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Hexameric Calgranulin C (S100A12) Binds to the Receptor for Advanced Glycated End Products (RAGE) Using Symmetric Hydrophobic Target-binding Patches

2006; Elsevier BV; Volume: 282; Issue: 6 Linguagem: Inglês

10.1074/jbc.m608888200

ISSN

1083-351X

Autores

Jingjing Xie, David S. Burz, Wei He, Igor Bronstein, Igor K. Lednev, Alexander Shekhtman,

Tópico(s)

Occupational exposure and asthma

Resumo

Calgranulin C (S100A12) is a member of the S100 family of proteins that undergoes a conformational change upon calcium binding allowing them to interact with target molecules and initiate biological responses; one such target is the receptor for advanced glycation products (RAGE). The RAGE-calgranulin C interaction mediates a pro-inflammatory response to cellular stress and can contribute to the pathogenesis of inflammatory lesions. The soluble extracellular part of RAGE (sRAGE) was shown to decrease the inflammation response possibly by scavenging RAGE-activating ligands. Here, by using high resolution NMR spectroscopy, we identified the sRAGE-calgranulin C interaction surface. Ca2+ binding creates two symmetric hydrophobic surfaces on Ca2+-calgranulin C that allow calgranulin C to bind to the C-type immunoglobulin domain of RAGE. Apo-calgranulin C also binds to sRAGE using a completely different surface and with substantially lower affinity, thus underscoring the role of Ca2+ binding to S100 proteins as a molecular switch. By using native gel electrophoresis, chromatography, and fluorescence spectroscopy, we established that sRAGE forms tetramers that bind to hexamers of Ca2+-calgranulin C. This arrangement creates a large platform for effectively transmitting RAGE-dependent signals from extracellular S100 proteins to the cytoplasmic signaling complexes. Calgranulin C (S100A12) is a member of the S100 family of proteins that undergoes a conformational change upon calcium binding allowing them to interact with target molecules and initiate biological responses; one such target is the receptor for advanced glycation products (RAGE). The RAGE-calgranulin C interaction mediates a pro-inflammatory response to cellular stress and can contribute to the pathogenesis of inflammatory lesions. The soluble extracellular part of RAGE (sRAGE) was shown to decrease the inflammation response possibly by scavenging RAGE-activating ligands. Here, by using high resolution NMR spectroscopy, we identified the sRAGE-calgranulin C interaction surface. Ca2+ binding creates two symmetric hydrophobic surfaces on Ca2+-calgranulin C that allow calgranulin C to bind to the C-type immunoglobulin domain of RAGE. Apo-calgranulin C also binds to sRAGE using a completely different surface and with substantially lower affinity, thus underscoring the role of Ca2+ binding to S100 proteins as a molecular switch. By using native gel electrophoresis, chromatography, and fluorescence spectroscopy, we established that sRAGE forms tetramers that bind to hexamers of Ca2+-calgranulin C. This arrangement creates a large platform for effectively transmitting RAGE-dependent signals from extracellular S100 proteins to the cytoplasmic signaling complexes. Calgranulin C (S100A12) is one of 21 members of the S100 family of EF-hand calcium-binding proteins identified to date (1Heizmann C.W. Fritz G. Schafer B.W. Front. Biosci. 