Structural Basis for Innate Immune Sensing by M-ficolin and Its Control by a pH-dependent Conformational Switch

Abstract

Ficolins are soluble oligomeric proteins with lectin-like activity, assembled from collagen fibers prolonged by fibrinogen-like recognition domains. They act as innate immune sensors by recognizing conserved molecular markers exposed on microbial surfaces and thereby triggering effector mechanisms such as enhanced phagocytosis and inflammation. In humans, L- and H-ficolins have been characterized in plasma, whereas a third species, M-ficolin, is secreted by monocytes and macrophages. To decipher the molecular mechanisms underlying their recognition properties, we previously solved the structures of the recognition domains of L- and H-ficolins, in complex with various model ligands (Garlatti, V., Belloy, N., Martin, L., Lacroix, M., Matsushita, M., Endo, Y., Fujita, T., Fontecilla-Camps, J. C., Arlaud, G. J., Thielens, N. M., and Gaboriaud, C. (2007) EMBO J. 24, 623–633). We now report the ligand-bound crystal structures of the recognition domain of M-ficolin, determined at high resolution (1.75–1.8 Å), which provides the first structural insights into its binding properties. Interaction with acetylated carbohydrates differs from the one previously described for L-ficolin. This study also reveals the structural determinants for binding to sialylated compounds, a property restricted to human M-ficolin and its mouse counterpart, ficolin B. Finally, comparison between the ligand-bound structures obtained at neutral pH and nonbinding conformations observed at pH 5.6 reveals how the ligand binding site is dislocated at acidic pH. This means that the binding function of M-ficolin is subject to a pH-sensitive conformational switch. Considering that the homologous ficolin B is found in the lysosomes of activated macrophages (Runza, V. L., Hehlgans, T., Echtenacher, B., Zahringer, U., Schwaeble, W. J., and Mannel, D. N. (2006) J. Endotoxin Res. 12, 120–126), we propose that this switch could play a physiological role in such acidic compartments. Ficolins are soluble oligomeric proteins with lectin-like activity, assembled from collagen fibers prolonged by fibrinogen-like recognition domains. They act as innate immune sensors by recognizing conserved molecular markers exposed on microbial surfaces and thereby triggering effector mechanisms such as enhanced phagocytosis and inflammation. In humans, L- and H-ficolins have been characterized in plasma, whereas a third species, M-ficolin, is secreted by monocytes and macrophages. To decipher the molecular mechanisms underlying their recognition properties, we previously solved the structures of the recognition domains of L- and H-ficolins, in complex with various model ligands (Garlatti, V., Belloy, N., Martin, L., Lacroix, M., Matsushita, M., Endo, Y., Fujita, T., Fontecilla-Camps, J. C., Arlaud, G. J., Thielens, N. M., and Gaboriaud, C. (2007) EMBO J. 24, 623–633). We now report the ligand-bound crystal structures of the recognition domain of M-ficolin, determined at high resolution (1.75–1.8 Å), which provides the first structural insights into its binding properties. Interaction with acetylated carbohydrates differs from the one previously described for L-ficolin. This study also reveals the structural determinants for binding to sialylated compounds, a property restricted to human M-ficolin and its mouse counterpart, ficolin B. Finally, comparison between the ligand-bound structures obtained at neutral pH and nonbinding conformations observed at pH 5.6 reveals how the ligand binding site is dislocated at acidic pH. This means that the binding function of M-ficolin is subject to a pH-sensitive conformational switch. Considering that the homologous ficolin B is found in the lysosomes of activated macrophages (Runza, V. L., Hehlgans, T., Echtenacher, B., Zahringer, U., Schwaeble, W. J., and Mannel, D. N. (2006) J. Endotoxin Res. 12, 120–126), we propose that this switch could play a physiological role in such acidic compartments. To protect themselves against infection, multicellular organisms have acquired innate immunity systems that rely upon the ability of a restricted pool of recognition molecules to sense conserved molecular patterns exposed at the surface of microbes and to elicit effector mechanisms designed to provide a first line of defense (1Janeway C.A. Immunol. Today. 