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• W2116742387 abstract "MAp19 is an alternative splicing product of the MASP-2 gene comprising the N-terminal CUB1-epidermal growth factor (EGF) segment of MASP-2, plus four additional residues at its C-terminal end. Like full-length MASP-2, it forms Ca2+-dependent complexes with mannan-binding lectin (MBL) and L-ficolin. The x-ray structure of human MAp19 was solved to a resolution of 2.5 Å. It shows a head to tail homodimer held together by interactions between the CUB1 module of one monomer and the EGF module of its counterpart. A Ca2+ ion bound to each EGF module stabilizes the dimer interfaces. A second Ca2+ ion is bound to the distal end of each CUB1 module, through six ligands contributed by Glu52, Asp60, Asp105, Ser107, Asn108, and a water molecule. Compared with its counterpart in human C1s, the N-terminal end of the MAp19 CUB1 module contains a 7-residue extension that forms additional inter-monomer contacts. To identify the residues involved in the interaction of MAp19 with MBL and L-ficolin, point mutants were generated and their binding ability was determined using surface plasmon resonance spectroscopy. Six mutations at Tyr59, Asp60, Glu83, Asp105, Tyr106, and Glu109 either strongly decreased or abolished interaction with both MBL and L-ficolin. These mutations map a common binding site for these proteins located at the distal end of each CUB1 module and stabilized by the Ca2+ ion. MAp19 is an alternative splicing product of the MASP-2 gene comprising the N-terminal CUB1-epidermal growth factor (EGF) segment of MASP-2, plus four additional residues at its C-terminal end. Like full-length MASP-2, it forms Ca2+-dependent complexes with mannan-binding lectin (MBL) and L-ficolin. The x-ray structure of human MAp19 was solved to a resolution of 2.5 Å. It shows a head to tail homodimer held together by interactions between the CUB1 module of one monomer and the EGF module of its counterpart. A Ca2+ ion bound to each EGF module stabilizes the dimer interfaces. A second Ca2+ ion is bound to the distal end of each CUB1 module, through six ligands contributed by Glu52, Asp60, Asp105, Ser107, Asn108, and a water molecule. Compared with its counterpart in human C1s, the N-terminal end of the MAp19 CUB1 module contains a 7-residue extension that forms additional inter-monomer contacts. To identify the residues involved in the interaction of MAp19 with MBL and L-ficolin, point mutants were generated and their binding ability was determined using surface plasmon resonance spectroscopy. Six mutations at Tyr59, Asp60, Glu83, Asp105, Tyr106, and Glu109 either strongly decreased or abolished interaction with both MBL and L-ficolin. These mutations map a common binding site for these proteins located at the distal end of each CUB1 module and stabilized by the Ca2+ ion. Studies performed over the past decade have led to the discovery of a novel route of complement activation termed the lectin pathway, which is increasingly recognized as an important component of innate antimicrobial host defense. This pathway is triggered by oligomeric lectins that recognize arrays of neutral carbohydrates on the surface of pathogens and share the ability to associate with enzymes called mannan-binding lectin-associated serine proteases (MASPs) 1The abbreviations used are: MASP, mannan-binding lectin-associated serine protease; CUB, module originally identified in complement proteins C1r/C1s, Uegf, and bone morphogenetic protein; EGF, epidermal growth factor; MBL, mannan-binding lectin.1The abbreviations used are: MASP, mannan-binding lectin-associated serine protease; CUB, module originally identified in complement proteins C1r/C1s, Uegf, and bone morphogenetic protein; EGF, epidermal growth factor; MBL, mannan-binding lectin. (1Fujita T. Nat. Rev. Immunol. 2002; 2: 346-353Crossref PubMed Scopus (567) Google Scholar, 2Holmskov U. Thiel S. Jensenius J.C. Annu. Rev. Immunol. 