Abstract
Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-terminal domain (NTD) of fibronectin (Fn), a number of streptococcal FnBPs also contain an upstream region (UR) that is closely associated with an NTD-binding region; UR binds to the adjacent gelatin-binding domain (GBD) of Fn. Previously, UR was shown to be required for efficient streptococcal invasion of epithelial cells. Here we show, using a Streptococcus zooepidemicus FnBP, that the UR-binding site in GBD resides largely in the 8F19F1 module pair. We also show that UR inhibits binding of a peptide from the α1 chain of type I collagen to 8F19F1 and that UR binding to 8F1 is likely to occur through anti-parallel β-zipper formation. Thus, we propose that streptococcal proteins that contain adjacent NTD- and GBD-binding sites form a highly unusual extended tandem β-zipper that spans the two domains and mediates high affinity binding to Fn through a large intermolecular interface. The proximity of the UR- and NTD-binding sequences in streptococcal FnBPs is consistent with a non-linear arrangement of modules in the tertiary structure of the GBD of Fn.
Highlights
Author’s Choice—Final version full access. 1 Both authors contributed to this work. 2 Supported by the British Heart Foundation. 3 Present address: Dept. of Chemical and Biological Sciences, School of
Binding to gelatin-binding domain (GBD) is mediated through a spacer domain ( known as an upstream region (UR)) on SfbI adjacent to and upstream of the FnBR region
Cell invasion assays demonstrated that the SfbI UR and FnBR regions fibronectin binding repeat; UR, upstream region; ESI, electrospray ionization; Isothermal Titration Calorimetry (ITC), isothermal calorimetry; HSQC, heteronuclear single-quantum coherence; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy
Summary
Expression and Purification of Recombinant 8F19F1—Unlabeled and uniformly 15N-labeled 8F19F1 module pair (Fn precursor residues 516 – 608) was expressed in Pichia pastoris using a procedure similar to that described previously [25]. Titrations with 8F19F1 and GBD were carried out in 10 mM NaH2PO4/K2HPO4, pH 7.4, at 25 °C Both the cell and syringe solutions were degassed at 20 °C for 15 min before use. Stirring speed was 307 rpm, and there was a delay of 360 s between injections For both titrations, a separate heat of dilution experiment was performed by injecting peptide into buffer. Hydroxyproline residues in the collagen peptide are indicated with an O. ferences in fluorescence anisotropy (FA) were simultaneously fit for all ment was performed at 37 °C in PBS (pH 7.3) after degassing 8F19F1 concentrations using the formula, both cell and syringe solutions at 30 °C for 15 min. A series of heteronuclear single-quantum coherence (HSQC) experiments with excitation sculpting for solvent suppression and where FA is the fluorescence anisotropy measured at each point, FA,final is the fluorescence anisotropy at saturation of competing ligand (FnZ peptide), [P] is the protein (8F19F1) concentration, [CL] is the concentration of competing ligand, KdCL is the affinity of the competing ligand, and KdBL is the affinity of bound ligand (collagen peptide, 4.5 M)
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