From the Department of Haematology, University of Cambridge, Cambridge Institute of Medical Research, Hills Road, Cambridge CB2 2XY, United Kingdom
Received for publication, November 15, 2002, and in revised form, January 24, 2003
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ABSTRACT |
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Polymerization of serpins commonly results from
mutations in the shutter region underlying the bifurcation of strands 3 and 5 of the A-sheet, with entry beyond this point being barred by a
H-bond network centered on His-334. Exposure of this histidine in
antithrombin, which has a partially opened sheet, allows polymerization and peptide insertion to occur at pH 6 or less when His-334 will be
predictably protonated with disruption of the H-bond network. Similarly, thermal stability of antithrombin is
pH-dependent with a single unfolding transition at pH 6, but there is no such transition when His-334 is buried by a fully
closed A-sheet in heparin-complexed antithrombin or in
The serpin family of serine protease inhibitors (1) provides a
clear example of the way in which dysfunction and disease can result
from conformational instability (2, 3). The inhibitory function of the
serpins is dependent on a triggered opening of the five-stranded A
The potential role of His-334 as a guardian of sheet opening is
indicated in the crystallographic structures of two serpins, antithrombin and heparin cofactor II (16, 17). Both of these serpins
are exceptional in having, in their native states, a partial opening of
the A-sheet with an initial insertion of the reactive loop (to a level
of P14, see Fig. 1a). In both, the hydrogen bonding between
strands 3 and 5 does not commence until His-334 (at the level of
insertion of P8 in cleaved serpins), which appears as the first barrier
to further opening of the sheet (Fig. 1b). The consequences
of the loss of this barrier and of opening the A-sheet are potentially
2-fold. It can allow the complete insertion of the uncleaved reactive
loop of the molecule to give the inactive latent form or it can allow
the incorporation of the reactive center loop of another molecule
resulting in serpin polymerization. Transition to the latent state
takes place as a physiological mechanism in the plasminogen
activator inhibitor-1 (18), which exceptionally among the serpins has a
glutamine rather than a histidine at 334 (19). Transition to the latent
form is also a significant pathological mechanism in the
conformationally unstable mutants of antithrombin in which a premature
conversion to the latent conformation can result in a catastrophic
decrease in antithrombin activity with massive thrombosis (20). More
commonly though, with most serpins such as
Materials--
Restriction enzymes and T4 DNA ligase were
purchased from New England Biolabs, and oligonucleotides were
synthesized by MWG-Biotech. The expression vector pQE31 and
Escherichia coli strain SG13009 (pREP4) were from Qiagen.
Isopropyl- Purification and Preparation of Native and Latent
Antithrombin--
Native antithrombin was purified from frozen plasma
by precipitation of plasma with dextran sulfate and calcium chloride
(23, 24) after which the supernatant was diluted with an equal volume of equilibration buffer (50 mM Tris-HCl, 10 mM
sodium citrate, 5 mM sodium EDTA, 0.4 M NaCl,
pH 7.4). This mixture was applied to a heparin-Sepharose affinity
chromatography column previously equilibrated with the same buffer, and
the column was thoroughly washed with equilibration buffer. The
antithrombin was eluted using a gradient from 0.4 to 2 M
NaCl in the equilibration buffer. Antithrombin peaks were further
purified by anion exchange chromatography on a HiTrap Q-Sepharose
column. Antithrombin was concentrated in 10 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 7.4, and
snap-frozen.
Latent antithrombin was prepared using the glycerol method (25).
Briefly, native antithrombin was incubated with 40% glycerol in 50 mM phosphate buffer (pH 6) for 24-36 h at 50 °C, which
gave nearly 100% latent formation. After incubation, the samples were diluted 4-fold with 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA loaded onto a HiTrap-Q-Sepharose column and washed
with the same buffer. Latent antithrombin was eluted with a 0-0.5
M NaCl gradient in 10 mM Tris-HCl, pH 7.4, and
1 mM EDTA. The concentration of antithrombin was determined
using an extinction coefficient of 6.5 (26).
Preparation of Recombinant Antitrypsin Variants--
Human
Complex Formation between Antitrypsin Variants and
Trypsin--
Stoichiometries and Rates of Inhibition--
Stoichiometries of
inhibition were determined by incubating increasing concentrations of
Thermal Stability--
Circular dichroism (CD) experiments were
performed using a JASCO J-810 spectropolarimeter. Samples of
Equilibrium Unfolding of Antithrombin--
Equilibrium unfolding
was monitored by spectrofluorimetry ( Polymerization of Antitrypsin Variants--
Antitrypsin variants
were incubated at 1 mg/ml in 20 mM Tris-HCl, 50 mM NaCl, pH 7.4, and 1 mM EDTA for 1 h at
temperature from 37 to 65 °C. Samples were loaded onto an 8% (w/v)
native gel. The proteins were visualized by staining with Coomassie Blue.
Peptide Insertion--
Antithrombin at 0.5 mg/ml or
Crystallization of Antithrombin/Peptides
Complexes--
Native antithrombin (1 mg/ml) was incubated with 2 mM of P14-8 peptide of antithrombin (acetyl-SEAAAS)
and a tri-peptide (formyl-MLF) at 37 °C for 24 h. Samples were
then washed several times with 10 mM Tris-HCl, pH 7.4, in a
concentrator to remove most of the free peptides, with concentration to
14 mg/ml. Crystallization was performed using hanging drop methods as
previously described (31) with the following modification.