2003; 21: 547-578Crossref PubMed Scopus (652) Google Scholar). One of these proteases (MASP-2) activates complement through cleavage of proteins C4 and C2 (3Vorup-Jensen T. Petersen S.V. Hansen A. Poulsen K. Schwaeble W. Sim R.B. Reid K.B. Davis S.J. Thiel S. Jensenius J.C. J. Immunol. 2000; 165: 2093-2100Crossref PubMed Scopus (174) Google Scholar, 4Rossi V. Cseh S. Bally I. Thielens N.M. Jensenius J.C. Arlaud G.J. J. Biol. Chem. 2001; 276: 40880-40887Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Three different lectins able to associate with the MASPs have been described: mannan-binding lectin (MBL), L-ficolin and H-ficolin (5Ikeda K. Sannoh T. Kawasaki N. Kawasaki T. Yamashina I. J. Biol. Chem. 1987; 262: 7451-7454Abstract Full Text PDF PubMed Google Scholar, 6Matsushita M. Fujita T. J. Exp. Med. 1992; 176: 1497-1502Crossref PubMed Scopus (558) Google Scholar, 7Matsushita M. Endo Y. Fujita T. J. Immunol. 2000; 164: 2281-2284Crossref PubMed Scopus (272) Google Scholar, 8Matsushita M. Kuraya M. Hamasaki N. Tsujimura M. Shiraki H. Fujita T. J. Immunol. 2002; 168: 3502-3506Crossref PubMed Scopus (170) Google Scholar). In serum, these three proteins are present as oligomers of homotrimeric subunits, each comprising N-terminal collagen-like fibers prolonged by carbohydrate recognition domains (2Holmskov U. Thiel S. Jensenius J.C. Annu. Rev. Immunol. 2003; 21: 547-578Crossref PubMed Scopus (652) Google Scholar). In addition to MASP-2, MASP-1 and MASP-3 have also been described (6Matsushita M. Fujita T. J. Exp. Med. 1992; 176: 1497-1502Crossref PubMed Scopus (558) Google Scholar, 9Thiel S. Vorup-Jensen T. Stover C.M. Schwaeble W. Laursen S.B. Poulsen K. Willis A.C. Eggleton P. Hansen S. Holmskov U. Reid K.B. Jensenius J.C. Nature. 1997; 386: 506-510Crossref PubMed Scopus (750) Google Scholar, 10Dahl M.R. Thiel S. Matsushita M. Fujita T. Willis A.C. Christensen T. Vorup-Jensen T. Jensenius J.C. Immunity. 2001; 15: 127-135Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). All MASPs exhibit modular structures homologous to those of C1r and C1s, the proteases of the C1 complex of complement, with an N-terminal CUB module (11Bork P. Beckmann G. J. Mol. Biol. 1993; 231: 539-545Crossref PubMed Scopus (519) Google Scholar), an epidermal growth factor (EGF)-like module of the Ca2+-binding type (12Campbell I.D. Bork P. Curr. Opin. Struct. Biol. 1993; 3: 385-392Crossref Scopus (331) Google Scholar), a second CUB module, two complement control protein modules (13Reid K.B. Bentley D.R. Campbell R.D. Chung L.P. Sim R.B. Kristensen T. Tack B.F. Immunol. Today. 1986; 7: 230-234Abstract Full Text PDF PubMed Scopus (187) Google Scholar), and a chymotrypsin-like serine protease domain. MASP-1 and MASP-3 are alternative splicing products of the MASP1/3 gene and share identical CUB1-EGF-CUB2-CCP1-CCP2 segments, but comprise different serine protease domains (10Dahl M.R. Thiel S. Matsushita M. Fujita T. Willis A.C. Christensen T. Vorup-Jensen T. Jensenius J.C. Immunity. 2001; 15: 127-135Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). In the same way, alternative splicing of the MASP-2 gene generates MBL-associated protein 19 (MAp19), a nonenzymic protein comprising the N-terminal CUB1-EGF segment of MASP-2 prolonged by four unique residues (14Stover C.M. Thiel S. Thelen M. Lynch N.J. Vorup-Jensen T. Jensenius J.C. Schwaeble W.J. J. Immunol. 1999; 162: 3481-3490PubMed Google Scholar, 15Takahashi M. Endo Y. Fujita T. Matsushita M. Int. Immunol. 1999; 11: 859-863Crossref PubMed Scopus (170) Google Scholar). Studies using recombinant human (16Thielens N.M. Cesh 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, 17Cseh S. Vera L. Matsushita M. Fujita T. Arlaud G.J. Thielens N.M. J. Immunol. 2002; 169: 5735-5743Crossref PubMed Scopus (60) Google Scholar, 18Zundel S. Cseh S. Lacroix M. Dahl M.R. Matsushita M. Andrieu J.-P. Schwaeble W.J. Jensenius J.C. Fujita T. Arlaud G.J. Thielens N.M. J. Immunol. 