C1r and C1s from Nile tilapia (Oreochromis niloticus): Molecular characterization, transcriptional profiling upon bacterial and IFN-γ inductions and potential role in response to bacterial infection.

The complement components C1r and C1s play a vital role in immunity with the activation of C1 complex in the classical complement pathway against pathogen infection. In this study, Nile tilapia (Oreochromis niloticus) C1r and C1s orthologs (OnC1r and OnC1s) were identified and characterized. The cDNA of OnC1r and OnC1s ORFs consisted of 1902 bp and 2100 bp of nucleotide sequence encoding polypeptides of 633 and 699 amino acids, respectively. The deduced OnC1r and OnC1s proteins both possessed CUB, EGF, CCP and SP domains, which were significantly homology to teleost. Spatial mRNA expression analysis revealed that the OnC1r and OnC1s were highly expressed in liver. After the in vivo challenges of Streptococcus agalactiae (S. agalactiae) and lipopolysaccharide (LPS), the mRNA expressions of OnC1r and OnC1s were significantly up-regulated in liver and spleen, which were consistent with immunohistochemical detection at the protein level. The up-regulation of OnC1r and OnC1s expressions were also demonstrated in head kidney monocytes/macrophages in vitro stimulated with LPS, S. agalactiae, and recombinant OnIFN-γ. Taken together, the results of this study indicated that OnC1r and OnC1s were likely to get involved in the immune response of Nile tilapia against bacterial infection.

Structure and activation of C1, the complex initiating the classical pathway of the complement cascade.

The complement system is an important antimicrobial and inflammation-generating component of the innate immune system. The classical pathway of complement is activated upon binding of the 774-kDa C1 complex, consisting of the recognition molecule C1q and the tetrameric protease complex C1r2s2, to a variety of activators presenting specific molecular patterns such as IgG- and IgM-containing immune complexes. A canonical model entails a C1r2s2 with its serine protease domains tightly packed together in the center of C1 and an intricate intramolecular reaction mechanism for activation of C1r and C1s, induced upon C1 binding to the activator. Here, we show that the serine protease domains of C1r and C1s are located at the periphery of the C1r2s2 tetramer both when alone or within the nonactivated C1 complex. Our structural studies indicate that the C1 complex adopts a conformation incompatible with intramolecular activation of C1, suggesting instead that intermolecular proteolytic activation between neighboring C1 complexes bound to a complement activating surface occurs. Our results rationalize how a multitude of structurally unrelated molecular patterns can activate C1 and suggests a conserved mechanism for complement activation through the classical and the related lectin pathway.

Characterization of rock bream (Oplegnathus fasciatus) complement components C1r and C1s in terms of molecular aspects, genomic modulation, and immune responsive transcriptional profiles following bacterial and viral pathogen exposure.

The complement components C1r and C1s play a crucial role in innate immunity via activation of the classical complement cascade system. As initiators of the pathogen-induced signaling cascade, C1r and C1s modulate innate immunity. In order to understand the immune responses of teleost C1r and C1s, Oplegnathus fasciatus C1r and C1s genes (OfC1r and OfC1s) were identified and characterized. The genomic sequence of OfC1r was enclosed with thirteen exons that represented a putative peptide with 704 amino acids (aa), whereas eleven exons of OfC1s represented a 691 aa polypeptide. In addition, genomic analysis revealed that both OfC1r and OfC1s were located on a single chromosome. These putative polypeptides were composed of two CUB domains, an EGF domain, two CCP domains, and a catalytically active serine protease domain. Phylogenetic analysis of C1r and C1s showed that OfC1r and OfC1s were evolutionary close to the orthologs of Pundamilia nyererei (identity = 73.4%) and Oryzias latipes (identity = 58.0%), respectively. Based on the results of quantitative real-time qPCR analysis, OfC1r and OfC1s transcripts were detected in all the eleven different tissues, with higher levels of OfC1r in blood and OfC1s in liver. The putative roles of OfC1r and OfC1s in response to pathogenic bacteria (Edwardsiella tarda and Streptococcus iniae) and virus (rock bream iridovirus, RBIV) were investigated in liver and head kidney tissues. The transcription of OfC1r and OfC1s was found to be significantly upregulated in response to pathogenic bacterial and viral infections. Overall findings of the present study demonstrate the potential immune responses of OfC1r and OfC1s against invading microbial pathogens and the activation of classical signaling cascade in rock bream.

