Structure of Complement C3(H2O) Revealed By Quantitative Cross-Linking/Mass Spectrometry And Modeling

Abstract

The slow but spontaneous and ubiquitous formation of C3(H2O), the hydrolytic and conformationally rearranged product of C3, initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. The structure of C3(H2O) has not been determined. Here we subjected C3(H2O) to quantitative cross-linking/mass spectrometry (QCLMS). This revealed details of the structural differences and similarities between C3(H2O) and C3, as well as between C3(H2O) and its pivotal proteolytic cleavage product, C3b, which shares functionally similarity with C3(H2O). Considered in combination with the crystal structures of C3 and C3b, the QCMLS data suggest that C3(H2O) generation is accompanied by the migration of the thioester-containing domain of C3 from one end of the molecule to the other. This creates a stable C3b-like platform able to bind the zymogen, factor B, or the regulator, factor H. Integration of available crystallographic and QCLMS data allowed the determination of a 3D model of the C3(H2O) domain architecture. The unique arrangement of domains thus observed in C3(H2O), which retains the anaphylatoxin domain (that is excised when C3 is enzymatically activated to C3b), can be used to rationalize observed differences between C3(H2O) and C3b in terms of complement activation and regulation.

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 unknown.

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) cross-link C3, C3(H₂O) and C3b with roughly equivalent overall efficiencies, and that broadly similar sets of cross-linked products were obtained. C, Sample identifiers and color coding used in this study. For example sample I-1 consists of an equimolar mixture of BS³-cross-linked C3 (blue) and BS³-d4-cross-linked C3(H₂O) (green). Pair-wise comparisons of C3(H₂O) against C3 (I-1 and I-2), and C3(H₂O) against C3b (red) (II-1 and II-2).

Fig. 3.

Comparison of cross-links in C3(H₂O) and C3. A, The 94 cross-links quantified from the C3(H₂O) versus C3 comparison fall into five groups: C3-unique (10), C3-enriched (1), C3(H₂O)-C3 mutual (57), C3(H₂O)-enriched (4) and C3(H₂O)-unique (22). B, The 57 C3(H₂O)-C3 mutual cross-links are displayed (as dark gray rods) on the crystal structure of C3 (PDB 2A73), reflecting the presence of similar structural features. C, Ten C3-unique cross-links (blue rods), one C3-enriched cross-link (cyan rod), four C3(H₂O)-enriched cross-links (dark green rods) and 22 C3(H₂O)-unique cross-links (green rods) are displayed on the crystal structure of C3, suggesting the locations of structural differences between C3(H₂O) and C3. ANA is colored in light blue.

Fig. 4.

Comparison of cross-links in C3(H₂O) and C3b. A, The 92 cross-links quantified from the C3(H₂O) versus C3b comparison fall into five groups: C3(H₂O)-unique (23), C3(H₂O)-enriched (4), C3(H₂O)-C3b mutual (57), C3b-enriched (1) and C3b-unique (7). B, The 57 C3(H₂O)-C3b mutual cross-links are displayed (as dark gray rods) on the crystal structure of C3b (PDB 2I07), reflecting similar structural features. C, Seven C3b-unique cross-links (red rods), one C3b-enriched cross-link (magenta rod), four C3(H₂O)-enriched cross-links (dark green rods) and 16 C3(H₂O)-unique cross-links (green rods) are displayed on the crystal structure of C3b, Note that C3(H₂O) residues that are cross-linked to ANA (absent from C3b) are shown as green spheres. The likely position of the C3(H₂O) ANA is indicated but the seven cross-links within ANA are not shown. These C3b or C3(H₂O)-unique cross-links suggest the locations of structural differences between C3(H₂O) and C3b. D, Five cross-links from 44^MG1 and TED (visualized in the C3b crystal structure PDB 2I07) indicate that three distinct sites on TED (marked as 1, 2, and 3) can be proximal with MG1, reflecting three different orientations of TED relative to the structural core (shown schematically in the three structures drawn at a smaller scale). Cross-links from MG1 to site 1 and 2 are mutual to C3b and C3(H₂O), whereas the cross-link to site 3 is unique to C3(H₂O). In the crystal structure of C3b, Cα -Cα distances between 44^MG1 and cross-linked residues at site 2 (1181^TED and 1195^TED) and site 3 (1049^TED) violate the BS3 cross-link limit (27.4 Å). These observations suggest mobility of CUB and TED with respect to MG1–6 in both C3b and C3(H₂O).

