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

N. Wakeham1, S. Terzyan1, P. Zhai1, J.A. Loy2,3, J. Tang2 and X.C. Zhang1,4

1 Crystallography Research Program and 2 Protein Studies Program, Oklahoma Medical Research Foundation, 825 N.E. 13th Street,Oklahoma City, OK 73104, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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, SK{alpha}, provides the complex with substrate recognition towards Pg. SK{alpha} 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 SK{alpha} 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 SK{alpha} (SK{alpha}{Delta}) complexed with the protease domain of Pg were determined. While the structure of SK{alpha}{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 SK{alpha}{Delta} triggered similar conformational changes in the Pg active site in both crystal forms.

Keywords: crystal structure/plasminogen activation/protein complex/streptokinase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptokinase (SK), a protein secreted by many hemolytic strains of streptococci, is a potent activator that converts human plasma plasminogen (Pg) to the fibrinolytic protease, plasmin (Pm). Thus, SK is widely used therapeutically for the treatment of acute myocardial infarction (Bachmann, 1994Go; Collen and Lijnen, 1995Go). This 414-residue protein contains three domains of similar size and folding (Conejero-Lara et al., 1998Go; Wang et al., 1998Go), referred to as {alpha}, ß and {gamma}, respectively. Human Pg is also a multidomain protein consisting of an N-terminal peptide, five kringle motifs and a C-terminal catalytic domain, which is homologous to trypsin and, once activated, possesses a broad substrate specificity (Bachmann, 1994Go; Parry et al., 2000Go). The catalytic domain alone is commonly referred to as micro-Pg (or micro-Pm) (i.e. µPg or µPm). Proteolytic activation of Pg in vivo is initiated with the cleavage of the Arg561–Va562 peptide bond by specific proteases tissue-type Pg activator or urokinase. SK, which does not possess intrinsic proteolytic activity, activates Pg by forming a tight binding 1:1 complex with a dissociation constant in the nanomolar range (Rodriguez et al., 1995Go; Conejero-Lara et al., 1998Go). With its activation bond intact, the Pg moiety in a SK:Pg activator complex expresses proteolytic activity, which catalyzes the conversion of other Pg molecules to Pm by proteolysis (Castellino, 1979Go). SK also forms an activator complex with Pm with at least 1000-fold higher affinity than its complex with Pg (Boxrud et al., 2000Go).

The crystal structure of the SK and µPm complex has an overall shape of a three-sided crater, with the active site of µPm at the bottom of the crater and the three domains of SK forming the rim (Wang et al., 1998Go). Although the three domains of SK are similar, evidence suggests that their functions in Pg activation are different. Various constructs of SK and Pg fragments have been studied in efforts to dissect the interactions between the two proteins (Reed et al., 1995Go; Rodriguez et al., 1995Go; Young et al., 1995Go; Conejero-Lara et al., 1998Go; Nihalani et al., 1998Go; Loy et al., 2001Go). These studies, together with the crystal structure of the SK:µPm complex, have provided a coherent picture about the functions of each individual SK domain in Pg activation. The main function of the SK{alpha} domain is to provide the SK:Pg complex with specificity to recognize substrate Pg molecules (Nihalani et al., 1998Go; Wang et al., 1998Go; Loy et al., 2001Go). The N-terminus of native SK is able to augment the formation of an active site within the Pg–SK complex, presumably by mimicking the neo-N-terminus generated from proteolytic activation (Wang et al., 1999aGo). The role of the SK{gamma} domain is to induce Pg active site formation through a ‘contact activation mechanism’ (Loy et al., 2001Go; Wu et al., 2001Go), in which an active conformation of Pg is attained by virtue of complex formation. For maximal efficiency of active site formation, however, both the native SK N-terminus and the SK{gamma} domain are required (Loy et al., 2001Go; Sazonova et al., 2001Go). The SKß domain may have multiple functions, including bridging the {alpha} and {gamma} domains, triggering a Pg conformational change from the closed form to an open form and binding of substrate Pg (Lin et al., 1996Go; Wang et al., 1998Go; Loy et al., 2001Go).

The SK{alpha} domain (residues 1–147) is structurally and functionally similar to another bacterial Pg activator, staphylokinase (SAK), produced by staphylococci (Rabijns et al., 1997Go; Parry et al., 2000Go). It is also homologous in amino acid sequence to the N-terminal domain of SK-uberis, a two-domain bovine-Pg activator produced by Streptococcus uberis (Johnsen et al., 2000Go). Both SAK and an isolated SK{alpha} domain require preexisting Pm to form a functional Pg activator complex (Grella and Castellino, 1997Go; Loy et al., 2001Go). However, SK{alpha} binds to Pg with a 10-fold higher KD than does SAK (Rodriguez et al., 1995Go; Arai et al., 1998Go) and is less effective in Pg activation (Loy et al., 2001Go). Full-length SK, on the other hand, is the most potent Pg activator in vitro (Lee et al., 1988Go), suggesting that there is cooperativity among its three domains. SK{alpha} contains a surface loop in the region of residues 45–70, which is absent in both SAK and SK-uberis. This loop is highly mobile and its conformation undefined in the crystal structure of the SK:µPm complex (Wang et al., 1998Go). In the present study, we investigated the effects of deletion in this loop on the amidolytic activity of the SK:Pg complex and on Pg activation. In parallel, SK{alpha} containing the same deletion was co-crystallized with µPg and two crystal structures of this complex were determined. Both structures reveal an unexpected new SK{alpha}–µPg interaction, which may shed light on the contact–activation mechanism brought about by the SK{gamma} domain within a normal SK:Pg complex.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutation construction