TFPI inhibits lectin pathway of complement activation by direct interaction with MASP-2.

The lectin pathway (LP) of complement has a protective function against invading pathogens. Recent studies have also shown that the LP plays an important role in ischemia/reperfusion (I/R)-injury. MBL-associated serine protease (MASP)-2 appears to be crucial in this process. The serpin C1-inhibitor is the major inhibitor of MASP-2. In addition, aprotinin, a Kunitz-type inhibitor, was shown to inhibit MASP-2 activity in vitro. In this study we investigated whether the Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI) is also able to inhibit MASP-2. Ex vivo LP was induced and detected by C4-deposition on mannan-coated plates. The MASP-2 activity was measured in a fluid-phase chromogenic assay. rTFPI in the absence or presence of specific monoclonal antibodies was used to investigate which TFPI-domains contribute to MASP-2 inhibition. Here, we identify TFPI as a novel selective inhibitor of MASP-2, without affecting MASP-1 or the classical pathway proteases C1s and C1r. Kunitz-2 domain of TFPI is required for the inhibition of MASP-2. Considering the role of MASP-2 in complement-mediated I/R-injury, the inhibition of this protease by TFPI could be an interesting therapeutic approach to limit the tissue damage in conditions such as cerebral stroke, myocardial infarction or solid organ transplantation.

The serine protease domain of MASP-3: enzymatic properties and crystal structure in complex with ecotin.

Mannan-binding lectin (MBL), ficolins and collectin-11 are known to associate with three homologous modular proteases, the MBL-Associated Serine Proteases (MASPs). The crystal structures of the catalytic domains of MASP-1 and MASP-2 have been solved, but the structure of the corresponding domain of MASP-3 remains unknown. A link between mutations in the MASP1/3 gene and the rare autosomal recessive 3MC (Mingarelli, Malpuech, Michels and Carnevale,) syndrome, characterized by various developmental disorders, was discovered recently, revealing an unexpected important role of MASP-3 in early developmental processes. To gain a first insight into the enzymatic and structural properties of MASP-3, a recombinant form of its serine protease (SP) domain was produced and characterized. The amidolytic activity of this domain on fluorescent peptidyl-aminomethylcoumarin substrates was shown to be considerably lower than that of other members of the C1r/C1s/MASP family. The E. coli protease inhibitor ecotin bound to the SP domains of MASP-3 and MASP-2, whereas no significant interaction was detected with MASP-1, C1r and C1s. A tetrameric complex comprising an ecotin dimer and two MASP-3 SP domains was isolated and its crystal structure was solved and refined to 3.2 Å. Analysis of the ecotin/MASP-3 interfaces allows a better understanding of the differential reactivity of the C1r/C1s/MASP protease family members towards ecotin, and comparison of the MASP-3 SP domain structure with those of other trypsin-like proteases yields novel hypotheses accounting for its zymogen-like properties in vitro.

Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites.

