From the
A thermostable mutation, F51L, at the hydrophobic core of human
The reactive center loop of the serpins is unusually
long and is able to adopt various conformations
(10, 11, 12, 13, 14, 15) .
Several biochemical studies and recent structural determinations show
that the reactive center loop of the active serpin is mobile and
partially inserted into the central A-sheet
(7, 14, 15, 16, 17) . On the
contrary, no insertion of the loop residue was observed with the active
form of
The ability of the reactive center loop to adopt various
conformations is associated with opening of the A-sheet, in which a
partial or complete insertion takes place. Some factors that may
regulate A-sheet opening have been identified by structural comparison
between the cleaved form of
Previously, we identified single amino acid substitutions in native
human
In an effort to understand the relationship between the
stress of the inhibitory serpins and the tertiary fold of the molecule,
we previously identified a stable but active variant (Phe-51
The effect of the F51L
mutation appears to be specific to the native state. Kinetic data in
Fig. 3
show clearly that the F51L mutation increased the
thermodynamic stability of
The defect of the S
The F51L mutation site does not
directly interact with the Z mutation site (at the tip of stand 5 of
the A-sheet), which is likely to be a critical site for partial
insertion of the loop into the A-sheet
(2) . Our results support
that structural features of the hydrophobic core are tightly linked
during folding to A-sheet closing and consequently to loop movement.
Identification of more sites that are critical for the A-sheet closing
of
We are grateful to Dr. Soon Jae Park for peptide
synthesis and to Dr. Jonathan Weissman for the gift of the pF(BLG)
vector.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-antitrypsin (
AT) increased the
conformational stability of the molecule by decreasing the unfolding
rate significantly without altering the refolding rate. The mutation
specifically influenced the transition between the native state and a
compact intermediate, which retained
70% of the far-UV CD signal,
but which had most of the fluorescence signal already dequenched. The
mutant
AT protein was more resistant than the
wild-type protein to the insertion of the tetradecapeptide mimicking
the sequence of the reactive center loop, indicating that the mutation
increases the closing of the central
-sheet, the A-sheet, in the
native state. The F51L mutation enhanced the folding efficiency of the
Z-type (E342K) genetic variation, which causes aggregation of the
molecule in the liver. It has been shown previously that the
aggregation of the Z protein occurs via loop-sheet polymerization, in
which the reactive center loop of one molecule is inserted into the
opening of the A-sheet of another molecule. Our results strongly
suggest that the hydrophobic core of
AT regulates the
opening-closing of the A-sheet and that certain genetic variations that
cause opening of the A-sheet can be corrected by inserting an
additional stable mutation into the hydrophobic core.
-Antitrypsin (
AT)
(
)
is a member of the serine protease inhibitor (serpin)
family, which shares a common tertiary structure composed of three
-sheets and several
-helices
(1, 2) . Two
salient features of the serpin structure are the stress of the native
conformation and the mobility of the reactive center loop, both of
which are considered to be critical for biological function. Inhibitory
serpins have a stressed conformation in which the reactive center loop
is open to proteolytic cleavage. The cleavage accompanies an
irreversible transition to a very stable relaxed form in which the
newly created N-terminal portion of the cleaved loop is completely
inserted as a strand of the major
-sheet, the A-sheet
(2, 3, 4) . Structural comparison of various
forms of serpins and thermostability studies of serpins complexed with
synthetic peptides carrying sequences of the reactive center loop
(5, 6, 7, 8) suggest that the
enhancement in stability appears to be mainly due to insertion of the
loop into the A-sheet, leading to an increase in the number of strands
in the sheet and the buried surface area. However, studies have
indicated that the stress of the native form of
AT is
not limited to the reactive center loop or the A-sheet, but may be
propagated throughout the molecule
(9, 10) . The
structural mechanism of the stress including the distribution is not
yet understood.
-antichymotrypsin
(10) . Although these
apparent contradicting data may argue for a distinctive inhibition
mechanism
(18, 19, 20) , the loop mobility
postulated by Carrell et al. (7) was confirmative.
AT and a noninhibitory
serpin, ovalbumin
(5) . Others have been identified by studies
on some genetic variations of
AT that cause
aggregation of the molecule in the liver. It was suggested that the Z
(E342K) or S
(S53F) mutation of
AT
enhances loop-sheet polymerization by opening the A-sheet more readily
and converting the molecule into a better acceptor for the loop
insertion from another molecule
(8, 21, 22) .