To assess the function of the 45–70 loop of SK, truncation mutants were generated in both full-length SK and the isolated SK{alpha} domain, denoted as SK{alpha} and SK{alpha}{Delta}, respectively. These mutant genes were obtained from modification of the previously described SK and SK{alpha} genes (Loy et al., 2001Go). Residues 48–59 were deleted using a nested polymerase chain reaction (PCR) method and the sense primer 5'-A ATT GAC CTA ACA TCA CGA CCT GCT {Delta} TCA AAA CCA TTT GCT ACT GAT AGT GG-3', where {Delta} marks the deletion position. The resulting amino acid sequence deletion in SK is HGGKTEQGLSPK. Recombinant SK{Delta} and SK{alpha}{Delta}, expressed in Escherichia coli, contain an extra methionine residue (Met0) at the N-terminus derived from the starting codon. In addition, both constructs contain a Trp to Ala substitution at position 6 which was inherited from the parental SK gene (Loy et al., 2001Go). It has been shown that this residue is located in the flexible N-terminal peptide and no effect of this point mutation on either amidolytic or Pg activator activities has been reported.

The human Pg catalytic domain (residues 542–791) containing an active-site Ser741 to Ala mutation (µPgS741A) has been described previously (Wang et al., 2000Go). This mutation eliminates potential proteolytic activity during crystallization experiments. An Arg561 to Ala mutation of µPg (µPgR561A), resistant to proteolytic activation, was constructed using a PCR primer, 5'-AAG AAA TGT CCT GGA GCG GTT GTG GGG GGG TGT G-3'. The double mutant µPgR561A,R719E was constructed using the primer 5'-TTT CTG AAT GGA GAA GTC CAA TCC ACC-3'. Each of the µPg recombinant proteins contains an N-terminal peptide of sequence MASMTGGQQMGRGSGS, adopted from the expression vector. All constructs were verified by DNA sequencing.

Protein purification and characterization

All SK and µPg variants were over-expressed as inclusion bodies from pET11 vector (Novagen, Madison, WI) in E.coli strain BL21(DE3). The refolding procedure was essentially the same as described previously (Loy et al., 2001Go). SK{Delta} and SK{alpha} were purified over a Sephacryl S-300-HR column (Pharmacia, Piscataway, NJ), full-length SK over a Sephacryl-200-HR, and SK{alpha}{Delta} over a Superdex-75, all in a buffer containing 20 mM Tris–HCl (pH 8.0) and 0.4 M urea. Each protein was further purified using Resource-Q anion-exchange chromatography (Pharmacia) in the same buffer and eluted with a 0–1.0 M NaCl gradient. The µPg mutants were purified over a Sephacryl-200-HR column in a buffer containing 20 mM HEPES (pH 7.0) and 0.4 M urea. Protein purities were confirmed on SDS–PAGE silver-stained Phastgels (Pharmacia) under both reduced and non-reduced conditions. Protein concentrations were analyzed using absorbance at 280 nm with the following extinction coefficients: 4868 (AU/M) for SK{alpha}, 32588 for SK, 50551 for µPg and its point mutation variants (Loy et al., 2001Go), 39 519 for SK{Delta} and 4960 for SK{alpha}{Delta} (determined by mass-spectroscopic amino acid analysis).

In addition, the qualities of the recombinant proteins were tested using functional assays (Loy et al., 2001Go). µPgR561A,R719E was compared with µPgR561A in active site formation using a SK construct containing the ß and {gamma} domains; the two µPg variants showed no difference in their amidolytic activity towards N-p-tosyl-glycine-proline-lysine-p-nitroanilide (N-p-tosyl-GPK-pNA; Sigma-Aldrich, St. Louis, MO) (data not shown). Since the R719E mutation is only relevant to the binding of the SK{alpha} domain (Wang et al., 1998Go), the above result indicates that the refolded µPgR561A,R719E sample was comparable to the µPgR561A sample used in other assays.

SDS–PAGE analysis of the SK:µPgR561A complex

The Met0–SK:µPgR561A complex was incubated at 1–200 µM concentrations in a buffer containing 0.1 M HEPES (pH 7.2) and 0.1% (w/v) polyethylene glycol 2000 (PEG 2K) at 22°C. Samples of the reaction mixtures were taken at different time points, up to 24 h, and analyzed by SDS–PAGE. Protein bands were visualized by either Coomassie blue or silver stain. The major degradation product was verified by N-terminal sequence analysis. The assay was repeated for both SK{Delta} and SK with the N-terminal methionine removed (see below).

