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• W2085452430 abstract "Dimerization of class II major histocompatibility complex (MHC) plays an important role in the MHC biological function. Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing specific T cell receptor Vβ elements. Here we have used structural, sedimentation, and surface plasmon resonance detection approaches to investigate the molecular interactions between MAM and the class II MHC molecule HLA-DR1 in the context of a hemagglutinin peptide-(306–318) (HA). Our results revealed that zinc ion can efficiently induce the dimerization of the HLA-DR1/HA complex. Because the crystal structure of the MAM/HLA-DR1/hemagglutinin complex in the presence of EDTA is nearly identical to the structure of the complex crystallized in the presence of zinc ion, Zn2+ is evidently not directly involved in the binding between MAM and HLA-DR1. Sedimentation and surface plasmon resonance studies further revealed that MAM binds the HLA-DR1/HA complex with high affinity in a 1:1 stoichiometry, in the absence of Zn2+. However, in the presence of Zn2+, a dimerized MAM/HLA-DR1/HA complex can arise through the Zn2+-induced DR1 dimer. In the presence of Zn2+, cooperative binding of MAM to the DR1 dimer was also observed. Dimerization of class II major histocompatibility complex (MHC) plays an important role in the MHC biological function. Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing specific T cell receptor Vβ elements. Here we have used structural, sedimentation, and surface plasmon resonance detection approaches to investigate the molecular interactions between MAM and the class II MHC molecule HLA-DR1 in the context of a hemagglutinin peptide-(306–318) (HA). Our results revealed that zinc ion can efficiently induce the dimerization of the HLA-DR1/HA complex. Because the crystal structure of the MAM/HLA-DR1/hemagglutinin complex in the presence of EDTA is nearly identical to the structure of the complex crystallized in the presence of zinc ion, Zn2+ is evidently not directly involved in the binding between MAM and HLA-DR1. Sedimentation and surface plasmon resonance studies further revealed that MAM binds the HLA-DR1/HA complex with high affinity in a 1:1 stoichiometry, in the absence of Zn2+. However, in the presence of Zn2+, a dimerized MAM/HLA-DR1/HA complex can arise through the Zn2+-induced DR1 dimer. In the presence of Zn2+, cooperative binding of MAM to the DR1 dimer was also observed. Major histocompatibility complex (MHC) 3The abbreviations used are: MHC, major histocompatibility complex; MAM, mycoplasma arthritidis-derived mitogen; SPR, surface plasmon resonance; HA, hemagglutinin peptide-(306–318); TCR, T cell receptor; APC, antigenpresenting cell; SAg, superantigen; SEA, staphylococcal enterotoxin A; AUC, analytical ultracentrifugation; SV, sedimentation velocity; HBS, Hepes-buffered saline; RU, resonance unit; r.m.s., root-mean-square.3The abbreviations used are: MHC, major histocompatibility complex; MAM, mycoplasma arthritidis-derived mitogen; SPR, surface plasmon resonance; HA, hemagglutinin peptide-(306–318); TCR, T cell receptor; APC, antigenpresenting cell; SAg, superantigen; SEA, staphylococcal enterotoxin A; AUC, analytical ultracentrifugation; SV, sedimentation velocity; HBS, Hepes-buffered saline; RU, resonance unit; r.m.s., root-mean-square. molecules can present a wide variety of antigens to T lymphocytes. Two classes, class I and class II MHC, recognized by CD8+ and CD4+ T lymphocytes, respectively, have been discovered. Class II MHC molecules are heterodimers, composed of an α-chain and a β-chain. These αβ heterodimers are highly polymorphic, type I integral membrane proteins, assembled in antigen-presenting cells (APCs) such as B cells and macrophages. Because the first crystal structure of a class II MHC molecule was reported in 1993 (1Brown J.H. Jardetzky T.S. Gorga J.C. Stern L.J. Urban R.G. Strominger J.L. Wiley D.C. Nature. 