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Substrate recognition by complement convertases revealed in the C5-cobra venom factor complex

2011; Springer Nature; Volume: 30; Issue: 3 Linguagem: Inglês

10.1038/emboj.2010.341

1460-2075

Nick S. Laursen, Kasper R. Andersen, Ingke Braren, Edzard Spillner, Lars Sottrup‐Jensen, G.R. Andersen,

Cellular transport and secretion

Article7 January 2011free access Substrate recognition by complement convertases revealed in the C5–cobra venom factor complex Nick S Laursen Nick S Laursen Department of Molecular Biology, Aarhus University, Aarhus, Denmark Search for more papers by this author Kasper R Andersen Kasper R Andersen Department of Molecular Biology, Aarhus University, Aarhus, Denmark Search for more papers by this author Ingke Braren Ingke Braren Institute of Biochemistry and Molecular Biology, Department of Chemistry, University of Hamburg, Hamburg, Germany Search for more papers by this author Edzard Spillner Edzard Spillner Institute of Biochemistry and Molecular Biology, Department of Chemistry, University of Hamburg, Hamburg, Germany Search for more papers by this author Lars Sottrup-Jensen Lars Sottrup-Jensen Department of Molecular Biology, Aarhus University, Aarhus, Denmark Search for more papers by this author Gregers R Andersen Corresponding Author Gregers R Andersen Department of Molecular Biology, Aarhus University, Aarhus, Denmark Search for more papers by this author Nick S Laursen Nick S Laursen Department of Molecular Biology, Aarhus University, Aarhus, Denmark Search for more papers by this author Kasper R Andersen Kasper R Andersen Department of Molecular Biology, Aarhus University, Aarhus, Denmark Search for more papers by this author Ingke Braren Ingke Braren Institute of Biochemistry and Molecular Biology, Department of Chemistry, University of Hamburg, Hamburg, Germany Search for more papers by this author Edzard Spillner Edzard Spillner Institute of Biochemistry and Molecular Biology, Department of Chemistry, University of Hamburg, Hamburg, Germany Search for more papers by this author Lars Sottrup-Jensen Lars Sottrup-Jensen Department of Molecular Biology, Aarhus University, Aarhus, Denmark Search for more papers by this author Gregers R Andersen Corresponding Author Gregers R Andersen Department of Molecular Biology, Aarhus University, Aarhus, Denmark Search for more papers by this author Author Information Nick S Laursen1, Kasper R Andersen1, Ingke Braren2, Edzard Spillner2, Lars Sottrup-Jensen1 and Gregers R Andersen 1 1Department of Molecular Biology, Aarhus University, Aarhus, Denmark 2Institute of Biochemistry and Molecular Biology, Department of Chemistry, University of Hamburg, Hamburg, Germany *Corresponding author. Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C, Aarhus DK-8000, Denmark. Tel: +45 89 42 50 24; Fax: +45 86 12 31 78; E-mail: [email protected] The EMBO Journal (2011)30:606-616https://doi.org/10.1038/emboj.2010.341 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Complement acts as a danger-sensing system in the innate immune system, and its activation initiates a strong inflammatory response and cleavage of the proteins C3 and C5 by proteolytic enzymes, the convertases. These contain a non-catalytic substrate contacting subunit (C3b or C4b) in complex with a protease subunit (Bb or C2a). We determined the crystal structures of the C3b homologue cobra venom factor (CVF) in complex with C5, and in complex with C5 and the inhibitor SSL7 at 4.3 Å resolution. The structures reveal a parallel two-point attachment between C5 and CVF, where the presence of SSL7 only slightly affects the C5–CVF interface, explaining the IgA dependence for SSL7-mediated inhibition of C5 cleavage. CVF functions as a relatively rigid binding scaffold inducing a conformational change in C5, which positions its cleavage site in proximity to the serine protease Bb. A general model for substrate recognition by the convertases is presented based on the C5–CVF and C3b–Bb–SCIN structures. Prior knowledge concerning interactions between the endogenous convertases and their substrates is rationalized by this model. Introduction Activation of the proteolytic complement cascade triggers