Removing the N-terminal methionine from recombinant SK variants

N-terminal Met0 of recombinant SK and SK{Delta} was removed for activity assays using the E.coli enzyme methionine aminopeptidase (Met-AP) (Lowther et al., 1999Go). The method adopted from the published procedure consisted of a reaction mixture of SK (40 µM) and Met-AP (2.0 µM) in a reaction buffer of 20 mM bis-tris-propane (pH 7.1), 50 mM KCl, and 2 mM CoCl2. The reactions were carried out at 30°C for 4 h and were stopped by the addition of 2 mM EDTA (pH 8.2) for 15 min at 22°C. The samples were dialyzed overnight against water at 4°C. The completeness of each reaction was determined using N-terminal sequence analysis. It is estimated that 100% Met0 was removed from SK, and 75% from SK{Delta}.

Amidolytic activity assay

The chromogenic substrate N-p-tosyl-GPK-pNA was used for amidolytic activity assays of the µPg variants. The absorption of the reaction product, p-nitrophenol, was monitored at 430 nm, which gave a lower background reading than at 406 nm. A linear relationship between the hydrolysis of N-p-tosyl-GPK-pNA and the absorbance at 430 nm was observed within the 0.0–0.5 AU range, with an extinction coefficient, {varepsilon}430, of 4714 AU/M determined experimentally using mass-spectroscopic analysis.

For the kinetic studies of enzymatic activity generated by the SK (or SK{Delta}) and µPgR561A complex, equimolar concentrations of SK and µPgR561A (0.1 or 0.5 µM) were pre-incubated in a quartz micro-cuvette for 3 min at 22°C in a buffer containing 0.1 M HEPES (pH 7.4) and 0.1% (w/v) PEG 2K. Various concentrations of substrate N-p-tosyl-GPK-pNA (0.0–7.5 mM) were added and the reactions were immediately monitored for substrate hydrolysis at 430 nm using the Beckman DU-640 spectrophotometer (Beckman Coulter, Inc., Fullerton, CA). PEG 2K was added to stabilize the enzyme and to reduce adsorption of the protein sample to the cuvette. The reaction rate of the linear phase of each assay was plotted as a function of substrate concentration. The KM and Vmax were calculated from the Michaelis–Menten equation using non-linear regression analysis (GraphPad Prism, San Diego, CA).

One-stage Pg activation assays

Pg activation by the SK variants was examined using one-stage activation assays. Native human-Pg was purified from plasma following previously published procedures (Deutsch and Mertz, 1970Go). To remove trace Pm, the Pg sample was further purified using an aprotinin agarose column (Sigma-Aldrich). SDS–PAGE analysis verified that the final purified sample consisted of Glu1-Pg. The active site concentration of human-Pg after activation was determined by the burst titration of Chase and Shaw (Chase and Shaw, 1969Go). SK or its variants (final concentration of 0.1–0.5 nM) was mixed with human-Pg (0.25 µM) and substrate N-p-tosyl-GPK-pNA (500 µM) in a buffer consisting of 0.1 M HEPES (pH 7.2) and 0.1% (w/v) PEG 2K. The generation of amidolytic activity was monitored at 22°C by measuring the increase in absorption at 406 nm. The absorption data from the rapid-rising phase of the assay was fitted to the equation, A = a(tt0)2, where A is the optical absorption, a is the acceleration rate and t0 is the so-called lag time for most SK to complex with Pm thus obtaining maximum Pg activator activity. Since only data from the rapid-rising phase was used, the result corresponds to the maximal stable activity of the complex. The acceleration rate, a, is related to Pg activator efficiency (kcat/KM) by the equation:

where [E] is the concentration of the Pg activator, SK:Pm; [S] is the concentration of substrate, human-Pg; RPm is the specific rate at which p-nitrophenol is produced by Pm; and {varepsilon}406 is the extinction coefficient of p-nitrophenol at 406 nm. Since the dissociation constant of the SK:Pm complex is in the picomolar range (Boxrud et al., 2000Go), under the condition of maximal stable activity the concentration of the SK:Pm complex is essentially the concentration of SK. The value of ([S]RPm{varepsilon}406) was experimentally determined to be 4.0x10–2 AU/s at 0.25 µM concentration of human-Pm: equimolar Pg and full-length recombinant SK (0.25 µM final) were incubated at 22°C for 5 min, N-p-tosyl-GPK-pNA was then added to 500 µM and the rate of absorption change was measured at 406 nm. In this assay applied to Met0–SK and Met0–SK{Delta}, the derived kcat/KM value was found to be approximately independent of the concentration of SK, but the lag time (t0) was inversely related to the concentration of SK (data not shown).

Binding affinity study using surface plasmon resonance

Dissociation constants between µPg variants and immobilized SK{alpha} or SK{alpha}{Delta} were determined by surface plasmon resonance using a BIAcore 1000 biosensor (BIAcore Inc., Sweden) as described previously (Loy et al., 2001Go). In these experiments, SK{alpha} and SK{alpha}{Delta} were separately immobilized onto the surface of a carboxylated dextran matrix sensor chip. Various concentrations of µPgR561A,S741A or µPgR561A,R719E (0.1–2.0 and 1.0–20.0 µM, respectively) were injected over the sensor surface and the binding interactions were recorded in the form of sensorgrams. Associations were measured during sample injection (80 s) and then dissociations were measured during injection of the running buffer alone (100 s). After each cycle, the sensor surface was regenerated by injecting 3.5 M urea in 0.1 M Tris–HCl (pH 7.2) followed by the running buffer. After subtraction of the non-specific refractive index component, the dissociation rate constants (koff) and association rate constants (kon) were calculated from the sensorgrams by non-linear fitting of the association and dissociation data based on a 1:1 complex model using BIAevaluation software version 3.0. Equilibrium dissociation constants were then calculated using the equation KD = koff/kon.

