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J. Biol. Chem., Vol. 279, Issue 28, 29391-29397, July 9, 2004

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The X-ray Structure of Human Mannan-binding Lectin-associated Protein 19 (MAp19) and Its Interaction Site with Mannan-binding Lectin and L-ficolin^*

Lynn A. Gregory, Nicole M. Thielens¶, Misao Matsushita||, Rikke Sorensen**, Gérard J. Arlaud¶, Juan Carlos Fontecilla-Camps, and Christine Gaboriaud

From the Laboratoire de Cristallographie et Cristallogénèse des Protéines and the ^¶Laboratoire d'Enzymologie Moléculaire, Institut de Biologie Structurale, 38027 Grenoble Cedex 1, France, the ^||Department of Applied Biochemistry and Institute of Glycobiology, Tokai University, Hiratsuka, Kanagawa 259-1292, Japan, and the ^**Department of Medical Microbiology and Immunology, University of Aarhus, DK 8000 Aarhus, Denmark

Received for publication, March 9, 2004 , and in revised form, April 20, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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)¹ (1, 2). One of these proteases (MASP-2) activates complement through cleavage of proteins C4 and C2 (3, 4). Three different lectins able to associate with the MASPs have been described: mannan-binding lectin (MBL), L-ficolin and H-ficolin (5–8). In serum, these three proteins are present as oligomers of homotrimeric subunits, each comprising N-terminal collagen-like fibers prolonged by carbohydrate recognition domains (2).

In addition to MASP-2, MASP-1 and MASP-3 have also been described (6, 9, 10). 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 (11), an epidermal growth factor (EGF)-like module of the Ca²⁺-binding type (12), a second CUB module, two complement control protein modules (13), 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 (10). 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 (14, 15). Studies using recombinant human (16–18) and rat (19) 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 (k_off) of the interaction (17, 18, 20).

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 (21, 22). 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Proteins—MBL and L-ficolin were purified from human serum as described by Zundel et al. (18) and Cseh et al. (17), 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 (16). The concentrations of the recombinant proteins were determined using absorption coefficients (A^1%_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 (23). 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^TM 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 (16) 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 (24).

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 (25). Crystallographic statistics for the native data set are given in Table I.

Modeling of the MBL Collagen-like Triple Helix—The human MBL collagen-like segment (positions 36–63) was modeled using the graphics program O (28). Regional differences based on the helical propensity of residues, as established from statistical analysis of collagen-like structures (30), were included in the model. The template Protein Data Bank file corresponding to imino-rich parameters (30) 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 (30) was used. The graphics program O (28) 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 (17). 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 (K_D) were calculated from the ratio of the dissociation and association rate constants (k_off/k_on). 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).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Overall Structure—Human MAp19 was produced in a baculovirus/insect cell expression system as described previously (16). 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 (21) as a search model, and refined to 2.5-Å resolution. The final R_work and R_free factors are 0.261 and 0.316, respectively, and the refined model has satisfactory stereochemistry (Table I). As expected from previous sedimentation velocity analyses (16), 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.

View larger version (49K): [in this window] [in a new window]   FIG. 1. Homodimeric structure of human MAp19. A, top view of the structure. B, side view of the structure. Molecules A and B are in red and green, respectively. Ca2+ ions are represented by golden spheres. The side chains of the residues involved in the inter-monomer interface (A) and the N-terminal interactions (B) are shown. Hydrogen bonds are represented as dotted lines. C, superimposition of the MAp19 and C1s CUB1-EGF monomeric structures (stereo view). The MAp19 and C1s structures are shown in red and yellow, respectively, and are superimposed onto their CUB1 module. Nt and Ct indicate N- and C-terminal ends.

The CUB1 Module and Ca²⁺-binding Site II—The MAp19 CUB1 module is organized in two four-stranded -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 (31). 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 C atoms) than to the C1s CUB1 module (root mean square deviation = 0.94 Å, based on 99 C 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).

View larger version (58K): [in this window] [in a new window]   FIG. 2. Structure-based sequence alignment of CUB1-EGF segments. The secondary structure elements and the numbering are those of human MAp19. Residues involved in the inter-monomer interface are colored orange and those involved in N-terminal interactions are green. Residues providing Ca2+ ligands are colored red, and marked with either closed circles (site I) or open circles (site II). Residues proposed to interact with MBL and L-ficolin are colored blue.

