Role of the streptokinase alpha-domain in the interactions of streptokinase with plasminogen and plasmin

The role of the streptokinase (SK) alpha-domain in plasminogen (Pg) and plasmin (Pm) interactions was investigated in quantitative binding studies employing active site fluorescein-labeled [Glu]Pg, [Lys]Pg, and [Lys]Pm, and the SK truncation mutants, SK-(55-414), SK-(70-414), and SK-(152-414). Lysine binding site (LBS)-dependent and -independent binding were resolved from the effects of the lysine analog, 6-aminohexanoic acid. The mutants bound indistinguishably, consistent with unfolding of the alpha-domain on deletion of SK-(1-54). The affinity of SK for [Glu]Pg was LBS-independent, and although [Lys]Pg affinity was enhanced 13-fold by LBS interactions, the LBS-independent free energy contributions were indistinguishable. alpha-Domain truncation reduced the affinity of SK for [Glu]Pg 2-7-fold and [Lys]Pg 相似文献   

Resolution of conformational activation in the kinetic mechanism of plasminogen activation by streptokinase

Streptokinase (SK) activates plasminogen (Pg) by specific binding and nonproteolytic expression of the Pg catalytic site, initiating Pg proteolysis to form the fibrinolytic proteinase, plasmin (Pm). The SK-induced conformational activation mechanism was investigated in quantitative kinetic and equilibrium binding studies. Progress curves of Pg activation by SK monitored by chromogenic substrate hydrolysis were parabolic, with initial rates (v(1)) that indicated no transient species and subsequent rate increases (v(2)). The v(1) dependence on SK concentration for [Glu]Pg and [Lys]Pg was hyperbolic with dissociation constants corresponding to those determined in fluorescence-based binding studies for the native Pg species, identifying v(1) as rapid SK binding and conformational activation. Comparison of [Glu]Pg and [Lys]Pg activation showed an approximately 12-fold higher affinity of SK for [Lys]Pg that was lysine-binding site dependent and no such dependence for [Glu]Pg. Stopped-flow kinetics of SK binding to fluorescently labeled Pg demonstrated at least two fast steps in the conformational activation pathway. Characterization of the specificity of the conformationally activated SK.[Lys]Pg* complex for tripeptide-p-nitroanilide substrates demonstrated 5-18- and 10-130-fold reduced specificity (k(cat)/K(m)) compared with SK.Pm and Pm, respectively, with differences in K(m) and k(cat) dependent on the P1 residue. The results support a kinetic mechanism in which SK binding and reversible conformational activation occur in a rapid equilibrium, multistep process.     

Coupling of conformational and proteolytic activation in the kinetic mechanism of plasminogen activation by streptokinase

Binding of streptokinase (SK) to plasminogen (Pg) induces conformational activation of the zymogen and initiates its proteolytic conversion to plasmin (Pm). The mechanism of coupling between conformational activation and Pm formation was investigated in kinetic studies. Parabolic time courses of Pg activation by SK monitored by chromogenic substrate hydrolysis had initial rates (v(1)) representing conformational activation and subsequent rates of activity increase (v(2)) corresponding to the rate of Pm generation determined by a specific discontinuous assay. The v(2) dependence on SK concentration for [Lys]Pg showed a maximum rate at a Pg to SK ratio of approximately 2:1, with inhibition at high SK concentrations. [Glu]Pg and [Lys]Pg activation showed similar kinetic behavior but much slower activation of [Glu]Pg, due to an approximately 12-fold lower affinity for SK and an approximately 20-fold lower k(cat)/K(m). Blocking lysine-binding sites on Pg inhibited SK.Pg* cleavage of [Lys]Pg to a rate comparable with that of [Glu]Pg, whereas [Glu]Pg activation was not significantly affected. The results support a kinetic mechanism in which SK activates Pg conformationally by rapid equilibrium formation of the SK.Pg* complex, followed by intermolecular cleavage of Pg to Pm by SK.Pg* and subsequent cleavage of Pg by SK.Pm. A unified model of SK-induced Pg activation suggests that generation of initial Pm by SK.Pg* acts as a self-limiting triggering mechanism to initiate production of one SK equivalent of SK.Pm, which then converts the remaining free Pg to Pm.     

