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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.
Highlights
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
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
C3(H2O) Versus C3 Comparison Confirms Structural Rearrangements in C3(H2O)—To interrogate the unknown arrangement of domains in C3(H2O), we first compared quantitative cross-linking/mass spectrometry (QCLMS) data for C3(H2O) and C3
Summary
Protein Preparation for Cross-linking—Plasma-derived human C3 and C3b were purchased from Complement Technology, Inc., Tyler, TX (and stored at Ϫ80 °C). Those cross-linked peptide pairs identified with fragment signals of both peptides in MS2 spectra were used to generate the list of identified cross-linked residue pairs and used for subsequent quantitation. The signal fold-change of a cross-linked peptide pair was calculated as log (C3/C3(H2O)), or log (C3b/ C3(H2O)). Signal fold-changes of all observed cross-linked peptide pairs were first normalized to their median. This corrected systematic errors introduced by minor differences in mixing ratios during sample preparation. The signal fold-change for a residue pair was calculated as the median of all its supporting cross-linked peptides. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium [52] (http://proteomecentral. proteomexchange.org) via the PRIDE partner repository with the data set identifier PXD003486
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