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 Sta phylococcus aureus that promotes binding of fibrinogen or fibrin to th e bacterial cell, Previous studies have shown that ClfA and the platel et integrin alpha(IIb)beta(3) recognize the same domain at the extreme C terminus of the fibrinogen gamma-chain. alpha(IIb)beta(3) interacti on with this domain is known to occur in close proximity to a Ca2+-bin ding EF-hand structure in the cr-subunit. Analysis of the primary stru cture of ClfA indicated the presence of a potential Ca2+-binding EF-ha nd-like motif at residues 310-321 within the fibrinogen-binding domain . Deletion mutagenesis and site-directed mutagenesis of this EF-hand i n recombinant truncated ClfA proteins (Clf40, residues 40-559; and Clf 41, residues 221-559) resulted in a significant reduction of affinity for native fibrinogen and a fibrinogen gamma-chain peptide. Furthermor e, Ca2+ (or Mn2+) could inhibit the binding of the fibrinogen gamma-ch ain peptide to Clf40-(40-559) and the adhesion of S. aureus cells to i mmobilized fibrinogen with an IC50 of 2-3 mns. In contrast, Mg2+ (or N a+) at similar concentrations had no effect on the ClfA-fibrinogen int eraction. Far-UV CD analysis of Clf40-(40-559) and Clf41-(221-559) in the presence of metal ions indicated Ca2+- and Mn2+-induced difference s in secondary structure, These data suggest that Ca2+ binds to an inh ibitory site(s) within ClfA and induces a conformational change that i s incompatible with binding to the C terminus of the gamma-chain of fi brinogen. Mutagenesis studies indicate that the Ca2+-dependent inhibit ory site is located within the EF-hand motif at residues 310-321.