Antithrombin-peptide complexes were mixed with equal amounts of latent
antithrombin and equilibrated against 10-20% PEG 4000 in 50 mM sodium cacodylate buffer, pH 6.8, and 0.2 M
NH4F, with or without 12% glycerol. Crystals grew to full
size in ~1 week.
Data Collection and Refinement--
Diffraction data to 2.8 Å were collected from a single frozen crystal at Daresbury
Synchrotron Radiation Source (station 14-2) and processed using
Mosflm (32) and Scala (33). Processing statistics are given in
the Table I. Since the crystal of the ternary peptide complex was essentially isomorphous with the structure of the previously solved antithrombin binary-peptide complex (34), the
original binary complex coordinates (PDB accession number 1BR8)
were used as the starting model. Refinement to 2.8 Å was performed in
crystallography NMR software (35) using the maximum
likelihood target of Pannu and Read (36). The model was built with O
(37). The final refined structure (Table I) contains all residues
except 1-4 and 24-44 in the latent molecule and 1-4, 28-41, and
381-383 in the peptide-annealed molecule. The coordinates and
structure factors have been deposited in the Protein Data Bank
(accession number 1LK6). Figures were made by using MolScript
(38), BOBSCRIPT (39), RASTER3D (40), and XtalView (41), and figures for
antithrombin (Fig. 1, a, b, c) were based on the highest resolution native
structure PDB 1E04.
Polymers, Latency, and Glycerol--
Prolonged heating
of antithrombin and pH Dependence of Transitions--
The incubation of antithrombin
at 50 °C for 16 h over a range of pH values (Fig.
2c) shows the ready formation of polymers up to pH 5.5 but
insignificantly so at pH values greater than 6. Above pH 6 there is a
much slower conformational transition, with the formation of latent
antithrombin (apparent as the heterodimer) and much less polymer
formation until pH 10. The effect of pH on the opening of the A-sheet
of serpins can also be assessed by the readiness with which they
complex with synthetic P14-3 peptides. This is clearly seen in Fig.
2d which shows the formation of the binary complex (seen as
a 7 M urea stable component) in antithrombin incubated for
12 h at 37° with the P14-3 peptide. The rapid formation of the
complex at pH 5 as compared with pH 6 and 7 indicates that the low pH
favors sheet opening and hence peptide annealment.
pH and Thermal Stability--
Serpins typically have a well
defined thermal transition with antithrombin having a melting point
(Tm) of 57.6 °C at pH 7.4. Antithrombin
differs, however, from the archetypal serpin
Recombinant Replacement of His-334--
To assess the contribution
of His-334 to the stability of Crystallographic Detail of His-334 Interaction with
Glycerol--
A crystallographic finding, reported here (PDB
accession number 1LK6), provides an unexpected insight with particular
relevance to His-334. The finding was incidental to the determination
of the structure of a ternary complex of antithrombin as part of a
larger2 series of
serpin-peptide complex structures. The ternary complex was formed by
incubation of antithrombin with P14-9 and P6-4 synthetic loop peptides
in the presence of glycerol. The 2.8 Å structure shows a glycerol
molecule sited in the position occupied in six-stranded antithrombin by
the side chain of threonine P8 (Fig. 1, d and e).
Critically, a hydroxyl of the glycerol forms a hydrogen bond with the
The findings here open a clearer understanding of the mechanisms
leading to aberrant conformational changes in the serpins. In
particular the pH dependence of these changes strongly supports structural evidence as to the critical role of His-334 in maintaining the metastable inhibitory conformation. In antithrombin, His-334 in
strand 5A is clearly seen as a barrier to further insertion of the
reactive loop, due to the bridging linkage of its imidazole side chain
to Asn-186 in strand 3A and to Ser-53 in the underlying In keeping with the conclusion that sheet opening is influenced by the
protonation of His-334, the thermal stability of antithrombin was shown
to be pH-dependent, with a characteristic transition at pH
6 (Fig. 3b). In comparison to this, there is no such pH transition in stability in An understanding of the mechanism controlling sheet opening is needed
for the design of therapeutic agents to prevent the pathological
polymerization of serpins. Although the backbone position of His-334 is
at the level of P8 of a fully inserted reactive center loop, the
imidazole side chain of the histidine, by its H-bonding to Asn-186,
effectively blocks entry to the sheet beyond the level of P12 (Fig. 1,
b and c). So entry of the reactive loop or its
homologue peptides, to level P10 and beyond, will disrupt the linkage
between s3A and s5A and allow full opening of the sheet. Thus insertion
of P14-9 or P14-8 peptides will enable the entry of the P7-3 sequence
of the reactive loop of another molecule into the lower half of the
opened sheet, with the resultant formation of polymers. But
normally there will be preferential entry of the molecule's own
reactive loop, facilitated by the ability of the side chain of its P8
threonine to re-form the H-bond network with His-334 of s5A and Asn-186
in s3A. The threonine at P8 is conserved in almost all serpins (13)
with a notable exception being in human A demonstration of the way insertion of the reactive loop to P9
disrupts the His-334-Asn-186 bond between s3 and s5A is seen in our
crystallographic structure of a complex of antithrombin with a P14-9
peptide (Fig. 1, d and e). In this structure,
stabilized by a further insertion of a P6-4 tripeptide, a gap has
opened between His-334 and the side chain of Asn-186 that is normally bridged by hydrogen bonding to the side chain of the P8 threonine. However, the unexpected finding in the structure is the presence in the