1993; 364: 33-39Crossref PubMed Scopus (2099) Google Scholar), crystal structures of more than 20 MHC class II molecules from both human and mouse have been determined. Some of these structures have revealed that the MHC class II heterodimer can self-associate to further form a(αβ)2 double dimer, termed the superdimer (2Schafer P.H. Pierce S.K. Jardetzky T.S. Semin. Immunol. 1995; 7: 389-398Crossref PubMed Scopus (45) Google Scholar). In the crystals the superdimer is arranged in such a way that allows simultaneous binding of two T cell receptors (TCRs) and two CD4 molecules (2Schafer P.H. Pierce S.K. Jardetzky T.S. Semin. Immunol. 1995; 7: 389-398Crossref PubMed Scopus (45) Google Scholar). Dimerization of class II MHC, either as preformed complexes on the APC membrane or as induced upon TCR engagement, appears consistent with many aspects of class II immunology, including signaling and T cell activation (2Schafer P.H. Pierce S.K. Jardetzky T.S. Semin. Immunol. 1995; 7: 389-398Crossref PubMed Scopus (45) Google Scholar, 3Chen Z.Z. McGuire J.C. Leach K.L. Cambier J.C. J. Immunol. 1987; 138: 2345-2352PubMed Google Scholar, 4Cambier J.C. Morrison D.C. Chien M.M. Lehmann K.R. J. Immunol. 1991; 146: 2075-2082PubMed Google Scholar). However, it remains controversial as to whether the crystallographic superdimer constitutes evidence that the superdimer exists as a physiologically relevant conformation of MHC class II (5Schafer P.H. Pierce S.K. Immunity. 1994; 1: 699-707Abstract Full Text PDF PubMed Scopus (67) Google Scholar, 6Roucard C. Garban F. Mooney N.A. Charron D.J. Ericson M.L. J. Biol. Chem. 1996; 271: 13993-14000Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 7Cherry R.J. Wilson K.M. Triantafilou K. O'Toole P. Morrison I.E. Smith P.R. Fernandez N. J. Cell Biol. 1998; 140: 71-79Crossref PubMed Scopus (57) Google Scholar, 8Hitzel C. Gruneberg U. van Ham M. Trowsdale J. Koch N. J. Immunol. 1999; 162: 4671-4676PubMed Google Scholar). In addition to peptide antigens, class II MHC can present a number of proteins, known as superantigens (SAgs), to T lymphocytes (9Li H. Llera A. Malchiodi E.L. Mariuzza R.A. Annu. Rev. Immunol. 1999; 17: 435-466Crossref PubMed Scopus (256) Google Scholar, 10Kotzin B.L. Leung D.Y. Kappler J. Marrack P. Adv. Immunol. 1993; 54: 99-166Crossref PubMed Scopus (572) Google Scholar). Upon binding to the MHC class II molecules on APCs, SAgs are recognized by TCRs carrying specific Vβ subsets, leading to polyclonal activation of a large portion (up to 20%) of T lymphocytes. SAgs have been hypothesized to play important roles in a number of human diseases, including food poisoning, toxic shock syndrome, and autoimmune diseases such as multiple sclerosis and rheumatoid arthritis (10Kotzin B.L. Leung D.Y. Kappler J. Marrack P. Adv. Immunol. 1993; 54: 99-166Crossref PubMed Scopus (572) Google Scholar, 11Abe J. Kotzin B.L. Jujo K. Melish M.E. Glode M.P. Kohsaka T. Leung D.Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4066-4070Crossref PubMed Scopus (318) Google Scholar, 12Conrad B. Weidmann E. Trucco G. Rudert W.A. Behboo R. Ricordi C. Rodriquez-Rilo H. Finegold D. Trucco M. Nature. 1994; 371: 351-355Crossref PubMed Scopus (312) Google Scholar, 13Renno T. Acha-Orbea H. Immunol. Rev. 1996; 154: 175-191Crossref PubMed Google Scholar, 14McCormick J.K. Yarwood J.M. Schlievert P.M. Annu. Rev. Microbiol. 