Streptokinase triggers conformational activation of plasminogen through specific interactions of the amino-terminal sequence and stabilizes the active zymogen conformation.

Cleavage of Arg(561)-Val(562) in plasminogen (Pg) generates plasmin (Pm) through a classical activation mechanism triggered by an insertion of the new amino terminus into a binding pocket in the Pg catalytic domain. Streptokinase (SK) circumvents this process and activates Pg through a unique nonproteolytic mechanism postulated to be initiated by the intrusion of Ile(1) of SK in place of Val(562). This hypothesis was evaluated in equilibrium binding and kinetic studies of Pg activation with an SK mutant lacking Ile(1) (SK(2--414)). SK(2--414) retained the affinity of native SK for fluorescein-labeled [Lys]Pg and [Lys]Pm but induced no detectable conformational activation of Pg. The activity of SK(2--414) was partially restored by the peptides SK(1--2), SK(1--5), SK(1--10), and SK(1--15), whereas Pg(562--569) peptides were much less effective. Active site-specific fluorescence labeling demonstrated directly that the active catalytic site was formed on the Pg zymogen by the combination of SK(1--10) and SK(2--414), whereas sequence-scrambled SK(1-10) was inactive. The characterization of SK(1--10) containing single Ala substitutions demonstrated the sequence specificity of the interaction. SK(1--10) did not restore activity to the further truncated mutant SK(55-414), which was correlated with the loss of binding affinity of SK(55--414) for labeled [Lys]Pm but not for [Lys]Pg. The studies support a mechanism for conformational activation in which the insertion of Ile(1) of SK into the Pg amino-terminal binding cleft occurs through sequence-specific interactions of the first 10 SK residues. This event and the preferentially higher affinity of SK(2--414) for the activated proteinase domain of Pm are thought to function cooperatively to trigger the conformational change and stabilize the active zymogen conformation.

Streptokinase binds preferentially to the extended conformation of plasminogen through lysine binding site and catalytic domain interactions.

Binding of streptokinase (SK) to plasminogen (Pg) activates the zymogen conformationally and initiates its conversion into the fibrinolytic proteinase, plasmin (Pm). Equilibrium binding studies of SK interactions with a homologous series of catalytic site-labeled fluorescent Pg and Pm analogues were performed to resolve the contributions of lysine binding site interactions, associated changes between extended and compact conformations of Pg, and activation of the proteinase domain to the affinity for SK. SK bound to fluorescein-labeled [Glu]Pg(1) and [Lys]Pg(1) with dissociation constants of 624 +/- 112 and 38 +/- 5 nM, respectively, whereas labeled [Lys]Pm(1) bound with a 57000-fold tighter dissociation constant of 11 +/- 2 pM. Saturation of lysine binding sites with 6-aminohexanoic acid had no effect on SK binding to labeled [Glu]Pg(1), but weakened binding to labeled [Lys]Pg(1) and [Lys]Pm(1) 31- and 20-fold, respectively. At low Cl(-) concentrations, where [Glu]Pg assumes the extended conformation without occupation of lysine binding sites, a 23-fold increase in the affinity of SK for labeled [Glu]Pg(1) was observed, which was quantitatively accounted for by expression of new lysine binding site interactions. The results support the conclusion that the SK affinity for the fluorescent Pg and Pm analogues is enhanced 13-16-fold by conversion of labeled [Glu]Pg to the extended conformation of the [Lys]Pg derivative as a result of lysine binding site interactions, and is enhanced 3100-3500-fold further by the increased affinity of SK for the activated proteinase domain. The results imply that binding of SK to [Glu]Pg results in transition of [Glu]Pg to an extended conformation in an early event in the SK activation mechanism.

Thrombin inhibitor design: X-ray and solution studies provide a novel P1 determinant.

