Structure and binding determinants of the recombinant kringle-2 domain of human plasminogen to an internal peptide from a group A Streptococcal surface protein

The X-ray crystal structure of a complex of a modified recombinant kringle-2 domain of human plasminogen, K2Pg[C4G/E56D/L72Y] (mK2Pg), containing an upregulated lysine-binding site, bound to a functional 30 residue internal peptide (VEK-30) from an M-type protein of a group A Streptococcus surface protein, has been determined by molecular replacement methods using K4Pg as a model, and refined at 2.7 A resolution to a R-factor of 19.5 %. The X-ray crystal structure shows that VEK-30 exists as a nearly end-to-end alpha-helix in the complex with mK2Pg. The final structure also revealed that Arg17 and His18 of VEK-30 served as cationic loci for Asp54 and Asp56 of the consensus lysine-binding site of mK2Pg, while Glu20 of VEK-30 coordinates with Arg69 of the cationic binding site of mK2Pg. The hydrophobic ligand-binding pocket in mK2Pg, consisting primarily of Trp60 and Trp70, situated between the positive and negative centers of the lysine-binding site, is utilized in a novel manner in stabilizing the interaction with VEK-30 by forming a cation-pi-electron-mediated association with the positive side-chain of Arg17 of this peptide. Additional lysine-binding sites, as well as exosite electrostatic and hydrogen bonding interactions involving Glu9 and Lys14 of VEK-30, were observed in the structural model. The importance of these interactions were tested in solution by investigating the binding constants of synthetic variants of VEK-30 to mK2Pg, and it was found that, Lys14, Arg17, His18, and Glu20 of VEK-30 were the most critical amino acid binding determinants. With regard to the solution studies, circular dichroism analysis of the titration of VEK-30 with mK2Pg demonstrated that the peptidic alpha-helical structure increased substantially when bound to the kringle module, in agreement with the X-ray results.This investigation is the first to delineate structurally the mode of interaction of the lysine-binding site of a kringle with an internal pseudo-lysine residue of a peptide or protein that functionally interacts with a kringle module, and serves as a paradigm for this important class of interactions.     

Effects on human plasminogen conformation and activation rate caused by interaction with VEK-30, a peptide derived from the group A streptococcal M-like protein (PAM)

In vertebrates, fibrinolysis is primarily carried out by the serine protease plasmin (Pm), which is derived from activation of the zymogen precursor, plasminogen (Pg). One of the most distinctive features of Pg/Pm is the presence of five homologous kringle (K) domains. These structural elements possess conserved Lys-binding sites (LBS) that facilitate interactions with substrates, activators, inhibitors and receptors. In human Pg (hPg), K2 displays weak Lys affinity, however the LBS of this domain has been implicated in an atypical interaction with the N-terminal region of a bacterial surface protein known as PAM (Pg-binding group A streptococcal M-like protein). A direct correlation has been established between invasiveness of group A streptococci and their ability to bind Pg. It has been previously demonstrated that a 30-residue internal peptide (VEK-30) from the N-terminal region of PAM competitively inhibits binding of the full-length parent protein to Pg. We have attempted to determine the effects of this ligand–protein interaction on the regulation of Pg zymogen activation and conformation. Our results show minimal effects on the sedimentation velocity coefficients (S°_20,w) of Pg when associated to VEK-30 and a direct relationship between the concentration of VEK-30 or PAM and the activation rate of Pg. These results are in contrast with the major conformational changes elicited by small-molecule activators of Pg, and point towards a novel mechanism of Pg activation that may underlie group A streptococcal (GAS) virulence.     

