From the Department of Biochemistry and Molecular Biology, University of Illinois at Chicago, Chicago, Illinois 60612-4316
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ABSTRACT |
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To probe the covalent serpin-proteinase complex,
we used wild-type and 4 new single cysteine variants (T85C, S121C,
D159C, and D298C) of 1-proteinase inhibitor
Pittsburgh. Cysteines in each variant could be labeled both in native
and proteinase-complexed
1-proteinase inhibitors.
Pre-reaction with 7-nitrobenz-2-oxa-1,3-diazole-chloride or fluorescein
prevented complex formation only with the D298C variant. Label at
Cys121 greatly increased the stoichiometry of inhibition
for thrombin and gave an emission spectrum that discriminated between
native, cleaved, and proteinase-complexed serpin and between complexes with trypsin and thrombin, whereas fluorophore at residue 159 on helix
F was almost insensitive to complex formation. Fluorescence resonance
energy transfer measurements for covalent and non-covalent complexes
were consistent with a location of the proteinase at the end of the
serpin distal from the original location of the reactive center loop.
Taken together, these findings are consistent with a serpin-proteinase
complex in which the reactive center loop is fully inserted into
-sheet A, and the proteinase is at the far end of the serpin from
its initial site of docking with the reactive center loop close to, but
not obscuring, residue 121.
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INTRODUCTION |
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Serpins are a family of widely distributed, structurally homologous proteins (1), many of which are inhibitors of serine proteinases (2). Whereas the many other families of protein inhibitors of serine proteinases, such as the Bowman-Birk, Kazal, and Kunitz families, inhibit target proteinases by forming tight non-covalent 1:1 complexes in which neither the proteinase nor the inhibitor undergoes significant structural change in most cases (3), serpins differ not only by apparently forming covalent 1:1 acyl enzyme complexes with their target proteinases (4), but by undergoing a major conformational change during, and as an essential part of, the inhibition process (5). Because of the requirement for conformational change as part of the inhibition mechanism, knowledge of the structure of the serpin-proteinase complex is critical for an understanding of how serpins inhibit their target proteinases through kinetic trapping of a normal covalent acyl enzyme intermediate on the proteinase substrate cleavage pathway.
A previous proposal that a major movement of the proteinase occurs
following cleavage of the scissile bond (6) has been supported by two
recent studies (7, 8). In one study (8) chemical cross-linking between
the proteinase and the serpin in the complex, together with a
measurement of the separation between P3 and P1' residues of the serpin
in the complex by fluorescence resonance energy transfer, was
consistent with a location of the proteinase half-way down the flank of
the serpin (Fig. 1) and in contact with helix F. The other study (7),
from this laboratory, used fluorescence resonance energy transfer
between fluorophores on the serpin 1-proteinase
inhibitor
(
1PI)1
Pittsburgh and the proteinase to compare the inter-fluorophore separation in the normal covalent serpin-proteinase complex with that
in the non-covalent complex with the non-functional anhydroproteinase. This study, although not able to precisely define the position of the
proteinase in the complex, demonstrated a movement of the proteinase of
at least 21 Å upon formation of the kinetically trapped covalent
complex.
We describe here more extensive mapping of this serpin-proteinase
complex by using wild-type 1PI Pittsburgh and 4 new
single cysteine variants. These well separated cysteines were used as follows: (i) to probe the accessibility of the cysteine in native and
proteinase-complexed serpin, (ii) to determine the effect of
derivatization of the cysteine on the ability to form covalent complex,
and (iii) for introduction of fluorophores, both as probes of the local
environment and for fluorescence resonance energy transfer
measurements. By these approaches we have been able to place further
constraints on the possible structures of the serpin-proteinase complex
and to show that it probably requires movement of the proteinase to the
bottom of the serpin and therefore full insertion of the cleaved
reactive center loop into
-sheet A. In this location the proteinase
is not in contact with the outer face of helix F. Our findings are thus
consistent with the model of Wright and Scarsdale (6).
