A bispecific inhibitor of complement and coagulation blocks activation in complementopathy models via a novel mechanism.

Inhibitors of complement and coagulation are present in the saliva of a variety of blood-feeding arthropods that transmit parasitic and viral pathogens. Here, we describe the structure and mechanism of action of the sand fly salivary protein lufaxin, which inhibits the formation of the central alternative C3 convertase (C3bBb) and inhibits coagulation factor Xa (fXa). Surface plasmon resonance experiments show that lufaxin stabilizes the binding of serine protease factor B (FB) to C3b but does not detectably bind either C3b or FB alone. The crystal structure of the inhibitor reveals a novel all ß-sheet fold containing 2 domains. A structure of the lufaxin-C3bB complex obtained via cryo-electron microscopy (EM) shows that lufaxin binds via its N-terminal domain at an interface containing elements of both C3b and FB. By occupying this spot, the inhibitor locks FB into a closed conformation in which proteolytic activation of FB by FD cannot occur. C3bB-bound lufaxin binds fXa at a separate site in its C-terminal domain. In the cryo-EM structure of a C3bB-lufaxin-fXa complex, the inhibitor binds to both targets simultaneously, and lufaxin inhibits fXa through substrate-like binding of a C-terminal peptide at the active site as well as other interactions in this region. Lufaxin inhibits complement activation in ex vivo models of atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH) as well as thrombin generation in plasma, providing a rationale for the development of a bispecific inhibitor to treat complement-related diseases in which thrombosis is a prominent manifestation.

Structure determination of an unstable macromolecular complex enabled by nanobody-peptide bridging.

Structure determination of macromolecular complexes is challenging if subunits can dissociate during crystallization or preparation of electron microscopy grids. We present an approach where a labile complex is stabilized by linking subunits though introduction of a peptide tag in one subunit that is recognized by a nanobody tethered to a second subunit. This allowed crystal structure determination at 3.9 Å resolution of the highly non-globular 320 kDa proconvertase formed by complement components C3b, factor B, and properdin. Whereas the binding mode of properdin to C3b is preserved, an internal rearrangement occurs in the zymogen factor B von Willebrand domain type A domain compared to the proconvertase not bound to properdin. The structure emphasizes the role of two noncanonical loops in thrombospondin repeats 5 and 6 of properdin in augmenting the activity of the C3 convertase. We suggest that linking of subunits through peptide specific tethered nanobodies represents a simple alternative to approaches like affinity maturation and chemical cross-linking for the stabilization of large macromolecular complexes. Besides applications for structural biology, nanobody bridging may become a new tool for biochemical analysis of unstable macromolecular complexes and in vitro selection of highly specific binders for such complexes.

Inhibition of the Complement Alternative Pathway by Chemically Modified DNA Aptamers That Bind with Picomolar Affinity to Factor B.

The complement system is a conserved component of innate immunity that fulfills diverse roles in defense and homeostasis. Inappropriate activation of complement contributes to many inflammatory diseases, however, which has led to a renewed emphasis on development of therapeutic complement inhibitors. Activation of complement component C3 is required for amplification of complement and is achieved through two multisubunit proteases called C3 convertases. Of these, the alternative pathway (AP) C3 convertase is responsible for a majority of the C3 activation products in vivo, which renders it an attractive target for inhibitor discovery. In this study, we report the identification and characterization of two related slow off-rate modified DNA aptamers (SOMAmer) reagents that inhibit formation of the AP C3 convertase by binding to the proprotease, factor B (FB). These aptamers, known as SL1102 (31 bases) and SL1103 (29 bases), contain uniform substitutions of 5-(N-2-naphthylethylcarboxyamide)-2'-deoxyuridine for deoxythymidine. SL1102 and SL1103 bind FB with K d values of 49 and 88 pM, respectively, and inhibit activation of C3 and lysis of rabbit erythrocytes under AP-specific conditions. Cocrystal structures of SL1102 (3.4 Å) and SL1103 (3.1 Å) bound to human FB revealed that SL1102 and SL1103 recognize a site at the juncture of the CCP1, CCP3, and vWF domains of FB. Consistent with these structures and previously published information, these aptamers inhibited FB binding to C3b and blocked formation of the AP C3 convertase. Together, these results demonstrate potent AP inhibition by modified DNA aptamers and expand the pipeline of FB-binding molecules with favorable pharmacologic properties.

Identification of intermolecular bonds between human factor B and Cobra Venom Factor important for C3 convertase stability.