Rapid-reaction kinetic characterization of the pathway of streptokinase-plasmin catalytic complex formation

Binding of the fibrinolytic proteinase plasmin (Pm) to streptokinase (SK) in a tight stoichiometric complex transforms Pm into a potent proteolytic activator of plasminogen. SK binding to the catalytic domain of Pm, with a dissociation constant of 12 pm, is assisted by SK Lys(414) binding to a Pm kringle, which accounts for a 11-20-fold affinity decrease when Pm lysine binding sites are blocked by 6-aminohexanoic acid (6-AHA) or benzamidine. The pathway of SK.Pm catalytic complex formation was characterized by stopped-flow kinetics of SK and the Lys(414) deletion mutant (SKDeltaK414) binding to Pm labeled at the active site with 5-fluorescein ([5F]FFR-Pm) and the reverse reactions by competitive displacement of [5F]FFR-Pm with active site-blocked Pm. The rate constants for the biexponential fluorescence quenching caused by SK and SKDeltaK414 binding to [5F]FFR-Pm were saturable as a function of SK concentration, reporting encounter complex affinities of 62-110 nm in the absence of lysine analogs and 4900-6500 and 1430-2200 nm in the presence of 6-AHA and benzamidine, respectively. The encounter complex with SKDeltaK414 was approximately 10-fold weaker in the absence of lysine analogs but indistinguishable from that of native SK in the presence of 6-AHA and benzamidine. The studies delineate for the first time the sequence of molecular events in the formation of the SK.Pm catalytic complex and its regulation by kringle ligands. Analysis of the forward and reverse reactions supports a binding mechanism in which SK Lys(414) binding to a Pm kringle accompanies near-diffusion-limited encounter complex formation followed by two slower, tightening conformational changes.     

Rapid Binding of Plasminogen to Streptokinase in a Catalytic Complex Reveals a Three-step Mechanism

Rapid kinetics demonstrate a three-step pathway of streptokinase (SK) binding to plasminogen (Pg), the zymogen of plasmin (Pm). Formation of a fluorescently silent encounter complex is followed by two conformational tightening steps reported by fluorescence quenches. Forward reactions were defined by time courses of biphasic quenching during complex formation between SK or its COOH-terminal Lys⁴¹⁴ deletion mutant (SKΔK414) and active site-labeled [Lys]Pg ([5-(acetamido)fluorescein]-d-Phe-Phe-Arg-[Lys]Pg ([5F]FFR-[Lys]Pg)) and by the SK dependences of the quench rates. Active site-blocked Pm rapidly displaced [5F]FFR-[Lys]Pg from the complex. The encounter and final SK·[5F]FFR-[Lys]Pg complexes were weakened similarly by SK Lys⁴¹⁴ deletion and blocking of lysine-binding sites (LBSs) on Pg kringles with 6-aminohexanoic acid or benzamidine. Forward and reverse rates for both tightening steps were unaffected by 6-aminohexanoic acid, whereas benzamidine released constraints on the first conformational tightening. This indicated that binding of SK Lys⁴¹⁴ to Pg kringle 4 plays a role in recognition of Pg by SK. The substantially lower affinity of the final SK·Pg complex compared with SK·Pm is characterized by a ∼25-fold weaker encounter complex and ∼40-fold faster off-rates for the second conformational step. The results suggest that effective Pg encounter requires SK Lys⁴¹⁴ engagement and significant non-LBS interactions with the protease domain, whereas Pm binding additionally requires contributions of other lysines. This difference may be responsible for the lower affinity of the SK·Pg complex and the expression of a weaker “pro”-exosite for binding of a second Pg in the substrate mode compared with SK·Pm.     

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 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.     

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.     

Full Time Course Kinetics of the Streptokinase-Plasminogen Activation Pathway

Our previously hypothesized mechanism for the pathway of plasminogen (Pg) activation by streptokinase (SK) was tested by the use of full time course kinetics. Three discontinuous chromogenic substrate initial rate assays were developed with different quenching conditions that enabled quantitation of the time courses of Pg depletion, plasmin (Pm) formation, transient formation of the conformationally activated SK·Pg* catalytic complex intermediate, formation of the SK·Pm catalytic complex, and the free concentrations of Pg, Pm, and SK. Analysis of full time courses of Pg activation by five concentrations of SK along with activity-based titrations of SK·Pg* and SK·Pm formation yielded rate and dissociation constants within 2-fold of those determined previously by continuous measurement of parabolic chromogenic substrate hydrolysis and fluorescence-based equilibrium binding. The results obtained with orthogonal assays provide independent support for a mechanism in which the conformationally activated SK·Pg* complex catalyzes an initial cycle of Pg proteolytic conversion to Pm that acts as a trigger. Higher affinity binding of the formed Pm to SK outcompetes Pg binding, terminating the trigger cycle and initiating the bullet catalytic cycle by the SK·Pm complex that converts the residual Pg into Pm. The new assays can be adapted to quantitate SK-Pg activation in the context of SK- or Pg-directed inhibitors, effectors, and SK allelic variants. To support this, we show for the first time with an assay specific for SK·Pg* that fibrinogen forms a ternary SK·Pg*·fibrinogen complex, which assembles with 200-fold enhanced SK·Pg* affinity, signaled by a perturbation of the SK·Pg* active site.     

