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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., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

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., (© 2010 The Authors Journal compilation © 2010 FEBS.)

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.

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.

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.

The first enzymatic event in the classical pathway of complement activation is autoactivation of the C1r subcomponent of the C1 complex. Activated C1r then cleaves and activates zymogen C1s. C1r is a multidomain serine protease consisting of N-terminal alpha region interacting with other subcomponents and C-terminal gammaB region mediating proteolytic activity. The gammaB region consists of two complement control protein modules (CCP1, CCP2) and a serine protease domain (SP). To clarify the role of the individual domains in the structural and functional properties of the gammaB region we produced the CCP1-CCP2-SP (gammaB), the CCP2-SP, and the SP fragments in recombinant form in Escherichia coli. We successfully renatured the inclusion body proteins. After renaturation all three fragments were obtained in activated form and showed esterolytic activity on synthetic substrates similar to each other. To study the self-activation process in detail zymogen mutant forms of the three fragments were constructed and expressed. Our major statement is that the ability of autoactivation and C1s cleavage is an inherent property of the SP domain. We observed that the CCP2 module significantly increases proteolytic activity of the SP domain on natural substrate, C1s. Therefore, we propose that CCP2 module provides accessory binding sites. Differential scanning calorimetric measurements demonstrated that CCP2 domain greatly stabilizes the structure of SP domain. Deletion of CCP1 domain from the CCP1-CCP2-SP fragment results in the loss of the dimeric structure. Our experiments also provided evidence that dimerization of C1r is not a prerequisite for autoactivation.

The catalytic properties of C1r, the protease that mediates activation of the C1 complex of complement, are mediated by its C-terminal region, comprising two complement control protein (CCP) modules followed by a serine protease (SP) domain. Baculovirus-mediated expression was used to produce fragments containing the SP domain and either 2 CCP modules (CCP1/2-SP) or only the second CCP module (CCP2-SP). In each case, the wild-type species and two mutants stabilized in the proenzyme form by mutations at the cleavage site (R446Q) or at the active site serine residue (S637A), were produced. Both wild-type fragments were recovered as two-chain, activated proteases, whereas all mutants retained a single-chain, proenzyme structure, providing the first experimental evidence that C1r activation is an autolytic process. As shown by sedimentation velocity analysis, all CCP1/2-SP fragments were dimers (5.5-5.6 S), and all CCP2-SP fragments were monomers (3.2-3.4 S). Thus, CCP1 is essential to the assembly of the dimer, but formation of a stable dimer is not a prerequisite for self-activation. Activation of the R446Q mutants could be achieved by extrinsic cleavage by thermolysin, which cleaved the CCP2-SP species more efficiently than the CCP1/2-SP species and yielded enzymes with C1s-cleaving activities similar to their active wild-type counterparts. C1r and its activated fragments all cleaved C1s, with relative efficiencies in the order C1r < CCP1/2-SP < CCP2-SP, indicating that CCP1 is not involved in C1s recognition.

The activation of the C1s-C1r-C1r-C1s tetramer in the C1 complex, which involves the cleavage of an Arg-Ile bond in the catalytic domains of the subcomponents, is a two-step process. First, the autolytic activation of C1r takes place, then activated C1r cleaves zymogen C1s. The Arg463Gln mutant of C1r (C1rQI) is stabilized in the zymogen form. This mutant was used to form a C1q-(C1s-C1rQI-C1r-C1s) heteropentamer to study the relative position of the C1r and C1s subunits in the C1 complex. After triggering the C1 by IgG-Sepharose, both C1s subunits are cleaved by the single proteolytically active C1r subunit in the C1s-C1rQI-C1r-C1s tetramer. This finding indicates that the tetramer is flexible enough to adopt different conformations within the C1 complex during the activation process, enabling the single active C1r to cleave both C1s, the neighboring and the sequentially distant one.

