Structural basis for the stabilization of the complementalternative pathway C3 convertase by properdinMartín Alcorloa, Agustín Tortajadaa,b, Santiago Rodríguez de Córdobaa,b,1, and Oscar Llorcaa,1

aCentro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain; and bCentro de Investigación Biomédica enEnfermedades Raras, 28040 Madrid, Spain

Edited by Douglas T. Fearon, University of Cambridge School of Clinical Medicine, Cambridge, United Kingdom, and approved July 5, 2013 (received forreview May 21, 2013)

Complement is an essential component of innate immunity. Itsactivation results in the assembly of unstable protease complexes,denominated C3/C5 convertases, leading to inflammation andlysis. Regulatory proteins inactivate C3/C5 convertases on hostsurfaces to avoid collateral tissue damage. On pathogen surfaces,properdin stabilizes C3/C5 convertases to efficiently fight infec-tion. How properdin performs this function is, however, unclear.Using electron microscopy we show that the N- and C-terminalends of adjacent monomers in properdin oligomers conform a curlyvertex that holds together the AP convertase, interacting withboth the C345C and vWA domains of C3b and Bb, respectively. Pro-perdin also promotes a large displacement of the TED (thioester-containing domain) and CUB (complement protein subcomponentsC1r/C1s, urchin embryonic growth factor and bone morphogeneticprotein 1) domains of C3b, which likely impairs C3-convertase in-activation by regulatory proteins. The combined effect of molecularcross-linking and structural reorganization increases stability of theC3 convertase and facilitates recruitment of fluid-phase C3 conver-tase to the cell surfaces. Our model explains how properdin medi-ates the assembly of stabilized C3/C5-convertase clusters, whichhelps to localize complement amplification to pathogen surfaces.

Complement is a crucial component of innate immunity. It isa first line defense mechanism against pathogens and it is

essential in the modulation of adaptive immune responses and toremove apoptotic cell debris and immune complexes (1). Acti-vation of complement results in the formation of unstable pro-tease complexes, named C3 convertases (C3bBb in the alternativepathway) (AP), which catalyze the cleavage of C3 to generate theactivated fragment, C3b. This exposes a reactive thioester thatattaches covalently to the target surfaces (opsonization), initiatingthe terminal pathway that causes cell lysis and generates in-flammation at the site of activation (1, 2).The complement AP is exquisitely regulated and pathological

conditions are associated with both loss-of-function variants ofthe regulatory molecules, as well as gain-of-function variants ofpropagating components of the pathway (2). Accelerated disso-ciation of the AP C3 convertase and inactivation of C3b arecritical steps to maintain complement homeostasis and to pre-vent nonspecific damage to self-cellular components whencomplement is activated. These activities are performed pri-marily by factor H (FH), in collaboration with the plasma serineprotease factor I (FI) (2). Self-tissues are also protected bymembrane-bound proteins that restrict complement activationby acting as cofactor for proteolytic inactivation of C3b by FI oraccelerating the dissociation of the C3bBb convertase. Thus, inhealth, spontaneous activation of C3 in plasma is kept at a lowlevel and further complement activation and C3b deposition isrestricted to targets lacking surface regulators. Recent advancesin understanding the structural basis of the assembly, activation,and regulation of the AP C3 convertase have provided importantinsights into the regulation of the AP and the pathogenic con-sequences of its dysregulation (2–4).Properdin is the only known complement regulator that

enhances the stability of the C3bBb convertase and the activity of

the AP. Properdin binds to C3bB and C3bBb more efficientlythan to C3b alone, stabilizing preformed C3bBb convertasecomplexes (5). Properdin is also a pattern-recognition moleculethat binds to negatively charged molecules on certain microbialsurfaces, apoptotic and necrotic cells, as well as cells undergoingmalignant transformation. Once bound to a surface, properdincan direct C3b deposition and C3bBb assembly, thus serving asa focal point for amplifying complement activation (6). Althoughthe importance of properdin has been somehow neglected, it playsimportant roles in antibacterial defense and in inflammatory orautoimmune diseases, as illustrated by the high vulnerability ofproperdin-deficient individuals to Neisseria meningitides infectionsand the reported role of properdin in a number of pathologicalconditions (7, 8).Properdin is a 53-kDa glycoprotein comprising seven con-

