Attenuating Lymphocyte Activity
2005; Elsevier BV; Volume: 280; Issue: 47 Linguagem: Inglês
10.1074/jbc.m507629200
ISSN1083-351X
AutoresDeanne M. Compaan, Lino C. Gonzalez, Irene Tom, Kelly M. Loyet, Dan Eaton, S.G. Hymowitz,
Tópico(s)T-cell and B-cell Immunology
ResumoFive CD28-like proteins exert positive or negative effects on immune cells. Only four of these five receptors interact with members of the B7 family. The exception is BTLA (B and T lymphocyte attenuator), which instead interacts with the tumor necrosis factor receptor superfamily member HVEM (herpes virus entry mediator). To better understand this interaction, we determined the 2.8-Å crystal structure of the BTLA-HVEM complex. This structure shows that BTLA binds the N-terminal cysteine-rich domain of HVEM and employs a unique binding surface compared with other CD28-like receptors. Moreover, the structure shows that BTLA recognizes the same surface on HVEM as gD (herpes virus glycoprotein D) and utilizes a similar binding motif. Light scattering analysis demonstrates that the extracellular domain of BTLA is monomeric and that BTLA and HVEM form a 1:1 complex. Alanine-scanning mutagenesis of HVEM was used to further define critical binding residues. Finally, BTLA adopts an immunoglobulin I-set fold. Despite structural similarities to other CD28-like members, BTLA represents a unique co-receptor. Five CD28-like proteins exert positive or negative effects on immune cells. Only four of these five receptors interact with members of the B7 family. The exception is BTLA (B and T lymphocyte attenuator), which instead interacts with the tumor necrosis factor receptor superfamily member HVEM (herpes virus entry mediator). To better understand this interaction, we determined the 2.8-Å crystal structure of the BTLA-HVEM complex. This structure shows that BTLA binds the N-terminal cysteine-rich domain of HVEM and employs a unique binding surface compared with other CD28-like receptors. Moreover, the structure shows that BTLA recognizes the same surface on HVEM as gD (herpes virus glycoprotein D) and utilizes a similar binding motif. Light scattering analysis demonstrates that the extracellular domain of BTLA is monomeric and that BTLA and HVEM form a 1:1 complex. Alanine-scanning mutagenesis of HVEM was used to further define critical binding residues. Finally, BTLA adopts an immunoglobulin I-set fold. Despite structural similarities to other CD28-like members, BTLA represents a unique co-receptor. Co-receptor signaling is an important mechanism of coordinating and tightly regulating immune response. For instance, activation of naïve T cells requires a second co-stimulatory signal in addition to stimulation of the T cell receptor by engagement with peptide-MHC complexes. Conversely, co-inhibitory signals are required to maintain T cell self-tolerance and prevent autoimmunity (1Sharpe A.H. Freeman G.J. Nat. Rev. 2002; 2: 116-126Crossref Scopus (1406) Google Scholar). The CD28-like family is one important class of co-receptors. These members of the immunoglobulin superfamily (IgSF) 2The abbreviations used are:IgSFimmunoglobulin superfamilyBTLAB and T lymphocyte attenuatorCRDcysteine-rich domainCTLA4cytotoxic T lymphocyte antigen 4ECDextracellular domaingDherpesvirus glycoprotein DHVEMherpesvirus entry mediatorLIGHThomologous to lymphotoxin, showing inducible expression, and competing with herpes simplex virus glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytesTNFSFtumor necrosis factor superfamilyTNFRSFtumor necrosis factor receptor superfamilyLTαlymphotoxin αNi-NTAnickel-nitriloacetic acidHPLChigh performance liquid chromatography function as either co-stimulators (CD28 and inducible T cell costimulator) or co-inhibitors (CTLA-4, programmed death-1, and BTLA) in modulating immune cell activity (2Greenwald R.J. Freeman G.J. Sharpe A.H. Annu. Rev. Immunol. 