Cobra venom factor (CVF) is the complement-activating protein in cobra venom. CVF is a structural and functional analog of complement component C3. CVF, like C3b, forms a convertase with factor B. This bimolecular complex CVF, Bb is an enzyme that cleaves C3 and C5. However, CVF, Bb exhibits significantly different functional properties from C3b,Bb. Whereas both, CVF, Bb and C3b, Bb exhibit spontaneous decay-dissociation into the respective subunits, thereby eliminating the enzymatic activity, the CVF, Bb convertase is physico-chemically far more stable, decaying with a half-life that is more than two orders of magnitude slower than that of C3b,Bb. In addition, CVF, Bb is completely resistant to inactivation by Factors H and I. These two properties of CVF, Bb allow continuous activation of C3 and C5, and complement depletion in serum. In order to understand the structural basis for the physico-chemical stability of CVF,Bb, we have created recombinant hybrid proteins of CVF and human C3, based on structural differences between CVF and human C3b in the C-terminal C345C domain. Here we describe three human C3/CVF hybrid proteins which differ in only one, two, or five amino acid residues from earlier described hybrid proteins. In all three cases, the hybrid proteins containing CVF residues form more stable convertases, and exhibit stronger complement-depletion activity than hybrid proteins with human C3 residues. Three bonds between CVF residues and Factor Bb residues could be identified by crystallographic modeling that contribute to the greater stability of the convertases.

Aliskiren inhibits renin-mediated complement activation.

Certain kidney diseases are associated with complement activation although a renal triggering factor has not been identified. Here we demonstrated that renin, a kidney-specific enzyme, cleaves C3 into C3b and C3a, in a manner identical to the C3 convertase. Cleavage was specifically blocked by the renin inhibitor aliskiren. Renin-mediated C3 cleavage and its inhibition by aliskiren also occurred in serum. Generation of C3 cleavage products was demonstrated by immunoblotting, detecting the cleavage product C3b, by N-terminal sequencing of the cleavage product, and by ELISA for C3a release. Functional assays showed mast cell chemotaxis towards the cleavage product C3a and release of factor Ba when the cleavage product C3b was combined with factor B and factor D. The renin-mediated C3 cleavage product bound to factor B. In the presence of aliskiren this did not occur, and less C3 deposited on renin-producing cells. The effect of aliskiren was studied in three patients with dense deposit disease and this demonstrated decreased systemic and renal complement activation (increased C3, decreased C3a and C5a, decreased renal C3 and C5b-9 deposition and/or decreased glomerular basement membrane thickness) over a follow-up period of four to seven years. Thus, renin can trigger complement activation, an effect inhibited by aliskiren. Since renin concentrations are higher in renal tissue than systemically, this may explain the renal propensity of complement-mediated disease in the presence of complement mutations or auto-antibodies.

The mechanism and modulation of complement activation on polymer grafted cells.

Cell surface engineering using polymers is a promising approach to address unmet needs and adverse immune reactions in the fields of transfusion, transplantation, and cell-based therapies. Furthermore, cell surface modification may minimize or prevent adverse immune reactions to homologous incompatible cells as the interface between the host immune system and the cell surface is modified. In this report, we investigate the immune system reaction, precisely the complement binding and activation on cell surfaces modified with a functional polymer, hyperbranched polyglycerol (HPG). We used red blood cells (RBCs) as a model system to investigate the mechanism of complement activation on cell surfaces modified with various forms of HPG. Using a battery of in vitro assays including: traditional diagnostic hemolytic assays involving sheep and rabbit erythrocytes, ELISAs and flow cytometry, we show that HPG modified RBCs at certain concentrations and molecular weights activate complement via the alternative pathway. We show that by varying the grafting concentration, molecular weight and the number of cell surface reactive groups of HPG, the complement activity on the cell surface can be modulated. HPGs with molecular weights greater than 28kDa and grafting concentrations greater than 1.0mM, as well as a high degree of HPG functionalization with cell surface reactive groups result in the activation of the complement system via the alternative pathway. No complement activation observed when these threshold levels are not exceeded. These insights may have an impact on devising key strategies in developing novel next generation cell-surface engineered therapeutic products for applications in the fields of cell therapy, transfusion and drug delivery. STATEMENT OF SIGNIFICANCE: Cell-surface engineering using functional polymers is a fast emerging area of research. Importantly modified cells are used in many experimental therapeutics, transplantation and in transfusion. The success of such therapies depend on the ability of modified products to avoid immune detection and subsequent rejection or removal. Polymer grafting has been shown to modulate immune response, however, there is limited knowledge available. Thus in this manuscript, we investigated the interaction of human complement, part of our innate immune system, by polymer modified cells. Our results provide important evidences on the mechanism of complement activation by the modified cells and also found ways to modulate the innate immune response. These results will have implications in development of next generation cell-based therapies.