served domains with homology to thrombospondin repeats(TSRs) of type I, and numbered TSR0 to TSR6 from the N- tothe C terminus (Fig. 1A) (9). Atomic structures for properdinhave not been resolved yet, but the structure of a double-TSRdomain from thrombospondin [Protein Data Bank (PDB) 3R6B]provides a reasonable model for TSR domains in properdin (10)(Fig. 1A). Each TSR comprises a folded core consisting of threeantiparallel strands (A, B, and C) held together by three disul-fides (11) (Fig. 1A). Human plasma contains a low concentrationof properdin (0.02 mg/mL) in the form of a polydisperse mixtureof oligomeric structures, mostly dimers, trimers, and tetramers(12). Examination of purified properdin using electron micros-copy (EM) revealed that each monomer forms an elongated rod-like molecule, which associates into cyclic polymers (13). Despiteearly work identifying a potential region in C3b interacting withproperdin (14), the structural basis for the AP C3 convertasestabilization by properdin is unknown. Using single-particle EM,image processing, 3D reconstruction techniques, and hybridmethods that combine electron microscopy and X-ray crystal-lography data (15), we propose a model for the 3D structure ofthe properdin–C3bBb complex.

ResultsIntricate Connections Between Properdin Monomers Assemble LargeOligomers. Human properdin was purified to homogeneity fromplasma by immunoaffinity followed by ionic exchange and sizeexclusion chromatography (Fig. 1B). The functional integrity ofthe purified properdin was verified using AP-dependent hemo-lytic assays with rabbit erythrocytes (Fig. S1). Properdin was

Author contributions: M.A., S.R.d.C., and O.L. designed research; M.A. and A.T. performedresearch; O.L. analyzed data; and S.R.d.C. and O.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The 3D-EM maps have been deposited in the Electron Microscopy DataBank database, www.emdatabank.org andwww.ebi.ac.uk/pdbe (EMD-2402 and EMD-2403).1To whom correspondence may be addressed. E-mail: ollorca@cib.csic.es or srdecordoba@cib.csic.es.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1309618110/-/DCSupplemental.

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observed in the electron microscope and found assembled intoseveral oligomeric species in which the elongated monomerswere connected at their ends. As previously reported (12, 13) themost common oligomers were triangle-shaped trimers, square-shaped tetramers, and pentagonal pentamers (Fig. 1C, and be-low). From these EM images we interpreted that the interactionbetween properdin monomers involves the N-terminal end ofone monomer and the C-terminal end of another, permitting theassembly of a variety of oligomers with the only restriction ofgeometrical constraints. Interestingly, we found that the angleformed by two adjacent monomers ranged from 60° in thetrimers to 108° in the pentamers, indicating a large flexibility inthe interaction between monomers.Analysis of the vertex of the properdin oligomers by single-

particle image processing methods revealed a complex structure.A total of 6,425 images of vertexes from the tetramers wereextracted from the micrographs to be sorted into homogenous

groups and averaged to improve the signal/noise ratio (Fig. 1D).The 3D structure of the vertex at 23.4-Å resolution revealeda connectivity between monomers that was very different fromthat proposed from X-ray scattering data and modeling (16)(Fig. 1E and Fig. S1). We modeled the number of TSR domainscomposed of this vertex by manually fitting the atomic structureof one of the homologous TSR domains from thrombospondin(PDB 3R6B) (10) into the EM density. Each properdin mono-mer comprises seven TSR domains and we found that four ofthese units could be accommodated into the vertex. Thus, eachproperdin monomer contributes four TSR domains for the as-sembly of two vertexes, at the N- and C-terminal end of eachmonomer, leaving three TSR units for the elongated connectionbetween vertexes. In agreement with this, we found that theaverage distance between vertexes, obtained from 150 images ofcomplexes, measured 14.3 ± 1.2 nm (Fig. 1F), which fits thelength spanned by three TSRs, assuming an averaged length of5 nm per TSR domain based on the atomic structure (10).

