Abstract
SFS is a non-anchored protein of Streptococcus equi subspecies equi that causes upper respiratory infection in horses. SFS has been shown to bind to fibronectin (FN) and block interaction of FN with type I collagen. We have characterized interactions of a recombinant 60-mer polypeptide, R1R2, with FN. R1R2 contains two copies of collagen-like 19-residue repeats. Experiments utilizing various FN fragments and epitope-mapped anti-FN monoclonal antibodies located the binding site to (8-9)FNI modules of the gelatin-binding domain. Fluorescence polarization and competitive enzyme-linked assays demonstrated that R1R2 binds preferentially to compact dimeric FN rather than monomeric constructs containing (8-9)FNI or a large dimeric FN construct that is constitutively in an extended conformation. In contrast to bacterial peptides that bind (2-5)FNI in addition to (8-9)FNI, R1R2 did not cause conformational extension of FN as assessed by a conformationally sensitive antibody. Equilibrium and stopped-flow binding assays and size exclusion chromatography were compatible with a two-step binding reaction in which each of the repeats of R1R2 interacts with one of the subunits of dimeric FN, resulting in a stable complex with a slow koff. In addition to not binding to type I collagen, the R1R2·FN complex incorporated less efficiently into extracellular matrix than free FN. Thus, R1R2 binds to FN utilizing features of compact soluble FN and in doing so interferes with the organization of the extracellular matrix. A similar bivalent binding strategy may underlie the collagen-FN interaction.
Highlights
Streptococcus equi expresses SFS protein that contains a disordered region, R1R2, able to inhibit the collagenfibronectin interaction
We report that high affinity binding of R1R2 to FN requires a bivalent interaction with 8-9FNI of both FN subunits preferentially displayed in compact rather than extended FN
R1R2 Blocks Binding of FN to Coated Type I Collagen—To show that the minimized R1R2 construct has the expected ability to block the FN-collagen interaction, we compared the ability of FN without or with R1R2 to bind adsorbed type I collagen at 37 °C in an enzyme-linked direct binding assay (Fig. 2)
Summary
Streptococcus equi expresses SFS protein that contains a disordered region, R1R2, able to inhibit the collagenfibronectin interaction. Bacterial surface proteins capture soluble FN, causing exposure of its integrin-binding site and allowing access of bacteria to host cell surface integrin, which leads to bacteria attachment and invasion of host cells (9 –11). These bacterial cell surface proteins include FNBPA of Staphylococcus aureus, SfbI of Streptococcus pyogenes, and BBK32 of Borrelia burgdorferi. Such proteins contain a disordered FN binding region that engages the FN70K domain comprising the N-terminal 1–9FNI and 1–2FNII modules of FN (Fig. 1A) [12, 13]. SFS lacks a sequence for anchoring to the cell surface [19], the VOLUME 290 NUMBER 8 FEBRUARY 20, 2015
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