2005; 23: 515-548Crossref PubMed Scopus (1963) Google Scholar). In general, these co-receptors are activated by members of the Ig containing B7 family (1Sharpe A.H. Freeman G.J. Nat. Rev. 2002; 2: 116-126Crossref Scopus (1406) Google Scholar). In addition to the CD28- and B7-like families of receptors and ligands, members of the TNF superfamilies of ligands and receptors (the TNFSF and TNFRSF respectively), such as OX40L-OX40, LIGHT-HVEM, CD27L-CD27, CD30L-CD30, and 4_1BBL-4_1BB, have also been reported to function as co-stimulators (3Watts T.H. Annu. Rev. Immunol. 2005; 23: 23-68Crossref PubMed Scopus (1131) Google Scholar). immunoglobulin superfamily B and T lymphocyte attenuator cysteine-rich domain cytotoxic T lymphocyte antigen 4 extracellular domain herpesvirus glycoprotein D herpesvirus entry mediator homologous to lymphotoxin, showing inducible expression, and competing with herpes simplex virus glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes tumor necrosis factor superfamily tumor necrosis factor receptor superfamily lymphotoxin α nickel-nitriloacetic acid high performance liquid chromatography Recently the CD28 family member BTLA was unexpectedly shown to bind and be activated by the TNFRSF member herpes virus entry mediator (HVEM, also known as TNFRSF14, HveA, ATAR, TR2, or LIGHTR) (4Gonzalez L.C. Loyet K.M. Calemine-Fenaux J. Chauhan V. Wranik B. Ouyang W. Eaton D.L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1116-1121Crossref PubMed Scopus (212) Google Scholar, 5Sedy J.R. Gavrieli M. Potter K.G. Hurchla M.A. Lindsley R.C. Hildner K. Scheu S. Pfeffer K. Ware C.F. Murphy T.L. Murphy K.M. Nat. Immunol. 2005; 6: 90-98Crossref PubMed Scopus (499) Google Scholar). This is the first example of cross-talk between the CD28 family and the TNFRSF. Whereas HVEM has been previously described as a co-stimulator triggered by the TNF-like ligands lymphotoxin α (LTα) and LIGHT (6Granger S.W. Rickert S. Cytokine Growth Factor Rev. 2003; 14: 289-296Crossref PubMed Scopus (106) Google Scholar), recent results from HVEM knock-out mice as well as the interaction between BTLA and HVEM are consistent with HVEM playing a co-inhibitory role (7Wang Y. Subudhi S.K. Anders R.A. Lo J. Sun Y. Blink S. Wang J. Liu X. Mink K. Degrandi D. Pfeffer K. Fu Y.X. J. Clin. Investig. 2005; 115: 711-717Crossref PubMed Scopus (155) Google Scholar). In addition to binding BTLA, LIGHT, and LTα, human HVEM is also a host cell receptor for herpes simplex virus 1 by binding to herpes simplex virus 1 glycoprotein D (gD) (8Montgomery R.I. Warner M.S. Lum B.J. Spear P.G. Cell. 1996; 87: 427-436Abstract Full Text Full Text PDF PubMed Scopus (1007) Google Scholar). Structurally, the connection between the IgSF family represented by BTLA and the TNFRSF proteins such as HVEM is unexpected. Crystal structures of CD28, CTLA-4, and programmed death-1 have revealed that these co-stimulatory and co-inhibitor receptors are all members of the immunoglobulin superfamily with each protein containing an extracellular IgV domain (9Evans E.J. Esnouf R.M. Manso-Sancho R. Gilbert R.J. James J.R. Yu C. Fennelly J.A. Vowles C. Hanke T. Walse B. Hunig T. Sorensen P. Stuart D.I. Davis S.J. Nat. Immunol. 2005; 6: 271-279Crossref PubMed Scopus (127) Google Scholar, 10Metzler W.J. Bajorath J. Fenderson W. Shaw S.Y. Constantine K.L. Naemura J. Leytze G. Peach R.J. Lavoie T.B. Mueller L. Linsley P.S. Nat. Struct. Biol. 1997; 4: 527-531Crossref PubMed Scopus (112) Google Scholar, 11Zhang X. Schwartz J.C. Guo X. Bhatia S. Cao E. Lorenz M. Cammer M. Chen L. Zhang Z.Y. Edidin M.A. Nathenson S.G. Almo S.C. Immunity. 