Coiled Coil Region of Streptokinase
-Domain Is Essential
for Plasminogen Activation*
Dung-Ho
Wu
,
Guey-Yueh
Shi,
Woei-Jer
Chuang,
Jung-Mao
Hsu,
Kung-Chia
Young§,
Chung-Wen
Chang, and
Hua-Lin
Wu¶
From the Departments of Biochemistry and § Medical
Technology, College of Medicine, National Cheng Kung University,
Tainan, Taiwan 701 and
Department of Pharmacy, Chia Nan
University of Pharmacy and Science, Tainan,
Taiwan 710, Republic of China
Received for publication, July 6, 2000, and in revised form, January 9, 2001
 |
ABSTRACT |
The specific functions of the amino acid residues
in the streptokinase (SK)
-domain were analyzed by studying the
interactions of human plasminogen (HPlg) and SK mutants prepared
by charge-to-alanine mutagenesis. SK with mutations of groups
of amino acids outside the coiled coil region of SK
-domain,
SKK278A,K279A,E281A,K282A, and
SKD360A,R363A had similar HPlg activator activities as
wild-type SK. However, significant changes of the functions of SK with
mutations within the coiled coil region were observed. Both
SKD322A,R324A,D325A and
SKR330A,D331A,K332A,K334A had decreased amounts of complex formation with microplasminogen and failed to activate HPlg.
SKD328A,R330A had a 21-fold reduced catalytic efficiency
for HPlg activation. The studies of SK with single amino acid mutation
to Ala demonstrate that Arg324, Asp325,
Lys332, and Lys334 play important roles in the
formation of a HPlg·SK complex. On the other hand, amino acid
residues Asp322, Asp328, and Arg330
of SK are involved in the virgin enzyme induction. Potential contact
between Lys332 of SK and Glu623 of human
microplasmin and strong interactions between Asp328 and
Lys330, Asp331 and Lys334, and
Asp322 and Lys334 of SK are noticed. These
interactions are important in maintaining a coiled coil conformation.
Therefore, we conclude that the coiled coil region of SK
-domain,
SK(Leu314-Ala342), plays very important roles
in HPlg activation by participating in virgin enzyme induction and
stabilizing the activator complex.
 |
INTRODUCTION |
Streptokinase (SK),1 a
potent human plasminogen (HPlg) activator, is a single chain secretory
protein produced by strains of
-hemolytic Streptococcus
(1-3). The HPlg and SK complex can activate HPlg to plasmin (HPlm)
which in turn catalyzes the hydrolysis of fibrin and dissolution of
blood clots. SK, therefore, has been used as a thrombolytic agent in
treatment of thromboembolic blockages in the blood vessels such as
acute myocardial infarction.
The NMR spectra of SK and the crystal structure of the catalytic domain
of HPlm (µPlm) complexed with SK demonstrate three structurally
autonomous domains in SK (4, 5). Based on the functional studies of
truncated SK peptides, we demonstrated that SK-(158-414) in complex
with equimolar HPlg could form amidolytically active virgin enzyme (6).
The NH2-terminal domain of SK, SK-(16-251), could interact
with substrate HPlg and render it to be activated by HPlg·SK
activator complex more effectively (7). We also demonstrate that HPlg
that binds to the COOH-terminal domains of SK functions as an enzyme to
catalyze the conversion of substrate HPlg that binds to the
NH2-terminal domain of SK to HPlm (7). The binding of HPlg
and SK is very complex since HPlg consists of five kringle domains and
a catalytic domain which have different affinity for SK (7). The
crystal structure of SK and µPlm complex revealed that SK consists of
three independent domains,
,
, and
, starting from the
NH2 terminus of SK (5). Many interaction sites are proposed
based on the crystal structure of SK·µPlm complex. Extensive
charged and hydrophobic interactions between the SK
-domain
especially the major coiled coil region and the strands 
1 and 
2
with µPlm are observed (5). Direct assistance by the SK
-domain in
the docking and processing of substrate HPlg by the activator complex
have been suggested (8).
The activation mechanism of HPlg by SK involving multiple interaction
steps was suggested (7, 9-15). SK forms a stable one to one complex
with HPlg and induces a conformational change of HPlg to become a
catalytically active enzyme that is named virgin enzyme. The HPlg·SK
virgin enzyme complex is converted to HPlm·SK that functions as an
enzyme activator to catalyze the activation of substrate HPlg to HPlm.
