(Received for publication, October 9, 1996, and in revised form, December 2, 1996)
From the Department of Haematology, University of Cambridge, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom
The reactive-site loops of serpins are
characterized by a defined mobility where the loop adopts a new
secondary structure as an essential part of the inhibitory process.
While the importance of mobility in the N-terminal region of the
reactive-site loop has been well studied, the role of mobility in the
C-terminal portion has not been investigated. The requirements for
mobility of the C-terminal portion of the reactive-site loop of
1-antitrypsin were investigated by creating a
disulfide bridge between the P
3 residue and residue 283 near the top of strand 2C; this disulfide would restrict the mobility
of the C-terminal portion of the reactive-site loop by locking together
strands 1 and 2 of the C
-sheet. The engineered disulfide bond had
no effect on the inhibitory activity of
1-antitrypsin,
indicating that there is no requirement for mobility in this region of
the molecule. Moreover, these results, coupled with those from
molecular modeling, indicate that insertion into the A
-sheet of the
intact reactive-loop beyond P12 is not rate-limiting for
the formation of the stable complex. The engineered disulfide bond
should also prove useful in the creation of more stable serpin
variants; for example, such a bond in plasminogen activator inhibitor-1
would prevent it from becoming latent by locking strand 1C onto the C
-sheet.
1-Antitrypsin
(
1-AT)1 is a member of the
serpin family of serine protease inhibitors. Members of this large
group of inhibitors are involved in the control of proteolysis in many
physiological processes including coagulation, fibrinolysis, complement
activation, and inflammation. Serpins are glycoproteins with a
molecular mass of approximately 50 kDa. They form a tight complex with
the active site of cognate serine proteases through an exposed peptide
loop of some 20 residues. Following the initial attack of the protease on the exposed peptide loop, serpins undergo significant conformational rearrangements resulting in the formation of an extremely stable bimolecular complex (1-4).
Early interactions between serpins and cognate proteases presumably
resemble those of the proteases with their natural peptide and
macromolecular substrates. Serpin reactive-site
loops2 share many features with the
cleavage sequences of natural substrates of serine proteases, including
sequence, surface accessibility, and flexibility. The requirements for
correct sequence and accessibility are relatively apparent; the need
for flexibility is less obvious. It has been proposed that the
structure of the transition state of good substrates of serine
proteases is closely homologous to that demonstrated by the so-called
"canonical" inhibitors, a class which includes the Kunitz, Kazal,
and ovomucoid third domain inhibitors (5, 6). These inhibitors have
reactive-site loops whose main-chain geometry is tightly restrained
into a specific conformation shared by all members of these families
between residues P3 and P3, both when free in
solution and when bound to the enzyme (7). This canonical conformation
allows a
-strand type of hydrogen bonding interaction between the
active site of the protease and the inhibitor; this structure is
believed to resemble the Michaelis complex formed early in the
proteolytic process. Natural proteolytic sites do not normally exist in
this configuration, yet must adopt it during the Michaelis step of the
reaction. It has been proposed by Hubbard et al. (8) that
the requirement for flexibility in proteolytic cleavage sites of
natural substrates is due to the distortions required to allow the
residues surrounding the scissile bond to adopt the Michaelis complex
structure, i.e. the canonical conformation. It is suggested
that these distortions would require substantial mobility of at least 5 residues on each side of the P1-P
1 bond
(8).
The nature of the serpin-enzyme complex is still a matter of great
debate. It has been variously proposed that a portion of the inhibitory
loop of serpins adopts a configuration similar to that of the canonical
inhibitors after docking with the protease (5, 9), that they resemble
the tetrahedral intermediate (10), and that the
P1-P1 peptide bond is cleaved in the complex with the P-side of the reactive-site loop fully inserted into the A
-sheet of the serpin (11-13). Each of these hypotheses requires that the serpin reactive-site loop adopts, at least transiently, a
Michaelis complex-like structure (i.e. similar to the
canonical configuration), and, according to the hypothesis of Hubbard
et al. (8) presented above, this requires mobility of at
least 5 residues on each side of the scissile bond. The presence of a
flexible reactive-site loop is both a conspicuous feature of serpins
and a notable point of difference between serine protease inhibitors of
the serpin family and those of the canonical classes. Up to 15 residues
on the N-terminal side of the P1-P
1 bond of the serpin reactive-site loop (residues P1 to
P15) constitute a very mobile region of the molecule, some
of which are believed to move from a solvent-accessible position to
being at least partially buried within the A
-sheet as an essential
part of the inhibitory process (11-14). Interfering with this mobility
has been demonstrated to adversely affect the inhibitory potential of
the serpin (15-18). In contrast, little is known about the role of the
P
side of the reactive-site loop. In the crystal structure of cleaved
1-antitrypsin, this region forms strand 1 of the C
-sheet from P
5 to P
10. Naturally occurring
mutations of strand 1C have been shown to interfere with normal serpin
activity (19, 20). In the crystal structures of two uncleaved members
of the serpin family, antithrombin (4) and plasminogen activator
inhibitor-1 (21), it has been demonstrated that strand 1C can detach
from the C
-sheet upon insertion of the P-side of the reactive-site
loop into the A
-sheet. In addition, molecular modeling indicates
that the N-terminal region of the reactive-site loop could not insert
extensively into the A
-sheet without either cleavage of the
P1-P
1 bond or the detaching of strand 1C from
the C
-sheet (22).