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MATERIALS AND METHODS |
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Site-directed Mutagenesis--
Site-directed mutagenesis was
carried out on a double-stranded pET16b plasmid (Novagen) containing
1PI cDNA, using the Quikchange method (Stratagene).
Double-stranded template DNA of two complementary primers containing
the mutation was annealed and extended with Pfu DNA
polymerase during thermal cycling. The pET16b plasmid contained an
N-terminally modified
1PI cDNA that lacked coding sequence for the first 5 residues (10), inserted between the NcoI and BamHI subcloning sites of the vector.
The sequences for the coding strands of the mismatch primers were as
follows (mismatch codons are underlined): M358R, 5'-GAG GCC ATA CCC
AGA TCT ATC CCC CCC; C232S, 5'-AAC ATC CAG CAC
AGC AAG AAG CTG TCC AG; T85C, G AAT TTC AAC CTC
TGT GAG ATT CCG GAG G; S121C, GGC CTG TTC CTC TGT GAG GGC CTG AAG; D159C, G AAA CAG ATC AAC
TGT TAC GTG GAG AAG GG; D298C, AC TGG AAC CTA TTG
TCT GAA GAG CGT CCT G. All
1PI variants used
in this study carried the Pittsburgh (M358R) mutation, and all except
the wild-type Pittsburgh variant carried the C232S mutation, so that
all variants contained only one free cysteine residue and all contained
a P1 arginine (Pittsburgh mutation (11-13)) that conferred high
affinity (5 nM) for the non-covalent complexes with
anhydrotrypsin (7). All mutations were confirmed by dideoxy sequencing
in the host plasmid.
Expression and Refolding of Recombinant 1PI
Variants--
The plasmids containing the mutated
1PI
genes were transformed into BL21 (DE3) cells (Novagen). 5 ml of an
overnight culture was used to inoculate 0.5 liters of LB broth
containing 125 mg/ml ampicillin. Cells were grown at 37 °C in a
shaking water bath to an A600 nm of 0.5. IPTG
(0.4 mM) was added to induce expression of
1PI, and the cells were allowed to grow for a further 2.5-3 h at 37 °C. Cells were harvested by centrifugation at
4000 × g for 10 min, and the cell pellet was collected
and washed twice with cold PBS. The cell pellet was resuspended in 10 ml of buffer A (50 mM Tris, pH 8.0, 50 mM NaCl,
and 1 mM EDTA) containing 1 mM
phenylmethylsulfonyl fluoride, 1 mM
-mercaptoethanol,
and 0.2 mg/ml lysozyme. The suspension was sonicated using four 30-s pulses. Inclusion bodies were harvested by spinning the lysed cells at
10,000 × g for 45 min. The pellet containing the
inclusion bodies was washed twice with buffer A containing 0.5% Triton
X-100 (Sigma). The inclusion bodies were finally dissolved in 10 ml of
buffer A containing 8 M guanidinium hydrochloride by
vortexing and sonication. Any non-solubilized inclusion bodies were
spun down at 10,000 × g for 15 min. The typical yield
of solubilized protein at this stage was ~20 mg, determined by BCA
assay, from 0.5-liter cell culture. Solubilized denatured
1PI was refolded by dropwise dilution over a period of
20 min of the 8 M guanidinium hydrochloride solution into
250 ml of a 4 °C solution of 10 mM sodium phosphate
buffer, pH 6.5, containing 1 mM EDTA and 1 mM dithiothreitol. The diluted solution was extensively dialyzed against
10 mM sodium phosphate buffer, pH 6.5, containing 1 mM EDTA to remove the guanidinium hydrochloride. After
dialysis the solution was centrifuged for 30 min at 4 °C and
8000 × g and filtered through a 0.2-µm filter to
remove any precipitated protein.