To be an effective HPlg activator, HPlg and SK must form a stable
activator complex, which can bind to the substrate HPlg and catalyze
its conversion to HPlm. Based on the studies of the truncated SK
peptides, we propose that the COOH-terminal half of SK is essential for
maintaining a stable HPlg·SK or HPlm·SK complex and induction of
virgin enzyme activity (6, 7). On the other hand, recent studies on the mechanism by which SK forms a virgin enzyme in HPlg have led to some
controversial conclusions (16, 17). The experiments with deletion of
Ile1 of SK (
Ile1-SK) and mutations of
Ile1 of SK have demonstrated that Ile1 of SK is
required for the nonproteolytic activation of HPlg by SK (16, 17). In
this "molecular sexuality hypothesis" model, the NH2
terminus, Ile1, of SK is proposed to form a salt bridge
with Asp740 of HPlg, which triggers a conformational change
to produce an active site in the HPlg moiety, and thereby plays an
essential role in the induction of virgin enzyme activity. It is most
likely that multiple binding or contact sites between SK and HPlg are required for the induction of virgin enzyme activity and that the
COOH-terminal half of SK (including all
-domain) is also essential
for induction of virgin enzyme activity as proposed by the "binding
activation" model of HPlg activation (6, 7, 18). The elimination of
one of the possible binding sites, such as
Ile1-SK, may
impair the induction of virgin enzyme activity (16). In this study, we
identified the amino acid residues in the SK
-domain which are
involved in either virgin enzyme activity or the formation of a stable
HPlg·SK complex by analyzing the functions of SK mutants with
Ala-scanning mutagenesis.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Enzymes used in DNA manipulation were purchased
from Roche Molecular Biochemicals, New England Biolabs, Stratagene, or
Promega Laboratories and were used according to the Cold Spring Harbor Manual (19) or the recommendations by the suppliers. The full-length SK
gene was obtained from Streptococcus equisimilis H46A (ATCC 12449) by the amplification with polymerase chain reaction as previously reported (6). Lys-Sepharose and CNBr-activated Sepharose 4B
were from Amersham Pharmacia Biotech; Macro-Prep High Q was from
Bio-Rad;
NH2-D-Val-Leu-Lys-p-nitroanilide
(S-2251) and p-nitrophenyl-p-guanidinobenzoate (NPGB) were obtained from Sigma. Bis-(sulfosuccinimidyl) suberate (BS3) was purchased from Pierce. Aprotinin was purchased
from Roche Molecular Biochemicals. All chemicals were of the highest
grade commercially available. SK antiserum was prepared in our
laboratory from mice.
Preparation of HPlg, µPlg, and Native SK--
HPlg was
prepared from pooled human plasma as described (6, 15). Forms 1 and 2 of native HPlg were separated by chromatography on Lys-Sepharose column
(20, 21). HPlg was passed through an aprotinin-substituted Sepharose 4B
column to remove possible trace amounts of HPlm contamination. The
method of preparing aprotinin-substituted Sepharose 4B was described in
a previous paper (15). Form 2 of HPlg was used throughout the
experiment. Human µPlg was made by incubating HPlg with HPlm in an
alkaline solution and purified by Lys-Sepharose and soybean trypsin
inhibitor-Sepharose 4B as described in previous reports (22-24).
Native SK (Behringwerke AG, Marburg, Germany) was further purified by
passing it through a Blue-Sepharose CL 6B column to remove serum
albumin (25).
Construction of SK Mutant Genes--
Thirteen SK mutants were
constructed in which clusters of two to four charged residues and a
specific single charged residue in the SK molecule between residues 278 and 363 were converted to Ala. Since we have found previously that
truncated wild-type SK-(16-378) could activate HPlg as efficiently as
the authentic SK (6), all the SK mutants were made with the length from
residues 16 to 378. Primers SK-S16, SK-AS378, and pairs of mutagenic
primers (as shown in Table I) were custom synthesized by Pan Asia
Hospital Supply Co. (Taiwan, Republic of China). SK-S16 and SK-AS378
were a pair of DNA primers covering the coding region of SK gene
encoding amino acid residues from 16 to 378. Their specific sequences
were as shown in Sequences 1 and 2,
For cloning convenience, BamHI recognition sequences
(underlined) were created. The mutagenic sense primer (as shown in
Table I), which contained a specific restriction site, and SK-AS378 primer, were used to amplify a part of SK DNA covering the mutated position to 1134 of the coding region of SK gene by a PCR technique. The mutagenic antisense primer and SK-S16 were used to amplify another
part of SK DNA covering nucleotide positions 48 to mutated point of
coding region of SK gene. These two amplified DNA fragments were
purified by agarose gel electrophoresis. The mutated SK gene was
constructed from these two pieces of DNA by denaturation, annealing,
and extension. SKR330A,D331A,K332A,K334A (abbreviated as
SK(330-334Ala)) gene was constructed by ExSiteTM PCR-based
Site-directed Mutagenesis kit (Stratagene). The 1.1-kilobase DNA
fragment was then ligated to pPCR-ScriptTM Amp SK(+) cloning vector
(Stratagene) and transformed into Epicurian coli
XL10-GoldTMKan ultracompetent cells. The mutations were confirmed by
the sequencing of the complete coding region using dideoxy sequencing
techniques (26). Both strands of each mutated gene were completely
sequenced to make sure no other PCR-initiated mutations had been introduced.
Expression and Purification of SK Mutants--
Mutated SK gene
fragments were subcloned in-frame into the overproducing plasmid pET-3a
(Novagen) at the BamHI site. Transformed bacteria cells,
Escherichia coli strain BL21(DE3)pLysS, were grown to
mid-log phase, and target gene expression was induced by adding 1 mM isopropyl-1-thio-
-D-galactopyranoside.
After 3 h, the expressing cells were harvested, washed, and
disintegrated. Then the target proteins were concentrated by ammonium
sulfate precipitation and purified to homogeneity by a high Q anion
exchange column (27). The expressed wild-type SK-(16-378) and all the
SK mutant proteins were sequenced, and the results showed that an
additional fusion peptide of 14 amino acid residues derived from the
pET-3 plasmid and BamHI restriction site was attached at
their NH2 termini.
Protein Concentration--
The protein concentrations were
determined spectrophotometrically using the following

values and molecular
weights, respectively: HPlg, 17.0 and 94,000; µPlg, 16.0 and 28,617;
SK-(16-387), 9.5 and 43,000.