cleavage of the homologous 185–200 kDa proteins C3, C4, and C5 (Supplementary Figure S1). Their proteolytic fragments mediate enhanced phagocytosis and pathogen lysis, clearance of immune complexes and apoptotic cells, inflammation and promote adaptive immune responses (Walport, 2001; Gasque, 2004). Three activation pathways converge at the formation of C3 convertases, cleaving C3 into C3a and C3b. The classical pathway (CP) is activated by surface-bound immune complexes, resulting in cleavage of C4 and thereby generation of C4b (Figure 1A). Proteolysis of C4 is also triggered via recognition of microbial carbohydrates in the lectin pathway (LP). C4b combines with C2 and subsequent cleavage of C2 to C2a results in generation of the LP/CP C3 convertase C4b2a. (C4b2a is the widely used short-hand nomenclature for the C4b–C2a complex in the complement field, and similar nomenclature is used for the remaining complexes in the following). The alternative pathway (AP) C3 convertase C3bBb is formed when factor B (fB) combines with C3b, after which fB is activated by factor D (fD) (Figure 1A). Normally the C3 convertases are surface anchored, as an internal thioester present in both nascent C3b and C4b can react with a surface nucleophile. However, a fluid-phase AP C3 convertase C3(H2O)Bb can also assemble after spontaneous hydrolysis of the C3 thioester and initiate the AP (Pangburn and Muller-Eberhard, 1983). Both the AP and CP C3 convertases may recruit an additional C3b molecule (Takata et al, 1987; Kinoshita et al, 1988; Pangburn and Rawal, 2002) to form a C5 convertase (C3bBb3b or C4b2a3b), which cleaves C5 to generate the large fragment C5b and the anaphylatoxin C5a (Figure 1A). Cobra venom factor (CVF) is a C3b homologue found in the venom of snakes from the genus Naja. Together with Bb, CVF assembles into a highly stable fluid phase convertase CVFBb cleaving both C3 and C5 (Vogel and Fritzinger, 2010). The release of C3a and C5a increases vascular permeability and blood flow, thereby promoting further transport of other snake venom components (Vogel and Fritzinger, 2010). Staphylococcus aureus secretes various proteins, which manipulate the functions of the complement system (Geisbrecht, 2008). One of these, SSL7, inhibits complement-mediated haemolytic and bactericidal activity by binding to C5 through its C-terminal β-grasp domain (Laursen et al, 2010). SSL7 is bifunctional and binds IgA through its N-terminal OB domain blocking the recognition of IgA by FcαRI (Ramsland et al, 2007). SSL7 binds to C5 at >70 Å from the cleavage site in C5 and possibly interferes with C5 recognition by convertases through IgA-dependent steric hindrance (Laursen et al, 2010). Figure 1.Complement activation and the structure of the C5–CVF complex. (A) The three activation pathways of complement (left) induce the assembly of the proteolytic C3 convertases and, after additional C3 cleavage, the C5 convertases. Factor B, C4, and C2 are cleaved by the pathway-specific serine proteases factor D, MASP-2, and C1s. CVFBb (right, green box) cleaves both C3 and C5. Abbreviations for the complexes: C4b2a, C4b–C2a complex; C3bBb, C3b–Bb complex; C4b2a3b, C4b–C2a–C3b complex; C3bBb3b, C3b–Bb–C3b complex; CVFBb, CVF–Bb complex; C3(H2O)Bb, C3(H2O)–Bb complex. (B) Stereo view of omit non-averaged 2mFo–DFc electron densities contoured at 1 σ around the loop Ser870–Lys882 in the C5 MG7 domain, which becomes ordered in the CVF complex in contrast to free C5. For residues with side chains shown, all atoms were omitted for map calculation. (C, D) Cartoon representation of C5 (C5a red, future Nt-α′ yellow, blue otherwise) in complex with CVF (Nt-γ yellow, otherwise green). This colouring scheme is used throughout unless otherwise noted. See also Supplementary Figure S1 for definition of domains and chains. Download figure Download PowerPoint To provide insight into the substrate recognition by the complement convertases, we studied the CVF–C5 complex. In contrast to C3b, CVF binds with high affinity to C5, which allowed us to crystallize the C5–CVF complex and determine its structure. Furthermore, to fully establish the mechanism of inhibition by SSL7, we determined the structure of the SSL7–C5–CVF complex. The