Structure of complement C3(H2O) revealed by quantitative cross-linking/mass spectrometry and modelling

The complement system performs immune surveillance, enabling rapid recognition and clearance of invading pathogens as well as apoptotic cells and particles threatening homeostasis (1). Multiple complement-activation pathways converge at the assembly of C3 convertases (2). These bimolecular proteolytic enzymes excise the anaphylatoxin domain (ANA1, corresponding to C3a) from the complement component C3 (184 kDa) leaving its activated form, C3b (175 kDa) (Fig. 1A). C3b can covalently attach, via a nascently exposed and activated thioester, to any nearby surface (3, 4) whereupon it undergoes rapid amplification (2). Fig. 1. Complement protein C3(H2O). A, Domain compositions of C3, C3b and C3(H2O). The thioester group in the TED is shown as a circle before (red) or after (gray) hydrolysis (25). B, Relationship between native C3, C3(H2O) and C3b. The structure of C3(H2O) is ... The complement system responds very swiftly to pathogens, independently of antibodies, due primarily to its “alternative pathway” of activation. This is initiated by spontaneous, although rare, conformational changes within C3 that are concerted with hydrolysis of its constitutively buried thioester linkage (5). Identical conformational changes accompany attack of the thioester by amines (6). The continuously and ubiquitously generated stable product, C3(H2O) (iC3 or C3N) does not bind to surfaces (as it no longer possesses a thioester group). Interestingly, C3(H2O) has been inferred to resemble C3b in many of its functional and structural features, despite its retention of the ANA (7–10) (Fig. 1A, ​,11B). The mature C3 molecule consists of two polypeptide chains (residues 1–645 in the β-chain and residues 650–1641 in the α-chain). A metaphor of a puppeteer holding a puppet has been used to describe the crystal structure of C3 (11) (supplemental Fig. S1 in Supplemental File). Macroglobulin domains (MGs) 1–6 and a “linking region” (LNK) adopt a key-ring like arrangement that forms the body of the puppeteer whereas MG7, MG8, and ANA form its shoulders, and a C345C domain equates to its head, joined to MG8 by an “anchor” region (the neck). A thioester-containing domain (TED) is the puppet, held at shoulder height by a CUB domain that forms the arm of the puppeteer. MGs 1–5, LNK, and half of MG6 are contributed by the β-chain whereas the remaining domains are coming from the α-chain. Comparing the crystal structures of C3 and C3b revealed significant domain rearrangements between them (11). Most dramatically, the CUB arm swings away from the shoulders toward the “feet” of the puppeteer (supplemental Fig. S1). As a result, the TED (i.e. the puppet) rotates and is repositioned. This is accompanied by exposure and activation of the thioester group, allowing attachment of C3b to surface-borne nucleophiles. The crystal structure of C3(H2O) has not been reported. New binding sites for complement components and cell-surface receptors are created in both nascent C3b and C3(H2O) (7, 12–18). Both proteins bind factor B that is subsequently cleaved to Bb. Importantly, both the resultant C3bBb and C3(H2O)Bb complexes are C3 convertases, generating further molecules of C3b and thereby stoking a positive-feedback loop. Because C3(H2O) (unlike C3b) is a spontaneously arising product of C3 domain rearrangements and thioester hydrolysis, C3(H2O)Bb (rather than C3bBb) is the initiating convertase of the alternative pathway of complement activation. Thus the constitutive presence of C3(H2O) ensures the alternative pathway can be activated quickly and indiscriminately allowing a rapid response to any cell not protected by the appropriate regulatory molecules such as factor H. Inappropriate regulation of complement activity is linked to many autoimmune, inflammatory and ischemia/reperfusion (I/R) injury-related diseases (19). It has been shown that hydrolysis of the thioester in C3 alone does necessarily result in transition to active C3(H2O) (20). Despite use of diverse methodologies (7, 9–13, 21–27), the remodeling of domains that underlies spontaneous formation of C3(H2O), and therefore triggers complement, are poorly understood. Current structural models of C3(H2O) rely on epitope-mapping (21), hydrogen-deuterium exchange (27), other biophysical solution studies (9) and negative-staining EM images (25). These indicate a “C3b-like” structure but do not provide direct evidence regarding placements of the ANA and TED relative to specific domains within the shoulders and body of the C3(H2O) molecule. It has been proposed that the ANA domain acts as a safety catch in native C3. Removal of the ANA triggers the dramatic structural transition into C3b (24). More knowledge of the C3(H2O) structure is required to test if the safety catch role of ANA (presumably displaced in C3(H2O) rather than removed, as in C3b) and subsequent domain reconfigurations are general mechanisms, relevant both to the spontaneous but rare hydrolytic C3 to C3(H2O) transition, and to the proteolytic cleavage-dependent but rapid C3 to C3b transition. Further understanding of this event depends on the ability to elucidate, in solution, the dynamic processes whereby the domains of a protein molecule are reorganized, following a triggering event, to form a new stable arrangement. Quantitative cross-linking/mass spectrometry (QCLMS) using isotope-labeled cross-linkers (Fig. 2A) has emerged as a new approach with which to elucidate the details of protein conformational changes (28–31). In this approach, chemical cross-linking captures proximities between amino acid residues and the residues involved are identified by mass spectrometry. Quantitative comparison of the cross-linking results obtained for two different conformations of a protein allows the details of the conformational change to be elucidated. We have developed a workflow for QCLMS analysis (32). In our benchmark study, we used QCLMS to accurately reveal differences and similarities between C3 and C3b in terms of the spatial arrangements of their domains (32). In another application, this technique successfully revealed conformational changes involved in maturation of the proteasome lid complex (33). Fig. 2. Quantitative CLMS analysis of C3, C3b and C3(H2O) in solution. A, The strategy of QCLMS using differentially isotope-labeled cross-linkers for comparing protein conformations. B, SDS-PAGE shows that BS3 (light cross-linker) and BS3-d4 (heavy cross-linker) ... Here we apply our QCLMS workflow, and an integrative modeling approach, to interrogate the unknown arrangement of domains in, C3(H2O), a key component of the complement alternative pathway. We combined knowledge of the crystal structures of C3 and C3b with QCLMS data sets for C3(H2O), C3 and C3b. We thus generated structural models for the conformational transition of C3 to C3(H2O) that are consistent with other biophysical studies and with previously observed functional similarities and differences between these proteins.