Factor B Is the Second Lipopolysaccharide-binding Protease Zymogen in the Horseshoe Crab Coagulation Cascade.

Factor B is a serine-protease zymogen in the horseshoe crab coagulation cascade, and it is the primary substrate for activated factor C, the LPS-responsive initiator of the cascade. Factor C is autocatalytically activated to α-factor C on LPS and is artificially converted to ß-factor C, another activated form, by chymotrypsin. It is not known, however, whether LPS is required for the activation of factor B. Here we found that wild-type factor B expressed in HEK293S cells is activated by α-factor C, but not by ß-factor C, in an LPS-dependent manner and that ß-factor C loses the LPS binding activity of factor C through additional cleavage by chymotrypsin within the N-terminal LPS-binding region. Surface plasmon resonance and quartz crystal microbalance analyses revealed that wild-type factor B binds to LPS with high affinity comparable with that of factor C, demonstrating that factor B is the second LPS-binding zymogen in the cascade. An LPS-binding site of wild-type factor B was found in the N-terminal clip domain, and the activation rate of a clip domain deletion mutant was considerably slower than that of wild-type factor B. Moreover, in the presence of LPS, Triton X-100 inhibited the activation of wild-type factor B by α-factor C. We conclude that the clip domain of factor B has an important role in localizing factor B to the surface of Gram-negative bacteria or LPS released from bacteria to initiate effective proteolytic activation by α-factor C.

Complement factor B mutations in atypical hemolytic uremic syndrome-disease-relevant or benign?

Atypical hemolytic uremic syndrome (aHUS) is a genetic ultrarare renal disease associated with overactivation of the alternative pathway of complement. Four gain-of-function mutations that form a hyperactive or deregulated C3 convertase have been identified in Factor B (FB) ligand binding sites. Here, we studied the functional consequences of 10 FB genetic changes recently identified from different aHUS cohorts. Using several tests for alternative C3 and C5 convertase formation and regulation, we identified two gain-of-function and potentially disease-relevant mutations that formed either an overactive convertase (M433I) or a convertase resistant to decay by FH (K298Q). One mutation (R178Q) produced a partially cleaved protein with no ligand binding or functional activity. Seven genetic changes led to near-normal or only slightly reduced ligand binding and functional activity compared with the most common polymorphism at position 7, R7. Notably, none of the algorithms used to predict the disease relevance of FB mutations agreed completely with the experimental data, suggesting that in silico approaches should be undertaken with caution. These data, combined with previously published results, suggest that 9 of 15 FB genetic changes identified in patients with aHUS are unrelated to disease pathogenesis. This study highlights that functional assessment of identified nucleotide changes in FB is mandatory to confirm disease association.

Gene cloning and function analysis of complement B factor-2 of Apostichopus japonicus.

In this study, a homologue of complement B factor (AjBf-2, GenBank ID: JN634069.1) was cloned and characterized from Apostichopus japonicus by using bioinformatics methods and molecular biotechnologies including homology cloning and RACE. The full-length cDNA of AjBf-2 was composed of 3261bp. The sequence shows 268bp in the 5'UT region, 395bp in the 3'UT region, and 2595 bp in the open reading frame. AjBf-2 gene encodes 865 amino acids. The deduced amino acids sequence and domain structure of AjBf-2 gene show significant similarity to the vertebrate Bf/C2 family protein. AjBf-2 is a mosaic protein. It has a deduced molecular mass of 96.8 kDa, with a conserved site for a D factor. AjBf-2 is composed of five short consensus repeats, a von Willebrand Factor domain, a serine protease domain and an Mg2+ binding site. It has eight consensus recognition sites for N-linked glycosylation and four cAMP- and cGMP-dependent protein kinase phosphorylation sites. Phylogenetic analysis of AjBf-2 compared with other species Bf shows that A. japonicus has a close evolutionary relationship with Strongylocentrotus purpuratus and Carcinoscorpius rotundicaud. It can be speculated that Bf in invertebrate is the ancestor of Bf in vertebrate. The result of RT-PCR shows that the AjBf-2 gene is expressed in every tested tissue of A. japonicus, and is especially high in the coelomocyte and the body wall. The expression tendency in coelomocyte and the body wall are approximately the same. After LPS induction, the expression of AjBf-2 gene peaks at 12 h in coelomocyte and 3 h in the body wall.

