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 Staphylococcus aureus collagen adhesin that is located in a region between amino acids Asp209 and Tyr233. Polyclonal antibodies raised against a recombinant form of the collagen adhesin inhibited the binding of collagen type II to S. aureus. When overlapping synthetic peptides mimicking 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 high concentrations inhibited the binding of collagen to S. aureus. A synthetic peptide derivative of CBD4 lacking 2 carboxyl-terminal residues (Asn232, Tyr233) had no inhibitory activity. The importance of these residues for collagen binding was confirmed by biospecific interaction analysis. Mutant adhesin proteins N232-->A and Y233-->A exhibited dramatic changes in collagen binding activity. The dominant dissociation rate for the binding of mutant adhesin protein N232-->A to immobilized collagen II decreased almost 10-fold, while the Y233-->A and the double mutant exhibited even more significant decreases in affinity and apparent binding ratio when compared to the wild type protein.
Patti
J M
JM
Albert B. Alkek Institute of Biosciences and Technology, Texas A&M University, Houston 77030, USA.
House-Pompeo
K
K
Boles
J O
JO
Garza
N
N
Gurusiddappa
S
S
Höök
M
M
D018829:Adhesins, Bacterial; D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D015374:Biosensing Techniques; D003094:Collagen / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D018829:Adhesins, Bacterial; D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D008024:Ligands; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C048165:adhesin, Staphylococcus aureus; D003094:Collagen