Identification of the Staphylococcus aureus MSCRAMM clumping factor B (ClfB) binding site in the alphaC-domain of human fibrinogen.
Clumping factor B (ClfB) of Staphylococcus aureus binds to cytokeratin 10 and to fibrinogen. In this study the binding site in human fibrinogen was localized to a short region within the C terminus of the Aalpha-chain. ClfB only bound to the Aalpha-chain of fibrinogen in a ligand-affinity blot and in solid-phase assays with purified recombinant fibrinogen chains. A variant of fibrinogen with wild-type Bbeta- and gamma-chains but with a deletion that lacked the C-terminal residues from 252-610 of the Aalpha-chain did not support adherence of S. aureus Newman expressing ClfB. A series of truncated mutants of the recombinant Aalpha-chain were tested for their ability to support adherence of S. aureus Newman ClfB(+), which allowed the binding site to be localized to a short segment of the unfolded flexible repeated sequence within the C terminus of the Aalpha-chain. This was confirmed by two amino acid substititions within repeat 5 of the recombinant Aalpha-chain which did not support adherence of Newman ClfB(+). Lactococcus lactis expressing ClfB mutants with amino acid substitutions (N256 and Q235) located in the putative ligand-binding trench between domains N2 and N3 of the A-domain were defective in adherence to immobilized fibrinogen and cytokeratin 10, suggesting that both ligands bind to the same or overlapping regions.
Walsh
Evelyn J
EJ
Microbiology Department, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland.
Miajlovic
Helen
H
Microbiology Department, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland.
Gorkun
Oleg V
OV
Department of Pathology and Laboratory Medicine, CB #7525, Brinkhous-Bullitt Building, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7525, USA.
Foster
Timothy J
TJ
Microbiology Department, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland.
D018829:Adhesins, Bacterial / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D003001:Cloning, Molecular; D005340:Fibrinogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013294:Lactococcus lactis / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013211:Staphylococcus aureus / Q000378:metabolism*
D018829:Adhesins, Bacterial; D011994:Recombinant Proteins; C434058:clfB protein, Staphylococcus aureus; D005340:Fibrinogen