Solution structure of the complex of VEK-30 and plasminogen kringle 2

The solution structure of the complex containing the isolated kringle 2 domain of human plasminogen (K2_Pg) and VEK-30, a 30-amino acid residue internal peptide from a streptococcal M-like plasminogen (Pg) binding protein (PAM), has been determined by multinuclear high-resolution NMR. Complete backbone and side-chain assignments were obtained from triple-resonance experiments, after which structure calculations were performed and ultimately refined by restrained molecular simulation in water. We find that, in contrast with the dimer of complexes observed in the asymmetric unit of the crystal, global correlation times and buoyant molecular weight determinations of the complex and its individual components showed the monomeric nature of all species in solution. The NMR-derived structure of K2_Pg in complex with VEK-30 presents a folding pattern typical of other kringle domains, while bound VEK-30 forms an end-to-end α-helix (residues 6–27) in the complex. Most of the VEK-30/K2_Pg interactions in solution occur between a single face of the α-helix of VEK-30 and the lysine binding site (LBS) of K2_Pg. The canonical LBS of K2_Pg, consisting of Asp54, Asp56, Trp60, Arg69, and Trp70 (kringle numbering), interacts with an internal pseudo-lysine of VEK-30, comprising side-chains of Arg17, His18, and Glu20. Site-specific mutagenesis analysis confirmed that the electrostatic field formed by the N-terminal anionic residues of the VEK-30 α-helix, viz., Asp7, and the non-conserved cationic residues of K2_Pg, viz., Lys43 and Arg55, play additional important roles in the docking of VEK-30 to K2_Pg. Structural analysis and kringle sequence alignments revealed several important features related to exosite binding that provide a structural rationale for the high specificity and affinity of VEK-30 for K2_Pg.     

The X-ray crystallographic structure of the angiogenesis inhibitor angiostatin

Angiogenesis inhibitors have gained much public attention recently as anti-cancer agents and several are currently in clinical trials, including angiostatin (Phase I, Thomas Jefferson University Hospital, Philadelphia, PA). We report here the bowl-shaped structure of angiostatin kringles 1-3, the first multi-kringle structure to be determined. All three kringle lysine-binding sites contain a bound bicine molecule of crystallization while the former of kringle 2 and kringle 3 are cofacial. Moreover, the separation of the kringle 2 and kringle 3 lysiner binding sites is sufficient to accommodate the alpha-helix of the 30 residue peptide VEK-30 found in the kringle 2/VEK-30 complex. Together the three kringles produce a central cavity suggestive of a unique domain where they may function in concert.     

The lack of binding of VEK-30, an internal peptide from the group A streptococcal M-like protein, PAM, to murine plasminogen is due to two amino acid replacements in the plasminogen kringle-2 domain

VEK-30, a 30-amino acid internal peptide present within a streptococcal M-like plasminogen (Pg)-binding protein (PAM) from Gram-positive group-A streptococci (GAS), represents an epitope within PAM that shows high affinity for the lysine binding site (LBS) of the kringle-2 (K2) domain of human (h)Pg. VEK-30 does not interact with this same region of mouse (m)Pg, despite the high conservation of the mK2- and hK2-LBS. To identify the molecular basis for the species specificity of this interaction, hPg and mPg variants were generated, including an hPg chimera with the mK2 sequence and an mPg chimera containing the hK2 sequence. The binding of synthetic VEK-30 to these variants was studied by surface plasmon resonance. The data revealed that, in otherwise intact Pg, the species specificity of VEK-30 binding in these two cases is entirely dictated by two K2 residues that are different between hPg and mPg, namely, Arg-220 of hPg, which is a Gly in mPg, and Leu-222 of hPg, which is a Pro in mPg, neither of which are members of the canonical K2-LBS. Neither the activation of hPg, nor the enzymatic activity of its activated product, plasmin (hPm), are compromised by replacing these two amino acids by their murine counterparts. It is also demonstrated that hPg is more susceptible to activation to hPm after complexation with VEK-30 and that this property is greatly reduced as a result of the R220G and L222P replacements in hPg. These mechanisms for accumulation of protease activity on GAS likely contribute to the virulence of PAM(+)-GAS strains and identify targets for new therapeutic interventions.     