The work presented in this manuscript investigates the requirement for
strand 1C mobility in serpin inhibitory function, using 1-antitrypsin as an model. The mobility of strand 1C was
restricted through the creation of a disulfide bridge between two
mutated residues Pro361
Cys and Ser283
Cys, present in positions P
3 and in an adjacent position
at the end of strand 2C, respectively (see Fig. 1). The disulfide bridge was successfully created and shown to have a minimal effect on
1-AT inhibitory function, consistent with there being no
general requirement for mobility in strand 1C in the inhibitory
mechanism of serpins.
The chromogenic substrate
N-MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide,
Staphylococcus aureus V8 protease, protease-free bovine serum albumin, and p-nitrophenyl
p-guanidinobenzoate were purchased from Sigma (Poole,
United Kingdom). Human leukocyte elastase (HLE) was prepared as
described previously (23). Restriction enzymes were obtained from New
England Biolabs (Beverly, MA), and oligonucleotides were synthesized by
members of the Dept. of Biochemistry, University of Cambridge.
Rifampicin was obtained as Rifadin ( Merion Merrell Dow Ltd., Uxbridge,
UK). All other chemicals were of the highest grade commercially
available.
All
antitrypsins were produced in Escherichia coli BL21 (DE3)
under the control of the T7 RNA polymerase promoter using the vector
pN15 previously described (24). The mutants were made by PCR,
exploiting the AvaI restriction site that lies at a position corresponding to the codons for residues 362 and 363, a PstI
site that was engineered silently into a position corresponding to codon 347, and a BstXI site that lies upstream of
Ser283. The Pro361
Cys oligonucleotide used
was: GAACTTGACCTCGGGG
GATAGACATGGG; the Ser283
Cys oligonucleotide used was:
CGATATCATCACCAAGTTCCTGGAAAATGAAGACAGAAGGT
TGCCAGCTTAC; the mutated bases are underlined. PCR was performed with these oligonucleotides using antitrypsin cDNA containing the silent PstI site as a template and the product cut with
BstXI and AvaI and cloned into p
N15 cut with
the same two enzymes. Thus, the wild-type fragment was removed and
replaced with a fragment containing the double mutation
Cys361-Cys283. The entire region amplified by
PCR was sequenced. To construct the
Cys361-Ser283 mutant, the
Cys361-Cys283 cDNA was cut with
BstXI and PstI, and a wild-type fragment of
1-AT cut with the same two enzymes was inserted,
effectively replacing the Cys283 codon by the wild-type
Ser283 codon.
E. coli BL21(DE3) was transformed with pN15 containing
the wild-type or mutant antitrypsin cDNA. Two 2-liter flasks, each containing 1 liter of 2 × TY media, were inoculated with 10 ml of
an overnight culture of transformed E. coli and grown at
37 °C. When the absorbance at 600 nm reached 0.6, isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 0.1 g/liter. After 30 min, rifampicin was added
to a final concentration of 100 µg/ml, and the culture was grown for
an additional 3 h.