P8 position of a glycerol molecule, oriented similarly to that of the
side chain of the threonine normally at P8. The glycerol, as with the
threonine, is hydrogen-bonded to His-334 with the precise bond
distances that ensure the anchoring of the imidazole ring to the main
chain of Ser-53. Thus the glycerol has in effect re-formed the bonding
network that stabilizes His-334. Although this new network does not
link to Asn-186 in s3A, the presence of the glycerol will impede the
insertion of external peptides in the P8-P7 position. This,
together with the potential for glycerol to also insert into other
vacant strand positions, provides an additional explanation for the
efficacy of glycerol in the protection of antithrombin against
polymerization (Fig. 2b). This protective effect of high
concentrations of glycerol had been assumed to be due to its decreasing
the rate of diffusion and hence of the collisions between individual
molecules required for polymerization. But whereas polymerization will
result from random intermolecular collisions, the monomeric
transformation to the latent form is due to the ordered entry of the
molecule's own reactive loop into the sheet. This entry of the
optimally oriented side chains of the reactive loop, including the P8
threonine, should readily displace any in situ glycerol
molecules. Thus the latent transition is less affected by the presence
of glycerol (Fig. 2b). Nor is it as dependent as
polymerization on the opening of the A-sheet as the latent transition
continues to occur at higher pH values when the His-334 network is
intact and the sheet is predictably closed (Fig. 2c). The
likely limiting factor in the latent transition is not so much the
opening of the A-sheet as the release of the intact loop by thermal
dissociation at its distal hinge, s1C (25, 45). This is consistent with
previous observation that replacement of glutamine with histidine at
334 in plasminogen activator inhibitor-1 does not decrease latent transition (46).
The prevention of polymerization by peptides that insert into the
opened A-sheet has been demonstrated in vitro and shown to
be selectively achievable (34, 47-50). The therapeutic challenge is to
convert these peptides into pharmacologically effective in
vivo agents. The identification here of a focal point for sheet opening gives encouragement as to the feasibility of designing smaller
and more effective peptide blockers of polymerization to prevent the
pathological polymerization of serpins. Specifically, the demonstration
that glycerol can readily act as a surrogate for the critical side
chain of the P8 threonine, opens the prospect of achieving an ultimate
aim in the field, the development of non-peptide blocking agents.
1-antitrypsin. Replacement of His-334 in
1-antitrypsin by a serine or alanine at pH 7.4 results
in the same polymerization and loop-peptide acceptance observed with
antithrombin at low pH. The critical role of His-334 and the
re-formation of its H-bond network by the conserved P8 threonine, on
the full insertion of strand 4, are relevant for the design of
therapeutic blocking agents. This is highlighted here by the
crystallographic demonstration that glycerol, which at high
concentrations blocks polymerization, can replace the P8 threonine and
re-form the disrupted H-bond network with His-334.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-sheet of the molecule to allow the insertion of the cleaved
reactive center loop as an additional strand in the center of the sheet
(4-6). As a consequence, the serpins are particularly vulnerable to
mutations affecting the critical region of the molecule underlying the
point of entry of the loop, between strands 3 and 5 of the sheet (7).
Mutations in this shutter region (Fig. 1a) allow the
aberrant opening of the A-sheet, with the risk of the insertion, into
its lower half, of the reactive loop of another molecule to give
intermolecular linkage and polymerization of the serpin. Even minor
changes in the shutter region of
1-antitrypsin (8) and
antichymotrypsin (9) result in their polymerization and intracellular
aggregation with consequent lung and liver disease and similarly with
C1-inhibitor (10) and antithrombin mutations (11) resulting in
angioedema and thrombosis. But the best example of the critical
function of this region comes from recent investigations of a novel
form of familial neurodegenerative disease due to the aggregation
within neurons of a brain-specific serpin, neuroserpin (12). The
polymerization and aggregation of neuroserpin results from mutations in
its shutter region. Two of these mutations affect residues previously
identified in other serpin diseases, Ser-53 and -56 (template numbering
(13) is used throughout this paper). The dysfunction accompanying
mutations of serines 53 and 56 is readily explicable by the alteration
in packing of residues underlying the A-sheet at the point where a
sliding movement opens the gap between strands 3 and 5A (2, 7). But a
third and novel mutation in neuroserpin (14, 15) has also focused
attention on histidine 334 at the point of bifurcation of strands 3 and
5 (Fig. 1, b and c). This conserved histidine is
centrally placed in the shutter region and is less directly involved in
the critical packing interactions of sheet opening than either serine
53 or 56, but surprisingly the mutation of His-334 results in a much
more severe neurodegenerative disease. An explanation for this is
likely to be the key H-bond network centered on His-334, which bridges
strands 3 and 5 of the A-sheet and notably links His-334 directly to
the base of Ser-53 and indirectly to Ser-56 (Fig. 1, b and
c).
