The crystal structure of the zymogen catalytic domain of complement protease C1r reveals that a disruptive mechanical stress is required to trigger activation of the C1 complex

C1r is the modular serine protease (SP) that mediates autolytic activation of C1, the macromolecular complex that triggers the classical pathway of complement. The crystal structure of a mutated, proenzyme form of the catalytic domain of human C1r, comprising the first and second complement control protein modules (CCP1, CCP2) and the SP domain has been solved and refined to 2.9 A resolution. The domain associates as a homodimer with an elongated head-to-tail structure featuring a central opening and involving interactions between the CCP1 module of one monomer and the SP domain of its counterpart. Consequently, the catalytic site of one monomer and the cleavage site of the other are located at opposite ends of the dimer. The structure reveals unusual features in the SP domain and provides strong support for the hypothesis that C1r activation in C1 is triggered by a mechanical stress caused by target recognition that disrupts the CCP1-SP interfaces and allows formation of transient states involving important conformational changes.     

The role of the individual domains in the structure and function of the catalytic region of a modular serine protease, C1r.

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.     

Monomeric structures of the zymogen and active catalytic domain of complement protease c1r: further insights into the c1 activation mechanism

C1r is the serine protease (SP) that mediates autoactivation of C1, the complex that triggers the classical complement pathway. We have determined the crystal structure of two fragments from the human C1r catalytic domain, each encompassing the second complement control protein (CCP2) module and the SP domain. The wild-type species has an active structure, whereas the S637A mutant is a zymogen. The structures reveal a restricted hinge flexibility of the CCP2-SP interface, and both are characterized by the unique alpha-helical conformation of loop E. The zymogen activation domain exhibits high mobility, and the active structure shows a restricted access to most substrate binding subsites. Further implications relevant to the C1r self-activation process are derived from protein-protein interactions in the crystals.     

Functional role of the linker between the complement control protein modules of complement protease C1s

C1s is the modular serine protease responsible for cleavage of C4 and C2, the protein substrates of the first component of C (C1). Its catalytic domain comprises two complement control protein (CCP) modules connected by a four-residue linker Gln340-Pro-Val-Asp343 and a serine protease domain. To assess the functional role of the linker, a series of mutations were performed at positions 340-343 of human C1s, and the resulting mutants were produced using a baculovirus-mediated expression system and characterized functionally. All mutants were secreted in a proenzyme form and had a mass of 77,203-77,716 Da comparable to that of wild-type C1s, except Q340E, which had a mass of 82,008 Da, due to overglycosylation at Asn391. None of the mutations significantly altered C1s ability to assemble with C1r and C1q within C1. Whereas the other mutations had no effect on C1s activation, the Q340E mutant was totally resistant to C1r-mediated activation, both in the fluid phase and within the C1 complex. Once activated, all mutants cleaved C2 with an efficiency comparable to that of wild-type C1s. In contrast, most of the mutations resulted in a decreased C4-cleaving activity, with particularly pronounced inhibitory effects for point mutants Q340K, P341I, V342K, and D343N. Comparable effects were observed when the C4-cleaving activity of the mutants was measured inside C1. Thus, flexibility of the C1s CCP1-CCP2 linker plays no significant role in C1 assembly or C1s activation by C1r inside C1 but plays a critical role in C4 cleavage by adjusting positioning of this substrate for optimal cleavage by the C1s active site.     