The antitrypsins were
purified from inclusion bodies as follows. The cells (10 g) were
resuspended in 30 ml of 50 mM Tris-HCl, pH 8.0, containing
300 mM NaCl, 10 mM EDTA, and 0.5% Triton X-100 and were lysed by three passages through a French Press at 16,000 p.s.i. The inclusion bodies and cellular debris were collected by
centrifugation at 5,000 × g for 20 min. This pellet
was resuspended in 30 ml of the above buffer, vortexed vigorously, and
centrifuged again. This procedure was repeated five times to wash the
inclusion bodies, three times in the same buffer, and twice in buffer
without the Triton X-100. The pellet was dissolved in 10 ml of 50 mM Tris-HCl, pH 8.0, 8 M guanidine
hydrochloride, 100 mM DL-dithiothreitol (DTT);
after purging with N2, the solution was incubated for
2 h. The antitrypsins were refolded by direct dropwise dilution into 1.6 liters of 50 mM Tris-HCl, pH 8.0, containing 5 mM DTT, at room temperature. The refolded protein was
loaded directly onto a 1.6 × 25 cm column of Q-Sepharose column
equilibrated in 50 mM Tris-HCl, pH 8.0, 50 mM
NaCl, 5 mM DTT at 4 °C, and the bound proteins eluted
using a 200-ml linear gradient from 50-250 mM NaCl. All
buffers were N2-purged. The purity was assessed by SDS-PAGE, and the antitrypsin-containing fractions were purged with
N2 and stored at 80 °C. For the preparation of the
oxidized form of the Cys361-Cys283 mutant, the
disulfide bond between Cys361 and Cys283 was
allowed to form during removal of DTT by dialysis for 16 h against
50 mM Tris-HCl, pH 8.0, 50 mM NaCl (4 × 5 liters).
The concentration of sulfhydryl
groups in antitrypsins was determined by the method of Ellman as
described in Ref. 25. The recombinant wild-type and oxidized
Cys361-Cys283 antitrypsins (0.03 µmol)
were diluted into a final volume of 6 ml of 80 mM sodium
phosphate buffer, pH 8.0, containing 130 mM EDTA and 2%
(w/v) SDS. 3 ml of this was reacted with 100 µl of 10 mM
5,5-dithiobis(2-nitrobenzoic acid) in 100 mM sodium phosphate buffer, pH 8.0, and incubated for 15 min at room temperature. After incubation, the absorbance of the 5,5
-dithiobis(2-nitrobenzoic acid)-treated solution was measured at 410 nm against the untreated antitrypsin blank solution. The concentration of free sulfhydryl in the
antitrypsin was calculated using the molar absorption value of 13,600 M
1 cm
1 for
2-nitro-5-thiobenzoate (25). Estimation of total cysteine content was
performed by amino acid analysis of the
Cys361-Cys283 antitrypsin at the Dept. of
Biochemistry, University of Cambridge.
The protein concentrations and inhibitory titers were determined as described previously (17). All assays were performed at 37 °C in 30 mM sodium phosphate buffer, pH 7.4, containing 0.16 M NaCl, 0.1% polyethylene glycol (Mr = 4000), 0.1% Triton X-100, and 500-600 µM N-MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide, using bovine serum albumin, polyethylene glycol (Mr = 8000)-coated plastic cuvettes. Slow-binding kinetics were performed using a Hewlett-Packard diode-array spectrophotometer. Each progress curve experiment consisted of 7 assays, with 1 in the absence of serpin and 6 others with increasing serpin concentrations. After initiation of the reactions by the addition of HLE, the production of p-nitroaniline was measured between 400 and 410 nm. Data points where substrate utilization was in excess of 10% of total substrate concentration were excluded from the analyses. Progress curve data were analyzed as described previously (17) to yield estimates for the association rate constant for the formation of the stable complex (kass). The dissociation constants (Ki) for the inhibitors could not be determined under the conditions used; however, considering the conditions of the experiment, an upper limit of 1 pM could be placed on all reactions. Progress curve experiments were performed at least three times, and the values reported represent the weighted mean of the determinations. Values of Km required for the calculation of kass were determined by standard initial velocity studies; an estimate of 85.5 ± 2.5 µM was obtained for N-MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide with HLE.
Analysis of Reaction Products by SDS-PAGEThe presence of
the disulfide bond between Cys361 and Cys283 in
the oxidized form of the Cys361-Cys283 protein
was confirmed by analysis of the migration of the protein on SDS-PAGE
before and after cleavage of the protein within the reactive-site loop.
1-AT and the mutant were incubated with S. aureus V8 protease in 50 mM
NH4HCO3 buffer, pH 7.8. This protease cleaves
the reactive-site loop of
1-AT at the P5
glutamic acid (Glu354) (26); when denatured, the C-terminal
fragment is released giving a reduction in the apparent size of the
protein on SDS-PAGE. In the double-cysteine mutant, however, the
C-terminal fragment should remain attached to the main body of the
molecule if the disulfide bond between Cys361 and
Cys283 was intact.