Crystallization

Two crystal forms (Forms I and II) were obtained for the SK{alpha}{Delta}:µPgS741A complex. The complex was prepared by mixing SK{alpha}{Delta} at a concentration of 18 mg/ml in a buffer containing 20 mM Tris–HCl (pH 8.0) and 0.4 M urea with µPgS741A at a concentration of 16 mg/ml in a buffer containing 20 mM HEPES (pH 7.5), 0.4 M urea and 0.02% (w/v) NaN3 in a 1:1 (v/v) ratio. Form I crystals were grown at 20°C from a hanging drop vapor diffusion experiment, in which the complex sample (2 µl) was mixed with an equal volume of the reservoir solution containing 1.0 M sodium acetate, 0.1 M HEPES (pH 7.5) and 50 mM cadmium sulfate. For the second crystal form (Form II), the same complex sample was equilibrated with a reservoir solution of 1.0 M Li2SO4, 50 mM HEPES (pH 7.0) and 10 mM magnesium acetate. Crystals were grown from a hanging drop set-up at 20°C. Crystals appeared in approximately 2 weeks. The above reservoir solutions containing an additional 30% glycerol were used as the cryo-solution to soak the crystals before being cooled in a 100 K nitrogen gas stream for data collection. Complete data sets were collected at 2.8 Å resolution for Form I and 2.3 Å resolution for Form II, from a MAR345 image-plate (Mar Research Inc., Norderstedt, Germany) and Riguku X-ray generator (Molecular Structure Co., Woodlands, TX) equipped with an Osmic mirror system (Osmic Inc., Troy, MI).

Structure determination

Crystal Form I belongs to the P6522 space group. One asymmetric unit contains one SK{alpha}{Delta} and one µPgS741A molecule with 56% solvent content. A solution for the µPg molecule was determined by the molecular replacement method using the program AMoRe (Navaza, 1994Go) and our previously determined µPgS741A crystal structure (PDB code: 1ddj) as a template. However, a molecular replacement search did not yield a convincing solution for the SK{alpha}{Delta} component. The initial sigma-A weighted phases (Read, 1986Go) calculated from the µPg partial structure were improved by electron density modification, including solvent flattening and histogram mapping, performed with the program DM in the CCP4 package (Bailey, 1994Go). A structural model of SK{alpha}{Delta} was built from this improved electron density map using the corresponding part of the SK:µPm complex structure (PDB code: 1bml) as a reference. The complex structure was refined with CNS (Brunger et al., 1998Go). The final refined model includes residues 544–791 of µPg, and residues Met0–Lys147 of SK{alpha}{Delta}. Most of the remaining part of the truncated 45–70 loop (i.e. residues 46, 47, 60–68) of SK{alpha}{Delta} is missing from the electron density map.

Crystal Form II belongs to the space group C2221. There is one molecule of each of the two proteins per crystallographic asymmetric unit with 47% solvent content. The phases were determined with the molecular replacement method using coordinates of µPg and SK{alpha}{Delta} from the Form I crystal. The final refined model of Form II includes residues 543–791 of µPg, and residues 14–147 of SK{alpha}{Delta}. The N-terminal residues prior to Asn14 and residues 47 and 60–68 in the truncated loop of SK{alpha}{Delta} are missing.


    Results
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 References
 
SK:µPgR561A complex auto-cleaves at the Lys59–Ser60 bond of SK moiety

SK is known to rapidly degrade upon complex formation with Pm or µPg (Wu et al., 1998; Boxrud et al., 2000Go). To investigate if the Met0–SK:µPgR561A complex was stable against proteolysis during assay, samples were taken at various incubation time after the complex formation and analyzed in SDS–PAGE (Figure 1Go). At 20 h, over 95% of SK had been converted into a 41 kDa fragment while µPgR561A remained unchanged. N-terminal sequence analysis established that cleavage occurred between Lys59 and Ser60 of SK, an autolytic site reported previously (Boxrud et al., 2000Go). An unaltered µPgR561A band confirmed that the mutation had prevented the conversion of µPg to µPm, therefore, the SK cleavage must have been carried out by the complex. The half-life for the cleavage of Met0–SK was estimated to be 6, 3.5 and 1.5 h for 50, 100 and 200 µM of complex, respectively. The cleavage of SK was much faster when the extra N-terminal methionine was removed. The half-life of 100 µM SK (with Met0 removed) in an equimolar mixture with µPgR561A was approximately 10 min. At a concentration of 0.5 µM, below which most of our activity assays were performed, the half-life of SK was longer than 30 min. Therefore, proteolytic degradation of SK within a SK-µPgR561A mixture would be negligible during assays using a short incubation time.