View larger version (38K): [in this window] [in a new window]   FIG. 3. Stereo view of Ca2+-binding site II. Oxygen atoms are shown in red, and nitrogen atoms in blue. The water molecule is represented as a light blue sphere. Ionic bonds are represented by dotted lines.

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 (21, 22), this molecule provides a link between the CUB1 and EGF modules, thereby stabilizing the inter-modular interface.

The Inter-monomer Interface—The interface between the two monomers involves a series of hydrophobic interactions, with in particular three major hydrophobic pockets (Fig. 1A). (i) A central pocket around the 2-fold symmetry axis of the dimer, involving Tyr⁴⁰, and Leu¹⁴⁵ from each monomer. Pro⁸ of molecule B is inserted in this pocket. (ii) An aromatic triad at each inter-monomer interface, comprising Phe¹² (from the CUB1 module), His¹⁴⁴, and Tyr¹⁴⁹ (from the EGF module). His¹⁴⁴ also coordinates Ca²⁺, and thereby provides a link between Ca²⁺-binding site I and the inter-monomer interface. (iii) A distal pocket at both ends of the interface, involving Phe²⁰, Pro²¹, and His⁴⁸ from the CUB1 module and Ala¹⁵⁴ from the EGF module. These three pockets are conserved in the C1s structure and, with a few exceptions, they involve homologous residues (22). An additional hydrophobic pocket not present in C1s, involving Tyr⁴⁵, Thr⁴⁷, Phe¹¹⁸, His¹⁴⁰, and His¹⁴², is found between the distal pocket and the aromatic triad. As a result, water molecules are excluded from the mid-to-distal region of the dimer interface, and hence the indirect hydrogen bond network found in this area in C1s (22) is not conserved in MAp19. Additional interactions between the CUB1 and EGF modules are provided by direct hydrogen bonds between the side chains of Tyr⁴⁵ and His¹⁴², and of Arg⁴³ and Asn¹⁴³ (Fig. 1A). As illustrated in Fig. 2, most of the residues involved in hydrophobic interactions and hydrogen bonds at the inter-monomer interface are either conserved or substituted by similar residues within the C1r/C1s/MASP family.

Mapping of the Interaction Site with MBL and L-ficolin—To locate the residues of MAp19 involved in the interaction with MBL and L-ficolin, a series of recombinant point mutants were produced and their binding properties were analyzed by surface plasmon resonance spectroscopy. We initially targeted various charged amino acid residues, of which some appeared to be conserved in the whole MASP family and absent in C1r and C1s. As summarized in Table II, most of these mutations (R41A, H55A, E58A, E78Q, D81N, and E133Q) had very little effect, if any, on the interaction with either MBL or L-ficolin. However, mutation of Glu⁸³ to Ala significantly increased the K_D values for both MBL and L-ficolin (Table II). A second series of mutations targeted residues of the CUB1 module either providing Ca²⁺ ligands (D60A and D105G) or located in the vicinity of Ca²⁺-binding site II (Y59A, Y106A, and E109A). All of these mutations markedly decreased the ability of MAp19 to associate with MBL and L-ficolin (Fig. 4, Table II). The Y59A, D105G, and Y106A mutations virtually abolished interaction with either protein. In contrast, the E109A and D60A mutations had more discriminating effects, the latter abolishing binding to L-ficolin, but only increasing 7-fold the K_D value for MBL. In most cases, the increases in K_D resulted from a decrease in the k_on value (Table II).

View larger version (28K): [in this window] [in a new window]   FIG. 4. Analysis by surface plasmon resonance spectroscopy of the interaction between the MAp19 variants and immobilized MBL or L-ficolin. A, interaction with MBL. B, interaction with L-ficolin. MBL and L-ficolin were immobilized on the sensor chip as described under "Experimental Procedures." Wild-type MAp19 and its Y59A, D60A, E83A, D105G, Y106A, and E109A mutants were each injected at a concentration of 50 nM.

View larger version (30K): [in this window] [in a new window]   FIG. 5. CD spectroscopy analysis of the MAp19 variants. A, far UV spectrum. B, near UV spectrum. Spectra were recorded at protein concentrations of 10–17 µM in 145 mM NaCl, 1 mM CaCl2, 50 mM triethanolamine hydrochloride, pH 7.4, as described under "Experimental Procedures." Each spectrum is the average of 30 scans. Molar ellipticities are shown.