Plasminogen Substrate Recognition by the Streptokinase-Plasminogen Catalytic Complex Is Facilitated by Arg253, Lys256, and Lys257 in the Streptokinase ��-Domain and Kringle 5 of the Substrate

Streptokinase (SK) conformationally activates the central zymogen of the fibrinolytic system, plasminogen (Pg). The SK·Pg* catalytic complex binds Pg as a specific substrate and cleaves it into plasmin (Pm), which binds SK to form the SK·Pm complex that propagates Pm generation. Catalytic complex formation is dependent on lysine-binding site (LBS) interactions between a Pg/Pm kringle and the SK COOH-terminal Lys⁴¹⁴. Pg substrate recognition is also LBS-dependent, but the kringle and SK structural element(s) responsible have not been identified. SK mutants lacking Lys⁴¹⁴ with Ala substitutions of charged residues in the SK β-domain 250-loop were evaluated in kinetic studies that resolved conformational and proteolytic Pg activation. Activation of [Lys]Pg and mini-Pg (containing only kringle 5 of Pg) by SK with Ala substitutions of Arg²⁵³, Lys²⁵⁶, and Lys²⁵⁷ showed decreases in the bimolecular rate constant for Pm generation, with nearly total inhibition for the SK Lys²⁵⁶/Lys²⁵⁷ double mutant. Binding of bovine Pg (BPg) to the SK·Pm complex containing fluorescently labeled Pm demonstrated LBS-dependent assembly of a SK·labeled Pm·BPg ternary complex, whereas BPg did not bind to the complex containing the SK Lys²⁵⁶/Lys²⁵⁷ mutant. BPg was activated by SK·Pm with a K_m indistinguishable from the K_D for BPg binding to form the ternary complex, whereas the SK Lys²⁵⁶/Lys²⁵⁷ mutant did not support BPg activation. We conclude that SK residues Arg²⁵³, Lys²⁵⁶, and Lys²⁵⁷ mediate Pg substrate recognition through kringle 5 of the [Lys]Pg and mini-Pg substrates. A molecular model of the SK·kringle 5 complex identifies the putative interactions involved in LBS-dependent Pg substrate recognition.Streptokinase (SK)⁶ activates the human fibrinolytic system by activating plasminogen (Pg) through a unique mechanism that is responsible for the use of SK as a thrombolytic drug and its role as a key pathogenicity factor in Group A streptococcal infection (1, 2). The crystal structure of SK bound to the catalytic domain of plasmin (μPm) shows that SK consists of three β-grasp, tightly folded domains, α, β, and γ, linked by flexible segments (3). In solution, SK is highly flexible and behaves hydrodynamically like three beads on a string (4). When bound to μPm, SK assumes a highly ordered structure resembling a three-sided crater surrounding the catalytic site that provides an exosite(s) for binding the catalytic domain of Pg as a substrate (3, 5). In the first step of the SK-mediated Pg activation pathway, SK binds the catalytic domain of the Pg zymogen in a rapid equilibrium process and inserts its NH₂-terminal Ile¹ residue into the NH₂-terminal binding cleft of Pg, activating the catalytic site nonproteolytically (6–10). Although structural proof is lacking, SK Ile¹ presumably forms a critical salt bridge with Asp^740(194) (plasminogen numbering; chymotrypsinogen numbering is in parentheses) that initiates conformational activation of the substrate binding site and oxyanion hole required for proteolytic activity (6, 8–10). The activated SK·Pg* complex binds a second molecule of Pg as a specific substrate and cleaves it at Arg^561(15)-Val^562(16) to form the fibrin-degrading proteinase, plasmin (Pm) (10–14). Proteolytic generation of Pm is propagated by formation of a high affinity SK·Pm complex that converts the remaining free Pg into Pm (5, 11).[Glu]Pg, the full-length form of Pg circulating in blood, consists of an NH₂-terminal PAN (Pg/Apple/Nematode (15, 16)) module, followed by five kringle domains (K1–K5), and the trypsin-like serine proteinase catalytic domain (17). Formation of the SK·Pg* and SK·Pm catalytic complexes and Pg substrate binding are inhibited by the lysine analog, 6-aminohexanoic acid (6-AHA), which binds to lysine-binding sites (LBS) located primarily in kringles K1, K4, and K5 of Pg and Pm (10, 11, 18–23). Cleavage of the Lys⁷⁷-Lys⁷⁸ peptide bond in [Glu]Pg by Pm releases the PAN module and generates the truncated form, [Lys]Pg. Formation of [Lys]Pg is accompanied by a conformational change of [Glu]Pg from a compact, closed α-conformation to a partially extended β-conformation with expression of higher affinity LBS for 6-AHA (24, 25). The fourth kringle module mediates a second conformational change, from the β-conformation to the extended γ-conformation (25).Binding of SK to [Glu]Pg is independent of LBS, with a dissociation constant of 100–150 nm, whereas formation of SK·[Lys]Pg is LBS-dependent with a 13–20-fold higher affinity that is reduced to that of [Glu]Pg by saturating concentrations of 6-AHA (10, 21). Activation of the catalytic domain in [Lys]Pm increases affinity for SK about 830-fold, which is reduced 11–20-fold by 6-AHA (5, 21). Interaction of the COOH-terminal Lys⁴¹⁴ residue of SK with a Pg/Pm kringle domain is responsible for the LBS-dependent enhancement of the affinity of SK·[Lys]Pg* and SK·Pm catalytic complex formation (22). Recent rapid reaction kinetic studies of the SK·Pm binding pathway demonstrated that interaction of Lys⁴¹⁴ with a Pm kringle enhances formation of an