Molecular cloning, characterization and expression of the complement component Bf/C2 gene in grass carp.

The complement system is an integral part of the host immune system and plays an immunoregulatory role at the interface between the innate and acquired immune responses. Factor B (Bf) serves as the catalytic subunit of complement C3 convertase in the alternative pathway (AP), while in the classical pathway (CP), this function is subjected to C2. In this study, we cloned and characterized the two Bf/C2 genes of grass carp, gcBf/C2A and gcBf/C2B. The gcBf/C2A and gcBf/C2B cDNA sequences are 2259 and 3004 bp in length, and the open reading frames (ORFs) of gcBf/C2A and gcBf/C2B were found to encode peptides of 752 and 837 amino acids, respectively. The genes share 30.7% amino acid identity with each other and 32.4-38.3% and 31.4-33% with the Bf and C2 genes in humans and mice. GcBf/C2A and gcBf/C2B were expressed in a wide range of grass carp tissues, with the highest level of expression of both genes detected in the liver. After a challenge with Aeromonas hydrophila, gcBf/C2A was significantly upregulated, especially at 4 h after infection, and the significantly higher expression of gcBf/C2B (27.3-fold) was found in the head kidney at 24 h post-challenge. The expression of gcBf/C2A was quickly upregulated at 1 day post-hatching and peaked at 5 days post-hatching. The maximum expression of gcBf/C2B was found at 1 day post-hatching. In conclusion, our data enables a better understanding of the physiological function of the Bf/C2 complement genes in vertebrates.

Access to the complement factor B scissile bond is facilitated by association of factor B with C3b protein.

Factor B is a zymogen that carries the catalytic site of the complement alternative pathway C3 convertase. During convertase assembly, factor B associates with C3b and Mg(2+) forming a pro-convertase C3bB(Mg(2+)) that is cleaved at a single factor B site by factor D. In free factor B, a pair of salt bridges binds the Arg(234) side chain to Glu(446) and to Glu(207), forming a double latch structure that sequesters the scissile bond (between Arg(234) and Lys(235)) and minimizes its unproductive cleavage. It is unknown how the double latch is released in the pro-convertase. Here, we introduce single amino acid substitutions into factor B that preclude one or both of the Arg(234) salt bridges, and we examine their impact on several different pro-convertase complexes. Our results indicate that loss of the Arg(234)-Glu(446) salt bridge partially stabilizes C3bB(Mg(2+)). Loss of the Arg(234)-Glu(207) salt bridge has lesser effects. We propose that when factor B first associates with C3b, it bears two intact Arg(234) salt bridges. The complex rapidly dissociates unless the Arg(234)-Glu(446) salt bridge is released whereupon conformational changes occur that activate the metal ion-dependent adhesion site and partially stabilize the complex. The remaining salt bridge is then released, exposing the scissile bond and permitting factor D cleavage.

Alternative complement pathway assessment in patients with atypical HUS.

The atypical Hemolytic Uremic Syndrome (aHUS) is a rare thrombotic microangiopathy leading to end stage renal disease in approximately 60% of patients. Over the last decade, a clear link has been demonstrated between this disease and defective complement regulation. The hallmark of the aHUS is the association with mutations in complement alternative pathway genes. Endothelial damage is related to complement dysregulation, but the exact mechanism is just starting to be elucidated. Screening for and characterization of mutations in the components of the C3 convertase (C3 and FB) or its regulators (FH, FI, MCP, and Thrombomodulin) or anti-FH antibodies has become an indispensable part of the disease's diagnostic. This review will initially summarize current knowledge on the understanding of complement activation and regulation, followed by a description on the genetic analysis as well as the methods used for complement protein quantification. Another part of this review will focus on the mechanisms of action of aHUS-associated mutations. We will emphasize on when and why some mutations lead to protein deficiency, while others result in - to dysfunctional but normally expressed proteins. Finally, we will discuss how the therapy of aHUS patients can be modified according to the functional consequences of each particular genetic defect.

Lessons from functional and structural analyses of disease-associated genetic variants in the complement alternative pathway.