2002; 7: D1356-D1368Crossref PubMed Google Scholar). Members of the S100 family have both intracellular and extracellular functions and participate in diverse cellular processes leading to cell growth and differentiation, cell cycle regulation, transcription, and signal transduction receptor activities (2Donato R. Microsc. Res. Tech. 2003; 60: 540-551Crossref PubMed Scopus (801) Google Scholar). This diversity is tailored in part by a distinct pattern of subcellular localization and tissue-specific expression. EF-hand calcium-binding proteins translate the physiological changes in calcium levels into specific cellular responses by undergoing a large conformational change that exposes a binding site recognized by downstream effectors, essentially acting as a molecular switch (3Lewit-Bentley A. Rety S. Curr. Opin. Struct. Biol. 2000; 10: 637-643Crossref PubMed Scopus (414) Google Scholar, 4Bhattacharya S. Bunick C.G. Chazin W.J. Biochim. Biophys. Acta. 2004; 1742: 69-79Crossref PubMed Scopus (209) Google Scholar).Most S100 proteins are noncovalent homodimers. Some members of the S100 family, such as S100A8 and S100A9, form heterodimers and heterotetramers (5Teigelkamp S. Bhardwaj R.S. Roth J. Meinardus-Hager G. Karas M. Sorg C. J. Biol. Chem. 1991; 266: 13462-13467Abstract Full Text PDF PubMed Google Scholar). Higher order homo-oligomers have also been reported (6Moroz O.V. Antson A.A. Dodson E.J. Burrell H.J. Grist S.J. Lloyd R.M. Maitland N.J. Dodson G.G. Wilson K.S. Lukanidin E. Bronstein I.B. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 407-413Crossref PubMed Scopus (96) Google Scholar, 7Strupat K. Rogniaux H. Van Dorsselaer A. Roth J. Vogl T. J. Am. Soc. Mass Spectrom. 2000; 11: 780-788Crossref PubMed Scopus (84) Google Scholar) and are suggested to play an important biological role in ligand targeting (8Moroz O.V. Dodson G.G. Wilson K.S. Lukanidin E. Bronstein I.B. Microsc. Res. Tech. 2003; 60: 581-592Crossref PubMed Scopus (64) Google Scholar). The basic structural and functional unit of the S100 proteins is a symmetric dimer comprised of two EF-hand subunits organized into an eight-helix bundle (4Bhattacharya S. Bunick C.G. Chazin W.J. Biochim. Biophys. Acta. 2004; 1742: 69-79Crossref PubMed Scopus (209) Google Scholar). The conformational change triggered by calcium binding asymmetrically affects the two EF-hand motifs of the subunit with the N-terminal EF-hand undergoing a relatively small change in conformation, whereas the change in the C-terminal EF-hand is much larger.This conformational rearrangement creates a shallow hydrophobic pocket. Structural studies of the ligated forms of S100 proteins showed that this hydrophobic surface can bind short peptides from the respective targets of S100 proteins (9Bhattacharya S. Large E. Heizmann C.W. Hemmings B. Chazin W.J. Biochemistry. 2003; 42: 14416-14426Crossref PubMed Scopus (82) Google Scholar, 10Rety S. Sopkova J. Renouard M. Osterloh D. Gerke V. Tabaries S. Russo-Marie F. Lewit-Bentley A. Nat. Struct. Biol. 1999; 6: 89-95Crossref PubMed Scopus (258) Google Scholar, 11Rety S. Osterloh D. Arie J.P. Tabaries S. Seeman J. Russo-Marie F. Gerke V. Lewit-Bentley A. Structure (Lond.). 2000; 8: 175-184Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 12Inman K.G. Yang R. Rustandi R.R. Miller K.E. Baldisseri D.M. Weber D.J. J. Mol. Biol. 2002; 324: 1003-1014Crossref PubMed Scopus (72) Google Scholar, 13Rustandi R.R. Baldisseri D.M. Weber D.J. Nat. Struct. Biol. 2000; 7: 570-574Crossref PubMed Scopus (286) Google Scholar). Surprisingly, despite utilizing a similar binding surface, different peptides bind to individual S100 proteins in different ways. For example, in the complexes determined by using x-ray crystallography involving S100A10 and a peptide from annexin II (10Rety S. Sopkova J. Renouard M. Osterloh D. Gerke V. Tabaries S. Russo-Marie F. Lewit-Bentley A. Nat. Struct. Biol. 1999; 6: 89-95Crossref PubMed Scopus (258) Google Scholar) and S100A11 and a peptide from annexin I (11Rety S. Osterloh D. Arie J.P. Tabaries S. Seeman J. Russo-Marie F. Gerke V. Lewit-Bentley A. Structure (Lond.). 