2001; 55: 77-104Crossref PubMed Scopus (563) Google Scholar). SAgs are known to bind to the MHC molecules in diverse ways. Currently, two sites have been identified on the MHC, one with a low binding affinity for SAgs and the other with a high affinity (9Li H. Llera A. Malchiodi E.L. Mariuzza R.A. Annu. Rev. Immunol. 1999; 17: 435-466Crossref PubMed Scopus (256) Google Scholar, 15Hudson K.R. Tiedemann R.E. Urban R.G. Lowe S.C. Strominger J.L. Fraser J.D. J. Exp. Med. 1995; 182: 711-720Crossref PubMed Scopus (148) Google Scholar, 16Hurley J.M. Shimonkevitz R. Hanagan A. Enney K. Boen E. Malmstrom S. Kotzin B.L. Matsumura M. J. Exp. Med. 1995; 181: 2229-2235Crossref PubMed Scopus (50) Google Scholar). The low affinity site (KD = 10–5 m) is on the MHC α1 domain, whereas the high affinity site (KD = 10–7 m), which is Zn2+-coordinated, is on the MHC β-chain. Some SAgs have a single MHC-II-binding site; for instance, staphylococcal enterotoxin B and toxic shock syndrome toxin-1 bind only to the MHC α-chain (17Jardetzky T.S. Brown J.H. Gorga J.C. Stern L.J. Urban R.G. Chi Y.I. Stauffacher C. Strominger J.L. Wiley D.C. Nature. 1994; 368: 711-718Crossref PubMed Scopus (498) Google Scholar, 18Kim J. Urban R.G. Strominger J.L. Wiley D.C. Science. 1994; 266: 1870-1874Crossref PubMed Scopus (247) Google Scholar). Others, such as SEA, bind to both low and high affinity sites on MHC-II (15Hudson K.R. Tiedemann R.E. Urban R.G. Lowe S.C. Strominger J.L. Fraser J.D. J. Exp. Med. 1995; 182: 711-720Crossref PubMed Scopus (148) Google Scholar, 19Kozono H. Parker D. White J. Marrack P. Kappler J. Immunity. 1995; 3: 187-196Abstract Full Text PDF PubMed Scopus (101) Google Scholar). In addition, it has been proposed that dimerization or oligomerization of the MHC antigens by SAgs is critical for T cell activation (20Li P.L. Tiedemann R.E. Moffat S.L. Fraser J.D. J. Exp. Med. 1997; 186: 375-383Crossref PubMed Scopus (63) Google Scholar, 21Papageorgiou A.C. Acharya K.R. Shapiro R. Passalacqua E.F. Brehm R.D. Tranter H.S. Structure (Camb.). 1995; 3: 769-779Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 22Baker H.M. Proft T. Webb P.D. Arcus V.L. Fraser J.D. Baker E.N. J. Biol. Chem. 2004; 279: 38571-38576Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 23Baker M. Gutman D.M. Papageorgiou A.C. Collins C.M. Acharya K.R. Protein Sci. 2001; 10: 1268-1273Crossref PubMed Scopus (28) Google Scholar, 24Sundstrom M. Abrahmsen L. Antonsson P. Mehindate K. Mourad W. Dohlsten M. EMBO J. 1996; 15: 6832-6840Crossref PubMed Scopus (94) Google Scholar). MAM is a potent T cell mitogen produced by Mycoplasma arthritidis (25Cole B.C. Curr. Top. Microbiol. Immunol. 1991; 174: 107-119PubMed Google Scholar), a natural pathogen of rodents. MAM can induce spontaneous chronic arthritis, which resembles human rheumatoid arthritis, in genetically susceptible strains of rodents. In vitro, administration of MAM can induce MHC class II-dependent T cell activation in a Vβ-restricted fashion, leading to B cell proliferation and differentiation and triggering cytokine expression (26Cole B.C. Atkin C.L. Immunol. Today. 1991; 12: 271-276Abstract Full Text PDF PubMed Scopus (139) Google Scholar). A structural study revealed that a MAM dimer can cross-link to two MHC molecules via interactions with the MHC α1 domains (27Zhao Y. Li Z. Drozd S. Guo Y. Mourad W. Li H. Structure (Camb.). 2004; 12: 277-288Abstract Full Text PDF PubMed Google Scholar). It was also reported that the superantigenic activity of MAM via MHC class II molecules is Zn2+-dependent (28Bernatchez C. Al-Daccak R. Mayer P.E. Mehindate K. Rink L. Mecheri S. Mourad W. Infect. Immun. 1997; 65: 2000-2005Crossref PubMed Google Scholar, 29Etongue-Mayer P. Langlois M.A. Ouellette M. Li H. Younes S. Al-Daccak R. Mourad W. Eur. J. Immunol. 