The human complement system is an important component of innate immunity. Complement-derived products mediate functions contributing to pathogen killing and elimination1. However, inappropriate activation of the system contributes to the pathogenesis of immunological and inflammatory diseases1. Complement component 3 (C3) occupies a central position because of the manifold biological activities of its activation fragments, including the major fragment, C3b, which anchors the assembly of convertases effecting C3 and C5 activation. C3 is converted to C3b by proteolysis of its anaphylatoxin domain2, by either of two C3 convertases. This activates a stable thioester bond, leading to the covalent attachment of C3b to cell-surface or protein-surface hydroxyl groups through transesterification3. The cleavage and activation of C3 exposes binding sites for factors B, H and I, properdin, decay accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46), complement receptor 1 (CR1, CD35) and viral molecules such as vaccinia virus complement-control protein4. C3b associates with these molecules in different configurations and forms complexes mediating the activation, amplification and regulation of the complement response1,4. Structures of C3 and C3c, a fragment derived from the proteolysis of C3b, have revealed a domain configuration, including six macroglobulin domains (MG1–MG6; nomenclature follows ref. 5) arranged in a ring, termed the β-ring5. However, because neither C3 nor C3c is active in complement activation and regulation, questions about function can be answered only through direct observations on C3b. Here we present a structure of C3b that reveals a marked loss of secondary structure in the CUB (for ‘complement C1r/C1s, Uegf, Bmp1’) domain, which together with the resulting translocation of the thioester domain provides a molecular basis for conformational changes accompanying the conversion of C3 to C3b. The total conformational changes make many proposed ligand-binding sites more accessible and create a cavity that shields target peptide bonds from access by factor I. A covalently bound N-acetyl-l-threonine residue demonstrates the geometry of C3b attachment to surface hydroxyl groups.
Large conformational changes in several domains are pivotal to the conversion of inactive C3 to active C3b. A major contributor to the structural stability of C3 is MG8, which is in close contact with the MG3, anaphylatoxin (ANA), amino-terminal region of the cleaved α-chain (residues 727–745) referred as α′NT (ANT), MG7, CUB and thioester (TED) domains, burying 198, 650, 90, 629, 180 and 1,002 Å2 of accessible surface area, respectively (Fig. 1a). Proteolytic removal of ANA thus destabilizes MG8. In addition, MG3 and ANT, which bury 244 and 395 Å2 of accessible area at the C3–ANA interface, are also destabilized. Neighbouring β-ring domains limit the movement of MG3 to a rotation of 15° and a translation of 4 Å. However, ANT rotates through 163° and flips to a new position 37 Å away (Fig. 1a), further destabilizing MG8. As a result, MG8 rotates through 66° and translates by 24 Å, veering away from CUB and TED (Fig. 1a). TED, released from packing confines, rotates through 164° and translates by 84 Å. TED movement is facilitated by the unravelling of the CUB β-sheet. In C3, CUB, an eight-stranded β-sheet, is composed of two half domains, namely CUBg (residues 912–962, strands β1–β5) and CUBf (1269–1330, β6–β8), constituting, respectively, part of C3g (933–979) and C3f (1281–1299). CUB and TED are iteratively inserted into the rest of C3: CUB between MG7 and MG8, and TED within the β5–β6 loop of CUB5. The N terminus of TED is thus connected to CUBg and its carboxy terminus to CUBf. In C3b, both CUBg and CUBf lose their β-sheet structure, with consequent spatial separation. CUBg elongates into a one-turn α-helix, a type I β-turn and a succession of 13 type IV β-turns, whereas CUBf extends into five successive type IV β-turns and two tandem γ-turns (Fig. 1b). Stretching of CUB subdomains in C3 is probably aided by lack of the disulphide bonds and metal-binding sites that stabilize CUB domains in other proteins6. Cumulatively, these structural changes make C3b more elongated and open than C3 (Fig. 2, and Supplementary Movie). As a direct consequence, putative binding sites for large ligands, such as factor B and properdin, are spatially dispersed, reducing potential steric collisions between them. Other C3 domains remain essentially unaltered in C3b and C3c. The overall folding of C3d is also conserved in C3, C3b, human7 and rat8 homologues.