Purification of Monomeric 1PI--
The refolded
1PI contained dimers and higher order oligomers in
addition to monomers. Monomeric
1PI was purified in two steps. The first step was ion exchange chromatography on DE52 (Whatman), using a linear 0-0.25 M NaCl gradient. This
gave a sharp early eluting peak for the monomer and a broader later
eluting peak for higher order aggregates. The pooled monomeric
fractions were dialyzed against 20 mM Bis-Tris-propane
buffer, pH 6.5, containing 1 mM EDTA and rechromatographed,
where necessary, on a MonoQ column equilibrated in the same buffer and
eluted by a 0-0.3 M NaCl gradient. Monomeric
1PI eluted as a very sharp single peak. The purity of
all preparations was confirmed by SDS-PAGE and by PAGE under non-denaturing conditions to confirm that all material was monomeric. Preparations were dialyzed against 20 mM sodium phosphate,
pH 7.4, 100 mM NaCl, 0.1 mM EDTA, 0.1%
PEG8000, quickly frozen in aliquots, and stored at
70 °C until
needed.
Preparation of Anhydrotrypsin and
-Trypsin--
Anhydrotrypsin was prepared from commercial
crystallized trypsin (Sigma) by alkaline
-elimination of the
phenylmethylsulfonyl fluoride adduct according to published procedures
(14). Following the reaction, the solution was treated with
Phe-Phe-Arg-chloromethyl ketone (20 µM) to inhibit any
remaining or regenerated active trypsin and acidified to pH 3.0.
-Anhydrotrypsin was purified from the reaction mixture by
chromatography on a soybean trypsin inhibitor affinity matrix. The
absence of proteolytic activity in the product was confirmed by
activity assay using the chromogenic trypsin substrate S-2222.
-Trypsin was prepared from TPCK-treated commercial trypsin by
affinity chromatography using the same soybean trypsin inhibitor
affinity matrix.
Preparative Labeling of 1PI with Fluorescein or
NBD--
All
1PI variants were labeled with fluorescein
by reaction of the single free cysteine with 5 iodoacetamido-fluorescein (Molecular Probes, Eugene, OR). The protein
(10-40 µM) was reacted with a 2-fold molar excess of
dithiothreitol for 15 min at room temperature and then with a
10-15-fold molar excess of 5-IAF. The reaction was allowed to proceed
overnight at 4 °C. Excess reagent was removed by dialysis for
24 h against 10,000 volumes of 20 mM sodium phosphate, pH 7.4, 100 mM NaCl, 0.1 mM EDTA, 0.1%
PEG8000. The extent of labeling was determined spectrophotometrically
using the absorbance at 495 nm for determination of the fluorescein
concentration and the absorbance at 280 nm, corrected for the
contribution from fluorescein at this wavelength, which was determined
to be 25% of the absorbance at 495 nm based on the spectrum of the
adduct of IAF with
-mercaptoethanol, to determine the protein
concentration. For all preparations the extent of labeling was close to
1 eq per mol or less, with a range from 0.59 to 1.06. This range
represents the determined stoichiometries for preparations made at
different times under somewhat different reactant concentrations and
does not necessarily reflect intrinsic differences in reactivity of the
various cysteines. Where comparisons of labeling efficiency are made
elsewhere, reactions were carried out under identical conditions for
each
1PI species. Extinction coefficients of 27,000 (15)
and 82,000 M
1 cm
1 were used for
1PI and fluorescein, respectively.
