The Tandem β-Zipper Model Defines High Affinity Fibronectin-binding Repeats within Staphylococcus aureus FnBPA

Abstract

Binding of the fibronectin-binding protein FnBPA fromStaphylococcus aureus to the human protein fibronectin has previously been implicated in the development of infective endocarditis, specifically in the processes of platelet activation and invasion of the endothelium. We recently proposed a model for binding of fibronectin to FnBPA in which the bacterial protein contains 11 potential binding sites (FnBPA-1 to FnBPA-11), each composed of motifs that bind to consecutive fibronectin type 1 modules in the N-terminal domain of fibronectin. Here we show that six of the 11 sites bind with dissociation constants in the nanomolar range; other sites bind more weakly. The high affinity binding sites include FnBPA-1, the sequence of which had previously been thought to be encompassed by the fibrinogen-binding A domain of FnBPA. Both the number and sequence conservation of the type-1 module binding motifs appears to be important for high affinity binding. The in vivo relevance of the in vitro binding studies is confirmed by the presence of antibodies in patients with S. aureus infections that specifically recognize complexes of these six high affinity repeats with fibronectin.Staphylococcus aureus is one of the most important bacterial pathogens to affect humans. Clinical manifestations of infection range from superficial skin infections (1) to life-threatening conditions, such as endocarditis (2, 3) and difficult to treat infections of the bones and joints (4, 5). The increasing virulence and antibiotic resistance exhibited by this major source of both community and hospital-acquired infection presents an urgent challenge. Furthering our understanding of the mechanism by which staphylococcal pathogenesis occurs is thus imperative for the development of novel therapeutic and preventative strategies.Since attachment to host tissue is a critical early step in infection, one particular group of targets for intervention is the microbial surface components recognizing adhesive matrix molecules (MSCRAMM) 4 family of surface-expressed adhesins (6, 7). These proteins exploit extracellular matrix proteins, such as fibronectin (Fn), using them as a bridge between the bacterial cell surface and host cell receptors that effect downstream signaling (8, 9). Although classically regarded as an exclusively extracellular pathogen, S. aureus has been shown to adhere to and invade several host cell types (10 -14), and the Fn-binding subfamily of MSCRAMMs (FnBPs) appears to be involved in this process (15). There is an emerging view that S. aureus can exist intracellularly, hijacking and invading host cells to establish persistence (16). Conceivably, this mechanism could facilitate rapid and effective bloodstream dissemination while allowing the bacterium to evade antibiotics and host immune surveillance, an apposite theory given the prevalence of bacterial metastasis (3) and infection relapse in staphylococcal disease (17).Fn is a large glycoprotein present in a soluble form in human plasma and other body fluids and in an insoluble form in...

Fibronectin Binds to and Induces Conformational Change in a Disordered Region of Leptospiral Immunoglobulin-like Protein B

“…The ␤-strand-rich conformation formed by LigBCen2NR after binding NTD observed via far UV CD spectroscopy suggests that the binding of LigBCen2NR to NTD might be also mediated by a ␤-zipper interaction. However, the entropy-driven interaction of LigBCen2NR and NTD is distinct from the enthalpy-driven binding of other bacterial proteins known to bind NTD via the ␤-zipper interaction (Table 1) (23,39,46). In addition, we are unable to identify substantial sequence similarity between LigBCen2NR and BBK32 or other Fn-binding proteins, so additional study is needed to further characterize the binding between LigBCen2NR and NTD.…”

“…The addition of NTD promotes the folding of LigBCen2NR from a disordered and extended structure to a folded structure. This finding is notable, since LigBCen2NR is located in the non-immunoglobulin-like region of LigB, as compared with other Fn-binding proteins, such as Staphylococcus aureus FnbpA and FnbpB (23), Streptococcus dysgalactiae FnBB (17), and Streptococcus pyogenes SfbI and SfbII (24). Thus, the binding mode appears to be similar to the known ␤-zipper mechanism but unique in sequence-specific interactions.…”

“…Additional Fn-binding proteins previously shown to be disordered include FNBD-D, the D1-D3 repeat region of FnbpA from S. aereus; FNBD-A and FNBD-B, the A1-A3 and B1-B3 repeat regions of FnbA and FnbB from S. dysgalactiae; and FNBD-P, the P1-P4 repeat regions of Sfb from S. pyogens (20). These Fn-binding regions change their conformation to a folded form upon binding to NTD (20,21,23). Similarly, upon binding to NTD, LigBCen2NR folds into a ␤-strand-rich structure, much like the D123 domain of FnbPA or the N-terminal of BBK32 (21).…”

“…Similarly, upon binding to NTD, LigBCen2NR folds into a ␤-strand-rich structure, much like the D123 domain of FnbPA or the N-terminal of BBK32 (21). High resolution structures of the complex between the B3 region of FnbB and the first and the second type I module of Fn (17,18) and the complex between the first or the fifth Fn binding region of FnbpA and the second to the fifth type I module of Fn (23,39,46) all indicate that a two ␤-strand complex, called a ␤-zipper, mediates those interactions. The binding of Sfb and BBK32 to NTD are also accomplished through the ␤-zipper interaction (23, 39, 46).…”

Turnabout is fair play

“…In contrast to other adhesins, which exclusively bind fibronectin and have evolved to bind this ligand with exceptionally high affinity, [20][21][22] MAM7 seems to bind to fibronectin relatively weakly (K D of 15 mM). This low affinity interaction is complemented by a second receptor, phosphatidic acid, resulting in an overall affinity of MAM7 for host cells that is very high (approx.…”