2004; 20: 337-347Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Based on sequence analysis, BTLA was also expected to contain an extracellular IgV domain. Similarly, the extracellular domains of B7-like proteins are comprised of Ig domains. Co-crystal structures of B7-1 and B7-2 bound to CTLA4 show that Ig domains from the receptor and ligand pack against each other forming a compact interface (12Schwartz J.C. Zhang X. Fedorov A.A. Nathenson S.G. Almo S.C. Nature. 2001; 410: 604-608Crossref PubMed Scopus (276) Google Scholar, 13Stamper C.C. Zhang Y. Tobin J.F. Erbe D.V. Ikemizu S. Davis S.J. Stahl M.L. Seehra J. Somers W.S. Mosyak L. Nature. 2001; 410: 608-611Crossref PubMed Scopus (373) Google Scholar). In contrast, the TNFSF and TNFRSF members are formed by very different structural elements and interact in a distinctive manner determined by the quaternary structure of TNF-like ligands. These proteins are homotrimeric or occasionally heterotrimeric proteins comprised of jelly-roll monomers. Multidomain TNFRSF family members are comprised of multiple pseudo-repeats of a cysteinerich motif. Structures of signaling complexes formed by TNF-like ligands and receptors show that the elongated receptors bind at monomer-monomer interfaces on the ligands in a manner much different from the compact B7-CD28-type interaction (14Bodmer J.L. Schneider P. Tschopp J. Trends Biochem. Sci. 2002; 27: 19-26Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). The BTLA-HVEM interaction is also unusual in that it represents the first example of a TNFRSF functioning as a "ligand" and is one of a handful of examples of a TNFRSF interacting with a non-TNF-like ligand. In addition to the BTLA-HVEM and gD-HVEM interactions, other TNFSFR, which bind unusual ligands, include the low affinity neurotrophin receptor p75, which binds the cystine knot growth factor NGF, and feline OX40, which acts as a co-receptor for the feline immunodeficiency virus (15Wiesmann C. de Vos A.M. Cell Mol. Life Sci. 2001; 58: 748-759Crossref PubMed Scopus (151) Google Scholar, 16Shimojima M. Miyazawa T. Ikeda Y. McMonagle E.L. Haining H. Akashi H. Takeuchi Y. Hosie M.J. Willett B.J. Science. 2004; 303: 1192-1195Crossref PubMed Scopus (157) Google Scholar). Crystal structures of the relevant complexes show that gD protein interacts primarily with the N-terminal cysteine-rich domain (CRD1) of HVEM on the surface opposite the TNFSF binding site (17Carfi A. Willis S.H. Whitbeck J.C. Krummenacher C. Cohen G.H. Eisenberg R.J. Wiley D.C. Mol. Cell. 2001; 8: 169-179Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Similarly, p75 uses the same respective surface on CRD1 and a part of CRD2 to bind NGF (18He X.L. Garcia K.C. Science. 2004; 304: 870-875Crossref PubMed Scopus (242) Google Scholar). Previous biochemical characterization suggests that BTLA also binds to HVEM on the CRD1 distal to the TNFSF binding site (4Gonzalez L.C. Loyet K.M. Calemine-Fenaux J. Chauhan V. Wranik B. Ouyang W. Eaton D.L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1116-1121Crossref PubMed Scopus (212) Google Scholar, 5Sedy J.R. Gavrieli M. Potter K.G. Hurchla M.A. Lindsley R.C. Hildner K. Scheu S. Pfeffer K. Ware C.F. Murphy T.L. Murphy K.M. Nat. Immunol. 