Preparative Labeling of -Trypsin and
-Anhydrotrypsin with
Tetramethylrhodamine Isothiocyanate--
-Trypsin and
-anhydrotrypsin were labeled with tetramethylrhodamine
isothiocyanate while immobilized on soybean trypsin inhibitor-agarose
beads (i) to permit equivalent reaction conditions for anhydrotrypsin
as for trypsin without concern for autodigestion by free trypsin, and
(ii) to provide a ready means of selecting only those labeled proteins
that were still active in binding to protein inhibitors. About 300 µl
of wet soybean trypsin inhibitor-agarose beads were equilibrated with
0.1 M sodium citrate buffer, pH 4.0, and then mixed with
700 µl of either
-trypsin or
-anhydrotrypsin, followed by
gentle rotation for 30 min at 4 °C. The beads were washed twice with
700 µl of 0.1 M citrate buffer, pH 4.0, to remove any
unbound protein and then four times with 700 µl of 0.1 M
sodium carbonate, pH 9.0, to raise the pH. The beads were resuspended in 700 µl of 0.1 M sodium carbonate, pH 9.0, and 10 µl
of a 10 mM solution of tetramethylrhodamine isothiocyanate
in N,N-dimethylformamide were added. The reaction
was allowed to proceed, with gentle rocking, at room temperature for
2-4 h, depending on the degree of labeling wanted. The beads were then
washed four times with 700 µl of 10 mM sodium carbonate,
pH 9.0, to remove excess reagent and any free protein. Labeled protein
was eluted with 500 µl of 0.2 M sodium citrate, pH 2.4. The eluate was dialyzed overnight against 1000 volume of 1 mM HCl containing 10 mM CaCl2 and
centrifuged at 14,000 × g for 10 min to remove any
precipitated material. The extent of labeling was determined
spectrophotometrically using extinction coefficients of 62,000 M
1 cm
1 at 550 nm and 35,800 M
1 cm
1 at 280 nm for
tetramethylrhodamine and trypsin, respectively. The absorbance at 280 nm was first corrected for the contribution at that wavelength from
tetramethylrhodamine, which was found empirically to be 28% of the
absorbance at 550 nm. The extent of label incorporation was 0.47 mol/mol for trypsin and, for two separate preparations of
anhydrotrypsin, 0.30 and 0.71 mol/mol.
Characterization of the Sites of Labeling on
-Trypsin--
The sites of labeling in
-trypsin were identified
by N-terminal sequencing of tetramethylrhodamine-labeled peptides
isolated from a tryptic digest of the labeled protein. 140 µg of
labeled
-trypsin was freeze-dried and dissolved in 50 µl of 8 M guanidinium hydrochoride, 50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA. Dithiothreitol was added
to 10 mM and the mixture incubated at room temperature for
30 min. Iodoacetic acid was added to 30 mM and allowed to react for 30 min at room temperature. The denatured labeled trypsin was
diluted into 600 µl of 50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA, 10 mM
CaCl2 containing 5 µg of active
-trypsin and digested in the dark at 37 °C for 2 h, after which an additional 5 µg
of
-trypsin was added. A second addition was made after a further 2 h, and the reaction was allowed to continue overnight. The
tryptic digest was chromatographed on a C-18 reverse phase column,
using a linear gradient from 80% buffer A (0.1% trifluoroacetic acid in water), 20% buffer B (0.1% trifluoroacetic acid, 90%
acetonitrile, 9.9% water) to 35% buffer A, 65% buffer B. Peaks were
monitored both for absorption at 280 nm and fluorescence at 550 nm. Two major peaks of approximately equal fluorescent intensity were obtained,
as well as several much smaller ones, and were submitted for N-terminal
sequence determination.
Assay for Ability of Labeled or Unlabeled 1PI
Variants to Form Covalent Complex--
The ability of the
1PI variants to form SDS-stable covalent complex with
trypsin was assayed by 10% SDS-PAGE of the reaction products and
visualization of the complex either by Coomassie staining for unlabeled
complex or by fluorescence intensity for labeled complex. Typically
2-3 µg of
1PI was reacted for 10 s with trypsin
at different molar ratios, ranging from 0.3:1 to 2:1
trypsin:
1PI, with
1PI fixed at 5-10
µM. This was sufficient time for the reaction to have
gone to >99% completion based on the published rate constant for this
reaction (7) and confirmed empirically by the absence of unreacted
serpin in lanes where the proteinase was in excess. This also confirmed
that the serpin was >95% active.
