Split intein-mediated backbone cyclization enhances the stability and activity of staphylokinase, a potent fibrin-selective plasminogen activator.

Staphylokinase (Sak), a small 15 kDa globular protein that is secreted by certain strains of Staphylococcus aureus, shows a potent fibrin-selective thrombolytic activity. Earlier work has shown that Sak could potentially become a low-cost alternative to currently used thrombolytic agents, such as tissue plasminogen activator (tPA). In attempts to improve its potential for clinical applications, numerous modifications of Sak have already been investigated. Here, we have characterized a novel Sak modification, cyclized Sak (cyc-Sak), which was prepared through split-intein mediated protein backbone cyclization. We have characterized the structure, stability and the activity of cyc-Sak using biophysical techniques, limited proteolysis studies and plasminogen (PG)-activation assays. Our results show that cyc-Sak possesses an identical structure, enhanced stability, resistance to proteolysis by exoproteases and improved PG-activation properties compared to its linear counterpart. It can be over-expressed with high yield in the cytoplasm of Escherichia coli and is easily purified in a two-step process. The intein-mediated cyclization occurs spontaneously in vivo during protein expression and does not necessitate further modification steps after purification of the protein. Furthermore, covalent Sak cyclization could be readily combined with other Sak modifications previously proposed, to generate an effective thrombolytic agent with lower immunogenicity and improved stability and activity.

C-terminal lysine residues enhance plasminogen activation by inducing conformational flexibility and stabilization of activator complex of staphylokinase with plasmin.

Staphylokinase (SAK), a potent fibrin-specific plasminogen activator secreted by Staphylococcus aureus, carries a pair of lysine at the carboxy-terminus that play a key role in plasminogen activation. The underlaying mechanism by which C-terminal lysins of SAK modulate its function remains unknown. This study has been undertaken to unravel role of C-terminal lysins of SAK in plasminogen activation. While deletion of C-terminal lysins (Lys135, Lys136) drastically impaired plasminogen activation by SAK, addition of lysins enhanced its catalytic activity 2-2.5-fold. Circular dichroism analysis revealed that C-terminally modified mutants of SAK carry significant changes in their beta sheets and secondary structure. Structure models and RING (residue interaction network generation) studies indicated that the deletion of lysins has conferred extensive topological alterations in SAK, disrupting vital interactions at the interface of SAK.plasmin complex, thereby leading significant impairment in its functional activity. In contrast, addition of lysins at the C-terminus enhanced its conformational flexibility, creating a stronger coupling at the interface of SAK.plasmin complex and making it more efficient for plasminogen activation. Taken together, these studies provided new insights on the role of C-terminal lysins in establishment of precise intermolecular interactions of SAK with the plasmin for the optimal function of activator complex.

Yersinia pestis Plasminogen Activator.

The Gram-negative bacterium Yersinia pestis causes plague, a fatal flea-borne anthropozoonosis, which can progress to aerosol-transmitted pneumonia. Y. pestis overcomes the innate immunity of its host thanks to many pathogenicity factors, including plasminogen activator, Pla. This factor is a broad-spectrum outer membrane protease also acting as adhesin and invasin. Y. pestis uses Pla adhesion and proteolytic capacity to manipulate the fibrinolytic cascade and immune system to produce bacteremia necessary for pathogen transmission via fleabite or aerosols. Because of microevolution, Y. pestis invasiveness has increased significantly after a single amino-acid substitution (I259T) in Pla of one of the oldest Y. pestis phylogenetic groups. This mutation caused a better ability to activate plasminogen. In paradox with its fibrinolytic activity, Pla cleaves and inactivates the tissue factor pathway inhibitor (TFPI), a key inhibitor of the coagulation cascade. This function in the plague remains enigmatic. Pla (or pla) had been used as a specific marker of Y. pestis, but its solitary detection is no longer valid as this gene is present in other species of Enterobacteriaceae. Though recovering hosts generate anti-Pla antibodies, Pla is not a good subunit vaccine. However, its deletion increases the safety of attenuated Y. pestis strains, providing a means to generate a safe live plague vaccine.

Oriented Immobilization on Gold Nanoparticles of a Recombinant Therapeutic Zymogen.

