Key points are not available for this paper at this time.
The homodimeric form of a recombinant cytokine interleukin-6 (IL-6D) is known to antagonize IL-6 signaling. In this study, spatially proximal residues between IL-6 chains in IL-6D were identified using a method for specific recognition of intermolecular cross-linked peptides. Our strategy involved mixing 1:1 15N-labeled and unlabeled (14N) protein to form a mixture of isotopically labeled and unlabeled homodimers, which was chemically cross-linked. This cross-linked IL-6D was subjected to proteolysis by trypsin and the generated peptides were analyzed by electrospray ionization time-of-flight mass spectrometry (MS). Molecular ions from cross-linked peptides of intermolecular origin are labeled with 15N/15N + 15N/14N + 14N/15N + 14N/14N yielding readily identified triplet/quadruplet MS peaks. All other peptide species are labeled with 15N + 14N yielding doublet peaks. Intermolecular cross-linked peptides were identified by MS, and cross-linked residues were identified. This intermolecular cross-link detection method, which we have designated “mixed isotope cross-linking” MIX may have more general application to protein-protein interaction studies. The pattern of proximal residues found was consistent with IL-6D having a domain-swapped fold similar to IL-10 and interferon-γ. This fold implies that IL-6D-mediated antagonism of IL-6 signaling is caused by obstruction of cooperative gp130 binding on IL-6D, rather than direct blocking of gp-130-binding sites on IL-6D. The homodimeric form of a recombinant cytokine interleukin-6 (IL-6D) is known to antagonize IL-6 signaling. In this study, spatially proximal residues between IL-6 chains in IL-6D were identified using a method for specific recognition of intermolecular cross-linked peptides. Our strategy involved mixing 1:1 15N-labeled and unlabeled (14N) protein to form a mixture of isotopically labeled and unlabeled homodimers, which was chemically cross-linked. This cross-linked IL-6D was subjected to proteolysis by trypsin and the generated peptides were analyzed by electrospray ionization time-of-flight mass spectrometry (MS). Molecular ions from cross-linked peptides of intermolecular origin are labeled with 15N/15N + 15N/14N + 14N/15N + 14N/14N yielding readily identified triplet/quadruplet MS peaks. All other peptide species are labeled with 15N + 14N yielding doublet peaks. Intermolecular cross-linked peptides were identified by MS, and cross-linked residues were identified. This intermolecular cross-link detection method, which we have designated “mixed isotope cross-linking” MIX may have more general application to protein-protein interaction studies. The pattern of proximal residues found was consistent with IL-6D having a domain-swapped fold similar to IL-10 and interferon-γ. This fold implies that IL-6D-mediated antagonism of IL-6 signaling is caused by obstruction of cooperative gp130 binding on IL-6D, rather than direct blocking of gp-130-binding sites on IL-6D. The interleukin-6 (IL-6) 1The abbreviations used for: IL, interleukin; CID, collision-induced dissociation; ESI, electrospray ionization; gp130, IL-6 receptor signaling subunit β chain; IL-6M, monomeric IL-6; IL-6D, dimeric IL-6; IL-6R, IL-6 receptor α-chain; MIX, mixed isotope cross-linking; MS, mass spectrometry; RP-HPLC, reversed-phase high-performance liquid chromatography; SEC, size-exclusion chromatography; BS3, bis(sulfosuccinimidyl)suberate. 