Identification of the Staphylococcus aureus MSCRAMM clumping factor B (ClfB) binding site in the αC-domain of human fibrinogen

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Abstract
Clumping factor B (ClfB) of Staphylococcus aureus binds to cytokeratin 10 and to fibrinogen. In this study the binding site in human fibrinogen was localized to a short region within the C terminus of the Aα-chain. ClfB only bound to the Aα-chain of fibrinogen in a ligand-affinity blot and in solid-phase assays with purified recombinant fibrinogen chains. A variant of fibrinogen with wild-type Bβ- and γ-chains but with a deletion that lacked the C-terminal residues from 252–610 of the Aα-chain did not support adherence of S. aureus Newman expressing ClfB. A series of truncated mutants of the recombinant Aα-chain were tested for their ability to support adherence of S. aureus Newman ClfB+, which allowed the binding site to be localized to a short segment of the unfolded flexible repeated sequence within the C terminus of the Aα-chain. This was confirmed by two amino acid substititions within repeat 5 of the recombinant Aα-chain which did not support adherence of Newman ClfB+. Lactococcus lactis expressing ClfB mutants with amino acid substitutions (N256 and Q235) located in the putative ligand-binding trench between domains N2 and N3 of the A-domain were defective in adherence to immobilized fibrinogen and cytokeratin 10, suggesting that both ligands bind to the same or overlapping regions.