2002; 32: 50-58Crossref PubMed Scopus (14) Google Scholar, 30Langlois M.A. El Fakhry Y. Mourad W. J. Biol. Chem. 2003; 278: 22309-22315Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). In this study, we have used structural, sedimentation, and surface plasmon resonance (SPR) detection approaches to investigate the molecular interactions between MAM and class II MHC molecule HLA-DR1, in the context of a hemagglutinin peptide (HA). Unexpectedly, we find that Zn2+ can induce the dimerization of a soluble class II MHC HLA-DR1/HA complex in solution. We also find that MAM binds the HLA-DR1/HA complex at a 1:1 stoichiometry, with a high affinity, and independent of zinc ion. Although Zn2+ is not directly involved in the binding between MAM and HLA-DR1, Zn2+-induced dimerization of the HLA-DR1/HA complex resulted in a dimerized MAM·MHC complex, in which cooperative binding of MAM to the HLA-DR1/HA complex dimer was observed. Protein Production—Soluble MAM was expressed as a glutathione S-transferase fusion protein as described previously (27Zhao Y. Li Z. Drozd S. Guo Y. Mourad W. Li H. Structure (Camb.). 2004; 12: 277-288Abstract Full Text PDF PubMed Google Scholar, 31Langlois M.A. Etongue-Mayer P. Ouellette M. Mourad W. Eur. J. Immunol. 2000; 30: 1748-1756Crossref PubMed Scopus (19) Google Scholar, 32Zhao Y. Li Z. Drozd S. Guo Y. Stack R. Hauer C. Li H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 353-356Crossref PubMed Scopus (5) Google Scholar). The HLA-DR1/HA complex was prepared using a refolding protocol as described (27Zhao Y. Li Z. Drozd S. Guo Y. Mourad W. Li H. Structure (Camb.). 2004; 12: 277-288Abstract Full Text PDF PubMed Google Scholar, 32Zhao Y. Li Z. Drozd S. Guo Y. Stack R. Hauer C. Li H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 353-356Crossref PubMed Scopus (5) Google Scholar, 33Frayser M. Sato A.K. Xu L. Stern L.J. Protein Expression Purif. 1999; 15: 105-114Crossref PubMed Scopus (87) Google Scholar). To generate biotinylated HLA-DR1 with covalently linked HA peptide for the BIAcore binding study, we made a new construct, using a two-step PCR protocol. Two sets of overlapping primers (forward, 5′-GGAATTCCATATGCCCAAGTATGTTAAGCAAAACACCCTGAAGTTGGCAACAGGAGGTGGTGGCTCACTA-3′ and 5′-GGAGGTGGTGGCTCACTAGTGGGAGGGGGCTCTGGAGGAGGTGGGTCCGGG-3′; reverse, 5′-GTCGTTCAGACCACCGCCGCTAGCTCCACCCCTAGTGGACTGTGCTCTCCATTCCACTGTGAG-3′ and 5′-CCCAAGCTTTCATTCGTGCCATTCGATTTTCTGAGCCTCGAAGATGTCGTTCAGACCACCGCCGCT-3′) were designed to amplify the DR1β, the linkers, the HA peptide-(306–318), and a BirA recognition peptide (34Beckett D. Kovaleva E. Schatz P.J. Protein Sci. 1999; 8: 921-929Crossref PubMed Scopus (544) Google Scholar). The amplified DNA was then ligated to the pET26b vector (Novagen) using the NdeI and HindIII sites (underlined in the primer sequences). The HA peptide was covalently linked to the N terminus of DR1β through a flexible linker (GGGGSLVGGGSGGGGS) similar to that used in our previous study (29Etongue-Mayer P. Langlois M.A. Ouellette M. Li H. Younes S. Al-Daccak R. Mourad W. Eur. J. Immunol. 2002; 32: 50-58Crossref PubMed Scopus (14) Google Scholar). The engineered DR1β also contained a C-terminal amino acid tag encoding a linker (QSTRGGASGG) and a signal sequence (GLNDIFEAQKIEWHE) for attaching biotin with the BirA enzyme (34Beckett D. Kovaleva E. Schatz P.J. Protein Sci. 1999; 8: 921-929Crossref PubMed Scopus (544) Google Scholar, 35Crawford F. Kozono H. White J. Marrack P. Kappler J. Immunity. 1998; 8: 675-682Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). Modified HA-DR1β was expressed, solubilized, and refolded with DR1α, using a protocol as described (27Zhao Y. Li Z. Drozd S. Guo Y. Mourad W. Li H. Structure (Camb.). 