2005; 6: 90-98Crossref PubMed Scopus (499) Google Scholar). To obtain a more detailed understanding of the novel interaction between the CD28-like protein BTLA and the TNFRSF member HVEM, we have determined the 2.8-Å crystal structure of the BTLA-HVEM complex. This structure shows that despite major structural differences between BTLA and gD, they bind to an overlapping site on HVEM using a similar β-sheet binding motif. We have used alanine-scanning mutagenesis and Scatchard assays to identify critical BTLA-binding residues on HVEM. Mutations that significantly reduced binding affinities were in close agreement with the crystal structure. Light scattering demonstrates that the recombinant extracellular domain of BTLA is monomeric and that BTLA and HVEM form a 1:1 complex in solution. Despite the homology and functional similarities between BTLA and the CD28 family, BTLA contains structurally unique features. Moreover, compared with the CD28-B7 binding site, BTLA uses a distinct surface to interact with HVEM. Finally, using the BTLA-HVEM structure, we propose a hypothetical model for a BTLA-HVEM-TNF ternary complex. Recombinant Protein Expression and Purification—DNA encoding human BTLA residues 26-137 (the initial methionine is residue 1) with the addition of a C-terminal His8 tag was expressed in Escherichia coli. Inclusion bodies from BTLA expressing E. coli were extracted under denaturing conditions, and the protein was purified on a Ni-NTA metal chelate column as described (19Kirchhofer D. Peek M. Li W. Stamos J. Eigenbrot C. Kadkhodayan S. Elliott J.M. Corpuz R.T. Lazarus R.A. Moran P. J. Biol. Chem. 2003; 278: 36341-36349Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Fractions were pooled and diluted to 50 μg/ml with buffer containing 0.1 m Tris (pH 8.6), 0.3 m NaCl, 20 mm glycine, 1 mm EDTA, 1 mm glutathione (oxidized), and 1 mm glutathione (reduced). The refolding mixture was incubated overnight at 2-8 °C, and the pH adjusted to pH 3.0 with trifluoroacetic acid. The acidified refolding mixture was loaded onto an RP-HPLC Vydac C4 column (1.0 × 25 cm) equilibrated with 0.1% (w/v) trifluoroacetic acid in water and eluted with a linear gradient of acetonitrile (from 15 to 55%) in 0.1% trifluoroacetic acid at 3 ml/min for a total of 35 min. Protein fractions were pooled, and the acetonitrile was removed by evaporation assisted by a gentle stream of N2. The RP-HPLC pool was buffer exchanged using a HiTrap Desalting column (Amersham Biosciences) equilibrated with buffer containing 10 mm HEPES (pH 6.8), 0.15 m NaCl. BTLA activity was evaluated using SPR (Biacore). BTLA-Fc fusion protein was expressed in Chinese hamster ovary cells as previously described (4Gonzalez L.C. Loyet K.M. Calemine-Fenaux J. Chauhan V. Wranik B. Ouyang W. Eaton D.L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1116-1121Crossref PubMed Scopus (212) Google Scholar). The BTLA-Fc construct contains a Genenase site (New England Biolabs) between the BTLA extracellular domain (ECD) and the Fc fusion. BTLA ECD was cleaved from the Fc domain by the addition of a 1:100 ratio of Genenase to BTLA-Fc in phosphate-buffered saline. After 2 h at room temperature, the reaction was quenched with a protease inhibitor mixture and loaded onto a protein A-Sepharose column. Cleaved BTLA was collected from the flow through. DNA encoding a truncated human HVEM ECD, residues 1-105 (residue numbered as in Protein Data Bank code 1JMA; residue 1 is the first residue in the mature protein), corresponding to the ordered portion of HVEM in the gD-HVEM complex, including CRD1, CRD2, and the N-terminal 22 residues of CRD3, was subcloned into the pET15b vector and subsequently cloned into the baculovirus secretion vector pAcGP67-B. Virus was made by co-transfection and three rounds of viral amplification in Sf9 cells. High titer virus was used to infect Hi5 insect cells, and protein was expressed for 3 days and the media treated as described (20Hymowitz S.G. Filvaroff E.H. Yin J.P. Lee J. Cai L. Risser P. Maruoka M. Mao W. Foster J. Kelley R.F. Pan G. Gurney A.L. de Vos A.M. Starovasnik M.A. EMBO J. 2001; 20: 5332-5341Crossref PubMed Scopus (439) Google Scholar) prior to being loaded onto a Ni-NTA-agarose column and eluted with 250 mm imidazole buffer. Thrombin was added (1 unit/mg of HVEM) to