1The abbreviations used for: IL, interleukin; CID, collision-induced dissociation; ESI, electrospray ionization; gp130, IL-6 receptor signaling subunit β chain; IL-6M, monomeric IL-6; IL-6D, dimeric IL-6; IL-6R, IL-6 receptor α-chain; MIX, mixed isotope cross-linking; MS, mass spectrometry; RP-HPLC, reversed-phase high-performance liquid chromatography; SEC, size-exclusion chromatography; BS3, bis(sulfosuccinimidyl)suberate. cytokine plays a critical role in host defense mechanisms such as T-cell activation, stimulation of B-cell differentiation, acute phase induction in hepatocytes, nerve cell differentiation, and osteoclast turnover (1Akira S. Taga T. Kishimoto T. Adv. Immunol. 1993; 54: 1-78Crossref PubMed Google Scholar). Abnormal IL-6 production is associated with a variety of diseases (2Jones S.A. Horiuchi S. Topley N. Yamamoto N. Fuller G.M. FASEB J. 2001; 15: 43-58Crossref PubMed Scopus (530) Google Scholar) such as rheumatoid arthritis (3Hirano T. Matsuda T. Turner M. Miyasaka N. Buchan G. Tang B. Sato K. Shimizu M. Maini R. Feldmann M. Kishimoto T. Eur. J. Immunol. 1988; 18: 1797-1801Crossref PubMed Scopus (656) Google Scholar), AIDS (4Nakajima K. Martinez Maza O. Hirano T. Breen E.C. Nishanian P.G. Salazar Gonzalez J.F. Fahey J.L. Kishimoto T. J. Immunol. 1989; 142: 531-536PubMed Google Scholar, 5Poli V. Balena R. Fattori E. Markatos A. Yamamoto M. Tanaka H. Ciliberto G. Rodan G.A. Costantini F. EMBO J. 1994; 13: 1189-1196Crossref PubMed Scopus (655) Google Scholar), osteoporosis (6Poli G. Bressler P. Kinter A. Duh E. Timmer W.C. Rabson A. Justement J.S. Stanley S. Fauci A.S. J. Exp. Med. 1990; 172: 151-158Crossref PubMed Scopus (420) Google Scholar, 7Jilka R.L. Hangoc G. Girasole G. Passeri G. Williams D.C. Abrams J.S. Boyce B. Broxmeyer H. Manolagas S.C. Science. 1992; 257: 88-91Crossref PubMed Scopus (1274) Google Scholar), psoriasis (8Grossman R.M. Krueger J. Yourish D. Granelli-Piperno A. Murphy D.P. May L.T. Kupper T.S. Sehgal P.B. Gottlieb A.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6367-6371Crossref PubMed Scopus (731) Google Scholar), multiple myeloma (9Bataille R. Jourdan M. Zhang X.G. Klein B. J. Clin. Invest. 1989; 84: 2008-2011Crossref PubMed Scopus (436) Google Scholar, 10Kawano M. Hirano T. Matsuda T. Taga T. Horii Y. Iwato K. Asaoku H. Tang B. Tanabe O. Tanaka H. Kuramoto A. Kishimoto T. Nature. 1988; 332: 83-85Crossref PubMed Scopus (1444) Google Scholar) and Kaposi's sarcoma (11Rettig M.B. Ma H.J. Vescio R.A. Pold M. Schiller G. Belson D. Savage A. Nishikubo C. Wu C. Fraser J. Said J.W. Berenson J.R. Science. 1997; 276: 1851-1854Crossref PubMed Scopus (412) Google Scholar). Thus the interactions between IL-6 and its associated receptors, the transmembrane glycoproteins IL-6R and gp130 (12Simpson R.J. Hammacher A. Smith D.K. Matthews J.M. Ward L.D. Protein Sci. 1997; 6: 929-955Crossref PubMed Scopus (296) Google Scholar), present an attractive target for therapeutic antagonists (13Nishimoto N. Kishimoto T. Yoshizaki K. Ann. Rheum. Dis. 2000; 59: i21-i27Crossref PubMed Google Scholar). IL-6 signaling is known to proceed via initial binding of IL-6 to the IL-6R to form a binary 1:1 complex. This binary complex interacts with gp130, later forming a signaling hexameric 2:2:2 complex comprising IL-6, IL-6R, and gp130 (14Ward L.D. Howlett G.J. Discolo G. Yasukawa K. Hammacher A. Moritz R.L. Simpson R.J. J. Biol. Chem. 1994; 269: 23286-23289Abstract Full Text PDF PubMed Google Scholar, 15Onishi M. Nosaka T. Kitamura T. Int. Rev. Immunol. 1998; 16: 617-634Crossref PubMed Scopus (20) Google Scholar).Previously, we have shown that a dimeric form of recombinant IL-6 (IL-6D) is a potent antagonist for IL-6 signaling (16Ward L.D. Hammacher A. Howlett G.J. Matthews J.M. Fabri L. Moritz R.L. Nice E.C. Weinstock J. Simpson R.J. J. Biol. Chem. 1996; 271: 20138-20144Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Recombinant IL-6D binds tightly to soluble IL-6R (sIL-6R) to form a 1:2 IL-6D(sIL-6R)2 complex (16Ward L.D. Hammacher A. Howlett G.J. Matthews J.M. Fabri L. Moritz R.L. Nice E.C. Weinstock