Since the loop insertion into the A-sheet always accompanies an
increase in stability, it is likely that the native stress of an
inhibitory serpin is associated with A-sheet opening and consequently
with loop mobility. However, the structural mechanism of the link
between native stress and opening of the A-sheet remains unclear.
AT that confer increased thermal and
conformational stability, but that maintain inhibitory activity
(23) . The substitution of Phe-51 with hydrophobic aliphatic
residues reduced heat-induced aggregation, and it is likely that the
mutation diminished loop-sheet polymerization. If this is the case, the
defect of some genetic variants of
AT that cause
loop-sheet polymerization may be suppressed by the thermostable
mutation. In the present study, a stable mutation, F51L, was further
characterized, and its effect on the Z or S
genetic
variation was examined. Since the Phe-51 mutation is located at the
hydrophobic core of the molecule, the present study addresses the
question regarding the link between the stability conferred by the
hydrophobic core and the A-sheet opening of the
AT
molecule.
Recombinant
Recombinant wild-type and F51L mutant
AT Proteins and
Plasmids
AT proteins produced in Escherichia coli were
purified from inclusion bodies after refolding and ion-exchange
chromatography as described previously
(23) . Concentrations of
AT were determined in 6
M guanidine
hydrochloride using a value of A1 cm1% = 4.3 at 280 nm,
calculated from the tyrosine and tryptophan content of the
AT protein
(24) and based upon
M
= 44,250. The Z-type (E342K) or
S
(S53F) mutation or its combination with the F51L
substitution was introduced into the
AT cDNA by
oligonucleotide-directed mutagenesis
(25) . The mutagenic
oligonucleotide was a 26-mer, and the mismatched codon was located in
the middle. For in vitro translation, the
AT
expression vector pF(BLG)AT was constructed in which the
AT cDNA was connected downstream of the T7 promoter
and the 5`-untranslated region of the rabbit
-globin gene.
Chemicals
Ultrapure urea was purchased from
Schwarz/Mann. [S]Methionine was purchased from
DuPont NEN. All other chemicals were reagent-grade. The buffer used for
folding experiments was 10 m
M phosphate, 50 m
M NaCl,
1 m
M EDTA, and 1 m
M
-mercaptoethanol (pH 6.5).
Urea-induced Equilibrium Unfolding
Transition
Equilibrium unfolding as a function of urea was
monitored by fluorescence spectroscopy and CD spectroscopy. The native
protein was incubated in 10 m
M potassium phosphate, 50 m
M NaCl, 1 m
M EDTA, 1 m
M -mercaptoethanol, and
various concentrations of urea (final pH 6.5) at 23 °C. Samples
were allowed to equilibrate for 8 h. Tryptophan fluorescence was
measured for each sample (Shimadzu RF-5000 fluorescence
spectrophotometer) with excitation at 280 nm and emission at 360 nm.
The CD signal was recorded (Jasco J-600) at 222 nm at 23 °C in a
cell with a 0.1-cm path length and a 1-nm bandwidth. The protein
concentration for the unfolding transition was 10 µg/ml for
fluorescence spectroscopy and 50 µg/ml for CD spectroscopy.
Experimental data of fluorescence measurement were fitted to a
two-state unfolding model as described previously
(23) .
Kinetics of Folding and
Unfolding
AT in the native or unfolded state
was subjected to structural change in various concentrations of urea,
and the time required to reach a new equilibrium was analyzed by
monitoring the fluorescence intensity (
= 280
nm and
= 360 nm). The final protein
concentration was 10 µg/ml. For refolding, the native protein was
unfolded in 7
M urea for 5 min, and refolding was initiated by
adding the unfolded protein to the designated urea concentrations. The
kinetic data were collected after manual mixing with a dead time of 15
s on average. The major kinetic phase was examined, and the data were
fitted to a two-state model to obtain the relaxation time:
F( t) = F( i)
e
Transverse Urea Gradient Gel Electrophoresis
Gels
were prepared with a gradient of 0-8
M urea
perpendicular to the direction of electrophoresis with an opposing
gradient of acrylamide from 15 to 11%
(26) . Four slab gels (100
80 mm) were prepared simultaneously in a multigel caster
(Hoefer Scientific Instruments) by using a gradient maker and a
single-channel peristaltic pump. The electrode buffer was 50 m
M Tris acetate, 1 m
M EDTA (pH 7.5). The native protein (20
µg in 100 µl), the protein unfolded in 8
M urea (10
min at room temperature), or the in vitro translation product
was applied across the top of the gel. The gels were run at a constant
current of 6 mA for 3 h at a controlled temperature of 23 °C.