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Fig. 1. Stability of the SK:µPgR561A complex. Equimolar (100 µM) µPgR561A and Met0–SK were mixed and incubated at 22°C. Aliquots were removed at various time points and analyzed by 20% SDS–PAGE, which was stained with Coomassie blue. SK' stands for the major SK component (i.e. residues 60–414) after proteolytic cleavage. The half-life of SK under the experimental conditions is estimated to be 3.5 h.

 
Similar activation experiments were carried out with SK{Delta} and µPgR561A. The SK{Delta}:µPgR561A complex did not display auto-cleavage after 24 h incubation in concentrations up to 100 µM. This indicates that the SK Lys59–Ser60 bond, which is absent in the SK{alpha} construct, is the only major site cleaved by a SK:µPg complex, although it may also be cleaved by Pm under other experimental conditions. Other SK fragments observed previously (Shi et al., 1994Go) are likely mediated by free Pm generated by the SK:Pg (Pm) complex. Whether the kringle domains in a SK:Pm complex would change the substrate specificity or substrate presentation remains to be determined.

The 45–70 loop of SK does not participate in active site formation of the activator complex

We studied the amidolytic activity of SK{Delta}:µPgR561A complex in order to assess a possible role of the 45–70 loop of SK during active site formation in a SK:Pg complex. Due to the R561A mutation at the Pg activation bond, the hydrolysis of substrate N-p-tosyl-GPK-pNA is a measure of the non-proteolytic activation of µPgR561A. We found that the KM and kcat values of both the Met0–SK:µPgR561A and Met0–SK{Delta}:µPgR561A complexes are virtually the same (Table IGo). Although the removal of Met0 at the N-terminus of SK and SK{Delta} increased the kcat values it did not significantly differentiate the kinetic parameters between the complexes (Table IGo). These results indicate that the 45–70 loop of SK plays no significant role in the contact activation mechanism regardless whether the native SK N-terminus is present or not.


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Table I. Kinetic data of the amidolytic activity of µPgR561A complexed with SK variants
 
The SK 45–70 loop plays an important role in the recognition of substrate Pg

We observed that Met0–SK{Delta} had a much decreased rate for Pg activation than that of Met0–SK (Figure 2Go and Table IIGo). This difference was maintained even after the removal of extra Met0, which also eliminates the lag time for both SK and SK{Delta} (Figure 2Go). The longer lag time in the Met0–SK{Delta}:Pg assay corresponds to the loss of both N-terminal insertion activation mechanism and substrate recognition, thus resulting in slower Pm production for forming SK:Pm complexes. These findings support the view that the SK 45–70 loop is involved in the recognition of substrate Pg.



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Fig. 2. Activation of Pg by SK variants. Activation of 0.25 µM human Pg by 0.25 nM Met0–SK{Delta}, Met0–SK and their Met0 removed versions (SK{Delta} and SK) was separately measured using one-stage activation assays at 22°C. A negative control without activator is also shown. The generation of Pm activity was monitored using substrate N-p-tosyl-GPK-pNA at 406 nm wavelength.

 

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Table II. Pg activation efficiency of SK variants
 
SK{alpha} as an isolated domain binds to the same Pg site as it does in full-length SK

The binding and dissociation rates of SK{alpha} and SK{alpha}{Delta} with µPgR561A, S741A was determined using surface plasmon resonance. The calculated association and dissociation rate constants (Table IIIGo) indicated that SK{alpha}{Delta} has approximately 3-fold higher KD towards µPg than does SK{alpha}. The KD of SK{alpha} towards µPgR561A,S741A is comparable with the previously reported value (65.8 nM) for SK{alpha} to µPgS741A (Loy et al., 2001Go).


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Table III. Affinity data for SK{alpha} variants binding to µPg variants
 
The residues involved in the binding of SK{alpha} to µPg have been defined by the crystal structure of the SK:µPm complex (Wang et al., 1998Go). Pg Arg719 which forms a buried salt bridge with SK Glu34 (Wang et al., 1998Go) has also been shown to play a critical role in SK:µPm complex formation (Dawson and Ponting, 1994Go). To verify the importance of this interaction in the SK{alpha}:µPg complex, we mutated Arg719 to Glu in µPgR561A to form a double mutant. In contrast to µPgR561A,S741A, µPgR561A,R719E showed no detectable binding to either SK{alpha} or SK{alpha}{Delta} (Table IIIGo). Since we had established that recombinant µPgR561A,R719E refolded correctly and had high purity, the absence of binding was attributed to the loss of Arg719. These observations also support the contention that an isolated SK{alpha} domain predominantly binds to the same site on µPg as does it in the context of a full-length SK. The lack of binding, thus the implied conclusion, persisted up to at least 20 µM in our experiments. As will be discussed below, at millimolar concentrations, however, SK{alpha}{Delta} and µPg were co-crystallized in a packing different from the interaction previously observed in the crystal structure of the SK:µPm complex (Wang et al., 1998Go).