Steady State Kinetic Parameters of Activation of HPlg by SK
Mutants--
A one-stage assay as described previously was used to
measure HPlg activation by SK mutants (28, 29). Briefly, HPlg at final
concentrations ranging from 0.04 to 4 µM was incubated
with 0.5 mM S-2251 in an assay cuvette containing 150 µl
of 0.05 M Tris buffer, pH 7.4, and 0.1 M NaCl.
Activation was initiated by adding an SK mutant of fixed concentration,
and the change in absorbance at 405 nm was monitored at 37 °C with a
Hitachi 330 spectrophotometer. The increments of absorbance between 10 and 300 s after addition of SK samples were used to measure the initial rate of HPlg activation. Initial reaction rates were determined from the slopes of plots of absorbance versus
t2, and double-reciprocal plots were then
constructed. HPlg activation parameters, Kplg
(the apparent Michaelis constant for the HPlg substrate) and
kplg (the catalytic rate constant of
activation), were calculated as described by Wohl et al.
(28). The
1M at 405 nm employed for
p-nitroanilide was 9559.
Active Site Titration--
The generation of active sites by SK
mutants in HPlg was determined by active site titration using the
fluorogenic substrate 4-methylumbelliferyl
p-guanidinobenzoate (MUGB, Fluka) in a Hitachi 850 fluorescence spectrophotometer as described (9, 30-32). Briefly, HPlg
(100 nM) was added to a cuvette containing 1 µM of MUGB in 50 mM Tris-HCl, 0.15 M NaCl, pH 7.4, at 25 °C. After 2 min, native SK (100 nM) or SK mutants (100 nM) or buffer alone was
added, and the increment of fluorescence was monitored at extinction
wavelength 365 nm and emission wavelength 445 nm. The stock solution of
MUGB (1 mM) was prepared in
NN-dimethylformamide before use and was diluted to 1 µM with the titration buffer.
Discontinuous Assays of Amidolytic Activity for Determination of
the Maximal Formation of HPlg·SK Complex--
The HPlm-free HPlg
samples used in this experiment were prepared either by passing HPlg
samples through an aprotinin column or by pretreatment with NPGB
immediately before experiments as described previously (10) to remove
possible trace amounts of HPlm contamination. Incubations of equimolar
HPlg and SK mutant proteins (final concentration, 2 µM)
were carried out at 25 °C in 10 mM Hepes/NaOH, pH 7.4. Aliquots were removed at various intervals to assay the amidolytic
activity and also for SDS-PAGE analysis. The amidolytic activity was
measured by adding aliquots of the HPlg·SK proteins (final
concentration, 0.2 µM) in an assay cuvette containing 0.5 mM S-2251 in 0.05 M Tris buffer, pH 7.4, and
0.1 M NaCl. The absorbance at 405 nm was monitored between 10 and 80 s. The initial reaction rate was calculated, and the duration to achieve maximal amidolytic activity was determined as
described by Chibber et al. (33).
Activation of HPlg by Catalytic Amounts of SK Proteins--
A
one-stage assay as described previously was used to measure HPlg
activation by SK proteins (28, 29). Human Glu-plasminogen (Glu-Plg)
(0.5 µM) was activated by incubation with a catalytic amount of SK protein (0.005 µM) at 37 °C in 50 mM Tris, 0.1 M NaCl, pH 7.4, containing 0.5 mM S-2251 as a substrate. The change in absorbance at 405 nm was monitored with a Hitachi 330 spectrophotometer. For the assay of
activation of HPlg by a catalytic amount of HPlm·SK complex,
equimolar HPlm and SK (0.125 µM each) were premixed at 4 °C for 2 min. HPlg (0.5 µM) was activated by
incubation with a catalytic amount of the preformed HPlm·SK complex
(0.005 µM) at 37 °C in 50 mM Tris, 0.1 M NaCl, pH 7.4, containing 0.5 mM S-2251 as a
substrate. The change in absorbance at 405 nm was monitored as described.
SDS-PAGE Analysis--
Protein samples were subjected to
SDS-PAGE according to the method of Laemmli (34).
Amino Acid Sequence Analysis--
The amino acid sequences of
the SK fragments were determined by Edman degradation in an Applied
Biosystems Sequencer (model 477A).
Cross-link of Human µPlg and SK Protein--
Human µPlg and
SK protein at a concentration of 5 µM were mixed at room
temperature for 2 min and followed by reaction with 0.1 mM
BS3 as cross-link reagent. After 60 min, the reaction was
stopped by adding ethanolamine (8 mM),
N-ethylmaleimide (3 mM) in sodium phosphate
buffer (8 mM, pH 7.4). Parallel samples containing either µPlg or SK were used as controls. The samples were then subjected to
SDS-PAGE in 10% acrylamide according to the method of Laemmli (34).
The electrophoresed proteins on the gel were transferred onto
Immobilon-P transfer membrane (Millipore), stained with Amido Black
and Western blotting with SK antiserum (35). The relative amount of the
cross-linked product was determined by scanning the Amido Black-stained
protein bands by a Bio-Imaging analyzer (Fuji, Japan) with a computer
program MacBAS version 2.4.
 |
RESULTS |
Thirteen mutants of recombinant SK in which clusters of two to
four charged residues and a specific single charged residue were
converted to Ala were prepared using the primers shown in Table
I. All the SK mutants were made with the
length from residues 16 to 378 since wild-type SK (16) could
activate HPlg as efficiently as the authentic SK (6). DNA sequencing
confirmed the expected nucleotide sequence changes. Homogeneous SK
proteins were obtained after ammonium sulfate precipitation and high Q
column chromatography. All the SK mutant proteins had the expected
molecular mass of 43 kDa as analyzed by SDS-PAGE.
