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   

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.     

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.     

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.     

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.     

Plasminogen binding with decapeptide and polypeptide fragments of streptokinase

The plasminogen binding with streptokinase decapeptides, modeling the primary structure of molecule, and chymotryptic fragments of streptokinase have been investigated. The immunoenzymatic assay has shown that plasminogen binds to all streptokinase fragments with the decreasing affinity in the set of fragments: 36 > 30 > 17 > 7 > 11 kDa. Location of the binding sites in streptokinase primary structure was performed using the immobilized decapeptides on plastic pins adopted to IEA. In the presence of 10 mM 6-aminohexanoic acid 11 sites for human Glu- and mini-plasminogens, pig and bovine plasminogens binding have been found. They were of the same location for human, bovine and pig plasminogens. 3 sites were located in plasminogen alpha-domain--T43-A72, N113-T126, Q133-V158, 5 sites in beta-domain--T163-L188, A203-S222, Q239-I264, Y275-L294, T315-L340, and 3 sites in gamma-domain--T361-R362, N377-E392, T397-N410. Participation of linear part of streptokinase polypeptide chain in plasminogen--streptokinase complex formation is suggested.     

Features of plasminogen activation in a complex with antiplasminogen monoclonal antibody IV-1C

Antiplasminogen monoclonal antibody IV-1c (IV-1c) with antigenic determinant in V709-G718 site of plasminogen (Pg) protease domain (Druzhina N.N. et al. 1996.) can induce catalytic activity in Pg moiety of the complex. Catalytic activity appeared in Pg-IV-1c complex after approximately 2 h lag-period. Rate of Lys-Pg activation was higher then that of Glu-Pg. Amidolytic activity of Pg-SK equimolar complex was completely inhibited by IV-1c at 2:1 = Pg:IV-1c molar ratio. At constant Glu-Pg concentration increasing of the IV-1c concentration to equimolar of Pg accelerated Pg activation. Subsequent increase of IV-1c concentration inhibited the Pg activation sharply. Increasing of Glu-Pg concentration at constant IV-1c one did not inhibit Glu-Pg activation in Pg-IV-1c complex. The rate dependence of Pg activation from Glu-Pg-IV-1c complex concentration curve had bell-shaped form with maximum at 500 nM. Electrophoretic analysis of components of Glu-Pg-IV-1c complex showed that Lys-Pg and Lys-Pm were not observed at 100 nM complex concentration for 6 h period of reaction. At 680 nM concentration Glu-Pg-IV-1c complex these forms appeared in initial moments of reaction activation after lag-period. Kinetic scheme and peculiarities of Pg activation reaction in Pg-IV-1c complex are discussed.     

Binding of the COOH-terminal lysine residue of streptokinase to plasmin(ogen) kringles enhances formation of the streptokinase.plasmin(ogen) catalytic complexes

Streptokinase (SK) activates human fibrinolysis by inducing non-proteolytic activation of the serine proteinase zymogen, plasminogen (Pg), in the SK.Pg* catalytic complex. SK.Pg* proteolytically activates Pg to plasmin (Pm). SK-induced Pg activation is enhanced by lysine-binding site (LBS) interactions with kringles on Pg and Pm, as evidenced by inhibition of the reactions by the lysine analogue, 6-aminohexanoic acid. Equilibrium binding analysis and [Lys]Pg activation kinetics with wild-type SK, carboxypeptidase B-treated SK, and a COOH-terminal Lys414 deletion mutant (SKDeltaK414) demonstrated a critical role for Lys414 in the enhancement of [Lys]Pg and [Lys]Pm binding and conformational [Lys]Pg activation. The LBS-independent affinity of SK for [Glu]Pg was unaffected by deletion of Lys414. By contrast, removal of SK Lys414 caused 19- and 14-fold decreases in SK affinity for [Lys]Pg and [Lys]Pm binding in the catalytic mode, respectively. In kinetic studies of the coupled conformational and proteolytic activation of [Lys]Pg, SKDeltaK414 exhibited a corresponding 17-fold affinity decrease for formation of the SKDeltaK414.[Lys]Pg* complex. SKDeltaK414 binding to [Lys]Pg and [Lys]Pm and conformational [Lys]Pg activation were LBS-independent, whereas [Lys]Pg substrate binding and proteolytic [Lys]Pm generation remained LBS-dependent. We conclude that binding of SK Lys414 to [Lys]Pg and [Lys]Pm kringles enhances SK.[Lys]Pg* and SK.[Lys]Pm catalytic complex formation. This interaction is distinct structurally and functionally from LBS-dependent Pg substrate recognition by these complexes.     

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.     

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.     

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.     

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.     

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.     

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.     