structures reveal that two distinct surface areas on both C5 and CVF mediate complex formation. CVF binding is shown to induce conformational changes in C5, which changes the position of the convertase cleavage site in C5, while complex formation with SSL7 results in a minor disturbance of the C5–CVF interface. The C5–CVF structure validates prior data concerning interactions between the endogenous convertases and their substrates and suggests a preferred orientation of surface-bound convertases. Results The structure of C5–CVF The endogenous C3 and C5 convertases are very large unstable macromolecular complexes with a lifetime of a few minutes and four out of five convertases are surface anchored. Crystallization of these convertases either alone or in complex with their substrates is therefore a challenging task. As an alternative we used CVF, which forms a very stable and soluble complex with C5 (Kd of 42 nM) (Rawal and Pangburn, 2000). We determined the crystal structures of the C5–CVF and the SSL7–C5–CVF complexes at 4.3 Å resolution (Figures 1 and 2; Table I; Supplementary Tables I and II). Despite the low resolution of the structures, the main-chain tracing is generally reliable due to the starting models of C5, C5–SSL7, and CVF determined at 3.1, 3.6, and 2.2 Å, respectively, used for structure determination (Figures 1B and 2A). Rigid body refinement and grouped B-factor refinement of the starting models reduced the Rfree to 29–30% without any manual rebuilding. More detailed conformational changes were then modelled in 2mFo–DFc electron density maps using a database of known structures for model building (Jones et al, 1991) before further positional refinement. Except for a few regions differing due to crystal packing, two-fold averaged 2mFo–DFc electron density maps were used for rebuilding of both complexes. For this reason, the assignment of regions involved in intermolecular contacts within the C5–CVF and the SSL7–C5–CVF complexes can be carried out with confidence. The modelled position of side chains must be judged with caution due to the low resolution of the diffraction data, and detailed intermolecular interactions such as hydrogen bonds and electrostatic interactions are difficult to assign unambiguously. Figure 2.The structure of the SSL7–C5–CVF complex. (A) Omit non-averaged 2mFo–DFc electron densities contoured at 1 σ from the SSL7–C5–CVF complex around the large helix in the OB domain of SSL7. For residues with side chains shown, all atoms were omitted for map calculation. (B) SSL7 binds through its β-grasp domain (yellow) to the C5 MG5 domain, but the β-grasp and the OB domain (red) also appear to interact with the CVF MG4 domain. (C) Comparison of the position of CVF (grey) in its complex with C5 (blue), and of CVF (green) in complex with SSL7–C5. Download figure Download PowerPoint Table 1. Data collection and refinement statistics C5–CVF SSL7–C5–CVF Data collection Space group P2221 P2221 Cell dimensions a, b, c (Å) 176.52, 179.20, 389.69 163.65, 181.96, 392.78 Resolution (Å) 50–4.3 (4.43–4.30)a 50–4.3 (4.42–4.30) Rsym 14.3 (62.5) 14.3 (52.5) I/σI 10.9 (3.1) 11.4 (3.7) Completeness (%) 94.2 (96.0) 96.8 (98.6) Redundancy 5.6 (5.6) 5.1 (5.1) Refinement Resolution (Å) 49.5–4.3 49.2–4.3 No. of reflections 79 835 77 924 Rwork/Rfree 22.6/26.3 22.4/25.5 No. of atoms Protein 45 184 48 212 Glycosylations 140 140 B factors Protein 196.33 171.40 Glycosylations 336.72 291.3 r.m.s. deviations Bond lengths (Å) 0.012 0.012 Bond angles (deg) 1.56 1.63 Values in parentheses are for the highest-resolution shell. One crystal was used for each dataset. Further details of statistics for data collection and refinement are found in Supplementary Tables I and II. C5 and CVF are arranged in a head-to-head manner and form a complex of dimensions 160 × 154 × 90 Å (Figure 1C and D) with the long axis of the two molecules aligned roughly in parallel. Two widely separated regions on C5 mediate binding to CVF and vice versa (Figure 3A and B; Supplementary Figure S2A and C. Note that prepro-numbering is used for all proteins.) Although many charged side chains are found at the intermolecular interface, the electrostatic potentials of the two proteins (Figure 3A and