The fibrinogen-binding MSCRAMM (clumping factor) of staphylococcus aureus Has a Ca²⁺-dependent inhibitory site

The clumping factor (ClfA) is a cell surface-associated protein of Staphylococcus aureus that promotes binding of fibrinogen or fibrin to the bacterial cell. Previous studies have shown that ClfA and the platelet integrin α(IIb)β₃ recognize the same domain at the extreme C terminus of the fibrinogen γ-chain. α(IIb)β(s) interaction with this domain is known to occur in close proximity to a Ca²⁺-binding EF-hand structure in the α- subunit. Analysis of the primary structure of ClfA indicated the presence of a potential Ca²⁺-binding EF-hand-like motif at residues 310-321 within the site-directed mutagenesis of this EF-hand in recombinant truncated ClfA proteins (Clf40, residues 40-559; and Clf41, residues 221-559) resulted in a significant reduction of affinity for native fibrinogen and a fibrinogen γ- chain peptide. Furthermore, Ca²⁺ (or Mn²⁺) could inhibit the binding of the fibrinogen γ-chain peptide to Clf40-(40-559) and the adhesion of S. aureus cells to immobilized fibrinogen with an IC₅₀ of 2-3 mm. In contrast, Mg²⁺ (or Na⁺) at similar concentrations had no effect on the ClfA- fibrinogen interaction. Far-UV CD analysis of Clf40-(40-559) and Clf41-(221- 559) in the presence of metal ions indicated Ca²⁺- and Mn²⁺-induced differences in secondary structure. These data suggest that Ca²⁺ binds to an inhibitory site(s) within ClfA and induces a conformational change that is incompatible with binding to the C terminus of the γ-chain of fibrinogen. Mutagenesis studies indicate that the Ca²⁺-dependent inhibitory site is located within the EF-hand motif at residues 310-321.