The crystal structures of proflavin and 6-fluorotryptamine thrombin have been completed showing binding of both ligands at the active site S1 pocket. The structure of proflavin:thrombin was confirmatory, while the structure of 6-fluorotryptamine indicated a novel binding mode at the thrombin active site. Furthermore, speculation that the sodium atom identified in an extended solvent channel beneath the S pocket may play a role in binding of these ligands was investigated by direct proflavin titrations as well as chromogenic activity measurements as a function of sodium concentration at constant ionic strength. These results suggested a linkage between the sodium site and the S1 pocket. This observation could be due to a simple ionic interaction between Asp189 and the sodium ion or a more complicated structural rearrangement of the thrombin S1 pocket. Finally, the unique binding mode of 6-fluorotryptamine provides ideas toward the design of a neutrally charged thrombin inhibitor.

Streptokinase binds to human plasmin with high affinity, perturbs the plasmin active site, and induces expression of a substrate recognition exosite for plasminogen.

Binding of streptokinase (SK) to plasminogen (Pg) conformationally activates the zymogen and converts both Pg and plasmin (Pm) into specific Pg activators. The interaction of SK with Pm and its relationship to the mechanism of Pg activation were evaluated in equilibrium binding studies with active site-labeled fluorescent Pm derivatives and in kinetic studies of SK-induced changes in the catalytic specificity of Pm. SK bound to fluorescein-labeled and native Pm with dissociation constants of 11 +/- 2 pm and 12 +/- 4 pm, which represented a 1,000-10,000-fold higher affinity than determined for Pg. Stoichiometric binding of SK to native Pm was followed by generation of a two-fragment form of SK cleaved at Lys(59) (SK'), which exhibited an indistinguishable affinity for labeled Pm, while a truncated, SK(55-414) species had a 120-360-fold reduced affinity. Binding of SK to native Pm was accompanied by a >50-fold enhancement in specificity for activation of Pg, which was paralleled by a surprising 2.6-10-fold loss of specificity of Pm for 8 of 11 tripeptide-pNA substrates. Further studies with Pm labeled at the active site with 2-anilinonaphthalene-6-sulfonic acid demonstrated directly that binding of SK to Pm resulted in expression of a new substrate binding exosite for Pg on the SK.Pm complex. It is concluded that SK activates Pg in part by preferential binding to the active zymogen conformation. High affinity binding of SK to Pm enhances Pg substrate specificity principally through emergence of a substrate recognition exosite.

Comparison of the active-site conformations of bovine alpha-thrombin and meizothrombin(desF1) by electron spin resonance.

The active site of the prothrombin activation intermediate meizothrombin(desF1) was probed using several fluorosulfonylphenyl spin labels specific for the active serine hydroxyl of serine proteases. The mobilities of the thrombin species inhibited with the nitroxide spin labels m-IV [4-(2,2,6,6-tetramethyl-piperidine-1-oxyl) -m-(fluorosulfonyl)benzamide] and m-V [3-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl) -m-(fluorosulfonyl)benzamide], which are sensitive to differences between alpha- and gamma-thrombin, were quite similar to that of alpha-thrombin. That is, no major conformational differences between meizothrombin(desF1) and alpha-thrombin were observed in this region of the extended active site. On the other hand, p-IV [4-(2,2,6,6-tetramethyl piperidine-1-oxyl)-p-(fluorosulfonyl)benzamide], p-V [3-(2,2,5,5-tetramethylpyrrolidine-1-oxyl) -p-(fluorosulfonyl)benzamide], and m-VII [N-[m- (fluorosulfonyl)phenyl]-4-N-(2,2,6,6-tetramethyl- piperidine-1-oxyl)urea], which probe an apolar binding region of bovine thrombin, exhibited large differences in mobility between alpha-thrombin and meizothrombin(desF1). The conformational consequences of indole binding to spin-labeled thrombin species demonstrated that both species also possess an indole-binding site. However, the nitroxide mobility changes upon indole binding to the spin-labeled protein derivative were somewhat different between the two thrombin species under study. In addition, the effects of the benzamidine binding were quite similar for each labeled protein. Thus is appears that, while both species posses a fully functional active site, the region in meizothrombin(desF1) probed by spin labels p-IV, p-V, and m-VII, which corresponds to the apolar binding region, differs in conformation from alpha-thrombin.