In order to determine whether the oxidized mutant was still capable of
making SDS-stable complexes, wild-type 1-AT and oxidized Cys361-Cys283 mutant (0.4 µM
each) were incubated with an equimolar amount of HLE in a volume of 20 µl at 25 °C for 2 min. SDS-gel loading buffer (5 µl) containing
2.5% SDS was added, and the sample was heated at 100 °C for 3 min
before analysis by SDS-PAGE.
The sequence of 1-AT contains
a single cysteine (Cys232). In order to restrict the
mobility of the C-terminal portion of the reactive-site loop, a
disulfide bond was engineered between the P3 residue
(Pro361
Cys) and a residue in strand 2C
(Ser283
Cys). Molecular modeling indicates that this
disulfide bond was feasible (Fig. 1) and that it would
lock the C-terminal end of the reactive-site loop including strand 1C
to the C
-sheet. The presence of the expected disulfide bond between
Cys361 and Cys283 was confirmed by sulfhydryl
titration assays and SDS-PAGE. Amino acid analysis of the
Cys361-Cys283 protein indicated the presence of
3.06 ± 0.07 cysteines per antitrypsin molecule, whereas only one
free sulfhydryl per antitrypsin molecule was detected using Ellman's
reagent, indicating that two of the cysteines participated in a
disulfide bridge. Given the known spatial arrangement of the three
cysteine residues it can be deduced that the disulfide bond is between
the residues Cys361 and Cys283, as shown in
Fig. 1. This was confirmed by cleavage of the oxidized Cys361-Cys283 mutant at position 354 by V8
protease and subsequent SDS-PAGE analysis of the protein which showed
that the C-terminal fragment of the
Cys361-Cys283 mutant remained attached when
denatured in the absence of reducing agents. After reduction, however,
SDS-PAGE separated the C-terminal fragment from the rest of the
molecule (Fig. 2). Non-reducing SDS-PAGE also showed
that the concentration of dimers formed by intermolecular disulfide
bonds was less than 5%. In addition, Fig. 2 shows that the intact
disulfide-bonded Cys361-Cys283 material has a
faster migration on SDS-PAGE than either the reduced material or the
wild-type material, consistent with an intramolecular disulfide bond
restricting complete unfolding during denaturation with SDS.
Inhibition of Human Leukocyte Elastase by Mutant Antitrypsins
Titration experiments with the oxidized form of
the Cys361-Cys283 antitrypsin showed that the
stoichiometry of inhibition of HLE was 1.1 relative to the wild-type.
Serpins have been shown to function as suicide inhibitors as shown in
Scheme 1 (11). After formation of an intermediate
complex (E·I), the pathway partitions between the
formation of the stable complex (E·I*) and the release of
cleaved inhibitor (I). The ratio of the rate constants for these two
reactions determines the stoichiometry of inhibition. Thus, the fact
that the stoichiometry of inhibition was not affected by the engineered
disulfide bond indicates that the relative rates of the inhibition and
cleavage pathways were not altered; i.e. the disulfide bond,
which restricts the mobility of strand 1C, does not affect the
partition of reaction products between the inhibitory and cleavage
pathways. In contrast, mutations at the N-terminal end of the
reactive-site loop can markedly affect this partitioning (15, 17,
18).
Scheme 1.
The Cys361-Cys283 mutant was still a potent
slow tight-binding inhibitor of HLE; restriction of the mobility of
strand 1C by the disulfide bond did not prevent the inhibitory process.
The association rate constant (kass) for
disulfide mutant was 0.21 × 107
M1 s
1 (Table I);
this value is about 5-fold lower than that of the recombinant wild-type
protein (1.20 × 107 M
1
s
1). In order to investigate further the reason for the
decrease in kass value, a mutant having only the
Pro361
Cys mutation was constructed. This mutant
(Cys361-Ser283) also had a reduced
kass value with HLE; the value obtained
(0.32 × 107 M
1
s
1) was similar to that obtained for
Cys361-Cys283 (Table I). This result
demonstrated that the 5-fold decrease in the association rate constant
of the double-cysteine mutant was not due to the restriction of the
mobility of strand 1C by the disulfide bond, but was predominantly due
to the replacement of the P
3 proline by cysteine.
|
Fig.
3 shows that the oxidized form of the
Cys361-Cys283 mutant formed typical SDS-stable
serpin-enzyme complexes, i.e. the disulfide bond does not
prevent this. The gels also show no appreciable generation of
reactive-center-cleaved 1-AT. Taken together with the
stoichiometry of inhibition value of 1.1 and observed
kass value, these data show that restricting the
mobility of strand 1C has no appreciable effect on the inhibitory
mechanism of
1-AT.
