based on the high resolution trigonal model unless specifically stated. The α-carbon trace of CD69 NKD is shown in Fig. 1 A. As predicted by its amino acid sequence, the overall structure of CD69 NKD displays the salient features of the CTLD fold. The domain, with overall dimensions of 44 × 32 × 30 Å, consists of two connected antiparallel β sheets and two α helices, like in the CRD of C-type animal lectins (1Drickamer K. Curr. Opin. Struct. Biol. 1999; 9: 585-590Crossref PubMed Scopus (535) Google Scholar) and mouse Ly49A NKD (47Tormo J. Natarajan K. Margulies D.H. Mariuzza R.A. Nature. 1999; 402: 623-631Crossref PubMed Scopus (235) Google Scholar) (Fig. 1 B). Strand β2 acts as a connection between the two β sheets formed by strands β0, β1, β5, β2, and β1′ in the lower part of the molecule (following the standard view for CTLD folds as shown in Fig. 1) and strands β2′, β2, β3, and β4 in the upper part. This portion of the molecule is also characterized by a long stretch, connecting strands β2′ and β3, lacking regular secondary structure. This region corresponds to the Ca2+-binding site of true C-type lectins. The two helices, α1 and α2, are located one on each side of the extended β structure. There are three intrachain disulfide bonds in CD69 NKD (Fig. 1), two of which (Cys113–Cys194 and Cys173–Cys186) correspond to the characteristic invariant disulfides found in all CTLDs. The third disulfide bond, Cys85–Cys96, connects a loop at the N terminus, which precedes the first β strand (β0), with strand β1 by linking two cysteines separated by 10 residues in this segment. This disulfide is only found in long-form C-type lectins (including lithostathine, tetranectin, and factors IX/X-binding protein) (4Day A.J. Biochem. Soc. Trans. 1994; 22: 83-88Crossref PubMed Scopus (153) Google Scholar) and appears to be present in most NKD domains (Fig.2). In members of the rodent Ly49 gene family, however, these two bonded cysteines are separated by only four residues and are located on contiguous β strands (47Tormo J. Natarajan K. Margulies D.H. Mariuzza R.A. Nature. 1999; 402: 623-631Crossref PubMed Scopus (235) Google Scholar) (Fig.1 B). Despite the low sequence identity between CD69 NKD and other CTLDs, which ranges from around 20% with CRDs of animal lectins to almost 30% for other NKDs, the overall structure of the domain is highly conserved. Superposition of CD69 with other CTLDs gives rise to r.m.s. deviations of between 1.2 Å for 100 equivalent Cα atoms with Ly49A and 1.4 Å for 95 equivalences with rat MBP (48Weis W.I. Kahn R. Fourme R. Drickamer K. Hendrickson W.A. Science. 1991; 254: 1608-1615Crossref PubMed Scopus (501) Google Scholar). The major differences among them occur at the N terminus, the position of helix α2, hairpin β2-β2′, the loop connecting it to strand β3, and hairpin β3-β4 (Fig. 1 B). These regions coincide with segments displaying higher variability in length and amino acid sequence among members of the NKD family (Fig. 2). Overall, CD69 NKD is more similar to long-form CTLDs, like tetranectin or lithostathine, and to other NKDs, like Ly49A. Although based on sequence identity the NKD structure closer to CD69 is that of the CD94 subunit of the NK-cell receptor CD94/NKG2 (around 28% identical), their superposition gives an r.m.s. deviation of 1.4 Å for 97 equivalent Cα positions. This relatively poorer match is mostly due to differences between helix α2 in CD69 and the equivalent region in CD94. In the crystal structure of the CD94 NKD homodimer, this helix is replaced by a loop that is involved in the dimerization interface (33Boyington J.C. Riaz A.N. Patamawenu A. Coligan J.E. Brooks A.G. Sun P.D. Immunity. 1999; 10: 75-82Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). CD94 forms, with distinct members of the NKG2 family, heterodimers that are involved in the recognition of the nonclassical MHC class I molecule HLA-E (49Braud V.M. Allan D.S. O'Callaghan C.A. Soderstrom K. D'Andrea A. Ogg G.S. Lazetic S. Young N.T. Bell J.I. Phillips J.H. Lanier L.L. McMichael A.J. Nature. 1998; 391: 795-799Crossref PubMed Scopus (1805) Google Scholar, 50Borrego F. Ulbrecht M. Weiss E.H. Coligan J.E. Brooks A.G. J. Exp. Med. 1998; 187: 813-818Crossref PubMed Scopus (599) Google Scholar, 51Lee N. Llano M. Carretero M. Ishitani A. Navarro F. López-Botet M. Geraghty D.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5199-5204Crossref PubMed Scopus (855) Google Scholar). The unraveling of this α helix in the crystal structure of CD94 NKD could be because of the formation of the homodimer, whose physiological role is uncertain. The amino acid sequence at this region retains a distribution of hydrophobic residues that appears suitable for the formation of an α helix, but analysis of sequence alignments reveals that, in CD94 and NKG2 proteins, this segment is two residues shorter than in the majority of NKD sequences. Whether the lack in CD94 of this α helix is due to the formation of the homodimer or consequence of the deletion at the C-terminal end of this segment would have to await the determina
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