From the Department of Oral Medicine and Diagnostic Sciences and Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, Chicago, Illinois 60612
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ABSTRACT |
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The reactive center loop of native
1-proteinase inhibitor has been reported to be in
a helical conformation and in a
-strand conformation by two
different studies. In the
-strand loop structure the P5
glutamic acid plays a unique role by stabilizing the loop in the
predicted optimal conformation for the interaction with target
proteinases and insertion into
-sheet A. We hypothesize here that
disrupting the interactions that stabilize the
-strand conformation
of the loop would result in changes in the inhibitory properties of the
serpin. In addition, our earlier studies on reactive center loop
mutants of
1-proteinase inhibitor suggested that the
P5 residue was important in stabilizing the
1-proteinase inhibitor-proteinase complexes. To address
these issues we made mutants of
1-proteinase inhibitor
with glycine, glutamine, or lysine at the P5 position and
measured the rates and stoichiometries of inhibition with trypsin and
human neutrophil elastase and the stabilities of the resulting
complexes. In most cases the rate of inhibition was reduced by about
half and the stoichiometry increased between 2- and 4-fold. The only
exception was for trypsin with the lysine variant where the
P5 was now the favored site of cleavage. These data show
that the P5 Glu is important in maintaining the reactive
center loop in a conformation optimal for interaction with the
proteinase and for a fast rate of loop insertion. The complexes formed
with trypsin and the variant serpins were less stable than that formed
with wild-type serpin and resulted in up to 33% regeneration of
trypsin activity over a period of 6 days, compared with 17% with wild
type. Thus, the P5 residue of
1-proteinase
inhibitor is important in all steps of the inhibitory mechanism in a
manner consistent with the structural role played by this residue in
the
-strand loop structure of native
1-proteinase inhibitor.
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INTRODUCTION |
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One of the best studied members of the serpin family of serine
proteinase inhibitors is 1-proteinase inhibitor
(
1-PI),1 which
is an important inhibitor of neutrophil elastase and is deficient in
emphysema (1-3).
1-PI inhibits its target serine proteinases by the branched suicide-substrate pathway (1, 4) characteristic of inhibitory serpins (Fig.
1). The structure of native
1-PI has been solved by two groups (5, 6). From one
study (6), the reactive center loop, which contains the scissile
P1-P1'
bond,2 was found to be in a
helical conformation similar to other native serpins, namely ovalbumin
and an
1-antichymotrypsin variant (8, 9). In the other,
more detailed, structure of native
1-PI (5) it was found
that the reactive center loop is in an extended
-strand conformation
similar to the canonical conformation found for other classes of
protein inhibitors of serine proteinases (10). In this structure the
P5 glutamic acid residue plays a crucial role in the
stabilization of the
-strand conformation of the reactive center
loop by forming hydrogen bonds with Arg-196 and the backbone amide of
Met-226. These residues, along with Arg-223, Lys-243, and Arg-281, are
part of a basic pocket. It was concluded that these interactions fixed
the loop in the optimal conformation for the interaction with
proteinases and for rapid loop insertion (5); therefore this
structurally important residue also has important mechanistic
implications. In this study we disrupted the stabilizing bonds present
in native wild-type
1-PI by making P5 Glu to
Gly, Gln, and Lys mutations. This allowed us to test the hypothesis
regarding the implications of the
-strand conformation of the
reactive center loop of
1-PI on the inhibition mechanism. We assessed the effect of these mutations on the rates and
stoichiometries of inhibition with trypsin and human neutrophil elastase.
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We showed previously that 1-PI mutants containing
the P6-P2 or the P12-P2
residues of ovalbumin formed complexes with trypsin which had less
stability compared with wild-type
1-PI, as measured by
the regeneration of trypsin activity from complexes (11). In these two
mutants the P5 Glu was changed to Gly. We suggested that in
complexes with wild-type
1-PI either the P5
Glu or the P3 Ile were in direct contact with the trypsin
and formed an important bond involved in the stabilization of the
complex, or that they interacted with adjacent residues in strand 5A,
or the loop connecting helix F with strand 3A, to stabilize the cleaved
loop inserted form of the serpin in the complex. As part of this
present study we addressed this hypothesis by testing the long term
stability of complexes formed with the previously discussed
P5 variants.