Direct immobilization of functional proteins on gold nanoparticles (AuNPs) affects their structure and function. Changes may vary widely and range from strong inhibition to the enhancement of protein function. More often though the outcome of direct protein immobilization results in protein misfolding and the loss of protein activity. Additional complications arise when the protein being immobilized is a zymogen which requires and relies on additional protein-protein interactions to exert its function. Here we describe molecular design of a glutathione-S-transferase-Staphylokinase fusion protein (GST-SAK) and its conjugation to AuNPs. The multivalent AuNP-(GST-SAK)n complexes generated show plasminogen activation activity in vitro. The methods described are transferable and could be adapted for conjugation and functional analysis of other plasminogen activators, thrombolytic preparations or other functional enzymes.

Engineered microparticles and nanoparticles for fibrinolysis.

Fibrinolytic agents including plasmin and plasminogen activators improve outcomes in acute ischemic stroke and thrombosis by recanalizing occluded vessels. In the decades since their introduction into clinical practice, several limitations of have been identified in terms of both efficacy and bleeding risk associated with these agents. Engineered nanoparticles and microparticles address some of these limitations by improving circulation time, reducing inhibition and degradation in circulation, accelerating recanalization, improving targeting to thrombotic occlusions, and reducing off-target effects; however, many particle-based approaches have only been used in preclinical studies to date. This review covers four advances in coupling fibrinolytic agents with engineered particles: (a) modifications of plasminogen activators with macromolecules, (b) encapsulation of plasminogen activators and plasmin in polymer and liposomal particles, (c) triggered release of encapsulated fibrinolytic agents and mechanical disruption of clots with ultrasound, and (d) enhancing targeting with magnetic particles and magnetic fields. Technical challenges for the translation of these approaches to the clinic are discussed.

The ß-domain of streptokinase affects several functionalities, including specific/proteolytic activity kinetics.

Streptokinase (SK) is a plasminogen activator which converts inactive plasminogen (Pg) to active plasmin (Pm), which cleaves fibrin clots. SK secreted by groups A, C, and G Streptococcus (SKA/SKC/SKG) is composed of three domains: SKα, SKß and SKγ. Previous domain-swapping studies between SK1/SK2b-cluster variants revealed that SKß plays a major role in the activation of human Pg. Here, we carried out domain-swapping between skcg-SK/SK2-cluster variants to determine the involvement of SKß in several SK functionalities, including specific/proteolytic activity kinetics, fibrinogen-bound Pg activation and α2 -antiplasmin resistance. Our results indicate that SKß has a minor to determining role in these diverse functionalities for skcg-SK and SK2b variants, which might potentially be accompanied by few critical residues acting as hot spots. Our findings enhance our understanding of the roles of SKß and hot spots in different functional characteristics of SK clusters and may aid in the engineering of fibrin-specific variants of SK for breaking down blood clots with potentially higher efficacy and safety.

Biochemical and structural analyses suggest that plasminogen activators coevolved with their cognate protein substrates and inhibitors.

Protein sequences of members of the plasminogen activation system are present throughout the entire vertebrate phylum. This important and well-described proteolytic cascade is governed by numerous protease-substrate and protease-inhibitor interactions whose conservation is crucial to maintaining unchanged protein function throughout evolution. The pressure to preserve protein-protein interactions may lead to either co-conservation or covariation of binding interfaces. Here, we combined covariation analysis and structure-based prediction to analyze the binding interfaces of urokinase (uPA):plasminogen activator inhibitor-1 (PAI-1) and uPA:plasminogen complexes. We detected correlated variation between the S3-pocket-lining residues of uPA and the P3 residue of both PAI-1 and plasminogen. These residues are known to form numerous polar interactions in the human uPA:PAI-1 Michaelis complex. To test the effect of mutations that correlate with each other and have occurred during mammalian diversification on protein-protein interactions, we produced uPA, PAI-1, and plasminogen from human and zebrafish to represent mammalian and nonmammalian orthologs. Using single amino acid point substitutions in these proteins, we found that the binding interfaces of uPA:plasminogen and uPA:PAI-1 may have coevolved to maintain tight interactions. Moreover, we conclude that although the interaction areas between protease-substrate and protease-inhibitor are shared, the two interactions are mechanistically different. Compared with a protease cleaving its natural substrate, the interaction between a protease and its inhibitor is more complex and involves a more fine-tuned mechanism. Understanding the effects of evolution on specific protein interactions may help further pharmacological interventions of the plasminogen activation system and other proteolytic systems.

Design of a DNA-Programmed Plasminogen Activator.