2000; 8: 175-184Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), the peptide is packed against helices 1 and 4 of S100A10. However, in the complexes determined using NMR involving S100B and the peptide from p53 (13Rustandi R.R. Baldisseri D.M. Weber D.J. Nat. Struct. Biol. 2000; 7: 570-574Crossref PubMed Scopus (286) Google Scholar), the p53 peptide makes contacts with helices 3 and 4, and in the complex between S100B and the peptide from NDR (nuclear Dbf2-related) kinase (9Bhattacharya S. Large E. Heizmann C.W. Hemmings B. Chazin W.J. Biochemistry. 2003; 42: 14416-14426Crossref PubMed Scopus (82) Google Scholar), the NDR peptide is mostly packed against helix 4. The observed structural differences suggest that the binding modes may be still different for the full-length S100 protein targets.The crystal structure of Ca2+-bound calgranulin C closely resembles structures of the S100/calgranulin family proteins, presenting a dimer of four-helix subunits (14Moroz O.V. Antson A.A. Murshudov G.N. Maitland N.J. Dodson G.G. Wilson K.S. Skibshoj I. Lukanidin E.M. Bronstein I.B. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 20-29Crossref PubMed Scopus (71) Google Scholar). The calciumbinding EF-hand motifs, helix 1-loop 1-helix 2 and helix 3-loop 3-helix 4 are linked by a short anti-parallel β-bridge, a common feature of the EF-hand proteins. Helices 2 and 3 are linked by the hinge region formed by loop 2. The hinge region is poorly conserved among S100 family members suggesting that specificity of target recognition may reside in this area. At low millimolar concentrations of Ca2+, calgranulin C can also form a hexamer consisting of three symmetrically positioned Ca2+-bound dimers (6Moroz O.V. Antson A.A. Dodson E.J. Burrell H.J. Grist S.J. Lloyd R.M. Maitland N.J. Dodson G.G. Wilson K.S. Lukanidin E. Bronstein I.B. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 407-413Crossref PubMed Scopus (96) Google Scholar).The S100 family members characteristically accumulate at sites of chronic inflammation. The mechanism by which calgranulin C modulates the course of inflammatory processes is related to its interaction with the receptor for advanced glycated products (RAGE) 2The abbreviations used are: RAGE, receptor for advanced glycation products; sRAGE, soluble RAGE; AGE, advanced glycation product; HSQC, heteronuclear single quantum coherence; PDB, Protein Data Bank; TNS, 6-(p-toluidino)naphthalene-2-sulfonate; ERK, extracellular signal-regulated kinase. 2The abbreviations used are: RAGE, receptor for advanced glycation products; sRAGE, soluble RAGE; AGE, advanced glycation product; HSQC, heteronuclear single quantum coherence; PDB, Protein Data Bank; TNS, 6-(p-toluidino)naphthalene-2-sulfonate; ERK, extracellular signal-regulated kinase. (15Hofmann M.A. Drury S. Fu C. Qu W. Taguchi A. Lu Y. Avila C. Kambham N. Bierhaus A. Nawroth P. Neurath M.F. Slattery T. Beach D. McClary J. Nagashima M. Morser J. Stern D. Schmidt A.M. Cell. 1999; 97: 889-901Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar). RAGE is a multiligand member of the immunoglobulin superfamily of cell surface molecules and displays a high degree of sequence homology to the neural adhesion molecule (N-CAM) (16Schmidt A.M. Hori O. Cao R. Yan S.D. Brett J. Wautier J.L. Ogawa S. Kuwabara K. Matsumoto M. Stern D. Diabetes. 1996; 45: 77-80Crossref PubMed Google Scholar, 17Neeper M. Schmidt A.M. Brett J. Yan S.D. Wang F. Pan Y.C. Elliston K. Stern D. Shaw A. J. Biol. Chem. 1992; 267: 14998-15004Abstract Full Text PDF PubMed Google Scholar). Based on sequence homology, RAGE contains an N-terminal V-type immunoglobulin domain followed by two C-type immunoglobulin domains, a trans-membrane helix and short cytoplasmic tail. The cytoplasmic tail is absolutely essential for intracellular signaling. A RAGE mutant lacking a cytoplasmic tail, dominant negative-RAGE, exhibits a strong dominant negative effect on RAGE-dependent signaling (15Hofmann M.A. Drury S. Fu C. Qu W. Taguchi A. Lu Y. Avila C. Kambham N. Bierhaus A. Nawroth P. Neurath M.F. Slattery T. Beach D. McClary J. Nagashima M. Morser J. Stern D. Schmidt A.M. Cell. 1999; 97: 889-901Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar, 18Yeh C.H. Sturgis L. Haidacher J. Zhang X.N. Sherwood S.J. Bjercke R.J. Juhasz O. Crow M.T. Tilton R.G. Denner L. Diabetes. 