B) do not suggest ionic interactions to be the driving force for the intermolecular interaction. In agreement with this, both the C5–CVF and the SSL7–C5–CVF complex were crystallized under similar solution conditions with high ionic strength. The largest of the two interfaces between C5 and CVF involves contacts between the MG4 and MG5 domains from both proteins (Figure 3C and D; Supplementary Figure S3A, B, E, and F) and has a calculated area of 1350 Å2 (Supplementary Table III). We will refer to this interface as the MG4–MG5 interface. Residues within the C5 MG4 domain interact exclusively with the CVF MG5 domain, while the C5 MG5 domain interacts mainly with CVF MG4 supplemented with a few contacts to CVF MG5 (Figure 3C and D; Supplementary Figure S3A and B). In C5, residues within the regions Ser419–Pro425, Thr470–Ile485, and Asp520–Asn527 are located in the vicinity of CVF residues Ser386–Thr389, Ile399–Leu404, Thr450–Lys467, and Arg498–Asn507. The MG4–MG5 interface is rather flat by nature and with few conformational changes in the participating regions compared with the structures of C5 and CVF alone. The other contact area, referred to as the MG7 interface, is formed between the C5 MG7 domain and the CVF MG6 and MG7 domains (Figure 3E and F; Supplementary Figure S3C, D, G, and H). It has a calculated area of 830 Å2 and appears to be more intricate than the MG4–MG5 interface. A hydrophobic core within the interface is formed by C5 Met853, Trp917, and Phe918 together with CVF residues Ile556, Met558, Val813, Leu904, and Trp905 (Figure 3E and F; Supplementary Figure S3C and D). The MG7 loop 871–882—disordered in the structure of free C5—forms an ordered loop resulting in putative contacts with CVF Tyr845, Gln901, and Glu902 (Figure 1B and 3E, F; Supplementary Figures S2 and S3). Contacts between this C5 loop and CVF are consistent with the decrease in CVFBb-mediated C5 cleavage after deletion of C5 Ser881–Gln886 (Low et al, 1999). Many C5 residues engaging in putative CVF interactions are well conserved in C5 and C3 (Supplementary Figure S2A and B), both substrates for CVFBb. CVF residues appearing to be in contact with C5, especially those presumably interacting with the C5 MG7 domain, are in general less conserved upon alignment with C3 (Supplementary Figure S2C), perhaps explaining the much higher affinity of CVF for C5 compared with C3b (Rawal and Pangburn, 2000). The conformation of C5b is apparently similar to that of C3b (Hadders et al, 2010), suggesting that in C5b the MG7 domain is located very differently relative to the C5 MG4–MG5 domains, and that the C5d and CUB domains are released from the MG8 domains and become exposed in C5b. Hence, C5 bound to CVF is kept in a conformation much closer to that of unbound C5 compared with C3b/C5b, and the conformational change between free and bound C5 is likely to be reversible. Importantly, CVF cannot interact with the MG4, MG5, and MG7 domains in the product C5b in the same manner as with the substrate C5, providing a mechanism for distinguishing substrate from product. Figure 3.C5–CVF interactions. (A) Surface representation of CVF with its structural domains individually coloured and residues contacting C5 coloured blue (left panel), CVF coloured according to conservation within CVF and mammalian C3 sequences (middle panel, see also Supplementary Figure S2C), and CVF with the electrostatic potential mapped to the surface (right panel). The view is related to that of Figure 1C by a 90° rotation around a vertical axis. (B) As in panel A, but with C5 residues interacting with CVF coloured blue (left), C5 coloured according to conservation (middle, see also Supplementary Figure S2A), and the electrostatic potential mapped on the surface of C5 (right). The view is related to that of panel A by a 180° rotation around a vertical axis. (C, D) Close-up of the MG4–MG5 interface between C5 and CVF in two orientations. (E, F) Details of the C5–CVF MG7 interface in two orientations. Download figure Download PowerPoint Conformational changes in C5 Complex formation with C5 has little influence on the conformation of CVF compared with either free CVF (Krishnan et al, 2009) or CVF in complex with fB (Janssen et al, 2009). Only