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 (SKΔK414) 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, SKΔK414 exhibited a corresponding 17-fold affinity decrease for formation of the SKΔK414·[Lys]Pg* complex. SKΔK414 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.


Streptokinase (SK) activates human fibrinolysis by inducing non-proteolytic activation of the serine proteinase zymogen, plasminogen (Pg), in the SK⅐Pg* catalytic complex. SK⅐Pg* 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 Lys 414 deletion mutant (SK⌬K414) demonstrated a critical role for Lys 414 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 Lys 414 . By contrast, removal of SK Lys 414 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, SK⌬K414 exhibited a corresponding 17-fold affinity decrease for formation of the SK⌬K414⅐[Lys]Pg* complex. SK⌬K414 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 Lys 414 to [Lys]Pg and [Lys]Pm kringles enhances SK⅐[Lys]Pg* and SK⅐[Lys]Pm catalytic complex formation. This interaction is distinct structurally and functionally from LBS-dependent Pg substrate recognition by these complexes.
Streptokinase (SK) 2 activates the human fibrinolytic system by activating the zymogen, plasminogen (Pg) to form the fibrin-degrading proteinase, plasmin (Pm) (1). The mechanism of SKactivated Pm formation is unique in that it is initiated by formation of an SK⅐Pg* complex in which the zymogen catalytic site is activated non-proteolytically (2)(3)(4)(5). SK⅐Pg* binds free Pg and converts it into Pm by intermolecular proteolytic cleavage (6). Pm binds tightly to SK in the catalytic mode (7,8) and SK⅐Pm propagates proteolytic Pg activation (6,9,10) through expression of a Pg substrate binding exosite (7). The mechanism is regulated by intrinsic differences in affinity of SK for [Glu]Pg, [Lys]Pg, and [Lys]Pm (5)(6)(7)(8)11).
[Glu]Pg is maintained in a compact conformation through intramolecular interaction of the NH 2 -terminal peptide with kringles 4 and 5 (13)(14)(15). Pm cleavage of the NH 2 -terminal peptide of [Glu]Pg generates the more reactive [Lys]Pg, which assumes an extended conformation with expression of enhanced lysine-binding site (LBS) interactions (12, 14 -16 (5-8, 11, 17-20). Recent studies of the Pg activation mechanism demonstrate that LBS interactions enhance SK⅐[Lys]Pg* catalytic complex formation and Pg substrate binding but are not absolutely required for these interactions (5,6). SK binding to the compact conformation of [Glu]Pg in the catalytic mode is LBS-independent (5,6,8,11).
Several studies have sought to define SK lysine residues that mediate its interactions with Pg and Pm kringles. The crystal structure of SK bound to the isolated Pm catalytic domain (micro-Pm) shows that SK consists of three homologous, independently folded ␤-grasp domains connected by flexible linking sequences (21). SK forms a "crater" around the Pm catalytic site which provides a surface for Pg substrate binding (21). Studies of SK domain truncation and deletion mutants, isolated domains, and point mutants have led to diverse interpretations, indicating that each of the SK domains may participate in kringle interactions (18 -20, 22, 23). Some studies support a role for the flexible 250-loop of the SK ␤-domain in LBS-dependent Pg substrate binding (19,20). By contrast, no studies have demonstrated the structural basis for the LBS dependence of catalytic complex formation.