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Table I
Sequence of PCR primers used for the construction of SK mutants
s represents sense; a represents antisense; underlined sequence
represents restriction site.
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HPlg activation assay by a catalytic amount of SK mutants was
performed to determine which mutation might affect the ability of SK to
form a functional HPlg activator complex with HPlg (Fig. 1). Five SK mutants,
SKK278A,K279A,E281A,K282A (abbreviated as SK(278-282Ala)),
SKD360A,R363A (SK(360-363Ala)), SKD331A,
SKK332A, and SKK334A induced rapid
activation of HPlg to HPlm as efficiently as the wild-type
SK-(16-378) (Fig. 1). In contrast, SKD322A,R324A,D325A (SK(322-325Ala)), SKR324A, and
SKR330A,D331A,K332A,K334A (SK(330-334Ala)) did not induce
measurable HPlm generation. SKD325A, SKD328A,
SKD328A,R330A (SK(328-330Ala)), and SKR330A
induced slow activation of HPlg with lag phases ranging between 2 and 4 min, whereas SKD322A induced rapid HPlg activation with a
lag phase slightly longer than that of the wild-type SK-(16-378) (Fig.
1). Kinetic assays revealed that the catalytic efficiencies (kplg/Kplg)
for HPlg activation of complexes of HPlg with SK(278-282Ala),
SK(360-363Ala), SKD331A, SKK332A,
SKK334A, and SKD322A were comparable with
wild-type SK-(16-378). In contrast, kplg/Kplg values of
SK(328-330Ala), SKD325A, and SKR330A were
14-21-fold lower than that of wild-type SK-(16-378), whereas
kplg/Kplg value of SKD328A was 5-fold lower (Table
II). In complex with SK, HPlg undergoes a
conformational change and become catalytically active without cleavage
of the activating peptide bond, which is the so-called virgin enzyme.
The rate of HPlg activation could be slowed down by lowering the
reaction temperature to 25 °C, and the virgin enzyme could be
observed. To determine whether these SK mutants could form a virgin
enzyme in the HPlg·SK complex, the active site generation was
monitored in the presence of an acylating agent MUGB that could also
inhibit any trace amounts of HPlm, as originally described by
McClintock and Bell (9). Wild-type SK-(16-378) could generate an
active site in HPlg in a one-to-one equimolar complex at 25 °C (Fig. 2A). Native SK could generate
an active site in HPlg more rapidly than wild-type SK-(16-378) (Fig.
2A). In contrast, no active site was titrated in equimolar
mixtures of HPlg with SK(322-325Ala), SK(328-330Ala),
SKD322A, SKD328A, and SKD330A (Fig.
2A). SKD331A, SKK332A, and
SKK334A in reaction with equimolar HPlg induced rapid formation of enzyme active sites, whereas SKR324A and
SKD325A showed a greater delay than wild-type SK-(16-378)
in the generation of active sites (Fig. 2A). To investigate
further the interaction of HPlg and SK mutants, amidolytic activity
measurement (Fig. 2B) and SDS-PAGE analysis (Fig.
2C) were performed to characterize the reaction products of
equimolar incubation of HPlg and SK in the absence of acylating agent
MUGB. HPlg samples used in this experiment were prepared by either
passing through an aprotinin column or pretreatment with NPGB to remove
trace amounts of HPlm as described, and the HPlg sample prepared by the
two methods gave the similar result on the amidolytic activity
measurement. Wild-type SK-(16-378) could form an amidolytically active
enzyme with HPlg in a equimolar complex at 25 °C (Fig.
2B). No cleavage of the activating peptide bond,
Arg561-Val562 was observed at up to 4 min
determined at intervals in a parallel experiment as shown by SDS-PAGE
analysis, but hydrolysis of S-2251 was observed (Fig. 2, B
and C). After reaction for more than 7 min, some HPlg was
hydrolyzed, and heavy and light chains of HPlm were detected, and some
SK-(16-378) was degraded to a 36-kDa peptide (Fig. 2C). The
36-kDa fragment of SK was SK-(60-378) as determined by
NH2-terminal amino acid sequence analysis and our previous finding (15). Native SK could develop an amidolytically active complex
with HPlg more rapidly than wild-type SK-(16-378) (Fig. 2B). However, no enzymatic activity was observed in
equimolar mixtures of HPlg with SK(322-325Ala), SK(328-330Ala),
SKD322A, SKD328A, and SKD330A (Fig.
2B), and no cleavage of either HPlg or SK was observed as
shown in SDS-PAGE analysis (Fig. 2C). Amidolytic activity
could be observed rapidly in equimolar mixtures of HPlg with
SKR324A and SKD325A, and HPlg was converted to
HPlm (Fig. 2, B and C). At the same time,
SKR324A and SKD325A were degraded to peptide
fragments of molecular masses less than 36 kDa as HPlm appeared (Fig.