the C-terminal C345C and the CUB domains have a slightly variable orientation relative to the remaining CVF molecule. But these discrepancies may easily be caused by differences in crystal packing interactions and the presence of factor B in the CVFB complex, and neither of the two domains are in direct contact with C5. Likewise, the conformation of C5-bound CVF is also close to that of C3b alone (Supplementary Figure S4) or C3b in complex with Bb and the bacterial convertase inhibitor SCIN (Rooijakkers et al, 2009). CVF residues 20–1473 can be superimposed onto CVF from its complex with fB with an r.m.s.d. of 1.34 Å over 876 Cα atoms, and superimposed with the structure of free CVF with an r.m.s.d. of 1.57 Å over 776 atoms. CVF residues 20–1473 from the C5–CVF complex can also be overlaid with C3b from its complex with Bb and SCIN with an r.m.s.d. of 1 Å for 824 Cα atoms. Hence, CVF seemingly acts as a relatively rigid scaffold for binding of both Bb and C5 in CVFBb. In contrast, C5 undergoes a significant overall conformational change upon CVF binding (Figure 4A and B; Supplementary Figure S5; Supplementary Animation 1). Residues 18–1510 from the structure of free C5 can be overlaid onto C5 from the CVF complex with an r.m.s.d. of 2.45 Å over 1307 Cα atoms. The conformational change within C5 can roughly be described as a concerted movement, with the C5a, MG7, CUB, C5d, and MG8 domains rotating by 18° relative to the MG1, MG4, and MG5 domains, a second structural entity that also remains largely unaffected compared with free C5. Located between these two structural entities, the MG3 domain rotates by 23° relative to either entity, while the domain pair MG2–MG6 turns by 10–12°. Separate from these effects is the major rearrangement that places the C5 C345C domain in a C3-like position (discussed below). The conformational rearrangement significantly changes the interdomain interactions of one end of the MG3 domain. In unbound C5 this end is associated with the MG8 domain, but in C5–CVF it instead appears to interact with the MG7 domain (Figure 4C and D). The movement of the MG3 domain also changes the position of the future Nt-α′ region, as this is sandwiched between the C5a and the MG3 domain (Figure 4E and F). Figure 4.Conformational changes in C5 upon binding to CVF. (A) Surface representation of free C5 with the MG3 and MG7 domains coloured grey and orange, respectively, and CVF shown in cartoon for comparison. Free C5 was placed by superposition of its MG4 and MG5 domains with the same domains in C5–CVF. (B) As in panel A, but for the C5–CVF complex. The C5 C345C domain is not shown in panels A and B, as its position in free C5 is governed by crystal packing. (C) Close-up of the interaction between the MG3 and MG8 domains in free C5. (D) In the CVF-bound state of C5 the MG3 domain is shifted towards the MG7 domain. (E) The future Nt-α′ chain (yellow) is sandwiched between C5a and the MG3 domain in free C5. (F) In the CVF-bound state of C5 the rotation of the MG3 domain induces conformational changes in Nt-α′. The two states of C5 in panels C–F were superimposed through the CUB, C5d, and MG8 domains. Download figure Download PowerPoint The structure of the SSL7–C5–CVF complex The S. aureus protein SSL7 binds with high affinity to IgA and C5, thereby inhibiting the binding of IgA to the FcαRI receptor, C5a generation, and C5-mediated serum killing of Escherichia coli (Langley et al, 2005; Laursen et al, 2010). We earlier showed that efficient SSL7-mediated inhibition of C5 cleavage by CVFBb required the presence of the SSL7-binding IgA Fc moiety, and suggested that this was due to steric hindrance preventing convertase binding to C5 (Laursen et al, 2010). To confirm this assumption, we determined the crystal structure of the SSL7–C5–CVF complex. As in C5–SSL7 (Laursen et al, 2010), SSL7 recognizes the C5–CVF complex by β-sheet pairing between its C-terminal β-grasp domain and the C5 MG5 domain, while the N-terminal OB domain of SSL7 is free to bind IgA. Within the SSL7–C5–CVF complex, both SSL7 domains apparently interact with the MG4 domain of CVF (Figure 2B and C). This causes a slight rotation of the CVF MG4 domain away from C5 MG5, which has little