title: A cluster of positively charged amino acids in the C4BP alpha-chain is crucial for C4b binding and factor I cofactor function.
abstract: C4b-binding protein (C4BP) is a regulator of the classical complement pathway, acting as a cofactor to factor I in the degradation of C4b. Computer modeling and structural analysis predicted a cluster of positively charged amino acids at the interface between complement control protein modules 1 and 2 of the C4BP alpha-chain to be involved in C4b binding. Three C4BP mutants, R39Q, R64Q/R66Q, and R39Q/R64Q/R66Q, were expressed and assayed for their ability to bind C4b and to function as factor I cofactors. The apparent affinities of R39Q, R64Q/R66Q, and R39Q/R64Q/R66Q for immobilized C4b were 15-, 50-, and 140-fold lower, respectively, than that of recombinant wild type C4BP. The C4b binding site demonstrated herein was also found to be a specific heparin binding site. In C4b degradation, the mutants demonstrated decreased ability to serve as factor I cofactors. In particular, the R39Q/R64Q/R66Q mutant was inefficient as cofactor for cleavage of the Arg937-Thr938 peptide bond in C4b. In contrast, the factor I mediated cleavage of Arg1317-Asn1318 bond was less affected by the C4BP mutations. In conclusion, we identify a cluster of amino acids that is part of a C4b binding site involved in the regulation of the complement system.
authors.affiliation: The Wallenberg Laboratory, Department of Clinical Chemistry, Lund University, University Hospital Malmö, S-205 02 Malmö, Sweden.
authors.forename: A M
authors.initials: AM
authors.lastname: Blom
authors.forename: J
authors.initials: J
authors.lastname: Webb
authors.forename: B O
authors.initials: BO
authors.lastname: Villoutreix
authors.forename: B
authors.initials: B
authors.lastname: Dahlbäck
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D015374:Biosensing Techniques; D002460:Cell Line; D002846:Chromatography, Affinity; D015935:Complement C4b / Q000235:genetics / Q000378:metabolism*; D017244:Complement Factor I / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D000596:Amino Acids; D015935:Complement C4b; D017244:Complement Factor I