From the Departament de Biologia Molecular i
Cel·lular, Centre d'Investigació i Desenvolupament-Consell
Superior d'Investigacions Científiques, Jordi Girona 18-26, 08034 Barcelona, Spain and the § Department of
Biochemistry, Trinity College, Dublin 2, Ireland.
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ABSTRACT |
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The three-dimensional structure of mouse liver
glutathione S-transferase P1-1 carboxymethylated at Cys-47
and its complex with
S-(p-nitrobenzyl)glutathione have been
determined by x-ray diffraction analysis. The structure of the modified
enzyme described here is the first structural report for a
Pi class glutathione S-transferase with no
glutathione, glutathione S-conjugate, or inhibitor bound.
It shows that part of the active site area, which includes helix B
and helix 310B, is disordered. However, the environment of
Tyr-7, an essential residue for the catalytic reaction, remains
unchanged. The position of the sulfur atom of glutathione is occupied
in the ligand-free enzyme by a water molecule that is at H-bond
distance from Tyr-7. We do not find any structural evidence for a
tyrosinate form, and therefore our results suggest that Tyr-7 is not
acting as a general base abstracting the proton from the thiol group of
glutathione. The binding of the inhibitor S-(p-nitrobenzyl)-glutathione to the
carboxymethylated enzyme results in a partial restructuring of the
disordered area. The modification of Cys-47 sterically hinders
structural organization of this region, and although it does not
prevent glutathione binding, it significantly reduces the affinity. A
detailed kinetic study of the modified enzyme indicates that the
carboxymethylation increases the Km for glutathione
by 3 orders of magnitude, although the enzyme can function efficiently
under saturating conditions.
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INTRODUCTION |
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Glutathione S-transferases
(GSTs)1 are a widely
distributed family of detoxifying enzymes (1). They catalyze the
nucleophilic attack of the sulfur atom of the tripeptide glutathione
(GSH) on electrophilic groups of a variety of hydrophobic compounds. Six different classes of mammalian soluble GSTs (Alpha, Mu, Pi, Theta,
Sigma, and Kappa) have been described, each one with different but
overlapping hydrophobic substrate specificity (2-7). The three-dimensional structure of representative enzymes of each class are
known (8-17). The different classes show different active site
structures, in particular at the hydrophobic subsite (for reviews see
Refs. 18 and 19). Despite these differences it is believed that they
all share a similar catalytic mechanism where the thiol group of
glutathione is activated to thiolate. The reactive ionized
S group attacks the electrophilic group of the
hydrophobic substrate leading to a glutathione S-conjugate
with increased aqueous solubility (20). How the activation of
glutathione is achieved by the enzyme is unclear. The three-dimensional
structures of Pi class GSTs complexed with different inhibitors have
shown that the only residue of the enzyme in close proximity to the
sulfur atom of glutathione is Tyr-7. Until the first GST structure was
solved (8), Cys-47 was considered a catalytic residue because its
modification inactivated the enzyme (21, 22). The three-dimensional
structure, however, revealed that Cys-47 is over 12 Å away from the
active pocket. This residue is located in helix 310B at the
C terminus of helix
B. This helical structure was found to have high
temperature factors and exposed hydrophobic residues (12), and the
hypothesis was put forward that it could be disordered in the
uncomplexed enzyme.
The hypothesis that the "free" enzyme (i.e. with no GSH
or GS-conjugates bound) has a structure different from that
seen when GSH or GS conjugates are bound has been implicated in a
number of studies. We currently have structures for mGST P1-1 with
S-hexylglutathione, S-(p-nitrobenzyl)glutathione (12), and
GSH2 bound, and in all of
these the thiol of Cys-47 is not accessible to solvent. Indeed this
appears to be a feature of all class Pi structures solved to date (8,
9, 13). This is important because some authors (23-25) have presented
a considerable body of evidence that implicates the availability of
cysteine 47 for mixed disulfide formation as a redox sensor. Cystine
forms a mixed disulfide with GST P1-1 in agreement with this hypothesis
(24). This is attractive as GST P1-1 is almost unique in having a thiol (Cys-47) that appears to be unreactive when GSH is bound to the enzyme
but that may be alkylated or oxidized in the absence of GSH (or
GS-conjugates). Indeed we have shown that the kinetics of protection
against alkylation by GS-conjugates are entirely consistent with such a
hypothesis (26). The simplest model to explain this behavior involves
at least two conformations for GST P1-1. For the "free" enzyme the
thiol of Cys-47 is reactive and exposed to solvent, whereas the binding
of GSH or GS-conjugates stabilizes a distinct conformation (seen in the
numerous crystal structures solved to date) where the thiol of Cys-47
becomes inaccessible to solvent and thiol reagents. It should be made
clear that binding of GS- conjugates does not physically occlude thiol
reagents from access to the thiol of Cys-47 because the side chain is
in a small hydrophobic pocket on the outer face of the
B-310B helices that form the GSH binding site. It has
not been possible to crystallize the "free" enzyme for GSTP1-1 to
date. We argued that alkylation of cysteine 47 may trap the "free"
enzyme in a distinct conformation that permits further analysis and
this has proved correct.