consequence for the putative contacts between the C5 MG4 and MG7 domains to CVF. This perturbation, however, probably explains the reduced affinity of immobilized SSL7–C5 for CVF (Supplementary Figure S6B) in a surface plasmon resonance (SPR) experiment compared with measurements of immobilized C5 (Rawal and Pangburn, 2000) and the slight inhibition of CVFBb-mediated C5 cleavage exerted by SSL7 alone (Laursen et al, 2010). Docking of IgA onto the SSL7–C5–CVF complex demonstrates massive steric hindrance between IgA and the CVF MG3 and MG4 domains (Supplementary Figure S7A). C5 and IgA bind to SSL7 with Kd values of 6 and 1 nM, respectively, and a trimeric SSL7–C5–IgA complex can be isolated (Ramsland et al, 2007; Laursen et al, 2010). Considering the in vivo concentrations of C5 and IgA, SSL7 most likely permanently binds both proteins simultaneously (Laursen et al, 2010). Although SSL7 alone inhibits C5 cleavage by CVFBb and the endogenous C5 convertases to some degree, full inhibition is only obtained in the presence of IgA (Bestebroer et al, 2010; Laursen et al, 2010). This is confirmed by our structure and SPR experiments indicating that although SSL7 weakens the binding of CVF to C5, it does not prevent the complex formation, and SSL7 only marginally influences the interaction between C3b and C5 (Supplementary Figure S6). In summary, our structures show how C5 and CVF interact in a head-to-head manner with the long axis of both proteins aligned approximately in parallel. The overall conformation of CVF is not significantly affected by interaction with the substrate, while a conformational change in C5 significantly changes the relative orientation of two surface areas in contact with CVF compared with free C5. Binding of SSL7 to the C5–CVF complex leads to a minor perturbation of the interaction between C5 and CVF, which is in agreement with the partial inhibition of C5 cleavage by SSL7 in the absence of IgA. Substrate recognition by CVFBb By a simple superposition of CVF from the C5–CVF complex with C3b from the C3b–Bb–SCIN complex (Rooijakkers et al, 2009), we have created a model reflecting how C5 and by homology C3 are recognized by CVFBb (Figure 5; Supplementary Figure S5). In the C5–CVFBb model, the peptide bond cleaved in C5 between Arg751 and Leu752 is located 19 Å away from its expected location in the catalytic site of Bb (Figure 5D), suggesting conformational changes in C5 and CVFBb relative to our model. The flexibility of the CVF C345C domain could contribute to bringing the Bb catalytic site to the substrate cleavage site as suggested (Rooijakkers et al, 2009). However, in the model, Bb loop regions flanking the entrance to the catalytic site are already within 5 Å of C5 (Figure 5C) and therefore large-scale rigid body movement (e.g., larger than 5 Å) of Bb appears to be not required. As an alternative, we suggest that the local conformational changes in C5 residues 741–769 may be important for accommodating C5 Arg751 in the Bb active site. In C3, α helix 4 in C3a is longer than in C5 (Figure 5E), and elongating this by 1–2 turns in C5 would significantly reduce the distance of residues upstream of Arg751 to the catalytic site. However, steric hindrance still appears to prevent C5 Arg751 from reaching the catalytic site, as residues Met754–Leu758 are sandwiched between C5a α helix 1 and the MG3 domain (Figure 4F). Such hindrance could be relieved if the helix is detached from the three other helices in C5a (Figure 5F) as recently observed in free C5a (Cook et al, 2010). The suggested local conformational changes in C5 could be triggered by contacts with Bb, which might explain why C5b, in contrast to C3b, cannot be generated by less-specific proteases like trypsin (Fredslund et al, 2008). Figure 5.Models for substrate–CVFBb complexes. (A) C5 (blue cartoon, C5a red) in complex with CVF (green surface) and Bb (sand-coloured surface) bound to CVF. The blue area on Bb marks the catalytic triad cleaving C5 Arg751–Leu752. (B) As in panel A, but with C3 taking the position of C5. This conformation of bovine C3 has a three-dimensional arrangement of the CVF contacting domains resembling that of C5 bound to CVF (Supplementary Figure S5C and D). (C) Three