initial rapid equilibrium SK·Pm encounter complex, succeeded by two sequential, tightening conformational changes, to achieve an overall dissociation constant of ∼12 pm (26). The Pg/Pm kringle domain responsible for the enhancement of SK·Pg* and SK·Pm complex formation is not known. Productive interaction of Pg as a substrate of the SK·Pg*/Pm complexes is also greatly inhibited by saturating 6-AHA (11). Kinetic and equilibrium binding studies of SK-mediated Pm formation resolved the conformational activation process from the coupled proteolytic generation of Pm (10, 11). The kinetic approach demonstrated that Lys⁴¹⁴ deletion reduced the affinity of formation of the SK·Pg* catalytic complex specifically, whereas the subsequent LBS-dependent proteolytic formation of Pm was unaffected, indicating that Pg substrate recognition is mediated by a structurally distinct region of SK and an unknown kringle (22).Previous structure-function studies have yielded diverse interpretations and conclusions regarding the structural basis of LBS-dependent Pg substrate recognition (23, 27–34). Each of the three domains of SK has been implicated in this regard (29, 30, 35, 36), and binding of two Pg molecules to the residue 1–59 sequence of the α-domain has been reported (36). In particular, segments 16–36, 41–48, 48–59, and 88–97 of the SK α-domain have been concluded to play a role in Pg substrate recognition (32, 33, 37, 38). For several SK mutants, a complex mixture of functional effects on their binding to [Glu]Pg and its conformational and proteolytic activation has been reported (28, 31, 33). Some of these effects may result from the inherent flexibility of SK when bound to Pg or Pm (39), and others may be due to the use of kinetic approaches that do not clearly discriminate between conformational and proteolytic activation.Some observations implicate a protruding hairpin loop called the 250-loop (residues Ala²⁵¹–Ile²⁶⁴) in the SK β-domain in Pg substrate recognition (27, 28, 31, 34). This loop is disordered in the structure of the SK·μPm complex but is ordered in the structure of the isolated β-domain (3, 40). Deletion of the 250-loop, Ala substitution of Lys²⁵⁶ and Lys²⁵⁷ at the apex of the loop, and substitution of multiple residues near and within the loop resulted in disparate effects on K_m and k_cat for [Glu]Pg activation (27, 28, 31). The conclusions of these studies were that Lys²⁵⁶ and Lys²⁵⁷ are involved in SK binding and conformational activation of [Glu]Pg in addition to proteolytic processing of Pg as a substrate. Some of these studies are problematic because the natural NH₂-terminal Ile¹ residue necessary for conformational activation is preceded either by an additional methionine (27, 31) or maltose-binding protein (28) in the recombinant SK species used.Because of the diverse conclusions regarding the functional properties of the 250-loop mutations and the possibility of other potential Pg substrate binding sites, the present studies were undertaken to resolve the function of residues in the 250-loop in LBS-dependent Pg substrate recognition by the SK·Pg* complex. The kringle domain of Pg involved in Pg substrate recognition has not been clearly identified but has been suggested to be K5 (27) on the basis that the isolated β-domain bound Pg (30) and K5 (29) in an LBS-dependent manner. Given the general specificity of Pg kringles for COOH-terminal Lys residues and zwitterionic ligands, such as 6-AHA, and the internal sequence of the 250-loop, it appeared possible that a pseudolysine motif on SK was involved. In the binding of a 30-residue peptide from plasminogen binding Group A streptococcal M-like protein (PAM), VEK-30, to K2 of Pg, Castellino and co-workers (41, 42) showed by crystallography and mutagenesis that residues with cationic (Arg and His) and anionic side chains (Glu) arranged spatially on a helix constituted a pseudolysine structure similar to 6-AHA that binds specifically to the LBS of K2. Additional evidence for pseudolysine structures in Pg binding comes from studies of α-enolase from Streptococcus pneumoniae, which has a 9-residue internal binding site for Pg containing essential basic (two Lys residues) and acidic (Asp and Glu residues) located on a surface loop (43, 44).To determine whether a similar SK structure is involved in [Lys]Pg substrate recognition, anionic and cationic residues in the 250-loop were substituted with Ala and characterized in kinetic studies using methods that resolve conformational and proteolytic activation. Studies with [Lys]Pg and mini-Pg, which contains only K5 and the catalytic domain, showed that Arg²⁵³, Lys²⁵⁶, and Lys²⁵⁷ facilitate LBS-dependent substrate recognition through interactions with K5. The absence of evidence for a pseudolysine structure in the 250-loop is compatible with the established atypical specificity of K5 for cationic ligands, such as benzamidine, N^α-acetyl-Lys-methyl ester, 6-aminohexane, and 5-aminopentane, in addition to zwitterionic ligands (19, 45–47). The studies resolve for the first time the structural features of SK that mediate the LBS-dependent interactions that enhance affinity of SK·Pg* and SK·Pm catalytic complex formation and those that facilitate binding of Pg as a substrate of these complexes.     