1-antitrypsin, the opening of the A-sheet predominantly
results in the formation of loop-sheet polymers with the insertion of
the P7-3 portion of the loop of one molecule into the lower half of the
A-sheet of the next molecule (21). Here we show how studies of both
types of transition in antithrombin and
1-antitrypsin
confirm the critical function of His-334 as a barrier to premature
opening of the A-sheet.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside was from Melford
Laboratories Ltd. (Suffolk, England). Kanamycin sulfate was from Roche
Molecular Biochemicals. Heparin-Sepharose, HiTrap Q-Sepharose, and
HiTrap chelating columns were from Amersham Biosciences. Trypsin
and ampicillin were from Sigma. The substrate S-2222 was from
Chromogenix. Peptides encoding P14-3 (acetyl-SEAAASTAVVIA) and P14-9
(acetyl-SEAAAS) of antithrombin reactive loop were synthesized by the
Department of Biochemistry, University of Cambridge, Cambridge, UK, and
an analogue peptide (FLEAIG) of P7-2 of antitrypsin reactive loop,
where proline at P2 position was replaced by a glycine, was synthesized
by MWG-Biotech. The peptide formyl-MLF was purchased from Sigma. High
affinity heparin pentasaccharide (H5*), which has an extra sulfate
group and higher affinity for antithrombin (22), was a gift from Dr.
Maurice Petitou (Sanofi Research, Toulouse, France).
1-antitrypsin cDNA was amplified by polymerase chain
reaction and inserted into the expression vector pQE31 as previously described (27). Mutagenesis was carried out by a two-step polymerase chain reaction. The recombinant
1-antitrypsin was
expressed with an MRSHHHHHH tag at the N terminus and purified from the
soluble fraction of E. coli lysate. Briefly, expression
plasmids were transformed into SG13009 (pREP4) cells and grown in 2 liters of 2× tryptone-yeast extract medium at 37 °C until
A600nm = 0.8-1.0; then
isopropyl-
-D-thiogalactopyranoside was added to a
concentration of 1 mM, and the culture was transferred to
30 °C for a further 3 h. The cells were collected by
centrifugation, resuspended in buffer A (25 mM phosphate
buffer, pH 8.0, 0.5 M NaCl, and 1 mM
-mercaptoethanol), and disrupted by sonication. The supernatant of
the cell lysate was loaded onto a HiTrap nickel-chelating column (5 ml), and after washing to baseline with buffer A, the bound protein
eluted as a shouldered peak with an imidazole gradient (0-0.2
M). The fractions containing antitrypsin were collected, dialyzed against buffer B (10 mM Tris-Cl, pH 8.0, 1 mM EDTA, and 1 mM
-mercaptoethanol), and
loaded onto a HiTrap Q-Sepharose column (5 ml). The column was then
washed with a NaCl gradient (0-0.5 M) in buffer B.
1-Antitrypsin was eluted as the major peak (second peak)
around 0.2 M NaCl. The fractions were pooled, concentrated,
and snap-frozen in liquid nitrogen. The protein concentrations were
determined using an extinction coefficient of 5.3 (28). All
1-antitrypsin variants were confirmed to be in pure
monomeric form by SDS and native PAGE.
1-Antitrypsin was mixed with trypsin (1:2
molar ratio) at room temperature (22 ± 1 °C) for 15 min.
Samples were then mixed with reduced SDS-loading buffer and heated at
95 °C for 5 min in a PCR block. SDS-gel electrophoresis (29) was
performed in a 12% gel, and the protein was visualized with Coomassie Blue.
1-antitrypsin with a fixed concentration of trypsin
(1µ M). Reactions were incubated at room temperature (22 ± 1 °C) for 30 min. The residual amidolytic activity was
determined by the addition of 0.1 mM S-2222 substrate in
PBS with 0.1% PEG 8000. Linear regression analysis of the decrease in
protease activity with an increasing concentration of
1-antitrypsin yielded the estimates for the
stoichiometry of inhibition as the intercept on the abscissa. The rates
of inhibition of trypsin by recombinant
1-antitrypsin
variants were determined at room temperature by a discontinuous assay
procedure as previously described (27). Briefly, under pseudo
first-order conditions, 10 µl of 0.5 µM
1-antitrypsin variants were mixed with 10 µl of
20 nM trypsin in PBS with 0.1% PEG 8000. The residual
protease activity was determined at timed intervals by diluting the
reaction mixture into 1 ml of the assay buffer containing 0.1 mM S-2222 substrate. The observed rate constant,
kobs, for the reaction was obtained from the
slope of a semilog plot of the residual protease activity against time,
and the second-order rate constant, kapp, was
calculated by dividing kobs by the initial
1-antitrypsin concentration.
1-antitrypsin or antithrombin were prepared in 0.1 M sodium acetate for pH 4-6, 0.1 M sodium phosphate for pH 6-8, 0.1 M Tris for pH 8-9, and 0.1 M glycine for pH 9-10. All buffers were filtered, and
samples were centrifuged before the experiment. Thermal unfolding
experiments were performed by monitoring the CD signal at 222 nm
between 25 and 95 °C using a heating rate of 2 °C/min at a
concentration of 0.25 mg/ml for antitrypsin variants and 0.5 mg/ml for
antithrombin. Melting points (Tm) were
calculated using an expression for a two-state transition as described
previously (30). All the results are the average of three experiments.
The pH-dependent antithrombin thermal melting points were
treated as a single transition between pH 5 and 8, and the
pKa of this transition was fitted by GraFit (version 3.0, Erithacus Software Ltd.).
ex = 290 nm,
em = 350 nm) in the presence of guanidine chloride (GdmCl).1 Antithrombin was
incubated in a buffer (50 mM phosphate or sodium acetate,
50 mM NaCl, and 1 mM EDTA) containing various
concentrations of GdmCl at 25 °C for about 16 h before spectral
measurements. The final concentration of protein was 20 µg/ml.
Equilibrium unfolding was fitted to a two-state model, and the
midpoints [GdmCl]1/2 of unfolding curves at different pH
were plotted against pH.
