title: The fibrinogen-binding MSCRAMM (clumping factor) of Staphylococcus aureus has a Ca2+-dependent inhibitory site.
abstract: 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 alphaIIbbeta3 recognize the same domain at the extreme C terminus of the fibrinogen gamma-chain. alphaIIbbeta3 interaction with this domain is known to occur in close proximity to a Ca2+-binding EF-hand structure in the alpha-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 fibrinogen-binding domain. Deletion mutagenesis and 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 gamma-chain peptide. Furthermore, Ca2+ (or Mn2+) could inhibit the binding of the fibrinogen gamma-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 gamma-chain of fibrinogen. Mutagenesis studies indicate that the Ca2+-dependent inhibitory site is located within the EF-hand motif at residues 310-321.
authors.affiliation: Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Republic of Ireland.
authors.forename: D P
authors.initials: DP
authors.lastname: O'Connell
authors.forename: T
authors.initials: T
authors.lastname: Nanavaty
authors.forename: D
authors.initials: D
authors.lastname: McDevitt
authors.forename: S
authors.initials: S
authors.lastname: Gurusiddappa
authors.forename: M
authors.initials: M
authors.lastname: Höök
authors.forename: T J
authors.initials: TJ
authors.lastname: Foster
mesh_terms: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002412:Cations; D002942:Circular Dichroism; D005340:Fibrinogen / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000378:metabolism*
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D002412:Cations; D011994:Recombinant Proteins; C048165:adhesin, Staphylococcus aureus; D005340:Fibrinogen; D002118:Calcium