2001; 50: 1495-1504Crossref PubMed Scopus (288) Google Scholar, 19Sousa M.M. Yan S.D. Stern D. Saraiva M.J. Lab. Investig. 2000; 80: 1101-1110Crossref PubMed Scopus (149) Google Scholar). This observation indicates that signaling molecules associate with the cytosolic domain of RAGE. A recent study revealed that ERK kinase binds directly to a site on the RAGE cytoplasmic tail suggesting its essential role in RAGE-mediated signaling (20Ishihara K. Tsutsumi K. Kawane S. Nakajima M. Kasaoka T. FEBS Lett. 2003; 550: 107-113Crossref PubMed Scopus (167) Google Scholar).RAGE was originally identified and characterized based on its ability to bind advanced glycation products (AGEs), adducts formed by glycoxidation that accumulate especially in disorders such as diabetes and renal failure (21Brownlee M. Annu. Rev. Med. 1995; 46: 223-234Crossref PubMed Scopus (1139) Google Scholar). Rather than working as a scavenger receptor to effectively uptake and dispose of AGEs, ligand-active RAGE causes cellular perturbations leading to an inflammatory response (22Wautier J.L. Zoukourian C. Chappey O. Wautier M.P. Guillausseau P.J. Cao R. Hori O. Stern D. Schmidt A.M. J. Clin. Investig. 1996; 97: 238-243Crossref PubMed Scopus (495) Google Scholar). The extracellular, naturally occurring splicing variant of RAGE, soluble RAGE (sRAGE) that includes three immunoglobulin domains (17Neeper M. Schmidt A.M. Brett J. Yan S.D. Wang F. Pan Y.C. Elliston K. Stern D. Shaw A. J. Biol. Chem. 1992; 267: 14998-15004Abstract Full Text PDF PubMed Google Scholar), was shown to alleviate the inflammatory effects resulting from the AGEs-RAGE interaction by sequestering RAGE-activating ligands (23Park L. Raman K.G. Lee K.J. Lu Y. Ferran Jr., L.J. Chow W.S. Stern D. Schmidt A.M. Nat. Med. 1998; 4: 1025-1031Crossref PubMed Scopus (1016) Google Scholar).At least seven members of the S100 family, calgranulin C, S100A1, S100A4, S100A11, S100A13, S100B, and S100P, were identified to be ligands of RAGE (15Hofmann M.A. Drury S. Fu C. Qu W. Taguchi A. Lu Y. Avila C. Kambham N. Bierhaus A. Nawroth P. Neurath M.F. Slattery T. Beach D. McClary J. Nagashima M. Morser J. Stern D. Schmidt A.M. Cell. 1999; 97: 889-901Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar, 24Hsieh H.L. Schafer B.W. Weigle B. Heizmann C.W. Biochem. Biophys. Res. Commun. 2004; 316: 949-959Crossref PubMed Scopus (91) Google Scholar, 25Donato R. Int. J. Biochem. Cell Biol. 2001; 33: 637-668Crossref PubMed Scopus (1308) Google Scholar). Calgranulin C binding to RAGE mediates the activation of endothelial cells, macrophages, and lymphocytes, cells central to the inflammatory response. By using specific inhibitors of different signaling pathways, it was found that phospholipase C, protein kinase C, Ca2+ fluxes, calmodulin-dependent kinase II, and mitogen-activated protein kinase/ERK kinase (MEK) are required for extracellular calgranulin C to initiate signal transduction through RAGE (25Donato R. Int. J. Biochem. Cell Biol. 2001; 33: 637-668Crossref PubMed Scopus (1308) Google Scholar). Besides S100 family proteins, a small protein shown to be involved in tumor growth, HMGB1, was also identified as a RAGE ligand (26Hori O. Brett J. Slattery T. Cao R. Zhang J. Chen J.X. Nagashima M. Lundh E.R. Vijay S. Nitecki D. Morser J. Stern D. Schmidt A.M. J. Biol. Chem. 1995; 270: 25752-25761Abstract Full Text Full Text PDF PubMed Scopus (1011) Google Scholar).Despite extensive in vivo studies of the functional role of RAGE, little is known about the structural organization of the receptor molecule and its interaction with physiological ligands. Biochemical characterization of RAGE-ligand interactions was limited to in vitro binding studies, which revealed that the N-terminal V domain of RAGE is involved in binding AGEs (27Schmidt A.M. Yan S.D. Stern D.M. Circulation. 