The Streptococcal Binding Site in the Gelatin-binding Domain of Fibronectin Is Consistent with a Non-linear Arrangement of Modules

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.

Borrelia burgdorferi Protein BBK32 Binds to Soluble Fibronectin via the N-terminal 70-kDa Region, Causing Fibronectin to Undergo Conformational Extension

“…Samples were prepared by dissolving uniformly 15 N-labeled 8 -9 FNI to a concentration of 0.2 and 0.85 mM, for binding studies and 8 -9 FNI (BBK32EN at 6.0 molar eq). Assignments for unbound 8 -9 FNI had been determined previously (17). Crystallography-A crystal of [2][3] FNI-BBK32TwL (0.6 mM/ 6.3 mM) was grown in 1.4 M sodium/potassium phosphate, pH 6.9, using sitting drop vapor diffusion.…”

“…Expression and purification of polyhistidine-tagged monomeric [1][2][3][4][5] FNI, N-3 FNIII, [6][7][8][9] FNI, [7][8][9] FNI, 7 FNI-1 FNIII, [1][2][3][4][5][6][7][8][9][10][11][12][13][14] FNIII, and 2-14 FNIII and dimeric 6 FNI-C, 1 FNIII-C, and 2 FNIII-C were accomplished using recombinant baculovirus and affinity chromatography as described previously (27)(28)(29). Expression and purification of unlabeled [2][3] FNI and 8 -9 FNI, and of uniformly 15 N-labeled 8 -9 FNI, were performed as described previously (13,17). Proteolytic FN70K was prepared as described previously (30).…”

“…These experiments were carried out as described previously (15,16) except that in enzyme-linked assays 20 mM Tris, 100 mM NaCl, pH 7.4, buffer was used, and in ITC experiments sodium phosphate buffer with 100 mM NaCl was used. ITC to study the interaction between BBK32EN and 8 -9 FNI and a con-trol experiment to determine the heat of dilution were performed as described previously (17).…”

“…Antibody inhibition studies indicate that the ␤-zipper formed by FUD may extend to encompass 9 FNI and the linker region between 9 FNI and 1 FNIII (16). Other studies have demonstrated that a peptide from FnBB of Streptococcus dysgalactiae uses the tandem ␤-zipper mechanism to bind to 1-2 FNI (13), as do FnZ (residues 370 -391) (17) and type I collagen (residues 778 -799) in binding to 8 FNI (18) (see Fig. 1).…”

“…Although FNBRs bind [1][2][3][4][5] FNI or [2][3][4][5] FNI, the "functional upstream domain" (FUD) comprising residues 423-471 of SfbI (16) and UR-FnZ-2 comprising residues 370 -428 of FnZ from Streptococcus equi subsp. zooepidemicus (17) bind to an extended site comprising modules [2][3][4][5] FNI and 8 FNI in a proposed extended tandem ␤-zipper (see Fig. 1).…”