Kringles of Pg and Pm bind zwitterionic COOH-terminal lysine residues and lysine analogues specifically, notably 6-aminohexanoic acid (6-AHA) (13,24). This is the basis for Pg binding by several proteins, including fibrin (25,26), antiplasmin (27), histidine-rich glycoprotein (28), and tetranectin (29). In fibrinolysis, LBS interactions with fibrin mediated by COOHterminal lysine residues localize and accelerate Pg activation * This work was supported by National Institutes of Health/NHLBI Grant HL056181 (to P. E. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1  and fibrin degradation by Pm (25,26,30) and protect fibrinbound Pm from inactivation by antiplasmin (31). Surprisingly, the fact that the COOH-terminal residue of SK is Lys 414 (32)

EXPERIMENTAL PROCEDURES
Wild-type SK and an SK Mutant Lacking the COOH-terminal Lys-Wild-type SK (wtSK) was prepared by methods described previously (33,34) or was expressed as a fusion protein with a tobacco etch virus (TEV) proteinase cleavage site (underlined) encoded before the wtSK protein, Met-His 6 -Ser-Ala-Gly-Gly-Ser-Pro-Trp-Asn-Glu-Asn-Leu-Try-Phe-Gln-SK Ile 1 -SK Ala 2 -SK Gly 3 . . . (His 6 -wtSK). From a pET30a(ϩ) vector backbone, a single nucleotide substitution mutated the P1 3 residue of a thrombin cleavage site (from Arg to Trp), which eliminated this unnecessary site and generated a NcoI restriction site. Flanking NcoI and XhoI restriction sites (underlined) where incorporated into the 5Ј-and 3Ј-PCR primers, respectively, with the sense primer, 5Ј-TCACTCCGCGGGTGGTA-GTCCATGGAACGAGAACCTGTATTTTCAGATTGCTG-GACCTGAGTGGCTG-3Ј, the same for wtSK and SK⌬K414 constructs, and the antisense primer, 5Ј-ATAATGGTGCTC-GAGTTATTTGTCGTTAGGGTTATCAGG-3Ј, only differed by the Lys 414 codon (bold). A construct that encoded a His 6tagged TEV proteinase was kindly provided by Dr. Laura Mizoue of the Vanderbilt University Center for Structural Biology and used to remove the His 6 -tag from the NH 2 terminus of wtSK.
His 6 -wtSK was expressed from Rosetta(DE3) pLysS cells induced with 20 g/liter lactose for 12-16 h at 37°C. Cells were harvested by centrifugation, resuspended in 50 mM Hepes, 125 mM NaCl, 1 mg/ml polyethylene glycol 8000, pH 7.4 (Buffer A) with 1 mM EDTA and 0.2% sodium azide, lysed by three cycles of sonication (ϳ45 s cycles) on ice, and centrifuged to clarify lysates. The pellet was resuspended in Buffer A containing 3 M NaSCN. The solubilized wtSK was dialyzed into 50 mM Hepes, 400 mM NaCl, 50 mM imidazole, pH 7.4 (Buffer B) and purified by Ni 2ϩ -iminodiacetic acid-Sepharose chromatography with a 50 -500 mM imidazole gradient in Buffer B. TEV proteinase was added to the eluted protein in a 1 to 5 molar ratio of enzyme to substrate. The reaction mixture was first dialyzed overnight into 50 mM Hepes, 300 mM NaCl, 1 mM dithiothreitol, 5% glycerol, pH 7.8 at 4°C, and subsequently dialyzed back into Buffer B. Uncleaved fusion protein, cleaved His 6 -tag, and the TEV proteinase bound to Ni 2ϩ -iminodiacetic acid-Sepharose, and wtSK was obtained from the column flow-through. wtSK was dialyzed against Buffer A without polyethylene glycol, quick-frozen, and stored at Ϫ80°C. SK⌬414K was prepared following an identical procedure. The correct NH 2 -terminal sequence for wtSK and SK⌬K414 was confirmed.
Fluorescence Equilibrium Binding-[Glu]Pg, [Lys]Pg, [Lys]Pm, and the active site-labeled fluorescein analogues were prepared as described (5,6,11). Fluorescence titrations were performed in Buffer A containing 1 mM EDTA and 1 M D-Phe-Phe-Arg-CH 2 Cl Ϯ 10 mM 6-AHA as described previously (5,6,33). Plasminogen Activation Kinetics-Coupled conformational and proteolytic activation of [Lys]Pg by wtSK and SK⌬K414 were quantitated as described previously (5,6). Fitting of parabolic progress curves of D-Val-Leu-Lys-pNA (VLK-pNA) hydrolysis at 200 M, in the presence of 15 nM [Lys]Pg and increasing wtSK and SK⌬K414 concentrations gave the initial rates (v 1 ) of VLK-pNA hydrolysis, reflecting conformational activation of the SK⅐Pg* complex, and the rates of activity increase (v 2 ), reflecting Pm generation. The SK dependences of v 1 and v 2 were analyzed using the simplified equations described previously (5,6).