1992; 13: 11-16Abstract Full Text PDF PubMed Scopus (1034) Google Scholar, 2Hoffmann J.A. Kafatos F.C. Janeway C.A. Ezekowitz R.A.B. Science. 1999; 284: 1313-1318Crossref PubMed Scopus (2152) Google Scholar). Among these molecules are the ficolins, a family of proteins found in a variety of animals ranging from invertebrates to mammals (3Fujita T. Matsushita M. Endo Y. Immunol. Rev. 2004; 198: 185-202Crossref PubMed Scopus (507) Google Scholar, 4Endo Y. Liu Y. Fujita T. Adv. Exp. Med. Biol. 2006; 586: 265-279Crossref PubMed Scopus (19) Google Scholar). Ficolins are oligomers of trimeric subunits, which are made of three identical polypeptide chains, comprising collagen-like triple helices prolonged by a globular recognition domain structurally related to the fibrinogen β and γ chains (5Matsushita M. Fujita T. Immunol. Rev. 2001; 180: 78-85Crossref PubMed Scopus (167) Google Scholar). Three ficolins have been identified in humans: L-ficolin and H-ficolin, which are both serum proteins, and M-ficolin, a secretory protein synthesized in bone marrow, lung, and spleen and by blood monocytes and neutrophils (6Endo Y. Matsushita M. Fujita T. Immunobiology. 2007; 212: 371-379Crossref PubMed Scopus (109) Google Scholar). L-ficolin is known to recognize various capsulated bacteria and exhibits binding specificity for diverse ligands, such as lipoteichoic acid (7Lynch N.J. Roscher S. Hartung T. Morath S. Matsushita M. Maennel D.N. Kuraya M. Fujita T. Schwaeble W.J. J. Immunol. 2004; 172: 1198-1202Crossref PubMed Scopus (221) Google Scholar), 1,3-β-d-glucan (8Ma Y.G. Cho M.Y. Zhao M. Park J.W. Matsushita M. Fujita T. Lee B.L. J. Biol. Chem. 2004; 279: 25307-25312Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), and the capsular antigen of type III group B streptococci (9Aoyagi Y. Adderson E.E. Min J.G. Matsushita M. Fujita T. Takahashi S. Okuwaki Y. Bohnsack J.F. J. Immunol. 2005; 174: 418-425Crossref PubMed Scopus (73) Google Scholar). H-ficolin has only been reported to bind to Aerococcus viridans (10Krarup A. Sørensen U.B. Matsushita M. Jensenius J.C. Thiel S. Infect. Immun. 2005; 73: 1052-1060Crossref PubMed Scopus (164) Google Scholar). In addition to pathogenic microorganisms, L-ficolin binds specifically to apoptotic HL60, U937, and Jurkat T cells, whereas binding of H-ficolin is restricted to apoptotic Jurkat T cells (11Kuraya M. Ming Z. Liu X. Matsushita M. Fujita T. Immunobiology. 2005; 209: 689-697Crossref PubMed Scopus (123) Google Scholar, 12Honoré C. Hummelshoj T. Hansen B.E. Madsen H.O. Eggleton P. Garred P. Arthritis Rheum. 2007; 56: 1598-1607Crossref PubMed Scopus (108) Google Scholar). The structures of the recognition domains of human L- and H-ficolins, alone and in complex with various ligands, have been solved by x-ray crystallography (13Garlatti V. Belloy N. Martin L. Lacroix M. Matsushita M. Endo Y. Fujita T. Fontecilla-Camps J.C. Arlaud G.J. Thielens N.M. Gaboriaud C. EMBO J. 2007; 24: 623-633Crossref Scopus (147) Google Scholar), revealing the structural determinants for their binding specificities. In addition to an outer S1 binding site, homologous to a site identified in the invertebrate tachylectin 5A (TL5A) 2The abbreviations used are: TL5A, tachylectin 5A; Neu5Ac, N-acetylneuraminic acid; r.m.s., root mean square; Mes, 4-morpholineethanesulfonic acid. (14Kairies N. Beisel H.G. Fuentes-Prior P. Tsuda R. Muta T. Iwanaga S. Bode W. Huber R. Kawabata S.