2004; 12: 277-288Abstract Full Text PDF PubMed Google Scholar, 32Zhao Y. Li Z. Drozd S. Guo Y. Stack R. Hauer C. Li H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 353-356Crossref PubMed Scopus (5) Google Scholar, 33Frayser M. Sato A.K. Xu L. Stern L.J. Protein Expression Purif. 1999; 15: 105-114Crossref PubMed Scopus (87) Google Scholar), with a modification that no synthetic HA peptide was added. Correctly folded HA/HLA-DR1 complex was purified, using ion exchange chromatography, on a Mono Q column (GE HealthCare); it was biotinylated with the BirA enzyme (Avidity), and size-purified on a Superdex 200 FPLC column (GE HealthCare). The extent of biotinylation was estimated with the EZ biotin quantitation kit (Pierce). Typically, 99% of the protein was biotinylated. Crystallization—Crystals of the MAM/HLA-DR1/HA complex were grown under the conditions described previously (27Zhao Y. Li Z. Drozd S. Guo Y. Mourad W. Li H. Structure (Camb.). 2004; 12: 277-288Abstract Full Text PDF PubMed Google Scholar, 32Zhao Y. Li Z. Drozd S. Guo Y. Stack R. Hauer C. Li H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 353-356Crossref PubMed Scopus (5) Google Scholar), except that EDTA was substitute for Zn(OAc)2. Crystals of the complex were also grown in the absence of both EDTA and Zn2+. To fully address whether zinc is involved in the complex formation, we soaked the complex crystals from the no-EDTA and no-Zn2+condition in the mother liquor containing 10 mm CdCl2. If a protein is known to bind zinc, cadmium ion is frequently used to substitute zinc for the determination of protein structure using the multiple isomorphous replacement method (36Kadima W. Biochemistry. 1999; 38: 13443-13452Crossref PubMed Scopus (10) Google Scholar, 37Hill C.P. Dauter Z. Dodson E.J. Dodson G.G. Dunn M.F. Biochemistry. 1991; 30: 917-924Crossref PubMed Scopus (62) Google Scholar). Microseeding was used to produce large crystals for x-ray diffraction data collection. X-ray Diffraction Data Collection, Structure Determination, and Refinement—Crystals of the complex obtained in the absence of zinc ion or in the presence of EDTA are isomorphous with the crystals previously obtained in the presence of zinc ion (27Zhao Y. Li Z. Drozd S. Guo Y. Mourad W. Li H. Structure (Camb.). 2004; 12: 277-288Abstract Full Text PDF PubMed Google Scholar, 32Zhao Y. Li Z. Drozd S. Guo Y. Stack R. Hauer C. Li H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 353-356Crossref PubMed Scopus (5) Google Scholar). Diffraction data for crystals of the MAM·DR1 (no-Zn2+), MAM·DR1 (EDTA), and MAM·DR1 (Cd2+) complexes were collected at 100 K at beamline X25 of the National Synchrotron Light Source at the Brookhaven National Laboratory (Table 1).TABLE 1Data collection, refinement, and model detailsMAM/HLA-DR1/HA complexNo-Zn2+EDTACd2+Data collection Cell dimensions (Ä)137.3 × 179.2 × 179137.7 × 179.6 × 180.0138.2 × 178.8 × 180.0 Resolution (Ä)41.4-2.840-3.044.6-2.6 Redundancy4.1 (3.6)5.2 (4.5)3.8 (3.1) Completeness (%)91.4 (90.6)89.8 (91.7)96.7 (82.5) Average I/σ(I)19.2 (2.9)17 (3.2)14.8 (1.7) Rsym (%)8.6 (44.1)12.4 (56.3)9.8 (56.4)Refinement Resolution limits (Ä)41.4-2.840-3.044.6-2.6 No. of reflections500714034566025 Rwork (%)27.221.123.4 Rfree (%)31.226.126.6 Non-H-atoms>Protein988898889888>PO4444>Water106118126 Average B (Ä2)45.137.548.0 Geometry>r.m.s.d. bondar.m.s.d. indicates root mean square deviation. length (Ä)0.0090.0080.008>r.m.s.d. bond angle (°)1.51.41.4a r.m.s.d. indicates root mean square deviation. Open table in a new tab The crystal structure of the MAM/HLA-DR1/HA complex at 2.6 Ä resolution (27Zhao Y. Li Z. Drozd S. Guo Y. Mourad W. Li H. Structure (Camb.). 