Analysis of binding sites on complement factor I that are required for its activity.
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.
Nilsson
Sara C
SC
Department of Laboratory Medicine, Medical Protein Chemistry, Malmö University Hospital, Lund University, S-205 02 Malmö, Sweden.
Nita
Izabela
I
Månsson
Lisa
L
Groeneveld
Tom W L
TW
Trouw
Leendert A
LA
Villoutreix
Bruno O
BO
Blom
Anna M
AM
D001665:Binding Sites; D002384:Catalysis; D003179:Complement C3b / Q000378:metabolism; D015935:Complement C4b / Q000378:metabolism; D017244:Complement Factor I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D040681:Structural Homology, Protein
D003179:Complement C3b; D015935:Complement C4b; D017244:Complement Factor I