J. Simpson R.J. J. Biol. Chem. 1996; 271: 20138-20144Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In contrast to the binary IL-6·sIL-6R complex, IL-6D(sIL-6R)2 binds gp130 weakly and does not show significant biological activity in the signal transducer and activator of transcription 3 (STAT3) phosphorylation assay (16Ward L.D. Hammacher A. Howlett G.J. Matthews J.M. Fabri L. Moritz R.L. Nice E.C. Weinstock J. Simpson R.J. J. Biol. Chem. 1996; 271: 20138-20144Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Natural (glycosylated) human IL-6 is also known to form a dimer that makes up a substantial part of IL-6 in blood or fibroblast secretions (17May L.T. Santhanam U. Sehgal P.B. J. Biol. Chem. 1991; 266: 9950-9955Abstract Full Text PDF PubMed Google Scholar, 18Fong Y. Moldawer L.L. Marano M. Wei H. Tatter S.B. Clarick R.H. Santhanam U. Sherris D. May L.T. Sehgal P.B. J. Immunol. 1989; 142: 2321-2324PubMed Google Scholar, 19Jablons D.M. Mule J.J. McIntosh J.K. Sehgal P.B. May L.T. Huang C.M. Rosenberg S.A. Lotze M.T. J. Immunol. 1989; 142: 1542-1547PubMed Google Scholar) and has also been shown to interact with membrane-bound IL-6R (15Onishi M. Nosaka T. Kitamura T. Int. Rev. Immunol. 1998; 16: 617-634Crossref PubMed Scopus (20) Google Scholar, 20Rose-John S. Hipp E. Lenz D. Legres L.G. Korr H. Hirano T. Kishimoto T. Heinrich P.C. J. Biol. Chem. 1991; 266: 3841-3846Abstract Full Text PDF PubMed Google Scholar, 59Wijdenes J. Clement C. Klein B. Morel-Fourrier B. Vita N. Ferrara P. Peters A. Mol. Immunol. 1991; 28: 1183-1192Crossref PubMed Scopus (46) Google Scholar). Recently, glycosylated natural human IL-6D, identified by immunoblotting and size exclusion chromatography, was shown to be a survival factor secreted by epithelial cells that inhibited the apoptosis of B-chronic lymphocytic leukemia cells (21Moreno A. Villar M.L. Camara C. Luque R. Cespon C. Gonzalez-Porque P. Roy G. Lopez-Jimenez J. Bootello A. Santiago E.R. Blood. 2001; 97: 242-249Crossref PubMed Scopus (46) Google Scholar). Significantly, recombinant human IL-6D fromEscherichia coli acted as a survival factor in a similar way (21Moreno A. Villar M.L. Camara C. Luque R. Cespon C. Gonzalez-Porque P. Roy G. Lopez-Jimenez J. Bootello A. Santiago E.R. Blood. 2001; 97: 242-249Crossref PubMed Scopus (46) Google Scholar). Taken together, these results suggest that natural and recombinant IL-6D may have similar biological activity.Elucidation of the IL-6D structure will be critical to understanding the basis of its antagonistic properties. Whereas the structure of IL-6 is known to be a 4-α-helical bundle (22Xu G.Y. Yu H.A. Hong J. Stahl M. McDonagh T. Kay L.E. Cumming D.A. J. Mol. Biol. 1997; 268: 468-481Crossref PubMed Scopus (65) Google Scholar), the structure of IL-6D is unknown. Previous biophysical studies of the sedimentation properties and the unfolding-dissociation relationship of IL-6D (23Matthews J.M. Hammacher A. Howlett G.J. Simpson R.J. Biochemistry. 1998; 37: 10671-10680Crossref PubMed Scopus (10) Google Scholar) have shown it is likely to form a metastable domain-swapped dimer (24Liu Y. Eisenberg D. Protein Sci. 2002; 11: 1285-1299Crossref PubMed Scopus (590) Google Scholar, 25Bennett M.J. Schlunegger M.P. Eisenberg D. Protein Sci. 1995; 4: 2455-2468Crossref PubMed Scopus (681) Google Scholar) in which adjacent subunits have the IL-6 structure, but contain interchanged α-helical bundle domain elements.Here, we investigate the arrangement of domain-swapped IL-6 chains within IL-6D using a technique based on cross-linking and mass spectrometry. Although the established techniques of x-ray crystallography and NMR spectroscopy yield high resolution data, often this takes months or years to obtain (26Liu Z. Sun C. Olejniczak E.T. Meadows R.P. Betz S.F. Oost T. Herrmann J. Wu J.C. Fesik S.W. Nature. 