Molecular Characterization of the Interaction of Staphylococcal Mscramms Clfa and Fbl with Fibrinogen

The ligand binding domain of the fibrinogen binding protein from Staphylococcus lugdunensis (Fbl) shares 60% sequence identity with clumping factor A (ClfA) of Staphylococcus aureus. Recombinant Fbl corresponding to the minimum fibrinogen-binding region (subdomains N2N3) was compared to ClfA for binding to fibrinogen. Fbl and ClfA had very similar affinities for fibrinogen by surface plasmon resonance. The binding site for Fbl in fibrinogen was localised to the extreme C-terminus of the fibrinogen γ-chain at the same site recognised by ClfA. Isothermal titration calorimetry (ITC) showed that Fbl and ClfA had very similar affinities for a peptide mimicking the C-terminal segment of the fibrinogen γ-chain. The peptide also inhibited binding of Fbl and ClfA to fibrinogen. A series of substituted γ-chain variant peptides behaved very similarly when used to inhibit ClfA and Fbl binding to immobilized fibrinogen. Both ClfA and Fbl bound to bovine fibrinogen with a lower affinity compared to human fibrinogen and did not bind detectably to ovine fibrinogen. The structure of the N2N3 subdomains of Fbl in complex with the fibrinogen γ-chain peptide was modelled based on the crystal structure of the N2N3 subdomains of ClfA:γchain peptide complex. Residues in the putative binding trench likely to be involved in fibrinogen binding were identified. Fbl variant proteins with alanine substitutions in key residues had reduced affinities for fibrinogen. Thus Fbl and ClfA bind the same site in fibrinogen by similar mechanisms. INTRODUCTION Staphylococcus aureus is an important human pathogen that colonizes the moist squamous epithelium in the anterior nares. It causes both superficial skin infections and more invasive diseases such as endocarditis, osteomyelitis and septic arthritis. Staphylococcus lugdunensis is a coagulase negative staphylococcus that is a commensal of the human skin. It can occasionally cause serious infections similar to those caused by S. aureus (1). S. lugdunensis expresses a fibrinogen binding surface protein (Fbl) with considerable similarity to clumping factor A (ClfA) of S. aureus (2,3). ClfA is a microbial surface component recognizing adhesive matrix molecules (MSCRAMM) that http://www.jbc.org/cgi/doi/10.1074/jbc.M109.062208 The latest version is at JBC Papers in Press. Published on December 10, 2009 as Manuscript M109.062208 Copyright 2009 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from