1997; : 37PubMed Google Scholar). It was also shown that the binding of AGEs to RAGE inhibited calgranulin C binding to RAGE (15Hofmann M.A. Drury S. Fu C. Qu W. Taguchi A. Lu Y. Avila C. Kambham N. Bierhaus A. Nawroth P. Neurath M.F. Slattery T. Beach D. McClary J. Nagashima M. Morser J. Stern D. Schmidt A.M. Cell. 1999; 97: 889-901Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar).Here we characterized the interaction between calgranulin C and sRAGE using native gel electrophoresis, chromatography, fluorescence and high resolution NMR spectroscopy. We showed that hexameric Ca2+-calgranulin C binding to sRAGE is mediated by the first C-type immunoglobulin-type domain of tetrameric RAGE, C1. Interaction surface mapping studies reveal that the apo-form of calgranulin C also binds to RAGE with comparatively low affinity, using a surface that is substantially different from the surface participating in the high affinity Ca2+-calgranulin C-RAGE interaction. These calciumdependent interactions substantiate the idea that Ca2+ binding is the trigger for a molecular switch that transmits structural rearrangements through the trans-membrane domain to the cytosolic tail of RAGE leading to signal transduction through the RAGE receptor.EXPERIMENTAL PROCEDURESPlasmid Construction—DNA coding for human sRAGE (amino acids 24-336), V domain (amino acids 24-129), C1 domain (amino acids 130-234), C2 domain (amino acids 235-336), VC1 domains (amino acids 24-234), and C1C2 domains (amino acids 130-336) was PCR-amplified from the human cDNA library (GenBank™ accession number BC020669) using Taq polymerase and oligonucleotides containing flanking 5′-NdeI and 3′-SalI restriction sites. DNA coding for full length human HMGB1 was PCR-amplified from the human cDNA library (GenBank™ accession number BC066889) using Taq polymerase and oligonucleotides containing flanking 5′-NcoI and 3′-SalI restriction sites. The restriction-digested PCR products of RAGE were ligated into the NdeI and SalI sites of expression vector pET28a (Novagen), and the restriction digested PCR product of HMGB1 was ligated into the NcoI and SalI sites of expression vector pRSF-1b (Novagen). Both plasmids confer kanamycin resistance. The resulting plasmids pET28sRAGE, pET28V, pET28C1, pET28C2, pET28VC1, and pET28C1C2 express cleavable, C-terminal His-tagged proteins. pRSF-HMGB1 plasmid expresses full-length human HMGB1 without additional tags.Expression and Purification of sRAGE and RAGE Domains—Plasmids expressing sRAGE or RAGE domains were transformed into Escherichia coli strain BL21(DE3) Codon+ (Novagen) for overexpression and purification. Cells were grown to ∼0.7 A600 at 37 °C in LB containing 35 mg/liter kanamycin, induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside, and grown overnight. Cells were harvested and resuspended in 50 mm Hepes-NaOH buffer (pH 7.0) containing 8 m urea and heat-lysed at 95 °C for 10 min. The lysate was centrifuged, and the supernatant loaded onto a His tag column. The column was washed with 50 mm Hepes buffer (pH 7.0), and the protein was allowed to renature on the column prior to eluting with 50 mm Hepes-NaOH (pH 7.0) containing 500 mm imidazole. Fractions containing the eluted protein were pooled, dialyzed