1-antitrypsin variants at 0.68 mg/ml were incubated at
37 °C in the presence of a 100-fold molar excess of the
P14-P3 12-mer peptide (acetyl-SEAAASTAVVIA) at different pH
values or the 6-mer peptide (FLEAIG) for different time intervals. The
results were analyzed by loading the samples onto an 8% (w/v) native
gel with 7 M urea.
X-ray data collection and refinement statistics
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Fig. 1.
Histidine 334 in the shutter region of
antithrombin. a, structure of native antithrombin (from
PDB 1E04) with reactive center loop in red, A- -sheet in
green, and strand 6 of B-
-sheet and helix B in
yellow. A red arrow indicates entry of the
reactive loop as strand 4. b, shows the hydrogen bonds
(dashed line) between main chains of the A-sheet. Histidine
334 forms the first hydrogen bonds between strands 3 and 5 of the
A-sheet. c, detailed interactions in the shutter region
(circled in a) showing oxygen atoms as red
balls, nitrogen atoms in blue, and the position of C
atoms in black. Hydrogen bonds are shown as cyan
dashed lines. The imidazole ring of His-334 forms side chain to
main chain interactions with Ser-53 and Asn-186 and with the side chain
of Asn-186. The hydroxyl of Ser-53 forms a hydrogen bond with the main
chain amine of Ser-56, while the hydroxyl of Ser-56 interacts with the
side chain of Asn-186. d, close-up of the shutter region of
antithrombin ternary complex (antithrombin with P14-9 and a
tripeptide). The glycerol in green occupies the P8 position
and hydrogen bonds to the imidazole of His-334 and the carbonyl group
of Phe-333 and carboxyl group of P9 residue of the P14-9 peptide.
e, stereo picture as in d showing
A
weighted 2Fo
Fc
electron density of the inserted peptides: P14-9 above and formyl-P6-4
below. The glycerol is separately buried between them in the position
usually occupied by the side chain of the P8 threonine. The key
H-bonds, to the imidazole and its bond to the amine of Ser-53, are
shown as dotted lines.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1-antitrypsin at temperatures
above 50 °C results not only in their polymerization (Fig.
2a) but also in a transition
of a proportion of molecules to the latent conformation. This
transition to the latent form is minor and barely detectable with
1-antitrypsin. However, latent antithrombin is readily
formed, though it may not be apparent due to the immediate formation of
a heterodimer (20) with reversible sheet C linkage of the latent
antithrombin to a molecule of active native antithrombin (Fig.
2b). With antithrombin, the addition of glycerol to the
incubation buffer suppresses polymer formation but allows the latent
transition (20), such that incubation at 50 °C of antithrombin in
40% glycerol results in the quantitative preparation of latent
antithrombin (25), free of polymers (Fig. 2b).
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Fig. 2.
pH-dependent conformational
changes of antithrombin. a, antithrombin and
antitrypsin were heated at 55 °C for 30 min. b,
antithrombin was incubated with 40% glycerol, 50 mM
phosphate buffer, pH 6.4, at 50 °C for up to 48 h.
L, latent antithrombin controls. c, native
antithrombin was incubated at 50 °C for 16 h at different pH
values. d, antithrombin was incubated at 37 °C with
100-fold molar excess of a P14-3 peptide (acetyl-SEAAASTAVVIA) at
different pH values. The samples were analyzed on an 8% 7 M urea gel to give clear separation of the
peptide-antithrombin binary complex (BC).
1-antitrypsin in that its Tm
undergoes an atypical change with pH (Fig.
3a) with a transition at
apparent pKa of 6.0 ± 0.1 (Fig. 3b). A transition at pH 6 was similarly observed with
guanidine chloride-induced antithrombin unfolding (data not shown). The inflection point at pH 6 suggested that the thermal stability of
antithrombin was dependent on the protonation of an individual histidine residue, with the likely candidate being His-334. This histidine is uniquely exposed in antithrombin, which has a partially opened A-sheet, with His-334 forming the first interlinking H-bonds between s3A and s5A (Fig. 1, b and c). By
comparison, in
1-antitrypsin, which is more thermally
stable with Tm 63.4, His-334 is protected by the
full closure of strands 3 and 5 in the A-sheet. To confirm that this
difference in pH dependence is related to the exposure of His-334, the
change in Tm was determined with a closed
A-sheet conformation of antithrombin. Closure of the A-sheet of
antithrombin was induced by addition of the core heparin
pentasaccharide, which has been shown to result in full closure of the
A-sheet with burial of His-334 to give a conformation of antithrombin
superimposable with that of
1-antitrypsin (31). As shown
in Fig. 3c, addition of the heparin pentasaccharide to
antithrombin results in an increased thermal stability with conversion
to a pH dependence curve similar to that of
1-antitrypsin and without the inflection at pH 6.
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Fig. 3.
pH-dependent thermal stability of
antithrombin and antitrypsin. A, the CD thermal melting
curves of antithrombin at different pH values: pH 7.6, black
solid line, pH 5.5, gray solid line, and pH 6, open circles. B and C, the variation
with the pH of thermal melting points of antithrombin alone
(closed circles), antithrombin with heparin high affinity
pentasaccharide (open circles), or antitrypsin (gray
closed triangles). Each point is an average of three measurements
with the standard error less than 0.2 °C. At low pH values,
antithrombin and antitrypsin become unstable at room temperature, so
the melting points were not calculated. The pKa of
the single transition of native antithrombin between pH 5 and 8 was
fitted with GraFit. The transitions between pH 4 and 5.5 of antitrypsin
and antithrombin-heparin pentasaccharide complexes were also fitted, but these
transitions do not represent the protonation of His-334 as a similar
transition was observed with the H334A mutant. D, diagram
showing how protonation disrupts the hydrogen bonding of His-334. The
nitrogen of His-334 imidazole ring forms hydrogen bonds with
carbonyl groups of Asn-186 and, prior to protonation, the
nitrogen
interacts with the main chain amine of Ser-53, with an optimal N-N
hydrogen bonding distance of 3.1 Å.