The binding of C1 (the first component of complement) to immune complexes leads to the autoactivation of C1r through the cleavage of the Arg463-Ile464 bond in the catalytic domain. Spontaneous activation of C1r (and C1) also occurs in the fluid phase, preventing the characterization of the zymogen form of C1r. To overcome this difficulty, the zymogen form of human C1r was stabilized by mutating the Arg in the Arg463-Ile464 bond to Gln. This mutant was designated as mutant QI. Recombinant C1r (wild type (wt) or mutant) was expressed in insect cells using serum-free medium in functionally pure form; therefore, the cell culture supernatant was suitable to reconstruct C1 for the hemolytic assay. Mutant QI was a stable, nonactivable zymogen and showed no hemolytic activity in reconstituted C1. However, this stable zymogen C1r mutant could form an active mixed dimer with the wt C1r, indicating that one active C1r subunit in the C1 complex is sufficient for the full activity of the entire complex. Our experiments also showed that the exchange of C1r monomers between the C1r dimers is completed in less than 16 h even at pH 7 and 4 degrees C. Two other mutants were also constructed by changing Arg463 to Lys, or Ile464 to Phe, and were designated as mutants KI and RF, respectively. Although these substitutions did increase the stability of the proenzyme in the cell culture supernatant, the mutant proteins retained their ability to autoactivate, and both had a wt-like hemolytic activity.

Our protein engineering studies on human C1r and C1s revealed important characteristics of the individual domains of these multidomain serine-proteases, and supplied evidence about the cooperation of the domains to create binding sites, and to control the activation process. We expressed the recombinant subcomponents in the baculovirus-insect cell system and checked the biological activity. Deletions and point mutants of C1r were constructed and C1r-C1s chimeras were also produced. Our deletion mutants demonstrated that the N-terminal CUB domain and the EGF-like domain of C1r together are responsible for the calcium dependent C1r-C1s interaction. It seems very likely that these two modules form the calcium-binding site of the C1r alpha-fragment and participate in the tetramer formation. The deletion mutants also demonstrated that the N-terminal region of the C1r molecule contains essential elements involved in the control of activation of the serine-protease module. The substrate specificity of the serine-protease is also determined by the five N-terminal noncatalytic domain of C1r/C1s chimera, which contains the catalytic domain of C1s preceded by the N-terminal region of C1r, could replace the C1r in the hemolytically active C1 complex. The C1s/C1r chimera, in which the alpha-fragment of the C1r was replaced for that of the C1s exibits both C1r- and C1s-like characteristics. We stabilized the zymogen form of human C1r by mutating the Arg(463)-Ile(464) bond. Using our stable zymogen C1r we showed that one active C1r in the C1 complex is sufficient for the full activity of the entire complex. Further experiment with this mutant could provide us with important information about the structure of the C1 complex.

The C1r subcomponent of the first component of complement is a complex, multidomain glycoprotein containing five regulatory or binding modules in addition to the serine protease domain. To reveal the functional role of the N-terminal regulatory domains, two deletion mutants of C1r were constructed. One mutant comprises the N-terminal half of domain I joined to the second half of the highly homologous domain III, resulting in one chimeric domain in the N-terminal region, instead of domains I-III. In the second mutant most of the N-terminal portion of domain I was deleted. Both deletion mutants were expressed in the baculovirus-insect cell expression system with yields typical of wild type C1r. Both mutants maintained the ability of the wild type C1r to dimerize. The folding and secretion of the recombinant proteins was not affected by these deletions, and C1-inhibitor binding was not impaired. The stability of the zymogen was significantly decreased however, indicating that the N-terminal region of the C1r molecule contains essential elements involved in the control of activation of the serine protease module. Tetramer formation with C1s in the presence of Ca2+ was abolished by both deletions. We suggest that the first domain of C1r is essential for tetramer formation, since the deletion of domain I from C1r impairs this interaction.

The availability of cDNA and genomic clones for the subcomponents of C1, as well as the recognition of the modular organization of serine-proteases have opened up exciting new possibilities for approaching structural problems. In this review the latest achievements of combined protein engineering, functional and structural studies are summarized. The concept of this research is to construct deletion, point and hybrid mutants of the highly homologous C1r and C1s subcomponents, to reveal the functional role of individual modules, map the interaction sites between subcomponents of the C1 complex and refine the structural model of C1. The first prerequisite of such an approach was the expression of the subcomponents in a eukaryotic system, in biologically active form. This was followed by expression of various mutants. Autographa californica nuclear polyhedrosis virus was used as vector to express human C1r and C1s in Spodoptera frugiperda cell culture and in lepidopteran larvae. The yield of expression was high enough to isolate recombinant subcomponents for structural and functional studies. Recombinant viruses containing the A-, B-, and C-chains of C1q were also constructed. The insect cells are able to beta-hydroxylate the Asn residue of the EGF domain in the C1r but with a low efficiency. It is clear now, that this post-translational modification does not play a role in the Ca2+ dependent C1r-C1s interaction. The results with deletion mutants of C1r show that both, domain I, and II are absolutely necessary for the tetramer formation and both have regulatory role in the autoactivation. The C1s alpha R hybrid does not dimerize in presence of Ca2+, however it can form a tetramer with C11(2) that can bind to C1q. This observation indicates that the function of the C1s alpha part in the hybrid is modulated by the C1r part (gamma B) of the molecule. The C1Rs hybrid behaves like C1r, providing haemolytically active C1 with C1q and C1s. This observations shows that the regulatory domains determine the high functional specificity of the serine-protease subcomponents of C1. In order to control the autoactivation process point mutant cDNAs were constructed by altering the Arg-Ile bond in the catalytic domain of the C1r. The Gln-Ile construction is a stable zymogen while the Arg-Phe mutant has a lower rate of autoactivation.