2004; 172: 4342-4350Crossref PubMed Scopus (75) Google Scholar) and rat (19Chen C.B. Wallis R. J. Biol. Chem. 2001; 276: 25894-25902Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) proteins have shown that MASP-1, MASP-2, and MASP-3 as well as MAp19 each associate as homodimers through their N-terminal CUB1-EGF moieties. Likewise, the MASPs and MAp19 each individually form Ca2+-dependent complexes with MBL and L-ficolin. The binding involves primarily the N-terminal CUB1-EGF moiety of each protein, but is strengthened by the CUB2 module, which decreases the dissociation rate constant (koff) of the interaction (17Cseh S. Vera L. Matsushita M. Fujita T. Arlaud G.J. Thielens N.M. J. Immunol. 2002; 169: 5735-5743Crossref PubMed Scopus (60) Google Scholar, 18Zundel S. Cseh S. Lacroix M. Dahl M.R. Matsushita M. Andrieu J.-P. Schwaeble W.J. Jensenius J.C. Fujita T. Arlaud G.J. Thielens N.M. J. Immunol. 2004; 172: 4342-4350Crossref PubMed Scopus (75) Google Scholar, 20Wallis R. Dodd R.B. J. Biol. Chem. 2000; 275: 30962-30969Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The structures of the CUB1-EGF-CUB2 segment of rat MASP-2 and of the CUB1-EGF domain of human C1s have recently been solved by x-ray crystallography, revealing homologous homodimeric structures (21Feinberg H. Uitdehaag J.C. Davies J.M. Wallis R. Drickamer K. Weiss W.I. EMBO J. 2003; 22: 2348-2359Crossref PubMed Scopus (88) Google Scholar, 22Gregory L.A. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2003; 278: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). We now report the crystal structure of human MAp19 and the identification by site-directed mutagenesis of its interaction site with MBL and L-ficolin. Proteins—MBL and L-ficolin were purified from human serum as described by Zundel et al. (18Zundel S. Cseh S. Lacroix M. Dahl M.R. Matsushita M. Andrieu J.-P. Schwaeble W.J. Jensenius J.C. Fujita T. Arlaud G.J. Thielens N.M. J. Immunol. 2004; 172: 4342-4350Crossref PubMed Scopus (75) Google Scholar) and Cseh et al. (17Cseh S. Vera L. Matsushita M. Fujita T. Arlaud G.J. Thielens N.M. J. Immunol. 2002; 169: 5735-5743Crossref PubMed Scopus (60) Google Scholar), respectively. Recombinant wild-type MAp19 and its variants were expressed using a baculovirus/insect cell system. All mutants were secreted in the culture medium, with yields ranging from 3 to 6 μg/ml, similar to those observed for wild-type MAp19. All variants were purified from the cell culture supernatants by anion-exchange chromatography on a Q-Sepharose Fast Flow column followed by gel filtration in the presence of Ca2+ ions on a TSK G3000 SWG column, as described previously for wild-type MAp19 (16Thielens N.M. Cesh 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). The concentrations of the recombinant proteins were determined using absorption coefficients (A1%1cm at 280 nm) of 11.4 (Y59A and Y106A mutants) and 12.0 (wild-type MAp19 and all other mutants), calculated by the method of Gill and von Hippel (23Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5048) Google Scholar). Molecular masses, calculated from the amino acid sequences, were as follows: wild-type MAp19, 19,087 Da; E78Q, D81N, and E133Q mutants, 19,086 Da; E58A, E83A, and E109A mutants, 19,029 Da; Y59A and Y106A mutants, 18,995 Da; MAp19 R41A, 19,002 Da; MAp19 H55A, 19,041 Da; MAp19 D60A, 19,043 Da. Site-directed Mutagenesis—The expression plasmids coding for all MAp19 mutants were generated using the QuikChange™ XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. The pFastBac1/MAp19 expression plasmid coding for wild-type MAp19 (16Thielens N.M. Cesh 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) was used as a template. Mutagenic oligonucleotides were purchased from MWG-BIOTECH (Courtaboeuf, France). The sequences of all mutants were confirmed by double-stranded DNA sequencing (Genome Express, Grenoble, France). Chemical Characterization of the Recombinant Proteins—Mass spectrometry analyses were performed using the matrix-assisted laser desorption ionization technique on a Voyager Elite XL instrument (Perseptive Biosystems, Cambridge, MA), under conditions described previously (24Lacroix M. Rossi V. Gaboriaud C. Chevallier S. Jaquinod M. Thielens N.M. Gagnon J. Arlaud G.J. Biochemistry. 