2C). These two SK mutants were extensively degraded to
smaller peptide fragments at longer incubation periods (Fig. 2C). The SK peptide fragments with molecular masses less
than 30 kDa had very little or no HPlg activator activity (15). As a
consequence, low HPlg activator activities of SKR324A and
SKD325A were observed (Fig. 1). HPlg was converted to HPlm,
and amidolytic activity was observed as soon as HPlm was formed in
equimolar mixtures of HPlg with SKD328A,
SKR330A, and SK(330-334Ala) (Fig. 2, B and
C). Less degradation of these SK mutants was observed, and
the major SK degradation product was a 36-kDa fragment (Fig. 2C). SKD331A, SKK332A, and
SKK334A in reaction with equimolar HPlg induced rapid
formation of amidolytic activity similar to that of SKR324A
and SKD325A (Fig. 2B). However, the patterns of SK degradation products were different. Less degradation of
SKD331A, SKK332A, and SKK334A with
the major SK degradation product of 36-kDa fragment was observed (Fig.
2C).

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Fig. 1.
Activation of HPlg by SK mutants.
HPlg (0.5 µM) was activated by incubation with a
catalytic amount (0.005 µM each) of native SK, wild-type
SK-(16-378), SK(278-282Ala), SK(360-363Ala),
SKD331A,SKK332A, or SKK334A ( );
SK(322-325Ala), SKR324A, or SK(330-334Ala) ( );
SKD325A (x); SKD328A ( ); SK(328-330Ala)
( ); SKR330A ( ); or SKD322A ( ) at
37 °C in 50 mM Tris, 0.1 M NaCl, pH 7.4, containing 0.5 mM S-2251 as a substrate. The change in
absorbance at 405 nm was monitored with a Hitachi 330 spectrophotometer.
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Table II
Steady state kinetic parameters of the activation of HPlg by native
SK and SK mutants
Values are the mean ± S.E. of three experiments.
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Fig. 2.
Interaction of equimolar HPlg and
SK. A, active site generation in HPlg by SK mutants.
HPlg (100 nM) was added to a cuvette containing 1 µM MUGB in 50 mM Tris-HCl, 0.15 M
NaCl, pH 7.4, at 25 °C. After 2 min, 100 nM native SK
( ); wild-type SK-(16-378) ( ); SK(322-325Ala), SK(328-330Ala),
SKD322A, SKD328A, or SKR330A ( );
SKR324A ( ); SKD325A ( );
SKD331A (×); SKK332A, SKK334A
( ); or buffer alone (+) was added, and the increment of fluorescence
was monitored at extinction wavelength at 365 nm and emission
wavelength at 445 nm. B, discontinuous assay for measuring
the amidolytic activity of equimolar complexes of HPlg and SK mutants.
HPlg (2 µM) was incubated with equimolar of native SK
( ); wild-type SK-(16-378) ( ); SK(322-325Ala), SK(328-330Ala),
or SKD322A ( ); SKR324A, SKD325A,
or SKD331A ( ); SKK332A, SKK334A
(×); SKD328A ( ); SKR330A ( ); or
SK(330-334Ala) ( ) at 25 °C in 10 mM Hepes/NaOH, pH
7.4. Aliquots (final concentration, 0.2 µM) were removed
at intervals for assay of amidolytic activity using S-2251 at a final
concentration of 0.5 mM. Amidolytic activity was expressed
as µM S-2251 hydrolyzed per min using an extinction
coefficient (1 M, 1 cm, 405 nm) of 9559. Arrows
indicate the formation of HPlm. C, SDS-PAGE analysis of
equimolar complexes of HPlg and SK mutants. Parallel samples in
incubations of B were also taken for SDS-PAGE
analysis.
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The stability HPlg and SK complex is essential for it to be an
effective HPlg activator, since the effective concentration of the
activator complex is dependent on the stability of the complex.
Dissociation of SK from the complex might render it to be degraded by
HPlm and consequently lose HPlg activator activity. The affinity and
stability of the equimolar µPlg·SK complex could be analyzed by the
cross-linked products of the one-to-one µPlg and SK mixture after
reaction with BS3 and separated by SDS-PAGE. A protein band
of 70 kDa corresponding to µPlg-SK cross-linked product was observed
in the incubations of µPlg with wild-type SK (16),
SK(328-330Ala), SKD322A, SKD328A, and
SKR330A (Fig. 3,
A-C). Reduced amount of the 70-kDa cross-linked product was
observed in SKD331A (~67% of the wild-type),
SKK332A, and SKD325A (~40% of the
wild-type), and SK(322-325Ala), SK(330-334Ala), SKR324A,
and SKK334A (
20% of the wild-type) (Fig. 3,
A-C). SKR324A and SKD325A were
extensively degraded in the incubations with µPlg as shown in the
Western blot analysis (Fig. 3B).

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Fig. 3.
Cross-link of µPlg
and SK mutants. Human µPlg and mutant SK protein at a
concentration of 5 µM each were cross-linked by reaction
with 0.1 mM BS3. The products were analyzed by
SDS-PAGE. The protein bands were transferred to Immobilon-P membrane,
stained with Amido Black (A) or with Western blot techniques
using anti-SK antibody (B). The relative amount of the
cross-linked product was determined by scanning the Amido Black-stained
protein bands by a Bio-Imaging analyzer (Fuji, Japan) with a computer
program MacBAS version 2.4 (C). Each result represents the
mean ± S.D. of three independent determinations. C1
represents wild-type or mutant SK + µPlg without BS3;
C2 represents wild-type or mutant SK + BS3.