2004; 12: 277-288Abstract Full Text PDF PubMed Google Scholar) was served as a starting model for structural refinement, using CNS (38Brunger 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 (16918) Google Scholar) with a protocol similar to that described previously (27Zhao Y. Li Z. Drozd S. Guo Y. Mourad W. Li H. Structure (Camb.). 2004; 12: 277-288Abstract Full Text PDF PubMed Google Scholar). Noncrystallographic symmetry restraints defined by pairs of individual domain were used throughout the refinement. The refinement statistics are summarized in Table 1. Analytical Ultracentrifugation (AUC) Sedimentation Velocity—Sedimentation velocity (SV) experiments were conducted at 20 °C in a Beckman Optima XL-I analytical ultracentrifuge at a rotor speed of 50,000 rpm. Double-sector cells were loaded with 400 μl of proteins samples and with 410 μl of reference solutions, respectively. Unless otherwise specified, the reference solution is Hepes-buffered saline (HBS) containing 10 mm Hepes buffer (pH 7.4), 150 mm NaCl. In certain experiments, 2 mm EDTA or ZnCl2 was added to both protein and reference solutions. Data were recorded with absorbance detection at wavelengths of 280, 294, and 300 nm for low, moderate, and high concentrations of proteins, respectively. Absorbance profiles were analyzed with the software SEDFIT (39Vistica J. Dam J. Balbo A. Yikilmaz E. Mariuzza R.A. Rouault T.A. Schuck P. Anal. Biochem. 2004; 326: 234-256Crossref PubMed Scopus (306) Google Scholar), using a model for continuous sedimentation coefficient distributions c(s) (40Schuck P. Perugini M.A. Gonzales N.R. Howlett G.J. Schubert D. Biophys. J. 2002; 82: 1096-1111Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar). Distributions were calculated with maximum entropy regularization at a predetermined confidence level of 1 S.D. AUC Sedimentation Equilibrium—Sedimentation equilibrium studies were conducted at a temperature of 20 °C and at three or four rotor speeds for each protein or mixture. The individual protein or protein mixture (100 μl) at various concentrations in HBS, with or without EDTA/ZnCl2, was loaded into an Epon double-sector centerpiece. Reference cells were loaded with 110 μl of reference solution. For the MAM/HLA-DR1/HA complex in the presence of 5 mm EDTA, mixtures of MAM and DR1 at various molar ratios and concentrations (5:2, 5:3, 15:1, and 1:4, in a real micromolar ratio of concentrations for MAM:HLA-DR1) were used for sedimentation equilibrium analysis with three rotor speeds (20,000, 25,000, and 30,000 rpm). For the complex with 1 mm ZnCl2, MAM and DR1, at 16:1 and 8:0.5 in a real micromolar ratio of concentrations, were analyzed with four rotor speeds (15,000, 20,000, 25,000, and 30,000 rpm). For DR1 alone, sedimentation equilibrium of DR1 was analyzed at concentrations of 1 and 5 μm with or without 1 mm ZnCl2, with three rotor speeds (18,000, 21,000, and 24,000 rpm). In the Zn2+ titration experiment for DR1 alone, 2 μm DR1 was used with various Zn2+ concentrations (0, 0.001, 0.01, 0.1, 0.3, 0.5, 0.7, and 1 mm). For MAM alone, the equilibrium experiment was conducted at protein concentrations of 2 and 15 μm, with or without 1 mm ZnCl2, and with three rotor speeds (20,000, 25,000, and 30,000 rpm). All equilibrium absorbance profiles were acquired at a 280-nm wavelength. The equilibrium sedimentation data were analyzed using the software SEDPHAT (39Vistica J. Dam J. Balbo A. Yikilmaz E. Mariuzza R.A. Rouault T.A. Schuck P. Anal. Biochem. 