Calculation of Stoichiometry of Inhibition--
The
stoichiometry of inhibition (SI) was calculated by scanning
densitometry of SDS-PAGE gels. Coomassie Blue-stained gels were
scanned, and the density of the bands corresponding to cleaved serpin
and complex were measured. The intensity of the band for complex was
corrected for the contribution from the proteinase, by assuming equal
staining of the serpin and trypsin per unit weight. This was justified
by a standard curve for trypsin and 1PI which showed
comparable staining for both proteins on a weight basis and a linear
dependence between amount of protein and band intensity in the range
used for the experiments. This method was considered accurate for SI
values in the range 1.1 to 5, corresponding to 91 to 20% complex, but
incapable of determining SI where complex bands were so faint as to be
not visible. SI values for fluorescein-labeled serpins were determined
in an analogous way, except intensity of the fluorescent bands
corresponding to cleaved and complexed bands were used, and no
correction was needed for contribution from (unlabeled) trypsin. No
error was thereby introduced for complex formed by unlabeled serpin. In
cases where the degree of fluorescein labeling was close to 100%, so
that all covalent complex was also fluorescent, or where labeling did
not affect complex formation, independent quantitation of SI by both
fluorescence and Coomassie Blue staining gave good agreement.
Ability to Label Cysteines in Covalent Complex--
5-6 µg of
either the Pittsburgh variant of 1PI or the cysteine
mutants (T85C, D159C, S121C and D298C) in a total volume of 20 µl
were reacted with 1 µg of
-trypsin (excess of
1PI
to ensure that complex was not degraded by excess proteinase) to form
the stable trypsin-
1PI complex. TLCK was added after a
few seconds to a final concentration of 25 µM. In all
reaction mixtures 1.5 µl of 1.8 mM IAF was added (final
concentration was about 100 µM), and the reaction was
allowed to proceed for 2 h at 4 °C. Dithiothreitol was added to
a final concentration of 1 mM, and the mixture was
incubated at room temperature for 10 min (to inactivate any unreacted
probe). The samples were then subjected to SDS-PAGE analysis (12%
acrylamide). A control reaction of IAF with TLCK-treated trypsin alone
was carried out and showed no labeling of trypsin under the conditions
used.
Fluorescence Measurements-- All fluorescence measurements were made on a SPEX fluorolog scanning fluorimeter. NBD spectra were acquired by exciting at 420 nm and scanning from 440 to 580 nm. Fluorescein and rhodamine spectra were recorded by exciting at 340 nm and scanning from 460 to 640 nm. All slit widths were 4 nm. Measurements were made at 25 °C. For time courses the emission signal was monitored at 515 nm, where the contribution of rhodamine fluorescence is negligible. For energy transfer measurements the labeled serpin was between 50 and 150 nM, and the proteinase was at 2-3 times the serpin concentration. 1 mM benzamidine was included in the cuvette as a competitive inhibitor of trypsin to slow down the reaction. NBD spectra were acquired at concentrations of 156 nM for the S121C variant and 400 nM for the D159C variant. The buffer used for all measurements was 20 mM sodium phosphate, pH 7.4, containing 100 mM NaCl, 0.1 mM EDTA, and 0.1% PEG8000.
To estimate the efficiency of energy transfer between fluorescein and rhodamine in the covalent complex, the fluorescence spectrum of the fluorescein-labeled serpin was recorded, and trypsin, either unlabeled or rhodamine-labeled, was then added to the cuvette in the presence of 1 mM benzamidine and the reaction followed by monitoring the change of fluorescein fluorescence at 515 nm. When a plateau was reached the fluorescence emission spectrum of the mixture was recorded. The amount of energy transfer for each variant was determined from the observed reduction in fluorescein fluorescence corrected for any contribution that arose solely from complex formation, which was determined from a control reaction using fluorescein-labeledPreparation and Fluorescence Spectra of Different NBD-labeled
Species--
NBD-labeled S121C variant was reacted with trypsin,
papain, or thrombin as described in the figure legend for Fig. 4. One aliquot of the reaction mixture was used for SDS-PAGE analysis, and
another aliquot was diluted to a final concentration of 156 nM 1PI and the emission fluorescence
spectrum recorded as described above. The Coomassie Blue-stained gel
was scanned and the density of the bands used to estimate both the
completeness of each reaction and the SI. Every reaction was found to
be more than 95% complete.