View larger version (38K): [in this window] [in a new window]   FIG. 6. The MBL/L-ficolin interaction site of human MAp19. A, top view of the MAp19 dimer structure. The closed and open circles indicate the two MBL-binding sites proposed by Feinberg et al. (21). B, proposed model of the interaction between MAp19 and MBL. The MAp19 mutations that inhibit interaction with MBL and L-ficolin are shown in red, and those that have no significant effect are shown in yellow. C, stereo view of Ca2+-binding site II and the proposed interaction site in MAp19.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

An interesting characteristic of the MAp19 homodimer lies in the peculiar topology of its two N-terminal segments, which, despite their identical amino acid sequences, exhibit strikingly different conformations and therefore interact with each other in an asymmetrical manner (Fig. 1B). Whether this structural feature is shared by rat MASP-2 remains to be determined, because the CUB1-EGF-CUB2 fragment crystallized by Feinberg et al. (21) starts at the Ser residue equivalent to Pro⁵ of human MAp19 (Fig. 2), the N-terminal segment Ser-Lys-Trp-Pro being disordered in the structure. It appears likely that the tight network of hydrophobic interactions contributed by the N-terminal extensions of MAp19 provides further stabilization of the dimeric structure. Nevertheless, as judged from the rat MASP-2 structure, it may be inferred that these interactions are not essential to the dimerization process. Interestingly, the N-terminal extension is strikingly different in the MASP-2/MAp19 and MASP-1/MASP-3 lineages (Fig. 2), suggesting that this region may contribute to the exclusive formation of homodimers through exclusion of unlike partners.

As previously observed in the human C1s CUB1-EGF structure (22), a Ca²⁺ ion is bound to the distal end of each CUB1 module of MAp19. In both proteins, Ca²⁺ is coordinated by six oxygen ligands, of which three (Glu⁵², Asp⁶⁰, and Asp¹⁰⁵ in MAp19; Glu⁴⁵, Asp⁵³, and Asp⁹⁸ in C1s) are common to both sites. This observation strengthens our initial proposal (22) that these three acidic residues, which are conserved in about two-thirds of the CUB module repertoire, define a particular CUB module subset with the ability to bind Ca²⁺. This conservation applies in particular to the CUB1 modules of the C1r/C1s/MASP family, which in addition also contain the two residues (Ser¹⁰⁷ and Asn¹⁰⁸) that provide additional Ca²⁺ ligands through their main chain carbonyl in MAp19. The fact that these latter two residues are present in C1s, but are not Ca²⁺ ligands, provides a further indication that Ca²⁺ binding sites have the ability to slightly adapt their coordination mode depending on their particular protein (and/or crystal) context (32).

As discussed previously in light of the C1s CUB1-EGF structure (22), the above considerations raise questions about the fact that no Ca²⁺ ion was seen in the CUB1 module of rat MASP-2 (21). To further investigate this question, we have superimposed the corresponding regions of human MAp19 and rat MASP-2. This analysis indicates that, whereas Glu⁵² and Asp⁶⁰ are positioned similarly in both proteins, the side chain of Asp¹⁰⁵ has a different orientation in rat MASP-2, its residues Ser¹⁰⁷ and Asn¹⁰⁸ being disordered (21). It appears very likely from these observations that Ca²⁺-binding site II is indeed present in the CUB1 module of rat MASP-2, but was not occupied to a significant extent in the x-ray structure solved by Feinberg et al. (21).

Analysis of the interaction properties of the various point mutants produced in this study provides unambiguous evidence that the MAp19-binding site for both MBL and L-ficolin lies at the distal end of the CUB1 module, in close vicinity of Ca²⁺-binding site II. As depicted in Fig. 6A, this site is different from those proposed by Feinberg et al. (21) on the basis of shape and symmetry considerations. On the other hand, our proposition is fully consistent with the observations that: (i) interaction of human MAp19 with either MBL or L-ficolin is abolished in the presence of EDTA (16, 17). This characteristic also applies to MASP-1, MASP-2, and MASP-3, although in the case of L-ficolin their binding ability is only partly sensitive to EDTA (17). (ii) A naturally occurring mutation D105G has been described recently in human MASP-2 and shown to result in a complete loss of interaction with MBL (33). As this same mutation, when introduced in MAp19, also abolishes its interaction with MBL, it may be inferred that, although the CUB2 domain of MASP-2 significantly stabilizes its interaction with MBL (17, 19, 20), the site identified here in the CUB1 module is the primary binding site.

