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.