2004; 326: 234-256Crossref PubMed Scopus (306) Google Scholar). Data analysis was performed by global least squares analysis of data from multiple concentrations and multiple rotor speeds, using conservation of mass constraints (39Vistica J. Dam J. Balbo A. Yikilmaz E. Mariuzza R.A. Rouault T.A. Schuck P. Anal. Biochem. 2004; 326: 234-256Crossref PubMed Scopus (306) Google Scholar). Affinity Measurement Using SPR—Affinity and kinetic analyses of the interactions between MAM and HLA-DR1/HA were determined using a BIAcore 3000 SPR instrument (BIAcore) at 25 °C. Biotinylated HLA-DR1/HA complex was immobilized (∼260 RU) onto a streptavidin (SA) sensor chip (BIAcore). The concentrations used for the injected MAM samples ranged from 8 μm to 32 nm, with 2-fold dilutions. A blank surface blocked by biotin was used as the control surface. To minimize nonspecific binding, we carried out all of the binding experiments in 10 mm Hepes buffer containing 1 m sodium chloride, 3.4 mm EDTA, 0.005% surfactant P20, at a flow rate of 30 μl/min. Pulses of 10 mm NaOH were used to regenerate both surfaces between injections. Association (kon) and dissociation (koff) rates, as well as the dissociation constant (KD), were obtained by global fitting of the SPR data from multiple concentrations to a simple 1:1 Langmuir binding model, using BIAevaluation software version 4.1. Crystal Structures of the MAM/HLA-DR1/HA Complex in the Presence of EDTA—Previously, we determined the crystal structure of a MAM/HLA-DR1/HA complex (27Zhao Y. Li Z. Drozd S. Guo Y. Mourad W. Li H. Structure (Camb.). 2004; 12: 277-288Abstract Full Text PDF PubMed Google Scholar). No Zn2+ could be detected coordinated into the crystals, even though the crystallization buffer contained 1 mm Zn(OAc)2 (27Zhao Y. Li Z. Drozd S. Guo Y. Mourad W. Li H. Structure (Camb.). 2004; 12: 277-288Abstract Full Text PDF PubMed Google Scholar, 32Zhao Y. Li Z. Drozd S. Guo Y. Stack R. Hauer C. Li H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 353-356Crossref PubMed Scopus (5) Google Scholar). However, zinc has been reported to be critical for the biological function of MAM (29Etongue-Mayer P. Langlois M.A. Ouellette M. Li H. Younes S. Al-Daccak R. Mourad W. Eur. J. Immunol. 2002; 32: 50-58Crossref PubMed Scopus (14) Google Scholar, 30Langlois M.A. El Fakhry Y. Mourad W. J. Biol. Chem. 2003; 278: 22309-22315Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 31Langlois M.A. Etongue-Mayer P. Ouellette M. Mourad W. Eur. J. Immunol. 2000; 30: 1748-1756Crossref PubMed Scopus (19) Google Scholar). To better understand the role of zinc in the biological activity of MAM, we determined the crystal structures of the binary complex under several contrasting conditions. The MAM/HLA-DR1/HA complex was successfully crystallized under conditions, similar to those previously described (32Zhao Y. Li Z. Drozd S. Guo Y. Stack R. Hauer C. Li H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 353-356Crossref PubMed Scopus (5) Google Scholar), but in the absence of Zn(OAc)2 and/or in the presence of 2 mm EDTA (see “Experimental Procedures”). These new crystals are isomorphous with those obtained under the Zn2+ condition. We also soaked the complex crystals in the mother liquor with 10 mm CdCl2. Diffraction data were collected to 2.8, 3.0, and 2.6 Ä resolution for the crystals without Zn2+, with EDTA, and with Cd2+, respectively. The crystal structures of these complex crystals were refined to Rfactor values of 27.2% (no Zn2+), 21.1% (EDTA), and 23.4% (Cd2+), with Rfree values of 31.2, 26.1, and 26.6%, respectively (Table 1). The structures determined from these crystals can be readily superposed with the structure of the complex crystal obtained in the presence of Zn2+ (Fig. 1) (27Zhao Y. Li Z. Drozd S. Guo Y. Mourad W. Li H. Structure (Camb.). 