Purification of the Properdin–C3bBb Convertase Complex. We as-sembled the complex between properdin and C3 convertase byincubating C3b, Factor B (FB), and Factor D (FD) in the pres-ence of properdin. In these experiments, we used the FB-D279Gmutant that increases the stability of C3 convertase (2). Themixture was resolved by gel filtration chromatography and themobility of the complex compared with that of properdin alone.We observed comigration of C3b, the Bb fragment of FB andproperdin in a large molecular weight species compared with theelution of C3bBb convertase alone. The purification of a stablecomplex containing C3bBb and properdin, which resisted purifi-cation, suggested properdin was contributing to stabilize theotherwise unstable C3bBb (17, 18) (Fig. 2A). The peak fractionwas observed in the electron microscope, revealing properdinoligomers decorated by extra densities corresponding to C3bBbconvertases (Fig. 2B). Interestingly, C3bBb convertase moleculeswere bound to properdin vertexes, revealing that the structureassembled by the oligomerization of two properdin monomerswas essential for C3bBb convertase recognition. C3bBb moleculesprotruded outwards from these vertexes. We tested several con-centrations of C3bBb convertase and found that the level ofvertex occupancy was dependent on the amount of C3bBb con-vertase used in the experiment (Fig. 2B). This indicates that eachproperdin oligomer has the potential to use all its vertexes to bindC3bBb convertase (Fig. 2C).

Properdin Cross-Links C3b and the Bb Fragment, Stabilizing the C3bBbConvertase. The basis for C3bBb convertase stabilization by pro-perdin was explored by processing 21,891 images of C3bBb con-vertase molecules at properdin vertexes extracted from themicrographs. These images were computationally classified to findand average those corresponding to similar views of the complex(Fig. 3A). These averages were extremely revealing comparedwith the crystal and EM structure of C3bBb convertase (17, 18).The macroglobulin (MG) ring and C345C domain from C3b, andthe von Willebrand factor type-A (vWA) and serine-protease (SP)domains from FB were clearly identified, as well as the properdinvertex contacting C345C and vWA (Fig. 3B). Other averages wereinterpreted as corresponding to additional views of the complexfrom a different angle and these were used for the 3D analysis ofthe complex (see below). Surprisingly, the most typical view of theproperdin–C3bBb convertase complex was found in two distinctsubtypes, either containing or not a strong globular density inthe proximity of MG3 domain (see below).Images of the properdin–C3bBb convertase complex were

then used to reconstruct its 3D structure. We found that twoconformations were coexisting in the dataset, which corre-sponded to those images that either contained or not a globulardensity in the proximity of the MG3 domain. The dataset was

Fig. 1. Structure of properdin oligomers by electron microscopy. (A) Sche-matic cartoon of the arrangement of TSR domains in a properdin monomer(Upper) and a view of the atomic structure of one homolog TSR domainfrom thrombospondin (PDB 3R6B, Lower) (10). Side chains of the proposedkey arginine and tryptophan residues are shown in blue and yellow, re-spectively. Disulfide bonds are shown in pink. (B) Final preparation of puri-fied properdin analyzed by SDS/PAGE. SS, silver staining; CS, Coomassiestaining; WB, Western blotting using polyclonal antibodies against pro-perdin. (C) Typical micrograph of properdin. Selected single molecule imagesfor several properdin oligomers are highlighted within a red square. (Scalebar, 26 nm.) (D) Reference-free 2D averages of properdin vertexes extractedfrom the micrographs reveal several views of the structure connecting twomonomers. (Scale bar, 7 nm.) (E) 3D structure of the properdin vertex andpseudoatomic model obtained by fitting a crystal structure of a TSR domainfrom thrombospondin (PDB 3R6B) (10) into the EM density. (Scale bar, 1.2nm.) (F) Carton representation of a properdin tetramer (Lower) and a rawimage for a properdin tetramer (Upper). Vertexes are represented as a bluecircle and the region whose distance was measured is indicated.

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consequently split in two subgroups (SI Materials and Methods).The 3D structure of the major population solved at 29.3-Å res-olution, corresponding to 66.6% of the particles, was interpretedby fitting the atomic structure of C3bBb convertase (PDB 2WIN)(17) into the EM map to generate a hybrid pseudoatomic modelof the complex (Fig. 3C). The Bb fragment together with theC345C domain had to be moved backward by ∼30° to fit into thedensity of the EM map, indicating a displacement compared withthe crystal structure. This is conceivable by the flexible linkerconnecting C345C to the MG ring, as observed in the EM imagesof C3bBb convertase (17, 18). The fitting revealed that a smallsegment of the MG ring corresponding to MG4 was not wellresolved in the reconstruction, which we interpret as an effect ofthe accumulation of staining agent in the center of the MG ringby the proximity of the globular density in MG3. Remarkably, wefound that the TED domain was missing at its expected locationin C3b, strongly suggesting that the globular density in the vi-cinity of the MG ring corresponds to the TED domain. The CUBdomain was also not found in properdin–C3bBb at the expectedlocation but a density nearby the TED domain was interpreted asthe CUB, further supporting the repositioning of the TED do-main (Fig. 3C).The structure revealed that properdin contacts both the

