title: Positively charged amino acids at the interface between alpha-chain CCP1 and CCP2 of C4BP are required for regulation of the classical C3-convertase.
abstract: C4b-binding protein (C4BP) is an abundant and potent down-regulator of complement activation. In its presence, the assembly of the classical pathway C3-convertase is prevented and its natural decay is accelerated. C4BP also acts as a cofactor to the serine proteinase factor I in the cleavage of C4b. C4BP contains repeats of small structural domains: complement control protein (CCP) modules. Previously, we constructed and purified nine recombinant C4BP molecules in which solvent exposed positively charged amino acids at the interface between CCP1 and CCP2 were mutated to polar glutamines. Several of these mutants showed lower binding ability for C4b. In the present investigation, the collection of mutants was tested with functional assays and we found a correlation between changes in the apparent affinity of C4BP mutants for C4b and their ability to down regulate the C3-convertase. Mutagenesis of R(39), K(63), R(64) and particularly H(67) resulted in impaired C4b binding paralleled by lost ability of the C4BP mutants to prevent C3-convertase assembly and to increase the decay rate of the C3-convertase. Furthermore, these amino acids were found to be crucial for the factor I cofactor activity of C4BP in fluid phase degradation of C4b. In conclusion, a cluster of positively charged amino acid residues at the CCP1-CCP2 interface is identified as functionally important in the regulation of the C3-convertase by C4BP.
authors.affiliation: Department of Clinical Chemistry, The Wallenberg Laboratory, Lund University, University Hospital Malmö, S-205 02 Malmö, Sweden. anna.blom@klkemi.mas.lu.se
authors.forename: A M
authors.initials: AM
authors.lastname: Blom
authors.forename: A F
authors.initials: AF
authors.lastname: Zadura
authors.forename: B O
authors.initials: BO
authors.lastname: Villoutreix
authors.forename: B
authors.initials: B
authors.lastname: Dahlbäck
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D050577:Complement C3-C5 Convertases / Q000737:chemistry / Q000378:metabolism*; D050716:Complement C4b-Binding Protein; D004912:Erythrocytes; D005340:Fibrinogen / Q000378:metabolism; D006649:Histocompatibility Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D012756:Sheep; D055672:Static Electricity
chemical_list: C055609:C4BPA protein, human; C489165:C4BPB protein, human; D050716:Complement C4b-Binding Protein; D006649:Histocompatibility Antigens; D005340:Fibrinogen; D050577:Complement C3-C5 Convertases