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SKD322A could not form virgin enzyme with HPlg; however, it
induced rapid HPlg activation (Figs. 1 and 2A). To determine
the mechanism responsible for the observation, we performed an
experiment in which HPlg was incubated with either 2-fold (to make a
HPlg:SK ratio of 1:2) or 1/2-fold (to make a HPlg:SK ratio of
2:1) concentration of SKD322A, and the amidolytic
activities and SDS-PAGE of the incubations were determined. A HPlg:SK
ratio of 1:2 is to ensure the formation of a one-to-one HPlg·SK
complex, and no free HPlg existed, whereas a HPlg:SK ratio of 2:1 is to
increase the chance for the formation of a HPlg·SK·HPlg ternary
complex. No cleavage of either HPlg or SKD322A was observed
in the 1:2 or 2:1 of HPlg:SK incubations at 25 °C for up to 7 min
(Fig. 4A). However, if the 2:1
ratio of HPlg:SK mixture was preincubated at 25 °C for 2 min and
shifted to 37 °C for various intervals, HPlg was converted to HPlm,
and at the same time the amidolytic activity could be detected (Fig. 4,
B and C). On the other hand, HPlg was not
converted to HPlm, and no enzyme activity was detected in the 1:2 ratio of HPlg:SK mixture under the same incubation condition as that of 2:1
mixture (Fig. 4, B and C).

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Fig. 4.
SDS-PAGE analysis and enzyme activity assay
of HPlg and SKD322A. HPlg (2 µM) was
incubated with 4 µM of SKD322A (to make a
HPlg:SKD322A ratio of 1:2) or with 1 µM of
SKD322A (to make a HPlg:SKD322A ratio of 2:1)
at 25 °C in 50 mM Tris, 0.1 M NaCl, pH 7.4. Aliquots were removed at intervals for SDS-PAGE analysis
(A). Parallel samples of A were preincubated at
25 °C for 2 min and shifted to 37 °C for further incubation and
for SDS-PAGE analysis (B). Parallel samples of
A were preincubated at 25 °C for 2, 4, and 7 min, and
aliquots (10-fold dilution) were removed for assay of amidolytic
activity at 37 °C in 50 mM Tris, 0.1 M NaCl,
pH 7.4, using S-2251 at a final concentration of 0.5 mM
(C). HPlg:SKD322A ratio of 1:2 preincubated at 25 °C for
2 min ( ); 4 min ( ); and 7 min ( ). HPlg:SKD322A
ratio of 2:1 preincubated at 25 °C for 2 min ( ); 4 min
( ); and 7 min ( ).
|
|
The HPlg activator activity of preformed
HPlm·SKD322A complex was also measured to determine the
stability of this complex. HPlm·SKD322A complex could
activate HPlg as effectively as wild-type SK-(16-387) (Fig.
5A). On the other hand, the
equimolar HPlm·SKR330A complex could only partially
activate HPlg (Fig. 5A), although the complex had similar
hydrolysis activity toward the small peptide substrate S-2251 as the
complexes of HPlm with wild-type SK-(16-378) and SKD322A
(Fig. 5B). The amidolytic activity of
HPlm·SKR330A increased compared with that of HPlm only
(Fig. 5B). Therefore, HPlm·SKD322A can serve
as a HPlg activator even though SKD322A cannot generate an
active site in HPlg·SK complex.

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|
Fig. 5.
A, activation of HPlg by
preformed HPlm and mutant SK complex. HPlg (0.5 µM) was
activated by incubation with HPlm (0.005 µM) and
equimolar concentration of wild-type SK-(16-378) ( ),
SKD322A ( ), SKR330A ( ), or HPlm alone
without SK ( ) at 37 °C in 50 mM Tris, 0.1 M NaCl, pH 7.4, containing 0.5 mM S-2251 as a
substrate. HPlm and mutant SK were preincubated at 4 °C for 2 min
before adding to the assay mixture. The change in absorbance at 405 nm
was monitored. B, amidolytic activity of HPlm (0.005 µM) in complexes with equimolar wild-type SK-(16-378)
( ), SKD322A ( ), or SKR330A ( ) was
measured at 37 °C in 50 mM Tris, 0.1 M NaCl,
pH 7.4, containing 0.5 mM S-2251 as a substrate. An
incubation with HPlm alone was used as a control ( ).
|
|
The interactions of the coiled coil region in the
-domain of SK and
that with HPlg are very important in the activation of HPlg by SK. The
coiled coil region of SK is intercalated between the
calcium-binding loop and the activation loop of HPlg (Fig. 6A). Potential hydrogen bonds
between side chains of SK Asp328 and Arg330,
and between side chains of SK Asp331 and Lys334
could affect the stability of the coiled coil structure of SK and the
activator activity (Fig. 6B). SK Lys334 also
forms hydrogen bond with SK Asp322 (Fig. 6C). SK
Lys332 is involved in potential hydrogen bond interaction
with Glu623 of µPlm, which may play an important role in
the formation of HPlg and SK complex (Fig. 6B). Thus,
mutations of these amino acids to Ala will disrupt the potential
hydrogen bonds or possible contacts and may cause instability of the
coiled coil conformation of SK and also interfere with the interaction
with HPlg.

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|
Fig. 6.
Ribbon diagram of
SK-µPlm binary complex. A,
overall structure of the complex. The -helix, -strands, and loop
region of µPlm are shown in red, blue, and
white, respectively. The SK is shown in yellow,
and the residues 314-342 of the coiled coil region in SK domain
are shown. B, potential interactions between
Lys332 of SK and Glu623 of µPlm and
intramolecular interactions among the residues in the coiled coil
region of SK -domain are shown. Lys332 of SK forms
potential hydrogen bond with Glu623 of µPlm. The
distance between the -amino group of Lys332 and the
-carboxyl group of Glu623 is 2.03 Å. The distances
between oxygen atom of -carboxyl group of Asp328 and
nitrogen atom of -amino group of Arg330 and those
between Asp331 and Lys334 are 2.14 and 2.06 Å, respectively. The pairs of charged amino acid residues are
within the distance of potential hydrogen bond formation. C,
interaction between Asp322 and Lys334 is shown.