Complement C1q is a hexameric molecule assembled from 18 polypeptide chains of three different types encoded by three genes. This versatile recognition protein senses a wide variety of immune and nonimmune ligands, including pathogens and altered self components, and triggers the classical complement pathway through activation of its associated proteases C1r and C1s. We report a method for expression of recombinant full-length human C1q involving stable transfection of HEK 293-F mammalian cells and fusion of an affinity tag to the C-terminal end of the C chain. The resulting recombinant (r) C1q molecule is similar to serum C1q as judged from biochemical and structural analyses and exhibits the characteristic shape of a bunch of flowers. Analysis of its interaction properties by surface plasmon resonance shows that rC1q retains the ability of serum C1q to associate with the C1s-C1r-C1r-C1s tetramer, to recognize physiological C1q ligands such as IgG and pentraxin 3, and to trigger C1r and C1s activation. Functional analysis of rC1q variants carrying mutations of LysA59, LysB61, and/or LysC58, in the collagen-like stems, demonstrates that LysB61 and LysC58 each play a key role in the interaction with C1s-C1r-C1r-C1s, with LysA59 being involved to a lesser degree. We propose that LysB61 and LysC58 both form salt bridges with outer acidic Ca(2+) ligands of the C1r and C1s CUB (complement C1r/C1s, Uegf, bone morphogenetic protein) domains. The expression method reported here opens the way for deciphering the molecular basis of the unusual binding versatility of C1q by mapping the residues involved in the sensing of its targets and the binding of its receptors.

Molecular determinants of the substrate specificity of the complement-initiating protease, C1r.

The serine protease, C1r, initiates activation of the classical pathway of complement, which is a crucial innate defense mechanism against pathogens and altered-self cells. C1r both autoactivates and subsequently cleaves and activates C1s. Because complement is implicated in many inflammatory diseases, an understanding of the interaction between C1r and its target substrates is required for the design of effective inhibitors of complement activation. Examination of the active site specificity of C1r using phage library technology revealed clear specificity for Gln at P2 and Ile at P1', which are found in these positions in physiological substrates of C1r. Removal of one or both of the Gln at P2 and Ile at P1' in the C1s substrate reduced the rate of C1r activation. Substituting a Gln residue into the P2 of the activation site of MASP-3, a protein with similar domain structure to C1s that is not normally cleaved by C1r, enabled efficient activation of this enzyme. Molecular dynamics simulations and structural modeling of the interaction of the C1s activation peptide with the active site of C1r revealed the molecular mechanisms that particularly underpin the specificity of the enzyme for the P2 Gln residue. The complement control protein domains of C1r also made important contributions to efficient activation of C1s by this enzyme, indicating that exosite interactions were also important. These data show that C1r specificity is well suited to its cleavage targets and that efficient cleavage of C1s is achieved through both active site and exosite contributions.

Roles of CUB and LDL receptor class A domain repeats of a transmembrane serine protease matriptase in its zymogen activation.

Matriptase is a type II transmembrane serine protease containing two complement proteases C1r/C1s-urchin embryonic growth factor-bone morphogenetic protein domains (CUB repeat) and four low-density lipoprotein receptor class A domains (LDLRA repeat). The single-chain zymogen of matriptase has been found to exhibit substantial protease activity, possibly causing its own activation (i.e. conversion to a disulfide-linked two-chain fully active form), although the activation seems to be mediated predominantly by two-chain molecules. Our aim was to assess the roles of CUB and LDLRA repeats in zymogen activation. Transient expression studies of soluble truncated constructs of recombinant matriptase in COS-1 cells showed that the CUB repeat had an inhibitory effect on zymogen activation, possibly because it facilitated the interaction of two-chain molecules with a matriptase inhibitor, hepatocyte growth factor activator inhibitor type-1. By contrast, the LDLRA repeat had a promoting effect on zymogen activation. The effect of the LDLRA repeat seems to reflect its ability to increase zymogen activity. The proteolytic activities were higher in pseudozymogen forms of recombinant matriptase containing the LDLRA repeat than in a pseudozymogen without the repeat. Our findings provide new insights into the roles of these non-catalytic domains in the generation of active matriptase.

Structure of the haptoglobin-haemoglobin complex.