remove the N-terminal His tag, and the solution was dialyzed overnight at 4 °C against 150 mm NaCl, 20 mm Tris (pH 8.0). The dialysate was concentrated and loaded onto an S-75 sizing column equilibrated in 150 mm NaCl, 20 mm Tris (pH 8). Fractions of purified HVEM were pooled and yielded ∼0.5 mg of protein/liter of Hi5 cells. As a control, a construct of full-length human HVEM ECD (hereafter referred to as HVEML) with a C-terminal His tag was also expressed and purified. This construct contains the same HVEM residues as that used by Carfi et al. (17Carfi A. Willis S.H. Whitbeck J.C. Krummenacher C. Cohen G.H. Eisenberg R.J. Wiley D.C. Mol. Cell. 2001; 8: 169-179Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar) and Whitbeck et al. (21Whitbeck J.C. Peng C. Lou H. Xu R. Willis S.H. Ponce de Leon M. Peng T. Nicola A.V. Montgomery R.I. Warner M.S. Soulika A.M. Spruce L.A. Moore W.T. Lambris J.D. Spear P.G. Cohen G.H. Eisenberg R.J. J. Virol. 1997; 71: 6083-6093Crossref PubMed Google Scholar). For crystallography, the BTLA-HVEM complex was made by adding excess HVEM residues 1-105 to E. coli-expressed BTLA and incubating at room temperature for 1 h. The complex was concentrated and purified from excess HVEM (residues 1-105) with an S-75 sizing column equilibrated in 150 mm NaCl, 20 mm Tris (pH 8.0). Fractions containing purified complex were pooled and concentrated to 9 mg/ml. DNA encoding LIGHT extracellular domain residues 91-240 was expressed and purified in the same manner as HVEM with a final purification on Superdex S-200 sizing equilibrated in 150 mm NaCl, 20 mm Tris (pH 8.0). Expression yielded greater than 1 mg of purified protein/liter of Hi5 cells. Crystallographic Data Collection and Structure Determination—Crystals of the BTLA-HVEM complex were grown by vapor diffusion at 19 °C using the sitting drop method. Crystals formed in drops containing protein solution were mixed with an equal volume of reservoir solution containing 2.0 m sodium formate, 0.1 m sodium acetate (pH 4.6). The resulting small, clustered crystals were used to seed new drops yielding larger, single crystals. The crystals were transferred briefly to a droplet containing reservoir solution with 30% glycerol before flash-cooling in liquid nitrogen. The crystals belonged to space group C2221, and the asymmetric unit contained two copies of the BTLA-HVEM complex. A data set to 2.8-Å resolution was measured from a single crystal at beam line 5.0.1 of the Berkeley Center for Structural Biology at the Advanced Light Source. The data were processed using the HKL package (22Otwinowsk Z. Minor W. Methods Enzymol. 1997; 276: 307-325Crossref Scopus (38617) Google Scholar). Structures of HVEM (chain B in PDB code 1JMA) and murine BTLA (PDB 1XAU; structure determined and deposited by D. Fremont and co-workers in 2004) were used as search models to determine the structure of the BTLA-HVEM complex by molecular replacement. Side chains, which differed between murine and human BTLA, were manually trimmed to C-β. The program Phaser (23Storoni L.C. McCoy A.J. Read R.J. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 432-438Crossref PubMed Scopus (1103) Google Scholar) gave a clear solution. A 2-fold noncrystallographic symmetry-averaged and solvent-flattened map using program dm (24Cowtan K. Main P. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 487-493Crossref PubMed Scopus (309) Google Scholar) revealed clear density for the missing hBTLA side chains. The model was refined with REFMAC5 (25Winn M.D. Murshudov G.N. Papiz M.Z. Methods Enzymol. 