Crystallography studies

To further study the structure of SK{alpha}{Delta} and its interaction with µPg, we determined the three-dimensional structures of the complex of SK{alpha}{Delta} and µPgS741A from two distinct crystal forms (I and II) (Figure 3Go). To our surprise, the SK{alpha}{Delta}–µPg interactions in these two crystal forms were totally different from that in the previously determined structure of the SK:µPm complex (Wang et al., 1998Go).The statistics of data collection and refinement are shown in Table IVGo. Coordinates of both crystal forms have been deposited to the Protein Databank (PDB) under codes 1l4d and 1l4z.



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Fig. 3. Stereo view of a ribbon diagram of the SK{alpha}{Delta}:µPg complex. µPg is shown in cyan, and SK{alpha}{Delta} in red. The coordinates are from the Form II crystal. Superimposed on the complex is full-length SK, shown in thin yellow wires, to illustrate a hypothetical stereo overlap of SK{alpha}{Delta} in this complex and the SK{gamma} domain in the SK:µPm complex (Wang et al., 1998Go). The SK {alpha}, ß and {gamma} domains are labeled as SKa, SKb, and SKg. The N- and C-termini are labeled as N and C, respectively. Positions of the visible ends of the 45–70 loop in both complex structures are labeled. Positions of the three critical point mutations in µPg are marked as dark blue spheres and are labeled. This Figure, in addition to Figures 4 and 5GoGo, was drawn with the program MolScript (Kraulis, 1991Go)

 

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Table IV. Crystallography data collection and refinement statistics
 
SK{alpha}{Delta} domain structures. Although residues 48–59 are absent in SK{alpha}{Delta}, its overall structures in both crystal forms are essentially identical to that of the SK{alpha} domain in the SK:µPm complex (Wang et al., 1998Go). The regions of secondary structure elements (Wang et al., 1999bGo; Parry et al., 2000Go) are as follows: ß1, residues 17–26; ß2, residues 32–33; ß2', residues 38–43; ß3, residues 73–75; {alpha}-helix {alpha}3,4, residues 76–89; ß4, residues 98–103; ß4', residues108–110; ß5, residues 115–118; ß6, residues 123–127; and ß7, residues 133–144. The root mean square deviation (r.m.s.d.) of C{alpha} atom coordinates is 0.8 Å (110 residues, using a 3 Å cut-off) between SK{alpha}{Delta} models of the two crystal forms, and 0.8 and 0.9 Å (101 residues) between Forms I/II and the SK{alpha} domain in the SK:µPm complex. Shortening of the 45–70 loop by 12 residues does not appear to reduce its mobility as most of the loop remains invisible in the new structures. In the Form I structure, a part of the N-terminal peptide (residues Met0–Ser12) links a few hydrogen bonds with strand ß2 of a neighboring SK{alpha}{Delta} molecule, resulting in an extension of its major ß-sheet. Having a different crystal packing, the N-terminal peptide in the Form II structure is mobile and could not be located in the electron density map. The largest conformational difference between SK{alpha}{Delta} and the SK{alpha} domain in full-length SK, aside from the 12-residue deletion and its flanking regions, is located in the loop region between helix {alpha}3,4 and strand ß4, which is part of interface in the SK:µPm complex (Wang et al., 1998Go). On the other hand, the conformations of this region in the new structures are nearly the same despite different crystal packings. Taken together, these observations suggest that an induced conformational change of the SK 90–96 loop had taken place upon complex formation between full-length SK and µPm.

µPg structures. The overall structures of µPg in Forms I and II are similar to each other, with a 0.6 Å RMSD for 239 C{alpha} atom pairs (3 Å cut-off). The largest conformational differences are located in two regions: (i) two closely located loops, residues 729(183)–732(186) and 766(220)–770(224) (chymotrypsin-numbering system in parenthesis); (ii) the autolysis loop [residues 688(144)–694(150)]. In (i), the C{alpha} atom coordinate shift of approximately 3 Å can be explained by the difference in crystal packings. In (ii), the coordinate shift of approximately 5 Å may be attributed to the slight difference in binding with SK{alpha}{Delta}. Not surprisingly, the present µPg structures have conformations more similar to those of free zymogen µPg (Peisach et al., 1999Go; Wang et al., 2000Go) (with a 2.0 Å overall r.m.s.d. between their C{alpha} atoms) than that of µPm in complex with SK (Parry et al., 1998Go; Wang et al., 1998Go) (with a 3.0 Å r.m.s.d.). In both crystal forms, the active site region of µPg assumes a non-functional conformation (Figure 4Go) in which the activation bond (Arg561(15)–Val562(16)) is clearly intact, the oxyanion hole is not formed and the ‘switch’ residue, Asp 740(194), is in the zymogen conformation. The S1 specificity pocket is essentially in the zymogen form, unoccupied by Asp735(185) which now protrudes into the solvent. The side chain of Trp731(215), normally blocking the S1 pocket in the zymogen structure, moves to a midway position towards its enzyme conformation. At such a position, its side chain stacks with the imidazole ring of the catalytic His603(57), keeping the latter in a non-functional rotamor. Another catalytic residue, Asp646(102), also assumes a non-functional rotamor. A disulfide bond, Cys737(191)–Cys765(219), which shapes the entrance of the S1 pocket, is in a position unlike that in either the enzyme or the zymogen.