Red cell haemoglobin is the fundamental oxygen-transporting molecule in blood, but also a potentially tissue-damaging compound owing to its highly reactive haem groups. During intravascular haemolysis, such as in malaria and haemoglobinopathies, haemoglobin is released into the plasma, where it is captured by the protective acute-phase protein haptoglobin. This leads to formation of the haptoglobin-haemoglobin complex, which represents a virtually irreversible non-covalent protein-protein interaction. Here we present the crystal structure of the dimeric porcine haptoglobin-haemoglobin complex determined at 2.9 Å resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected ß-strand swap between two complement control protein (CCP) domains, defining a new fusion CCP domain structure. The haptoglobin serine protease domain forms extensive interactions with both the α- and ß-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the αß dimer is highly overlapping with the interface between the two αß dimers that constitute the native haemoglobin tetramer. Several haemoglobin residues prone to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin-haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin-haemoglobin to the macrophage scavenger receptor CD163 (ref. 3) protrudes from the surface of the distal end of the complex, adjacent to the associated haemoglobin α-subunit. Small-angle X-ray scattering measurements of human haptoglobin-haemoglobin bound to the ligand-binding fragment of CD163 confirm receptor binding in this area, and show that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of multimeric haptoglobin-haemoglobin for CD163 (ref. 4).

A molecular dynamics study of C1r and C1s dimers: implications for the structure of the C1 complex.

Complement is an important part of the immune system. It is initiated through three different pathways known as the classical, lectin, and alternative pathway. The multimolecular C1 complex of the classical pathway consists of a subcomponent, C1q, which binds to a tetramer comprising two C1r and two C1s proteases. A detailed description of the structure of the C1 complex is essential to fully understand how the complex acts on pathogens. A variety of different models have been proposed, which differ mainly in the way the proteases interact with C1q. In this study, we have used a combination of homology-based structure prediction and molecular dynamics to predict a partial structure of the C1s/C1r/C1r/C1s tetramer. For computational expediency the study was restricted to the CUB(1) -EGF-CUB(2) domains which are directly involved in the formation of the tetramer and its interaction with C1q; the catalytic fragments (CCP(1) -CCP(2) -SP), which mediate C1 activation and subsequent cleavage of substrates, were omitted. A systematic molecular dynamics (MD) study of several possible dimeric combinations suggest that the tetramer is formed when a pair of C1r/C1s dimers form a "doughnut" via a C1s/C1s head-to-tail interaction, which is stabilized by several putative salt bridges at the dimer interface. This result is consistent with biochemical data which have shown that self assembly requires the formation of C1r-C1s contacts and that electrostatic interactions play a key role. Furthermore, it identifies a number of putative binding residues that can be tested using site-directed mutagenesis.

Interaction between separated consecutive complement control modules of human C1r: implications for dimerization of the full-length protease.

Complement control protein modules (CCP) typically mediate protein:protein interaction during immune response in vertebrates. Using NMR chemical shift perturbation mapping, we present previously lacking experimental evidence for intermolecular interactions between the CCP1 and CCP2 modules of the human C1r serine protease (SP). The identified interface is clearly distinct from that observed in the covalently linked CCP1-CCP2 pair. Structural models of the CCP1-CCP2-SP segments of two C1r molecules built on the basis of shift perturbation data are fully consistent with an extended interaction interface and suggests the possibility of a structural rearrangement as a switch between functional states of human C1r.

Intermodule cooperativity in the structure and dynamics of consecutive complement control modules in human C1r: structural biology.

The modular C1r protein is the first protease activated in the classical complement pathway, a key component of innate immunity. Activation of the heteropentameric C1 complex, possibly accompanied by major intersubunit re-arrangements besides proteolytic cleavage, requires targeted regulation of flexibility within the context of the intramolecular and intermolecular interaction networks of the complex. In this study, we prepared the two complement control protein (CCP) modules, CCP1 and CCP2, of C1r in their free form, as well as their tandem-linked construct, CCP1CCP2, to characterize their solution structure, conformational dynamics and cooperativity. The structures derived from NMR signal dispersion and secondary chemical shifts were in good agreement with those obtained by X-ray crystallography. However, successful heterologus expression of both the single CCP1 module and the CCP1CCP2 constructs required the attachment of the preceding N-terminal module, CUB2, which could then be removed to obtain the properly folded proteins. Internal mobility of the modules, especially that of CCP1, exhibited considerable changes accompanied by interfacial chemical shift alterations upon the attachment of the C-terminal CCP2 domain. Our NMR data suggest that in terms of folding, stability and dynamics, CCP1 is heavily dependent on the presence of its neighboring modules in intact C1r. Therefore, CCP1 could be a focal interaction point, capable of transmitting information towards its neighboring modules.