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RESULTS |
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Accessibility of Engineered Cysteines in Native and
Proteinase-complexed Variants--
Previous studies on a recombinant
Pittsburgh variant of 1PI (P1 Met
Arg) have shown
that the cysteine at position 232 is quite accessible to nucleophiles
(7), consistent with its exposed location in the crystal structure of
1PI. We found here that cysteine 232 is also accessible
in the complex, since it could be comparably labeled with 5-IAF while
in complex with both
-trypsin and thrombin, as judged by the
intensity of fluorescence associated with the band of complex on
SDS-PAGE (Table I, gel not shown). To
carry out similar accessibility studies at different sites on the
serpin, we created four new variants, each containing a single free
cysteine at strategic locations on the serpin surface (Fig.
1). The choice of sites was guided by
proposed models for the serpin-proteinase complex (6, 8, 9), with the
aim of creating one or more variants that had a cysteine that might be
accessible when the serpin was uncomplexed but inaccessible in
complex.
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Effect of Cysteine Modification on Complex Formation--
We also
determined whether covalent modification of any of the cysteines with
fluorescein, before reaction with proteinase, affected the
subsequent ability of the serpin to inhibit -trypsin, to establish
if the presence of a bulky group impeded movement of the proteinase
during its migration to its final position in the complex. All variants
except D298C could still form covalent complex with
-trypsin, as
judged by the formation of covalent complex visible on SDS-PAGE (gel
not shown). The SI values, estimated from quantitation of the bands of
complex and cleaved
1PI on the gels (not shown), were
close to 1 for variants 85, 121, 159, and 232 labeled with fluorescein
(Table I), showing that the relative fluxes along the inhibitory and
substrate branches of the serpin pathway had not been significantly
affected by the labeling. For the D298C variant, the attachment of
fluorescein resulted in a sufficiently large reduction in the flux
along the inhibitory branch of the serpin pathway (16) that no covalent complex was detectable by SDS-PAGE examined by fluorescence. Instead, the only fluorescent product was substrate-cleaved
1PI.
A comparison of the different outcomes of the reactions of the 121 and
298 variants is shown in Fig. 2.
Attachment of the smaller NBD fluorophore also resulted in a greatly
increased SI for the 298 variant. However, since NBD fluorescence could
not be seen on the SDS gel and some unlabeled variant was still present
and competent to form complex, we cannot be sure that the inhibition
pathway was completely blocked. The fluorescein-labeled S121C variant,
while showing a normal SI with
-trypsin of close to 1, gave an SI of
>5 with thrombin (Table I). Thus, depending on the proteinase used to
form complex, labeling of either of the positions 121 and 298 resulted
in perturbations of the SI.
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Sensitivity of Fluorescent Label to Complex Formation--
The
sensitivity of fluorophores at each of the cysteine sites to
proteinase-induced changes was examined. Fluorescein at positions 85, 159, and 232 was little perturbed by formation of complex with
-trypsin (Table II) (spectra not
shown). Fluorescein at 121, however, gave a 20% reduction in intensity
and a red shift of ~3 nm upon complex formation. Since this
fluorophore gave almost no change upon substrate-like cleavage with
papain (Fig. 3), it seems that it is the
presence of the proteinase, rather than simply reactive center loop
insertion and accompanying conformational changes, that results in the
20% enhancement of fluorescein fluorescence in the covalent complex.
Although fluorescein at position 298 showed a 100% fluorescence
enhancement upon reaction with
-trypsin (Fig. 3 and Table II), the
species was cleaved loop-inserted
1PI rather than
covalent complex with
-trypsin, since the label blocked the
inhibitory pathway (see above).