The fact that Asp⁶⁰ and Asp¹⁰⁵ each provide a Ca²⁺ ligand raises the question whether these residues are directly involved in the interaction with MBL and L-ficolin, or are required solely because they stabilize, through Ca²⁺ binding, the distal end of the CUB1 module in the conformation that is appropriate for binding. A comparative analysis of the elution profiles of the MAp19 mutants upon purification by anion-exchange chromatography (see "Experimental Procedures") provides interesting information in this respect. As anticipated from the loss of a negative charge, each of the mutants E58A, E78Q, D81N, E83A, E109A, and E133Q eluted significantly earlier than wild-type MAp19. In contrast, the D105G mutant eluted at a position similar to that of wild-type MAp19, and D60A was significantly delayed, indicating an increase in the overall negative charge of the molecule. A plausible explanation for these observations is that these latter two mutations destabilize, possibly to different extents, the Ca²⁺-binding site, hence releasing the Ca²⁺ ion and therefore unmasking the carboxyl group of the other two acidic residues engaged in Ca²⁺ binding. Based on these considerations, it appears likely that Asp⁶⁰ and Asp¹⁰⁵ are not directly involved in the interaction with MBL and L-ficolin but, like the other Ca²⁺ ligands, are essential to stabilize the protein scaffold that maintains the binding site.

On the other hand, it may be postulated that Tyr⁵⁹, Tyr¹⁰⁶, and Glu¹⁰⁹, on the distal extremity of the Ca²⁺-binding site, and Glu⁸³, located in the more distant loop 7 (Fig. 6C), each directly participate in the interaction with MBL and L-ficolin. Whereas Tyr¹⁰⁶ is specific of the MASP-2/MAp19 lineage, Tyr⁵⁹, Glu⁸³, and Glu¹⁰⁹ as well as the residues involved in Ca²⁺-binding site II are conserved in the CUB1 module of the MASPs identified so far in different species. This strongly supports the hypothesis that the MBL/L-ficolin-binding site identified in the present study applies not only to MAp19 and MASP-2, but also to MASP-1 and MASP-3. This hypothesis appears consistent with the recent report (29) that rat MBL associates with all MASPs through similar sites located in the same area of the collagen-like domain. Our finding that MAp19 shares a common binding site for MBL and L-ficolin is in agreement with the observation that most of the residues that form the core of the proposed MASP-binding site in MBL are also present in the ficolins (29). Thus, in the current state of our knowledge, it appears likely that MBL and L-ficolin associate with the MASPs through similar mechanisms involving homologous sites in either partner.

A three-dimensional model of the homotrimeric triple-helical segment of human MBL containing the MASP-binding site proposed by Wallis et al. (29) was constructed as described under "Experimental Procedures" and docked onto the MBL-binding site identified in the present study. As illustrated in Fig. 6B, the spacing between residues Glu⁸³ and Glu¹⁰⁹ in MAp19 is similar to that between residues Arg⁴⁷ and Lys⁵⁵ in the MBL triple helix model. As Arg⁴⁷ and Lys⁵⁵ are both likely candidates for the interaction (29), a tempting hypothesis is that these make salt bridges with Glu⁸³ and Glu¹⁰⁹, respectively (Fig. 6B). If this model is correct, additional hydrophobic interactions may involve Tyr⁵⁹ and Tyr¹⁰⁶ of MAp19 and Leu⁵⁶ of MBL. In the proposed interaction, the two opposite collagen helices of MBL are oriented in such a way that they form an angle of 80–90° at the hinge, a value that appears consistent with electron microscopy pictures of MBL (34). We believe that this model provides a sound basis for further investigation, e.g. by site-directed mutagenesis, of the assembly of MBL- and ficolin-MASP complexes.

	FOOTNOTES

 
^* This work was supported by the Commissariat à l'Energie Atomique, the Centre National de la Recherche Scientifique, and the Université Joseph Fourier, Grenoble. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1SZB [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Both authors contributed equally to the work.

To whom correspondence should be addressed. Tel.: 33-4-38-78-95-99; Fax: 33-4-38-78-51-22; E-mail: christine.gaboriaud{at}ibs.fr.

¹ The 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.

	ACKNOWLEDGMENTS

 
We thank Hadar Feinberg and William Weis for providing the rat MASP-2 CUB1-EGF-CUB2 coordinates, Philippe Carpentier for help with the use of the x-ray diffraction equipment at BM14, ESRF, and Vincent Forge for help with CD and fluorescence analyses.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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