C345C domain in C3b and the vWa domain in Bb (Fig. 3C),indicating that properdin stabilizes the C3bBb convertase byholding together the two components of this enzymatic complex.The structure also suggested that properdin would interact withthese two domains more efficiently when the Bb fragment is inthe conformation found in C3bBb convertase than the closedconformation of the C3bB proconvertase. We confirmed this

hypothesis after observing that properdin did not interact withthe complex between C3b homolog cobra venom factor (CVF)and FB, because CVF and FB form a tight complex, but FB ismaintained in its closed conformation (19) (Fig. 3D).

Properdin Promotes a Relocation of the TED Domain. The 3Dstructure of the minor population of properdin–C3bBb con-vertase complexes revealed essentially the same structural fea-tures of the most abundant conformation albeit at lowerresolution, but the TED domain was at its expected location inC3b (Fig. 4 A and B). Thus, the TED domain was found in twopositions in the context of the properdin–C3bBb convertasecomplex, at an approximate 1:2 ratio between the two species(33.3 vs. 66.6%). We searched for this conformation of the TEDdomain in the C3b preparation used for these studies by col-lecting single molecule images, which were classified and aver-aged as before (Fig. 4C). We found that only 3.5% of 5,000images of C3b showed this unusual conformation, whereas∼73% corresponded to the TED position described in the crystalstructure (2–4). Interestingly, ∼18% of the images revealed al-ternative locations for the TED domain. As a whole, theseresults indicate that properdin stimulates the rearrangement ofthe TED domain in C3b.

Properdin Interferes with C3bBb Convertase Decay. The reposi-tioning of the TED domain, and presumably also the CUB do-main to which it is tethered, is predicted to remove essentialstructural determinants for the interaction of C3b with the com-plement regulators FH, decay-accelerating factor (DAF), andcomplement receptor 1 (CR1), turning the properdin–C3bBbcomplex less prone to accelerated decay compared with C3bBb

Fig. 2. Purification and electron microscopy of the properdin–C3bBb convertase complex. (A) Chromatograms (Upper) and silver-stained SDS/PAGE (Lower)for the fractions of size-exclusion chromatography experiments performed in a Superdex 200 gel-filtration column (GE Healthcare) using properdin, C3b, FD,and either wild-type FB or the FB-D279G mutant. Chromatograms show profiles for properdin injected alone (P, blue line), the incubation of C3b, FB, and FDto assemble a C3bBb convertase (C3bBb, magenta discontinuous line), and the incubation of properdin, C3b, FB-D279G, and FD to assemble a properdin–C3bBb convertase complex (PC3bBb, green line). Lower shows SDS/PAGE of selected fractions from the chromatographies above. (Left) Assembly of a C3bBbconvertase (C3bBb, magenta discontinuous line). (Right) Properdin–C3bBb convertase complex (PC3bBb, green line). (Center) SDS/PAGE of a chromatographyanalyzing the interaction of properdin with the C3bB proconvertase. The input to the gel-filtration column is indicated as IN, and C3b, FB, and properdin areloaded as controls. Chains of C3b detected in the SDS/PAGE are indicated. The formation of the properdin–C3bBb convertase complex is revealed by theadvanced elution of C3bBb (factions 10–15) in the presence of properdin compared with the elution of C3bBb convertase alone (C3bBb, fractions 17–21), aswell as the appearance of a new band corresponding to the FB fragment Bb (labeled Bb) resulting from the proteolysis of the input FB (labeled FB). Thefraction selected for EM analysis is labeled. (B) Representative micrograph corresponding to properdin–C3bBb convertase complexes collected at two ex-perimental conditions generating partial (Left) or high occupancy (Right) of properdin by C3bBb convertase. Selected C3bBb convertase molecules bound toproperdin have been labeled with an open arrow. Black arrows stand for unbound C3bBb convertase molecules. (Scale bar, 14 nm.) (C) Gallery of raw imagesof properdin–C3bBb convertase complexes selected from the micrographs and panelled according to the oligomeric state of properdin, and showing, fromleft to right, increased occupancy of properdin vertexes with C3bBb convertase molecules. (Scale bar, 14 nm.)