Complement is an essential component of innate immunity and a major trigger of inflammatory responses. A critical step in complement activation is the formation of the C3 convertase of the alternative pathway (AP), a labile bimolecular complex formed by activated fragments of the C3 and factor B components that is fundamental to provide exponential amplification of the initial complement trigger. Regulation of the AP C3 convertase is essential to maintain complement homeostasis in plasma and to protect host cells and tissues from damage by complement. During the last decade, several studies have associated genetic variations in components and regulators of the AP C3 convertase with a number of chronic inflammatory diseases and susceptibility to infection. The functional characterization of these protein variants has helped to decipher the critical pathogenic mechanisms involved in some of these complement related disorders. In addition, these functional data together with recent 3D structures of the AP C3 convertase have provided fundamental insights into the assembly, activation and regulation of the AP C3 convertase.

Structures of C3b in complex with factors B and D give insight into complement convertase formation.

Activation of the complement cascade induces inflammatory responses and marks cells for immune clearance. In the central complement-amplification step, a complex consisting of surface-bound C3b and factor B is cleaved by factor D to generate active convertases on targeted surfaces. We present crystal structures of the pro-convertase C3bB at 4 angstrom resolution and its complex with factor D at 3.5 angstrom resolution. Our data show how factor B binding to C3b forms an open "activation" state of C3bB. Factor D specifically binds the open conformation of factor B through a site distant from the catalytic center and is activated by the substrate, which displaces factor D's self-inhibitory loop. This concerted proteolytic mechanism, which is cofactor-dependent and substrate-induced, restricts complement amplification to C3b-tagged target cells.

C3, C5, and factor B bind to chitosan without complement activation.

Chitosan is a polycationic and biocompatible polysaccharide composed of glucosamine and N-acetyl glucosamine that is chemotactic for neutrophils and stimulates wound repair through mechanisms that remain unclear. It was previously shown that chitosan depletes complement proteins from plasma, suggesting that chitosan activates complement. Complement activation leads to cleavage of C5 to produce C5a, a neutrophil chemotactic factor. Here, we tested the hypothesis that chitosan generates C5a in human whole blood, citrated plasma, and serum. C5a fragment appeared in coagulating whole blood, and mixtures of chitosan-glycerol phosphate/whole blood, in parallel with platelet and thrombin activation. However, in plasma and serum, thrombin and chitosan-GP failed to generate C5a, although native C3, C5, and factor B adsorbed noncovalently to insoluble chitosan particles incubated in citrated plasma, serum, EDTA-serum and methylamine-treated plasma. By surface plasmon resonance, pure C3 adsorbed to chitosan. The profile of serum factors associating with chitosan was consistent with a model in which anionic blood proteins with a pI lower than the pK(0) 6.78 of chitosan (the upper limit of chitosan pK(a)) associate electrostatically with cationic chitosan particles. Zymosan, a yeast ghost particle, activated complement in serum and citrated plasma, but not in EDTA-serum or methylamine plasma, to generate fluid-phase C5a, while C3b formed covalent cross-links with zymosan-associated proteins and became rapidly cleaved to iC3b, with factor Bb stably associated. These data demonstrate that chitosan is a nonreactive biomaterial that does not directly activate complement, and provide a novel basis for predicting anionic serum protein-chitosan interactions.

Coexistence of closed and open conformations of complement factor B in the alternative pathway C3bB(Mg2+) proconvertase.

Complement factor B (fB) circulates in plasma as a proenzyme that, upon binding to C3b in the presence of Mg(2+), is cleaved by factor D to produce Ba and Bb fragments. Activated Bb remains bound to C3b organizing the alternative pathway C3 convertase (C3bBb). Recently, we have visualized the stable C3bB(Ni(2+)) proconvertase using electron microscopy, revealing a large conformational change of the C3b-bound fB likely exposing the fD-cleavage site. In contrast, the crystal structure of the proconvertase formed by human fB and the cobra venom factor reveals fB in the closed conformation of the proenzyme. In this study, we have used single-particle electron microscopy and image processing to examine the C3bB(Mg(2+)) proconvertase. We describe two C3bB(Mg(2+)) conformations, one resembling cobra venom factor, likely representing the loading state of fB to C3b, and another identical with C3bB(Ni(2+)). These data illustrate the coexistence of C3b-bound fB in closed and open conformations that either exist in equilibrium or represent structural transitions during the assembly of the C3bB proconvertase.

Insights into complement convertase formation based on the structure of the factor B-cobra venom factor complex.

