IDENTIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF THE LIGAND-BINDINGDOMAIN OF THE COLLAGEN ADHESIN FROM STAPHYLOCOCCUS-AUREUS

We have recently shown that the expression of a collagen adhesin is bo th necessary and sufficient to mediate the attachment of Staphylococcu s aureus to cartilage a complex collagen-containing substrate [Switals ki, L. M., Patti, J. M., Butcher, W., Gristina, A. G., Speziale, P., & Hook, M. (1993) Mol. Microbiol. 7, 99-107]. We now report on the loca lization of the ligand binding site within the 135-kDa S. aureus colla gen adhesin. Using deletion mutagenesis in combination with Western li gand blot and direct binding assays, the collagen binding domain (CBD) was localized to a 168 amino acid long segment [CBD(151-318)] within the N-terminal portion of the adhesin. Using biospecific interaction a nalysis, pepsin-digested bovine type II collagen was found to contain eight binding sites for CBD(151-318); two binding sites were of ''high '' affinity (K(d) = 3 muM) and six sites were of low affinity (K(d) = 30 muM). Short truncations in the terminal flanking regions of CBD(151 -318) resulted in two CBDs (180-318 and 151-297) that lacked collagen binding activity. Analysis by circular dichroism of the recombinant CB Ds in the far UV revealed similar secondary structures, predominantly beta-sheet, whereas the near-UV spectra indicated dramatic changes in the degree of intermolecular packing (tertiary structure). The deduced amino acid sequence of the ligand binding domain of the collagen adhe sin is presented.