The distance between oxygen atom of -carboxyl group of
Asp322 and nitrogen atom of -amino group of
Lys334 is 1.92 Å (adapted from Wang et al.
(5)).
|
|
 |
DISCUSSION |
In the present study, the amino acid residues in the SK
-domain, which are involved in interaction with HPlg, were
investigated by construction of mutants in which clusters of two to
four charged amino acids and a specific single charged residue were
mutagenized to Ala. The formation of the HPlg and SK complex, the
ability to induce virgin enzyme activity, and the HPlg activator
activity of the SK mutants were evaluated. This kind of approach has
been used to study the structure-function relationships in other HPlg activators, e.g. staphylokinase (36), tissue-type
plasminogen (Plg) activator (37), and urokinase-type Plg activator
(38).
Thirteen SK mutants were prepared, two of which had the mutations
outside the coiled coil region in the SK
-domain. One mutant, SK(360-363Ala), had the similar HPlg activator activity as wild-type SK, and the other mutant, SK(278-282Ala), had even better activator activity (Table II) than the wild-type SK. These results are
consistent with the observations by x-ray crystallography that there is
no direct contact by these amino acids with µPlm, which functions as
an activator enzyme (5). These amino acid residues may not be involved
in the substrate HPlg binding either since the Kplg values of
these SK mutants do not show a significant change (Table II).
SK mutants with mutations of charged amino acid residues to Ala in the
coiled coil region of the SK
-domain resulted in significant changes
in the functions of SK. Two SK mutants with multiple charged amino
acids to Ala mutations in the coiled coil region, including SK(322-325Ala) and SK(330-334Ala), failed to activate HPlg (Fig. 1).
This result is consistent with the previous report that the ability of
rSKK332A,K334A to activate or cleave HPlg was reduced by
34-fold (39). Both SK(322-325Ala) and SK(330-334Ala) had reduced
capability to form a stable complex with µPlg and had the least HPlg
activator activity (Fig. 1 and Fig. 3). On the other hand,
SK(328-330Ala) could form a stable complex with µPlg and activate
HPlg with a lag time and with the catalytic efficiency 21-fold lower
than that of wild-type SK-(16-378) (Fig. 1, Fig. 3, and Table II). It
appears that the groups of charged amino acids in the coiled coil
region of SK play important but different roles in the activation of
HPlg.
We further analyzed the effect of mutation of single charged
amino acid to Ala in the SK coiled coil region on the activation of
HPlg. The mutants could be classified into three different categories
according to their effects on the interactions with HPlg (Table
III). The SK mutants, including
SKR324A, SKD325A, SKD331A, SKK332A, and SKK334A, had reduced capability to
form a complex with µPlg (Fig. 3 and Table III). SKR324A
and SKD325A had a markedly reduced HPlg activator activity
(Fig. 1), and the SK molecule in the one-to-one HPlg:SK mixture was
rapidly degraded to smaller peptides with molecular masses less than 30 kDa (Fig. 2C). However, SKD331A,
SKK332A, and SKK334A in the one-to-one HPlg:SK
mixture were degraded to a 36-kDa peptide that was stable for at least 20 min (Fig. 2C). Arg324 and
Asp325 in the SK
-domain may play important roles in the
formation of a stable complex with µPlm, although no direct
interaction of these residues with µPlm was observed in the crystal
structure of the complex (5). The possibility that mutations of these two residues may disturb the coiled coil structure cannot be excluded. SK mutants that cannot form stable complex with HPlg tend to be degraded fast in the presence of HPlm and lose their activator activity. Mutations at Lys332 or Lys334 also
caused decrease of µPlg·SK complex formation. However, in reaction
with HPlg, SKK332A or SKK334A was degraded much
slower, and the major SK degradation product was a 36-kDa fragment,
which still had the HPlg activator activity (15). Mutations at
Lys332 or Lys334 would prevent the cleavage of
the peptide bonds of 332-333 or 334-335. The stability of the complex
of µPlg with SKK332A or SKK334A was declined;
however, they still could activate HPlg because the slow inactivation
of SK molecule may compensate for the effect of loss of the stability
of the complex. SK(330-334Ala) with combined mutation at these
residues will result in a further decrease in the capability of complex
formation with HPlg and the loss of activator activity to a greater
extent.
Mutations at Asp328 and Arg330 belong to
another category. SKD328A, SKR330A, and
SK(328-330Ala) formed stable complexes with µPlg as with wild-type
SK (Fig. 3). However, no virgin enzyme activity was detected in
equimolar mixture of HPlg and SK(328-330Ala) and SKD328A
or SKR330A (Fig. 2A), and a delayed amidolytic
activity was observed as soon as HPlm was formed in equimolar mixtures of HPlg with SKD328A or SKR330A (Fig. 2,
B and C). These mutants also had a lag period in
initiation as well as lower efficiency in HPlg activation (Fig. 1 and
Tables II and III). SK, on binding to HPlm, simply modifies the
substrate specificity of the enzyme HPlm (40). HPlm·SK is an
efficient Plg activator, although HPlm itself has no such activity.
