Analysis of the interactions between streptokinase domains and human plasminogen

Abstract

The contrasting roles of streptokinase (SK) domains in binding human Glul-plasminogen (Plg) have been studied using a set of proteolytic fragments, each of which encompasses one or more of SK's three structural domains (A, B, C). Direct binding experiments have been performed using gel filtration chromatography and surface plasmon resonance. The latter technique has allowed estimation of association and dissociation rate constants for interactions between Plg and intact SK or SK fragments. Each of the SK fragments that contains domain B (fragments A2-B-C, A2-B, B-C, and B) binds Plg with similar affinity, at a level approximately 100-to 1,000-fold lower than intact SK. Experiments using 10 mM 6-aminohexanoic acid or 50 mM benzamidine demonstrate that either of these two lysine analogues abolishes interaction of domain B with Plg. Isolated domain C does not show detectable binding to Plg. Moreover, the additional presence of domain C within other SK fragments (B-C and A2-B-C) does not alter significantly their affinities for Plg. In addition, 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 SK and the two-chain complex AI . A2-B-C) show a significantly higher affinity for 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 appear independently to possess affinity for Plg, they function cooperatively to establish the additional interactions with Plg to form an efficient native-like Plg activator complex.Keywords: fibrinolysis; lysine-binding site; plasminogen; serine protease; streptokinase; zymogen activation Streptococcus equisimilis streptokinase (SK) is a single-chain secreted protein of 414 amino acids (Malke et al., 1985), which indirectly causes the activation of the blood plasma zymogen human plasminogen (Plg) to the fibrinolytic enzyme plasmin (Christensen, 1945) via active-site formation within Plg. This proceeds without proteolytic cleavage by means of a rapid and avid binding

Involvement of a Nine-residue Loop of Streptokinase in the Generation of Macromolecular Substrate Specificity by the Activator Complex through Interaction with Substrate Kringle Domains

“…In binary interaction studies, it was evident that when soluble nSK/SK del254 -262 was added to immobilized HPG, a rapid and avid SK⅐HPG binary complex formation occurs. The dissociation of this complex is very slow due to the high stability of the SK⅐HPG complex, as has been observed by others also (15). After allowing the complex to dissociate maximally (ϳ20 min), the dissociation base line becomes stable, which remains unaffected even after washing with 2.5 mM EACA.…”

“…Interestingly, experiments performed by immobilizing nSK/ SK del254 -262 on carboxymethyldextran cuvettes using standard amino-coupling procedures, as shown by other groups also (15,33), yielded similar binding constants for nSK and SK del254 -262 (data not shown), indicating that the coupling procedure per se does not interfere significantly in the binding interaction between SK and HPG.…”

“…It is quite intriguing that the ␤ domain provides a major portion of the intermolecular affinity of SK for HPG necessary for the formation of the tightly held equimolar activator complex (15) and at least some of the affinity required for the (transient) interaction of the latter with substrate HPG, as the present study indicates. This arouses curiosity as to whether this domain would, by itself, possess HPG activator activity, however compromised at a quantitative level, since two seemingly fundamental requirements for a single-domain HPG activator protein (viz.…”

“…However, a clear cut identification of a structural epitope/element in conferring substrate HPG affinity onto the SK⅐PG activator complex has not been demonstrated until now. Of the three domains of SK, the central ␤ domain displays maximal affinity for HPG (15), the N-terminal ␣ domain displays relatively lesser affinity for HPG with the ␥ domain showing much less affinity of for HPG. 2 Solution and structural studies suggest that both ␣ and ␤ domains are involved in the substrate recognition phenomenon (8,12).…”

Resolution of Conformational Activation in the Kinetic Mechanism of Plasminogen Activation by Streptokinase

“…A number of studies have examined the binding interactions by various experimental approaches but have resulted in a very broad range of dissociation constants, varying from 28 pM to 220 nM for [Glu]Pg (25)(26)(27)(28)(29)(30)(31)(32), for example, where the higher affinities may result from Pm generation in SK/Pg mixtures. Our equilibrium binding studies have employed active site-labeled fluorescent Pg and Pm analogs to quantitate SK binding in the absence of proteolytic reactions (21,28,33,34).…”