Engineering streptokinase for generation of active site-labeled plasminogen analogs

We previously demonstrated that streptokinase (SK) can be used to generate active site-labeled fluorescent analogs of plasminogen (Pg) by virtue of its nonproteolytic activation of the zymogen. The method is versatile and allows stoichiometric and active site-specific incorporation of any one of many molecular probes. The limitation of the labeling approach is that it is both time-consuming and low yield. Here we demonstrate an improved method for the preparation of labeled Pg analogs by the use of an engineered SK mutant fusion protein with both COOH- and NH₂-terminal His₆ tags. The NH₂-terminal tag is followed by a tobacco etch virus proteinase cleavage site to ensure that the SK Ile¹ residue, essential for conformational activation of Pg, is preserved. The SK COOH-terminal Lys⁴¹⁴ residue and residues Arg253–Leu260 in the SK β-domain were deleted to prevent cleavage by plasmin (Pm) and to disable Pg substrate binding to the SK·Pg^∗/Pm catalytic complexes, respectively. Near elimination of Pm generation with the SKΔ(R253–L260)ΔK414–His₆ mutant increased the yield of labeled Pg 2.6-fold and reduced the time required more than 2-fold. The versatility of the labeling method was extended to the application of Pg labeled with a near-infrared probe to quantitate Pg receptors on immune cells by flow cytometry.     

Enhanced plasminogen activation by staphylokinase in the presence of streptokinase beta/betagamma domains: plasminogen kringles play a role

Presence of isolated beta or betagamma domains of streptokinase (SK) increased the catalytic activity of staphylokinase (SAK)-plasmin (Pm) complex up to 60%. In contrast, fusion of SK beta or betagamma domains with the C-terminal end of SAK drastically reduced the catalytic activity of the activator complex. The enhancement effect mediated by beta or betagamma domain on Pg activator activity of SAK-Pm complex was reduced greatly (45%) in the presence of isolated kringles of Pg, whereas, kringles did not change cofactor activity of SAK fusion proteins (carrying beta or betagamma domains) significantly. When catalytic activity of SAK-microPm (catalytic domain of Pm lacking kringle domains) complex was examined in the presence of isolated beta and betagamma domains, no enhancement effect on Pg activation was observed, whereas, enzyme complex formed between microplasmin and SAK fusion proteins (SAKbeta and SAKbetagamma) displayed 50-70% reduction in their catalytic activity. The present study, thus, suggests that the exogenously present beta and betagamma interact with Pg/Pm via kringle domains and elevate catalytic activity of SAK-Pm activator complex resulting in enhanced substrate Pg activation. Fusion of beta or betagamma domains with SAK might alter these intermolecular interactions resulting in attenuated functional activity of SAK.     