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Fig. 4. Stereo view of a structural comparison of µPm and µPg. The active sites and autolysis loops are shown after superimposing the entire catalytic domains. The backbone trace of the free µPg structure [PDB ID: 1ddj (Wang et al., 2000Go)] is shown in blue, µPm [PDB ID: 1bml (Wang et al., 1998Go)] in red, and µPg from the SK{alpha}{Delta}:µPg complex (Form II) in green. Positions of some key residues are labeled as the amino acid name (single letter) followed by Pg residue number. The chymotrypsin numbers are included in parenthesis for the catalytic residues.

 
Novel SK{alpha}-µPg interaction. In spite of different crystal packings, the two co-crystal forms share a similar µPg–SK{alpha}{Delta} interaction, each with a large buried surface area of 1280 Å2 in Form I and 1460 Å2 in Form II. This zipper-like interaction mode involves structural motifs of ß-turn1,2 (SK, residues 27–31), activation loop [Pg, residues 559(14)–565(20)], 105 loop (SK, residues 104–107), autolysis loop [Pg, residues 685(141)–697(155)] and 120 loop (SK, residues 118–123) (see Figure 3Go). Side chains of Thr691(147) and Phe692(148) at the tip of the Pg autolysis loop become deeply buried in a hydrophobic pocket in SK formed by the N-terminus of helix {alpha}3,4, strand ß4' itself and loops flanking it (Figure 5Go). While the zipper interaction between µPg and SK{alpha}{Delta} is the same in the two crystal forms, their SK{alpha}{Delta}:µPg complexes are, however, not identical. Superimposing the µPg molecules would result in an 8 Å translation in the mass center and a 39° rotation between the two SK{alpha}{Delta} partners. The hinge axis is located in the SK{alpha}{Delta}–µPg interface and passes roughly through the C{alpha} atoms of Thr25 and Ser105 of SK. In either structure, no symmetry related µPg molecules are found in an enzyme–substrate-like spatial relationship. In addition, µPg and SK{alpha}{Delta} molecules are found in neither an enzyme–cofactor interaction, as that observed in the SK:µPm crystal structure (Wang et al., 1998Go), nor a cofactor–substrate interaction, as that observed in the µPm:SAK:µPm ternary complex (Parry et al., 1998Go).



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Fig. 5. Stereo view of the knot-and-hole interface between SK{alpha}{Delta} and µPg. The backbone trace of µPg is shown in cyan and SK{alpha}{Delta} in red. Side chains involved are shown as stick models and are colored based on atom types.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
SK from Streptococcus equisimilis contains a surface loop (residues 45–70) in its N-terminal domain, which is susceptible to protease digestion. In fact, the peptide bond at SK Lys59–Ser60 is among the first to be cleaved upon mixing SK with Pg (Parrado et al., 1996Go). In the crystal structure of the SK:µPm complex (Wang et al., 1998Go), this bond was cleaved and most of the loop was invisible in the electron density map. This loop is, however, located inside the projected binding pocket for substrate Pg in the SK:µPm complex. Our data from comparative studies of SK{Delta} and wild-type SK demonstrated that the SK 45–70 loop is important for Pg activation (Figure 2Go and Table IIGo). A similar conclusion was reached by others from mutagenesis studies on the N-terminal region of this loop (Nihalani et al., 1998Go; Kim et al., 2000Go). As a working hypothesis, it seems probable that this loop or its proteolytically cleaved form forms a flexible arm in the SK:Pg activator complex which wraps around the substrate Pg molecule. Thus, a truncated loop results in a non-productive conformation for substrate recognition.

SK activation of Pg in a complex involves two mechanisms, namely N-terminal insertion and contact activation. The extra N-terminal methionine (i.e. Met0) in recombinant SK was therefore used to dissect the functional role of the 45–70 loop in each mechanism. In the presence of Met0, the insertion mechanism is impaired, leaving the contact activation as the only viable mechanism (Loy et al., 2001Go). The fact that the activation complex with SK{Delta} has almost identical kinetic parameters for amidolytic activity as the complex containing native SK suggests that the 45–70 loop of SK plays no role in the contact–activation mechanism. On the other hand, clear kinetic differences between SK{Delta} and SK in the absence of the extra N-terminal Met0 (Table IGo) illustrates that the 45–70 loop plays a positive albeit accessory role in the N-terminal insertion mechanism.

The new crystal structures presented here illustrate that the removal of half of the 45–70 loop not only does not impair the folding but the resulting SK{alpha}{Delta} has the same overall conformation of SK{alpha}. This observation is in agreement with the hypothesis that the 45–70 loop is an insertion to the canonical ß-grasp folding during evolution (Parry et al., 2000Go). Based on the reduced affinity of SK{alpha}{Delta} to µPg, however, one may argue that the loop region may play some roles in facilitating a conformation of SK{alpha} domain that permits optimal binding interactions between the two proteins; however, such a possibility seems remote because of the identical overall structures of SK{alpha}{Delta} and SK{alpha}.