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Fluorescence Resonance Energy Transfer between Serpin and
Proteinase--
We have previously shown that there is a very large
difference in fluorescence resonance energy transfer between covalent and non-covalent complexes of rhodamine-labeled trypsin (or
anhydrotrypsin) and 1PI labeled at Cys232
with fluorescein, consistent with a large change in position of the
proteinase as a result of covalent complex formation (7). In the
present study we used the new cysteine-containing variants to extend
such measurements to define better the location of the proteinase in
the covalent complex. Before doing this we determined the sites of
attachment of the rhodamine label by peptide mapping of a tryptic
digest of labeled
-trypsin. This showed that, although less than an
average of one label was incorporated per trypsin, the label was
distributed over several sites. Two major peptides, one accounting for
16 and one 20% of the rhodamine fluorescence, were sequenced and shown
to correspond to peptides starting with sequences
XLXAP and VCNYV, respectively. This identified
the labeled lysines as 159 and 239.
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DISCUSSION |
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We have described here the use of the Pittsburgh variant of
1PI and four new single cysteine-containing derivatives
to map the covalent complex that this serpin forms with proteinases. The advantage of the Pittsburgh variant over wild-type
1PI is that its affinity for anhydrotrypsin is high
enough to allow examination of both non-covalent and covalent
complexes. Previous studies using fluorescence resonance energy
transfer between fluorophores on trypsin and
1PI
Pittsburgh had shown that the proteinase undergoes a major change in
location from its initial site of docking with the reactive center loop
(7). However, because only a single distance constraint was used it was
not possible to distinguish between three different models for the
complex, each of which involves a very different extent of proteinase
translocation. Whisstock et al. (9) have proposed a modest
movement of the proteinase from the initial site of docking, with
insertion of the reactive center loop into
-sheet A up to P12.
Wilczynska et al. (8) have proposed a greater movement,
involving alignment of the proteinase with the flank of helix F and the
cleaved reactive center loop inserted up to P5. The third proposal,
from Wright and Scarsdale (6), involves the proteinase moving
completely to the end of the serpin distal from the initial docking
site, which requires complete insertion of the cleaved reactive center loop in a manner similar to that of substrate-cleaved serpin. Our
present findings, taken as a whole, are consistent with the third model
of full loop insertion and full movement of the proteinase (Fig.
6). The basis for this conclusion is as
follows. (i) If complex formation involves complete insertion of the
cleaved, but still covalently bound, reactive center loop into
-sheet A, it becomes immediately understandable why cysteine at 298 can be modified both in native and proteinase-complexed
1PI yet not permit complex formation when labeled
beforehand, since the reactive center loop, with the large proteinase
covalently bound, would need to insert past this residue. A large
group, such as fluorescein, might impede such insertion, thereby
allowing only substrate cleavage to occur, as observed. (ii) With
proteinase at the distal end of the serpin in covalent complex
(bottom in Fig. 6) and at the proximal end in non-covalent
complex (top in Fig. 1), the relative efficiency of
fluorescence resonance energy transfer should be greater in the anhydro
complex than in the covalent complex for fluorophore at 232, but the
reverse for fluorophore at 121, as was found. The expectation for
position 85 is also that it would be closer in the covalent than the
non-covalent complex and thus give higher efficiencies of fluorescence
resonance energy transfer in the covalent complex, again as found.
(iii) The proteinase would be expected to be close to residue 121 but
not obscure it. Introduction of label at this position might therefore
influence the rate of achievement of the final complex, through steric
effects and thus affect the SI. This is indeed the case for position
121, where the effect on SI is dependent both on the size of the label and the size of the proteinase. Thrombin, which is ~50% larger than
trypsin, gives a much higher SI than does trypsin for both the
fluorescein and NBD derivatives, although the effect is less for the
smaller NBD, consistent with steric clashes during complex formation
being most pronounced for the larger proteinase with the larger label.