2000; 408: 1004-1008Crossref PubMed Scopus (540) Google Scholar, 27Ban N. Nissen P. Hansen J. Moore P.B. Steitz T.A. Science. 2000; 289: 905-920Crossref PubMed Scopus (2775) Google Scholar). Techniques in mass spectrometry (MS) combined with cross-linking (28Henry C.M. Chem. Eng. News. 2001; 78: 22-36Crossref Scopus (7) Google Scholar, 29Wallon G. J. M. L. EMBO J. 2000; PubMed Scopus Google Scholar, Tang N. J.C. C.M. G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar) or other techniques such as J.L. Biochemistry. 2001; PubMed Scopus Google N. E. K. 2002; 16: PubMed Scopus Google Scholar) have been for of Tang N. J.C. C.M. G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar) or protein J. S. E.C. M. Chem. 2000; PubMed Scopus Google Scholar, A. K. Biochemistry. 2001; PubMed Scopus Google Scholar). chemically or cross-linking a protein complex Chem. 1990; PubMed Scopus Google Scholar), by of the cross-linked complex and MS of the peptide mixture B. M. Biol. 1998; PubMed Scopus Google Scholar). peptides be identified by mass the pattern by mass spectrometry adjacent protein and of of or protein are and not of protein Tang N. J.C. C.M. G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar). these have as a to x-ray or NMR for of between G. J. M. L. EMBO J. 2000; PubMed Scopus Google Scholar) and the of within J. S. E.C. M. Chem. 2000; PubMed Scopus Google the of cross-linking high and the of protein the of peptide species that are from the of cross-linked makes it to intermolecular cross-linked peptides from MS This has been by that MS of cross-linked species within complex peptide A. K. Biochemistry. 2001; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, M. P. M. M. P. P. Protein Sci. 2000; PubMed Scopus Google Scholar, P. H. U. H. S. Chem. 2001; PubMed Scopus Google Scholar). the of a 1:1 mixture of and cross-linking readily mass detection of cross-linked species by the P. H. U. H. S. Chem. 2001; PubMed Scopus Google Scholar). for interactions between these in that to and peptides. This results in cross-linked peptide species that not yield on intermolecular such as cross-linked peptides or peptides by cross-linking Tang N. J.C. C.M. G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar, 2002; 16: PubMed Scopus Google Scholar). these cross-linking studies of such as IL-6D, cross-linked peptides in from M. P. M. M. P. P. Protein Sci. 2000; PubMed Scopus Google we present a method for intermolecular cross-linked peptides in the IL-6 that we have designated mixed isotope cross-linking to IL-6D, the MIX method of 15N-labeled and unlabeled (14N) IL-6, combined as a 1:1 mixture and to form a of and 15N-labeled IL-6D. and mass peptide on this mixture intermolecular cross-linked peptides to be identified as form or mass of the of peptides within these cross-linked peptides. In cross-linked and peptides are as doublet mass peaks. This to between and cross-linked species makes the MIX technique a for intermolecular the application of this technique to proximal intermolecular residues within the homodimeric form of IL-6 and to the of domain based on the known structure of monomeric human interleukin-6 (22Xu G.Y. Yu H.A. Hong J. Stahl M. McDonagh T. Kay L.E. Cumming D.A. J. Mol. Biol. 1997; 268: 468-481Crossref PubMed Scopus (65) Google Scholar). The interleukin-6 (IL-6) 1The abbreviations used for: IL, interleukin; CID, collision-induced dissociation; ESI, electrospray ionization; gp130, IL-6 receptor signaling subunit β chain; IL-6M, monomeric IL-6; IL-6D, dimeric IL-6; IL-6R, IL-6 receptor α-chain; MIX, mixed isotope cross-linking; MS, mass spectrometry; RP-HPLC, reversed-phase high-performance liquid chromatography; SEC, size-exclusion chromatography; BS3, bis(sulfosuccinimidyl)suberate. 