surface-exposed loops (blue) in Bb are located close to the C5 MG3 domain (grey) and C5a (red), while a fourth loop (green) potentially interacts with the disordered loop (green dashed line, residues 1388–1396) in C5 MG8 (green) or the disordered C5a loop (red dashed line, residues 744–750). (D) Close-up of the suggested C5–Bb interface with C5a in red and the future Nt-α′ in yellow. In Bb the catalytic triad side chains are labelled D, H, and S. (E) As in panel D, but with the docked C3 molecule pointing the cleaved bond Arg746–Ser747 further towards the catalytic site. (F) Model suggesting how relocation of α helix 1 in C5a could allow Nt-α′ to undergo a conformational change. Download figure Download PowerPoint As CVFBb also cleaves C3 (Vogel and Fritzinger, 2010), C3 is likely to adopt a conformation closely related to that of C5 in complex with CVF. Remarkably, a conformation of bovine C3 strongly resembling that of C5 bound to CVF has apparently been stabilized by packing in crystals of bovine C3 (Fredslund et al, 2006). A second copy of bovine C3 and human C3 (Janssen et al, 2005) does not adopt a conformation resembling CVF-bound C5 (Supplementary Figure S5). Superimposing this particular conformation of bovine C3 on C5 in our C5–CVFBb model allows us to investigate how C3 may bind to CVFBb (Figure 5B). In comparison with the C5–CVFBb model, the scissile peptide bond Arg746–Ser747 in bovine C3 (Figure 5E) is apparently free to reach the catalytic site. Hence, fewer local conformational changes around the scissile bond would be required in C3 compared with C5 after binding to CVFBb, which is consistent with the ≈100-fold faster turnover of C3 compared with C5 for CVFBb and C3bBb (Rawal and Pangburn, 2000). In conclusion, the derived C5–CVFBb model suggests that CVF binding places the C5 MG7 and C5a domains more or less correctly relative to Bb, which might explain why Bb contributes little to CVFBb affinity for C5 (Rawal and Pangburn, 2000). Modest overall repositioning of Bb relative to C5 in combination with local conformational changes in C5 residues 741–769 and the Bb loops facing C5 are then probably sufficient to place Arg751 in the Bb catalytic site possibly combined with a movement of C5a α helix 1. The endogenous C3 and C5 convertases In terms of sequence homology, CVF is 49% identical to human C3 compared with, for example, 77% sequence identity between human and bovine C3. In combination with the functional and structural similarities between CVF and C3b, we expect that C3b in the AP convertases binds the substrates (C3 and C5) in a manner analogously to how CVF recognizes C5. By further extrapolation—which is justified by the pronounced functional similarities between the AP and the CP convertases—we also expect this to apply to C4b in the CP convertases. We therefore propose a general model for substrate–convertase recognition applicable to both C3 and C5 convertases. In this model, the MG4–MG5 domains of C3b/C4b interact with the MG4–MG5 domains of the substrates C3 and C5 at one interface (the MG4–MG5 interface), while the MG6–MG7 domains of C3b/C4b interact with the substrate MG7 domain at a second interface (the MG7 interface). This proposal is compatible with a variety of experimental results. With respect to the AP C3 convertase, C3b–C3b interactions mediated by the MG4 and MG5 domains are present in the C3b–Bb–SCIN structure, which strongly resemble the MG4–MG5 interface observed in the C5–CVF structure (Supplementary Figure S8), and these interactions have already been suggested to mirror convertase–C3 interactions (Rooijakkers et al, 2009). The involvement of the MG4–MG5 interface in convertase–substrate interactions is in agreement with the inhibitory effects and structural data on compstatin (Janssen et al, 2007) and CRIg (Wiesmann et al, 2006), as these would prevent substrate binding to the AP convertase due to steric hindrance (Supplementary Figure S7B). Likewise, the involvement of the C3b MG4 domain in substrate recognition consistently explains the effects upon mutation of C3 Met373 to a threonine, which leads to AP dysfunction (Sfyroera et al, 2010). At the substrate level, the C3 Met373 mutation leads to reduced C3 cleavage by the A