C3b is a key component of all three pathways of complement activation. In the alternative pathway (AP), C3b provides the noncatalytic subunits of both C3 convertase and C5 convertases, whereas in the classical (CP) and lectin (LP) pathways it provides one of two noncatalytic subunits of the C5 convertase. Covalent attachment of C3b to an AP-activating surface forms the nidus of the C3 convertase, whereas its attachment to a C3 convertase changes the latter’s specificity to C5 convertase. Both attachments are mediated through the reactive carbonyl of Gln 991, which, serving as the acyl donor, esterifies hydroxyl groups on acceptors. In C3, Gln 991 forms a buried thioester with Cys 988; on removal of ANA, His 1104 attacks this thioester to form an acyl-imidazole intermediate, freeing the thiolate of Cys 988, which catalyses the transfer of the acyl group of Gln 991 to an acceptor hydroxyl1. His 1104 in C3 is too far (11.7 Å) from the thioester to form the acyl-imidazole intermediate, and its approach is blocked by the TED–MG8 interface5. In C3b, proteolytic removal of ANA has evidently resulted in a rotation of TED, fully exposing the thioester to solvent and eliminating the hindrance to the approach of His 1104 to Cys 988 and Gln 991. The resulting activated acyl group of Gln 991 has esterified the γ-hydroxyl of the N-acetyl-l-threonine (AcT) present in the medium. The attached AcT (Supplementary Fig. 1) is geometrically similar to free AcT (ref. 9), except at the site of attachment to Gln 991. The magnitude of TED translation is comparable to the more than 50 Å estimated from X-ray scattering measurements1. One energy source driving structural changes between C3 and C3b might be hydrolysis of the high-energy buried thioester bond5, in a manner similar to that in α2-macroglubulin, a related protein10. Residues 944–1273 in CUB and TED, which show the greatest change between C3 and C3b, correspond to those identified by NMR11. Relocation of TED in C3b positions its cell-surface-binding region opposite the β-ring domains5, placing a string of proposed binding sites for protein ligands in an array between the β-ring and the cell surface (Fig. 3a).
One such important ligand is factor B, a serine protease, which initiates the formation of AP C3 convertase (C3bBb) by associating with activator surface-bound C3b. The resulting C3bB is activated through cleavage by factor D protease and is stabilized by the binding of properdin12. The stabilized convertase generates numerous C3b molecules, both by self-amplification and by the amplification of CP/LP through C3 convertase, which also generate C3b, which could form additional AP convertase complexes. Antibody and peptide binding13 and site-directed mutagenesis14 indicated a possible factor-B-binding site within residues 730–739 in the ANT domain of C3b. Mutation of 730DE and 736EE significantly decreased the binding of C3b to factor B (ref. 14). ANT, nearly completely buried in C3, switches to the surface of C3b (Fig. 3b). This motion, combined with movements of MG7 and MG8, makes residues 730DE and 736EE, buried in C3, fully accessible in C3b (Fig. 3c). However, the dispositions of ANT in C3b and C3c are essentially identical (Fig. 1a), although C3c does not bind factor B or support convertase assembly. Therefore, although ANT may provide one site for factor B binding, additional sites absent or obstructed on C3c must exist on C3b. Studies on a cobra-venom cleavage fragment of C3 (C3o) indicate that C3o might contact factor B through residues 933–942 in CUBg15. Unlike C3c, C3o lacks residues 727–736 but retains the 933–942 region, and supports the formation of the AP C3 convertase. Residues 933–942 are in the middle of CUBg, the unravelling of which exposes them to ligand binding in C3b (Fig. 3a). These observations indicate that factor B might contact both ANT and parts of CUBg. In addition, studies with an anti-C3d antibody indicate that factor B might also interact with C3d, emphasizing the complex nature of interactions between C3b and factor B (ref. 16). Structure-guided mutational studies are apparently needed to describe the topology of factor-B-binding sites on C3b more accurately. The AP convertase might be stabilized by tripartite interactions between C3b, properdin and Bb17, with C3b interacting with properdin through residues 1403–1435 (ref. 18). A combination of removal of ANA and transformations in TED and MG8 exposes the pair of β-strands formed by 1403–1435 (in MG8) to ligands (Fig. 3b). Although accompanied by only a small change in solvent accessibility (184 Å2), its repositioning could eliminate barriers for ligand binding.
