ANALYSIS OF THE INTERACTIONS BETWEEN STREPTOKINASE DOMAINS AND HUMAN PLASMINOGEN

The contrasting roles of streptokinase (SK) domains in binding human G lu1-plasminogen (Plg) have been studied using a set of proteolytic fra gments, each of which encompasses one or more of SK's three structural domains (A, B, C). Direct binding experiments have been performed usi ng gel filtration chromatography and surface plasmon resonance. The la tter technique has allowed estimation of association and dissociation rate constants for interactions between Plg and intact SR or SK fragme nts. Each of the SR fragments that contains domain B (fragments A2-B-C , A2-B, B-C, and B) binds Plg with similar affinity, at a level approx imately 100- to 1,000-fold lower than intact SK. Experiments using 10 mM 6-aminohexanoic acid or 50 mM benzamidine demonstrate that either o f these two lysine analogues abolishes interaction of domain B with Pl g. Isolated domain C does not show detectable binding to Plg. Moreover , the additional presence of domain C within other SK fragments (B-C a nd A2-B-C) does not alter significantly their affinities for Plg. In a ddition, Plg-binding by a noncovalent complex of two SK fragments that contains domains A and B is similar to that of domain B. By contrast, species containing domain B and both domains A and C (intact SR and t he two-chain complex A1.A2-B-C) show a significantly higher affinity f or Plg, which could not be completely inhibited by saturating amounts of 6-AHA. These results show that SK domain B interacts with Plg in a lysine-dependent manner and that although domains A and C do not appea r independently to possess affinity for Plg, they function cooperative ly to establish the additional interactions with Plg to form an effici ent native-like Plg activator complex.