C5 convertase of the alternative complement pathway: covalent linkage between two C3b molecules within the trimolecular complex enzyme

C5 convertase of the alternative C pathway is a complex enzyme consisting of three C fragments--one molecule of a major fragment of factor B (Bb) and two molecules of a major fragment of C3 (C3b). Within this C3bBbC3b complex, the first C3b binds covalently to the target surface, and Bb, which bears a catalytic site, binds noncovalently to the first C3b. In the present investigation, we studied the nature of the convertase that is assembled on E surfaces and obtained evidence that the second C3b binds directly to the alpha'-chain of the first through an ester bond rather than to the target surface. Thus, the alternative pathway C5 convertase could be described as a trimolecular complex in which Bb binds noncovalently to a covalently linked C3b dimer. We also obtained evidence that not only the second C3b but also the first C3b participates in binding C5, that is, covalently-linked C3b dimer acts as a substrate-binding site. Because of this two-site binding, the convertase has a much higher affinity for C5 than the surrounding monomeric C3b molecules. Based on this evidence, a new model of the alternative pathway C5 convertase is proposed. Covalent association of two subunits and the bivalent binding of the substrate are then common properties of the alternative and classical pathway C5 convertases.     

Covalent binding of C3b to C4b within the classical complement pathway C5 convertase. Determination of amino acid residues involved in ester linkage formation.

C5 convertase of the classical complement pathway is a protein complex consisting of C4b, C2a, and C3b. Within this complex C3b binds to C4b via an ester linkage. We now present evidence that the covalent C3b-binding site on human C4b is Ser at position 1217 of C4. We also show that formation of the covalently linked C4b.C3b complex occurs in the mouse complement system and that the C3b-binding site on mouse C4b is Ser at position 1213 which is homologous to Ser-1217 of human C4. Therefore, covalent binding of C3b to a single specific site on C4b within the classical pathway C5 convertase is likely a common phenomenon in the mammalian complement system. Specific noncovalent association of metastable C3b with C4b would occur first, leading to reaction of the thioester with a specific hydroxy group. This is supported by two lines of experimental evidence, one which shows that a mutant C4 that does not make a covalent linkage with C3b is still capable of forming C5 convertase and a second in which the C4b.C3b complex has been demonstrated by cross-linking erythrocytes bearing this C5 convertase.     

The conversion of human complement component C5 into fragment C5b by the alternative-pathway C5 convertase.

The cleavage of human complement component C5 to fragment C5b by the alternative pathway C5 convertase was studied. The alternative-pathway C5 convertase on zymosan can be represented by the empirical formula zymosan--C3b2BbP. Both properdin-stabilized C3 and C5 convertase activities decay with a half life of 34 min correlating with the loss of the Bb subunit. The C5 convertase functions in a stepwise fashion: first, C5 binds to C3b and this is followed by cleavage of C5 to C5b. The capacity to bind C3b is a stable feature of component C5, as C5b also has this binding capacity. Component C5, unlike component C3, does not form covalent bonds with zymosan after activation, and C5 is not inhibited by amines. Therefore C5, although similar in structure to C3, does not appear to contain the internal thioester group reported for C3 and C4.     

Alternative complement pathway activation fragment Ba binds to C3b. Evidence that formation of the factor B-C3b complex involves two discrete points of contact

Alternative complement pathway C3 convertase formation involves the cleavage of C3b-associated factor B into fragments Ba and Bb. Whereas Bb, in complex with C3b, has proteolytic specificity toward native C3, the function of the Ba moiety in the formation and/or decay of alternative complement pathway C3 convertase is uncertain. Therefore, we have examined the effect of purified Ba fragment on both fluid-phase and surface-bound enzymatic activity and showed that whereas Ba could inhibit the rate of C3 convertase formation, the rate of intrinsic decay remained unaffected. A specific, metal ion-independent interaction between Ba and C3b was subsequently demonstrated by use of the cross-linking reagent dithiobis(succinimidyl propionate). When cell-associated 125I-B was activated by D, the dissociation of Bb fragment displayed simple first-order kinetics with a half-time of 2.4 min, this value being in reasonable agreement with the hemolytically determined decay rate of 1.8 min. In contrast, most of the Ba fragment undergoes rapid dissociation, but there is also evidence to suggest the establishment of a new equilibrium due to the ability of Ba to rebind to C3b. Cumulatively, these data are consistent with a model in which the attachment of intact B to C3b is mediated by two points of contact, one being in the Ba domain and the other in the Bb domain. Due to avidity effects, each of these interactions could be of relatively low intrinsic affinity, and the characteristic unidirectionality of alternative complement pathway C3 convertase decay may simply result from the low intrinsic association of "univalent" Bb for the C3b subunit.     

