A cluster of positively charged amino acids in the alpha-chain of C4b-binding protein (C4BP) is pivotal for the regulation of the complement system and the interaction with bacteria

C4b-binding protein (C4BP) is a potent inhibitor of the classical pathway o f complement. Structural analysis of our 3D model of the C4BP alpha -chain suggested that a cluster of positively charged amino acids at the interface between complement control protein (CCP) modules 1 and 2 could be involved in ligand binding, Nine C4BP mutants, where the positively charged amino a cids were changed to glutamines, were expressed. We found that all of these displayed lower apparent affinity for C4b and that the site was also a spe cific heparin-binding site. The mutants demonstrated decreased ability to s erve as factor I cofactors in a degradation of C4b, Also, their capacity to prevent the assembly of C3-convertase and to accelerate its decay were dec reased. In conclusion, we found a binding site on C4BP that is functionally important for the regulation of C3-convertase. Many strains of Streptococcus pyogenes bind C4BP, via surface M proteins, w hich plays an important role in pathogenesis. Using the set of C4BP mutants we identified a key recognition surface for M proteins which overlaps with the C4b-binding site. The analysis of all mutants and inhibition with mono clonal antibodies indicated that the binding sites for C4b and M proteins a re only overlapping but not identical. Furthermore, we found that the molec ular mechanisms involved in these two interactions differ, since the bindin g between M proteins and C4BP is relatively insensitive to salt in contrast to the C4BP-C4b-binding. We suggest that binding between C4b and C4BP is g overned mostly by electrostatic interactions, while additional non-covalent forces cause tight binding of C4BP to streptococcal M proteins.