The data were also analyzed by fitting of the family of progress curves as a function of SK concentration with a more complete mechanism including both SK⅐Pg*-and SK⅐Pm-catalyzed Pg activation pathways under bimolecular reaction conditions (Scheme 1). For this analysis, the K m values for chromogenic substrate hydrolysis by Pm, SK⅐Pg*, and SK⅐Pm were fixed at the previously determined values, whereas the corresponding k cat values were allowed to vary within the experimental error of their determination to optimize the fit. KЈ A for SK⅐Pm binding was fixed at 12 pM (7). The fitted parameters were K A for SK⅐Pg* formation and the bimolecular rate constants for Pm generation by SK⅐Pg* (k Pg ) and SK⅐Pm (k Pm ) (Scheme 1) (5, 6). Nonlinear least squares fitting was performed with SCIENTIST (MicroMath) or DYNAFIT (35). Error estimates represent the 95% confidence interval.

Binding of Native SK and CpB-treated SK to [Lys]
Pg-Native SK was treated with CpB to remove the COOH-terminal lysine residue, under conditions where there was no detectable degradation of SK observable by SDS-gel electrophoresis (not shown). Titrations of [5F]FFR-[Lys]Pg with native SK and CpB-SK were performed in the absence and presence of saturating 6-AHA to evaluate the effect of CpB treatment on the LBS dependence of SK affinity (Fig. 1A) Pg with wtSK and SK⌬K414 were performed in the absence and presence of 10 mM 6-AHA (Fig. 1B). In the absence and presence of 6-AHA, wtSK bound labeled [Lys]Pg with K D 28 Ϯ 6 nM and 520 Ϯ 70 nM, respectively, and with ⌬F max /F o of Ϫ23 Ϯ 1% and Ϫ21 Ϯ 1%. Like CpB-treated SK, SK⌬K414 bound to labeled [Lys]Pg with weaker affinity than wtSK, with K D 610 Ϯ 200 nM and 750 Ϯ 350 nM in the absence and presence of 6-AHA, respectively, and ⌬F max /F o of Ϫ24 Ϯ 2% and Ϫ24 Ϯ 3%. Native and wtSK bound with indistinguishable affinity to labeled Pg, and their LBS-dependent losses of affinity in the presence of 6-AHA were the same (Fig. 1, A and  B). Deletion of the COOH-terminal lysine decreased the weakening effect of 6-AHA on affinity from 19-to 1.2-fold. This demonstrated that the SK mutant lacking the COOH-terminal lysine exhibited a selective loss of LBS-dependent affinity for labeled [Lys]Pg.
Binding of wtSK and SK⌬K414 to [Glu]Pg-Titrations of [5F]FFR-[Glu]Pg with wtSK and SK⌬K414 were performed in the absence and presence of 10 mM 6-AHA (Fig. 1C). In the absence and presence of 6-AHA, wtSK bound labeled [Glu]Pg with K D 930 Ϯ 120 nM and 471 Ϯ 140 nM, respectively, and with ⌬F max /F o Ϫ37 Ϯ 1% and Ϫ19 Ϯ 1%. SK⌬K414 bound labeled [Glu]Pg with K D 634 Ϯ 160 nM and 560 Ϯ 150 nM in the absence and presence of 6-AHA, respectively, and with ⌬F max /F o Ϫ30 Ϯ 2% and Ϫ19 Ϯ 1%. Although the amplitudes of the fluorescence changes were decreased by 6-AHA, the affinities of wtSK and SK⌬K414 for labeled [Glu]Pg in the presence or absence of 6-AHA were indistinguishable and LBS-independent. This was consistent with the LBS independence of SK binding to [Glu]Pg in the compact conformation (5,8,11) and an independence of the affinity on deletion of Lys 414 .