Mapping surface accessibility of the C1r/C1s tetramer by chemical modification and mass spectrometry provides new insights into assembly of the human C1 complex.

C1, the complex that triggers the classic pathway of complement, is a 790-kDa assembly resulting from association of a recognition protein C1q with a Ca(2+)-dependent tetramer comprising two copies of the proteases C1r and C1s. Early structural investigations have shown that the extended C1s-C1r-C1r-C1s tetramer folds into a compact conformation in C1. Recent site-directed mutagenesis studies have identified the C1q-binding sites in C1r and C1s and led to a three-dimensional model of the C1 complex (Bally, I., Rossi, V., Lunardi, T., Thielens, N. M., Gaboriaud, C., and Arlaud, G. J. (2009) J. Biol. Chem. 284, 19340-19348). In this study, we have used a mass spectrometry-based strategy involving a label-free semi-quantitative analysis of protein samples to gain new structural insights into C1 assembly. Using a stable chemical modification, we have compared the accessibility of the lysine residues in the isolated tetramer and in C1. The labeling data account for 51 of the 73 lysine residues of C1r and C1s. They strongly support the hypothesis that both C1s CUB(1)-EGF-CUB(2) interaction domains, which are distant in the free tetramer, associate with each other in the C1 complex. This analysis also provides the first experimental evidence that, in the proenzyme form of C1, the C1s serine protease domain is partly positioned inside the C1q cone and yields precise information about its orientation in the complex. These results provide further structural insights into the architecture of the C1 complex, allowing significant improvement of our current C1 model.

Calcium-dependent conformational flexibility of a CUB domain controls activation of the complement serine protease C1r.

C1, the first component of the complement system, is a Ca(2+)-dependent heteropentamer complex of C1q and two modular serine proteases, C1r and C1s. Current functional models assume significant flexibility of the subcomponents. Noncatalytic modules in C1r have been proposed to provide the flexibility required for function. Using a recombinant CUB2-CCP1 domain pair and the individual CCP1 module, we showed that binding of Ca(2+) induces the folding of the CUB2 domain and stabilizes its structure. In the presence of Ca(2+), CUB2 shows a compact, folded structure, whereas in the absence of Ca(2+), it has a flexible, disordered conformation. CCP1 module is Ca(2+)-insensitive. Isothermal titration calorimetry revealed that CUB2 binds a single Ca(2+) with a relatively high K(D) (430 mum). In blood, the CUB2 domain of C1r is only partially (74%) saturated by Ca(2+), therefore the disordered, Ca(2+)-free form could provide the flexibility required for C1 activation. In accordance with this assumption, the effect of Ca(2+) on the autoactivation of native, isolated C1r zymogen was proved. In the case of infection-inflammation when the local Ca(2+) concentration decreases, this property of CUB2 domain could serve as subtle means to trigger the activation of the classical pathway of complement. The CUB2 domain of C1r is a novel example for globular protein domains with marginal stability, high conformational flexibility, and proteolytic sensitivity. The physical nature of the behavior of this domain is similar to that of intrinsically unstructured proteins, providing a further example of functionally relevant ligand-induced reorganization of a polypeptide chain.

Analysis of human C1q by combined bottom-up and top-down mass spectrometry: detailed mapping of post-translational modifications and insights into the C1r/C1s binding sites.