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alone (4, 20). Similarly, the repositioning of the TED domainshould impair the interaction between C3b and membrane co-factor protein (MCP or CD46), whereas changes in the CUBdomain could affect Factor I (FI) binding. The combined effectof these changes should slow down the FI-dependent proteolyticinactivation of C3b. We tested this hypothesis by assemblinga C3bBb convertase through the incubation of C3b, FB, and FDin the absence or presence of properdin (Fig. 5A). Next, solubleversions of MCP and FI were added and incubated for 15 min inall cases, to allow for cofactor activity, and the intact C3b wasquantified by measuring the ratio between α′ chain/β chain ofC3b. We observed that the amount of intact C3b remaining afterthe incubation was significantly increased in the presence ofproperdin in a dose-dependent manner (Fig. 5B). These datashow that properdin also contributes to stabilize the C3bBbconvertase by interfering the interaction of C3b with the com-plement regulators, which should impact both their accelerateddecay and factor-I mediated cofactor activities.

DiscussionA major point of regulation in the activation of complement isaltering the stability of the alternative pathway C3bBb con-vertase. Down-regulation to control homeostasis and preventtissue damage is provided by a number of plasma and mem-brane-associated regulators that accelerate the dissociation ofthe C3bBb complex (3). In contrast, properdin is the only com-plement regulator that stabilizes the C3bBb convertase, which

may be critical to tip the balance in favor of amplification onmicrobial pathogens (9). The molecular bases of the properdinfunctions are still poorly understood. Using EM single-particleimage processing methods we describe here a structural modelfor the properdin–C3bBb complex supporting that properdinstabilizes the C3bBb convertase by holding together the twocomponents of the AP C3 convertase, C3b and Bb, and bypromoting a large displacement of the TED domain that likelyinterferes with the decay of C3 convertase by complement down-regulators.Properdin oligomerizes by a complex interplay between N- and

C-terminal ends of two elongated monomers forming a curlyvertex structure (Fig. 6A). This interaction allows the assembly ofoligomers containing a variable number of monomers, and themaximum number of units that could be accommodated peroligomer is probably only limited by conformational and geo-metrical restrains. Our structural model for properdin is differentfrom that proposed by Sun et al. based on X-ray scattering andmodeling data (16). Their model showed connections betweenthe N- and C-terminal ends of properdin but the 3D structure ofthese contact points is very different from the structure of thevertexes that we have resolved using EM (Fig. S1). In addition,their model proposed that the properdin oligomers were partiallycollapsed, whereas we find well-defined triangular and squared-shaped molecules supporting full extension of the properdinmonomers. Although we cannot rule out an effect on the con-formation of properdin by the carbon surface used as support film

Fig. 3. Structure of the properdin–C3bBb convertasecomplex. (A) Representative reference free 2D aver-ages of C3bBb convertase molecules bound to pro-perdin vertexes (Right), compared with a view of thecrystallographic and EM structures of C3bBb con-vertase (Left) (PDB 2WIN) (17). Each domain has beencolored differently and labeled. (Scale bar, 5 nm.) (B)Selected average of the properdin–C3bBb convertasecomplex. Different domains and regions are labeled.(Scale bar, 5 nm.) (C) Two views of the structure of theproperdin–C3 convertase complex at 29.3-Å resolu-tion. A pseudoatomic model of the properdin–C3convertase complex was obtained by fitting theatomic structure of C3bBb convertase (PDB 2WIN) (17)into the EM structure. The MG ring is displayedin blue. C345, CUB, and TED domains are colored inorange, red, and green, respectively. vWA and SPdomains from the Bb fragment are colored in pink.Densities corresponding to properdin vertex are la-beled with asterisks. (Scale bar, 2 nm.) (D) Fractionsfrom a size-exclusion chromatography loaded withthe incubation of CVF, FB-D279 mutant, properdin,and FD were analyzed by SDS/PAGE. Properdindoes not interact with CVF-FB in the conditionstested, as revealed by the absence of comigrationof the CVFB complex with properdin. Inset, UpperRight corner shows an average of images obtainedfor the purified CVFB complex using electron mi-croscopy. (Scale bar, 5 nm.)

