Paths reunited: Initiation of the classical and lectin pathways of complement activation

Abstract

Understanding the structural organisation and mode of action of the initiating complex of the classical pathway of complement activation (C1) has been a central goal in complement biology since its isolation almost 50 years ago. Nevertheless, knowledge is still incomplete, especially with regard to the interactions between its subcomponents C1q, C1r and C1s that trigger activation upon binding to a microbial target. Recent studies have provided new insights into these interactions, and have revealed unexpected parallels with initiating complexes of the lectin pathway of complement: MBL-MASP and ficolin-MASP. Here, we develop and expand these concepts and delineate their implications towards the key aspects of complement activation via the classical and lectin pathways.

Figure 1. Schematic representations of the individual components of the initiating complexes of lectin and classical complement pathways

Top, C1q, MBL and ficolins (not shown) resemble bouquets, comprising N-terminal collagen-like domains linked to C-terminal target-recognition domains (carbohydrate-recognition, antibody-binding and fibrinogen-like, respectively). Middle, domain organisations of C1rs heterotetramers and MASP homodimers. C1r-C1s and MASP-MASP interactions are conserved (black boxes), so the C1rs tetramer is effectively equivalent to two MASP dimers linked through the CCP-SP domains of C1r. Bottom, crystal structures of MASP-1 CUB1-EGF-CUB2 (green; PDB: 3DEM (Teillet et al., 2008)) and C1s CUB1-EGF (orange and purple; PDB: 1NZI, (Gregory et al., 2003)) fragments. Each EGF domain binds a Ca²⁺ ion (red sphere) aiding binding to the CUB1 domain of its partner. Additional Ca²⁺ ions (red spheres) are co-ordinated within the CUB domains and organize the loops that form the binding sites for the MBL/C1q stalks. Putative binding residues are coloured red and yellow for large and lesser effects on binding, respectively. In vivo, C1s does not form homodimers, but each chain instead binds to a C1r polypeptide through homologous interactions.

Figure 2. C1 and MBL-MASP complexes

Top (top) and side (bottom) views of C1 and MBL-MASP complexes derived from modelling and binding studies. Only the CUB1-EGF-CUB2 domains of C1r and C1s (purple and orange) and MASP (green) are shown. In both cases the CCP1-CCP2-SP domains are located inside the complex, on top of the CUB1-EGF-CUB2 domains. Binding sites are: C1q (blue), MBL (dark blue), C1rs (yellow) and MASP (orange). Notably, interactions of C1q with C1rs and MBL with MASP are analogous, with four binding sites on each MASP dimer and three on each C1r-C1s (CUB2 of C1s lacks a binding site), making a total of six sites on a C1rs heterotetramer.

Figure 3. Strained-to-relaxed model of C1 activation

Phase 1: Zymogen C1. The active sites of C1r are kept apart through the interactions of one SP domain with the CCP domains of its partner. Binding to C1q induces strain, pulling the C1q stalks closer together (as indicated by the arrows). Although C1r and C1s are zymogens, their SP domains are relatively exposed in circulating complexes, so are accessible to reversible binding by C1-INH (Ziccardi, 1985). However, irreversible binding and inhibition by C1-INH only occurs upon activation of C1r and/or C1s and serves to neutralise the low level of spontaneous activation that occurs in solution (Ziccardi, 1982). The CCP-SP domains of C1s are not shown for clarity. Phase 2: target binding induced C1r autoactivation. Multiple simultaneous interactions of the C1q heads with an activating surface (e.g., IgG bound to bacterial surfaces) enable the stalks to move apart again (see arrows), releasing the induced strain within the complex. The CCP-SP domains of C1r spring apart allowing autocatalysis of one polypeptide by its partner. Side and top views are shown. Phase 3: C1s activation. Flexibility at the inter-domain CUB2-CCP junctions allows the C1r SP domain to cleave C1s, converting it to the active form. Phase 4: Activated C1. The catalytic domains of C1s, and possibly C1r, move out into solution, where the former activate downstream substrates C4 and C2. Once C1 is bound to a surface, C1-INH can only access the SP domains of C1s and/or C1r after they have moved outside of the complex.

Figure 4. Conformational changes in C1 and MBL-MASP complexes

Side and top views of complexes during activation and fully activated. Autoactivation of C1r and MASPs occurs inside the complexes. Flexibility at the interdomain CUB2-CCP1 junctions would then allow the CCP-protease domains to move outside the cones created by the C1q/MBL stalks and into solution where they would be able to activate downstream substrates C4 and C4b2.