Although the functional specificity and catalytic versatility of enzymes have been exploited in numerous settings, controlling the spatial and temporal activity of enzymes remains challenging. Here we describe an approach for programming the function of streptokinase (SK), a protein that is clinically used as a blood "clot buster" therapeutic. We show that the fibrinolytic activity resulting from the binding of SK to the plasma proenzyme plasminogen (Pg) can be effectively regulated (turned "OFF" and "ON") by installing an intrasteric regulatory feature using a DNA-linked protease inhibitor modification. We describe the design rationale, synthetic approach, and functional characterization of two generations of intrasterically regulated SK-Pg constructs and demonstrate dose-dependent and sequence-specific temporal control in fibrinolytic activity in response to short predesignated DNA inputs. The studies described establish the feasibility of a new enzyme-programming approach and serves as a step toward advancing a new generation of programmable enzyme therapeutics.

Exploring the potential of a structural alphabet-based tool for mining multiple target conformations and target flexibility insight.

Protein flexibility is often implied in binding with different partners and is essential for protein function. The growing number of macromolecular structures in the Protein Data Bank entries and their redundancy has become a major source of structural knowledge of the protein universe. The analysis of structural variability through available redundant structures of a target, called multiple target conformations (MTC), obtained using experimental or modeling methods and under different biological conditions or different sources is one way to explore protein flexibility. This analysis is essential to improve the understanding of various mechanisms associated with protein target function and flexibility. In this study, we explored structural variability of three biological targets by analyzing different MTC sets associated with these targets. To facilitate the study of these MTC sets, we have developed an efficient tool, SA-conf, dedicated to capturing and linking the amino acid and local structure variability and analyzing the target structural variability space. The advantage of SA-conf is that it could be applied to divers sets composed of MTCs available in the PDB obtained using NMR and crystallography or homology models. This tool could also be applied to analyze MTC sets obtained by dynamics approaches. Our results showed that SA-conf tool is effective to quantify the structural variability of a MTC set and to localize the structural variable positions and regions of the target. By selecting adapted MTC subsets and comparing their variability detected by SA-conf, we highlighted different sources of target flexibility such as induced by binding partner, by mutation and intrinsic flexibility. Our results support the interest to mine available structures associated with a target using to offer valuable insight into target flexibility and interaction mechanisms. The SA-conf executable script, with a set of pre-compiled binaries are available at http://www.mti.univ-paris-diderot.fr/recherche/plateformes/logiciels.

Amorphous protein aggregates stimulate plasminogen activation, leading to release of cytotoxic fragments that are clients for extracellular chaperones.

The misfolding of proteins and their accumulation in extracellular tissue compartments as insoluble amyloid or amorphous protein aggregates are a hallmark feature of many debilitating protein deposition diseases such as Alzheimer's disease, prion diseases, and type II diabetes. The plasminogen activation system is best known as an extracellular fibrinolytic system but was previously reported to also be capable of degrading amyloid fibrils. Here we show that amorphous protein aggregates interact with tissue-type plasminogen activator and plasminogen, via an exposed lysine-dependent mechanism, to efficiently generate plasmin. The insoluble aggregate-bound plasmin is shielded from inhibition by α2-antiplasmin and degrades amorphous protein aggregates to release smaller, soluble but relatively hydrophobic fragments of protein (plasmin-generated protein fragments (PGPFs)) that are cytotoxic. In vitro, both endothelial and microglial cells bound and internalized PGPFs before trafficking them to lysosomes. Clusterin and α2-macroglobulin bound to PGPFs to significantly ameliorate their toxicity. On the basis of these findings, we hypothesize that, as part of the in vivo extracellular proteostasis system, the plasminogen activation system may work synergistically with extracellular chaperones to safely clear large and otherwise pathological protein aggregates from the body.

Two Isoforms of Yersinia pestis Plasminogen Activator Pla: Intraspecies Distribution, Intrinsic Disorder Propensity, and Contribution to Virulence.