Our structure of C3b also provides insight into regulatory events in complement function. Complement activation is regulated largely through manipulation of the stability of C3 convertase and C5 convertase. In blood, AP C3 convertase and C5 convertase are rapidly dissociated, and their C3b component is proteolysed by factor I, a serine protease. Both dissociation and proteolysis are mediated by regulatory proteins through their decay acceleration and factor I cofactor activities, respectively. Regulatory proteins are largely made up of repeating units, each about 60 residues long, called short consensus repeats or complement control protein modules19. Both decay acceleration and factor I cofactor activities involve the specific binding of regulatory proteins to C3b, with a strong dependence on ionic strength. Structural studies20 and site-specific mutagenesis21 have indicated that positive charges on regulators, and negative charges on C3b, might have a key function in this interaction. It is therefore intriguing to observe that C3b has a larger negative surface-charge density than C3 (Fig. 4a). Negative charge augmentation is partly a result of an increase in 1,230 Å2 of accessible surface area for acidic side chains (Asp, Glu and Tyr) of CUB and TED domains in C3b in comparison with C3. Exposed acidic residues might also provide additional anchoring points for regulators. Most regulators bind to multiple clusters of acidic residues on C3b. For example, factor H recognizes two such clusters on C3b: on residues 744–754 in ANT22 and on residues 1187–1249 in TED23. The distance between these is about 99 Å in C3b. Electron microscopy and solution scattering24 data indicate a separation of 85–295 Å for C3b-binding sites in factor H. Thus, the size and flexibility of factor H could easily enable it to span these sites separated by 99 Å. Vaccinia virus complement-control protein (VCP) binds to C3dg (residues 933–1281)25 but not to C3d, indicating that the presence of acidic clusters in CUBg (Fig. 3a) might provide anchoring points for positively charged areas in VCP20,26. Cofactor activity depends on the formation of a C3b–cofactor–factor-I complex, followed by conformational changes that permit the cleavage of C3b by factor I (ref. 1). Three peptide bonds, Arg 1281-Ser 1282, Arg 1298-Ser 1299 and Arg 932-Glu 933, in C3b are cleaved successively, resulting in a complete loss of the ability to form a C3 convertase1. Rearrangement of the CUB domain results in the sequestration of all three scissile bonds in a cavity formed by the β-ring, TED and CUBg (Fig. 4b). Shielding of the scissile bonds in this cavity hinders direct access by factor I, a large two-chain glycoprotein27. Our structure of C3b therefore shows the necessity for a conformational change in C3b to enable factor I to access the scissile bonds. Moreover, the surface of C3b surrounding the scissile bonds is largely negatively charged (Fig. 4a), making the approach and interaction with factor I, which has a negatively charged Asp 90 at its substrate-binding site, unfavourable27. Thus, in addition to facilitating conformational changes required for hydrolysis, positively charged cofactors might also electrostatically assist the approach and docking of C3b with factor I by reducing the negative charge on C3b. Structural characterization of this central component of complement is likely to provide a basis for a better understanding of complement function and for the potential therapy of complement-related diseases.
Crystallization, structure determination and refinement
C3b was prepared by trypsin cleavage of C3 and purified as described28. The cleavage was performed in the presence of 10 mM AcT to provide a nucleophile for covalent attachment of the side-chain carbonyl of Gln 991, on activation of the internal thioester3. Esterification was confirmed through mass spectroscopic analysis on tryptic digests of modified C3b. Modified protein was stored at a concentration of 7.7 mg ml-1 in 20 mM Tris-HCl pH 7.5, and a protein inhibitor cocktail. Crystals were obtained through vapour diffusion from 2 μl of the protein solution mixed with an equal volume of well solution. Wells contained 200 mM Tris-HCl pH 7.5, 100 mM NaCl, 20 mM LiCl and 15% PEG6000. Although crystals could also be grown with C3b prepared without AcT, they did not diffract beyond 6.5 Å. AcT modification improved diffraction to 2.3 Å. Crystals had a solvent content of 79% and were mechanically fragile, but reflection data set to 2.3 Å could be assembled from partial measurements on four crystals. Data were measured on flash-frozen crystals at 100 K on the ID22 beamline (Advanced Photon Source) at 0.9791 Å. Statistics for data processing with the HKL2000 program package (ref. 29) are given in Supplementary Table 1. The structure was determined through molecular replacement with Phaser30, by using fragments of the structures of C3c and C3 (ref. 5) as search models. The β-ring of C3c was first located; remaining domains of C3c and TED domain from C3 were then added sequentially, alternating with rigid-body refinement. CUB domains were built into difference maps phased on the rest of the structure. The structure of C3b, consisting of 1,564 residues, has been refined in REFMAC30 to an R value of 18.0 with a free R value of 19.4. Representative geometric parameters are summarized in Supplementary Table 1 and an overall view of the structure is shown in Fig. 2. There are 20 residues in disallowed regions of the Ramachandran map, and fewer cis peptides than in C3 (ref. 5).
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We thank J. Chrzas for help with data measurement, K. Judge for technical assistance, the UAB Mass Spectroscopy Facility for mass spectra, and L. DeLucas for support. This research was supported by the NIH (H.M.K.M. and S.V.L.N.). G.J.K. is an International Senior Wellcome Trust Fellow for Biomedical Sciences in South Africa. Author Contributions K.G. and H.M.K.M. performed the crystallization and data measurement; A.A.A. and H.M.K.M. conducted the structure solution and refinement, and A.A.A., H.M.K.M., J.E.V., S.V.L.N. and G.J.K. performed the analysis.
Coordinates and structure factors are deposited in the Protein Data Bank under accession number 2HR0. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.
Catalytic residues and Experimental electron density. (JPG 97 kb)
Data collection and refinement statistics (DOC 40 kb)
A representation of the dynamics of C3 conversion to C3b and binding of the latter to microbial surfaces. (WMV 3471 kb)
This file contains text to accompany the above Supplementary Figure and Supplementary Movie. (DOC 21 kb)
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Abdul Ajees, A., Gunasekaran, K., Volanakis, J. et al. The structure of complement C3b provides insights into complement activation and regulation. Nature 444, 221–225 (2006). https://doi.org/10.1038/nature05258
Retraction Note: The structure of complement C3b provides insights into complement activation and regulation
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