2003; 374: 300-321Crossref PubMed Scopus (683) Google Scholar) with tight noncrystallographic symmetry restraints on residues 34-144 of BTLA and residues 4-83 of HVEM. The last 10 residues of BTLA were not included in the noncrystallographic symmetry restrains as they differed significantly because of the involvement of the C-terminal His tag in crystal packing contacts. Additional density was observed for a metal ion interacting with the His tag of symmetry related copies of chain A. This ion was modeled as a Ni(II) and is likely an artifact because of purification of BTLA by Ni-NTA affinity chromatography. Refinement and model statistics are shown in TABLE ONE. The coordinates for the BTLA-HVEM complex have been deposited in the Protein Data Bank and assigned access code 2AW2.TABLE ONECrystallographic data collection and refinement statisticsBTLA-HVEMData collection Space groupC2221 Resolution (Å)50-2.80 (2.90-2.80)aNumbers in parentheses refer to the highest resolution shell. Unit cell constants (Å)a = 50.5 b = 168.2 c = 149.4 RsymbRsym=∑|I−〈I〉/∑I. 〈I 〉 is the average intensity of symmetry related observations of a unique reflection.0.052 (0.455)aNumbers in parentheses refer to the highest resolution shell. No. observations62,260 Unique reflections15,639 Completeness (%)99.9 (100)aNumbers in parentheses refer to the highest resolution shell. 〈I/σI 〉13.4 (2.3)aNumbers in parentheses refer to the highest resolution shell. Asymmetric unit2 BTLA-HVEM complexesRefinement Resolution (Å)30-2.8 Final RcR=∑|Fo−Fc|/∑Fo. Rfree is calculated as R, but for 10% of the reflections which have been excluded from refinement.Rfree (%)23.1, 27.8 Root mean square deviation bonds (Å)0.007 Root mean square deviation angles (°)1.04 Root mean square deviation bonded Bs (Å2)1.8 Ramachandran plot (%)dPercentage of residues in the most favored, additionally allowed, generously allowed, and disallowed regions of a Ramachandran plot.86.5; 12.4; 0.8; 0.3a Numbers in parentheses refer to the highest resolution shell.b Rsym=∑|I−〈I〉/∑I. 〈I 〉 is the average intensity of symmetry related observations of a unique reflection.c R=∑|Fo−Fc|/∑Fo. Rfree is calculated as R, but for 10% of the reflections which have been excluded from refinement.d Percentage of residues in the most favored, additionally allowed, generously allowed, and disallowed regions of a Ramachandran plot. Open table in a new tab Alanine Scanning Mutagenesis and Cell Binding Affinity Assays—The QuikChange site-directed mutagenesis kit (Stratagene) was used as recommended by the manufacturer to generate single alanine mutations in HVEM. Mutant HVEM sequences were confirmed by DNA sequencing. Recombinant BTLA-Fc was iodinated by the lactoperoxidase (Biotrend) method and LIGHT by the IODO-GEN (PerkinElmer Life Sciences) method. Displacement binding studies were done as previously described (4Gonzalez L.C. Loyet K.M. Calemine-Fenaux J. Chauhan V. Wranik B. Ouyang W. Eaton D.L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1116-1121Crossref PubMed Scopus (212) Google Scholar) with 0.5 nm labeled BTLA and varying concentrations of unlabeled protein. LIGHT binding was confirmed with 0.5 nm labeled LIGHT with or without 1000-fold excess of unlabeled protein. LIGHT binding to alanine mutants was normalized as a percentage of wild type binding. AD-293 cells were transiently transfected with either wild type or alanine mutant HVEM cDNA as previously described (4Gonzalez L.C. Loyet K.M. Calemine-Fenaux J. Chauhan V. Wranik B. Ouyang W. Eaton D.L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1116-1121Crossref PubMed Scopus (212) Google Scholar) using pRK mock-transfected cells as a negative control. Expression of wild type and alanine mutant HVEM was confirmed by flow cytometry as previously described using fluorescein isothiocyanate-conjugated mouse anti-human HVEM (clone 122, MBL) in comparison to an isotype control (BD Biosciences). The HVEM antibody epitope is