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Crystal structure of the CUB1-EGF-CUB2 region of mannose-binding protein associated serine protease-2

2003; Springer Nature; Volume: 22; Issue: 10 Linguagem: Inglês

10.1093/emboj/cdg236

1460-2075

H. Feinberg, Joost C.M. Uitdehaag, Jason M. Davies, Russell Wallis, Kurt Drickamer, William I. Weis,

Galectins and Cancer Biology

Article15 May 2003free access Crystal structure of the CUB1-EGF-CUB2 region of mannose-binding protein associated serine protease-2 Hadar Feinberg Hadar Feinberg Departments of Structural Biology and of Molecular and Cellular Physiology, Stanford University School of Medicine, 299 Campus Drive West, Stanford, CA, 94305-5126 USA Search for more papers by this author Joost C.M. Uitdehaag Joost C.M. Uitdehaag Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Jason M. Davies Jason M. Davies Departments of Structural Biology and of Molecular and Cellular Physiology, Stanford University School of Medicine, 299 Campus Drive West, Stanford, CA, 94305-5126 USA Search for more papers by this author Russell Wallis Russell Wallis Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Kurt Drickamer Kurt Drickamer Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author William I. Weis Corresponding Author William I. Weis Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Hadar Feinberg Hadar Feinberg Departments of Structural Biology and of Molecular and Cellular Physiology, Stanford University School of Medicine, 299 Campus Drive West, Stanford, CA, 94305-5126 USA Search for more papers by this author Joost C.M. Uitdehaag Joost C.M. Uitdehaag Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Jason M. Davies Jason M. Davies Departments of Structural Biology and of Molecular and Cellular Physiology, Stanford University School of Medicine, 299 Campus Drive West, Stanford, CA, 94305-5126 USA Search for more papers by this author Russell Wallis Russell Wallis Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Kurt Drickamer Kurt Drickamer Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author William I. Weis Corresponding Author William I. Weis Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Author Information Hadar Feinberg1, Joost C.M. Uitdehaag2, Jason M. Davies1, Russell Wallis2, Kurt Drickamer2 and William I. Weis 2 1Departments of Structural Biology and of Molecular and Cellular Physiology, Stanford University School of Medicine, 299 Campus Drive West, Stanford, CA, 94305-5126 USA 2Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU UK ‡H.Feinberg and J.C.M.Uitdehaag contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:2348-2359https://doi.org/10.1093/emboj/cdg236 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Serum mannose-binding proteins (MBPs) are C-type lectins that recognize cell surface carbohydrate structures on pathogens, and trigger killing of these targets by activating the complement pathway. MBPs circulate as a complex with MBP-associated serine proteases (MASPs), which become activated upon engagement of a target cell surface. The minimal functional unit for complement activation is a MASP homodimer bound to two MBP trimeric subunits. MASPs have a modular structure consisting of an N-terminal CUB domain, a Ca2+-binding EGF-like domain, a second CUB domain, two complement control protein modules and a C-terminal serine protease domain. The CUB1-EGF-CUB2 region mediates homodimerization and binding to MBP. The crystal structure of the MASP-2 CUB1-EGF-CUB2 dimer reveals an elongated structure with a prominent concave surface that is proposed to be the MBP-binding site. A model of the full six-domain structure and its interaction with MBPs suggests mechanisms by which binding to a target cell transmits conformational changes from MBP to MASP that allow activation of its protease activity. Introduction Complement-mediated neutralization of pathogens by lysis or opsonization can be initiated through multiple pathways. The classical and lectin pathways both start by specific recognition of the target cell. In the classical pathway, antibodies provide the recognition