In Vitro Translation of Cloned Genes
In vitro transcription was performed with the SP6/T7 transcription system
(Boehringer Mannheim) at 37 °C for 1 h. In vitro translation was performed with a rabbit reticulocyte lysate system
(Promega). A typical translation reaction mixture (final volume of 25
µl) contained 20 µCi of [S]methionine,
0.5 µg of mRNA, and 17.5 µl of reticulocyte lysate treated with
micrococcal nuclease. The labeling reaction was carried out at 30
°C for 1 h and was chased with unlabeled methionine (2
m
M).
Binary Complex Formation
The binary complex of the
wild-type and F51L AT proteins with the
tetradecapeptide of the reactive center loop sequence
(Ac-Thr-Glu-Ala-Ala-Gly-Ala-Met-Phe-Leu-Glu-Ala-Ile-Val-Met-OH) was
formed by incubating the native protein (0.24 mg/ml) in 10 m
M phosphate, 50 m
M NaCl, and 1 m
M EDTA (pH 6.5)
with a 230-fold molar excess of the peptide at 40 °C for various
period up to 6 h. The degree of complex formation was determined by
13.5% polyacrylamide gel electrophoresis in the absence of urea. The
buffer system was identical to the urea gradient gel electrophoresis
system described above.
Effect of the F51L Mutation on Thermodynamic, Kinetic,
and Structural Aspects of the Conformational Transition of
A previous study on urea-induced
reversible unfolding of AT
AT showed that the F51C
mutation increased conformational stability toward the denaturant in a
concentration-independent manner
(23) . The thermostable
substitution at Phe-51 was further characterized using the F51L
mutation as a prototype to avoid possible complications involved with
the cysteine residue in the F51C mutation. The F51L mutation also
increased stability toward urea as demonstrated by the equilibrium
unfolding transitions monitored by fluorescence and CD signals
(Fig. 1, A and B). Although the transition
monitored by the fluorescence signal was fitted well to the two-state
unfolding model (Fig. 1 A) to give midpoints of
transition at 1.9 ± 0.1 and 2.8 ± 0.1
M urea for
the wild-type and mutant proteins, respectively, the transition
monitored by the CD signal (Fig. 1 B) indicates that the
unfolding of
AT is multiphasic. The increase in
equilibrium stability by the F51L mutation was also observed in the
transverse urea gradient gel electrophoresis. Fig. 2shows that
two major transitions of
AT were detected during
unfolding and refolding of
AT in urea. A major effect
of the F51L mutation was a shift in the transition point to a higher
urea concentration in the initial unfolding step, which was also
confirmed in the refolding transition. The midpoints of the transition
of the wild-type and F51L mutant proteins were approximately 2 and 3
M urea, respectively. It appears that the mutation
specifically influenced the initial stage of unfolding and had no
effect on the later unfolding transition at a higher urea
concentration.
Figure 1:
A, urea-induced
unfolding of the wild-type ( WT; ) and F51L mutant
(
) recombinant
AT proteins as measured by the
increase in fluorescence emission intensity at 360 nm (
= 280 nm; excitation and emission slit widths of 5 nm for
both). Samples were equilibrated for 8 h in 10 m
M potassium
phosphate, 50 m
M NaCl, 1 m
M EDTA, 1 m
M
-mercaptoethanol (pH 6.5), and various concentrations of urea
at 23 °C. The protein concentration was 10 µg/ml. The
transition midpoints of the wild-type and F51L mutant proteins were 1.9
± 0.1 and 2.8 ± 0.1
M, respectively, as
determined by fitting the data to a two-state unfolding model.
B, urea-induced unfolding of the wild-type (
) and F51L
mutant (
) recombinant
AT proteins as measured by
the change in the CD signal. The loss of CD ellipticity at 222 nm was
monitored using a 0.1-cm path length cell as a probe of structural
change in the
AT protein. The protein concentration
was 50 µg/ml.
Figure 2:
Transverse urea gradient gel
electrophoresis of the wild-type ( WT) and F51L
AT proteins. The native protein ( Unfolding)
or the protein unfolded in 8
M urea ( Refolding) was
applied across the top of the gel. Electrophoresis was carried out at 6
mA of constant current for 3 h at 23 °C. Protein bands were
visualized by staining with Coomassie Blue.