1-antitrypsin, variants
were expressed recombinantly with substitutions of His-334 by serine
and alanine and with the variants named here as Wt for the wild type
and H334S and H334A, respectively, for the variants. Both
1-antitrypsin variants retain their inhibitory activity
and form stable complexes with trypsin identical to those of Wt (Fig.
4a), and rates and
stoichiometries of trypsin inhibition were unchanged (data not shown).
Incubation of H334A for 1 h at a range of temperatures showed
substantial polymerization at 45 °C, some 5-10 °C prior to
equivalent polymerization of the recombinant Wt
1-antitrypsin (Fig. 4b). The H334S variant
had a smaller increase in polymerization, intermediate between the Wt
and H334A forms. The more ready opening of the A-sheet of H334A
versus Wt
1-antitrypsin is shown by the
increased rate of peptide (P7-2) insertion with the H334A variant (Fig.
4c). The inherent change in stability of the three forms is
reflected in their thermal stability, with a decrease from a
Tm of 63.4 °C in Wt
1-antitrypsin to 59.6 °C in H334S and to 58.2 °C
in H334A. The H334A mutant has a similar pH-dependent
Tm curve to those of antitrypsin and
antithrombin-heparin pentasaccharide complexes (data not shown).
This indicates that the transitions between pH 4 and 5.5 (Fig. 3,
b and c) do not represent the protonation of
His-334, which is consistent with the burial of His-334 both in
antitrypsin and heparin-pentasaccharide-complexed antithrombin.
View larger version (77K):
[in a new window]
Fig. 4.
PAGE of antitrypsin variants.
a, complex formations between antitrypsin variants (WT,
H334S, and H334A) and trypsin. Antitrypsin was mixed with excess
trypsin (1:2 molar ratio) at room temperature for 15 min and then
heated with reduced SDS loading buffer and analyzed by SDS-PAGE. Due to
excess of trypsin and antitrypsin-trypsin complexes are susceptible to
cleavage, the bands marked with arrows are cleaved AT-T
complex (cpx*). b, polymerization of antitrypsin
variants. Antitrypsin variants were incubated between 37 and 65 °C
for 1 h and analyzed on a native PAGE. c,
antitrypsin-peptide binary complex (BC) formation. Wild type
antitrypsin and H334A were incubated with 100-fold molar excess of
peptide (FLEAIG) for up to 48 h and analyzed by a 7 M
urea PAGE.
nitrogen of the His-334 imidazole with the bond length of 2.9 Å being identical to that formed by the hydroxyl of the P8 threonine.
Moreover in each case the His-334 imidazole is oriented such that its
nitrogen is maintained at an optimal hydrogen bond distance of 3.1 Å from the main chain amine of Ser-53. But as well as this specific
linkage to His-334, a series of hydrogen bonds are also formed with
surrounding structures including the main chain oxygen of Phe-333 and
the carboxyl group of the P9 residue. Subsequently, we have also shown
crystallographically that glycerol insertion, as in Fig. 1,
d and e, takes place even upon rapid exposure of
formed crystals to glycerol just prior to diffraction.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-sheet (Fig.
1, b and c). The significance of these
interactions is highlighted by the identification of mutations in the
shutter region causing familial dementias: at His-334, Ser-53, and also at Ser-56, which bonds to Asn-186 (14, 15). The mutations predictably
result in a laxity in sheet opening at 37 °C similar to that induced
by incubation of the normal protein at 50 °C. The consequence of
such incubation (Fig. 2, a-c) is the formation of either
intermolecular loop-sheet linkages to give polymers or the monomeric
transition to the latent conformation with a full insertion of the
reactive loop into the A-sheet. The demonstration here that both
polymerization and peptide annealing of antithrombin occurred at pH
below 6 (Fig. 2, c and d) together with the
structurally known exposure of His-334 in antithrombin, indicates that
sheet opening is facilitated by the protonation of His-334. This
protonation of the
nitrogen of the imidazole will break the
hydrogen bond that anchors His-334 to Ser-53 in the underlying B-sheet
(Fig. 3d). The accompanying acquisition of a positive charge
will disfavor the burying of the imidazole with the combined effects
giving disruption of the hydrogen bonds that link strands 3 and 5 of the A-sheet.
1-antitrypsin in which the
histidine is buried in a tightly closed A-sheet. Moreover, when
antithrombin is converted to a form with a similarly closed A-sheet, by
complexing with heparin, its Tm curve loses the
inflection at pH 6 and reverts to the typical serpin response as seen
with
1-antitrypsin (Fig. 3c). Although there
are four histidines in antithrombin, only His-334 undergoes a radical
change in environment, due to burying, on heparin activation. Evidence
that His-334 has the same protective function against aberrant sheet
opening in
1-antitrypsin is provided by recombinant
substitutions at 334 with alanine (H334A) and serine (H334S). These
substituted variants of
1-antitrypsin have properties similar to that of antithrombin when its pH value is decreased to below
6. The H334A and H334S variants of
1-antitrypsin, as compared with the wild type recombinant, have a large decrease in
Tm and more readily form polymers and accept
synthetic loop-peptides (Fig. 4). The shutter control of sheet opening
is a delicately tuned mechanism, and even slight perturbations may have
disastrous functional consequences. For example, two relevant natural
antithrombin variants with minor shutter mutations and a decrease in
Tm of just over 1 °C result in severe
episodic thrombosis (24, 42).
