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

Abstract

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 Ca(2+)-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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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 (K_d > 40 µM) was <0.1 in all experiments, and probably reflects Ca²⁺-induced protein precipitation.

Fig. 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.

Fig. 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.

Fig. 6. Ca²⁺ dependence of MASP-2 binding to MBP-A dimers analyzed by sedimentation velocity. Top: apparent sedimentation distributions [g(s*)] of mixtures of the CUB-1-EGF-CUB-2 fragment of MASP-2 (0.38 mg/ml) and MBP-A dimers (0.075 mg/ml) in the presence of increasing concentrations of CaCl₂. The apparent sedimentation coefficients (s*) of the uncomplexed components were 4.5 S and 5.0 S for the MASP-2 fragment and MBP-A dimer, respectively. Bottom: the average sedimentation coefficient of each mixture plotted as a function of Ca²⁺ concentration.

Fig. 7. Model of MASP-2 and interaction with the MBP dimer. (A) The N-termini of two molecules of human C1r CCP1-CCP2-SP (Protein Data Bank accession code ID 1GPZ) were positioned near the C-termini of the CUB1-EGF-CUB2 dimer, preserving symmetry about the 2-fold axis of the dimer (arrow). The CUB1-EGF-CUB2 dimer is colored blue and gray, Ca²⁺ in magenta, and the CCP1-CCP2-SP chains in dark and light green. (B) Changes were applied to the junctions between domains as indicated by the arrows in order to bring the catalytic sites of the SP domains into proximity of one another. (C) Model of a dimer of trimeric MBP subunits bound to the MASP2 model shown in (A). Coordinates of the trimeric coiled-coil neck and lectin region of rat MBP-A (blue, PDB ID 1RTM) were placed at the C-terminus of a model of the collagen-like region (red) generated from a collagen peptide [(Pro-Pro-Gly)₁₀]₃ (Berisio et al., 2002) (Protein Data Bank accession code ID 1K6F); the dashed lines represent the flexible joint between these two regions of the MBP primary structure. The green arrows point to the Ca²⁺ (magenta) at which carbohydrate ligands from a pathogen bind. The gap in the collagen connected by three dashed lines represents the interruption in the collagen consensus sequence, where the trimeric stalks splay away from the kink. Other dashed lines in the model represent protein links between the different domains. (D) Same as (C), but rotated ∼90° about the vertical axis. (E) The dimer observed in the crystal structure of the human C1r CCP1-CCP2-SP zymogen (Protein Data Bank accession code ID 1GPZ), proposed to correspond to an inactive form due to blockage of the catalytic site of one SP by CCP1 of the other protomer (Budayova-Spano et al., 2002b); colored as in (A)–(D). (F) Model of inactive MASP-2 made by moving CUB2 relative to CUB1-EGF (curved arrow) such that the N-terminal ends of the C1r CCP1-CCP2-SP zymogen dimer are near the C-termini of the CUB1-EGF-CUB2, bound to two MBP trimers. The MBP dimer model is that shown in (C) and (D). (G) Same as (F), but rotated ∼90° about the vertical axis.