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• W2012105542 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 drop vapor diffusion method by mixing equal volumes of the protein solution and of a reservoir solution composed either of 11% polyethylene glycol 4000, 5% isopropyl alcohol, 0.1 m Hepes, pH 7.0, or of 23% polyethylene glycol 4000, 0.32 m lithium sulfate, 0.1 m Mes, pH 5.6. M-ficolin-ligand complexes were obtained by soaking crystals obtained at pH 7.0 in a cryoprotecting solution composed of 11–14% polyethylene glycol 4000, 15% polyethylene glycol 400, 0.1 m Hepes, and 500 mm ligand (GlcNAc, GalNAc, or Neu5Ac) just before flash-cooling the crystal in liquid nitrogen. Data collection was performed at different ESRF beamlines (ID23eh2, ID14eh4, or ID14eh2), as stated in Table 1. Diffraction data were processed using either MOSFLM from CCP4 (22Collaborative Computational Project 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19762) Google Scholar) or XDS (23Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3230) Google Scholar). Complete crystallographic data statistics are provided in Table 1. The two ligand-free M-ficolin structures obtained at pH 7.0 and 5.6 were solved by molecular replacement, using the L-ficolin structure (Protein Data Bank code 2j1g) as a search model. Model rebuilding was performed using the graphic program Coot (24Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23349) Google Scholar). Refinements were carried out with Refmac5 (25Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 247-255Crossref Scopus (13865) Google Scholar). The quality of the map allowed construction of all but the first N-terminal residue of the recombinant fragment at pH 7.0. The N-terminal extremity of M-ficolin exhibits various conformations depending on crystal environment. In the structure obtained at pH 5.6, the segment 278–285 looks disordered in each molecule. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession codes 2jhm, 2jhk, 2jhi, 2jhl, 2jhh; see Tables 1 and 2).TABLE 1Crystallographic data collection statisticsProtein Data Bank codeESRF beamlineResolution rangeaData corresponding to the last resolution shell are indicated in parenthesesSpbSpace groupUnit cell dimensionsObserved reflectionsaData corresponding to the last resolution shell are indicated in parenthesesUnique reflectionsaData corresponding to the last resolution shell are indicated in parenthesesRed.aData corresponding to the last resolution shell are indicated in parentheses,cRedundancyComp.aData corresponding to the last resolution shell are indicated in parentheses,dCompletenessI/SigIaData corresponding to the last resolution shell are indicated in parenthesesRsymaData corresponding to the last resolution shell are indicated in parenthesesÅÅ%%2jhmID14-eh21.52–56.8 (1.52–1.60)H3a = b = 73.71, c = 124.6198,652 (9529)36,615 (4482)2.7 (2.1)94.3 (94.3)11.3 (3.0)7.4 (27.7)2jhkID23-eh21.75–23.7 (1.75–1.80)H3a = b = 73.75, c = 124.89146,501 (11,878)25,519 (2072)5.7 (5.72)99.3 (98.7)17.7 (4.2)6.3 (27.4)2jhiID23-eh21.80–28.3 (1.80–1.85)H3a = b = 73.97, c = 126.47135,776 (10,669)23,757 (1862)5.7 (5.72)99.3 (97.7)10.4 (3.0)9 (36.4)2jhlID14-eh41.75–28.0 (1.75–1.80)H3a = b = 73.93, c = 124.83137,933 (11,099)24,703 (1973)5.6 (5.6)98.9 (97.6)23.5 (4.9)4.6 (24.4)2jhhID23-eh21.70–28.0 (1.70–1.75)P3a = b = 69.40, c = 77.63258,896 (20,733)45,425 (3642)5.7 (5.7)98.7 (95.5)14.9 (3.9)7.3 (36.9)a Data corresponding to the last resolution shell are indicated in parenthesesb Space groupc Redundancyd Completeness Open table in a new tab TABLE 2Information about the structures solved and their crystallographic refinement statisticsPDB codepHLigandResolution high/lowaData corresponding to the last resolution shell are indicated in parenthesesRwork/RfreeaData corresponding to the last resolution shell are indicated in parenthesesr.m.s.deviation bond/angleMean B factor protein/ligandRamachandran FR/AAR/GAR/DRaData corresponding to the last resolution shell are indicated in parentheses,bFR, favored region; AAR, additional allowed region; GAR, generously allowed region; DR, disallowed regionÅ%Å/degreesÅ2%2jhm71.52–56.79 (1.52–1.56)18.1/20.7 (21.9/25.6)0.009/1.1911.784.5/15.5/0.0/0.02jhk7GlcNAc1.75–23.71 (1.75–1.81)17.3/18.6 (20.4/23.9)0.008/1.1721.0/42.185.0/15.0/0.0/0.02jhi7GalNAc1.80–28.35 (1.80–1.86)19.6/21.4 (23.8/31.7)0.01/1.2325.3/36.785.0/15.0/0.0/0.02jhl7Neu5Ac1.75–27.98 (1.75–1.81)21.0/23.9 (23.2/23.7)0.008/1.2427.4/36.084.5/15.5/0.0/0.02jhh5.61.70–28.03 (1.70–1.74)21.9/25.1 (26.0/30.0)0.009/1.2722.587.3/12.4/0.3/0.0a Data corresponding to the last resolution shell are indicated in parenthesesb FR, favored region; AAR, additional allowed region; GAR, generously allowed region; DR, disallowed region Open table in a new tab In order to determine the three-dimensional structure of the fibrinogen-like recognition domain of human M-ficolin, the segment corresponding to this domain (residues 80–297 of mature M-ficolin) was expressed in a baculovirus/insect cells system. As assessed by mass spectrometry, a single species with a mass of 24,549 ± 12 Da was purified, accounting for the unmodified polypeptide chain (calculated value 24,553 Da). Selection of the best diffracting crystals among several crystallization hits obtained at two different pH values allowed us to solve to the five x-ray structures presented here (Table 2). The Ligand-free and Ligand-bound Structures Solved at Neutral pH—The ligand-free structure obtained at pH 7.0 was solved by molecular replacement 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 starting model and refined to 1.5 Å resolution (Table 2). The protein is homotrimeric, with crystallographic 3-fold symmetry (Fig. 1A). As expected from the amino acid sequence conservation of the interprotomer interfaces in ficolins, this assembly is very similar to those 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). Likewise, homologous Ca2+ binding sites are found in the most external part of the trimer, with a distance of 65 Å between the Ca2+ ions, as observed in L-ficolin (Fig. 1, A and D). Ca2+ coordination in M-ficolin involves two water molecules, both carboxylate oxygens of Asp233, one of the side-chain oxygens of Asp235, and the main-chain carbonyl oxygens of Ser237 and Ser239. M-ficolin and L-ficolin have highly similar overall protomer structures, with an r.m.s. deviation value of 0.5 Å for 211 superposed Cα atoms (Fig. 1B). Only minor structural differences are observed (e.g. for the free N-terminal end and at position 170). A major functionally relevant feature of the structure is the cis-conformation of the Asp253–Cys254 peptide bond, already observed in H- and L-ficolins (Fig. 1C), as well as in the homologous invertebrate lectin TL5A (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 sharp contrast with the trans-conformation seen in the M-ficolin structure recently reported by Tanio et al. (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 activity. In order to gain structural insights into the binding specificity of M-ficolin, the crystals obtained at neutral pH were soaked into ligand-containing solutions. Three different structures of M-ficolin in complex with GlcNAc, GalNAc, and Neu5Ac were thus solved and refined up to 1.75 Å resolution (Table 2). Each of these three ligands was bound to the S1 site, in the vicinity of the Ca2+ binding site. These two sites are located in the P domain (residues 218–288), which forms the external part of the homotrimeric structure (Fig. 1). The ligand-free and ligand-bound structures obtained at neutral pH are very similar, with a mean overall r.m.s. deviation of 0.2 ± 0.03 Å. This novel M-ficolin structural conformation, endowed with ligand recognition ability, will be referred to as the “binding state.” An Evolutionarily Conserved N-Acetyl-binding Pocket—The detailed interactions of the three ligands with site S1 observed at pH 7.0 are depicted in Fig. 2 (A–C). A common set of three different interactions stabilizes the ligand acetamido group: (i) its methyl group is in Van der Waals contacts with the surrounding hydrophobic pocket formed by Phe245, His255, Tyr271, Ala272, and Tyr283; (ii) its carbonyl oxygen is hydrogen-bonded to the backbone NH group of Cys254 and His255; (iii) its nitrogen atom is hydrogen-bonded to the hydroxyl group of Tyr271. This latter interaction