function, which leads to activation of the C1r and C1s proteases, which in turn activate complement components C4 and C2 (Arlaud et al., 2002). Complement component C1q links antibody binding to C1r and C1s activation, by binding to the Fc region of IgM or IgG and to C1r and C1s. In the lectin pathway, mannose-binding protein (MBP, also referred to as mannose-binding lectin) recognizes cell-surface carbohydrate structures and directly activates MBP-associated serine proteases (MASPs), which then activate C4 and C2 (Weis et al., 1998). The serum form of MBP is composed of two, three or four subunits, each of which is a trimer of identical polypeptides (Wallis and Drickamer, 1999). The trimeric subunits are elongated molecules, consisting of N-terminal collagen-like domains and C-terminal carbohydrate-recognition domains (CRDs). Between these two regions, a short coiled-coil of α-helices holds the CRDs in a fixed orientation so that they can bind carbohydrate arrays on target cell surfaces (Weis and Drickamer, 1994). At the extreme N-terminus of the polypeptide, disulfide bonds link polypeptides within and between trimeric subunits (Wallis and Drickamer, 1999). There are three known MASPs, denoted MASP-1, -2 and -3 (Takayama et al., 1994; Thiel et al., 1997; Dahl et al., 2001). Each MASP consists of six modular protein domains. The N-terminal three domains, comprising two CUB domains (Bork and Beckmann, 1993) flanking a single epidermal growth factor (EGF)-like domain, are responsible for interaction with MBP (Wallis and Dodd, 2000; Chen and Wallis, 2001). Two complement control repeats (CCPs, also known as sushi domains or short consensus repeats) link this portion of the molecule to the C-terminal serine protease (SP) domain. MASP-1 and MASP-2 are distinct gene products. MASP-3 is an alternatively spliced product of the MASP-1 gene, which is identical to MASP-1 except in the SP domain (Dahl et al., 2001). Although all three MASPs can form complexes with MBP, there is conflicting evidence about their roles in complement activation. There is general agreement that MASP-2 can activate C4 and C2 (Vorup-Jensen et al., 2000; Rossi et al., 2001). The role of MASP-1 is less clear: in one report, MASP-1 displays little to no activity on C2, C4 or C3 (Rossi et al., 2001), whereas MASP-1 was reported to activate C2 and C3 directly in other studies (Matsushita et al., 1998, 2000). The physiological role of MASP-3 is not known, but it may downregulate the activity of MASP-2 by competing for binding to MBP (Dahl et al., 2001). Dimers of trimeric MBP subunits are the smallest oligomers that fix complement (Chen and Wallis, 2001). The dimers form a 1:1 complex with homodimers of MASP-1 or MASP-2. Thus, a complex consisting of two MBP subunits and two MASP polypeptides represents the minimal complement-fixing unit. Calcium ions play two distinct roles in this complex. Weak Ca2+-binding sites, with dissociation constants of ∼0.1–1 mM (Kawasaki et al., 1983; Taylor and Summerfield, 1987; Weis et al., 1991; Ng and Weis, 1998), form an essential part of the carbohydrate-binding site in the CRDs of MBP. Ca2+ is also required for the interaction between MASPs and MBP (Wallis and Dodd, 2000; Chen and Wallis, 2001; Thielens et al., 2001); in this case, the affinity for Ca2+ is higher. MASP constructs spanning the CUB1-EGF-CUB2 region are proteolytically sensitive in the absence of Ca2+, suggesting that Ca2+ stabilizes the structure and relative orientations of these domains (Wallis and Dodd, 2000). There are strong parallels between the MBP–MASP complex and the C1qrs complex. Like MBP, C1q consists of multimers of a trimeric subunit that is held together by lateral association of N-terminal collagen-like domains (Arlaud et al., 2002). Interruptions in the collagen-like domains of both MBP and C1q create bends in the collagen triple helices, giving the oligomeric structures the overall appearance of bouquets. The C1r and C1s components associate with the collagen-like domain of C1q and have the same domain organization as the MASPs, and each can form homodimers. C1r and C1s interact to form a C1r2C1s2 