The crystal structures reported here represent a novel binding mode between SK{alpha}{Delta} and µPg. Since the full-length 45–70 loop in SK{alpha} would not directly prevent this binding, the new binding mode does not seem to be the consequence of the loop truncation. Nevertheless, the new crystal structures improve our understanding on the interactions of these proteins. Comparing the crystal structures of µPm (Parry et al., 1998Go; Wang et al., 1998Go), free µPg (Peisach et al., 1999Go; Wang et al., 2000Go) and µPg in the SK{alpha}{Delta}:µPg complexes, the largest structural differences occur in the three loops surrounding the active site: the 760 loop [also called the S1 entrance frame, residues 760(214)–765(219)], the oxyanion stabilizing loop [residues 737(191)–740(194)] and the autolysis loop (see Figure 4Go). The autolysis loop is where µPg/µPm contacts with SK in the SK:µPm (Wang et al., 1998Go) and the two SK{alpha}{Delta}:µPg complexes. Such contacts probably influence the conformation of its immediate neighbor, the oxyanion stabilizing loop. Since the oxyanion stabilizing loop and its neighbor, the 760 loop, are linked by a disulfide bond between Cys737(191) and Cys765(219), the movement of the oxyanion stabilizing loop must also carry along through the 760 loop. These movements also carry functional consequences. The oxyanion stabilizing loop is responsible for the formation of a catalytic competent oxyanion hole and the negative charge at the bottom of the S1 specificity pocket. The 760-loop conformation is responsible for the formation of the entrance of the S1 pocket. Asp740(194), which assumes two distinct conformations associated with the zymogen and enzyme forms, is an important switch residue to the conformation of these structural elements. In free µPg, the Asp740(194) side chain hydrogen bonds to the backbone amide groups of Trp685(141) and Gly686(142), while in µPm it forms a buried salt bridge with the neo-N-terminus generated during proteolytic activation. In the new structures, the conformational changes in the autolysis loop towards the enzyme form appear to cause a rotation of the peptide bond plane between residues 685(141) and 686(142), resulting in disruption of the hydrogen bond between Asp740(194) and Gly686(142). Although the detailed structural movements are not given in the crystal structures of this system, the common theme of SK{gamma} and SK{alpha}{Delta} binding to µPg suggests that binding to the autolysis loop triggers conformational changes in the active site region. The autolysis loop forms a cover for the activation pocket with the stem of this loop in direct contact with Asp740(194). Contacting with SK may cause the N-terminal stem of the autolysis loop [i.e. residues 686(142)–688(144)] to adopt a more enzyme-like conformation. It is noteworthy that the SK{gamma} domain in full-length SK and the isolated SK{alpha}{Delta} domain interact with the autolysis loop in different ways, resulting in different extents of conformational changes in the µPg active site. SK{gamma} pushes the autolysis loop towards the activation pocket, while SK{alpha}{Delta} has the tip of the autolysis loop inserted into a deep pocket. This pocket in SK{alpha}{Delta} has no equivalence in the homologous structure of SAK, excluding the possibility that SAK might use a similar binding mechanism to trigger active site formation in Pg. It may be reasonable to expect that such extensive interactions between components would have biological relevance. We suspect that it may reflect another function of SK{alpha} unrelated to Pg activation in the course of evolution, although it is also possible that the complex formation is purely an artificial result induced by the crystallization conditions.

Based on the new SK{alpha}{Delta}:µPg complex crystal structures and a better understanding of the mechanism this enzyme system utilizes, it is conceivable to engineer a new protein that specifically binds to the autolysis loop region of Pg, thus competing with the SK{gamma} domain and blocking SK mediated Pg activation. Such proteins are potentially useful to prevent Pg activation associated with Streptococcus invasions. It is noteworthy that SK{Delta} remains as an active Pg activator with an increased half-life. This is consistent with the report that conversion of Lys59 to Glu is sufficient for extending the functional half-life of SK (Wu et al., 1998). However, such a simple point mutation may introduce a new epitope for human antibodies (Parhami-Seren et al., 1997Go), which would not be a problem if the loop is cleaved or truncated. Therapeutic thrombolytic agents, such as SK and tissue-type Pg activator, have short half-lives; it is considered by some to be problematic for managing treatment. Providing alternative and improved management of thrombolytic therapy is desired in order to gain maximal benefits for the restoration of blood flow following acute myocardial infarction and other blood clotting disorders. Recently, attention has been given to develop clot-dissolving agents with both prolonged half-lives and reduced antigenicity. The results presented in this report provide information for creating novel forms of SK as potent as the native, but with the potential for more stringent applications.


    Notes
 
3 Present address: ProteomTech Inc., 655 Research Parkway, Suite 575, Oklahoma City, OK 73104, USA. Back

4 To whom correspondence should be addressed. E-mail: zhangc{at}omrf.ouhsc.edu Back


    Acknowledgments
 
We thank A.Howerton for technical assistance. PCR primer synthesis and N-terminal sequencing analysis were performed by the Molecular Biology Resource Facility, William K. Warren Medical Research Institute, University of Oklahoma Medical Center. This work was supported by NIH grant HL60626 and resources of the Oklahoma Medical Research Foundation.


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Received March 20, 2002; revised May 1, 2002; accepted May 6, 2002.





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