2004; 12: 277-288Abstract Full Text PDF PubMed Google Scholar). The root-mean-square (r.m.s.) deviations in the C-α positions of all amino acids among these structures are within 0.35 Ä. For the Cd2+-soaked crystals, no Cd2+ ion could be located in the electron density map. These structures could suggest that the Zn2+ ion is not directly involved in the interaction between MAM and class II MHC molecules. MAM Binds to the HLA-DR1/HA Complex with a 1:1 Stoichiometry, in the Absence of Zn2+—To further investigate the molecular interaction between MAM and class II MHC molecule, we performed sedimentation velocity analytical ultracentrifugation with individual proteins and mixtures (Fig. 2). Previously, we demonstrated that MAM exists as a monomer at low protein concentration (≤11 μm) and that it can dimerize at high concentrations (≥25 μm) independent of Zn2+ (41Li H. Zhao Y. Guo Y. Vanvranken S.J. Li Z. Eisele L. Mourad W. Mol. Immunol. 2007; 44: 763-773Crossref PubMed Scopus (5) Google Scholar). The MAM monomer showed a single peak with a sedimentation coefficient (S) of about 2.1 (Fig. 2A), whereas the S value for a MAM dimer is 3.0 (41Li H. Zhao Y. Guo Y. Vanvranken S.J. Li Z. Eisele L. Mourad W. Mol. Immunol. 2007; 44: 763-773Crossref PubMed Scopus (5) Google Scholar). In the absence of Zn2+ ion, the HLA-DR1/HA complex showed a large peak, corresponding to a DR1 monomer, at 3.3 S (Fig. 2A). The sedimentation coefficient distribution of a mixture of 6 μm MAM and 6 μm HLA-DR1/HA showed two peaks at 2.1 and 4.0 S, respectively. The peak at 2.1 S corresponds to residue-unbound MAM monomer, whereas the peak at 4.0 S represents some species other than the unbound HLA-DR1/HA complex (at 3.3 S). This result demonstrates an interaction between the two molecules. Indeed, the value of the new peak at 4.0 S is very close to the S value (4.1 S) predicted by hydrodynamic modeling of a MAM/HLA-DR1/HA complex with a 1:1 stoichiometry. Increasing the protein concentrations of MAM (to 30 μm) and HLA-DR1/HA (to 10 μm) resulted in a peak for the sedimentation coefficient at 4.0 S, similar to that for the MAM·DR1 complex at low protein concentrations (Fig. 2). Addition of EDTA to the MAM/HLA-DR1/HA complex did not alter the 4.0 S peak position (Fig. 2B). These experiments demonstrated that MAM forms a complex with the HLA-DR1/HA complex in a 1:1 stoichiometry. Because addition of EDTA to the MAM/HLA-DR1/HA complex mixture did not change the sedimentation profile, we conclude that formation of the MAM/HLA-DR1/HA complex is not dependent on divalent metal ions such as Zn2+. MAM Binds to the HLA-DR1/HA Complex with High Affinity, in the Absence of Zn2+—By the SPR technique, we measured the affinity and kinetics of MAM binding to the HLA-DR1/HA complex. We produced a C-terminal biotinylated DR1 molecule using a strategy similar to that described by Wu et al. (42Wu L.C. Tuot D.S. Lyons D.S. Garcia K.C. Davis M.M. Nature. 2002; 418: 552-556Crossref PubMed Scopus (234) Google Scholar) (Fig. 3A). A construct was made to express the β-chain of HLA-DR1 with an HA peptide covalently linked to its N terminus through a linker peptide similar to what we and others described previously (29Etongue-Mayer P. Langlois M.A. Ouellette M. Li H. Younes S. Al-Daccak R. Mourad W. Eur. J. Immunol. 2002; 32: 50-58Crossref PubMed Scopus (14) Google Scholar, 43Fremont D.H. Hendrickson W.A. Marrack P. Kappler J. Science. 1996; 272: 1001-1004Crossref PubMed Scopus (332) Google Scholar). In addition, an amino acid tag encoding a signal sequence for attaching biotin with the BirA enzyme was attached to the C terminus of the DR1 β-chain through a p" @default.
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