We found that the variants had reduced rates of enzyme inhibition and
increased stoichiometries of inhibition (2-3-fold) showing that
mutations of the P5 residue influenced steps both before, and after, the branch point in the mechanism, i.e. rates of
proteinase interaction and of loop insertion. This is in agreement with
the hypothesis of the importance of the reactive center loop
conformation on serpin inhibitory activity as predicted from the
-strand loop structure (5). We also found that complexes between the
variants and trypsin showed regeneration of trypsin faster than with
wild-type
1-PI, in agreement with our hypothesis. Thus,
we have identified that the P5 Glu is an important residue
in the mechanism of proteinase inhibition of
1-PI, in a
predictable manner consistent with its important stabilizing role known
from the
-strand structure of native
1-PI.
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EXPERIMENTAL PROCEDURES |
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Materials--
Restriction enzymes were obtained from Pharmacia
Biotech Inc., oligonucleotides were synthesized by DNA Agency (Malvern,
PA), Sequenase was from U. S. Biochemical Corp., the pET16b vector was
from Novagen (Madison, WI), the Quick Change site-directed mutagenesis
kit was from Stratagene (La Jolla, Ca), human neutrophil elastase (HNE)
was from Athens Research (Athens, GA),
methoxysuccinyl-(Ala)2-Pro-Val-p-nitroanilide and phenylmethylsulfonyl fluoride were from Sigma, and S-2222 was from
Chromogenix (Franklin, OH). -Trypsin was purified from L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin (Sigma) on soybean trypsin inhibitor-agarose as reported
previously (12).
Mutagenesis, Expression, Refolding, and Purification of
Recombinant 1-Proteinase Inhibitor--
The wild-type
1-PI cDNA with deletion of the first five codons was
contained in a NcoI-XbaI fragment in the pET16b
vector (11). Site-directed mutagenesis of the P5 residue
from glutamic acid to glutamine, glycine, or lysine was performed using
the Quick Change site-directed mutagenesis kit following the
manufacturer's protocol. The oligonucleotides were 19 bases in length
and spanned the P8 residue codon to the first base of the
P2 codon. The codon for glutamic acid was GAG and the
mutated codons were CAG, GGG, and AAG, respectively.
Determination of the Stoichiometry of Inhibition for Trypsin and
Elastase--
Stoichiometry of inhibition (SI) values for the
inhibition of trypsin were determined by incubating different
concentrations of wild-type and the P5 variants of
1-PI for 2 h or 5 h at 25 °C, with 8 nM trypsin in 0.1 M Hepes, pH 7.4, 0.1 M NaCl, 10 mM CaCl2 and 0.1% PEG
8000. The residual amidolytic activity was determined by the addition
of 100 µM S-2222. Linear regression analysis of the
decrease in proteinase activity with increasing concentration of
1-PI yielded the estimates for the stoichiometry of
inhibition as the intercept on the abscissa. SI values for the
inhibition of HNE were determined in a similar fashion by incubating
different concentrations of wild-type and the P5 variants
of
1-PI for 30 min or 1 h at 25 °C, with 5 nM HNE in 0.1 M Hepes, pH 8.0, 0.5 M NaCl, and 0.1% PEG 8000. The residual amidolytic
activity was determined by the addition of 200 µM
methoxysuccinyl-(Ala)2-Pro-Val-p-nitroanilide. The obtained values for SI were adjusted by a correcting factor corresponding to the percentage of monomeric species present for the
1-PI species tested.
Rates of Inhibition of Trypsin and Elastase by Wild-type and
Variant 1-PI--
The rate of inhibition of trypsin by
recombinant
1-PI was determined at 25 °C by use of a
discontinuous assay procedure. Under pseudo-first order conditions, 150 or 200 nM wild-type
1-PI, 300 nM
or 1 µM P5G-
1-PI, 600 nM or 1.54 µM
P5Q-
1-PI, and 735 nM or 1.12 µM P5K-
1-PI were incubated
with 12.5 nM trypsin in 0.1 M Hepes, pH 7.4, 0.1 M NaCl, 10 mM CaCl2, and 0.1%
PEG 8000. The residual trypsin activity was determined at various time
by diluting the reaction mixture into the assay buffer containing 100 µM S-2222. The pseudo-first order constant,
kobs for the reaction was obtained from the
slope of a semi-log plot of the residual trypsin activity against time,
and the second order rate constant, kapp, was
determined by kobs/[I0], where
[I0] is the initial serpin concentration.