It has been shown previously that several endemic Y. pestis isolates with limited virulence contained the I259 isoform of the outer membrane protease Pla, while the epidemic highly virulent strains possessed only the T259 Pla isoform. Our sequence analysis of the pla gene from 118 Y. pestis subsp. microtus strains revealed that the I259 isoform was present exclusively in the endemic strains providing a convictive evidence of more ancestral origin of this isoform. Analysis of the effects of the I259T polymorphism on the intrinsic disorder propensity of Pla revealed that the I259T mutation slightly increases the intrinsic disorder propensity of the C-terminal tail of Pla and makes this protein slightly more prone for disorder-based protein-protein interactions, suggesting that the T259 Pla could be functionally more active than the I259 Pla. This assumption was proven experimentally by assessing the coagulase and fibrinolytic activities of the two Pla isoforms in human plasma, as well as in a direct fluorometric assay with the Pla peptide substrate. The virulence testing of Pla-negative or expressing the I259 and T259 Pla isoforms Y. pestis subsp. microtus and subsp. pestis strains did not reveal any significant difference in LD50 values and dose-dependent survival assays between them by using a subcutaneous route of challenge of mice and guinea pigs or intradermal challenge of mice. However, a significant decrease in time-to-death was observed in animals infected with the epidemic T259 Pla-producing strains as compared to the parent Pla-negative variants. Survival curves of the endemic I259 Pla+ strains fit between them, but significant difference in mean time to death post infection between the Pla-strains and their I259 Pla+ variants could be seen only in the isogenic set of subsp. pestis strains. These findings suggest an essential role for the outer membrane protease Pla evolution in Y. pestis bubonic infection exacerbation that is necessary for intensification of epidemic process from endemic natural focality with sporadic cases in men to rapidly expanding epizootics followed by human epidemic outbreaks, local epidemics or even pandemics.

Soluble urokinase plasminogen activator receptor and incidence of venous thromboembolism.

Raised plasma levels of the soluble urokinase plasminogen activator receptor (suPAR) have been associated with increased incidence of cardiovascular diseases. Whether suPAR is associated with venous thromboembolism (VTE) is largely unknown. The purpose of the present study was to investigate the relationship between suPAR and incidence of VTE in a cohort study. suPAR was measured in 5,203 subjects (aged 46-68 years, 58 % women) from the general population, who participated in the Malmö Diet and Cancer (MDC) study between 1991 and 1994. Incident cases of VTE were identified from the Swedish patient register during a mean follow-up of 15.7 years. Of 5,203 subjects with measurements of suPAR, 239 had VTE during follow-up (127 venous thrombosis, 86 lung embolism, 26 both). Incidence of VTE was significantly higher in subjects with suPAR levels in the top quartile. Adjusted for age and sex, the HR (4th vs 1st quartile) was 1.74 (95 %CI: 1.2-2.6, p for trend=0.003). After adjustments for risk factors, the HR was 1.66 (95 %CI: 1.1-2.5, p for trend=0.016). High level of suPAR was a risk indicator for incidence of VTE in this population-based cohort study. The causal relationships between suPAR and VTE remain to be explored.

[Screening of Producers of Proteinases with Fibrinolytic and Collagenolytic Activities among Micromycetes].

Screening for producers of proteinases with fibrinolytic (plasmin-like and plasminogen-activating) and collagenolytic activities-was carried out among 83 strains of microscopic fungi belonging to various ecological groups. Entomopathogenic micromycetes secreted proteinases with higher fibrinolytic and collagenolytic activity than saprotrophic, potentially phytopathogenic, and epiphytic strains. Micromycete strains possessing proteolytic enzymes with collagenase activity were revealed, as well as the strains producing proteinases with plasmin-like activity. None of the strains studied secreted proteinases possessing only plasminogen-activating activity. Tolypocladium inflatum k1 was found to be a producer of extracellular proteinases with high plasminogen-activating, plasmin-like, and collagenolytic activities. The specific plasminogen-activating activity of T. inflatum k1 was shown to be 20% higher than its plasmin-like activity.

Fibrinolytic and procoagulant activities of Yersinia pestis and Salmonella enterica.

Pla of the plague bacterium Yersinia pestis and PgtE of the enteropathogen Salmonella enterica are surface-exposed, transmembrane ß-barrel proteases of the omptin family that exhibit a complex array of interactions with the hemostatic systems in vitro, and both proteases are established virulence factors. Pla favors fibrinolysis by direct activation of plasminogen, inactivation of the serpins plasminogen activator inhibitor-1 and α2-antiplasmin, inactivation of the thrombin-activable fibrinolysis inhibitor, and activation of single-chain urokinase. PgtE is structurally very similar but exhibits partially different functions and differ in expression control. PgtE proteolysis targets control aspects of fibrinolysis, and mimicry of matrix metalloproteinases enhances cell migration that should favor the intracellular spread of the bacterium. Enzymatic activity of both proteases is strongly influenced by the environment-induced variations in lipopolysaccharide that binds to the ß-barrel. Both proteases cleave the tissue factor pathway inhibitor and thus also express procoagulant activity.