1-antitrypsin
where there is a methionine (this explains why the P14-3 homologue
peptide of antithrombin more readily inserts into
1-antitrypsin than does its own P14-3 peptide (43)). The
clear preference for a threonine at P8 is also shown in another
particularly relevant structure, that of a shutter mutant (L55P) of
1-antichymotrypsin (44). This pathological mutant is
seen in a frozen transitional form, with the reactive loop inserted to
P12 but with further entry blocked by the insertion into the P8
position of a threonine from the adjacent F helix. Intriguingly this
threonine at position 165 at end of the F helix is invariantly
conserved in the inhibitory serpins.
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FOOTNOTES |
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* 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.
The atomic coordinates and the structure factors (code 1LK6) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed. Tel.: 44-1223-336831;
Fax: 44-1223-336827; E-mail: awz20@cus.cam.ac.uk.
Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M211663200
2 A. Zhou, P. E. Stein, J. A. Huntington, and R. W. Carrell, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: GdmCl, guanidine chloride; Wt, wild type.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Gettins, P. G. W., Patston, P. A., and Olson, S. T. (1996) Serpins: Structure, Function, and Biology , R. G. Landes, Austin, TX |
2. | Stein, P. E., and Carrell, R. W. (1995) Nat. Struct. Biol. 2, 96-113[Medline] [Order article via Infotrieve] |
3. |
Carrell, R. W.,
and Lomas, D. A.
(2002)
N. Engl. J. Med.
346,
45-53 |
4. | Loebermann, H., Tokuoka, R., Deisenhofer, J., and Huber, R. (1984) J. Mol. Biol. 177, 531-556[Medline] [Order article via Infotrieve] |
5. |
Stratikos, E.,
and Gettins, P. G.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
4808-4813 |
6. | Huntington, J. A., Read, R. J., and Carrell, R. W. (2000) Nature 407, 923-926[CrossRef][Medline] [Order article via Infotrieve] |
7. | Whisstock, J. C., Skinner, R., Carrell, R. W., and Lesk, A. M. (2000) J. Mol. Biol. 296, 685-699[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Seyama, K.,
Nukiwa, T.,
Takabe, K.,
Takahashi, H.,
Miyake, K.,
and Kira, S.
(1991)
J. Biol. Chem.
266,
12627-12632 |
9. | Poller, W., Faber, J.-P., Weidinger, S., Tief, K., Scholz, S., Fischer, M., Olek, K., Kirchgesser, M., and Heidtmann, H.-H. (1993) Genomics 17, 740-743[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Eldering, E.,
Verpy, E.,
Roem, D.,
Meo, T.,
and Tosi, M.
(1995)
J. Biol. Chem.
270,
2579-2587 |
11. | Lane, D. A., Bayston, T., Olds, R. J., Fitches, A. C., Cooper, D. N., Millar, D. S., Jochmans, K., Perry, D. J., Okajima, K., Thein, S. L., and Emmerich, J. (1997) Thromb. Haemost. 77, 197-211[Medline] [Order article via Infotrieve] |
12. | Davis, R. L., Shrimpton, A. E., Holohan, P. D., Bradshaw, C., Feiglin, D., Collins, G. H., Sonderegger, P., Kinter, J., Becker, L. M., Lacbawan, F., Krasnewich, D., Muenke, M., Lawrence, D. A., Yerby, M. S., Shaw, C. M., Gooptu, B., Elliott, P. R., Finch, J. T., Carrell, R. W., and Lomas, D. A. (1999) Nature 401, 376-379[CrossRef][Medline] [Order article via Infotrieve] |
13. | Huber, R., and Carrell, R. W. (1989) Biochemistry 28, 8951-8966[Medline] [Order article via Infotrieve] |
14. | Davis, R. L., Shrimpton, A. E., Carrell, R. W., Lomas, D. A., Gerhard, L., Baumann, B., Lawrence, D. A., Yepes, M., Kim, T. S., Ghetti, B., Piccardo, P., Takao, M., Lacbawan, F., Muenke, M., Sifers, R. N., Bradshaw, C. B., Kent, P. F., Collins, G. H., Larocca, D., and Holohan, P. D. (2002) Lancet 359, 2242-2247[CrossRef][Medline] [Order article via Infotrieve] |
15. | Lomas, D. A., and Carrell, R. W. (2002) Nat. Rev. Genet. 3, 759-768[CrossRef][Medline] [Order article via Infotrieve] |
16. | Schreuder, H. A., de Boer, B., Dijkema, R., Mulders, J., Theunissen, H. J., Grootenhuis, P. D., and Hol, W. G. (1994) Nat. Struct. Biol. 1, 48-54[Medline] [Order article via Infotrieve] |
17. |
Baglin, T. P.,
Carrell, R. W.,
Church, F. C.,
Esmon, C. T.,
and Huntington, J. A.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
11079-11084 |
18. | Mottonen, J., Strand, A., Symersky, J., Sweet, R. M., Danley, D. E., Geoghegan, K. F., Gerard, R. D., and Goldsmith, E. J. (1992) Nature 355, 270-273[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Irving, J. A.,
Pike, R. N.,
Lesk, A. M.,
and Whisstock, J. C.