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in electron microscopy, compared with the structure describedfrom measurements in solution, we believe the EM conformationcould reflect closer the situation on cellular surfaces.Each vertex is the structural unit of recognition and stabili-

zation of C3bBb convertase by holding together the C345C andthe vWA domains from C3b and Bb, respectively (Fig. 6A). Thus,oligomerization, which requires the involvement of the N- and

the C-terminal TSRs, is essential for interaction with the C3bBbconvertase. This model is consistent with previous evidence show-ing that properdin binds equivalently to C3b, iC3b, and C3c (21),which we now justify by the interaction of properdin with theC345C domain, and with experiments showing that properdininteracts with FB (22). Early studies using a combination of cy-anogen bromide (CNBr) digestions and synthetic peptide syn-thesis, have proposed that the properdin binding site in C3b islocated within a 34-amino acid peptide region in the MG8 do-main of the α′-chain, spanning residues 1402–1435 (14). Ourmodel of the structure of the properdin–C3bBb complex does notshow contacts compatible with that region in C3b. To explainthese apparently conflicting data we like to suggest that theinteractions between C3b, FB, and properdin transit throughseveral intermediates so that properdin may initially recognizesites in C3b that are distinct from those conforming the fullyassembled properdin–C3bBb complex.The structure of each of the properdin vertexes could be

interpreted as a combination of TSR0 from one monomer andTSR4-5-6 from another (TSR0/TSR4-5-6), or the alternativeoptions TSR0-1/TSR5-6 and TSR0-1-2/TSR6, but we could notdiscriminate between these three options at the resolution ofthese studies (Fig. 6B). The studies by Higgins et al. (23) pro-posed functions for several TSR domains in properdin by char-acterizing recombinant properdin lacking single specific TSRs.TSR0, TSR1, and TSR2 were not included in those studies. Theysuggested that TSR4 and TSR5 were required for C3b bindingand C3bBb convertase stabilization, respectively. These resultsare not in conflict with our findings but they need to be re-analyzed, as any mutation affecting oligomerization would in-directly affect C3bBb binding. A vertex comprising TSR0 fromone monomer and TSR4-5-6 (TSR0/TSR4-5-6) from anothermonomer would agree with the published results. Importantly,the TSR4 and TSR5 mutants were also affected in oligomeri-zation, not assembling as trimers and tetramers. This could beinterpreted as TSR4 and TSR5 being part of the vertex, but al-ternatively, if the mutations affect oligomerization indirectly,they could fail to stabilize the C3bBb convertase as a conse-quence of the oligomerization defect rather than by being in-volved in C3bBb binding. The involvement of TSR6 at the vertexin stabilization of C3bBb is supported by the disease-associatedY387D mutation in TSR6, which produces normal plasma levelsof properdin, which assembles oligomer lacking the capability tostabilize the C3bBb convertase (24).A remarkable finding is the positioning of the TED domain in

two alternative locations, one in agreement with the crystalstructure of C3b and an alternative location in the vicinity of theMG3 domain, and the accompanying relocation of the CUBdomain attached to the TED. Such movements appear to be anintrinsic property of C3b, as we find a similar conformation ina small percentage of C3b molecules, but certainly properdinturns this alternative conformation into the major species. Inagreement with our findings, previous EM studies by Nishidaet al. found a proportion of C3b molecules in this alternativeconformation (25). In addition, recent FRET data obtained forC3b in complex with SCR1–4 from FH suggested some degree ofmobility of the TED domain (26). Large displacements of theCUB-TED region seem feasible as these take place during thestructural transition from C3 to C3b (4) and also from C3b toiC3b (27).The rearrangement of the TED removes essential structural

determinants for recognition of C3b by some regulators such asFH and MCP (4). SCR1–4 of FH interacts with C3b as anelongated string of monomers and this causes decay of C3 con-vertase, as revealed in the crystal structure of this complex (20).Movements of the TED domain in iC3b, a proteolytic fragment ofC3b, have been shown to disrupt FH binding and consequentiallyiC3b is not regulated by FH (27). Similarly, the rearrangements of