contained in all HVEM point mutants. Expression levels of point mutants are normalized to that of wild type HVEM as denoted by the percentage of cells above a given mean fluorescence threshold determined by isotype control antibody staining. Light Scattering—Molar Mass determination was carried out using a Agilent 1100 series (Agilent, Palo Alto, CA) HPLC system in line with a Wyatt MiniDawn MALS (multiangle light scattering) detector (Wyatt Technology, Santa Barbara, CA). Concentration measurements were made using an online Wyatt OPTILAB DSP interferometric refractometer (Wyatt Technology). Astra software (Wyatt Technology) was used for light scattering data acquisition and processing. Either a Shodex 803 or a S75 10/300 column (Amersham Biosciences) equilibrated with filtered phosphate-buffered saline (pH 7.2) was used with a flow rate of 1 ml/min. Both the light scattering unit and the refractometer were calibrated as per the manufacturer's instructions. A value of 0.180 ml/g was assumed for the dn/dc ratio of the protein. Measuring the signal from monomeric bovine serum albumin normalized the detector responses. The temperature of the light scattering unit was maintained at 25 °C, and the temperature of the refractometer was kept at 35 °C. The column and all external connections were at ambient temperature (20-25 °C). Recombinant BTLA produced in Chinese hamster ovary cells, the purified HVEM-BTLA complex used for crystallization, and HVEML was loaded at 1.0 mg/ml. The structure of the human BTLA-HVEM complex was solved by molecular replacement using the structures of the HVEM ECD (PDB code 1JMA, chain B) and the 1.8-Å structure of murine BTLA ECD (PDB code 1XAU) as search models with the program Phaser (23Storoni L.C. McCoy A.J. Read R.J. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 432-438Crossref PubMed Scopus (1103) Google Scholar). A solution was found with two copies of the BTLA-HVEM complex forming the asymmetric unit. The structure was manually rebuilt and refined to an R/Rfree of 23.1 and 27.8%, respectively (TABLE ONE, Fig. 1). The final model consists of BTLA residues 34-137 and HVEM residues 5-92 and 95-102. In both copies of BTLA, an additional 4-6 residues from the C-terminal His tag were well ordered in the electron density and are included in the model. Noncrystallographic symmetry restraints were used throughout the refinement and the two copies of the BTLA-HVEM complex are very similar. This structure reveals that human BTLA, like murine BTLA (PDB code 1XAU), is a compact IgG domain composed of two flat β-sheets, which are formed by strands B, E, and D in one sheet and strands A′,G, F, C, and C′ in the other (Fig. 1B). The sheets are buttressed by three disulfides (residues 72-79, 34-63, and 58-115). The Cys72-Cys79 disulfide connects the C and C′ strands; the Cys34-Cys63 disulfide joins the N-terminal region preceding strand A′ to the CD loop, and the Cys58-Cys115 disulfide connects the B and F strands. This disulfide is completely buried in the hydrophobic core of BTLA and is part of the "Y-corner" motif, DX(G/A)DXYXC. The B-F strand disulfide and the Y-corner motif are both highly conserved features in IgSF domains. In addition to the Cys58-Cys115 disulfide, the Cys34-Cys63 disulfide is also conserved in murine BTLA. The Cys72-Cys79 disulfide is not conserved in all murine BTLA alleles (see below). This disulfide is not present in the variant of murine BTLA, which was crystallized (PDB code 1XAU) in which the cysteine corresponding to Cys79 is replaced by a tryptophan. The sequence of human BTLA contains three putative N-linked glycosylation sites. Expression of recombinant BTLA in eukaryotic cells results in a protein with significant glycosylation that is unsuitable for crystallization. For the structural studies, recombinant BTLA was expressed in E. coli cells to produce a protein without glycosylation. Examination of the BTLA-HVEM complex (below) shows that the putative N-linked glycosylation sites are away from the binding site and would not be expected to affect the interaction between BTLA and HVEM (Fig. 1). Analysis of the interaction between BTLA expressed in E. coli and HVEM by surface plasmon resonance shows that glycosylation is not required for HVEM binding. 