C3 convertase of the alternative complement pathway. Demonstration of an active, stable C3b, Bb (Ni) complex

The purposes of this study were to demonstrate the C3 convertase complex, C3b, Bb (EC 3.4.21.47), of the alternative pathway of complement by ultracentrifugation and to determine whether the metal ion required for enzyme formation is present in the active enzyme complex. It has been shown previously that C3b,Bb formed with Ni2+ rather than Mg2+ exhibits enhanced stability. Using sucrose density gradient ultracentrifugation, an enzymatically active C3b,Bb(Ni) complex could be demonstrated which has a sedimentation coefficient of 10.7 S and which is stable in 10 mM EDTA. Upon formation of the enzyme with the radioisotope 63Ni2+, the ultracentrifugal distribution of the metal correlated with that of the enzyme complex. The molar ratio of Ni to C3b,Bb was 1:1. Displacement of Ni by Mg during formation of the enzyme indicated that both metals may bind to the same site in the enzyme. Binding of 63Ni to the catalytic site bearing fragment Bb was significantly stronger than its binding to C3b or to the zymogen, Factor B. It is proposed that there is one metal-binding site in the C3b,Bb enzyme which is not susceptible to chelation by EDTA and which is located in the Bb subunit.     

Residual hemolytic and proteolytic activity expressed by Bb after decay-dissociation of C3b,Bb

Bb (Mr = 63,000) is the catalytic site-bearing subunit of the C3 convertase of the alternative complement pathway, C3b,Bb, which is dissociated from the complex upon decay of the enzyme. Because purified Bb induced certain leukocyte activities, we examined whether it expresses residual hemolytic or proteolytic activity. Hemolytic activity of Bb was tested by using Factor B- or Factor D-depleted normal human serum and rabbit or sheep erythrocytes. Proteolytic activity of Bb was assessed by using purified C3 or C5 as substrates and SDS-PAGE to detect protein cleavage. Bb expressed metal-dependent hemolytic activity that was approximately 100-fold lower than that of Factor B. This activity could be inhibited by Factor H and enhanced by properdin. Low but statistically significant binding of 125I-labeled Bb to C3b on erythrocytes was demonstrated. Monoclonal antibodies that bind to Bb but not to intact Factor B inhibited the Bb hemolytic activity. Purified Bb cleaved C3 to C3a and C3b, as evidenced by the appearance of the alpha'-chain of C3b. It also cleaved C5 to C5a and C5b when cobra venom factor was present in the reaction mixture. Metal ions were required for expression of proteolytic activity, and Ni supported the activity better than Mg. These results indicate that decayed Bb has residual C3 and C5 cleaving activity and hemolytic activity, expression of which appears to require its association with C3b, C3(H2O), or cobra venom factor. These observations may aid in explaining the mechanism of action of Bb on leukocytes.     

Isolation and characterization of a 33,000-dalton fragment of complement Factor B with catalytic and C3b binding activity

Factor B is the zymogen of the catalytic site bearing subunit Bb of the C3/C5 convertase of the alternative pathway of complement. In this study, the location of the C3b binding site and the catalytic site within the Bb subunit were investigated. When human Factor B was treated with porcine elastase, fragments with respective molecular weights of 36,000, 35,000, 33,000, 31,000, and 25,000 were generated. Binding studies showed that only the 33,000-dalton fragment was capable of binding to C3b. The 33,000-dalton fragment was purified using fast protein liquid chromatography and found to be part of the Bb fragment upon testing with monoclonal antibody 15-6-19-1. Amino-terminal amino acid sequence analysis of the 33,000-dalton fragment placed it in the C-terminal half of Bb. The fragment expressed esterolytic activity as evidenced by cleavage of the synthetic substrate N alpha-acetyl-glycyl-L-lysine methyl ester and restored alternative pathway activity in Factor B-depleted serum. Its hemolytic activity was approximately 60-fold lower than that of Factor B. Comparative binding studies in the presence of metal ions using zymosan-C3b showed that the 33,000-dalton fragment bound to C3b with higher affinity than Factor B. Addition of the fragment to human serum inhibited alternative pathway activation by rabbit erythrocytes due to its high affinity for C3b and its low hemolytic activity compared to Factor B. These results show that the C-terminal 33,000-dalton portion of Bb contains not only the enzymatic site of Bb but also a C3b binding site which confers hemolytic activity upon the fragment. The observation that the fragment inhibited alternative pathway activation suggests that a synthetic peptide may be constructed that exhibits negative regulator activity in the alternative pathway.     