Also it has been proposed that Tyr-7 might be in the tyrosinate form in the free enzyme acting as a general base and activating the thiol group of glutathione by proton abstraction (8, 27, 28) when the substrate binds to the active site. However, to date no structure of an unliganded Pi class GST has been reported, and Dirr et al. (18) have reviewed a number of observations inconsistent with the tyrosinate hypothesis.
We have solved the three-dimensional structure of the mouse liver GST
P1-1 enzyme modified by iodoacetic acid at Cys-47 to form the
carboxymethylated enzyme (referred to as CM-mGST P1-1 from now on) in
an unliganded form and with
S-(p-nitrobenzyl)glutathione bound at the active
site. The carboxymethylated enzyme shows a complete disorder in
the B-310B region with a partial destruction of the
active site architecture. However, the environment of Tyr-7 remains
unchanged in the free enzyme with no indication of an ionized state for
this residue. On the other hand the binding of the inhibitor,
S-(p-nitrobenzyl)glutathione, results in a
limited restructuring of the disordered area. The results presented
here provide a structural explanation for the effect of the chemical modification of Cys-47 and suggest that Tyr-7 is not acting as a
general base. It is presently unclear whether carboxymethylation is
locking the enzyme in a conformer of the native enzyme, and further
work is required to investigate this.
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EXPERIMENTAL PROCEDURES |
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Modified mouse liver GST P1-1 was prepared by incubating 18 mg
of the native enzyme (26) with 200 mM iodoacetic acid in 100 mM sodium phosphate, pH 8, in a final volume of 10 ml
for 40 min and then removing excess iodoacetic acid by gel filtering on
Sephadex G-25 equilibrated in 10 mM sodium phosphate, pH 7. The modified enzyme does not bind to glutathione-Sepharose, so to
ensure that any residual native enzyme was removed, the modified preparation was passed through this affinity resin, and the protein that eluted in the wash through was pooled, concentrated using an
Amicon filter, and stored at 20 °C. This material, homogenous by
the criteria of SDS/polyacrylamide gel electrophoresis and exhibiting a
pI of 8.2 (29) exhibits a marked increase in the KmGSH (see
below).
Suitable crystals of CM-mGST P1-1 were grown by vapor diffusion at room
temperature from hanging drops prepared by mixing 3 µl of protein
solution (7 mg/ml) and 3 µl of precipitant solution containing 33 mM Hepes buffer, pH 7, 64 mM CaCl2,
and 8.3% PEG 4000. The drops were equilibrated against a reservoir of
19% PEG 4000, 0.15 M CaCl2, 75 mM
Hepes buffer. Long prismatic crystals appeared after 1 week.
Three-dimensional diffraction data were collected up to 1.9 Å resolution, at 4 °C, using the X11 station, the European Molecular
Biology Laboratory at the Deutsches Elektronensynchrotron ( = 0.937 Å). Because of the intensity decay three crystals of approximate size
0.7 × 0.2 × 0.2 mm were necessary to collect a full data
set. Images were taken in one-degree rotation steps with the detector
set at 165-mm distance. The crystals are tetragonal space group
I4, with unit cell dimensions a = b = 132.5 Å and c = 63.2 Å, and have
one dimer/asymmetric unit. The data were processed with the XDS package
(30) and merged together with the program PROTEIN (31). Intensity data
statistics are given in Table I.