tetramer that binds to C1q. Binding of C1q to antibodies activates C1r, which in turn activates C1s for action on the downstream complement components (Arlaud et al., 2002). In contrast, binding of the different MASPs to MBP appears to be mutually exclusive. MASP-2 is activated upon binding of MBP to cell surfaces, and subsequently activates C2 and C4, even in the absence of MASP-1/3 (Vorup-Jensen et al., 2000; Chen and Wallis, 2001). A recently published structure of the C-terminal portion of C1r has provided insights into the mechanism of activation of the SP domain (Budayova-Spano et al., 2002b). However, no information about the structure of the N-terminal domains of any of this family of molecules has been reported. In the work reported here, the structure of the three-domain fragment of MASP-2 that forms the MBP-binding region has been determined, revealing its probable mechanism for interaction with MBP and providing an explanation for the Ca2+ dependence of this interaction. Combining the various structures that are now known, it is possible to develop a plausible model for the activation complex between MBP and MASP. Results and discussion Structure determination The rat MASP-2 CUB1-EGF-CUB2 region (residues 1–280) was expressed in Chinese hamster ovary cells with a C-terminal His6 tag as described previously (Wallis and Dodd, 2000). Crystals of this material were grown in the presence of Ca2+ near neutral pH, and the structure was determined at 2.7 Å resolution (Table I). The asymmetric unit of the crystals contains one dimer. The CUB1 domain comprises residues 1–118, the EGF-like domain spans residues 119–163 and CUB2 contains residues 164–280. Residues 1–4 and 103–106 in CUB1, 125–131 in the EGF-like domain, and 218–221, 223–224 and 279–280 in CUB2, and the C-terminal His6 tag, are disordered, as are the side chains of seven other residues. Of the two N-linked glycosylation sites, the first GlcNAc residue is observed at Asn84 of CUB1, whereas no carbohydrate is visible at Asn266 of CUB2. The structure confirms the presence of the β-hydroxylation of Asn139 that is characteristic of many Ca2+-binding EGF domains. Table 1. Crystallographic statistics (A) Data collection Native K2PtCl4 Wavelength (Å) 0.979 1.06238 Resolution (Å) (last shell) 40–2.7 (2.77–2.7) 62–3.0 (3.08–3.0) Rsyma 0.086 (0.346) 0.074 (0.232) % completeness 98.7 (99.6) 97.2 (99.0) % anomalous completeness 96.0 (97.3) Average redundancy 2.8 4.8 (B) SIRAS phasing (to 4 Å) Isomorphous phasing powerc 0.86 Anomalous phasing powerc 0.93 Figure of merit 0.358 Figure of merit (4.17–4.00 Å) 0.287 R-factorb 0.248 Rfreeb 0.282 Average B-factor CUB1/EGF/CUB2 (Å2) 32.7/34.5/58.6 Bond length r.m.s.d. (Å) 0.0069 Angle r.m.s.d. (°) 1.4 Ramachandran plot (% in most favored/allowed/generous/disallowed regions) 81.3/16.8/1.4/0.5 a Rsym = ΣhΣi(|Ii(h)| − | |)/ΣhΣi Ii(h), where Ii(h) is the observed intensity and is the mean intensity obtained from multiple measurements. b R-factor and Rfree = Σ||Fo| − |Fc||/Σ|Fo|, where |Fo| is the observed structure factor amplitude and |Fc| is the calculated structure factor amplitude for the working and test sets, respectively. c Phasing power = <|FH|>/E, where <|FH|> is the r.m.s. calculated heavy atom scattering amplitude and E is the estimated lack of closure error. Protomer structure and Ca2+ binding The MASP-2 CUB1-EGF-CUB2 region has the shape of an elongated letter 'C' (Figure 1), with approximate dimensions 100 × 35 × 35 Å. Both CUB and EGF-like domains have elongated shapes, and the small interfaces between each pair of domains in the monomer occur at the ends of the domains. The first two domains are very well defined in the electron density, whereas the CUB2 domain is less well defined, as reflected in its much higher average temperature factor (Table IC). The CUB1 and EGF domains have more crystal- or non-crystallographic symmetry-related contacts than does the CUB2 domain, which may explain these differences. The relatively poor order of the CUB2 domain may also reflect limited flexibility between it and the EGF-like domain. In contrast, the interface between