The influence of membrane components on regulation of alternative pathway activation by decay-accelerating factor.

Decay-accelerating factor (DAF) is a C regulatory protein which functions in membranes to inhibit autologous C activation on cell surfaces. A liposome model was used to study the mechanism of DAF action and examine the effects of membrane-bound glycophorin and LPS on the regulatory activity of DAF. Liposomes were incubated in MgEGTA-treated human serum and activation of the alternative pathway measured by C3b binding. Liposomes composed of phosphatidylcholine, phosphatidylethanolamine, and cholesterol activated the alternative pathway in proportion to their content of PE. Incorporation of 10(-7) mol/mol phospholipid of either human E or HeLa cell-derived DAF inhibited C activation by liposomes containing 40% phosphatidylethanolamine by 50%, an efficiency comparable to that observed in intact E. HeLa DAF that had been treated with phosphatidylinositol-specific phospholipase C to remove its glycolipid anchor had no effect on C activation by liposomes at concentrations as high as 10(-5) mol/mol phospholipid. Incorporation of DAF into liposomes prepared with bound C3b inhibited the deposition of additional C3b by C3bBbP. However, the incorporated DAF increased the amount of Bb generated from B in the presence of D indicating that accelerated decay of the convertase was the primary effect of DAF. Similarly, treatment of intact human E with anti-DAF decreased the amount of Bb generated by the alternative pathway convertase. To study the effects of other membrane components on DAF activity, liposomes were prepared with purified human glycophorin A or LPS. In glycophorin liposomes the presence of PE was required to activate the alternative pathway and DAF inhibited this activation. In contrast, LPS liposomes bound C3b independently of PE and the incorporation of DAF had no effect. These results demonstrate that within a membrane, DAF's inhibitory activity on the alternative pathway C3 convertase is mediated independently of other membrane proteins, that in this model the major activity of DAF is to accelerate convertase decay, and that the presence of other membrane molecules that may serve as C3 acceptors can circumvent DAF function.     

Polymerization of factor B of the alternative complement pathway via disulfide bond(s) in the presence of Cu2+ and stimulation by C3b, the major fragment of C3

Factor B(B) of the alternative complement pathway has been found to dimerize via disulfide bond(s) in the presence of CuCl2. Poly B has no B activity. The Bb fragment was also dimerized, indicating that one free sulfhydryl group on the Bb portion might be involved in polymerization. The Ba fragment was not dimerized. C3b, the major fragment of C3, has the capacity to stimulate polymerization of B. Incubation of C3b, B and factor D in the presence of Mg2+ and Cu2+ resulted in the formation of poly B and diminished cleavage of B. These results suggest that polymerization of B due to Cu2+ might be partly responsible for the impairment of C3 convertase activity of the alternative pathway.     

The low C5 convertase activity of the C4A6 allotype of human complement component C4.

We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.     