1. C1r and C1s cDNAs were placed downstream the strong polyhedrin promoter in the Autographa californica nuclear polyhedrosis virus and the recombinant proteins were expressed in insect cells, in biologically active form. The yield of expression is high enough to get recombinant components for chemical and functional studies (5 micrograms/ml cell culture supernatant). 2. The biological activity and the post-translational modifications of the recombinant subcomponents were checked. The rC1r and rC1s proved to be biologically active in the hemolytic assay, although their glycosylations were different compared to that of the serum proteins. The insect cells are able to beta-hydroxylate the Asn residue of the EGF domain in the C1r but with a low efficiency. It is clear now, that this post-translational modification does not play a role in the Ca2+ dependent C1r-C1s interaction. 3. Two deletion mutants of C1r cDNA were constructed in order to clarify the role of domain I and II. The results show that both, domain I, and II are absolutely necessary for the tetramer formation and both have a regulatory role in the autoactivation. The autoactivation of the mutants is accelerated significantly. 4. Hybrid cDNA constructions were also made, and one of them was expressed. In the C1s alpha R hybrid the C1s alpha part cannot dimerize in presence of Ca2+, but it can form a tetramer with C1r2, that can bind to C1q. This observation indicates that the function of the C1s alpha part in the hybrid is modulated by the C1r part (gamma B) of the molecule. 5. In order to control the autoactivation process point mutant cDNAs were constructed through altering the Arg-Ile bond in the catalytic domain of the C1r. The Gln-Ile construction is a stable zymogen while the Arg-Phe mutant has a lower rate of autoactivation. These results do justify our approach of using domain-domain interchange, domain deletion and point mutations in combination, to reveal the structural background of C1 function at intramolecular level.

Spodoptera frugiperda cells are the hosts of wild type and recombinant virus in the baculovirus insect cell expression system. The expression of the foreign gene could be enhanced by the addition of ecdysteroids and increased amount of recombinant protein was secreted into the medium. The time and concentration dependence of this effect was followed in the case of 20-hydroxyecdysone-, makisterone and ecdysone. 20-hydroxyecdysone proved to be the most efficient, producing a three fold increase in the level of recombinant protein secreted into the medium, as it was measured by ELISA. This effect was also confirmed by tracing the L-(35S)methionine incorporation into the gene product. Makisterone was also effective in stimulation, while ecdysone proved to be ineffective.

The gene of human C1r has been expressed in a baculovirus-insect-cell system via the pAc373 transplacement vector. The full-length cDNA copy was inserted into the pAc373 vector downstream from the strong polyhedrin promoter of the baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda cells were cotransfected with the resultant plasmid, pAcC1r, and the wild-type AcNPV DNA. Recombinant viruses, which drove the expression of C1r protein, were selected by plaque morphology and ELISA. Insect cells infected with the recombinant virus produced and secreted human C1r protein, at a level of 1-2 mg/l of medium. The expressed C1r was isolated from the medium by chromatofocusing. On reducing gels only a single Coomassie-staining band was observed, and this band migrated at 80-83 kD characteristic of the unactivated C1r proenzyme. Its identification as C1r was immunologically confirmed on Western blots. C1 reconstituted from purified C1r expressed in insect cells together with human C1q and C1s proved biologically active in a hemolytic assay. Thus, the baculovirus-insect-cell system is capable of expressing and secreting a sophisticated, multifunctional human complement subcomponent in its biologically activatable form.