is mediated by a water molecule in the case of GlcNAc, whereas the Tyr271 side chain slightly moves toward the ligand to provide a direct hydrogen bond in the case of GalNAc and Neu5Ac. Tyr271 is thus the only flexible component of the binding site (Fig. 2D). As illustrated in Fig. 2E, S1 is highly homologous to the GlcNAc binding site of the distantly related invertebrate lectin TL5A (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). Both the hydrophobic pocket and the unusual cis-conformation of the Asp253-Cys254 peptide bond (Arg218-Cys219 in TL5A) are conserved, the latter being essential to correctly position the two consecutive backbone NH groups for appropriate interaction with the acetamido oxygen. These characteristics are also highly conserved in mammalian ficolins, except for a slightly different hydrophobic pocket in human H-ficolin (Fig. 1D). Interestingly, the replacement of Tyr271 by a phenylalanine in L-ficolin (Fig. 2F) could explain the lack of binding of N-acetylated ligands in its S1 site, where an acetate molecule is often bound instead (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). Recognition of the three N-acetylated carbohydrates by M-ficolin also involves additional hydrogen-bonding interactions with their sugar rings, but those supplemental interactions depend on the nature of the ligand. In the case of GlcNAc, the 1-OH oxygen is hydrogen-bonded to the backbone carbonyl group of His255 (Fig. 2B). This differs from the recognition of GlcNAc by TL5A, where the 1-OH oxygen forms hydrogen bonds with the guanidinium nitrogen of Arg186 and with the hydroxyl group of Tyr248 (Fig. 1E). More distant polar interactions are observed in the case of GalNAc, with a water-mediated hydrogen bond between 4-OH and the backbone oxygen of Asp253 and a direct interaction between 1-OH and the hydroxyl group of Tyr283 (Fig. 2A). Structural Basis of Sialic Acid Recognition—The structure of the M-ficolin-Neu5Ac complex reveals a more extensive network of polar interactions required to recognize this bulkier molecule (Fig. 2C). The neuraminic group is hydrogen-bonded through the 8-OH oxygen to the hydroxyl group of Tyr283 (Fig. 2C). Further stabilization is achieved by direct and water-mediated hydrogen bonds between the 7-OH oxygen and the backbone oxygen and nitrogen of Asp253, respectively. Interestingly, as illustrated by the superposition of the M- and L-ficolin structures (Fig. 2F), steric hindrance may explain why most ficolins do not interact with sialic acids. Indeed, two small residues in the vicinity of the S1 site, Gly221 and Ala256 in M-ficolin, are replaced in L-ficolin by the bulkier residues, phenylalanine and threonine, respectively. These two residues reduce the size of the binding pocket, thereby limiting its access to large carbohydrate molecules such as NeuNAc (Fig. 2F). Sequence alignments of mammalian ficolins show that, except for mouse ficolin B and human M-ficolin, both positions are occupied by bulkier residues (Fig. 1D). The S1 Binding Site Is Disrupted and Exhibits Increased Flexibility at Acidic pH—It was recently reported that the GlcNAc binding activity of M-ficolin is pH-sensitive, and the structure of its recognition domain, obtained at pH 5.6, was found to exhibit inactive loop conformations around 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). Such differences might have been either a direct consequence of the acidic pH of the crystallization solution or a possible artifact linked to the introduction of a 23-residue-long C-terminal tag in the recombinant domain (26Tanio M. Kondo S. Sugio S. Kohno T. Acta Crystallogr. F Struct. Biol. Crystalliz. Commun. 