Fig. 4. Positioning of the TED domain in the properdin–C3bBb convertasecomplex. (A) Representative 2D averages of the minor conformation of theproperdin–C3bBb convertase complex. These show that the TED domain isnot in the proximities of the MG3 domain, but in the location found in thecrystal structure of C3b. (Scale bar, 5 nm.) (B) One view of the structure ofthe minor conformation of the properdin–C3bBb convertase complex at33.0-Å resolution. Densities corresponding to properdin vertex are labeledwith asterisks. (Scale bar, 2 nm.) (C) Processing and classification of images ofC3b revealed that most molecules show the TED domain in the classicalconformation, whereas a small percentage of molecules display the TEDdomain in alternative conformations. An arrow points to the TED domainplaced close to the MG3 domain, found in 3.5% of the images analyzed. Aview from the crystal structure of C3b (PDB 2I07) is shown to help compar-ison with the EM images. Each domain has been colored differently andlabeled. (Scale bar, 5 nm.)

Fig. 5. C3bBb convertase decay in the presence of MCP, FI, and properdin.(A) C3bBb convertase was formed by incubating C3b, FB, and FD in the ab-sence (P) (−) or the presence of two amounts of properdin (P) (0.9 μg + and1.8 μg ++) and incubated with MCP and FI. SDS/PAGE shows the result of thisreaction after incubating for 15 min. Each experiment was performed induplicate. (B) The amount of C3b remaining after incubation was estimatedby quantifying the ratio between α′ chain/β chain of C3b. Experiments la-beled as 1–4 correspond to the matching experiment in A (0.9 μg of P, grayand 1.8 μg of P, black). Error bars indicate the mean ± SD of two in-dependent experiments.

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the TED domain that we find in the properdin–C3bBb convertasecomplex would limit C3 convertase regulation by FH, MCP, andother regulators that use similar mechanisms, such as DAF.These effects probably combine with the consequences of thechanges observed in the CUB domain that are predicted toaffect the interaction of C3b and FI (4). In agreement with thisinterpretation, we observed a reduced FI-dependent cofactoractivity of MCP for the proteolysis of C3b in the presence ofproperdin. We speculate this will contribute, in addition to the

holding of C3b and Bb together, to enhance the complementresponses in vivo.

Materials and MethodsGeneration and Purification of Properdin–C3bBb Convertase Complexes. C3band properdin were purified from plasma and FB from the supernatant ofCHO cells. C3b (5 μg), FB (10 μg, FB or FB-D279G), and properdin (P, 1 μg)were incubated for 35 min at room temperature in 20 mM Hepes (pH 7.5),75 mM NaCl and 5 mM MgCl2 at a final molar ratio P:C3b:FB 0.7:1:4. Sub-sequently, 100 ng of FD (Calbiochem) was added and the mixture wasinjected in a Superdex 200 column (GE Healthcare). Fractions were analyzedusing 10% (wt/vol) SDS/PAGE.

Electron Microscopy and Image Processing. A few microliters of freshly puri-fied complexes were adsorbed onto carbon-coated grids and negativelystained with 2% (wt/vol) uranyl formate. Micrographs were recorded usinga JEOL 1230 transmission electron microscope and a TemCam-F416 detectorfrom Tietz Video and Image Processing Systems (TVIPS) using EM-TOOLS(TVIPS). Images were collected at a final magnification of 54,926×. UsingEMAN (28), 6,425 images for properdin vertexes and 21,891 images forC3bBb convertases bound to a propedin vertex were selected and processed.Ab initio templates for refinement were obtained using the command“e2initial model” in EMAN2 (28) and the random conical tilt (RCT) method.

For further details, see SI Materials and Methods.

ACKNOWLEDGMENTS.We thank our colleagues at the laboratory of S.R.d.C.for help in the purification of C3b, FB, and properdin. This work was fundedby the Autonomous Region of Madrid (S2010/BMD-2316 to S.R.d.C. andO.L.), the Ramón Areces Foundation (O.L.), and the Spanish government(SAF2011-22988 to O.L. and SAF2011-26583 to S.R.d.C.). O.L. is additionallysupported by Red Temática de Investigación Cooperativa en Cáncer (RD06/0020/1001), and S.R.d.C. is also supported by the Fundación Renal IñigoAlvarez de Toledo and the Seventh Framework Programme European UnionProject EURenOmics (European Consortium for High-Throughput Research inRare Kidney Diseases). M.A. is a Sara Borrell Fellow from the Instituto deSalud Carlos III (CD09/00282).

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