Determination of the Cleavage Sites in Wild-type and Variant
1-PI by HNE and Trypsin--
For all of the
1-PI variants, cleavage was done using HNE and trypsin,
and characterization of the cleavage site was performed by
NH2-terminal sequence analysis of the cleavage reaction
mixture after stopping the reaction by addition of phenylmethylsulfonyl fluoride to 5 mM and freezing until analysis. Automated
Edman degradation was carried out in an Applied Biosystems 477A
sequencer, by the protein sequencing facility in the Department of
Biochemistry, University of Illinois-Chicago. All
1-PI
variants and wild-type
1-PI were cleaved by HNE (4.5:1
ratio) in 0.1 M Hepes, pH 8.0, 0.5 M NaCl, and
0.1% PEG 8000 for 10 min at 25 °C. All
1-PI variants and wild-type
1-PI were cleaved by trypsin (4:1 ratio)
in 0.1 M Hepes, pH 7.4, 0.1 M NaCl, 10 mM CaCl2, and 0.1% PEG 8000 for 15 min at
25 °C.
Stability of Complexes between 1-PI and
Trypsin--
16 nM wild-type
1-PI, 33.5 nM P5G-
1-PI, 43.5 nM
P5Q-
1-PI, or 161 nM
P5K-
1-PI were incubated with 12.5 nM trypsin in 0.1 M Hepes, pH 7.4, 0.1 M NaCl, 10 mM CaCl2 and 0.1% PEG
8000 at 25 and 37 °C. The residual trypsin activity was determined
at various time by diluting the reaction mixture into the assay buffer containing 100 µM S-2222. Note that the amount of
1-PI in the incubations corresponds to active protein
determined as described above by correcting for the amount of inactive
polymer in each sample. To standardize all incubations so that the same
amount of functional serpin was in each, the concentration of trypsin (12.5 nM) was multiplied by the SI for the reaction to
determine the concentration of active serpin that would give complete
inhibition. This value was then multiplied by 1.3 to give a small
excess of serpin to ensure that the condition of complete inhibition
could be met.
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RESULTS AND DISCUSSION |
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Inhibition of Trypsin and HNE--
The aims of this study were
2-fold. First, to test the effect of disrupting the native conformation
of the reactive center loop on the inhibitory activity of
1-PI. From the two known native structures of
1-PI, the one describing a
-strand conformation predicted it would have direct mechanistic implications. We disrupted this optimal conformation by introducing changes at P5, the
crucial residue for stabilization (5). The second aim was to test
whether these same changes impair the stability of the resulting
complexes with trypsin. Thus, variants of
1-PI with
changes at the P5 residue from glutamic acid to glutamine,
glycine, or lysine were expressed in E. coli. Glutamine was
chosen to remove the charge but keep the same size residue, glycine was
chosen, as this residue is present in ovalbumin and the
1-PI-ovalbumin chimeras studied previously (11), and
lysine was chosen to introduce a positive charge at this position. The
rates and SI values of trypsin and HNE by the
1-PI-P5 variants were measured (Table
I). Amino-terminal sequencing of reaction
mixtures of the variants with HNE showed that the only detectable
cleavage site was at the P1-P1' bond. Therefore
multiplication of kapp by SI gives
k'app, which corresponds to the rate of
formation of [EI], i.e. the steps prior to the branch point (1). In all cases k'app was reduced
by about half. Therefore, the P5 residue can affect the
initial interaction with the proteinase as judged by the reduction in
k'app and the steps after the branch point,
namely loop insertion, as judged by the increase in SI. In the case of
trypsin with P5K-
1-PI, the SI appeared to be
close to 10. However, amino-terminal sequencing of this reaction
mixture revealed that the predominant site of cleavage was not at the
P1-P1' bond but at the
P5-P4 bond, consequently k'app could not be determined. Amino-terminal
sequencing of reaction mixtures with both trypsin and HNE showed that
in all other cases the only cleavage site was the
P1-P1' bond. Overall, the data show that it is
the loss of the glutamic acid that is critical rather than the gain of
an unfavorable amino acid. This supports the idea that the
P5 Glu is necessary for stabilizing the canonical
-strand conformation of the reactive center loop through
interactions with the basic pocket (see Introduction) and that loss of
these interactions alters the conformation of the loop to one that is no longer optimal for interaction with the proteinase and for loop
insertion.
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Stability of Complexes with Trypsin--
To test the second
hypothesis that P5 Glu is important in the kinetic
stabilization of 1-PI-proteinase complexes, we
investigated the long term stability of complexes formed by trypsin
with the variants. Wild-type or variant
1-PI were
incubated with trypsin at a 1.3-fold excess of inhibitor over the SI.
In this way each incubation had the same amount of functional serpin.
The reaction mixtures were incubated at either 25 or 37 °C for up to
6 days and aliquots assayed for enzyme activity throughout this time period. Control experiments under identical conditions showed that
trypsin in the absence of serpin remained fully active throughout the
time course. At 37 °C the complex with wild-type
1-PI
was stable at 21 h, whereas the complexes with the glycine,
glutamine, and lysine variants were reactivating with 4, 8, and 12.5%
of the trypsin activity being regenerated, respectively (Fig.