The catalytic activities of HPlm·SKR330A and
HPlm·SK, as measured by the rate of hydrolysis of small peptide substrate, were similar and were higher than that of HPlm alone (Fig.
5B). However, the activation of HPlg by SKR330A
or preformed HPlm·SKR330A complex was slower than the
wild-type SK (Fig. 1 and Fig. 5A). The Kplg of HPlg
activation was significantly higher for SKR330A and
SK(328-330Ala) (Table II), suggesting that Arg330 may be
involved in binding of substrate HPlg. Therefore, mutation at
Arg330 will reduce its HPlg activator activity and impair
the virgin enzyme induction, although it could form a HPlg·SK complex
that was proteolytically resistant. Mutation at Asp328 had
similar effects as that of Arg330, although
SKD328A had a relatively higher efficiency in HPlg activation.
SKD322A is another very unique SK mutant that can form a
stable complex with µPlg with no virgin enzyme activity (Fig. 2 and Fig. 3). However, SKD322A could activate HPlg as
efficiently as wild-type SK with a delay of onset of HPlg activation
(Fig. 1). The delayed time was reduced if the complex of HPlm and
SKD322A was used as the activator (Fig. 5A). The
delayed activation was observed in the activation of HPlg by
staphylokinase which could not form a virgin enzyme with HPlg, and
HPlg·staphylokinase complex cannot activate HPlg (36, 41). Mutation
at Asp322 only inhibits the virgin enzyme formation, but
the complex stability and activator activity are not compromised. Thus,
Asp322 should be very essential for induction of the virgin
enzyme activity but is not for the complex stability of SK and µPlg.
The result of the 2:1 ratio of HPlg:SK incubation (Fig. 4, B
and C) indicated that if HPlg·SK·HPlg ternary complex
was formed, the HPlg could be converted to HPlm directly without the
formation of the virgin enzyme. The observation is consistent with the
proposal that the close proximity of two HPlg molecules in the
HPlg·SK·HPlg complex may accelerate the autoactivation of HPlg
forming HPlm·SK without the prior formation of HPlg*·SK virgin
enzyme complex (16). The side chain of Asp322 forms a
potential hydrogen bond with the
-amino group of Lys334
to form a loop (Fig. 6C). Kinetic analysis of HPlg
activation by SK mutants (Table II) showed that both the Kplg
and kplg of SKD322A were higher than those of wild-type SK,
probably because no virgin enzyme was involved in the activation of
HPlg by SKD322A, and the activation might not follow the
same reaction steps.
In conclusion, the functional studies of SK mutants demonstrate that
amino acid residues in the coiled coil region of
SK(Leu314-Ala342) are involved in virgin
enzyme induction, stability of the HPlg·SK complex, and the
activation reaction. Mutations at Arg324,
Asp325, Lys332, and Lys334 to Ala
reduced the formation of the HPlg·SK complex. Mutation at
Asp322 to Ala abolished virgin enzyme activity but had no
effect on HPlg activation. Mutations at Asp328 and
Arg330 resulted in impairment of virgin enzyme formation,
lower efficiency of HPlg activation without affecting the formation of
µPlg·SK complex. Therefore, the amino acid residues
Arg324, Asp325, Lys332, and
Lys334 are very critical for the formation of the activator
complex, Asp322, Asp328, and Arg330
are involved in induction of virgin enzyme activity, and
Arg330 is involved in modulating the binding of substrate
HPlg. Analysis of the crystal structure data on the coiled coil region
of SK
-domain and human µPlm indicates hydrogen bond interaction
between Lys332 of SK and Glu623 of µPlm (Fig.
6B). Strong interactions between Asp328 and
Arg330, Asp331 and Lys334, and
Asp322 and Lys334 are also noticed (Fig. 6,
B and C). The conformation of the coiled coil
region of SK
-domain is essential in stabilizing the formation of
HPlg·SK complex and in inducing the virgin enzyme. Studies of the
interaction between SK
- and
-domains and HPlg are in progress.
This study will lead to identifying the essential amino acid residues
in SK molecule as a Plg activator and better understanding of the
reaction mechanism. This knowledge may be applied to create an SK
molecule with designed properties for improving the clinical application of SK as a thrombolytic agent.
 |
FOOTNOTES |
*
This work was supported by Grants NSC-86-2314-B006-011,
NSC-87-2316-B006-010, and NSC-87-2316-B006-002-M42 from the National Science Council, Republic of China.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Biochemistry, Medical College, National Cheng Kung University, Tainan, Taiwan, Republic of China. Tel.: 886-6-2353535-5541; Fax:
886-6-2741694; E-mail: halnwu@mail.ncku.edu.tw.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M005935200
 |
ABBREVIATIONS |
The abbreviations used are:
SK, streptokinase;
HPlg, human plasminogen;
HPlm, human plasmin;
Plg, plasminogen;
PAGE, polyacrylamide gel electrophoresis;
µPlg, human
microplasminogen, plasminogen fragment consisting of
Lys531-Asn791;
µPlm, human microplasmin;
SK(278-282Ala), SKK278A,K279A,E281A,K282A;
SK(360-363Ala), SKD360A,R363A;
SK(322-325Ala), SKD322A,R324A,D325A;
SK(328-330Ala), SKD328A,R330A;
SK(330-334Ala), SKR330A,D331A,K332A,K334A;
BS3, Bis-(sulfosuccinimidyl) suberate;
NPGB, p-nitrophenyl-p-guanidinobenzoate;
MUGB, 4-methylumbelliferyl p-guanidinobenzoate.
 |
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