1The abbreviations used for: IL, interleukin; CID, collision-induced dissociation; ESI, electrospray ionization; gp130, IL-6 receptor signaling subunit β chain; IL-6M, monomeric IL-6; IL-6D, dimeric IL-6; IL-6R, IL-6 receptor α-chain; MIX, mixed isotope cross-linking; MS, mass spectrometry; RP-HPLC, reversed-phase high-performance liquid chromatography; SEC, size-exclusion chromatography; BS3, bis(sulfosuccinimidyl)suberate. cytokine plays a critical role in host defense mechanisms such as T-cell activation, stimulation of B-cell differentiation, acute phase induction in hepatocytes, nerve cell differentiation, and osteoclast turnover (1Akira S. Taga T. Kishimoto T. Adv. Immunol. 1993; 54: 1-78Crossref PubMed Google Scholar). Abnormal IL-6 production is associated with a variety of diseases (2Jones S.A. Horiuchi S. Topley N. Yamamoto N. Fuller G.M. FASEB J. 2001; 15: 43-58Crossref PubMed Scopus (530) Google Scholar) such as rheumatoid arthritis (3Hirano T. Matsuda T. Turner M. Miyasaka N. Buchan G. Tang B. Sato K. Shimizu M. Maini R. Feldmann M. Kishimoto T. Eur. J. Immunol. 1988; 18: 1797-1801Crossref PubMed Scopus (656) Google Scholar), AIDS (4Nakajima K. Martinez Maza O. Hirano T. Breen E.C. Nishanian P.G. Salazar Gonzalez J.F. Fahey J.L. Kishimoto T. J. Immunol. 1989; 142: 531-536PubMed Google Scholar, 5Poli V. Balena R. Fattori E. Markatos A. Yamamoto M. Tanaka H. Ciliberto G. Rodan G.A. Costantini F. EMBO J. 1994; 13: 1189-1196Crossref PubMed Scopus (655) Google Scholar), osteoporosis (6Poli G. Bressler P. Kinter A. Duh E. Timmer W.C. Rabson A. Justement J.S. Stanley S. Fauci A.S. J. Exp. Med. 1990; 172: 151-158Crossref PubMed Scopus (420) Google Scholar, 7Jilka R.L. Hangoc G. Girasole G. Passeri G. Williams D.C. Abrams J.S. Boyce B. Broxmeyer H. Manolagas S.C. Science. 1992; 257: 88-91Crossref PubMed Scopus (1274) Google Scholar), psoriasis (8Grossman R.M. Krueger J. Yourish D. Granelli-Piperno A. Murphy D.P. May L.T. Kupper T.S. Sehgal P.B. Gottlieb A.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6367-6371Crossref PubMed Scopus (731) Google Scholar), multiple myeloma (9Bataille R. Jourdan M. Zhang X.G. Klein B. J. Clin. Invest. 1989; 84: 2008-2011Crossref PubMed Scopus (436) Google Scholar, 10Kawano M. Hirano T. Matsuda T. Taga T. Horii Y. Iwato K. Asaoku H. Tang B. Tanabe O. Tanaka H. Kuramoto A. Kishimoto T. Nature. 1988; 332: 83-85Crossref PubMed Scopus (1444) Google Scholar) and Kaposi's sarcoma (11Rettig M.B. Ma H.J. Vescio R.A. Pold M. Schiller G. Belson D. Savage A. Nishikubo C. Wu C. Fraser J. Said J.W. Berenson J.R. Science. 1997; 276: 1851-1854Crossref PubMed Scopus (412) Google Scholar). Thus the interactions between IL-6 and its associated receptors, the transmembrane glycoproteins IL-6R and gp130 (12Simpson R.J. Hammacher A. Smith D.K. Matthews J.M. Ward L.D. Protein Sci. 1997; 6: 929-955Crossref PubMed Scopus (296) Google Scholar), present an attractive target for therapeutic antagonists (13Nishimoto N. Kishimoto T. Yoshizaki K. Ann. Rheum. Dis. 2000; 59: i21-i27Crossref PubMed Google Scholar). IL-6 signaling is known to proceed via initial binding of IL-6 to the IL-6R to form a binary 1:1 complex. This binary complex interacts with gp130, later forming a signaling hexameric 2:2:2 complex comprising IL-6, IL-6R, and gp130 (14Ward L.D. Howlett G.J. Discolo G. Yasukawa K. Hammacher A. Moritz R.L. Simpson R.J. J. Biol. Chem. 1994; 269: 23286-23289Abstract Full Text PDF PubMed Google Scholar, 15Onishi M. Nosaka T. Kitamura T. Int. Rev. Immunol. 