2006; 62: 652-655Crossref PubMed Scopus (9) Google Scholar). To investigate this question, our own construct, corresponding solely to the fibrinogen-like recognition domain of M-ficolin, was crystallized at pH 5.6, and its structure was solved and refined to a resolution of 1.7 Å (Table 2). Although 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 resulting structure is similar (mean subunit r.m.s. deviation value of 0.7 Å), with some differences mostly arising from changes in the ligand-binding region, as illustrated in Fig. 3B. This additional M-ficolin structure also clearly shows the Asp253-Cys254 peptide bond in a trans-conformation, which drastically modifies the positioning of the His255 side chain (Fig. 3B). In addition to this cis-trans conformational change, the acidic pH induces large displacements (>10 Å) of Tyr271 and Tyr283, both essential for ligand binding (Fig. 3, A and B). Thus, with the exception of Phe245, all residues making up S1 are extensively displaced at acidic pH, resulting in a conformation clearly inappropriate for ligand binding. This acidic conformation is significantly different from the structures obtained at neutral pH, with a mean subunit r.m.s. deviation of 2.14 ± 0.45 Å, a value that increases significantly to 3.36 ± 0.64 Å when only the ligand-binding region is considered. This acidic conformation will be therefore referred to as the “nonbinding” state. The conformational transition from the binding to the nonbinding state involves the concerted displacement of four surface segments or loops, namely L1 (218–224), L2 (253–258), L3 (264–274), and L4 (278–288). Except Phe245, all of the residues defining S1 are included in loops L2–L4, and L1 includes Gly221, which, as stated above, is probably the key determinant for the specificity toward sialic acid. The increased flexibility of these four loops at acidic pH was assessed by analyzing both the gaps in the crystallographic models, which correspond to disordered segments (Table 3), and the mean B factor in these loops according to the experimental context (Tables 4 and 5).TABLE 3Gaps corresponding to disordered segments in the structures determined at pH 5.6GapProtein Data Bank codeSubunitLoop277–2882JHHaPresent studyCL4277–2862JHHaPresent studyFL4262–268bResidue numbering corresponding to the present study2D39cTanio et al. (18)AL379–81bResidue numbering corresponding to the present study2D39cTanio et al. (18)B260–268bResidue numbering corresponding to the present study2D39cTanio et al. (18)BL3274–281bResidue numbering corresponding to the present study2D39cTanio et al. (18)BL479–82bResidue numbering corresponding to the present study2D39cTanio et al. (18)Ca Present studyb Residue numbering corresponding to the present studyc Tanio et al. (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) Open table in a new tab TABLE 4Detailed analysis of the mean B factor value in loops L1–L4 of the different structures and its comparison with the overall subunit value Loop segments are as follows: L1, 218–224; L2, 253–258; L3, 264–274; L4, 278–288.Protein Data Bank codeSubunitpHLigandMean B factor L1BLRaBLR, B loop ratio = (mean B factor loop – overall subunit B)/overall subunit B × 100 L1Mean B factor L2BLRaBLR, B loop ratio = (mean B factor loop – overall subunit B)/overall subunit B × 100 L2Mean B factor L3BLRaBLR, B loop ratio = (mean B factor loop – overall subunit B)/overall subunit B × 100 L3Mean B factor L4BLRaBLR, B loop ratio = (mean B factor loop – overall subunit B)/overall subunit B × 100 L4Overall subunit B factor2JHMbPresent studyF7.0None11.33–2.88.923.314.625.012.46.711.72JHKbPresent studyF7.0GlcNAc18.89–10.016.7–20.329.439.925.119.621.02JHIbPresent studyF7.0GalNAc24.4–3.620.120.530.420.227.69.125.32JHLbPresent studyF7.0Neu5Ac27.18–0.822.119.332.117.230.310.627.42JHHbPresent studyF5.6none24.79.331.941.233.648.927.320.722.62JHHbPresent studyC5.6None25.312.631.038.233.950.728.526.922.52D39cTanio et al. (18)A5.6None40.4127.021.219.129.867.830.269.817.82D39cTanio et al. (18)B5.6none41.296.835.167.634.363.828.837.520.92D39cTanio et al. (18)C5.6None19.4–12.237.669.632.345.628.829.822.2a BLR, B loop ratio = (mean B factor loop – overall subunit B)/overall subunit B × 100b Present studyc Tanio et al. (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) Open table in a new tab TABLE 5Averaged B loop ratio values for loops L1–L4 depending on pH Loop segments are as follows: L1, 218–2" @default.
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• W2012105542 title "Structural Basis for Innate Immune Sensing by M-ficolin and Its Control by a pH-dependent Conformational Switch" @default.
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