C1q is a subunit of the C1 complex, a key player in innate immunity that triggers activation of the classical complement pathway. Featuring a unique structural organization and comprising a collagen-like domain with a high level of post-translational modifications, C1q represents a challenging protein assembly for structural biology. We report for the first time a comprehensive proteomics study of C1q combining bottom-up and top-down analyses. C1q was submitted to proteolytic digestion by a combination of collagenase and trypsin for bottom-up analyses. In addition to classical LC-MS/MS analyses, which provided reliable identification of hydroxylated proline and lysine residues, sugar loss-triggered MS(3) scans were acquired on an LTQ-Orbitrap (Linear Quadrupole Ion Trap-Orbitrap) instrument to strengthen the localization of glucosyl-galactosyl disaccharide moieties on hydroxylysine residues. Top-down analyses performed on the same instrument allowed high accuracy and high resolution mass measurements of the intact full-length C1q polypeptide chains and the iterative fragmentation of the proteins in the MS(n) mode. This study illustrates the usefulness of combining the two complementary analytical approaches to obtain a detailed characterization of the post-translational modification pattern of the collagen-like domain of C1q and highlights the structural heterogeneity of individual molecules. Most importantly, three lysine residues of the collagen-like domain, namely Lys(59) (A chain), Lys(61) (B chain), and Lys(58) (C chain), were unambiguously shown to be completely unmodified. These lysine residues are located about halfway along the collagen-like fibers. They are thus fully available and in an appropriate position to interact with the C1r and C1s protease partners of C1q and are therefore likely to play an essential role in C1 assembly.

Early complement proteases: C1r, C1s and MASPs. A structural insight into activation and functions.

C1r, C1s and the mannose-binding lectin-associated serine proteases (MASPs) are responsible for the initiation of the classical- and lectin pathway activation of the complement system. These enzymes do not act alone, but form supramolecular complexes with pattern recognition molecules such as C1q, MBL, and ficolins. They share the same domain organization but have different substrate specificities and fulfill different physiological functions. In the recent years the rapid progress of structural biology facilitated the understanding of the molecular mechanism of complement activation at atomic level. In this review we summarize our current knowledge about the structure and function of the early complement proteases, delineate the latest models of the multimolecular complexes and present the functional consequences inferred from the structural studies. We also discuss some open questions and debated issues that need to be resolved in the future.

Identification of the C1q-binding Sites of Human C1r and C1s: a refined three-dimensional model of the C1 complex of complement.

The C1 complex of complement is assembled from a recognition protein C1q and C1s-C1r-C1r-C1s, a Ca(2+)-dependent tetramer of two modular proteases C1r and C1s. Resolution of the x-ray structure of the N-terminal CUB(1)-epidermal growth factor (EGF) C1s segment has led to a model of the C1q/C1s-C1r-C1r-C1s interaction where the C1q collagen stem binds at the C1r/C1s interface through ionic bonds involving acidic residues contributed by the C1r EGF module (Gregory, L. A., Thielens, N. M., Arlaud, G. J., Fontecilla-Camps, J. C., and Gaboriaud, C. (2003) J. Biol. Chem. 278, 32157-32164). To identify the C1q-binding sites of C1s-C1r-C1r-C1s, a series of C1r and C1s mutants was expressed, and the C1q binding ability of the resulting tetramer variants was assessed by surface plasmon resonance. Mutations targeting the Glu(137)-Glu-Asp(139) stretch in the C1r EGF module had no effect on C1 assembly, ruling out our previous interaction model. Additional mutations targeting residues expected to participate in the Ca(2+)-binding sites of the C1r and C1s CUB modules provided evidence for high affinity C1q-binding sites contributed by the C1r CUB(1) and CUB(2) modules and lower affinity sites contributed by C1s CUB(1). All of the sites implicate acidic residues also contributing Ca(2+) ligands. C1s-C1r-C1r-C1s thus contributes six C1q-binding sites, one per C1q stem. Based on the location of these sites and available structural information, we propose a refined model of C1 assembly where the CUB(1)-EGF-CUB(2) interaction domains of C1r and C1s are entirely clustered inside C1q and interact through six binding sites with reactive lysines of the C1q stems. This mechanism is similar to that demonstrated for mannan-binding lectin (MBL)-MBL-associated serine protease and ficolin-MBL-associated serine protease complexes.