Binding and activation of plasminogen on immobilized immunoglobulin G. Identification of the plasmin-derived Fab as the plasminogen-binding fragment

We have found that tissue plasminogen activator catalyzes the binding of plasminogen (Pg) to immunoglobulin G (IgG) immobilized on a surface. This enhancement is due to the formation of plasmin, since plasmin treatment of immobilized IgG produced a 20-fold increase in Pg binding. Pg binding is lysine site dependent and reversible. The augmentation of Pg binding by plasmin is specific as other proteases produced significantly less or no effect. Immobilized plasmin-treated IgG also specifically binds Pg in plasma. IgG-immobilized Pg is activated by tissue plasminogen activator, and a significant portion of the plasmin formed remains bound to the IgG. The Pg reactive species in a plasmin-treated IgG digest was identified as the Fab fragment by chromatography utilizing the immobilized high affinity lysine-binding site of plasminogen. Specificity of the interaction was further demonstrated by immunoblot-ligand analysis which demonstrated that the plasmin-derived Fab fragment bound Pg whereas papain-derived Fab or plasmin-derived Fc fragments did not. These data suggest that Pg binds to the new COOH-terminal lysine residue of the plasmin-derived Fab. Pg also binds to an immobilized immune complex following plasmin treatment. These findings indicate that surface-bound IgG localizes plasminogen thus extending the spectrum of activity of the plasmin system to immunologic reactions.     

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.     

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.     

Enhancement through mutagenesis of the binding of the isolated kringle 2 domain of human plasminogen to omega-amino acid ligands and to an internal sequence of a Streptococcal surface protein.

In the background of the recombinant K2 module of human plasminogen (K2(Pg)), a triple mutant, K2(Pg)[C4G/E56D/L72Y], was generated and expressed in Pichia pastoris cells in yields exceeding 100 mg/liter. The binding affinities of a series of lysine analogs, viz. 4-aminobutyric acid, 5-aminopentanoic acid, epsilon-aminocaproic acid, 7-aminoheptanoic acid, and t-4-aminomethylcyclohexane-1-carboxylic acid, to this mutant were measured and showed up to a 15-fold tighter interaction, as compared with wild-type K2(Pg) (K2(Pg)[C4G]). The variant, K2(Pg)[C4G/E56D], afforded up to a 4-fold increase in the binding affinity to these same ligands, whereas the K2(Pg)[C4G/L72Y] mutant decreased the same affinities up to 5-fold, as compared with K2(Pg)[C4G]. The thermal stability of K2(Pg)[C4G/E56D/L72Y] was increased by approximately 13 degrees C, as compared with K2(Pg)[C4G]. The functional consequence of up-regulating the lysine binding property of K2(Pg) was explored, as reflected by its ability to interact with an internal sequence of a plasminogen-binding protein (PAM) on the surface of group A streptococci. A 30-mer peptide of PAM, containing its K2(Pg)-specific binding region, was synthesized, and its binding to each mutant of K2(Pg) was assessed. Only a slight enhancement in peptide binding was observed for K2(Pg)[C4G/E56D], compared with K2(Pg)[C4G] (K(d) = 460 nM). A 5-fold decrease in binding affinity was observed for K2(Pg)[C4G/L72Y] (K(d) = 2200 nM). However, a 12-fold enhancement in binding to this peptide was observed for K2(Pg)[C4G/E56D/L72Y] (K(d) = 37 nM). Results of these PAM peptide binding studies parallel results of omega-amino acid binding to these K2(Pg) mutants, indicating that the high affinity PAM binding by plasminogen, mediated exclusively through K2(Pg), occurs through its lysine-binding site. This conclusion is supported by the 100-fold decrease in PAM peptide binding to K2(Pg)[C4G/E56D/L72Y] in the presence of 50 mM 6-aminohexanoic acid. Finally, a thermodynamic analysis of PAM peptide binding to each of these mutants reveals that the positions Asp(56) and Tyr(72) in the K2(Pg)[C4G/E56D/L72Y] mutant are synergistically coupled in terms of their contribution to the enhancement of PAM peptide binding.     

Lipoprotein a inhibits streptokinase-mediated activation of human plasminogen

Lipoprotein a [Lp(a)] inhibits human plasminogen (Pg) conversion to plasmin (Pm) by streptokinase- (SK-) mediated activation. Kinetic and binding studies indicate that Lp(a) inhibits Pg activation by competitive and uncompetitive inhibition. Lp(a) competes with Pg for SK and forms a stable complex. Lp(a) does not, however, inhibit Pg activation by the proteolytic SK-Pm complex. The SK-Pg and SK-Pg(act) intermediate complexes are possible targets of the Lp(a) uncompetitive inhibition. The competitive inhibition constant (Kic) is 45 nM or 14 mg/dL, and the uncompetitive inhibition constant (Kiu) is 140 nM or 42 mg/dL, corresponding to physiologic and pathophysiologic Lp(a) concentrations, respectively.     

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.