Functional roles of streptokinase C-terminal flexible peptide in active site formation and substrate recognition in plasminogen activation

The bacterial protein streptokinase (SK) activates human plasminogen (Pg) into the fibrinolytic protease plasmin (Pm). Roughly 40 residues from the SK C-terminal domain are mobile in the crystal structure of SK complexed with the catalytic domain of Pm, and the functions of this C-tail remain elusive. To better define its roles in Pg activation, we constructed and characterized three C-terminal truncation mutants containing SK residues 1-378, 1-386, and 1-401, respectively. They exhibit gradually reduced amidolytic activity and Pg-activator activity, as well as marginally decreased binding affinity toward Pg, as more of the C-terminus is deleted. As compared with full-length SK, the shortest construct, SK(1-378), exhibits an 80% decrease in amidolytic activity (k(cat)/K(M)), an 80% decrease in Pg-activator activity, and a 30% increase in the dissociation constant toward the Pg catalytic domain. The C-terminal truncation mutations did not attenuate the resistance of the SK-Pm complex to alpha(2)-antiplasmin. Attempts at using a purified C-tail peptide to rescue the activity loss of the truncation mutants failed, suggesting that the integrity of the SK C-terminal peptide is important for the full function of SK.     

Epsilon amino caproic acid inhibits streptokinase-plasminogen activator complex formation and substrate binding through kringle-dependent mechanisms

Lysine side chains induce conformational changes in plasminogen (Pg) that regulate the process of fibrinolysis or blood clot dissolution. A lysine side-chain mimic, epsilon amino caproic acid (EACA), enhances the activation of Pg by urinary-type and tissue-type Pg activators but inhibits Pg activation induced by streptokinase (SK). Our studies of the mechanism of this inhibition revealed that EACA (IC(50) 10 microM) also potently blocked amidolytic activity by SK and Pg at doses nearly 10000-fold lower than that required to inhibit the amidolytic activity of plasmin. Different Pg fragments were used to assess the role of the kringles in mediating the inhibitory effects of EACA: mini-Pg which lacks kringles 1-4 of Glu-Pg and micro-Pg which lacks all kringles and contains only the catalytic domain. SK bound with similar affinities to Glu-Pg (K(A) = 2.3 x 10(9) M(-1)) and to mini-Pg (K(A) = 3.8 x 10(9) M(-)(1)) but with significantly lower affinity to micro-Pg (K(A) = 6 x 10(7) M(-)(1)). EACA potently inhibited the binding of Glu-Pg to SK (K(i) = 5.7 microM), but was less potent (K(i) = 81.1 microM) for inhibiting the binding of mini-Pg to SK and had no significant inhibitory effects on the binding of micro-Pg and SK. In assays simulating substrate binding, EACA also potently inhibited the binding of Glu-Pg to the SK-Glu-Pg activator complex, but had negligible effects on micro-Pg binding. Taken together, these studies indicate that EACA inhibits Pg activation by blocking activator complex formation and substrate binding, through a kringle-dependent mechanism. Thus, in addition to interactions between SK and the protease domain, interactions between SK and the kringle domain(s) play a key role in Pg activation.     

Degradation of streptokinase and the catalytic properties of the plasmin-streptokinase complex

Streptokinase (SK) interacts with human plasminogen (Pg) or plasmin (Pm) with formation of Pg-SK or Pm-SK complex. Pm-SK complex manifests a fibrinolytic, amidolytic and Pg activator activity. SK in complex with Pm isn't stable and so capable to be hydrolysed rapidly. We investigated a correlation between molecular form of SK and catalytic properties of equimolar Pm-SK complex during preincubation at 20 degrees C. It was found out that amidolytic activity of Pm-SK complex was not changing for 5 hours and decreased to the initial Pm value after 24 hours. During this time alpha 2-antiplasmin (alpha 2-AP) has any effect on amidolytic activity of the complex. Fibrinolytic activity of Pm-SK complex makes up 20% of the initial Pm value and wasn't changing within the investigated period. Pg activator activity was decreasing rapidly to 30-40% of the initial one within few minutes from the moment of Pm-SK complex formation. It was 10-20% of that initial after 24 hours. The decrease in Pg activator activity of Pm-SK complex correlated with the initial very rapid conversion of 47 kDa SK to 36 kDa SK within few minutes and following more slow conversion of SK in 31, 25 and 15 kDa fragments after 5 hours. alpha 2-AP didn't influence on the Pg activator activity of Pm-SK complex but eliminated its fibrinolytic activity completely. It was supposed that alpha 2-AP inhibited fibrinolytic activity of Pm-SK complex similarly to 6-aminohexanoic acid by preventing Pm-SK complex binding to fibrin polymer.     