NMR Backbone Dynamics of VEK-30 Bound to the Human Plasminogen Kringle 2 Domain

To gain insights into the mechanisms for the tight and highly specific interaction of the kringle 2 domain of human plasminogen (K2_Pg) with a 30-residue internal peptide (VEK-30) from a group A streptococcal M-like protein, the dynamic properties of free and bound K2_Pg and VEK-30 were investigated using backbone amide ¹⁵N-NMR relaxation measurements. Dynamic parameters, namely the generalized order parameter, S², the local correlation time, τ_e, and the conformational exchange contribution, R_ex, were obtained for this complex by Lipari-Szabo model-free analysis. The results show that VEK-30 displays distinctly different dynamic behavior as a consequence of binding to K2_Pg, manifest by decreased backbone flexibility, particularly at the binding region of the peptide. In contrast, the backbone dynamics parameters of K2_Pg displayed similar patterns in the free and bound forms, but, nonetheless, showed interesting differences. Based on our previous structure-function studies of this interaction, we also made comparisons of the VEK-30/K2_Pg dynamics results from different kringle modules complexed with small lysine analogs. The differences in dynamics observed for kringles with different ligands provide what we believe to be new insights into the interactions responsible for protein-ligand recognition and a better understanding of the differences in binding affinity and binding specificity of kringle domains with various ligands.     

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.     

Dimerization Is Not a Determining Factor for Functional High Affinity Human Plasminogen Binding by the Group A Streptococcal Virulence Factor PAM and Is Mediated by Specific Residues within the PAM a1a2 Domain

A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2_hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97–125 + Tyr), interacted specifically with isolated K2_hPg. However, the binding strength of VEK30 (K_D = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (K_D = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH₂ and COOH termini of VEK30. VEK64 (PAM residues 83–145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2_hPg with nearly the same affinity as PAM (K_D = 1–2 nm). However, addition of two PAM residues (Arg¹²⁶-His¹²⁷) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2_hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg¹¹³, His¹¹⁴, Glu¹¹⁶, Arg¹²⁶, His¹²⁷), mutation of which reduced PAM binding affinity for K2_hPg by ∼1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence.     

Mechanism of inhibitory effect of angiostatin on plasminogen activation by its physiologic activators

The influence of angiostatin K1-4.5--a fragment of the heavy chain of plasmin and a powerful inhibitor of angiogenesis--on kinetic parameters (k(Pg) and K(Pg)) of human Glu-plasminogen activation under the action of urokinase (uPA) not having affinity for fibrin and fibrin-specific tissue plasminogen activator (tPA) was investigated. Angiostatin does not affect the k(Pg) value, but increases the value K(Pg) urokinase plasminogen activation. A decrease in the k(Pg) value and an increase in the K(Pg) value were found for fibrin-stimulated plasminogen activation by tPA with increasing concentrations of angiostatin. The obtained results show that angiostatin is competitive inhibitor of the uPA activator activity, while it inhibits the activator activity of tPA by mixed type. Such an influence ofangiostatin on the kinetic constants ofthe urokinase plasminogen activation suggests that angiostatin dose dependent manner replaces plasminogen in the binary enzyme-substrate complex uPA-Pg. In case of fibrin-stimulated plasminogen activation by tPA, both zymogen and tPA are bound to fibrin with formation of the effective triple tPA-Pg-fibrin complex. Angiostatin replaces plasminogen both from the fibrin surface and from the enzyme-substrate tPA-Pg complex that leads to a decrease in k(Pg) and an increase in K(Pg) of plasminogen activation. Inhibition constants by angioststin (Ki) of plasminogen-activator activities of uPA and tPA determined by Dixon method were found to be 0.59 +/- 0.04 and 0.12 +/- 0.05 microM, respectively.     

The effects of ligand binding on the backbone dynamics of the kringle 1 domain of human plasminogen

The internal motions of the backbone nitrogen atoms of the kringle 1 domain of human plasminogen (K1(Pg)) were examined in the absence and presence of the ligand, epsilon-aminocaproic acid. These dynamic properties were determined from (15)N NMR relaxation data in terms of the extended model-free parameters. The model of isotropic reorientation was found sufficient to account for overall molecular tumbling for both apo and EACA-bound K1(Pg). The global rotational correlation time (tau(m)) for apo-K1(Pg) was 5.87(+/-0.01) ns, while the tau(m) for ligand-bound K1(Pg) was 5.20(+/-0.01) ns, suggesting that perhaps some small degree of aggregation occurred in the apo form of the kringle module. Complexation of K1(Pg) with ligand mainly reduced those internal motions that occurred on a 100 ps to 5 ns time-scale. The magnitude of the chemical exchange was also attenuated upon ligand binding. These data are consistent with studies employing other approaches that suggest that the binding pocket is preformed in K1(Pg).     