1997; 36: 6270-6282Crossref PubMed Scopus (43) Google Scholar). Circular Dichroism—CD measurements were performed on a JASCO J-810 spectropolarimeter using a 1.0-mm path length quartz cell. Spectra were recorded over the 200–320 nm range at 0.5-nm intervals at a scan speed of 50 nm/min and corrected for the contribution of the buffer (145 mm NaCl, 1 mm CaCl2, 50 mm triethanolamine hydrochloride, pH 7.4). The concentrations of the MAp19 variants were as follows: wild-type, 17.2 μm; Y59A, 17.2 μm; E83A, 13 μm; D105G, 17.2 μm; Y106A, 17.6 μm; E109A, 10.1 μm. Crystallization and Data Collection—Recombinant MAp19 was concentrated to 2.5 mg/ml in 145 mm NaCl, 1 mm CaCl2, 50 mm triethanolamine HCl, pH 7.4. Crystals suitable for x-ray data collection were obtained at 20 °C by the hanging drop vapor diffusion method by mixing equal volumes of the protein solution and a reservoir solution composed of 3% (w/v) PEG 8000 and 0.1 m Tris-HCl, pH 8.5. They were transferred to a cryoprotectant solution containing 15% (w/v) PEG 8000, 21.75% glycerol, and 0.1 m Tris-HCl, and then flash cooled in liquid nitrogen. A native data set indexed in the space group P43212 was measured at the European Synchrotron Radiation Facility (ESRF) beamline BM14 to a resolution of 2.50 Å. The images were processed and the reflections scaled using the program XDS (25Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3225) Google Scholar). Crystallographic statistics for the native data set are given in Table I.Table IData collection (collected at BM14, ESRF) and refinement statisticsSpace groupP43212Unit cell (Å)a = 67.75, b = 67.75, c = 187.92(°)α = 90.00, β = 90.00, γ = 90.00λ (Å)0.946Resolution (Å)17-2.50Rsym0.047 (0.357)aStatistics for high resolution bin (2.6-2.5 Å) are in parentheses.% Completeness98.2 (93.8)Redundancy6.5 (6.5)I/σ (I) average23.7 (5.1)No. of unique reflections15692 (1489)Model statisticsFinal resolution (Å)2.5No. of residues333No. of water molecules146No. of ions4Root mean square deviation χ2 bonds (Å)0.0068Root mean square deviation χ2 angles (°)1.39Rwork0.261Rfree0.316a Statistics for high resolution bin (2.6-2.5 Å) are in parentheses. Open table in a new tab Structure Determination and Refinement—The structure of MAp19 was determined using the molecular replacement method. The rotational and translational searches were carried out using the program AMORE (26Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5028) Google Scholar). Whereas initial searches using the human C1s CUB1-EGF structure (22Gregory L.A. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2003; 278: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) were unsuccessful, a molecular replacement solution was obtained using the CUB1-EGF fragment of rat MASP-2 in the dimeric form (Protein Data Bank accession code 1NT0) (21Feinberg H. Uitdehaag J.C. Davies J.M. Wallis R. Drickamer K. Weiss W.I. EMBO J. 2003; 22: 2348-2359Crossref PubMed Scopus (88) Google Scholar). Rigid body refinement with the program CNS (27Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar) further improved the orientation and position of each of the domains of the search model. Additional refinement of the model was carried out with CNS and model rebuilding was performed using the graphics program O (28Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13009) Google Scholar). The quality of the map allowed construction of 333 residues of a total of 340 in the asymmetric unit. Residues Thr1 (molecule A), Thr1–Leu2 (molecule B), and Ser169–Leu170 (molecules A and B) are disordered. Residue Pro21 is in the cis configuration. The atomic coordinates have been deposited in the Protein Data Bank under the code 1SZB. Modeling of the MBL Collagen-like Triple Helix—The human MBL collagen-like segment (positions 36–63) was modeled using the graphics program O (28Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13009) Google Scholar). Regional differences based on the helical propensity of residues, as established from statistical analysis of collagen-like structures (30Rainey J.K. Goh M.C. Protein Sci. 2002; 11: 2748-2754Crossref PubMed Scopus (60) Google Scholar), were included in the model. The template Protein Data Bank file