There are two ways of increasing the free energy of
stabilization of a protein: by stabilizing the native state or by
destabilizing the unfolded state. Kinetic studies of unfolding and
refolding monitored by the fluorescence signal revealed that the F51L
mutation retarded the unfolding rate substantially, while the refolding
rate was not affected by the mutation (Fig. 3). This result
indicates that the mutation increased the free energy of stabilization
by decreasing the energy level of the native state rather than by
increasing the energy level of the partially unfolded conformation.
Figure 3:
Kinetics of unfolding and refolding of
the wild-type ( and
) and F51L (
and
)
recombinant
AT proteins. The relaxation time of
urea-dependent refolding (
and
) and unfolding (
and
) monitored by the fluorescence signal was plotted.
AT in the native or unfolded state was subjected to
structural change in various concentrations of urea, and the time to
reach a new equilibrium was analyzed by monitoring the fluorescence
intensity (
= 280 nm and
= 360 nm). The final protein concentration was 10
µg/ml. For refolding, the native protein was unfolded in 7
M urea for 5 min, and refolding was initiated by the addition of the
unfolded protein to urea at the designated
concentrations.
The transition influenced by the mutation in urea denaturation may
correspond to the transition from the native to the open conformation,
in which the major sheet, the A-sheet, is able to accept the reactive
center loop of another molecule. To test this, the complex formation
was examined between the native AT protein and the
tetradecapeptide that mimics the reactive center loop sequence. Fig. 4
shows that the kinetics of the complex formation with the peptide was
retarded by the F51L mutation, indicating that the mutant protein is
more resistant to the peptide insertion.
The F51L Substitution Increases the Folding Efficiency of
the Z-type Variant
If the F51L mutation truly enhances closing
of the A-sheet, it might compensate for the defect of those genetic
mutations that induce A-sheet opening. The effect of the F51L mutation
on Z- or S-type
AT was examined by
monitoring the folding transition on urea gradient gel electrophoresis
with in vitro translation products (Fig. 5). Most of the
nascent Z or S
polypeptides were accumulated at the
intermediate state. However, on combination with the F51L mutation,
part of the population of the Z type, but not the S
type, could fold into the native state. The gel patterns of the
wild-type and F51L
AT proteins synthesized in
vitro were very similar to those of the purified proteins
(Fig. 2), indicating that most of these translation products are
successfully folded. The results show that the folding defect of Z-type
AT was partially relieved by the F51L mutation. The
defect caused by the S
mutation was not suppressed by
the F51L mutation.
Figure 5:
Effect
of the F51L mutation on the defect of Z and S
variants. The translation products of
AT labeled with
[
S]methionine for 1 h carrying the indicated
mutations were analyzed by urea gradient gel electrophoresis. The
protein bands were visualized by
autoradiography.
aliphatic hydrophobic residues) of human
AT, which
mapped on the B- sheet in the hydrophobic core of the molecule
(23) . The existence of such a mutation supports the notion that
the stress of the native serpin is not limited to the A-sheet, but may
be distributed over the molecule
(9, 10) .
Stability Increase in
What is the nature
of the transition influenced by the F51L mutation, which was
demonstrated in the unfolding transition monitored by fluorescence and
CD spectroscopies and urea gradient gel electrophoresis (Figs. 1 and
2)? The equilibrium unfolding of AT by Enhancing the
A-sheet Closing from the Hyrophobic Core
AT was multiphasic,
involving at least two slow steps, although the fluorescence signal
could detect only the initial unfolding that fits to the two-state
model. The F51L mutation shifted the first unfolding transition to a
higher urea concentration. Various data indicate that this transition
reflects unfolding of native
AT to an open
conformation, in which the major sheet (A-sheet) is open to be able to
accept the loop insertion. It was shown that loop-sheet polymerization
could be induced with wild-type
AT under mild
denaturing conditions
(7) . In addition, the thermostable
mutation that shifted this transition (Figs. 1 and 2) was shown
previously to retard the aggregation rate
(23) . The transition
midpoints of the wild-type and mutant
AT proteins in
the urea gradient gel system were approximately 2 and 3
M,
respectively, which are close to those obtained by fluorescence
spectroscopy (Fig. 1 A). Trp-194, the major contributor
of the signal among two tryptophans
(27, 28) , is likely
to be a sensitive probe for the A-sheet opening because it is located
at the top of strand 3 of the A-sheet, being hydrogen-bonded with
residues in strand 5 of the A-sheet both in the intact cleaved
(2) and model intact
(29)
AT proteins.
