Fibronectin-binding protein A (FnBPA) plays a crucial role in the pathogenesis of Staphylococcus aureus by binding to the host proteins fibronectin (Fn) and fibrinogen (Fg) found in the extracellular matrix and blood plasma, initiating tissue adherence, host cell invasion, and attachment to implanted materials. Although the interactions between FnBPA and host ligands Fn and Fg have been studied extensively using domains or segments of the proteins, the combined regulation of multiple ligands binding to FnBPA is poorly understood, and the underlining molecular mechanism of invasion is still not clear. Here, structure–activity relationships and regulation of FnBPA-dependent host interaction were investigated using a set of biochemical and biophysical techniques. With surface plasmon resonance (SPR) as the primary approach, the binding studies were performed using purified recombinant and native full-length proteins as well as a non-purified system (blood plasma). By capturing full-length FnBPA on a biosensor in the same orientation as on the S. aureus cell wall, SPR binding analysis show that circulating plasma can bind tightly to FnBPA with stronger affinity than that of Fn- and Fg-binding, also Fn and Fg can bind to FnBPA simultaneously. The results demonstrate that Fg binds to full-length FnBPA with 100-fold stronger affinity than previously reported, presumably through unidentified binding sites in both Fg and FnBPA. Furthermore, the structure analysis and binding study indicate that Fg conformation and structural orientation of surface-associated FnBPA are critical for high-affinity interaction between Fg and FnBPA. The proteolytic cleavage of N1 domain and low pH affect the folding of N1 domain and Fg-binding activity of FnBPA, suggesting N1 fold-back conformation is required for fully active Fg-binding. Moreover, the strong interaction between FnBPA and Fn induces conformational changes in Fn and exposes cryptic integrin binding sites, resulting in an enhanced Fn-α5β1 interaction for host cell signaling. In addition, SPR binding analysis indicates that amino acid substitutions, which reflect disease-related polymorphisms found in FnBPA-9, alter the Fn-binding reaction and, in turn, impact the formation and function of the ligand complex within specific pathological contexts. The regulation and biological consequences of FnBPA ligand-binding activities revealed in this study are likely to have important implications for S. aureus interactions with the host in vivo. This study provides mechanistic insights into the FnBPA-dependent ligand binding, activation, and host cell signaling