Crystal structure of the kringle 2 domain of tissue plasminogen activator at 2.4-A resolution.

The crystal structure of the kringle 2 domain of tissue plasminogen activator was determined and refined at a resolution of 2.43 A. The overall fold of the molecule is similar to that of prothrombin kringle 1 and plasminogen kringle 4; however, there are differences in the lysine binding pocket, and two looping regions, which include insertions in kringle 2, take on very different conformations. Based on a comparison of the overall structural homology between kringle 2 and kringle 4, a new sequence alignment for kringle domains is proposed that results in a division of kringle domains into two groups, consistent with their proposed evolutionary relation. The crystal structure shows a strong interaction between a lysine residue of one molecule and the lysine/fibrin binding pocket of a noncrystallographically related neighbor. This interaction represents a good model of a bound protein ligand and is the first such ligand that has been observed in a kringle binding pocket. The structure shows an intricate network of interactions both among the binding pocket residues and between binding pocket residues and the lysine ligand. A lysine side chain is identified as the positively charged group positioned to interact with the carboxylate of lysine and lysine analogue ligands. In addition, a chloride ion is located in the kringle-kringle interface and contributes to the observed interaction between kringle molecules.     

Binding of the NG2 proteoglycan to kringle domains modulates the functional properties of angiostatin and plasmin(ogen)

Interactions of the developmentally regulated chondroitin sulfate proteoglycan NG2 with human plasminogen and kringle domain-containing plasminogen fragments have been analyzed by solid-phase immunoassays and by surface plasmon resonance. In immunoassays, the core protein of NG2 binds specifically and saturably to plasminogen, which consists of five kringle domains and a serine protease domain, and to angiostatin, which contains plasminogen kringle domains 1-3. Apparent dissociation constants for these interactions range from 12 to 75 nm. Additional evidence for NG2 interaction with kringle domains comes from its binding to plasminogen kringle domain 4 and to miniplasminogen (kringle domain 5 plus the protease domain) with apparent dissociation constants in the 18-71 nm range. Inhibition of plasminogen and angiostatin binding to NG2 by 6-aminohexanoic acid suggests that lysine binding sites are involved in kringle interaction with NG2. The interaction of NG2 with plasminogen and angiostatin has very interesting functional consequences. 1) Soluble NG2 significantly enhances the activation of plasminogen by urokinase type plasminogen activator. 2) The antagonistic effect of angiostatin on endothelial cell proliferation is inhibited by soluble NG2. Both of these effects of NG2 should make the proteoglycan a positive regulator of the cell migration and proliferation required for angiogenesis.     

Cell surface adenosine deaminase binds and stimulates plasminogen activation on 1-LN human prostate cancer cells

Adenosine deaminase (ADA) is expressed intracellularly by all cells, but in some tissues, it is also associated with the cell surface multifunctional glycoprotein CD26/dipeptidyl peptidase IV. By modulating extracellular adenosine, this "ecto-ADA" may regulate adenosine receptor signaling implicated in various cellular functions. CD26 is expressed on the surface of human prostate cancer 1-LN cells acting as a receptor for plasminogen (Pg). Since ADA and Pg bind to CD26 at distinct but nearby sites, we investigated a possible interaction between these two proteins on the surface of 1-LN cells. Human ADA binds to CD26 on the surface 1-LN cells and immobilized CD26 isolated from the same cells with similar affinity. In both cases, ADA binding is diminished by mutation of ADA residues known to interact with CD26. ADA was also found to bind Pg 2 in the absence of CD26 via the Pg kringle 4 (K4) domain. In the presence of 1-LN cells or immobilized CD26, exogenous ADA enhances conversion of Pg 2 to plasmin by 1-LN endogenous urinary plasminogen activator (u-PA), as well as by added tissue Pg Activator (t-PA), suggesting that ADA and Pg bind simultaneously to CD26 in a ternary complex that stimulates the Pg activation by its physiologic activators. Consistent with this, in melanoma A375 cells that bind Pg, but do not express CD26, the rate of Pg activation was not affected by ADA. Thus, ADA may be a factor regulating events in prostate cancer cells that occur when Pg binds to the cell surface and is activated.     