the CUB1 and EGF-like domains appears to be made rigid by Ca2+ binding to the latter (see below). Figure 1.Overall view of the rat MASP-2 CUB1-EGF-CUB2 protomer. CUB1 is shown in blue, the EGF-like domain in gray, CUB2 in red, Ca2+ in magenta and disulfide bonds in yellow. Download figure Download PowerPoint The only other known CUB domain structures are those of seminal plasma spermadhesins (Romao et al., 1997; Romero et al., 1997; Varela et al., 1997). Both the spermadhesin and MASP-2 CUB domains are β sandwich structures that contain two four-stranded antiparallel β-sheets (Figure 2A–D). The N-terminal portion of the spermadhesin polypeptides has two additional β strands, each of which forms a fifth parallel strand at the edge of a sheet (Figure 2C and D). Using the spermadhesin strand nomenclature, the MASP-2 CUB1 domain lacks both β1 and β2 strands, whereas the CUB2 domain contains the β2 strand. A small β hairpin is present in the loop between β7 and β8 of CUB1. All of the CUB domains contain a disulfide bond between the β5–β6 and the β7–β8 loops. The spermadhesin and the MASP-2 CUB2 domains have an additional disulfide bond linking the polypeptide preceding β2–β4 (Figure 2B and C). Superposition of the common eight-stranded core of acidic seminal fluid protein (aSFP) (Romao et al., 1997) and the MASP-2 CUB domains gives a root mean square deviation (r.m.s.d.) of 1.16 Å for CUB1 and 1.63 Å for CUB2 (64 Cα positions). Superposition of the two MASP-2 domains gives an r.m.s.d. of 1.45 Å. A structure-based sequence alignment based on comparison of aSFP and the MASP domains is presented in Figure 2E. Figure 2.The CUB domain. (A and B) Ribbon diagrams of MASP-2 CUB1 and CUB2. The color scheme is the same as Figure 1. (C) Ribbon diagram of bovine aSFP (Romao et al., 1997) (Protein Data Bank accession code ID 1SFP). (D) CUB domain topology. The MASP-2 CUB1 domain is shown in gray. β strands 1 and 2 appear in aSFP and other spermadhesin CUB domains, and β strand 2 is present in the MASP-2 CUB2 domain. β′ and β″ are two short strands formed from an extended loop found in CUB1 between β strands 7 and 8. (E) Structure-based sequence alignment of CUB domains. The secondary structure of MASP-2 CUB1 is shown above the sequences, and the secondary structure of aSFP is shown below. Highlighted residues are: green, compact dimer interface; pink, CUB1-EGF interface; yellow, cysteines in disulfide bonds. Phe36 is in both the dimer and EGF interfaces. The sequences and their SwissProt identifiers are: rat MASP-2, Q9JJS8; rat MASP-1, Q9JJS9; human C1r, P00736; human C1s, P09871; pig seminal plasma glycoprotein chain a (SPP-a), P35495; cow acidic seminal fluid protein, P29392. Download figure Download PowerPoint The MASP-2 EGF-like domain is very similar to other Ca2+-binding EGF-like domains (Figure 3). These domains feature an extended polypeptide followed by a long β hairpin and often, as in the MASP-2 domain, a second, smaller β hairpin near the C-terminus of this domain. The structure is stabilized by three disulfide bonds found in all EGF-like domains. Superposition of 31 structurally equivalent Cα positions between the MASP-2 and the Ca2+-binding EGF-like domain from factor IX (Rao et al., 1995) gives an r.m.s.d. of 1.65 Å. Figure 3.The EGF-like domain. (A and B) The EGF-like domains of MASP-2 and human coagulation factor IX (Protein Data Bank accession code ID 1EDM). Ca2+ is in magenta, disulfide bonds in yellow. Residues that contribute coordination bonds are shown in ball-and-stick representation, with red oxygen atoms and blue nitrogen atoms. In (B), Asn58 from a symmetry-related EGF domain is shown with green bonds. (C) Structure-based sequence alignment. Symbols above and below the alignments are: *, amino acid side chain Ca2+ ligand; ∧, main-chain carbonyl oxygen Ca2+ ligand; #, β-hydroxylated side chain Ca2+ ligand; !, side chain Ca2+ ligand from symmetry-related molecule (factor IX). Highlighted residues are: green, compact dimer interface; pink, CUB1-EGF interface; yellow, cysteines in disulfide bonds. SwissProt identifiers for rat MASP and human C1r and C1s sequences are