Streptococcus pyogenes fibronectin-binding Protein (SfbX), expressed by emm12 and emm49 strains of S. pyogenes, binds fibronectin through COOH-terminal interactions of the protein.1 SfbX NH2-terminal residues 54–229 share 21% identity to staphylocoagulase (SC) residues 107–321 from Tager 104 strain, which increases to 35% homology when conservative substitutions are considered. SC is a non-proteolytic activator of the central coagulation zymogen prothrombin (ProT). Our structure of the prethrombin 2 complex with a fully active SC fragment (SC(1-325)) showed that SC(1-325) consisted of two α-helical bundle domains and that the NH2-terminal dipeptide is critical for ProT activation via the molecular sexuality mechanism.2 In this mechanism, SC inserts its NH2-terminal Ile1 into the Asp194 pocket of the ProT catalytic domain, inducing conformational activation of the catalytic site. On the basis of its homology, SfbX has been postulated to be a member of the SC family of zymogen activator and adhesion proteins. Plasmids encoding a His6-tagged SfbX(1-312) tobacco etch virus proteinase-cleavable fusion protein and the viral proteinase were co-transformed into E. coli to enable generation of the native SfbX NH2-terminus (Ile-Ser-Asn) during purification. SfbX(1-312) was purified by affinity chromatography on ProT-Affigel and Ni2+-iminodiacetic acid-Sepharose. Active site-specific fluorescent probe labeling of a mixture of ProT and SfbX(1-312) showed covalent labeling of the ProT zymogen, demonstrating that SfbX(1-312) is a non-proteolytic activator of ProT. Incubation of ProT with the NH2-terminally blocked SfbX(1-312) fusion protein did not result in labeling of the active site, indicating that the native NH2-terminus is required for activation, and suggesting that SfbX(1-312) activates ProT through the molecular sexuality mechanism. In ProT activation assays measured by the appearance of chromogenic substrate activity, SfbX(1-312) activated ProT weakly and was more effective in activating prethrombin 1, lacking the fragment 1 domain of ProT. Preliminary binding studies using ProT and thrombin labeled at the catalytic site with fluorescence probes demonstrated binding of SfbX(1-312). SfbX(1-312) decreased thrombin activity toward D-Phe-Pip-Arg-pNA by 75%, with an apparent KD of ~20 nM, indicating that the thrombin catalytic site is perturbed by SfbX binding. Clotting assays showed that neither SfbX(1-312) nor a mixture of SfbX(1-312) and prethrombin 1 clotted human fibrinogen or plasma. Addition of 5 μM SfbX(1-312) to a factor V-dependent plasma clotting assay increased clotting times, indicating that SfbX(1-312) has an inhibitory effect. We conclude that SfbX(1-312) activates ProT conformationally, possibly through the molecular sexuality mechanism. SfbX(1-312) binds tightly to thrombin, perturbing its catalytic site, but does not clot human fibrinogen or plasma. Further studies are needed to determine whether ProT is the pathophysiological target zymogen of SfbX(1-312), to delineate the natural substrate of the SfbX(1-312)·ProT complex, and to elucidate the role of SfbX in the pathology of S. pyogenes infection.
Disclosure: No relevant conflicts of interest to declare.