Functional characterization of complement proteases C1s/mannan-binding lectin-associated serine protease-2 (MASP-2) chimeras reveals the higher C4 recognition efficacy of the MASP-2 complement control protein modules

C1s and mannan-binding lectin-associated serine protease-2 (MASP-2) are the proteases that trigger the classical and lectin pathways of complement, respectively. They have identical modular architectures and cleave the same substrates, C2 and C4, but show markedly different efficiencies toward C4. Multisite-directed mutagenesis was used to engineer hybrid C1s/MASP-2 molecules where either the complement control protein (CCP) modules or the serine protease (SP) domain of C1s were swapped for their MASP-2 counterparts. The resulting chimeras (C1s(MASP-2 CCP1/2) and C1s(MASP-2 SP), respectively) were expressed and characterized chemically and functionally. Whereas C1s(MASP-2 SP) was recovered as an active enzyme, C1s(MASP-2 CCP1/2) was produced in a proenzyme form and was susceptible to activation by C1r, indicating that the activation properties of the chimeras were dictated by the nature of their SP domain. Similarly, each activated chimera had an esterolytic activity characteristic of its own SP domain and cleaved C2 with an efficiency comparable with that of their parent C1s and MASP-2 proteases. Both chimeras cleaved C4, but whereas C1s(MASP-2 SP) and C1s had Km values in the micromolar range, C1s(MASP-2 CCP1/2) and MASP-2 had Km values in the nanomolar range, resulting in 21-27-fold higher kcat/Km ratios. Thus, the higher C4 cleavage efficiency of MASP-2 arises from a higher substrate recognition efficacy of its CCP modules. Remarkably, C1s(MASP-2 CCP1/2) retained C1s ability to associate with C1r and C1q to form a pseudo-C1 complex and to undergo activation within this complex, indicating that the C1s-CCP modules have no direct implication in either function.     

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.

Structured summary

MINT-8045767: CCP1 (uniprotkb:P00736) and CCP2 (uniprotkb:P00736) bind (MI:0407) by nuclear magnetic resonance (MI:0077)     

Quantitative Characterization of the Activation Steps of Mannan-binding Lectin (MBL)-associated Serine Proteases (MASPs) Points to the Central Role of MASP-1 in the Initiation of the Complement Lectin Pathway

Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is ∼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process.     

The structure of MBL-associated serine protease-2 reveals that identical substrate specificities of C1s and MASP-2 are realized through different sets of enzyme-substrate interactions

A family of serine proteases mediates the proteolytic cascades of several defense mechanisms in vertebrates, such as the complement system, blood coagulation and fibrinolysis. These proteases usually form large complexes with other glycoproteins. Their common features are their modular structures and restricted substrate specificities. The lectin pathway of complement, where mannose-binding lectin (MBL) recognizes the carbohydrate structures on pathogens, is activated by mannose-binding lectin-associated serine protease-2 (MASP-2). We present the 2.25A resolution structure of the catalytic fragment of MASP-2 encompassing the second complement control protein module (CCP2) and the serine protease (SP) domain. The CCP2 module stabilizes the structure of the SP domain as demonstrated by differential scanning calorimetry measurements. The asymmetric unit contains two molecules with different CCP-SP domain orientations, reflecting increased modular flexibility at the CCP2/SP joint. This flexibility may partly explain the ability of the MASP-2 dimer to perform all of its functions alone, whereas the same functions are mediated by the much larger C1r2-C1s2 tetramer in the C1 complex of the classical pathway. The main scaffold of the MASP-2 SP domain is chymotrypsin-like. Eight surface loops determine the S1 and other subsite specificities. Surprisingly, some surface loops of MASP-2, e.g. loop 1 and loop 2, which form the S1 pocket are similar to those of trypsin, and show significant differences if compared with those of C1s, indicating that the nearly identical substrate specificities of C1s and MASP-2 are realized through different sets of enzyme-substrate interactions.     