Binding of wtSK and SK⌬K414 to [Lys]Pm-To determine whether the COOH-terminal lysine of SK also interacted with [Lys]Pm kringles, titrations of [5F]FFR-Pm with wtSK and SK⌬K414 were performed in the absence and presence of 10 mM 6-AHA (Fig. 1D). wtSK bound labeled [Lys]Pm with K D 19 Ϯ 7 pM, consistent with the previously reported affinity (7) and ⌬F max /  ously (5,6). Simultaneous fits of the direct titration and the competitive titration data in Fig. 2 by the cubic binding equation gave a K D of 8.8 Ϯ 4.3 nM for wtSK ( Fig. 2A), which was increased to 124 Ϯ 71 nM by 6-AHA (Fig. 2B). SK⌬K414 bound to native [Lys]Pg with indistinguishable values of 157 Ϯ 66 nM and 106 Ϯ 40 nM in the absence and presence of 6-AHA (Fig.  2C). The values for wtSK were in good agreement with the previously determined dissociation constants of 10 Ϯ 3 nM and 115 Ϯ 32 nM for native SK in the absence and presence of 6-AHA, respectively (5).
Plasminogen Activation Kinetics-Determination of the affinities of wtSK and SK⌬K414 for native [Lys]Pg was necessary to interpret the kinetics of native [Lys]Pg activation by the SK mutant. The kinetics of [Lys]Pg activation were examined by analysis of reaction progress curves in the presence of VLK-pNA, monitored by hydrolysis of the chromogenic substrate. As previously detailed (5, 6), the parabolic progress curves were resolved into an initial rate of substrate hydrolysis (v 1 ) representing the activity of the conformationally activated SK⅐Pg* complex and the rate of acceleration (v 2 ) representing the subsequent proteolytic generation of Pm. The dependences of v 1 and v 2 on SK concentration were analyzed as described under "Experimental Procedures." A second method was also used for the analysis in which families of progress curves collected as a function of SK concentration were fit by numerical integration of the rate equations for the complete mechanism, including both SK⅐Pg*-and SK⅐Pm-catalyzed sequential reactions (Scheme 1 and see "Experimental Procedures").
Analysis of the hyperbolic v 1 dependence on SK concentration in the absence of 6-AHA gave apparent K A of 2 Ϯ 1 nM and 68 Ϯ 27 nM for wtSK and SK⌬K414, respectively (Fig. 3A). In the presence of 6-AHA, the formation of the wtSK⅐[Lys]Pg* complex was weakened to 43 Ϯ 12 nM (22-fold), whereas the affinity of SK⌬K414 was indistinguishable at 87 Ϯ 28 nM, indicating that formation of the SK⌬K414⅐[Lys]Pg* catalytic complex was LBS-independent. Analysis of the bimodal v 2 dependence on wtSK concentration in the absence of 6-AHA gave a  in the absence (f) and presence (Ⅺ) of 10 mM 6-AHA. Lines represent the fits by the equations described previously (5,6) with the parameters given in the "Results and Discussion." Activation reactions were performed and analyzed as described under "Experimental Procedures." similar affinity of 1.5 Ϯ 0.1 nM for SK⅐[Lys]Pg* formation. The v 2 dependence of SK⌬K414 was shifted to higher SK concentration, due to coupling between the formation of SK⌬K414⅐[Lys]Pg* and Pm generation, corresponding to K A 63 Ϯ 54 nM for SK⌬K414⅐[Lys]Pg* formation. Values of K A determined kinetically in the absence and presence of 6-AHA were in good agreement with the dissociation constants determined above by competitive binding. The bimolecular rate constants (k Pg ), representing Pg substrate binding and Pm formation, were indistinguishable for wtSK and SK⌬K414, as indicated by the same v 2 maxima. In the presence of 6-AHA, v 2 was decreased 20-fold for wtSK and an indistinguishable ϳ25fold for SK⌬K414. Analysis of reaction progress curves by numerical integration of the rate equations gave K A 3.2 Ϯ 0.1 nM and 88 Ϯ 7 nM for wtSK and SK⌬K414, respectively, in the absence of 6-AHA, and indistinguishable k Pg of 0.0006 Ϯ 0.0001 nM Ϫ1 s Ϫ1 , whereas in the presence of 6-AHA, K A for wtSK and SK⌬K414 increased to 39 Ϯ 1 nM (12-fold) and 131 Ϯ 8 nM (1.5-fold), respectively.
In Pm kringles enhances SK⅐Pg* and SK⅐Pm catalytic complex formation. The Pg substrate interaction is concluded to be mediated by a distinct SK structure responsible for Pg substrate recognition by the SK⅐Pg* and SK⅐Pm complexes. Further studies will be required to clarify the SK structure responsible for the LBS dependence of Pg substrate recognition.