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13519-13524Crossref PubMed Scopus (121) Google Scholar), three additional sites, called S2, S3, and S4, were discovered in L-ficolin. Together, these new sites define a continuous recognition surface able to sense various acetylated and neutral carbohydrate markers in the context of extended polysaccharides, as found on microbial or apoptotic surfaces (13Garlatti V. Belloy N. Martin L. Lacroix M. Matsushita M. Endo Y. Fujita T. Fontecilla-Camps J.C. Arlaud G.J. Thielens N.M. Gaboriaud C. EMBO J. 2007; 24: 623-633Crossref Scopus (147) Google Scholar). Recombinant M-ficolin shows a marked preference for acetylated compounds, as also observed for L-ficolin (15Frederiksen P.D. Thiel S. Larsen C.B. Jensenius J.C. Scand. J. Immunol. 2005; 62: 462-473Crossref PubMed Scopus (94) Google Scholar) and binds neoglycoproteins bearing GlcNAc, GalNAc, and sialyl-N-acetyllactosamine (16Liu Y. Endo Y. Iwaki D. Nakata M. Matsushita M. Wada I. Inoue K. Munakata M. Fujita T. J. Immunol. 2005; 175: 3150-3156Crossref PubMed Scopus (208) Google Scholar). Binding to the smooth type LT2 strain of Salmonella typhimurium and to Streptococcus aureus has been reported, but only binding to the latter could be inhibited by GlcNAc (17Teh C. Le Y. Lee S.H. Lu J. Immunology. 2000; 101: 225-232Crossref PubMed Scopus (138) Google Scholar). The structure of the recognition domain of human M-ficolin was recently reported, but this turned out to be in a conformation devoid of ligand binding activity (18Tanio M. Kondo S. Sugio S. Kohno T. J. Biol. Chem. 2007; 282: 3889-3895Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Here we report five novel x-ray structures of this domain, namely a ligand-free and three ligand-bound structures obtained at pH 7.0 plus an inactive form obtained at pH 5.6. For the first time, these provide the structural basis for the recognition function of M-ficolin and reveal how it is subject to a pH-dependent conformational switch. Recombinant Protein Production and Purification—The DNA segment encoding the C-terminal residues 80–297 of mature human M-ficolin was amplified using VentR polymerase and the pMT/Bip/V5-HisA plasmid containing the full-length cDNA (16Liu Y. Endo Y. Iwaki D. Nakata M. Matsushita M. Wada I. Inoue K. Munakata M. Fujita T. J. Immunol. 2005; 175: 3150-3156Crossref PubMed Scopus (208) Google Scholar) as a template, according to established procedures. This segment starts at the first residue following the collagen-like sequence. The DNA was cloned in frame with the melittin signal peptide of the pNT-Bac baculovirus transfer vector (19Rossi V. Bally I. Thielens N.M. Esser A.F. Arlaud G.J. J. Biol. Chem. 1998; 273: 1232-1239Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), and the recombinant baculovirus was generated using the Bac-to-Bac™ system (Invitrogen Corp.) and amplified as described previously (20Thielens N.M. Cseh S. Thiel S. Vorup-Jensen T. Rossi V. Jensenius J.C. Arlaud G.J. J. Immunol. 2001; 166: 5068-5077Crossref PubMed Scopus (114) Google Scholar). High Five cells were infected with the recombinant virus for 96 h at 27 °C. The protein was purified from culture supernatants by ion exchange chromatography on a Q-Sepharose Fast Flow column (GE Healthcare) equilibrated in 50 mm triethanolamine-HCl, pH 7.6, using a linear gradient to 250 mm NaCl. Mass spectrometry analysis was performed using the matrix-assisted laser desorption ionization technique under conditions described previously (21Teillet F. Dublet B. Andrieu J.P. Gaboriaud C. Arlaud G.J. Thielens N.M. J. Immunol. 2005; 174: 2870-2877Crossref PubMed Scopus (109) Google Scholar). Crystallization, Structure Determination, and Refinement—The protein was concentrated to 6 mg/ml in 145 mm NaCl, 50 mm triethanolamine-HCl, pH 7.6. Several crystallization hits were obtained using the high throughput crystallization facility at EMBL, Grenoble. Crystals were reproducibly obtained at 20 °C using the hanging by of the protein and of a of pH or of Mes, pH 5.6. were obtained by obtained at pH 7.0 in a of and mm ligand GalNAc, or the crystal in was performed at or as in were using from Biol. PubMed Scopus Google Scholar) or W. J. Scopus Google Scholar). are in The ligand-free M-ficolin structures obtained at