The X-ray crystal structure of full-length human plasminogen

Plasminogen is the proenzyme precursor of the primary fibrinolytic protease plasmin. Circulating plasminogen, which comprises a Pan-apple (PAp) domain, five kringle domains (KR1-5), and a serine protease (SP) domain, adopts a closed, activation-resistant conformation. The kringle domains mediate interactions with fibrin clots and cell-surface receptors. These interactions trigger plasminogen to adopt an open form that can be cleaved and converted to plasmin by tissue-type and urokinase-type plasminogen activators. Here, the structure of closed plasminogen reveals that the PAp and SP domains, together with chloride ions, maintain the closed conformation through interactions with the kringle array. Differences in glycosylation alter the position of KR3, although in all structures the loop cleaved by plasminogen activators is inaccessible. The ligand-binding site of KR1 is exposed and likely governs proenzyme recruitment to targets. Furthermore, analysis of our structure suggests that KR5 peeling away from the PAp domain may initiate plasminogen conformational change.     

Mutations in alpha-chain of C4BP that selectively affect its factor I cofactor function

C4b-binding protein (C4BP) inhibits all pathways of complement activation, acting as a cofactor to the serine protease factor I (FI) in the degradation of activated complement factors C4b and C3b. C4BP is a disulfide-linked polymer of seven alpha-chains and a unique beta-chain, the alpha- and beta-chains being composed of eight and three complement control protein (CCP) domains, respectively. In previous studies we have localized cofactor activity and binding of C4b to alpha-chain CCP1-3 of C4BP, whereas the binding of C3b required additionally CCP4. Likewise, introduced point mutations that decreased binding of C4b/C3b caused a decrease in cofactor activity. In the present study, we describe two mutants of C4BP, K126Q/K128Q and F144S/F149S, clustered on alpha-chain CCP3, which selectively lost their ability to act as cofactors in the cleavage of both C4b and C3b. Both mutants show the same binding affinity for C4b/C3b as measured by surface plasmon resonance and have the same inhibitory effect on formation and decay of the classical pathway C3-convertase as the wild type C4BP. It appears that C4b and C3b do not undergo the same conformational changes upon binding to the C4BP mutants as during the interaction with the wild type C4BP, which then results in the observed loss of the cofactor activity.     

Crystal structure of the catalytic domain of human complement c1s: a serine protease with a handle

C1s is the highly specific modular serine protease that mediates the proteolytic activity of the C1 complex and thereby triggers activation of the complement cascade. The crystal structure of a catalytic fragment from human C1s comprising the second complement control protein (CCP2) module and the chymotrypsin-like serine protease (SP) domain has been determined and refined to 1.7 A resolution. In the areas surrounding the active site, the SP structure reveals a restricted access to subsidiary substrate binding sites that could be responsible for the narrow specificity of C1s. The ellipsoidal CCP2 module is oriented perpendicularly to the surface of the SP domain. This arrangement is maintained through a rigid module-domain interface involving intertwined proline- and tyrosine-rich polypeptide segments. The relative orientation of SP and CCP2 is consistent with the fact that the latter provides additional substrate recognition sites for the C4 substrate. This structure provides a first example of a CCP-SP assembly that is conserved in diverse extracellular proteins. Its implications in the activation mechanism of C1 are discussed.     

Human complement component C1s. Partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation

Human C1s proenzyme (Mr 83 000) was isolated by a rapid two-stage method involving affinity chromatography of C1 on IgG-Sepharose and isolation of subcomponent C1s by ion-exchange chromatography on DEAE-Sephacel. Single-chain C1s proenzyme was activated to two-chain C1s with self-activated C1r. After reduction and S-carboxamidomethylation the heavy chain of C1s (Mr 57 000) was isolated by ion exchange chromatography on DEAE-Sephacel. Cleavage of C1s heavy chain with CNBr yielded five fragments whose N-terminal sequences were determined. The alignment of the fragments within the heavy chain was established by tryptic peptides containing methionine. C1s heavy chain comprises about 470 amino acid residues and 42% of its sequence was determined. An intrachain sequence homology and a homology to the alpha 2 chain of human haptoglobin were identified. The C-terminal CNBr fragment comprising 44 amino acid residues was completely sequenced. From BNPS-skatole cleavage of reduced and alkylated C1s proenzyme a fragment was isolated which overlaps the C1s heavy and light chain parts and which contains the peptide bond cleaved during activation. The results show that this is an Arg-Ile bond and that under standard conditions of activation no peptide material is liberated from this portion of the molecule. The sequence data and homology to two-chain serine proteases indicate a single interchain disulfide bond in C1s.     