into 10 mm Hepes-NaOH (pH 6.5), 100 mm NaCl, concentrated to 0.2-0.5 mm, and loaded onto a Sephadex 200 size exclusion column (Amersham Biosciences) equilibrated with the same buffer. Fractions containing the eluted protein were pooled and concentrated to 0.3-1.0 mm using centricones (Millipore). All concentrations cited for sRAGE and its domains refer to total monomer concentration. Purity was estimated to be >90% by Coomassie-stained SDS-PAGE.Expression and Purification of HMGB1—Plasmid expressing full-length human HMGB1, pRSF-HMGB1, was transformed into E. coli strain BL21(DE3) Codon+ (Novagen) for overexpression and purification. Cells were grown to ∼0.7 A600 at 37 °C in LB containing 35 mg/liter kanamycin and induced for 3 h with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside. Cells were harvested and resuspended in 50 mm Tris-NaOH buffer (pH 8.0), 500 mm NaCl, 20 mm EDTA, 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride and sonicated on ice for 10 min. The lysate was centrifuged, and the supernatant was loaded onto a DEAE-cellulose column (Sigma) equilibrated in the same buffer. The flow-through fraction of supernatant was collected and mixed with solid ammonium sulfate (50% of maximum solubility at 0 °C). The supernatant was dialyzed against 20 mm Tris-NaOH (pH 8.0) and loaded on a Q-Sepharose column (Amersham Biosciences) equilibrated with the same buffer. Protein was eluted at 1 ml/min with a linear gradient of 20 mm Tris/HCl (pH 8.0) to 20 mm Tris/HCl (pH 8.0), 1 m NaCl over 30 min. Fractions containing the eluted protein were pooled, dialyzed into NMR buffer (10 mm Hepes-NaOH (pH 6.5), 100 mm NaCl, 0.02% (w/v) NaN3), and concentrated to 0.3-1.0 mm using centricones (Millipore). Purity was estimated to be >90% by Coomassie-stained SDS-PAGE.Labeling, Expression, and Purification of Calgranulin C—Human calgranulin C (S100A12; amino acids 1-92) was expressed using pQE60calC (14Moroz O.V. Antson A.A. Murshudov G.N. Maitland N.J. Dodson G.G. Wilson K.S. Skibshoj I. Lukanidin E.M. Bronstein I.B. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 20-29Crossref PubMed Scopus (71) Google Scholar); this construct contains four additional amino acids (MGGS) at the N terminus. To uniformly label calgranulin C for NMR spectroscopy, pQE60calC was transformed into E. coli strain BL21(DE3) Codon+ (Novagen). For U-15N-labeling, cells were grown at 37 °C in minimal medium (M9) containing 35 mg/liter kanamycin and 1 g/liter [15N]ammonium chloride as the sole nitrogen source. For U-15N,13C-labeling, cells were grown at 37 °C in M9 medium containing 35 mg/liter kanamycin, 1 g/liter [15N]ammonium chloride, and 0.5 g/liter [13C]glucose instead of unlabeled glucose as the sole carbon source. Cells were grown to ∼0.6 A600, induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside, grown for 15 h, harvested, and resuspended in 50 mm Tris/HCl (pH 7.5), 300 mm NaCl, prior to lysis. The supernatant was diluted 1:1 with low salt buffer (50 mm Tris/HCl (pH 7.5), 10 mm CaCl2) and clarified prior to loading onto a phenyl-FF column (Amersham Biosciences). Protein was eluted at 1 ml/min with a linear gradient of 50 mm Tris/HCl (pH 7.5), 150 mm NaCl, 5 mm CaCl2 to 50 mm Tris/HCl (pH 7.5), 100 mm NaCl, 5 mm EDTA over 30 min. Fractions containing the eluted protein were pooled and dialyzed into 20 mm Tris/HCl (pH 8.0). The dialyzed protein was loaded onto an uno_Q1 column (Bio-Rad) and eluted at 2 ml/min with a linear gradient of 20 mm Tris/HCl (pH 8.0) to 20 mm Tris/HCl, 200 mm NaCl (pH 8.0) over 30 min. Fractions containing the eluted protein were pooled, dialyzed into NMR buffer, and concentrated to a final concentration of 0.3-0.5 mm using centricones. All concentrations cited for calgranulin C refer to total monomer concentration. Purity was estimated to be >95% by Coomassie-stained PAGE.Protein Pulldown Assay—10 μ l of 10 μm sRAGE in binding buffer (10 mm Hepes-NaOH (pH 6.5), 100 mm NaCl) was mixed with 10 μl of 20 μm HMGB1 in the same buffer. For calgranulin C, 10 μl of 10 μm sRAGE in 10 mm Hepes-NaOH (pH 6.5), 100 mm NaCl, 5 mm CaCl2, and 50 mm imidazole were mixed with 10 μl of 20 μm Ca2+-calgranulin C in the same buffer. 