Fig. 5.

Quantified cross-links reveal unique structural features in C3(H₂O). A, Combining pair-wise comparisons of C3(H₂O) versus C3 and C3(H₂O) versus C3b resulted in 101 quantified cross-links, falling into six categories as summarized in the Venn diagram. B, The 48 cross-links that were observed in C3, C3b and C3(H₂O) are drawn as black rods in the crystal structures of C3 (PDB 2A73) and C3b (PDB 2I07). These cross-links reflect structural features that are preserved in all three proteins. C, Cα-Cα distances are shown for the three C3-unique, six C3(H₂O)-unique and seven C3(H₂O)-C3 mutual (but missing from C3b) cross-links, as measured in the C3 structure (PDB 2A73). These cross-links all involve residues of ANA. The theoretically maximum cross-linkable distance was subtracted, and the data plotted, showing that most of the C3(H₂O)-unique cross-links are incompatible with the relative position of ANA as seen in C3. D, In the “shoulder” region of the C3 structure are shown 14 C3(H₂O)-C3 mutual cross-links (cyan rods) and nine C3(H₂O)-unique cross-links (green rods) (C-α atoms of cross-linked residues shown as spheres). The domains in this region of C3(H₂O) must be rearranged relative to their positions in C3. E, In the “shoulder” region of C3 are shown ten C3-unique cross-links (blue rods), indicating structural features of C3 that are likely absent in C3(H₂O). F, Cross-linking partners of ANA residues in C3(H₂O) are highlighted (blue spheres) in the C3b crystal structure (PDB 2I07). This suggests a likely location of ANA relative to the rest of the C3(H₂O) structure (cf. Fig. 3C). G, Detailed view of six C3b-unique cross-links (red) within the “shoulder” region (four from Ser⁷²⁷, the α′ chain N terminus in C3b) drawn on the C3b structure (PDB 2I07). These cross-links suggest structural features of C3b that are likely to be missing from C3(H₂O). H, A model (center) of a unique arrangement, in C3(H₂O), for ANA, the neighboring MG7 and MG8 domains, and the region of C3(H₂O) corresponding to α′-NT of C3b. The arrangements of these domains in C3 and C3b structures are shown for reference. I, A schematic to represent the domain architecture of C3(H₂O), inferred from the quantified cross-linking data and the crystal structures of C3 and C3b, with the putative binding site for factor B indicated. In the inset, the domains that are C3-like (blue) or C3b-like (red) in arrangement, or display features unique to C3(H₂O) (green), are highlighted. This inferred structure can be compared with the integrative model in Fig. 6.

Fig. 6.

An integrative model for C3(H₂O). A, The positions and structures of domains treated as rigid bodies in our modeling of C3, C3b and C3(H₂O) structures. Horizontal bars are color-coded to indicate the positions of domains/rigid bodies within the primary sequences. The red insertions were treated as flexible strings of beads that allow mobility between the rigid bodies. B–D, The models of C3, C3b, and C3(H₂O) (obtained as described under “Experimental Procedures”) are displayed using the localization densities of their domains (see (A)), shown as transparent surfaces. Backbone traces of models are also shown. The models of C3 and C3b compare well with the corresponding crystallographic structures (PDB 2A73 and PDB 2I07) that are shown alongside at a reduced scale. E, The accuracy and the precision of C3, C3b, and C3(H₂O) domains. The accuracy is the average root mean squared distance (r.m.s.d) (Cα atoms) of the cluster of solutions with respect to the corresponding crystallographic structure (PDB 2A73 for C3 or PDB 2I07 for C3b). The precision is the average r.m.s.d (Cα atoms) of the cluster of solutions with respect to the cluster center (defined under “Experimental Procedures”). Both accuracy and precision are computed after alignment of solutions on the rigid body MG1–6_α_′NT. F, Contact map for cluster B of solutions of C3(H₂O) model (gray shades) overlaid with the cross-links map (colored circles). Green and orange circles are satisfied and violated cross-links, respectively (a satisfied cross-link is defined when the Cα -Cα distance is below 35 Å (44)).