Characterization of recombinant mannan-binding lectin-associated serine protease (MASP)-3 suggests an activation mechanism different from that of MASP-1 and MASP-2

Mannan-binding lectin (MBL)-associated serine proteases (MASP-1, -2, and -3) are homologous modular proteases that each associate with MBL and L- and H-ficolins, which are oligomeric serum lectins involved in innate immunity. To investigate its physicochemical, interaction, and enzymatic properties, human MASP-3 was expressed in insect cells. Ultracentrifugation analysis indicated that rMASP-3 sedimented as a homodimer (s(20,w) = 6.2 +/- 0.1 S) in the presence of Ca(2+), and as a monomer (s(20,w) = 4.6 +/- 0.1 S) in EDTA. As shown by surface plasmon resonance spectroscopy, it associated with both MBL (K(D) = 2.6 nM) and L-ficolin (K(D) = 7.2 nM). The protease was produced in a single-chain, proenzyme form, but underwent slow activation upon prolonged storage at 4 degrees C, resulting from cleavage at the Arg(430)-Ile(431) activation site. Activation was prevented in the presence of protease inhibitors iodoacetamide and 1,10-phenanthroline but was not abolished upon substitution of Ala for the active site Ser(645) of MASP-3, indicating extrinsic proteolysis. In contrast, the corresponding mutations Ser(627)-->Ala in MASP-1 and Ser(618)-->Ala in MASP-2 stabilized the latter in their proenzyme form. Likewise, the MASP-1 and MASP-2 mutants were each activated by their active counterparts, but MASP-3 S645A was not. Activated MASP-3 did not react with C1 inhibitor; had no activity on complement proteins C2, C4, and C3; and only cleaved the N-carboxybenzyloxyglycine-L-arginine thiobenzyl ester substrate to a significant extent. Based on these observations, it is postulated that MASP-3 activation and control involve mechanisms that are different from those of MASP-1 and -2.     

Histidine decarboxylase of Lactobacillus 30a. Hydroxylamine cleavage of the -seryl-seryl- bond at the activation site of prohistidine decarboxylase

Conversion of the pi subunit of prohistidine decarboxylase to the alpha beta subunits of the active enzyme proceeds by a nonhydrolytic, monovalent cation-dependent, serinolysis reaction in which the hydroxyl oxygen of serine 82 of the pi chain is incorporated into the carboxyl group at the COOH terminus (serine 81) of the beta chain. Serine-82 becomes the pyruvate residue at the NH2 terminus of the alpha chain (Recsei, P.A., Huynh, Q. K., and Snell, E.E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 973-977). The unusual reactivity of this particular -Ser-Ser- bond is demonstrated by its sensitivity to 1 M hydroxylamine, which cleaves the native proenzyme under mild conditions (pH 8.0, 37 degrees C) to yield a modified beta chain with serine hydroxamate at the COOH terminus (Ser-81) and a modified alpha chain containing serine (Ser-82 of the proenzyme) rather than pyruvate at the NH2 terminus. Neither an -Asn-Gly- bond nor other -Ser-Ser- bonds in the proenzyme were cleaved under these conditions. The reaction also did not occur with the denatured enzyme or with model peptides, indicating that the enhanced reactivity is a result of the particular conformation at this position in the native protein. The reaction with the native proenzyme proceeded optimally at pH 7.5-8.0 with a half-time (30 min) substantially less than that (3.5-4.5 h) required for the activation reaction and was not increased in rate by addition of K+. Correspondingly, preincubation of the proenzyme at pH 8.0 in the absence of both hydroxylamine and K+ modestly increased the rate of activation when K+ was subsequently added. Although these findings do not exclude other mechanisms, they are all consistent with and most easily explained by rearrangement of the pi chain to form an internal ester intermediate prior to the beta-elimination that occurs during activation to yield the alpha and beta chains of the mature enzyme.     

