Complement factor H (FH) attenuates C3b molecules tethered via their thioester domains to self-surfaces and thereby protects host tissues. FH is a cofactor for initial C3b proteolysis that ultimately yields a surface-attached fragment (C3d), corresponding to the thioester domain. We used NMR and X-ray crystallography to study the C3d:FH19–20 complex in atomic detail. NMR further identified glycosaminoglycan-binding residues in FH module 20 of the C3d:FH19–20 complex. Mutagenesis justified the merging of the C3d:FH19–20 structure with an existing C3b:FH1–4 crystal structure. The merged structure was concatenated with the available FH6–8 crystal structure and new SAXS-derived FH1–4, FH8–15 and FH15–19 envelopes. The combined data suggests a bent-back FH molecule, binding via its termini to two sites on one C3b molecule and simultaneously to adjacent polyanionic host-surface markers.

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We have used the interaction between module 7 of complement factor H (CFH approximately 7) and a fully sulfated heparin tetrasaccharide to exemplify a new approach for studying contributions of basic side chains to the formation of glycosaminoglycan (GAG)-protein complexes. We first employed HISQC and H(2)CN NMR experiments to monitor the side-chain resonances of lysines and arginines in (15)N, (13)C-labeled protein during titrations with a fully sulfated heparin tetrasaccharide under physiological conditions. Under identical conditions and using (15)N-labeled protein, we then cross-linked tetrasaccharide to CFH approximately 7 and confirmed the 1:1 stoichiometry by FT-ICR-MS. We subsequently characterized this covalent protein-GAG conjugate by NMR and further MS techniques. MALDI-TOF MS identified protein fragments obtained via trypsin digestion or chemical fragmentation, yielding information concerning the site of GAG attachment. Combining MS and NMR data allowed us to identify the side chain of K405 as the point of attachment of the cross-linked heparin oligosaccharide to CFH approximately 7. On the basis of the analysis of NMR and MS data of the noncovalent and cross-linked CFH approximately 7-tetrasaccharide complexes, we conclude that the K446 side chain is not essential for binding the tetrasaccharide, despite the large chemical shift perturbations of its backbone amide (15)N and (1)H resonances during titrations. We show that R444 provides the most important charge-charge interaction within a C-terminal heparin-binding subsite of CFH approximately 7 whereas side chains of R404, K405, and K388 are the predominant contributors to an N-terminal binding subsite located in the immediate vicinity of residue 402, which is implicated in age-related macular degeneration (AMD).