50 mm imidazole was included in the binding buffer to avoid weak interactions between Ca2+-calgranulin C and Ni2+ beads. After incubating for 10 min, His tag beads (Amersham Biosciences) equilibrated in the binding buffer were added into the protein solution. The beads were washed three times with the binding buffer. The protein absorbed on the His tag beads was analyzed using Coomassie-stained SDS-PAGE.Fluorescence Titrations—Fluorescence titrations were performed using an LS-55 luminescence spectrometer (PerkinElmer Life Sciences). All measurements were made at 25 °C using a 1 × 1-cm quartz cell. The excitation wavelengths were 255 nm for tyrosine fluorescence and 365 or 370 nm for TNS fluorescence; excitation and emission slit widths were 5 and 10 nm, respectively. The fluorescence intensity (F) at a fixed wavelength was corrected for sample dilution upon addition of titrant. Fluorescence data were analyzed using GRAMS (Thermo Galactic) and SPSS (Lead Technologies, Inc.) software.Ca2+ binding to calgranulin C was measured using two fluorescence probes. For tyrosine fluorescence experiments, a 2.5 μm solution of calgranulin C in 10 mm Hepes-NaOH (pH 7.4) was titrated with a stock solution of CaCl2 (2 mm in distilled water); for TNS fluorescence experiments, a solution containing 2.5 μm calgranulin C and 20 μm TNS in 10 mm Hepes-NaOH (pH 7.4) was titrated with the stock solution of CaCl2. Corrected fluorescence intensity was fit to Equation 1 to estimate the apparent Ca2+ binding constant, K, for both tyrosine and TNS fluorescence titrations, F=f1N-W-1K+W-N-+1K2+4NK2N+f2N+W+1K-W-N-+1K2+4NK2N(Eq. 1) where f1 is the fluorescence at zero Ca2+ concentration; f2isthe fluorescence of the Ca2+-calgranulin C complex; N is total protein concentration, and W is the total Ca2+ concentration.C1C2 binding to calgranulin C was measured by performing TNS fluorescence titrations. A solution containing 3 μm calgranulin C and 10 μm TNS in 10 mm Hepes-NaOH (pH 6.8) plus 400 μm CaCl2 was titrated with a stock solution of C1C2 (44 μm in 10 mm Hepes-NaOH (pH 6.8)). Corrected fluorescence intensity was fit to Equation 1 to estimate the apparent C1C2-binding constant K. In this case f1 is the TNS fluorescence at zero C1C2 concentration; f2 is the TNS fluorescence of the [Ca2+-calgranulin C]-C1C2 complex; W is the concentration of C1C2, and N is the concentration of Ca2+-calgranulin C.Gel Filtration—Calgranulin C and sRAGE were equilibrated in 10 mm Hepes-NaOH (pH 8.3), 100 mm NaCl, and 2 mm CaCl2. 50-μl samples of calgranulin C (200 μm) and sRAGE (500 μm) were loaded onto a Superdex™ 200 10/300 GL gel filtration column (Amersham Biosciences), pre-equilibrated with the same buffer, and eluted at a flow rate of 0.5 ml/min. The column was calibrated using gel filtration standards (Bio-Rad), which contain bovine thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa), and vitamin B12 (1,350 Da). The axial ratio of Ca2+-calgranulin C was calculated based on the crystallographic coordinates (14Moroz O.V. Antson A.A. Murshudov G.N. Maitland N.J. Dodson G.G. Wilson K.S. Skibshoj I. Lukanidin E.M. Bronstein I.B. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 20-29Crossref PubMed Scopus (71) Google Scholar) using the HYDRO software package (28Garcia de la Torre J. Navarro S. Lopez Martinez M.C. Diaz F.G. Lopez Cascales J.J. Biophys. J. 1994; 67: 530-531Abstract Full Text PDF PubMed Scopus (276) Google Scholar).Native PAGE—Native gels containing 12, 10.5, and 9% acrylamide were prepared in 0.375 m Tris/HCl (pH 8.8) (29Bollag D. Rozycki M. Edelstein S. Protein Methods. 