Thrombolytic and Antiplatelet Effects of a Novel Plasminogen Activator from the Venom of Gloydius Brevicaudus Viper.

AIM: To investigate the thrombolytic and antiplatelet effects of a novel plasminogen activator from the venom of the Gloydius brevicaudus viper (GBV-PA) in vitro and in vivo. METHODS: Thrombolytic experiments were performed in rabbit models of ear vein thrombosis and carotid artery thrombosis and in dog model of acute cerebral infarction. Inhibition of thrombus formation was evaluated in rat inferior vena cava thrombosis model and ferric chloride-induced arterial thrombosis. In vitro, we assayed the antithrombotic effect of GBV-PA on rabbit blood clots, euglobulin lysis time (ELT) of rabbit plasma, and ADP-induced platelet aggregation. RESULTS: GBV-PA intravenous administration significantly reduced vascular recanalization times of rabbit ear veins thrombosis and thrombus weight of rabbit carotid artery thrombosis. The arterial recanalization rates were dose- and time-dependently improved after the administration of GBV-PA in canine acute cerebral infarction model. Thrombus length and weight were significantly reduced by GBV-PA both in rat inferior vena cava and ferric chloride-induced arterial thrombosis models. Thrombus formation in the blood of rabbits that were administered of GBV-PA was also inhibited. GBV-PA radically reduced plasma ELT of the rabbit's blood clots. ADP-induced platelet aggregation was inhibited by GBV-PA in a dose-dependent manner with a half-maximal inhibitory concentration of 19.9 µg/mL. CONCLUSION: This study demonstrates that GBV-PA is a thrombolytic and antiplatelet agent. It has significant antithrombotic effects on various in vitro and in vivo experimental models of thrombosis. The mechanisms that underline its antithrombotic effects were related to GBV-PA's capabilities of increasing fibrinolytic activity and inhibition of platelet aggregation.

Bilobed shape of PadA reveals the connectivity from single to multi-domain bacterial plasminogen activators.

The bacterial plasminogen activator, PadA activates bovine, ovine and caprine plasminogen but remains inert toward human plasminogen. It shows high sequence homology with human plasminogen activator, staphylokinase (SAK) but generates active-site in bovine plasminogen non-proteolytically, similar to streptokinase (SK). To examine the structural requirements for the function of this unique cofactor, attempts were made to visualize solution structure of the PadA using small-angle X-ray scattering (SAXS) data and compare its shape profile with structural models based on crystal structures of staphylokinase and streptokinase domains. The bilobal shape solved for the PadA matched closely with the structural model of α-domain of SK rather than its sequence homolog, SAK. The SAXS based solution structure of the PadA exhibited an extra volume and high mobility around Y(90)DKAEK(95) and P(104)ITES(108) loop regions that were found to play a crucial role in its cofactor function. Structure and sequence analysis of bacterial cofactors and mammalian plasminogens displayed evolutionary conservation of crucial complimentary amino acids required for making a functional binary activator complex between bacterial plasminogen activators and their cognate partner plasminogen. These studies highlighted the importance of structure-function related evolutionary strategies adopted by bacteria for exploiting mammalian plasminogen activation system and its understanding may help in designing and the development of new thrombolytic agents for clinical interventions.

[Streptokinase and Staphylokinase: Differences in the Kinetics and Mechanism of Their Interaction with Plasminogen, Inhibitors and Fibrin].