corresponding to imino-rich parameters (30Rainey J.K. Goh M.C. Protein Sci. 2002; 11: 2748-2754Crossref PubMed Scopus (60) Google Scholar) was used for modeling most of the triple helix, except for segments 45–50 and 54–56, where the alternative template file designed for imino-deficient segments (30Rainey J.K. Goh M.C. Protein Sci. 2002; 11: 2748-2754Crossref PubMed Scopus (60) Google Scholar) was used. The graphics program O (28Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13009) Google Scholar) was then used to search for a plausible model of MAp19 interaction with two modeled MBL triple helices. Although different sets of MAp19 orientations were tried initially, only one major hypothetical mode of interaction was found to be fully compatible with both the present mutagenesis data and previously published experimental information (see “Discussion”). Surface Plasmon Resonance Spectroscopy and Data Evaluation—Surface plasmon resonance spectroscopy analyses were performed using a BIAcore 3000 instrument (BIAcore AB, Uppsala, Sweden). MBL and L-ficolin/P35 were immobilized on the surface of a CM5 sensor chip (BIAcore AB) using the amine coupling chemistry as described previously (17Cseh S. Vera L. Matsushita M. Fujita T. Arlaud G.J. Thielens N.M. J. Immunol. 2002; 169: 5735-5743Crossref PubMed Scopus (60) Google Scholar). Binding of the wild-type and mutant MAp19 species was measured over 16,000 resonance units of immobilized L-ficolin or 9,000 resonance units of immobilized MBL, at a flow rate of 20 μl/min in 145 mm NaCl, 1 mm CaCl2, 50 mm triethanolamine hydrochloride, pH 7.4, containing 0.005% surfactant P20 (BIAcore AB). Equivalent volumes of each MAp19 sample were injected in parallel over a surface with immobilized bovine serum albumin to serve as blank sensorgrams for subtraction of the bulk refractive index background. Regeneration of the surfaces was achieved by injection of 10 μl of 1 m NaCl, 20 mm EDTA. Data were analyzed by global fitting to a 1:1 Langmuir binding model of both the association and dissociation phases for several concentrations simultaneously, using the BIAevaluation 3.1 software (BIAcore AB). The apparent equilibrium dissociation constants (KD) were calculated from the ratio of the dissociation and association rate constants (koff/kon). Each MAp19 variant was analyzed at six different concentrations, ranging from 5 to 60 nm for wild-type MAp19 and all mutants except D60A and E83A (25–250 nm), E109A (50–500 nm), Y59A, D105G, and Y106A (100–1200 nm). Overall Structure—Human MAp19 was produced in a baculovirus/insect cell expression system as described previously (16Thielens N.M. Cesh 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). Mass spectrometry analysis of the recombinant protein yielded a value of 19,088 ± 9 Da, in full agreement with the amino acid sequence (calculated mass: 19,087.1 Da). The crystal structure of MAp19 was solved by molecular replacement using the rat MASP-2 CUB1-EGF structure (21Feinberg H. Uitdehaag J.C. Davies J.M. Wallis R. Drickamer K. Weiss W.I. EMBO J. 2003; 22: 2348-2359Crossref PubMed Scopus (88) Google Scholar) as a search model, and refined to 2.5-Å resolution. The final Rwork and Rfree factors are 0.261 and 0.316, respectively, and the refined model has satisfactory stereochemistry (Table I). As expected from previous sedimentation velocity analyses (16Thielens N.M. Cesh 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), MAp19 associates as a Ca2+-dependent homodimer (Fig. 1). The two monomers interact in a head to tail manner, through major contacts between the CUB1 module of one monomer and the EGF module of its counterpart. The dimer contains four Ca2+ ions, one at each CUB1-EGF interface (site I) and one bound to the distal end of each CUB1 module (site II) (Fig. 1A). The structure has a length of 95 Å and a width of 20–45 Å. Overall, the assembly is reminiscent of those observed for the CUB1-EGF segments of human C1s and rat MASP-2 (21Feinberg H. Uitdehaag J.C. Davies J.M. Wallis R. Drickamer K. Weiss W.I. EMBO J. 2003; 22: 2348-2359Crossref PubMed Scopus (88) Google Scholar, 22Gregory L.A. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2003; 278: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). However, a detailed comparison of the three structures reveals differences in the relative positioning of their CUB1 and EGF modules. Thus, superimposition of the MAp19 and C1s monomeric structures on their CUB1 domains reveals extensive displacement of the EGF modules, resulting in a distance of ∼10 Å between their distal ends, corresponding to a rotation of ∼25° about the long axis of the CUB domains (Fig. 1C). Similarly, a displacement of EGF modules is observed when the human MAp19 and rat MASP-2 monomers are compared, although the rotation angle is only about 14° in that case. In both instances, the flexibility originates from a hinge located near Glu122, at the boundary between the CUB1 and EGF modules (Fig. 1C). Additional differences are observed when the dimeric structures are compared. Thus, there are significant variations in the relative positioning of the CUB1 module of one monomer and the EGF module of its counterpart, with rotation angles of 15–19° between MAp19 and the other two dimers. Interestingly, in contrast to the C1s structure (22Gregory L.A. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2003; 278: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), MAp19 does not feature a groove in the region where the four modules meet, the corresponding space being partly filled by loops 10 of both EGF modules (Fig. 1B). A comparison in this respect with the rat MASP-2 CUB1-EGF dimer is not possible, because both loops 10 are disordered in the structure (21Feinberg H. Uitdehaag J.C. Davies J.M. Wallis R. Drickamer K. Weiss W.I. EMBO J. 2003; 22: 2348-2359Crossref PubMed Scopus (88) Google Scholar). The CUB1 Module and Ca2+-binding Site II—The MAp19 CUB1 module is organized in two four-stranded β-sheets, each made of anti-parallel strands, providing further evidence that this topology, also observed in the corresponding module of human C1s and rat MASP-2, defines a particular CUB domain subset distinct from the plasma spermadhesins (31Romero A. Romao J.J. Varela P.F. Kölln L. Dias J.M. Carvalho A.L. Sanz L. Topfer-Petersen E. Calvete J.J. Nat. Struct. Biol. 1997; 5: 458-463Google Scholar). As expected from sequence homologies, the MAp19 CUB1 module is structurally closer to its counterpart in rat MASP-2 (root mean square deviation = 0.56 Å, based on 106 Cα atoms) than to the C1s CUB1 module (root mean square deviation = 0.94 Å, based on 99 Cα atoms). Compared with C1s, MAp19 contains a 7-residue extension at its N-terminal end (Figs. 1C and 2). Except for Thr1 of molecule A and residues Thr1 and Leu2 of molecule B, the corresponding segments are well defined in the structure and closely interact with each other. As illustrated in Fig. 1B, the interactions mainly involve van der Waals contacts between residues Pro5, Trp7, Pro8, Glu9, and Pro10 of molecule A and residues Leu3, Gly4, Pro8, Glu9, Pro10, and Val11 of molecule B. Other interactions are mediated by Trp7 of molecule A, which forms a hydrogen bond with the main chain carbonyl group of Glu9 of molecule B, and additional van der Waals contacts with residues Ala36, Pro37, and Ala121 of molecule B. Remarkably, these interactions are asymmetrical and the two N-terminal segments exhibit quite different conformations in the structure (Fig. 1B). A Ca2+ ion (site II) is bound to the distal end of each CUB1 module (Fig. 1). In molecule A, the Ca2+ ion is coordinated by six oxygen ligands, namely one of the side chain oxygens of Glu52, Asp60, and Asp105, the main chain carbonyl oxygen of Ser107, the side chain carbonyl oxygen of Asn108, and a water molecule (Fig. 3). The bond distances are in average 2.4 Å, consistent with the value determined for known Ca2+-binding sites (32Harding M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1432-1443Crossref PubMed Scopus (234) Google Scholar). The coordination involves the same protein ligands in molecule B, but the water molecule is missing. A comparison with the homologous site previously observed in C1s (22Gregory L.A. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2003; 278: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), reveals several significant differences. (i) The residues equivalent to Ser107 and Asn108 of MAp19 are not Ca2+ ligands in C1s. (ii) The C1s residue Asp53 contributes two ligands instead of one for the homologous residue Asp60 of MAp19. (iii) Whereas Asp105 of MAp19 binds Ca2+ through its side chain, its counterpart Asp98 in C1s binds through its main chain carbonyl. (iv) Two water molecules are present in the C1s Ca2+ binding site. On the other hand, as observed in C1s, the Ca2+ ion is the central element of a network of interactions that connect together loops L3, L5, and L9, thereby extensively stabilizing the distal end of the MAp19 CUB1 module. In this respect, it is interesting to note that Tyr24 is H-bonded to the side chain carboxyl of Asp60 and the main chain nitrogen of Glu52, and thereby stabilizes loop L3 in the same way as does its counterpart Tyr17 in C1s (22Gregory L.A. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2003; 278: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Ca2+-binding Site I—The MAp19 EGF module has a -fold similar to that described for other EGF-like modules (12Campbell I.D. Bork P. Curr. Opin. Struct. Biol. 1993; 3: 385-392Crossref Scopus (331) Google Scholar), with one major and one minor anti-parallel double-stranded β-sheets (Fig. 1, A and B). Loop 10, which is disordered in the rat MASP-2 structure (21Feinberg H. Uitdehaag J.C. Davies J.M. Wallis R. Drickamer K. Weiss W.I. EMBO J. 2003; 22: 2348-2359Crossref PubMed Scopus (88) Google Scholar), is structurally well defined in MAp19 and exhibits a rather extended conformation (Fig. 1B), different from that of the corresponding loop of C1s (22Gregory L.A. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2003; 278: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The remainder of the MAp19, C1s, and rat MASP-2 EGF modules show root mean square deviation values of 0.61–0.69 Å, indicative of high structural homology. The Ca2+ ion bound to each EGF module (site I) is coordinated by seven oxygen ligands, including a water molecule and six ligands provided by the EGF module, namely one of the side chain oxygens of Asp123 and Glu126, the side chain carbonyl of Asn143, and the main chain carbonyl of Ile124, His144, and Gly147. These residues are strictly homologous to those providing Ca2+ ligands in C1s. In contrast, as discussed previously (22Gregory L.A. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2003; 278: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), the Ca2+-binding site observed in the rat MASP-2 structure only involves five coordination ligands (21Feinberg H. Uitdehaag J.C. Davies J.M. Wallis R. Drickamer K. Weiss W.I. EMBO J. 2003; 22: 2348-2359Crossref PubMed Scopus (88) Google Scholar). As observed in the C1s structure (22Gregory L.A. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2003; 278: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), Asn143 lacks β-hydroxylation, providing further evidence that post-translational modification of this residue to erythro-β-hydroxyasparagine is not achieved in baculovirus/insect cell systems and is not essential for Ca2+ binding. In molecule A, the water molecule involved in Ca2+ coordination also forms hydrogen bonds with three of the other Ca2+ ligands (Asp123, Glu126, and Gly147) and with Gly39 in loop 4 of the CUB1 module. Thus, as observed in the C1s and rat MASP-2 structures (21Feinberg H. Uitdehaag J.C. Davies J.M. Wallis R. Drickamer K. Weiss W.I. EMBO J. 2003; 22: 2348-2359Crossref PubMed Scopus (88) Google Scholar, 22Gregory L.A. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2003; 278: 32157-32164Abstract F" @default.
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• W2116742387 title "The X-ray Structure of Human Mannan-binding Lectin-associated Protein 19 (MAp19) and Its Interaction Site with Mannan-binding Lectin and L-ficolin" @default.
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