title: Identification and characterization of the C3 binding domain of the Staphylococcus aureus extracellular fibrinogen-binding protein (Efb).
abstract: The secreted Staphylococcus aureus extracellular fibrinogen-binding protein (Efb) is a virulence factor that binds to both the complement component C3b and fibrinogen. Our laboratory previously reported that by binding to C3b, Efb inhibited complement activation and blocked opsonophagocytosis. We have now located the Efb binding domain in C3b to the C3d fragment and determined a disassociation constant (Kd) of 0.24 microM for the Efb-C3d binding using intrinsic fluorescence quenching assays. Using truncated, recombinant forms of Efb, we also demonstrate that the C3b binding region of Efb is located within the C terminus, in contrast to the fibrinogen binding domains that are located at the N-terminal end of the protein. Enzyme-linked immunosorbent assay-type binding assays demonstrated that recombinant Efb could bind to both C3b and fibrinogen simultaneously, forming a trimolecular complex and that the C-terminal region of Efb could inhibit complement activity in vitro. In addition, secondary structure analysis using circular dichroism spectroscopy revealed that the C-terminal, C3b binding region of Efb is composed primarily of alpha-helices, suggesting that this domain of Efb represents a novel type of C3b-binding protein.
authors.affiliation: Center for Extracellular Matrix Biology, Texas A&M University System Health Science Center, Albert B. Alkek Institute of Biosciences and Technology, Houston, Texas 77030-7552, USA.
authors.forename: Lawrence Y L
authors.initials: LY
authors.lastname: Lee
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Liang
authors.forename: Magnus
authors.initials: M
authors.lastname: Höök
authors.forename: Eric L
authors.initials: EL
authors.lastname: Brown
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D015153:Blotting, Western; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003167:Complement Activation; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005340:Fibrinogen / Q000737:chemistry; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013211:Staphylococcus aureus / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C114014:Efb protein, Staphylococcus aureus; D011994:Recombinant Proteins; D005340:Fibrinogen