(2000)
Genome Res.
10,
1845-1864 |
20. |
Zhou, A.,
Huntington, J. A.,
and Carrell, R. W.
(1999)
Blood
94,
3388-3396 |
21. |
Sivasothy, P.,
Dafforn, T. R.,
Gettins, P. G.,
and Lomas, D. A.
(2000)
J. Biol. Chem.
275,
33663-33668 |
22. | Petitou, M., and van Boeckel, C. A. (1992) Fortschr. Chem. Org. Naturst. 60, 143-210[Medline] [Order article via Infotrieve] |
23. | McKay, E. J. (1981) Thromb. Res. 21, 375-382[Medline] [Order article via Infotrieve] |
24. | Bruce, D., Perry, D. J., Borg, J.-Y., Carrell, R. W., and Wardell, M. R. (1994) J. Clin. Invest. 94, 2265-2274[Medline] [Order article via Infotrieve] |
25. | Carrell, R. W., Huntington, J. A., Mushunje, A., and Zhou, A. (2001) Thromb. Haemost. 86, 14-22[Medline] [Order article via Infotrieve] |
26. | Nordenman, B., Nystrom, C., and Bjork, I. (1977) Eur. J. Biochem. 78, 195-203[Abstract] |
27. |
Zhou, A.,
Carrell, R. W.,
and Huntington, J. A.
(2001)
J. Biol. Chem.
276,
27541-27547 |
28. | Pannell, R., Johnson, D., and Travis, J. (1974) Biochemistry 13, 5439-5445[Medline] [Order article via Infotrieve] |
29. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
30. |
Dafforn, T. R.,
Mahadeva, R.,
Elliott, P. R.,
Sivasothy, P.,
and Lomas, D. A.
(1999)
J. Biol. Chem.
274,
9548-9555 |
31. |
Jin, L.,
Abrahams, J. P.,
Skinner, R.,
Petitou, M.,
Pike, R. N.,
and Carrell, R. W.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
14683-14688 |
32. | Leslie, A. W. G. (1992) Joint CCP4 and ESF-EACMB Newsletter on Protein Crystallography , Vol. 26 , Daresbury Laboratory, Warrington, UK |
33. | Evans, P. R. (1993) in Proceedings of the CCP4 Study Weekend: Data Collection and Processing (Sawyer, L., Isaacs, N., and Bailey, S., ed) Vol. 114-122, Daresbury Laboratory, Warrington, UK |
34. | Skinner, R., Chang, W. S., Jin, L., Pei, X., Huntington, J. A., Abrahams, J. P., Carrell, R. W., and Lomas, D. A. (1998) J. Mol. Biol. 283, 9-14[CrossRef][Medline] [Order article via Infotrieve] |
35. | Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve] |
36. | Pannu, N. S., and Read, R. J. (1996) Acta Crystallogr. Sect. A 52, 659-668[CrossRef] |
37. | Jones, T. A., Zou, J.-Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve] |
38. | Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef] |
39. | Esnouf, R. M. (1999) Acta Crystallogr. Sect. D Biol. 55, 938-940[CrossRef] |
40. | Merritt, E. A., and Bacon, D. J. (1997) Methods Enzymol. 277, 505-524 |
41. | McRee, D. E. (1999) J. Struct. Biol. 125, 156-165[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Beauchamp, N. J.,
Pike, R. N.,
Daly, M.,
Butler, L.,
Makris, M.,
Dafforn, T. R.,
Zhou, A.,
Fitton, H. L.,
Preston, F. E.,
Peake, I. R.,
and Carrell, R. W.
(1998)
Blood
92,
2696-2706 |
43. | Chang, W.-S. W., Wardell, M. R., Lomas, D. A., and Carrell, R. W. (1996) Biochem. J. 314, 647-653[Medline] [Order article via Infotrieve] |
44. |
Gooptu, B.,
Hazes, B.,
Chang, W. S.,
Dafforn, T. R.,
Carrell, R. W.,
Read, R. J.,
and Lomas, D. A.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
67-72 |
45. |
Im, H.,
Woo, M. S.,
Hwang, K. Y.,
and Yu, M. H.
(2002)
J. Biol. Chem.
277,
46347-46354 |
46. |
Hansen, M.,
Busse, M. N.,
and Andreasen, P. A.
(2001)
Eur. J. Biochem.
268,
6274-6283 |
47. | Lomas, D. A., Evans, D. L., Finch, J. T., and Carrell, R. W. (1992) Nature 357, 605-607[CrossRef][Medline] [Order article via Infotrieve] |
48. | Schulze, A. J., Baumann, U., Knof, S., Jaeger, E., Huber, R., and Laurell, C. B. (1990) Eur. J. Biochem. 194, 51-56[Abstract] |
49. |
Mahadeva, R.,
Dafforn, T. R.,
Carrell, R. W.,
and Lomas, D. A.
(2002)
J. Biol. Chem.
277,
6771-6774 |
50. | Xue, Y., Bjorquist, P., Inghardt, T., Linschoten, M., Musil, D., Sjolin, L., and Deinum, J. (1998) Structure 6, 627-636[CrossRef][Medline] [Order article via Infotrieve] |