pH 7.0 and 5.6 were solved by molecular using the L-ficolin structure as a was performed using the P. K. Biol. 2004; PubMed Scopus Google Scholar). were out with Biol. Scopus Google Scholar). The of the of but the first residue of the recombinant at pH The of M-ficolin exhibits various conformations on crystal In the structure obtained at pH the segment in The and structure have been in the Protein and to the resolution are in to the resolution are in to the resolution are in to the resolution are in to the resolution are in to the resolution are in to the resolution are in to the resolution are in in a new the structures solved and their to the resolution are in to the resolution are in B to the resolution are in additional to the resolution are in additional in a new In to the structure of the fibrinogen-like recognition domain of human M-ficolin, the segment to this domain 80–297 of mature was in a cells by a with a of was for the polypeptide of the crystallization hits obtained at pH to to the five x-ray structures The and at ligand-free structure obtained at pH 7.0 was solved by molecular using L-ficolin (13Garlatti V. Belloy N. Martin L. Lacroix M. Matsushita M. Endo Y. Fujita T. Fontecilla-Camps J.C. Arlaud G.J. Thielens N.M. Gaboriaud C. EMBO J. 2007; 24: 623-633Crossref Scopus (147) Google Scholar) as a model and to resolution The protein is with from the acid of the in ficolins, this is to previously described for the L- and H-ficolin recognition domains (13Garlatti V. Belloy N. Martin L. Lacroix M. Matsushita M. Endo Y. Fujita T. Fontecilla-Camps J.C. Arlaud G.J. Thielens N.M. Gaboriaud C. EMBO J. 2007; 24: 623-633Crossref Scopus (147) Google Scholar). homologous binding sites are found in the of the with a of between the as observed in L-ficolin and in M-ficolin both of one of the of and the of and M-ficolin and L-ficolin have with an of for structural are observed for the and at of the structure is the of the peptide observed in and as as in the homologous invertebrate (14Kairies N. Beisel H.G. Fuentes-Prior P. Tsuda R. Muta T. Iwanaga S. Bode W. Huber R. Kawabata S.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13519-13524Crossref PubMed Scopus (121) Google Scholar). This is in with the in the M-ficolin structure recently reported by (18Tanio M. Kondo S. Sugio S. Kohno T. J. Biol. Chem. 2007; 282: 3889-3895Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), which is devoid of ligand binding In to structural insights into the binding specificity of M-ficolin, the obtained at neutral pH were into Three structures of M-ficolin in complex with GlcNAc, GalNAc, and were solved and to resolution of these three ligands was to the S1 site, in the of the binding sites are in the domain which the of the structure The ligand-free and ligand-bound structures obtained at neutral pH are with a mean of This novel M-ficolin structural with ligand recognition be to as the of the three ligands with site S1 observed at pH 7.0 are in of three the ligand its group is in with the by and its is to the group of and its is to the group of This latter is by a in the of GlcNAc, whereas the the ligand to provide a in the of and is the only of the binding site in S1 is homologous to the GlcNAc binding site of the related invertebrate (14Kairies N. Beisel H.G. Fuentes-Prior P. Tsuda R. Muta T. Iwanaga S. Bode W. Huber R. Kawabata S.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13519-13524Crossref PubMed Scopus (121) Google Scholar). the and the of the peptide in are the latter to the for with the are also conserved in ficolins, for a in human H-ficolin the of by a in L-ficolin could the of binding of ligands in its S1 site, an is (13Garlatti V. Belloy N. Martin L. Lacroix M. Matsushita M. Endo Y. Fujita T. Fontecilla-Camps J.C. Arlaud G.J. Thielens N.M. Gaboriaud C. EMBO J. 2007; 24: 623-633Crossref Scopus (147) Google Scholar). of the three carbohydrates by M-ficolin also additional with their but on the of the In the of GlcNAc, the is to the group of This differs from the recognition