Although multiple lines of evidence have suggested that the
mobility of strand 1C may be important to the function of serpins (8,
19, 20), the data presented in this work indicate that, in
1-AT at least, restricting the mobility of strand 1C did
not significantly affect the inhibitory activity of the molecule. The
hypothesis presented by Hubbard et al. (8) concerning the requirement for mobility suggested that distortion of up to 5 residues
on either side of the P1-P
1 bond is required
for the formation of the canonical structure of the P2 to
P
2 residues. This would be required for any of the current
models of the serpin-enzyme inhibition reaction: the formation of the
Michaelis complex (10), the canonical form (5, 9), or cleavage of the
reactive-site loop (11-13). However, it is apparent from the data
presented here that in
1-AT mobility beyond residue
P
3 on the P
side of the scissile bond is not required for
the formation of the stable serpin-enzyme complex.
Although mobility of the P3 residue was not required for
complex formation, contacts between the active site of HLE and this residue appear to play a small role. Mutation of the P
3
proline to cysteine led to a 4-fold decrease in the
kass value. A similar decrease was observed with
the disulfide mutant. These results suggest that the decreases in
kass are due to loss of contacts with the
P
3 proline, since the chemical nature of the cystine in
Cys361-Cys283 and cysteine in
Cys361-Ser283 are different. Alternatively, the
P
3 proline could encourage a conformation of the
reactive-site loop that facilitates rapid complex formation.
Biochemical evidence now suggests that while in the stable complex the
hinge region of the serpin (residues P8 to P15)
is at least partially inserted into the A -sheet (14); the extent of
the insertion is still a matter of debate, but it appears that insertion at least as far as P12 is required (15, 27).
Recent modeling studies have indicated that insertion of the hinge
region into the A
-sheet to P12 or beyond requires
either the unlinking of strand 1C from the C
-sheet or cleavage
within the reactive-site loop (22). Data presented in this work
indicate that the uncoupling of strand 1C from the C
-sheet is not
necessary for the inhibitory function of
1-AT. This
observation may be interpreted in diverse ways: either extensive
insertion of the hinge region beyond P12 is not a
requirement for inhibitory function, or that insertion beyond
P12 occurs and the P1-P
1 bond is
cleaved in the complex, as suggested by two recent publications (12,
13). If the P1-P
1 bond is indeed cleaved in
the complex, and the reactive-site loop is fully inserted into the A
-sheet as strand 4A (12, 13), then the data presented in this work
indicate that cleavage occurs before insertion of the hinge region
beyond P12. It is now widely accepted that serpins operate
as suicide inhibitors (see Scheme 1), where after the formation of an
initial complex, this complex may lead to the concurrent generation of
either a stable serpin-enzyme complex (E·I*) or to the
generation of reactive-site loop cleaved serpin and active enzyme
(E + I
) (16-18, 27-29). Two processes are probably
involved in the conversion of the initial complex to the final stable
complex: insertion of the hinge region of the reactive-site loop into
the A
-sheet and cleavage of the P1-P
1 bond
(12, 13). Insertion of the hinge region appears to be the rate-limiting
process in the formation of the stable complex; mutations in the hinge
region of serpins reduce the rate at which the stable complex is formed
from the intermediate E·I, presumably by slowing down the
rate of insertion of the hinge region into the A
-sheet (18, 27),
and, thus, the insertion of the hinge region appears to be the
rate-limiting step in the formation of the stable complex
(E·I*). The presence of a disulfide bond preventing the
removal of strand 1C from the C
-sheet did not reduce the rate of
formation of the inhibited complex. Although an attack on the
P1-P
1 bond by the catalytic triad of the
serine protease appears to be essential for the formation of the final stable serpin-enzyme complex, the data suggest that this attack is not
a rate-limiting step and that it occurs after the rate-limiting step,
i.e. after initiation of insertion of the hinge region into the A
-sheet.
The disulfide mutant described here has implications for pharmaceutical
intervention in human medicine of the important regulatory systems
controlled by serpins such as coagulation, fibrinolysis, inflammation,
and complement activation. It has been discovered that serpins may
loose activity by converting to a latent form where the reactive-site
loop has inserted into the A -sheet fully as strand 4A; this
insertion is permitted because strand 1C has detached from the C
-sheet (22). The presence of a disulfide bond such as that described
in this work could be used to abolish the formation of a latent form of
a serpin such as plasminogen activator inhibitor-1 (21), or
antithrombin (4), thus increasing their utility without necessarily
affecting the inhibition of cognate proteases.
This paper is dedicated to the memory of Stuart R. Stone.