given in the Figure 2 legend; the human factor IX sequence is SwissProt P00740. (D) The CUB1-EGF interface. The CUB1 domain is shown in blue, the EGF-like domain in gray. Residues involved in Ca2+ binding and packing interactions between the domains are shown in ball-and-stick representation. The Ca2+ is shown as a magenta sphere, with coordination bonds shown in pink. Download figure Download PowerPoint The single Ca2+ is bound near the N-terminal end of the EGF-like domain (Figure 3A). Only five coordination ligands are observed. Relative to the pentagonal bipyramidal Ca2+ coordination geometry observed in the factor IX EGF-like domain (Rao et al., 1995; Figure 3B), the two apical positions are occupied by main chain carbonyl oxygen atoms of Val120 and Tyr140. The side chains of Asp119 and Asn139, residues highly conserved amongst Ca2+-binding EGF-like domains (Stenflo et al., 2000; Figure 3C), each provides a coordination ligand. The second oxygen atom of the Asp119 side chain is too far (2.9 Å) from the Ca2+ to be considered a coordination ligand, but at the 2.7 Å resolution limit of these crystals this possibility cannot be ruled out. In factor IX, both carboxylate oxygen atoms of Asp64, the equivalent of MASP-2 Asn139, coordinate the Ca2+. Surprisingly, Glu122, which is also conserved amongst these domains and the equivalent Gln50 of which provides a coordination ligand in the factor IX domain (Rao et al., 1995), does not interact with the Ca2+; its coordination position is approximately replaced by a water molecule. In the crystal structure of factor IX, the final coordination position is occupied by a symmetry-related asparagine residue, whereas this position is vacant in the present structure. Ca2+-binding EGF-like domains have a low affinity for Ca2+, but the intrinsic mM affinity increases by up to three orders of magnitude in the presence of an adjacent protein domain (Stenflo et al., 2000). For example, the CUB1-EGF construct of C1r displays roughly 300× higher affinity for Ca2+ than the isolated EGF-like module (Thielens et al., 1999). The affinity of the MASP-2 CUB1-EGF-CUB2 fragment for Ca2+ was measured by isothermal titration calorimetry (Figure 4). Stoichiometric binding (occupancy 1.1 ± 0.2) with a Kd of 6.3 ± 2.6 μM, presumably corresponding to the conserved site in the EGF-like domain, is followed by a much weaker partial binding that is manifest as precipitation. Within one protomer, residues Glu118, Asp119, Val120 and Gly142, which lie in or near the Ca2+ site, also participate in interactions with Gly35, Phe36 and Arg37 in the CUB1 domain (Figure 3C and D). The water molecule that serves as a direct Ca2+ coordination ligand also forms hydrogen bonds with residues Gly35 of the CUB1 domain and Glu122 of the EGF-like domain. Figure 4.Ca2+-binding to the CUB-1-EGF-CUB-2 fragment of MASP-2 analyzed by isothermal titration calorimetry. Top: representative experiment showing heat release as a solution of CaCl2 was added to the MASP-2 fragment in 40 aliquots over 300 min. Bottom: data were fitted to a model in which there are two Ca2+-binding sites on each MASP fragment: a high-affinity site with an estimated occupancy (1.3) consistent with one Ca2+ on each MASP protomer and a lower affinity site. The occupancy of the low-affinity binding site (Kd > 40 μM) was <0.1 in all experiments, and probably reflects Ca2+-induced protein precipitation. Download figure Download PowerPoint There are also extensive interactions between the CUB1 and the EGF-like domains in the dimer interface (see below), including a hydrogen bond between Arg39 and the β-OH group of the Ca2+ ligand β-hydroxyAsn139 (Figure 5). Virtually all of the residues observed in the MASP-2 CUB1-EGF interface are conserved in character in MASP-1, C1r and C1s (Figures 2E and 3C). When Ca2+ is removed from Ca2+-binding EGF-like domains, the structure around the Ca2+ site undergoes significant conformational rearrangements (Rao et al., 1995; Stenflo et al., 2000). The intimate involvement of Asp119, Val120 and β-hydroxyAsn139 in both the CUB1 interface and Ca2+ ligation suggests that Ca2+ binding is