Crystal structure of the CUB1-EGF-CUB2 domain of human MASP-1/3 and identification of its interaction sites with mannan-binding lectin and ficolins.

MASP-1 and MASP-3 are homologous proteases arising from alternative splicing of the MASP1/3 gene. They include an identical CUB(1)-EGF-CUB(2)-CCP(1)-CCP(2) module array prolonged by different serine protease domains at the C-terminal end. The x-ray structure of the CUB(1)-EGF-CUB(2) domain of human MASP-1/3, responsible for interaction of MASP-1 and -3 with their partner proteins mannan-binding lectin (MBL) and ficolins, was solved to a resolution of 2.3A(.) The structure shows a head-to-tail homodimer mainly stabilized by hydrophobic interactions between the CUB(1) module of one monomer and the epidermal growth factor (EGF) module of its counterpart. A Ca(2+) ion bound primarily to both EGF modules stabilizes the intra- and inter-monomer CUB(1)-EGF interfaces. Additional Ca(2+) ions are bound to each CUB(1) and CUB(2) module through six ligands contributed by Glu(49), Asp(57), Asp(102), and Ser(104) (CUB(1)) and their counterparts Glu(216), Asp(226), Asp(263), and Ser(265) (CUB(2)), plus one and two water molecules, respectively. To identify the residues involved in interaction of MASP-1 and -3 with MBL and L- and H-ficolins, 27 point mutants of human MASP-3 were generated, and their binding properties were analyzed using surface plasmon resonance spectroscopy. These mutations map two homologous binding sites contributed by modules CUB(1) and CUB(2), located in close vicinity of their Ca(2+)-binding sites and stabilized by the Ca(2+) ion. This information allows us to propose a model of the MBL-MASP-1/3 interaction, involving a major electrostatic interaction between two acidic Ca(2+) ligands of MASP-1/3 and a conserved lysine of MBL. Based on these and other data, a schematic model of a MBL.MASP complex is proposed.

The initiating proteases of the complement system: controlling the cleavage.

The complement system is a vital component of the host immune system, but when dysregulated, can also cause disease. The system is activated by three pathways: classical, lectin and alternative. The initiating proteases of the classical and lectin pathways have similar domain structure and employ similar mechanisms of activation. The C1r, C1s and MASP-2 proteases have the most defined roles in the activation of the system. This review focuses on the mechanisms whereby their interaction with substrates and inhibitors is regulated.

Revisiting the mechanism of the autoactivation of the complement protease C1r in the C1 complex: structure of the active catalytic region of C1r.

C1r is a modular serine protease which is the autoactivating component of the C1 complex of the classical pathway of the complement system. We have determined the first crystal structure of the entire active catalytic region of human C1r. This fragment contains the C-terminal serine protease (SP) domain and the preceding two complement control protein (CCP) modules. The activated CCP1-CCP2-SP fragment makes up a dimer in a head-to-tail fashion similarly to the previously characterized zymogen. The present structure shows an increased number of stabilizing interactions. Moreover, in the crystal lattice there is an enzyme-product relationship between the C1r molecules of neighboring dimers. This enzyme-product complex exhibits the crucial S1-P1 salt bridge between Asp631 and Arg446 residues, and intermolecular interaction between the CCP2 module and the SP domain. Based on these novel structural information we propose a new split-and-reassembly model for the autoactivation of the C1r. This model is consistent with experimental results that have not been explained adequately by previous models. It allows autoactivation of C1r without large-scale, directed movement of C1q arms. The model is concordant with the stability of the C1 complex during activation of the next complement components.