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.     

Kringle-kringle interactions in multimer kringle structures.

The crystal structure of a monoclinic form of human plasminogen kringle 4 (PGK4) has been solved by molecular replacement using the orthorthombic structure as a model and it has been refined by restrained least-squares methods to an R factor of 16.4% at 2.25 A resolution. The X-PLOR structure of kringle 2 of tissue plasminogen activator (t-PAK2) has been refined further using PROFFT (R = 14.5% at 2.38 A resolution). The PGK4 structure has 2 and t-PAK2 has 3 independent molecules in the asymmetric unit. There are 5 different noncrystallographic symmetry "dimers" in PGK4. Three make extensive kringle-kringle interactions related by noncrystallographic 2(1) screw axes without blocking the lysine binding site. Such associations may occur in multikringle structures such as prothrombin, hepatocyte growth factor, plasminogen (PG), and apolipoprotein [a]. The t-PAK2 structure also has noncrystallographic screw symmetry (3(1)) and mimics fibrin binding mode by having lysine of one molecule interacting electrostatically with the lysine binding site of another kringle. This ligand-like binding interaction may be important in kringle-kringle interactions involving non-lysine binding kringles with lysine or pseudo-lysine binding sites. Electrostatic intermolecular interactions involving the lysine binding site are also found in the crystal structures of PGK1 and orthorhombic PGK4. Anions associate with the cationic centers of these and t-PAK2 that appear to be more than occasional components of lysine binding site regions.     

Regulation of nonproteolytic active site formation in plasminogen

Streptokinase may be less effective at saving lives in patients with heart attacks because it explosively generates plasmin in the bloodstream at sites distant from fibrin clots. We hypothesized that this rapid plasmin generation is due to SK's singular capacity to nonproteolytically generate the active protease SK x Pg*, and we examined whether the kringle domains regulate this process. An SK mutant lacking Ile-1 (deltaIle1-SK) does not form SK x Pg*, although it will form complexes with plasmin that can activate plasminogen. When compared to SK, deltaIle1-SK diminished the generation of plasmin in plasma by more than 30-fold, demonstrating that the formation of SK x Pg* plays an important role in SK activity in the blood. The rate of SK x Pg* formation (measured by an active site titrant) was much slower in Glu-Pg, which contains five kringle domains, than in Pg forms containing one kringle (mini-Pg) or no kringles (micro-Pg). In a similar manner, Streptococcus uberis Pg activator (SUPA), an SK-like molecule, generated SUPA x Pg* much slower with bovine Pg than bovine micro-Pg. The velocity of SK x Pg* formation was regulated by agents that influence the conformation of Pg through interactions with the kringle domains. Chloride ions, which maintain the compact Pg conformation, hindered SK x Pg* formation. In contrast, epsilon-aminocaproic acid, fibrin, and fibrinogen, which induce an extended Pg conformation, accelerated the formation of SK x Pg*. In summary, the explosive generation of plasmin in blood or plasma, which diminishes SK's therapeutic effects, is attributable to the formation of SK x Pg*, and this process is governed by kringle domains.     

Structure and Function Characterization of the a1a2 Motifs of Streptococcus pyogenes M Protein in Human Plasminogen Binding

Plasminogen (Plg)-binding M protein (PAM) is a group A streptococcal cell surface receptor that is crucial for bacterial virulence. Previous studies revealed that, by binding to the kringle 2 (KR2) domain of host Plg, the pathogen attains a proteolytic microenvironment on the cell surface that facilitates its dissemination from the primary infection site. Each of the PAM molecules in their dimeric assembly consists of two Plg binding motifs (called the a1 and a2 repeats). To date, the molecular interactions between the a1 repeat and KR2 have been structurally characterized, whereas the role of the a2 repeat is less well defined. Here, we report the 1.7-Å x-ray crystal structure of KR2 in complex with a monomeric PAM peptide that contains both the a1 and a2 motifs. The structure reveals how the PAM peptide forms key interactions simultaneously with two KR2 via the high-affinity lysine isosteres within the a1a2 motifs. Further studies, through combined mutagenesis and functional characterization, show that a2 is a stronger KR2 binder than a1, suggesting that these two motifs may play discrete roles in mediating the final PAM-Plg assembly.     