3B). The complex with
wild-type
1-PI started to reactivate at 24 h. Reactivation reached a plateau at 43 h for the variants and at 72 h for the wild-type. The maximum reactivation was 17% for
wild-type
1-PI, 24% for
P5G-
1-PI, 31% for
P5Q-
1-PI, and 33% for
P5K-
1-PI. At 25 °C the reactivation was
slower, reaching a plateau for all the
1-PI species
after 120 h of incubation (Fig. 3A). In an earlier study (11) we found that the reactivation of the complex of trypsin
with an
1-PI mutant containing the
P6-P2 residues of ovalbumin (which includes a
glycine at P5) was much faster and occurred to a greater
extent (75% reactivation in 48 h). This suggests that the
P5 residue is only partially responsible for the decrease
in complex stability and that other residues in this region also play a
role. In contrast, preliminary studies showed that complexes with HNE
were much more stable than those with trypsin (not shown). The longer
term stability of the
1-PI-HNE complex compared with the
1-PI-trypsin complex are consistent with earlier
observations (15, 16). Part of the reason for this difference might be
because human
1-PI and bovine trypsin are not a
naturally occurring pair, whereas human
1-PI and HNE are. In the latter case the inhibitor and the enzyme would have co-evolved to give optimum interactions, whereas in the former case
there would be no requirement for this to occur.
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The Role of the P5 Residue in Proteinase
Inhibition and Complex Stability--
The P5 Glu makes
important structural contacts in both the intact -strand
conformation and in the cleaved form of
1-PI (5, 17).
Therefore, it was likely that mutating this residue would lead to
changes in the inhibitory properties of the serpin. From the similar
decrease in kapp and
k'app for all three variants with both
proteinases it can be concluded that removal of the glutamic acid
(rather than addition of positive charge in the case of the lysine
variant) reduced the rate at which trypsin and HNE interacted with the
serpin. With this residue changed, the interactions that stabilize the
canonical conformation of the loop can no longer occur, hence the loop
probably adopts a different conformation, such as a helical one, as
this might now be the most stable conformation for the loop to adopt in
this context (18). This conformation is no longer optimal for binding to the proteinase, hence k'app is reduced. The
results are in agreement with the predictions made from the native
structure of
1-PI describing the loop in a canonical
conformation (5). According to Fig. 1, the serpin and the enzyme first
form a reversible Michaelis complex and then a reaction takes place to
give the intermediate [EI]. This latter step involves the
formation of optimal subsite interactions between the serpin and the
enzyme, such as through the P1' and S1'
subsites (19), as well as the initiation of loop insertion (20, 21).
Thus, by changing the conformation of the reactive center loop by loss
of the glutamic acid at P5, both steps might be reduced.
The increase in SI represents a reduction in the rate of loop insertion
into
-sheet A. The basic pocket surrounding P5 in the
native molecule is critical for stabilizing the canonical structure of
the loop with the hinge region ready to insert as strand 4 of
-sheet
A. Part of the driving force for loop insertion might come from the
disruption of this basic pocket, which occurs during the serpin
conformational change (5). In the case of the variants this driving
force would be lost as the interaction with the basic pocket probably
does not occur. Presumably then, the rate of loop insertion would be
slowed down enough to have an influence on the SI.
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ACKNOWLEDGEMENTS |
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We thank Dr. Peter Gettins, Dr. Steve Olson, and Dr. James Huntington for their helpful comments.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL-49242.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.
Present address: MRC Centre, Hills Rd., Cambridge CB2 2QH, United
Kingdom.
§ To whom correspondence should be addressed: Dept. of Oral Medicine and Diagnostic Sciences (MC 838), College of Dentistry, University of Illinois at Chicago, 801 S. Paulina St., Chicago, IL 60612. Tel.: 312-996-8554; Fax: 312-413-1604.
1
The abbreviations used are: 1-PI,
1-proteinase inhibitor;
P5G-
1-PI,
P5Q-
1-PI, and
P5K-
1-PI,
1-PI mutants with
the P5 residue changed to glycine, glutamine, or lysine,
respectively; SI, stoichiometry of inhibition; HNE, human neutrophil
elastase; PEG, polyethylene glycol.
2 Using the nomenclature of Schechter and Berger (7) the P1-P1' peptide bond is that which is cleaved by the proteinase. Residues NH2-terminal to this bond are designated P2, P3, and so on, and those COOH-terminal are designated P2', P3', and so on.
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REFERENCES |
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