1998; 16: 617-634Crossref PubMed Scopus (20) Google Scholar). we have shown that a dimeric form of recombinant IL-6 (IL-6D) is a potent antagonist for IL-6 signaling (16Ward L.D. Hammacher A. Howlett G.J. Matthews J.M. Fabri L. Moritz R.L. Nice E.C. Weinstock J. Simpson R.J. J. Biol. Chem. 1996; 271: 20138-20144Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Recombinant IL-6D binds tightly to soluble IL-6R (sIL-6R) to form a 1:2 IL-6D(sIL-6R)2 complex (16Ward L.D. Hammacher A. Howlett G.J. Matthews J.M. Fabri L. Moritz R.L. Nice E.C. Weinstock J. Simpson R.J. J. Biol. Chem. 1996; 271: 20138-20144Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In contrast to the binary IL-6·sIL-6R complex, IL-6D(sIL-6R)2 binds gp130 weakly and does not show significant biological activity in the signal transducer and activator of transcription 3 (STAT3) phosphorylation assay (16Ward L.D. Hammacher A. Howlett G.J. Matthews J.M. Fabri L. Moritz R.L. Nice E.C. Weinstock J. Simpson R.J. J. Biol. Chem. 1996; 271: 20138-20144Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Natural (glycosylated) human IL-6 is also known to form a dimer that makes up a substantial part of IL-6 in blood or fibroblast secretions (17May L.T. Santhanam U. Sehgal P.B. J. Biol. Chem. 1991; 266: 9950-9955Abstract Full Text PDF PubMed Google Scholar, 18Fong Y. Moldawer L.L. Marano M. Wei H. Tatter S.B. Clarick R.H. Santhanam U. Sherris D. May L.T. Sehgal P.B. J. Immunol. 1989; 142: 2321-2324PubMed Google Scholar, 19Jablons D.M. Mule J.J. McIntosh J.K. Sehgal P.B. May L.T. Huang C.M. Rosenberg S.A. Lotze M.T. J. Immunol. 1989; 142: 1542-1547PubMed Google Scholar) and has also been shown to interact with membrane-bound IL-6R (15Onishi M. Nosaka T. Kitamura T. Int. Rev. Immunol. 1998; 16: 617-634Crossref PubMed Scopus (20) Google Scholar, 20Rose-John S. Hipp E. Lenz D. Legres L.G. Korr H. Hirano T. Kishimoto T. Heinrich P.C. J. Biol. Chem. 1991; 266: 3841-3846Abstract Full Text PDF PubMed Google Scholar, 59Wijdenes J. Clement C. Klein B. Morel-Fourrier B. Vita N. Ferrara P. Peters A. Mol. Immunol. 1991; 28: 1183-1192Crossref PubMed Scopus (46) Google Scholar). Recently, glycosylated natural human IL-6D, identified by immunoblotting and size exclusion chromatography, was shown to be a survival factor secreted by epithelial cells that inhibited the apoptosis of B-chronic lymphocytic leukemia cells (21Moreno A. Villar M.L. Camara C. Luque R. Cespon C. Gonzalez-Porque P. Roy G. Lopez-Jimenez J. Bootello A. Santiago E.R. Blood. 2001; 97: 242-249Crossref PubMed Scopus (46) Google Scholar). Significantly, recombinant human IL-6D fromEscherichia coli acted as a survival factor in a similar way (21Moreno A. Villar M.L. Camara C. Luque R. Cespon C. Gonzalez-Porque P. Roy G. Lopez-Jimenez J. Bootello A. Santiago E.R. Blood. 2001; 97: 242-249Crossref PubMed Scopus (46) Google Scholar). Taken together, these results suggest that natural and recombinant IL-6D may have similar biological of the IL-6D structure will be critical to understanding the basis of its antagonistic properties. Whereas the structure of IL-6 is known to be a 4-α-helical bundle (22Xu G.Y. Yu H.A. Hong J. Stahl M. McDonagh T. Kay L.E. Cumming D.A. J. Mol. Biol. 1997; 268: 468-481Crossref PubMed Scopus (65) Google Scholar), the structure of IL-6D is unknown. Previous biophysical studies of the sedimentation properties and the unfolding-dissociation relationship of IL-6D (23Matthews J.M. Hammacher A. Howlett G.J. Simpson R.J. Biochemistry. 1998; 37: 10671-10680Crossref PubMed Scopus (10) Google Scholar) have shown it is likely to form a metastable domain-swapped dimer (24Liu Y. Eisenberg D. Protein Sci. 2002; 11: 1285-1299Crossref PubMed Scopus (590) Google Scholar, 25Bennett M.J. Schlunegger M.P. Eisenberg D. Protein Sci. 1995; 4: 2455-2468Crossref PubMed Scopus (681) Google Scholar) in which adjacent subunits have the IL-6 structure, but contain interchanged α-helical bundle domain we investigate the arrangement of domain-swapped IL-6 chains within IL-6D using a technique based on cross-linking and mass spectrometry. Although the established techniques of x-ray crystallography and NMR spectroscopy yield high resolution data, often this takes months or years to obtain (26Liu Z. Sun C. Olejniczak E.T. Meadows R.P. Betz S.F. Oost T. Herrmann J. Wu J.C. Fesik S.W. Nature. 