(iv) With proteinase at the bottom of the serpin and in the vicinity of
residue 121, the emission spectrum of fluorophore at this position
should be responsive to complex formation but in a different way than
from simple loop insertion. Indeed NBD at position 121 can discriminate
between non-covalent complex, covalent complexes with different
proteinases, and cleaved
1PI. The large changes in NBD
fluorescence for cleavage or covalent complex formation contrast with
the absence of perturbation for formation of the anhydro complex and
thus strongly indicate that the covalent complex is very different from
that of the non-covalent complex with respect to perturbation of the
serpin structure. In this regard, the covalent complex is much more
similar to cleaved, loop-inserted serpin, as is expected for the model
of Wright and Scarsdale (6). It is important to note that NBD label at
121 shows a 6-nm blue shift when complex is formed with trypsin but no
such wavelength shift in papain-cleaved
1PI. Similarly,
the intensity of the NBD emission spectrum of the thrombin complex is
much higher than for that of the thrombin-cleaved serpin. Both of these
results indicate that the presence of the proteinase, either trypsin or
thrombin, causes significant spectral changes that are distinct from
changes caused solely by loop insertion and associated conformational
change. Although our results are most consistent with full insertion of
the reactive center loop and placement of the proteinase at the distal
end of the serpin, they do not allow a precise positioning of the
proteinase, except that it must be close enough to position 121 to
perturb fluorophore at this position but not so close as to prevent
labeling of cysteine in the S121C variant when in complex with either
-trypsin or thrombin.
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The model of Wright and Scarsdale (6) is also the one that is
philosophically most consonant with the requirements and restrictions
for stable serpin-proteinase complex formation. Thus, a given serpin
can often form SDS-stable complexes with many different proteinases
that differ greatly in size and shape. The Pittsburgh variant used here
is a good example, in that it can inhibit trypsin, elastase, thrombin,
C1s, factor XIIa, plasmin, and urokinase (18, 19). Similarly a given
proteinase may be able to form complexes with different serpins.
Thrombin is inhibited by antithrombin, heparin cofactor II, protease
nexin 1, plasminogen activator inhibitor 1, and protein C inhibitor. It
is hard to conceive a model for the complex that involves a specific
interaction between serpin and proteinase in the final trapped complex
and can yet accommodate such a wide array of different proteinases. The
model favored here, however, has as the only requirement a
conformationally strained acyl ester linkage between the P1 residue at
the very bottom of -sheet A and the active site serine of the
proteinase, for which there is some experimental evidence (20-22).
This is common to all pairs of serpin-proteinase complexes.
Another satisfying aspect of such a model is that it has been found
experimentally that the P1-P1' bond must be exactly 14 residues from
the hinge point for insertion into -sheet A, which makes it just
long enough upon complete insertion to have residues P2 and P1 protrude
from the end of the sheet and provide enough of a linker to reach into
the proteinase active site and thereby to impose a particular
non-optimal conformation for the acyl group in the proteinase-active
site. If the scissile bond were closer to this hinge point the acyl
intermediate could not be trapped by such a full insertion mechanism.
If the scissile bond were further away the resulting peptide that
extends beyond
-sheet A would be so long that there could be no
constraint imposed on the conformation of the acyl ester linkage, no
disruption of the catalytic site of the proteinase, and hence no
kinetic trap.
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ACKNOWLEDGEMENT |
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We thank Dr. Steven Olson, University of Illinois at Chicago, for comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL49234.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.
Supported by a University Fellowship from the University of
Illinois at Chicago.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, M/C 536, University of Illinois at Chicago, 1819-1853 West Polk St., Chicago, IL 60612-4316. Tel.: 312-996-5534; Fax: 312-413-8769; E-mail: pgettins{at}tigger.cc.uic.edu.
1
The abbreviations used are: 1PI,
1-proteinase inhibitor; 5-IAF,
5-iodoacetamidofluorescein; NBD, 7-nitrobenz-2-oxa-1,3-diazole; SI,
stoichiometry of inhibition, defined as the number of moles of serpin
required to inhibit 1 mol of proteinase by formation of SDS-stable
complex; TLCK, tosyl-lysyl chloromethyl ketone; PAGE, polyacrylamide
gel electrophoresis; Bis-Tris,
2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.
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REFERENCES |
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