Identification and biochemical characterization of the ligand binding domain of the collagen adhesin from Staphylococcus aureus.
We have recently shown that the expression of a collagen adhesin is both necessary and sufficient to mediate the attachment of Staphylococcus aureus to cartilage, a complex collagen-containing substrate [Switalski, L. M., Patti, J. M., Butcher, W., Gristina, A. G., Speziale, P., & Höök, M. (1993) Mol. Microbiol. 7, 99-107]. We now report on the localization of the ligand binding site within the 135-kDa S. aureus collagen adhesin. Using deletion mutagenesis in combination with Western ligand 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 analysis, pepsin-digested bovine type II collagen was found to contain eight binding sites for CBD(151-318); two binding sites were of "high" affinity (Kd = 3 microM) and six sites were of low affinity (Kd = 30 microM). 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 CBDs 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 adhesin is presented.
Patti
J M
JM
Institute of Biosciences and Technology, Texas A & M University, Houston 77030.
Boles
J O
JO
Höök
M
M
D018829:Adhesins, Bacterial; D000595:Amino Acid Sequence; D000818:Animals; D001422:Bacterial Adhesion; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002645:Chickens; D002942:Circular Dichroism; D003094:Collagen / Q000378:metabolism*; D017931:DNA Primers; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013211:Staphylococcus aureus / Q000378:metabolism*
D018829:Adhesins, Bacterial; D017931:DNA Primers; D011994:Recombinant Proteins; C048165:adhesin, Staphylococcus aureus; D003094:Collagen