Functional role of the noncatalytic subunit of complement C5 convertase

The C5 convertase is a serine protease that consists of two subunits: a catalytic subunit which is bound in a Mg2+-dependent complex to a noncatalytic subunit. To understand the functional role of the noncatalytic subunit, we have determined the C5-cleaving properties of the cobra venom factor-dependent C5 convertase (CVF, Bb) made with CVF purified from the venom of Naja naja (CVFn) and Naja haje (CVFh) and compared them to those for two C3b-dependent C5 convertases (ZymC3b,Bb and C3b,Bb). A comparison of the kinetic parameters indicated that although the four C5 convertases (CVFn,Bb, ZymC3b,Bb, CVFh,Bb, and C3b,Bb) had similar catalytic rate constants (kcat = 0.004-0.012 s-1) they differed 700-fold in their affinity for the substrate as indicated by the Km values (CVFn,Bb = 0.036 microM, ZymC3b,Bb = 1.24 microM, CVFh,Bb = 14.0 microM, and C3b,Bb = 24 microM). Analysis of binding interactions between C5 and the noncatalytic subunits (CVFh or C3b, or CVFn) using the BIAcore, revealed dissociation binding constants (Kd) that were similar to the Km values of the respective enzymes. The kinetic and binding data demonstrate that the binding site for C5 resides in the noncatalytic subunit of the enzyme, the affinity for the substrate is solely determined by the noncatalytic subunit and the catalytic efficiency of the enzyme appears not to be influenced by the nature of this subunit.     

The crystal structure of C2a, the catalytic fragment of classical pathway C3 and C5 convertase of human complement

The multi-domain serine protease C2 provides the catalytic activity for the C3 and C5- convertases of the classical and lectin pathways of complement activation. 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 C-terminal fragment C2a consisting of a serine protease (SP) and a von Willebrand factor type A (vWFA) domain, remains attached to C4b, forming the C3 convertase, C4b2a. Here, we present the crystal structure of Mg(2+)-bound C2a to 1.9 A resolution in comparison to its homolog Bb, the catalytic subunit of the alternative pathway C3 convertase, C3bBb. Although the overall domain arrangement of C2a is similar to Bb, there are certain structural differences. Unexpectedly, the conformation of the metal ion-dependent adhesion site and the position of the alpha7 helix of the vWFA domain indicate a co-factor-bound or open conformation. The active site of the SP domain is in a zymogen-like inactive conformation. On the basis of these structural features, we suggest a model for the initial steps of C3 convertase assembly.     

Inhibition of the function of activated properdin by squid chondroitin sulfate E glycosaminoglycan and murine bone marrow-derived mast cell chondroitin sulfate E proteoglycan

We compared the relative capacities of two over-sulfated glycosaminoglycans, heparin and chondroitin sulfate E, to alter the function of native properdin (nP) and activated properdin (aP) in the formation and stabilization of the amplification C3 convertase (C3b,Bb). Heparin was more active on a weight basis than chondroitin sulfate E in inhibiting the formation of C3b,Bb without or with nP, but had no influence on the decay of a pre-formed convertase, either unstabilized or stabilized with nP or aP. In contrast, chondroitin sulfate E was over 10-fold more active than heparin in preventing the formation of C3b,Bb in the presence of aP, and gave dose-related acceleration of decay of pre-formed C3b,Bb,aP but not of unstabilized or nP-stabilized pre-formed convertase. The inhibitory effect of both glycosaminoglycans on the formation of C3b,Bb in the presence of nP or aP was less when the number of C3b sites per target cell was increased. The preferential action of chondroitin sulfate E on the function of aP during the formation and decay of C3b,Bb,aP as compared to C3b,Bb,nP implies functional differences in the two forms of P even when they have been incorporated into C3b,Bb. The equal potency, when adjusted for uronic acid content, of chondroitin sulfate E proteoglycan isolated from the T cell-dependent, bone marrow-derived murine mast cell and of chondroitin sulfate E glycosaminoglycan from squid reveals that the linkage of the glycosaminoglycan to a peptide core does not diminish its regulatory action on the alternative complement pathway.     

Further evidence for the antibody nature of C3 nephritic factor (C3NeF).

C3 nephritic factor (C3NeF), found in the sera of some patients with membranoproliferative glomerulonephritis, has been shown to be composed of two heavy and two light chains, like IgG; in addition it shares antigenic determinants with IgG. Purified C3NeF binds to the amplification convertase of complement, C3b,Bb, and thereby prevents decay of its C3-cleaving potential. The capability of C3NeF to bind to C3b,Bb was used as a means for purifying C3NeF to homogeneity. The investigation described in this report suggests that binding of C3NeF to C3b,Bb occurs via the Fab portion of the molecule. Pepsin treatment of eight C3NeF preparations resulted in an average loss of 76% of C3NeF functional activity. Papain treatment induced a loss of approximately 90%. The decrease in functional activity could be attributed to the accelerated rate of dissociation of 125I-F(ab')2 and 125I-Fab fragments from stabilized cell-bound C3b,Bb. The dissociation rate of 125I-F(ab')2 from C3b,Bb was comparable with the decay of the functional activity of C3b,Bb stabilized by F(ab')2 or Fab fragments of C3NeF. Although these results suggest that the stabilizing activity of C3NeF is mediated by the Fab portion of the molecule, it was found that the Fc portion also contributes to its functional activity.     