a critical feature in determining the relative positions of the CUB1 and EGF-like domains in MASPs, C1r and C1s. Figure 5.The CUB1-EGF-CUB2 dimer. (A) The compact dimer. Left, view down the 2-fold symmetry axis [indicated by the ○ sign]. Right, view perpendicular to the 2-fold axis. The proposed MBP binding sites are indicated by solid or dashed circles, the diameters of which roughly correspond to that of a collagen triple helix. The distance between the sites in the dimer is indicated. (B) The extended dimer. Left, view perpendicular to the 2-fold axis (indicated by the arrow). Right, view down the 2-fold axis. The location of the proposed binding site [(A), solid circles] is shown; the filled circle indicates that one collagen helix would be antiparallel to the other. (C) The compact dimer interface. The two protomers are shown in blue and gray. Side chains participating in direct packing or hydrogen-bonding interactions are shown in ball-and-stick representation. Hydrogen bonds are shown in dashed green lines. The Ca2+ are shown as pink spheres. Water-mediated interactions have been omitted for clarity. Download figure Download PowerPoint Both MASP-1 and MASP-2 display increased resistance to trypsinolysis in the presence of Ca2+ (Wallis and Dodd, 2000). The proteolytically sensitive site at Arg173 is in the N-terminal region of the CUB2 domain, and forms part of the interface between the EGF-like and CUB2 domains. These data suggest that the binding of Ca2+ to the EGF-like domain may also influence the position of the CUB2 domain and thereby contribute to the formation of the MBP-binding site. Dimer structure and MBP binding The MASP-2 CUB1-EGF-CUB2 fragment studied here mediates dimerization of the protein and binding to MBP (Wallis and Dodd, 2000; Chen and Wallis, 2001). The crystallographic asymmetric unit contains a dimer and, combined with the packing of molecules in the crystal lattice, there are two distinct dimers present in the crystal. In the first, lateral association of the CUB1 and EGF-like domains about a 2-fold symmetry axis creates a relatively compact dimer (Figure 5A). The other dimer interface involves only the CUB1 domain, where antiparallel pairing of the β4 strands from two protomers creates a highly extended structure (Figure 5B). This pairing is distinct from the spermadhesin PSI/II dimer, which forms by antiparallel strand pairing on the opposite edge of the sandwich, using both sheets (Romero et al., 1997). Sedimentation equilibrium studies demonstrate that dimerization requires at most the first two domains and is independent of Ca2+ (Wallis and Dodd, 2000), but it has not been possible to produce the CUB1 domain in isolation to assess whether it alone can dimerize. Analytical ultracentrifugation was used to determine which of the two crystallographically observed dimers corresponds to the solution configuration of the CUB1-EGF-CUB2 fragment. The s20,w of the MASP-2 fragment determined by sedimentation velocity is 4.55 ± 0.10 S. Sedimentation coefficients calculated from the atomic coordinates of the two dimers gives values of 4.53 S for the more compact dimer (Figure 5A) and 4.18 S for the extended dimer (Figure 5B), indicating that the compact dimer is the solution species. Inspection of the interfaces also favors the compact dimer. Obligate oligomer interfaces tend to have significant numbers of non-polar interactions (Janin et al., 1988; Jones and Thornton, 1995). The compact dimer interface is larger (1844 Å2 total buried surface area) and features the packing of a number of hydrophobic side chains (Figure 5C), in contrast to the almost exclusively polar interactions that characterize the smaller (1540 Å2 total buried surface area) extended dimer interface (data not shown). The residues in the compact dimer interface are highly conserved among MASPs, C1r and C1s (Figures 2E and 3C). Finally, as discussed below, the symmetry of this dimer also favors its assignment as the solution species. Unless otherwise noted, the remainder of the discussion refers to the compact dimer. The CUB1-EGF-CUB2 dimer features two prominent concave surfaces