title: The complement regulator C4b-binding protein (C4BP) interacts with both the C4c and C4dg subfragments of the parent C4b ligand: evidence for synergy in C4BP subsite binding.
abstract: C4b-binding protein (C4BP) is a multimeric serum protein that is a potent regulator of the classical and lectin complement pathways. The binding site for C4b has been localized to complement control protein (CCP) domains 1-3 of the C4BP alpha-chain and, in particular, to a cluster of positively charged amino acids predicted to be at the interface between CCP 1 and CCP 2. To determine the regions of C4b contributing to C4BP binding, we have examined via surface plasmon resonance technology the binding of the C4c and C4dg subfragments of C4b to C4BP. At half-physiologic ionic strength, specific and saturable binding was observed for both C4c and C4dg. C4c exhibited much greater ionic strength sensitivity in its binding than did C4dg. Analysis of the effect on binding of the subfragments to various C4b-binding-defective C4BP mutants, together with cross-competition experiments, suggests that the subsites in C4BP for C4c and C4dg are adjacent, but distinct. Additionally, we observed synergy in subsite filling such that the presence of C4dg enhanced the extent of C4c binding over its basal level, and vice versa. The enhanced binding of C4c in the presence of C4dg was not due to an increase in affinity but rather reflected a 2-3-fold increase in the number of sites capable of binding C4c. This suggests the existence of a conformational equilibrium between high- and low-affinity states in the C4c binding subsite within each C4BP subunit, an equilibrium which is shifted in favor of the high-affinity state by the filling of the C4dg subsite.
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors.forename: Elisa
authors.initials: E
authors.lastname: Leung
authors.forename: Anna M
authors.initials: AM
authors.lastname: Blom
authors.forename: Liliana
authors.initials: L
authors.lastname: Clemenza
authors.forename: David E
authors.initials: DE
authors.lastname: Isenman
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003181:Complement C4 / Q000737:chemistry*; D050716:Complement C4b-Binding Protein / Q000737:chemistry* / Q000235:genetics; D006493:Heparin / Q000737:chemistry; D006801:Humans; D008024:Ligands; D009154:Mutation; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry; D020349:Surface Plasmon Resonance
chemical_list: D003181:Complement C4; D050716:Complement C4b-Binding Protein; D008024:Ligands; D010446:Peptide Fragments; D006493:Heparin