Decay-accelerating factor must bind both components of the complement alternative pathway C3 convertase to mediate efficient decay

Decay-accelerating factor (DAF; CD55) inhibits the complement (C) cascade by dissociating the multimolecular C3 convertase enzymes central to amplification. We have previously demonstrated using surface plasmon resonance (Biacore International) that DAF mediates decay of the alternative pathway C3 convertase, C3bBb, but not of the inactive proenzyme, C3bB, and have shown that the major site of interaction is with the larger cleavage subunit factor B (Bb) subunit. In this study, we dissect these interactions and demonstrate that the second short consensus repeat (SCR) domain of DAF (SCR2) interacts only with Bb, whereas SCR4 interacts with C3b. Despite earlier studies that found SCR3 to be critical to DAF activity, we find that SCR3 does not directly interact with either subunit. Furthermore, we demonstrate that properdin, a positive regulator of the alternative pathway, does not directly interact with DAF. Extending from studies of binding to decay-accelerating activity, we show that truncated forms of DAF consisting of SCRs 2 and 3 bind the convertase stably via SCR2-Bb interactions but have little functional activity. In contrast, an SCR34 construct mediates decay acceleration, presumably due to SCR4-C3b interactions demonstrated above, because SCR3 alone has no binding or functional effect. We propose that DAF interacts with C3bBb through major sites in SCR2 and SCR4. Binding to Bb via SCR2 increases avidity of binding, concentrating DAF on the active convertase, whereas more transient interactions through SCR4 with C3b directly mediate decay acceleration. These data provide new insights into the mechanisms involved in C3 convertase decay by DAF.     

A covalent dimer of complement C4b serves as a subunit of a novel C5 convertase that involves no C3 derivatives

A C intermediate, LAC14, was prepared from TNP-aminocaproyl liposomes sensitized with anti-TNP antibody (Ab) and purified human C1 and C4. LAC14, containing radiolabeled C4, was analyzed by SDS-PAGE followed by autoradiography, and yielded a 210-kDa band and a predominant 400-kDa band. The 210-kDa band consisted of monomeric C4b bound to low molecular mass acceptors. The 400-kDa band was comprised of a 200-kDa moiety, as well as beta- and gamma-chains of C4. The 200-kDa moiety contained neither C1 nor sensitizing Ab, but it was largely decreased by treatment with NH2OH to the 90-kDa moiety with the mobility corresponding to the alpha'-chain of C4b. A covalent dimer of C4b, therefore, is the predominant form of C4b deposited on liposomes sensitized with antibody. The C4b-C4b dimer formed rapidly (within 5 min) followed by slow dissociation into monomers. The LAC14 bearing the C4b dimer but not the monomer was lysed, although with relatively low efficiency, by the addition of oxyC2 and EDTA-supplemented C3-deficient serum (C3DS), and, furthermore, LAC142 possessed the ability to convert C5 into C5a and C5b. Moreover, lysis was inhibited not by anti-C3 Ab but by anti-C4 Ab. In other experiments, the dimer served as an element of C3 convertase, as well. These findings imply that the C4b dimer, when complexed with C2, expresses C3/C5 convertase activity without participation of C3, and may provide a molecular mechanism whereby sera from patients with complete C3 deficiency retain the ability to induce C-mediated cytolysis.     