that are large enough to accommodate a collagen-like triple helix, which including side chains has a diameter of ∼12 Å (Emsley et al., 2000). The first is formed by the 'C' shape of the protomer. A collagen triple helix can be placed in the middle of each protomer surface, where it would contact all three domains (Figure 5A, solid circles). These two sites are separated by ∼30 Å. A second concave surface is formed by CUB1 of one protomer and the EGF-like and CUB2 domains of the other protomer (Figure 5A, dashed circles); these two sites are separated by ∼65 Å. No mutational data are available that would localize the MBP binding site on the CUB1-EGF-CUB2 surface, and either of the proposed arrangements would be consistent with the observation that all three domains are required for MBP binding. However, the fact that the only subfragment of CUB1-EGF-CUB2 that displays even weak binding to MBP is the CUB1-EGF fragment suggests that the primary interactions involve these two domains (Chen and Wallis, 2001; Thielens et al., 2001). Of the two proposed binding sites shown in Figure 5A, the first (solid circles) has more extensive contact with the CUB1 and EGF-like domains, whereas much of the interaction surface in the second site (dashed circles) comes from CUB2, making it less likely. Electron microscopy of C1 complexes indicates that the C1r2C1s2 complex binds to the collagen-like portion of C1q just C-terminal to a kink created by an interruption in the Gly-X-Y sequence pattern (Strang et al., 1982; Weiss et al., 1986; Arlaud et al., 2002). Given the similarity of the two systems, it is likely that MASPs interact with MBPs in the comparable region. In MBPs, the N-terminal cysteine-rich domains and the first portion of the collagen-like region associate laterally in a parallel orientation, so that past the kink the individual trimeric subunits splay apart from one another but still run in a roughly parallel direction. Therefore, the collagen-like region of MBP must bind to the MASP dimer in an approximately parallel orientation. The 2-fold axis that generates the compact dimer is parallel to either proposed binding site, which would imply a parallel orientation for the bound MBP trimeric stalks. It is worth noting that for the first binding site (Figure 5A, solid circles), the 2-fold rotation axis of the alternative extended dimer in the crystals would put the two collagen-like stalks in a non-physiological, antiparallel arrangement (Figure 5B). In isolated C1q, the average angle between individual trimeric subunits past the kink is ∼40–50° (Schumaker et al., 1981). MBP bouquets look very similar to C1q, and the interruption in the Gly-X-Y pattern is the point at which the individual trimeric stalks kink and splay away from each other (Lu et al., 1990). Each Gly-X-Y triplet is 8.65 Å long in a standard collagen structure. Ignoring irregularities in the structure near the kink, the 12 Gly-X-Y repeats in the individual collagenous region of the stalks would be 103 Å long. Inspection of MBP sequences indicates that the most highly conserved region lies in the first five repeats following the kink. If MASPs bind in this region and two trimeric stalks are separated by 45°, the sites would be ∼30 Å apart at repeat 5, consistent with the proposed binding site in the middle of the protomer (Figure 5A, solid circles). It is likely that at least one Gly-X-Y repeat is irregular following the kink, so this spacing might be accommodated even closer to the kink. On the other hand, if the site were located near the C-terminal portion of the stalk, the other proposed binding sites (Figure 5A, dashed circles) could be accommodated. Given the sequence conservation and the electron microscopic data for the location of C1r/C1s on C1q (Strang et al., 1982), we favor the first alternative, with its smaller separation of sites as well as the smaller contribution of CUB2 to the binding surface (see above). High affinity binding to MBPs requires the first three MASP domains and is strictly dependent on Ca2+ (Wallis and Dodd, 2000; Chen and Wallis, 2001; Thielens et al., 2001). Analytical ultrace