2nd Ed. Wiley-Liss, Inc., New York1996: 155-173Google Scholar). All samples were prepared in 10 mm Hepes-NaOH (pH 6.5), 100 mm NaCl, 4 mm CaCl2, and 5% glycerol. Concentrations were 200 μm for calgranulin C and 20 μm for sRAGE. Binding reactions containing 20 μm sRAGE and 5, 10, 15, and 20:1 mole ratios of calgranulin C were allowed to equilibrate for 30 min at 4 °C prior to loading on pre-heated (20 min, 200 V) gels. The gels were loaded under full voltage (200 V) and run at 4 °C for 3 h at 200 V using standard Tris-glycine running buffer (pH 8.3) supplemented with 4 mm CaCl2. Gels were stained using Imperial protein stain (Pierce). The data were processed according to Ref. 29Bollag D. Rozycki M. Edelstein S. Protein Methods. 2nd Ed. Wiley-Liss, Inc., New York1996: 155-173Google Scholar. Molecular weight standards used were the same as those used in the column chromatography experiments. All experiments were repeated three times.NMR Experiments—NMR experiments were performed on a Bruker Avance spectrometer, operating at a 1H frequency of 700 MHz and equipped with a cryoprobe. All NMR data were collected at 25 °C. Protein samples of calgranulin C, with concentrations ranging from 0.3 to 0.5 mm, were dissolved in NMR buffer (10 mm Hepes-NaOH (pH 6.5), 100 mm NaCl, 0.02% (w/v) NaN3). The solution conditions used in these experiments are comparable with those expected to be found in vivo. To prepare Ca2+-calgranulin C, a solution of 1 m CaCl2 was titrated into a 0.5 mm [U-13C,15N]calgranulin C solution until the molar ratio of metal:protein was 6:1. No changes in the NMR spectrum of the protein were detected at higher molar ratios. Gradient diffusion experiments to obtain rotational correlation times were performed using the pulse sequence described previously (30Ferrage F. Zoonens M. Warschawski D.E. Popot J.L. Bodenhausen G. J. Am. Chem. Soc. 2003; 125: 2541-2545Crossref PubMed Scopus (69) Google Scholar). To perform the titration experiments with sRAGE, a solution of 0.5 mm [U-15N]calgranulin C or [U-15N]Ca2+-calgranulin C was titrated into 0.2 mm sRAGE in three steps to yield sRAGE to calgranulin C molar ratios of 1:1, 1:6, and 1:10, respectively. Titration experiments with C1C2, V domain, and C2 domain of sRAGE were performed by titrating 1mm solutions of C1C2, V domain, and C2 domain into 0.5 mm [U-15N]calgranulin C or [U-15N]Ca2+-calgranulin C solutions. The results of the titration were monitored by 1H{15N} HSQC. Over the course of titration, the signal to noise ratio of the peaks that did not show any changes was kept constant by adjusting the number of scans. The [U-15N]calgranulin C sample did not show any changes in the 1H{15N} HSQC spectra upon 3- and 10-fold dilutions. The 1H{15N} HSQC spectrum of [U-15N]Ca2+-calgranulin C sample showed a slight decrease in line broadening upon 10-fold dilution. The HNCACB, CBCA-(CO)NH, HNCO, HNCACO, and 15N-edited nuclear Overhauser effect spectroscopy experiments were collected using previously described sequences. All spectra were processed using TOPSPIN (Bruker, Inc), and assignments were made using CARA (31Masse J.E. Keller R. J. Magn. Reson. 2005; 174: 133-151Crossref PubMed Scopus (74) Google Scholar).Titrating C1C2 into apo-calgranulin C and Ca2+-calgranulin C results in free calgranulin C and a large, 200-kDa, complex in an equilibrium characterized by the lifetimes of two states. Because the affinity of apo-calgranulin C to C1C2 is low (Kd ∼140 μm), we expect the fast exchange regime, koff ≫

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