of GlcNAc by TL5A, the with the of and with the group of are observed in the of GalNAc, with a between and the of and a between and the group of of structure of the complex reveals a of to recognize this The group is the to the group of is by and between the and the and of as by the of the and L-ficolin structures ficolins with residues in the of the S1 site, and in M-ficolin, are in L-ficolin by the and residues the of the binding thereby its to carbohydrate molecules such as of ficolins for mouse ficolin B and human M-ficolin, both are by residues The S1 Binding and at was recently reported that the GlcNAc binding activity of M-ficolin is and the structure of its recognition domain, obtained at pH was found to inactive conformations the S1 binding site (18Tanio M. Kondo S. Sugio S. Kohno T. J. Biol. Chem. 2007; 282: 3889-3895Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). have been a of the acidic pH of the crystallization or a to the of a C-terminal in the recombinant domain M. Kondo S. Sugio S. Kohno T. Biol. 2006; 62: PubMed Scopus Google Scholar). To this to the fibrinogen-like recognition domain of M-ficolin, was at pH and its structure was solved and to a resolution of this new crystal form differs from the one reported previously (18Tanio M. Kondo S. Sugio S. Kohno T. J. Biol. Chem. 2007; 282: 3889-3895Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), the structure is of Å), with from in the as in This additional M-ficolin structure also shows the peptide in a which the of the In addition to this conformational the acidic pH of and both for ligand binding and with the of residues S1 are at acidic in a conformation for ligand This acidic conformation is from the structures obtained at neutral with a mean of a that to only the is This acidic conformation be to as the The conformational from the binding to the nonbinding the of surface or namely and of the residues S1 are in and as is the for the specificity acid. The of these at acidic pH was by both the in the which to and the mean B in these according to the context and to in the structures determined at pH to the to the to the to the to the to the (18Tanio M. Kondo S. Sugio S. Kohno T. J. Biol. Chem. 2007; 282: 3889-3895Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) in a new analysis of the mean B in of the structures and its comparison with the are as B B B B B B B B B B B B B B B B B B B B (18Tanio M. Kondo S. Sugio S. Kohno T. J. Biol. Chem. 2007; 282: 3889-3895Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) in a new B for on pH are as in a new be in are found in and in the structures determined at pH 5.6. This a high of these at this which the that the with the neutral pH structure analysis of the of the mean B of with the mean B of the is in This is to as the is for in the M-ficolin structures means that the B is in the with the of the The and are observed for in the structure determined by (18Tanio M. Kondo S. Sugio S. Kohno T. J. Biol. Chem. 2007; 282: 3889-3895Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), a of under these This is in with the observed for at neutral pH in this is a To a of the pH-dependent in in the B for on the pH revealing a at pH 5.6 with 7.0 for We have previously solved the crystal structure of the fibrinogen-like recognition domains of human L- and H-ficolins (13Garlatti V. Belloy N. Martin L. Lacroix M. Matsushita M. Endo Y. Fujita T. Fontecilla-Camps J.C. Arlaud G.J. Thielens N.M. Gaboriaud C. EMBO J. 2007; 24: 623-633Crossref Scopus (147) Google Scholar) and now report the structure of the domain of This additional that this type of domain as a structure that is between the in the globular of protein C. J. A. Lacroix M. C. D. D. Fontecilla-Camps J.C. Arlaud G.