2000; 408: 1004-1008Crossref PubMed Scopus (540) Google Scholar, 27Ban N. Nissen P. Hansen J. Moore P.B. Steitz T.A. Science. 2000; 289: 905-920Crossref PubMed Scopus (2775) Google Scholar). Techniques in mass spectrometry (MS) combined with cross-linking (28Henry C.M. Chem. Eng. News. 2001; 78: 22-36Crossref Scopus (7) Google Scholar, 29Wallon G. J. M. L. EMBO J. 2000; PubMed Scopus Google Scholar, Tang N. J.C. C.M. G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar) or other techniques such as J.L. Biochemistry. 2001; PubMed Scopus Google N. E. K. 2002; 16: PubMed Scopus Google Scholar) have been for of Tang N. J.C. C.M. G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar) or protein J. S. E.C. M. Chem. 2000; PubMed Scopus Google Scholar, A. K. Biochemistry. 2001; PubMed Scopus Google Scholar). chemically or cross-linking a protein complex Chem. 1990; PubMed Scopus Google Scholar), by of the cross-linked complex and MS of the peptide mixture B. M. Biol. 1998; PubMed Scopus Google Scholar). peptides be identified by mass the pattern by mass spectrometry adjacent protein and of of or protein are and not of protein Tang N. J.C. C.M. G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar). these have as a to x-ray or NMR for of between G. J. M. L. EMBO J. 2000; PubMed Scopus Google Scholar) and the of within J. S. E.C. M. Chem. 2000; PubMed Scopus Google Scholar). the of cross-linking high and the of protein the of peptide species that are from the of cross-linked makes it to intermolecular cross-linked peptides from MS This has been by that MS of cross-linked species within complex peptide A. K. Biochemistry. 2001; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, M. P. M. M. P. P. Protein Sci. 2000; PubMed Scopus Google Scholar, P. H. U. H. S. Chem. 2001; PubMed Scopus Google Scholar). the of a 1:1 mixture of and cross-linking readily mass detection of cross-linked species by the P. H. U. H. S. Chem. 2001; PubMed Scopus Google Scholar). for interactions between these in that to and peptides. This results in cross-linked peptide species that not yield on intermolecular such as cross-linked peptides or peptides by cross-linking Tang N. J.C. C.M. G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar, 2002; 16: PubMed Scopus Google Scholar). these cross-linking studies of such as IL-6D, cross-linked peptides in from M. P. M. M. P. P. Protein Sci. 2000; PubMed Scopus Google Scholar). we present a method for intermolecular cross-linked peptides in the IL-6 that we have designated mixed isotope cross-linking to IL-6D, the MIX method of 15N-labeled and unlabeled (14N) IL-6, combined as a 1:1 mixture and to form a of and 15N-labeled IL-6D. and mass peptide on this mixture intermolecular cross-linked peptides to be identified as form or mass of the of peptides within these cross-linked peptides. In cross-linked and peptides are as doublet mass peaks. This to between and cross-linked species makes the MIX technique a for intermolecular the application of this technique to proximal intermolecular residues within the homodimeric form of IL-6 and to the of domain based on the known structure of monomeric human interleukin-6 (22Xu G.Y. Yu H.A. Hong J. Stahl M. McDonagh T. Kay L.E. Cumming D.A. J. Mol. Biol. 1997; 268: 468-481Crossref PubMed Scopus (65) Google Scholar). D. E. A. L. R. L. J. and G. F. for on of the and A. and G. E. for critical on the
Taverner et al. (Fri,) studied this question.