Finally, the nascent polypeptides carrying the Z or S
mutation, which causes loop-sheet polymerization in vivo (21, 22) , were arrested at this intermediate state
(Fig. 5). Although recent structural determination of
antithrombin dimer suggested another kind of polymerization with the
C-sheet
(14, 15) , C-sheet polymerization of
AT has not been reported. These data indicate that the
folding intermediate detected in the present study is likely to be an
open state of the A-sheet, slightly less compact than the native form,
with tryptophan fluorescence being fully dequenched, but containing
70% of the native far-UV CD signal.
AT by lowering the energy
level of the native state, indicating that the native state of the
molecule is specifically changed by the F51L mutation. In addition, the
native fluorescence intensity of the F51L mutant protein was lower than
that of the wild-type protein for the same amount of protein, while the
unfolded values for both were similar (Fig. 1 A), indicating
that the native state of the F51L mutant protein is more packed near
Trp-194. If the native state of the F51L mutant
AT
protein is more stably closed in the A-sheet than that of the wild-type
protein, it would be more resistant to the insertion by the peptide of
the reactive center loop sequence. The complex formation experiment in
Fig. 4
shows that this is indeed the case, suggesting strongly
that the change in the A-sheet opening is one of the physical changes
caused by the F51L mutation. In the crystal structure of cleaved
AT
(2) , Phe-51 is located in strand 6 of the
B-sheet in the hydrophobic core, interacting not only with conserved
hydrophobic residues (Phe-372 and Phe-384) in strands 5 and 4 of the
B-sheet, but also with some residues (Ala-336 and Leu-338) in strand 5
of the A-sheet. The mutations at Phe-51 may regulate the
opening-closing of the A-sheet through interactions with some of these
residues. By decreasing the side chain volume
(23) , the stable
mutations may provide a more favorable packing in the native form of
mutant proteins.
Figure 4:
Complex formation of the wild-type
( WT) and F51L AT proteins with a peptide of
the reactive center loop sequence. The native
AT
protein was incubated with a 14-mer peptide (Ac-TEAAGAMFLEAIVM-OH) in
10 m
M phosphate (pH 6.5), 50 m
M NaCl, and 1 m
M EDTA (protein concentration of 0.24 mg/ml; protein/peptide ratio
of 1:230; total volume of 10 µl) at 40 °C for 0-6 h. The
reaction samples were analyzed by native gel electrophoresis. The gel
electrophoresis condition was as described for Fig. 2, except that the
gel contained 13.5% polyacrylamide without any
urea.
Suppression of the Z-type Folding Defect by the F51L
Substitution
The proposed mechanism for the F51L mutation is
exactly opposite of that postulated for the genetic defect of the Z
(E342K) or S(S53F) mutation, which was attributed to
the opening of the A-sheet
(21, 22) . A major defect of
these genetic mutations is the arrest of the final folding of the
nascent chains from the intermediate to the native state
(Fig. 5). Upon combination with the F51L mutation, however, some
of the nascent Z polypeptides, but not the S
polypeptides, could fold into the native state. The folding
arrest could be due to a destabilization of the mutant native protein
or alternatively to a kinetic retardation of folding. The Z type could
fold into the native form, although very slowly, with a folding rate on
the order of several hours.
(
)
The F51L mutation
allowed part of the population of the Z polypeptides to fold properly,
while the rest of the Z polypeptides remained at the intermediate
state. It appears that competition exists between the productive
folding pathway and a kinetic trap: while the Z mutation induces a
partitioning into the folding trap, a more favorable partitioning into
the productive pathway is induced by the F51L mutation. It is likely
that the F51L mutation, by shifting the equilibrium toward the
production of the native form, decreased the amount of the intermediate
that can be trapped.
mutation
could not be suppressed by the F51L mutation. This may be because the
conformational stability of the S
protein is
drastically affected. The S
mutation occurs at the N
terminus of the B-helix that is comprised in the hydrophobic core of
the molecule. Replacement of Ser-53, which initiates the helix and the
side chain O
hydrogen-bonded to the backbone nitrogen
of Ser-56
(2) , with bulky phenylalanine is likely to have a
drastic effect on the stability. The results in Fig. 5are
consistent with the S
mutation causing a more severe
aggregation of the molecule than the Z-type mutation
(22) . Our
results suggest that some, although not all, genetic variations that
cause a secretion block by enhancing the A-sheet opening can be
corrected by inserting an appropriate stable mutation at the
hydrophobic core. Indeed, a preliminary investigation of the yeast
secretion system revealed that the secretion block of Z-type
AT can be partially relieved by the F51L
mutation.
(
)
AT will be essential for understanding the
mechanism of the native stress of the serpins.
AT,
-antitrypsin; serpin, serine protease inhibitor.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.