CRITICAL RESIDUES IN THE LIGAND-BINDING SITE OF THE STAPHYLOCOCCUS-AUREUS COLLAGEN-BINDING ADHESIN (MSCRAMM)

We have identified a discrete collagen-binding site within the Staphyl ococcus aureus collagen adhesin that is located in a region between am ino acids Asp(209) and Tyr(233). Polyclonal antibodies raised against a recombinant form of the collagen adhesin inhibited the binding of co llagen type II to S. aureus. When overlapping synthetic peptides mimic king segments of the adhesin fragment were tested for their ability to neutralize the inhibitory activity of the antibody only one peptide, CBD4 was found to be active. CBD4 bound directly to collagen and at hi gh concentrations inhibited the binding of collagen to S. aureus, A sy nthetic peptide derivative of CBD4 lacking 2 carboxyl-terminal residue s (Asn(232) Tyr(233)) had no inhibitory activity. The importance of th ese residues for collagen binding was confirmed by biospecific interac tion analysis, Mutant adhesin proteins N-232 --> A and Y-233 --> A exh ibited dramatic changes in collagen binding activity. The dominant dis sociation rate for the binding of mutant adhesin protein N-232 --> A t o immobilized collagen II decreased almost 10-fold, while the Y-233 -- > A and the double mutant exhibited even more significant decreases in affinity and apparent binding ratio when compared to the wild type pr otein.