C3 adsorbed to a polymer surface can form an initiating alternative pathway convertase

Contact between blood and a biomaterial surface induces an immediate complement-mediated inflammatory response. Under these conditions, the alternative pathway of complement is often initiated and amplified on the biomaterial surface. Adsorption of a protein such as C3 to a polymer surface induces conformational changes in the protein. Based on the expression on adsorbed C3 of conformational neoepitopes specific for bound C3 fragments, we have hypothesized that adsorbed C3 is able to bind factor B and form a functional C3,Bb convertase. Using a quartz crystal microbalance to monitor binding of proteins to a polymer surface, we have demonstrated that a functional C3-containing alternative pathway convertase can be formed, in particular, in the presence of properdin. These data indicate that adsorption of C3 induces conformational changes that turn C3 into a C3b-like molecule that is able to participate in the functioning of the alternative convertase, and they suggest a new mechanism for complement activation on a biomaterial surface.     

Formation of classical C3 convertase during the alternative pathway of human complement activation

The fluid phase C3 convertase of the alternative pathway of human complement activation has been constructed from the isolated C3 component and from purified factors B and D. The enzyme was able to activate the isolated components C4 and C2 in the presence of C4 but had no effect on C2 in the absence of C4. The C4 and C2 activation was monitored by the loss of their hemolytic activity during the incubation with the alternative fluid phase C3 convertase. The activation of C4 and C2 components by the membrane-bound alternative C3 convertase formed on red cells (EC3bBb) was followed by the formation of C3 convertase of the classic pathway--EC4b2a. This resulted in the enhancement of hemolysis.     

Guinea pig erythrocytes, after their contact with influenza virus, acquire the ability to activate the human alternative complement pathway through virus-induced desialation of the cells

Guinea pig erythrocytes that had been exposed to influenza A virus activated the alternative complement pathway in whole human serum in the absence of natural antibodies. Because all virus particles were eluted from the treated cells, activation was not dependent on antiviral antibodies or on virus particles themselves. The relative capacity of treated erythrocytes to activate the alternative pathway was dependent on the amount of virus to which the cells had been exposed and was directly related to the amount of sialic acid removed from the erythrocyte membrane during incubation with either whole virus particles or purified viral sialidase. C3b bound to cells that had been treated with virus, and P-stabilized amplification convertase sites P,C3b,Bb formed on these cells, exhibited increased resistance to the action of the regulatory proteins beta-1H and C3b Ina compared with C3b and P,C3b,Bb on untreated, nonactivating cells. The acquired resistance of the cell-bound, P-stabilized amplification convertase to decay-dissociation by beta-1H was directly related to the activating capacity of the treated cells in whole serum (r = 0.95) and to the amount of sialic acid removed from the cells by the virus (r = 0.98). Desialation represents a specific alteration of the cell surface by which a nonimmune host, through activation of the alternative pathway, may deposit C3b on a target cell that had been exposed to influenza virus and may lyse virus virus-modified cells during orthomyxovirus infections.     

Molecular dissection of interactions between components of the alternative pathway of complement and decay accelerating factor (CD55)

The complement regulatory protein decay accelerating factor (DAF; CD55), inhibits the alternative complement pathway by accelerating decay of the convertase enzymes formed by C3b and factor B. We show, using surface plasmon resonance, that in the absence of Mg(2+), DAF binds C3b, factor B, and the Bb subunit with low affinity (K(D), 14 +/- 0.1, 44 +/- 10, and 20 +/- 7 microm, respectively). In the presence of Mg(2+), DAF bound Bb or the von Willebrand factor type A subunit of Bb with higher affinities (K(D), 1.3 +/- 0.5 and 2.2 +/- 0.1 microm, respectively). Interaction with the proenzyme C3bB was investigated by flowing factor B across a C3b-coated surface in the absence of factor D. The dissociation rate was dependent on the time of incubation, suggesting that a time-dependent conformational transition stabilized the C3b-factor B interaction. Activation by factor D (forming C3bBb) increased the complex half-life; however, the enzyme became susceptible to rapid decay by DAF, unlike the proenzyme, which was unaffected. A convertase assembled with cobra venom factor and Bb was decayed by DAF, albeit far less efficiently than C3bBb. DAF did not bind cobra venom factor, implying that Bb decay is accelerated, at least in part, through DAF binding of this subunit. It is likely that DAF binds the complex with higher affinity/avidity, promoting a conformational change in either or both subunits accelerating decay. Such analysis of component and regulator interactions will inform our understanding of inhibitory mechanisms and the ways in which regulatory proteins cooperate to control the complement cascade.