Immune protection by the complement system critically depends on assembly of C3 convertases on the surface of pathogens and altered host cells. These short-lived protease complexes are formed through pro-convertases, which for the alternative pathway consist of the complement component C3b and the pro-enzyme factor B (FB). Here, we present the crystal structure at 2.2-A resolution, small-angle X-ray scattering and electron microscopy (EM) data of the pro-convertase formed by human FB and cobra venom factor (CVF), a potent homologue of C3b that generates more stable convertases. FB is loaded onto CVF through its pro-peptide Ba segment by specific contacts, which explain the specificity for the homologous C3b over the native C3 and inactive products iC3b and C3c. The protease segment Bb binds the carboxy terminus of CVF through the metal-ion dependent adhesion site of the Von Willebrand factor A-type domain. A possible dynamic equilibrium between a 'loading' and 'activation' state of the pro-convertase may explain the observed difference between the crystal structure of CVFB and the EM structure of C3bB. These insights into formation of convertases provide a basis for further development of complement therapeutics.

Cloning and molecular characterization of two complement Bf/C2 genes in large yellow croaker (Pseudosciaena crocea).

Complement components factor B and C2 are two crucial proteases in the alternative pathway (AP) and classical pathway (CP). Two Bf/C2 cDNAs, LycBf/C2A and LycBf/C2B were isolated from the large yellow croaker (Pseudosciaena crocea) by suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). Through sequence alignment and computer 3D modeling analysis, we found that both of the deduced proteins contain three complement control protein (CCP) modules, a von Willebrand factor A (vWFA) domain, and one serine protease (SP) domain. Both structural analysis and phylogenetic analyses suggested that LycBf/C2A is more like human factor B than human C2 while LycBf/C2B is more human C2-like. After that, RT-PCR assay showed that LycBf/C2A and LycBf/C2B were mostly expressed in liver, albeit detectable in other tissues. Finally, after being infected with attenuated live Vibrio anguillarum strain, the expression level of LycBf/C2A and LycBf/C2B were found remarkably up-regulated in liver, spleen and kidney, indicating that the two complement factors play a pivotal role in the immune response to bacterial challenge in large yellow croaker.

The structure of C2b, a fragment of complement component C2 produced during C3 convertase formation.

The second component of complement (C2) is a multi-domain serine protease that provides catalytic activity for the C3 and C5 convertases of the classical and lectin pathways of human complement. The formation of these convertases requires the Mg(2+)-dependent binding of C2 to C4b and the subsequent cleavage of C2 by C1s or MASP2, respectively. The crystal structure of full-length C2 is not yet available, although the structure of its C-terminal catalytic segment C2a has been determined. The crystal structure of the N-terminal segment C2b of C2 determined to 1.8 A resolution presented here reveals the arrangement of its three CCP domains. The domains are arranged differently compared with most other CCP-domain assemblies, but their arrangement is similar to that found in the Ba part of the full-length factor B structure. The crystal structures of C2a, C2b and full-length factor B are used to generate a model for C2 and a discussion of the domain association and possible interactions with C4b during formation of the C4b-C2 complex is presented. The results of this study also suggest that upon cleavage by C1s, C2a domains undergo conformational rotation while bound to C4b and the released C2b domains may remain folded together similar to as observed in the intact protein.

3D structure of the C3bB complex provides insights into the activation and regulation of the complement alternative pathway convertase.

Generation of the alternative pathway C3-convertase, the central amplification enzyme of the complement cascade, initiates by the binding of factor B (fB) to C3b to form the proconvertase, C3bB. C3bB is subsequently cleaved by factor D (fD) at a single site in fB, producing Ba and Bb fragments. Ba dissociates from the complex, while Bb remains bound to C3b, forming the active alternative pathway convertase, C3bBb. Using single-particle electron microscopy we have determined the 3-dimensional structures of the C3bB and the C3bBb complexes at approximately 27A resolution. The C3bB structure shows that fB undergoes a dramatic conformational change upon binding to C3b. However, the C3b-bound fB structure was easily interpreted after independently fitting the atomic structures of the isolated Bb and Ba fragments. Interestingly, the divalent cation-binding site in the von Willebrand type A domain in Bb faces the C345C domain of C3b, whereas the serine-protease domain of Bb points outwards. The structure also shows that the Ba fragment interacts with C3b separately from Bb at the level of the alpha'NT and CUB domains. Within this conformation, the long and flexible linker between Bb and Ba is likely exposed and accessible for cleavage by fD to form the active convertase, C3bBb. The architecture of the C3bB and C3bBb complexes reveals that C3b could promote cleavage and activation of fB by actively displacing the Ba domain from the von Willebrand type A domain in free fB. These structures provide a structural basis to understand fundamental aspects of the activation and regulation of the alternative pathway C3-convertase.