Analysis of Binding Sites on Complement Factor I That Are Required for Its Activity

Abstract

The central complement inhibitor factor I (FI) degrades activated complement factors C4b and C3b in the presence of cofactors such as C4b-binding protein, factor H, complement receptor 1, and membrane cofactor protein. FI is a serine protease composed of two chains. The light chain comprises the serine protease domain, whereas the heavy chain contains several domains; that is, the FI and membrane attack complex domain (FIMAC), CD5, low density lipoprotein receptor 1 (LDLr1) and LDLr2 domains. To understand better how FI acts as a complement inhibitor, we used homology-based models of FI domains to predict potential binding sites. Specific amino acids were then mutated to yield 16 well expressed mutants, which were then purified from media of eukaryotic cells for functional analyses. The Michaelis constant (K m ) of all FI mutants toward a small substrate was not altered, whereas some mutants showed increased maximum initial velocity (V max ). All the mutations in the FIMAC domain affected the ability of FI to degrade C4b and C3b irrespective of the cofactor used, whereas only some mutations in the CD5 and LDLr1/2 domains had a similar effect. These same mutants also showed impaired binding to C3met. In conclusion, the FIMAC domain appears to harbor the main binding sites important for the ability of FI to degrade C4b and C3b.

Structural basis for complement factor I control and its disease-associated sequence polymorphisms

“…It is interesting that several mutations of fI that affect function are surface exposed [Lys51Ala, Arg62Ala, and the aHUS-associated Ala222Gly (21,36,37)]. We therefore predict that these residues may map to the cofactor or C3b binding sites.…”

“…However, the nonsubstratelike, broad-spectrum protease inhibitor diisopropyl fluorophosphate is able to react with the active site serine only if fI is preincubated with C3b (19). This requirement strongly suggests that substrate-induced remodeling of the active site is key to fI To gain insight into previously unexplained disease-associated gene polymorphisms and mutations known to alter cofactorassisted C3b/C4b cleavage by fI (21,22,32,33), we mapped them to our crystal structure (Fig. 4 and SI Appendix, Table S2).…”

“…4A). More importantly, all the mutants that display a significantly increased rate of activity toward synthetic or natural substrates (21,22) cluster at the interface between the two chains, centered around 216-223 529-536 Fig. 3.…”

“…Second, it is not known how fI acquires its exquisite specificity for the selected sites of cleavage in C3b and C4b (20). Third, a number of fI point mutants have been engineered that show, against synthetic or natural substrates, increased rates of activity with respect to the wild-type enzyme (21,22), but no explanation for these observations has been proposed. Finally, because homology modeling of the whole fI is rendered unfeasible by its multidomain architecture, the understanding of fI gene polymorphisms associated with disease also is fraught with uncertainty.…”

Molecular characterization of two novel cases of complete complement inhibitor Factor I deficiency

“…Unfortunately we could not gain access to patient fibroblasts to verify if this occurs in the currently studied patients. In a previous report of structure--function relationships of FI, we constructed mutant K249Q/Q259R/E270Q (or K267Q/Q277R/E288Q including signal sequence), which caused misfolding and lack of secretion of this recombinant protein (Nilsson et al, 2010b). In Patient 2, E288 lies next to mutated D289.…”