A Molecular Switch Governs the Interaction between the Human Complement Protease C1s and Its Substrate,Complement C4

The complement system is an ancient innate immune defense pathway that plays a front line role in eliminating microbial pathogens. Recognition of foreign targets by antibodies drives sequential activation of two serine proteases, C1r and C1s, which reside within the complement Component 1 (C1) complex. Active C1s propagates the immune response through its ability to bind and cleave the effector molecule complement Component 4 (C4). Currently, the precise structural and biochemical basis for the control of the interaction between C1s and C4 is unclear. Here, using surface plasmon resonance, we show that the transition of the C1s zymogen to the active form is essential for C1s binding to C4. To understand this, we determined the crystal structure of a zymogen C1s construct (comprising two complement control protein (CCP) domains and the serine protease (SP) domain). These data reveal that two loops (492–499 and 573–580) in the zymogen serine protease domain adopt a conformation that would be predicted to sterically abrogate C4 binding. The transition from zymogen to active C1s repositions both loops such that they would be able to interact with sulfotyrosine residues on C4. The structure also shows the junction of the CCP1 and CCP2 domains of C1s for the first time, yielding valuable information about the exosite for C4 binding located at this position. Together, these data provide a structural explanation for the control of the interaction with C1s and C4 and, furthermore, point to alternative strategies for developing therapeutic approaches for controlling activation of the complement cascade.     

Insight into the intermolecular recognition mechanism involved in complement component 4 activation through serine protease-trypsin

Serine protease cleaved-complement component 4 (C4) at sessile loop, which is significant for completion of lectin and classical complement pathways at the time of infections. The co-crystalized structure of C4 with Mannose-binding protein-associated serine protease 2 (MASP2) provided the structural and functional aspects of its interaction and underlined the C4 activation by MASP2. The same study also revealed the significance of complement control protein (CCP) domain through mutational study, where mutated CCP domain led to the inhibition of C4 activation. However, the interaction of trypsin serine domain with C4α sessile loop revealed another aspect of C4 activation. The human C4 cleavage by Trypsin (Tryp) in a control manner was explored but not yet revealed the identification of cleaved fragments. Hence, the present study investigated the Tryp mediated C4 activation using computational approach (protein–protein docking and molecular dynamics simulation) by comparing with the co-crystalized structure of C4-MASP2. Docking result identified the crucial interacting residues Gly219, Gln178, and Asn102 of Tryp catalytic pocket which were interacting with Arg756 and Glu759 (sessile loop) of α-Chain (C4) in a similar manner to C4-MASP2 co-crystallized complex. Moreover, MD simulation results and mutational study underlined the conformational rearrangements in the C4 due to the Tryp interaction. Comparative analysis of C4 alone, C4-Tryp, and C4-MASP2 revealed the impact of Tryp on C4 was similar as MASP2. These studies designate the role of sessile loop in the interaction with serine domain, which could be useful to understand the various interactions of C4 with other complement components.     

Studies on the mechanism of formation of the pyruvate prosthetic group of phosphatidylserine decarboxylase from Escherichia coli