3L. Gonzalez, unpublished data. The BTLA-HVEM Complex—The BTLA-HVEM complex consists of a single globular BTLA interacting with the membrane distal region of rod-shaped HVEM (Fig. 1). BTLA binds HVEM using two short segments: an N-terminal extension preceding strand A′ (residues 35-43) and the short G° strand (residues 118-128). The HVEM binding site for BTLA consists almost exclusively of residues from CRD1. BTLA residues 35-43 interact with HVEM residues 26-33, which form the "tip" of HVEM CRD1 distal to the C terminus. This loop in HVEM leads to a strand (residues 33-38) that, together with the G° strand from BTLA, makes the heart of the binding interface. These two strands form a small anti-parallel intermolecular β-sheet. This interaction is primarily mediated by main chain hydrogen bonds and includes relatively few side chain contacts. These two interactions, in conjunction with small contribution from the BTLA CC′ loop, generate an interface that buries ∼1800 Å2 of solvent accessible surface area, which is contributed equally by both binding partners. This complex positions the C termini of the two proteins in opposite directions consistent with the BTLA-HVEM complex forming between proteins resident on different cells. Moreover, as full-length HVEM includes an additional ∼60 residues prior to the transmembrane helix, it is also possible that the binary BTLA-HVEM interaction could occur between proteins residing on the same cell. Sequence Polymorphism in Murine BTLA—Three different BTLA alleles have been isolated from 23 mice strains. These alleles have been labeled BALB/c-like, MRL/lpr-like, and C57BL/6-like according to the strains from which they were derived (26Hurchla M.A. Sedy J.R. Gavrieli M. Drake C.G. Murphy T.L. Murphy K.M. J. Immunol. 2005; 174: 3377-3385Crossref PubMed Scopus (158) Google Scholar). The crystal structure of murine BTLA is of the BALB/c-like variant. The BALB/c and MLR/lpr alleles differ at only one amino acid, whereas the C57BL/6-like allele differs from the other two by 10 or 11 amino acids, respectively, in the extracellular Ig domain (26Hurchla M.A. Sedy J.R. Gavrieli M. Drake C.G. Murphy T.L. Murphy K.M. J. Immunol. 2005; 174: 3377-3385Crossref PubMed Scopus (158) Google Scholar). The structural and functional consequences of these differences can be predicted based on the structure of the human BTLA-HVEM complex. The most interesting difference is the polymorphism between Trp and Cys at position 85 corresponding to the human residue Cys79 (hereafter human BTLA residue numbers will follow in parentheses). In the C57BL/6 allele, the presence of a Cys at this position suggests that a disulfide analogous to the Cys72-Cys79 disulfide in hBTLA will be formed. The disulfide containing variant may be more stable as solvent-exposed unpaired cysteine residues can lead to inappropriate oxidation, oligomerization, or misfolding. Another interesting difference between the alleles is that in the C57BL/6 variant, glutamines replace a glutamate and an arginine at positions 72 (h66) and 102 (h93), respectively, resulting in exchange of a solvent-exposed salt bridge with a potential hydrogen bonding interaction. Three of the remaining
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