Role of lysine binding sites in activation of plasminogen by streptokinase

The function of lysine-binding sites in kringle domains K1-4 and K5 of plasminogen (Pg) during its activation by streptokinase (SK) was studied. Activation rates of Glu- and Lys-Pg exceed activation rate of mini- and micro-Pg 26 and 40 times, respectively. 6-Animohexanoic acid (6-AHA) in concentrations from 10(-5) to 10(-2) M inhibits activation of Glu-, Lys- and mini-Pg and does not impact the activation of micro-Pg. Complete inhibition of Lys-Pg activation occurs with presence of 10(-3) M 6-AHA while 90% inhibition of mini-Pg activation and 70% inhibition of Glu-Pg activation occur with 10(-2) M 6-AHA. Isolated kringles K1-3 and K4 of Pg inhibit activation of Glu-Pg by SK and concentrations [I]50 are 4.0 and 8.1 x 10(-6) M, respectively. Catalytic activity of Glu-Pg-SK, Lys-Pg-SK and Pm-SK complexes with respect to S 2251 is not inhibited by 6-AHA in concentrations from 10(-5) to 10(-2) M. Activation of substrate Pg by Pm-SK complex is also inhibited by 6-AHA in concentrations from 10(-5) to 10(-2) M; however, this effect of inhibition is significantly weaker than that with activation by SK. Cleavage of C-terminal Lys or chemical modification of NH2-groups of amino acid residues in SK molecule also results in the decrease of the Glu-Pg activation rate. Lysin-binding sites in K1-4 and K5 of Pg molecule are important at different steps of Pg activation process which includes formation of equimolar complex; structural reorganizations resulted in formation of active center in Pg; and binding of substrate Pg with Pg-SK complex. Lysin-binding sites in K1-4 of Pg are necessary for maintenance of high rate of Pg activation by SK.     

Effects of deletion of streptokinase residues 48-59 on plasminogen activation

Streptokinase (SK) is a thrombolytic agent widely used for the clinical treatment of clotting disorders such as heart attack. The treatment is based on the ability of SK to bind plasminogen (Pg) or plasmin (Pm), forming complexes that proteolytically activate other Pg molecules to Pm, which carries out fibrinolysis. SK contains three major domains. The N-terminal domain, SKalpha, provides the complex with substrate recognition towards Pg. SKalpha contains a unique mobile loop, residues 45-70, absent in the corresponding domains of other bacterial Pg activators. To study the roles of this loop, we deleted 12 residues in this loop in both full-length SK and the SKalpha fragment. Kinetic data indicate that this loop participates in the recognition of substrate Pg, but does not function in the active site formation in the activator complex. Two crystal structures of the deletion mutant of SKalpha (SKalpha(delta)) complexed with the protease domain of Pg were determined. While the structure of SKalpha(delta) is essentially the same as this domain in full-length SK, the mode of SK-Pg interaction was however different from a previously observed structure. Even though mutagenesis studies indicated that the current complex represents a minor interacting form in solution, the binding to SKalpha(delta) triggered similar conformational changes in the Pg active site in both crystal forms.     

Structure-function analysis of the streptokinase amino terminus (residues 1-59)

