Structural organization of the fibrinogen-binding region of the clumping factor B MSCRAMM of Staphylococcus aureus

The clumping factor B (ClfB) of Staphylococcus aureus is a surface protein that binds to fibrinogen (Ni Eidhin, D., Perkins, S., Francois, P., Vaudaux , P., Hook, M., and Foster, T. J., 1998 Mol. Microbiol. 30, 245-257). The l igand-binding activity is located in the similar to 500-residue A-region (r esidues 44-542), which represents the N-terminal half of the MSCRAMM protei n. We now hypothesize that the ClfB A-region is composed of three subdomain s, which we have named N1, N2, and N3, respectively. To examine this hypoth esis, we expressed recombinant forms of the individual putative subdomains, the tandem motifs N12 and N23, and the full-length A-region N123. Far UV c ircular dichroism spectra showed that each subdomain is composed mainly of beta -sheets with little or no discernible alpha -helices. Heat-induced unf olding of individual subdomains occurred with a single state transition and was reversible, indicating that the subdomains can fold as discreet units. Gel permeation chromatography indicated that N2, N3, and N23 are globular. In contrast, domain Nl appeared to be elongated and conferred a somewhat e longated structure on segments containing this subdomain (i.e. N12 or N123) . N123, N12, and N23 all bound to fibrinogen, but N23 had a higher affinity for fibrinogen than that observed for the full-length A-region; N123 or fo r N12. However, an extended N terminus of N23 was required for ligand bindi ng. A form of N23 that was generated by proteolytic processing and lacked t he N-terminal extension was unable to bind fibrinogen. Recombinant forms of individual subdomains did not bind fibrinogen. The addition of recombinant N23 effectively inhibited ClfB-mediated bacterial adherence to fibrinogen, and N123 caused some reduction in bacterial attachment, whereas N12 was es sentially inactive. Antibodies raised against the central N2 domain of the A-region were the most effective at inhibiting bacterial adhesion to immobi lized fibrinogen, although anti-N3 or anti-N1 antibodies also caused some r eduction in ClfB-mediated adherence to fibrinogen.