An internal histidine residue from the bacterial surface protein, PAM, mediates its binding to the kringle-2 domain of human plasminogen.

The determinants of binding of a peptide lacking C-termini-exposed lysine residues to a kringle domain were investigated using an up-regulated lysine binding kringle (K2Pg[C4G/E56D/K72Y]) of plasminogen and a peptide (a1-PAM) with a sequence derived from a surface-exposed M-like streptococcal protein. Significant kringle-induced chemical shifts in a His side-chain of a1-PAM were revealed by two-dimensional NMR. Further studies using isothermal titration calorimetry (ITC) provided support for the involvement of His12 in the peptide/ protein complex. In an effort to screen a1-PAM-derived truncation peptides, a combinatorial mixture, a1deltaa2-PAM[H12X] (where X=Pro, Arg, His, Trp, Lys, Ala, Phe, Asp and Gly), was analyzed using the surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI) platform. The major peptide that remained bound to the surface of the K2Pg[C4G/ E56D/K72Y]-containing chip was that containing His12, corresponding to the wild-type sequence. Minor peaks, representing binding, were obtained for Lys12-, Arg12- and Trp12-containing peptides. Individual peptides containing these amino acids were then examined using ITC and the binding constants obtained correlated with the relative strengths of binding estimated from the SELDI-based screen.     

Disruption of interkringle disulfide bond of plasminogen kringle 1-3 changes the lysine binding capability of kringle 2, but not its antiangiogenic activity

Kringle 1-3 of human plasminogen is a potent inhibitor of endothelial cell proliferation. To understand a possible role for the unique cystine bridge between kringle 2 and kringle 3, we disrupted the interkringle disulfide bond by mutating Cys(169) and Cys(297) to serine residues. The yield of the mutant during the refolding process was decreased significantly. Anti-endothelial cell proliferative activity of the mutant was similar to that of the wild type. There was no significant difference in in vivo antiangiogenic activity between the wild type and the mutant in chorioallantoic membrane assay. However, in the mutant, the weak lysine binding capability of kringle 2 was not detected and its mobility in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis is different from that of the wild type. These results support the notion that the overall antiangiogenic function of angiostatin is mediated by individual kringles, and suggest that the lysine binding capability of kringle 2 is likely not important for the antiangiogenic activity of kringle 1-3.     

Crystal and molecular structure of human plasminogen kringle 4 refined at 1.9-A resolution.

The crystal structure of human plasminogen kringle 4 (PGK4) has been solved by molecular replacement using the bovine prothrombin kringle 1 (PTK1) structure as a model and refined by restrained least-squares methods to an R factor of 14.2% at 1.9-A resolution. The K4 structure is similar to that of PTK1, and an insertion of one residue at position 59 of the latter has minimal effect on the protein folding. The PGK4 structure is highly stabilized by an internal hydrophobic core and an extensive hydrogen-bonding network. Features new to this kringle include a cis peptide bond at Pro30 and the presence of two alternate, perpendicular, and equally occupied orientations for the Cys75 side chain. The K4 lysine-binding site consists of a hydrophobic trough formed by the Trp62 and Trp72 indole rings, with anionic (Asp55/Asp57) and cationic (Lys35/Arg71) charge pairs at either end. With the adjacent Asp5 and Arg32 residues, these result in triply charged anionic and cationic clusters (pH of crystals at 6.0), which, in addition to the unusually high accessibility of the Trp72 side chain, serve as an obvious marker of the binding site on the K4 surface. A complex intermolecular interaction occurs between the binding sites of symmetry-related molecules involving a highly ordered sulfate anion of solvation in which the Arg32 side chain of a neighboring kringle occupies the binding site.