Comparative in vitro study of the kinetics of various reactions involved in the process of thrombolysis initiated by streptokinase (SK) and staphylokinase (STA) was carried out. It was shown that at the interaction of an equimolar ratio of plasminogen (Pg) with SK or STA the rate of formation and the specific esterase activity of the complex plasmin (Pm) · SK are higher than those of the complex Pm · STA. The catalytic efficiency (kcat/Km) of hydrolysis of the chromogenic plasmin substrates by Pm · SK complex was 2 times higher than by Pm · STA complex. In the absence of fibrin catalytic efficiency (kPg/K(Pg)) of activation of Glu-plasminogen and Lys-plasminogen glycoform II by Pm · SK complex was higher than by Pm · STA complex, but the pres- ence of fibrin increased kPg/K(Pg)) activation of both plasminogens by Pm · STA complex significantly stronger than by Pm · SK complex due to the decrease in K(Pg)). In contrast to STA (15.5 kDa), SK molecule (47 kDa) creates significant steric hindrances for the interaction of plasmin in Pm · SK complex with protein inhibi- tors. In addition, SK caused greater fibrinogen degradation than STA. It is shown that Pm · SK and Pm · STA complexes lyse fibrin clots in buffer with similar rates, while the rate of lysis of plasma clots, immersed in plas- ma, by Pm · STA complex are significantly higher than those by Pm · SK complex. It was revealed that the species specificity of STA and S K is determined mainly by the rate of formation and the efficiency of Pm · SK and Pm · STA complexes in the activation of autologous plasminogen. The lysis efficiency of plasma clots of mammals fell in the series: human > dog > rabbit for SK and the dog > human > rabbit for STA. The results show that in the purified system SK is a more effective activator of plasminogen than STA. In the system con- taining fibrin and α2-AP, the activator and fibrinolytic activities of STA are higher than those of SK, due to the increased stability in plasma and fibrin specificity of STA, the fast reaction of the complex Pm · STA with α2AP and the ability of the STA to recyclization in the presence of α2AP.

Oxidative refolding of rPA in l-ArgHCl and in ionic liquids: A correlation between hydrophobicity, salt effects, and refolding yield.

The ionic liquid 1-ethyl-3-methyl imidazolium chloride (EMIM Cl) and the amino acid l-arginine hydrochloride (l-ArgHCl) have been successfully used to improve the yield of oxidative refolding for various proteins. However, the molecular mechanisms behind the actions of such solvent additives-especially of ionic liquids-are still not well understood. To analyze these mechanisms, we have determined the transfer free energies from water into ionic liquid solutions of proteinogenic amino acids and of diketopiperazine as peptide bond analogue. For EMIM Cl and 1-ethyl-3-methyl imidazolium diethyl phosphate, which had a suppressive effect on protein refolding, as well as for l-ArgHCl favorable interactions with amino acid side chains, but no favorable interactions with the peptide backbone could be observed. A quantitative analysis of other ionic liquids together with their already published effects on protein refolding showed that only solvent additives within a certain range of hydrophobicity, chaotropicity and kosmotropicity were effective for the refolding of recombinant plasminogen activator.

The effects of N-ethyl-N'-methyl imidazolium chloride on the solubility, stability and aggregation of tc-rPA.

The ionic liquid N-ethyl-N'-methyl imidazolium chloride (EMIMCl) has been described as being very efficient in promoting refolding of the recombinant plasminogen activator rPA. Our study reveals that molar concentrations of EMIMCl increase the solubility of native and unfolded proteins due to favorable interactions with amino acid side chains rather than favorably interacting with the peptide backbone. This delicate balance of favorable interactions with side chains and unfavorable interactions with the peptide backbone provides a molecular explanation of how EMIMCl suppresses protein aggregation and simultaneously promotes refolding. By contrast, high concentrations of EMIMCl denature proteins because of a reduced water content and strong favorable interactions with amino acid side chains. This denatured species is not soluble and aggregates because, in contrast to the classical denaturants, guanidine hydrochloride and urea, EMIMCl does not solubilize the peptide backbone. STRUCTURED DIGITAL ABSTRACT: PNP and PNP bind by molecular sieving (1, 2, 3, 4).

The ß-domain of cluster 2b streptokinase is a major determinant for the regulation of its plasminogen activation activity by cellular plasminogen receptors.

Cluster 2b streptokinase (SK2b), secreted by invasive skin-trophic strains of Streptococcus pyogenes (GAS), is a human plasminogen (hPg) activator that optimally functions when human plasma hPg is bound, via its kringle-2 domain, to cognizant bacterial cells through the a1a2 domain of the major cellular hPg receptor, Plasminogen-binding group A streptococcal M-like protein (PAM). Another class of streptokinases (SK1), secreted primarily by GAS strains that possess affinity for pharyngeal infections, does not require PAM-bound hPg for optimal activity. We find herein that replacement of the central ß-domain of SK2b with the same module from SK1 reduces the dependency of SK2b on PAM, and the converse is true when the ß-domain of SK1 is replaced with this same region of SK2b. These data suggest that simple evolutionary shuttling of protein domains in GAS can be employed by GAS to rapidly generate strains that differ in tissue tropism and invasive capability and allow the bacteria to survive different challenges by the host.