Phosphatidylserine decarboxylase from Escherichia coli uses a pyruvate group as the enzyme cofactor (Satre, M., and Kennedy, E. P. (1978) J. Biol. Chem. 253, 479-483). Comparison of the DNA sequence of the psd gene with the partial amino acid sequence of the mature gene product suggests that the two nonidentical subunits of the mature enzyme are formed by cleavage of a proenzyme resulting in the conversion of Ser-254 to an amino-terminal pyruvate residue (Li, Q.-X., and Dowhan, W. (1988) J. Biol. Chem. 263, 11516-11522). The cleavage of the wild-type proenzyme occurs rapidly with a half-time on the order of 2 min. When Ser-254 is changed to cysteine (S254C), threonine (S254T), or alanine (S254A) by site-directed mutagenesis, the rate of processing of the proenzyme and the production of the functional enzyme are drastically affected. Proenzymes with S254C or S254T are cleaved with a half-time of around 2-4 h while the S254A proenzyme does not undergo processing. The reduced processing rate for the mutant proenzymes is consistent with less of the functional enzyme being made. Mutants encoding the S254C and S254T protein produce 16 and 2%, respectively, of the activity of the wild-type allele but can still complement a temperature-sensitive mutant in the psd locus. There is no detectable activity or complementation observed with the S254A protein. These results are consistent with the hydroxyl group of Ser-254 playing a critical role in the cleavage of the peptide bond between Gly-253 and Ser-254 of the prophosphatidylserine decarboxylase and support the mechanism proposed by Snell and coworkers (Recsei and Snell (1984) Annul Rev. Biochem. 53, 357-387) for the formation of the prosthetic group of pyruvate-dependent decarboxylases.     

Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase

Human 8-oxoguanine-DNA glycosylase (OGG1) is the major enzyme for repairing 8-oxoguanine (8-oxoG), a mutagenic guanine base lesion produced by reactive oxygen species (ROS). A frequently occurring OGG1 polymorphism in human populations results in the substitution of serine 326 for cysteine (S326C). The 326 C/C genotype is linked to numerous cancers, although the mechanism of carcinogenesis associated with the variant is unclear. We performed detailed enzymatic studies of polymorphic OGG1 and found functional defects in the enzyme. S326C OGG1 excised 8-oxoG from duplex DNA and cleaved abasic sites at rates 2- to 6-fold lower than the wild-type enzyme, depending upon the base opposite the lesion. Binding experiments showed that the polymorphic OGG1 binds DNA damage with significantly less affinity than the wild-type enzyme. Remarkably, gel shift, chemical cross-linking and gel filtration experiments showed that S326C both exists in solution and binds damaged DNA as a dimer. S326C OGG1 enzyme expressed in human cells was also found to have reduced activity and a dimeric conformation. The glycosylase activity of S326C OGG1 was not significantly stimulated by the presence of AP-endonuclease. The altered substrate specificity, lack of stimulation by AP-endonuclease 1 (APE1) and anomalous DNA binding conformation of S326C OGG1 may contribute to its linkage to cancer incidence.     

Analysis of binding sites on complement factor I using artificial N-linked glycosylation

Factor I (FI) is a serine protease that inhibits all complement pathways by degrading activated complement components C3b and C4b. FI functions only in the presence of several cofactors, such as factor H, C4b-binding protein, complement receptor 1, and membrane cofactor protein. FI is composed of two chains linked by a disulfide bridge; the light chain comprises only the serine protease (SP) domain, whereas the heavy chain contains the FI membrane attack complex domain (FIMAC), CD5 domain, and low density lipoprotein receptor 1 (LDLr1) and LDLr2 domains. To better understand how FI inhibits complement, we used homology-based three-dimensional models of FI domains in an attempt to identify potential protein-protein interaction sites. Specific amino acids were then mutated to yield 20 recombinant mutants of FI carrying additional surface-exposed N-glycosylation sites that were expected to sterically hinder interactions. The Michaelis constant (K(m)) of all FI mutants toward a small substrate was not increased. We found that many mutations in the FIMAC and SP domains nearly abolished the ability of FI to degrade C4b and C3b in the fluid phase and on the surface, irrespective of the cofactor used. On the other hand, only a few alterations in the CD5 and LDLr1/2 domains impaired this activity. In conclusion, all analyzed cofactors form similar trimolecular complexes with FI and C3b/C4b, and the accessibility of FIMAC and SP domains is crucial for the function of FI.