Streptokinase (SK) binds to plasminogen (Pg) to form a complex that converts substrate Pg to plasmin. Residues 1-59 of SK regulate its capacity to induce an active site in bound Pg by a nonproteolytic mechanism and to activate substrate Pg in a fibrin-independent manner. We analyzed 24 SK mutants to better define the functional properties of SK-(1-59). Mutations within the alphabeta1 strand (residues 17-26) of SK completely prevented nonproteolytic active site induction in bound Pg and rendered SK incapable of protecting plasmin from inhibition by alpha2-antiplasmin. However, when fibrin-bound, the activities of alphabeta1 strand mutants were similar to that of wild-type (WT) SK and resistant to alpha2-antiplasmin. Mutation of Ile1 of SK also prevented nonproteolytic active site induction in bound Pg. However, unlike alphabeta1 strand mutants, the functional defect of Ile1 mutants was not relieved by fibrin, and complexes of Ile1 mutants and plasmin were resistant to alpha2-antiplasmin. Plasmin enhanced the activities of alphabeta1 strand and Ile1 mutants, suggesting that SK-plasmin complexes activated mutant SK.Pg complexes by hydrolyzing the Pg Arg561-Val562 bond. Mutational analysis of Glu39 of SK suggested that a salt bridge between Glu39 and Arg719 of Pg is important, but not essential, for nonproteolytic active site induction in Pg. Deleting residues 1-59 rendered SK dependent on plasmin and fibrin to generate plasminogen activator (PA) activity. However, the PA activity of SK-(60-414) in the presence of fibrin was markedly reduced compared with WT SK. Despite its reduced PA activity, the fibrinolytic potency of SK-(60-414) was greater than that of WT SK at higher (but not lower) SK concentrations due to its capacity to deplete plasma Pg. These studies define mechanisms by which the SK alpha domain regulates rapid active site induction in bound Pg, contributes to the resistance of the SK-plasmin complex to alpha2-antiplasmin, and controls fibrin-independent Pg activation.     

Domain interactions between streptokinase and human plasminogen.

Plasmin (Pm), the main fibrinolytic protease in the plasma, is derived from its zymogen plasminogen (Plg) by cleavage of a peptide bond at Arg(561)-Val(562). Streptokinase (SK), a widely used thrombolytic agent, is an efficient activator of human Plg. Both are multiple-domain proteins that form a tight 1:1 complex. The Plg moiety gains catalytic activity, without peptide bond cleavage, allowing the complex to activate other Plg molecules to Pm by conventional proteolysis. We report here studies on the interactions between individual domains of the two proteins and their roles in Plg activation. Individually, all three SK domains activated native Plg. While the SK alpha domain was the most active, its activity was uniquely dependent on the presence of Pm. The SK gamma domain also induced the formation of an active site in Plg(R561A), a mutant that resists proteolytic activation. The alpha and gamma domains together yielded synergistic activity, both in Plg activation and in Plg(R561A) active site formation. However, the synergistic activity of the latter was dependent on the correct N-terminal isoleucine in the alpha domain. Binding studies using surface plasmon resonance indicated that all three domains of SK interact with the Plg catalytic domain and that the beta domain additionally interacts with Plg kringle 5. These results suggest mechanistic steps in SK-mediated Plg activation. In the case of free Plg, complex formation is initiated by the rapid and obligatory interaction between the SK beta domain and Plg kringle 5. After binding of all SK domains to the catalytic domain of Plg, the SK alpha and gamma domains cooperatively induce the formation of an active site within the Plg moiety of the activator complex. Substrate Plg is then recognized by the activator complex through interactions predominately mediated by the SK alpha domain.     

Modifying effect of antiplasminogen monoclonal antibody IV-1c on human plasmin catalytic properties

Antiplasminogen monoclonal antibody IV-1c (IV-1c) binds to Val 709-Gly 718 site of plasminogen (Pg) protease domain, which is far removed from the active site. Pg-IV-1c complex formation induces catalytic activity in proenzymes active site. Influence of IV-1c binding to plasmin (Pm) on Pm catalytic properties has not been investigated yet. Data on catalytic properties of Pm in equimolar Pm-IV-1c complex are presented. It was found that Pm and mini-Pm amidolytic and caseinolytic activity was twice as high as in Pm-IV-1c and mini-Pm-IV-1c complexes. 20 mM 6-AHA and 100 mM arginine did not influence this rise. The increase of amidolytic activity is connected with reduction of K(m) of S 2251 hydrolysis reaction for Pm and mini-Pm from 0.125 and 0.43 to 0.05 and 0.23 mM, correspondingly. Kcat remains almost the same. Fibrinolytic and fibrinogenolytic activity of Pm in Pm-IV-1c complex decreased to 20% of initial value alpha 2-Antiplasmin inhibited Pm activity in complex Pm-IV-1c by 80%. Pm-IV-1c complex did not activate free Pg, but activated equimolar Pg-IV-1c complex. Affinity of IV-1c to Pm and Pg was the same as C50 approximately 1.5 nM. Binding of Pm with IV-1c in a complex: a) leads to increase of Pm active site affinity to LMW substrates; b) causes steric hindrances for fibrin/fibrinogen access to Pm active site; c) proceeds with the same affinity for Pm and Pg, that indicates to invariable Val 709-Gly 718 site conformation after Pg transition in Pm.