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and the structure of the carbohydrate recognition domain of S. Ezekowitz Biol. PubMed Scopus Google Scholar). In to a the recognition domains of ficolins conserved and are in of The structures determined at neutral pH provide at the into the structural determinants in the recognition by M-ficolin of its three known ligands, GlcNAc, GalNAc, and of these three molecules binds to the outer site S1 homologous to that described in TL5A, and binding in a of with the ligand to that previously described for recognition of GlcNAc by (14Kairies N. Beisel H.G. Fuentes-Prior P. Tsuda R. Muta T. Iwanaga S. Bode W. Huber R. Kawabata S.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13519-13524Crossref PubMed Scopus (121) Google Scholar). it that M-ficolin has the binding of TL5A, with a specificity for a of This is in with which the ability to recognize acetylated molecules its S1 site but has acquired additional binding sites and that bind these in a (13Garlatti V. Belloy N. Martin L. Lacroix M. Matsushita M. Endo Y. Fujita T. Fontecilla-Camps J.C. Arlaud G.J. Thielens N.M. Gaboriaud C. EMBO J. 2007; 24: 623-633Crossref Scopus (147) Google Scholar). In line with (16Liu Y. Endo Y. Iwaki D. Nakata M. Matsushita M. Wada I. Inoue K. Munakata M. Fujita T. J. Immunol. 2005; 175: 3150-3156Crossref PubMed Scopus (208) Google Scholar), the structure of the complex between the M-ficolin recognition domain and that M-ficolin specifically this property is by the of and at in the of site of the acid The known ficolins, this structural is only by human M-ficolin and mouse ficolin B provides a the ability to recognize sialylated is restricted to these proteins, which are both secreted by the (16Liu Y. Endo Y. Iwaki D. Nakata M. Matsushita M. Wada I. Inoue K. Munakata M. Fujita T. J. Immunol. 2005; 175: 3150-3156Crossref PubMed Scopus (208) Google Scholar, Y. N. Liu Y. Iwaki D. Takahashi M. Fujita T. Nakata M. Matsushita M. 2005; PubMed Scopus Google Scholar). This be related to the whereas acid is found on such as the surface capsular of group B Streptococcus A.F. A. V. J. 2007; PubMed Scopus Google Scholar), it is also a of it is to that the of L- and H-ficolins to recognize acid is designed to recognition of cells by these proteins in In M-ficolin the ability to sense in compartments. acid is found at high at the surface of immune cells, with for a mm on B cells B.E. N. J.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: PubMed Scopus (133) Google Scholar), M-ficolin could also play a role in or immune a role established for recognition such as S.I. Y. Immunol. 2006; PubMed Scopus Google Scholar) or V. R. Y. Immunol. 2000; PubMed Scopus Google Scholar). of the pH-sensitive from this study is that the activity of M-ficolin is subject to a pH-dependent switch. The conformational from the binding to the nonbinding the of surface namely and with as a conserved a of was from the pH of the GlcNAc binding activity of M-ficolin, it has been that this is related to in the of of its residues (18Tanio M. Kondo S. Sugio S. Kohno T. J. Biol. Chem. 2007; 282: 3889-3895Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). comparison of the structures of the binding and nonbinding reveals in the by the chains of and In the binding is to the of and to the of to the of the comprising the In this is to the group of The third also the and are at acidic pH with an in these the of and the of the it the by the ligand in the structures of Finally, the the in the pH-sensitive conformational switch by this study only the the ligand binding site This that this switch that the mouse of M-ficolin was recently found in the lysosomes of activated macrophages T. B. U. Schwaeble W.J. D.N. J. Endotoxin Res. 2006; PubMed Scopus Google Scholar), it is to that this is in ligand the M-ficolin secreted upon bind to its and as a complex with its The pH in the lysosomes be to the conformational in the of the This be to to that described for such as the S. K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) or the family G. L. K. K. J. Science. PubMed Scopus Google Scholar, N. Sci. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). M-ficolin be or to be In the residues that are to play a role in the conformational switch could be to their We the at for in the of