Analogous Interactions in Initiating Complexes of the Classical and Lectin Pathways of Complement

Abstract

The classical and lectin pathways of complement activation neutralise pathogens and stimulate key immunological processes. Both pathways are initiated by collagen-containing, soluble pattern-recognition molecules associated with specific serine proteases. In the classical pathway, C1q binds to antibody-antigen complexes or bacterial surfaces to activate C1r and C1s. In the lectin pathway, mannan-binding lectin (MBL) and ficolins bind to carbohydrates on pathogens to activate MASP-2. To characterise the interactions leading to classical pathway activation, we have analysed binding between human C1q, C1r and C1s, which associate to form C1, using full-length and truncated protease components. We show that C1r and C1s bind to C1q independently. The CUB1-EGF fragments contribute most towards binding, but CUB2 of C1r, but not of C1s, is also important. Each C1rs tetramer presents a total of six binding sites, one for each of the collagenous domains of C1q. We also demonstrate that subcomponents of the lectin and classical pathways cross-interact. Thus, although the stoichiometries of complexes differ, interactions are analogous, with equivalent contacts between recognition and protease subcomponents. Importantly, these new data are contrary to existing models of C1, and enable us to propose a new model, using MBL-MASP interactions as a template.

Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation

“…The structure is compatible with previous mutagenesis data that highlight the importance of the lysine residue in the collagen and Glu45 and Asp98 in C1s, and explains the Ca 2+ dependence of binding (11,12). It shares a number of similarities with the complex between the CUB2 domain of MASP-1 bound to a collagen-like peptide of MBL of the lectin pathway (15).…”

“…In addition, there is overall charge complementarity, with the binding regions of CUB1 and CUB2 having net negative and positive charges, respectively. A C1s-C1s interaction at the center of the C1 complex has been predicted in previous molecular dynamics and modeling studies (11,12,17) but has not been observed directly. In the absence of C1q, the interaction must be weak because only C1s dimers are detected in solution (corresponding to the X-shaped dimers), but is stabilized when C1r-C1s tetramers bind to C1q, explaining how the elongated proteases form the compact structures observed in electron micrographs of the C1 complex (9).…”

“…For cocrystallization, we designed a short collagen-like peptide containing the sequence Hyp-GlyLys-Leu-Gly-Pro at the center. We have shown that C1r and C1s recognize this sequence and that the lysine residue at the center of the motif is essential for binding (12).…”

“…Calcium plays a central role in the assembly and is necessary for binding of C1r to C1s and of the protease tetramer to C1q (10). Recent work has identified separate binding sites for C1q in the CUB1 and CUB2 domains of C1r and in CUB1 of C1s, providing a total of six sites on a C1r-C1s tetramer, one for each collagenous stem (11,12). In this study, we have determined the structures of a series of fragments of C1s, including C1s bound to a collagen-like peptide from C1q and a tetramer that reveals how the proteases pack together at the center of the complex.…”

Deciphering Complement Receptor Type 1 Interactions with Recognition Proteins of the Lectin Complement Pathway

“…Three MASPs have been identified, MASP-1, MASP-2, and MASP-3, among which only MASP-1 and MASP-2 are essential for complement activation (7), whereas the role of MASP-3 still requires further elucidation. The three MASPs and the C1s-C1r-C1r-C1s tetramer interact in a similar way with the collagen-like stalks of the recognition proteins, which allows in vitro cross-interaction between subcomponents of the classical and lectin pathways (8,9). Following recognition of a target, the activated C1 and MBL/ficolin-MASP proteolytic complexes cleave complement components C4 and C2 to generate the C3 convertase responsible for cleavage of C3 and subsequent opsonization of the target by C3 fragments such as C3b.…”