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Crystals of CM-mGST P1-1 complexed with
S-(p-nitrobenzyl)glutathione were grown at room
temperature from hanging drops. The best crystals appeared by mixing 3 µl of protein solution (7 mg/ml), 1 µl of 20% PEG 8000 in 0.1 M MOPS buffer, pH 6.4, and 0.2 M of ammonium
sulfate and 2 µl of inhibitor saturated solution in 0.1 M
MES buffer at pH 7.5. The drops were equilibrated against 10% PEG
8000, 50 mM MOPS buffer. After 10 days needle-like crystals grew to a size of 0.6 × 0.2 × 0.1 mm. The crystals
diffracted weakly on a rotating anode. However, a 3 Å data set could
be collected at the BW7B station, the European Molecular Biology
Laboratory at the Deutsches Elektronensynchrotron ( = 0.869 Å), at
4 °C. The crystals are orthorhombic space group
P212121 and with cell dimensions
a = 62.45, b = 132.69, and
c = 214.47 Å and have three protein dimers in the
asymmetric unit. The data were processed with DENZO (32). Intensity
data statistics are given in Table I.
The coordinates from the mGST P1-1 enzyme as in the
S-(p-nitrobenzyl)glutathione complex structure
(Protein Data Bank code 1glq) (12) were used as a starting model for
the determination of both structures by molecular replacement with
AMoRe (33). The solution for the unliganded form was found using two
independent monomers as initial model and a resolution range of 12-4
Å. After the rigid body refinement routine of AMoRe, the correlation
coefficient (for Fobs) was 0.75, and the
R-factor was 0.303. A combined x-ray and energy minimization refinement
followed with XPLOR (34), including cycles of simulated annealing. In
the refinement process 10% of the reflections were set aside for the
R-free calculation (35). Fourier electron density maps were calculated
at 3 Å resolution with coefficients 2Fo Fc and Fo
Fc and displayed using TURBO (36). Most of the
molecule fitted correctly in the electron density except for residues
35-51, where the electron density was absent in the maps contoured at 1 and 2
, respectively. No protein atoms could be located in this
area. Further refinement cycles followed adding solvent molecules and
slowly increasing the resolution up to 2.0 Å. Atomic isotropic B
factors were refined with restraints imposed on atoms directly bonded.
At this stage the R-cryst was 0.197, and the R-free was 0.236. A final
run of positional and B-factor refinement was carried out including all
data consisting in 26183 reflections (Fobs > 2
Fobs) in the resolution range 8.0-2.0 Å.
This resulted in a final R-factor of 0.195 (Table
II). The final structure consists of 385 protein residues of 418 amino acids of the chemical sequence and 150 solvent molecules.
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To solve the structure of CM-mGST P1-1 complexed with
S-(p-nitrobenzyl)glutathione, three dimers of
mGST P1-1 were used as initial model. After the rigid body refinement
routine of AMoRe, a correlation coefficient (for
Fobs) of 0.53 and an R-factor of 43.7% were
obtained for the correct solution. Refinement of the structure followed
with MAIN (37), omitting the area corresponding to the -helix B that
was disordered in the unliganded structure. Initially, the
noncrystallographic symmetry (NCS) operators relating the two monomers
in each dimer and the three dimers of the asymmetric unit were
determined from the molecular model. With these symmetry operators and
masks build around the molecules, the map was subject to 6-fold density
averaging and solvent flattening. Iterative cycles of density averaging
and refinement of the NCS operators followed, maximizing the
correlation among the six monomers. After this procedure, inspection of
the active sites in 3 Å resolution Fourier maps showed clear electron
density corresponding to the inhibitor molecules. The inhibitor
S-(p-nitrobenzyl)glutathione was fitted in the
electronic density (Fig. 1). Also part of
the disordered area in the unliganded enzyme now showed clear density, and the protein model was build accordingly. Further refinement of the
model using NCS restraints was performed with MAIN giving an R-factor
of 0.27. Simulated annealing and positional refinement with XPLOR
followed, setting aside 5% of the reflections for R-free calculation.
After NCS-restrained B-factor refinement, the R-cryst was 0.246, and
the R-free 0.277 (Table II). Because of the limited resolution of this
data set, we decided to conclude the refinement process at this point,
not including solvent molecules and not breaking the NCS restraints to
maintain a reasonable parameter/data ratio. The final structure
consists of three protein dimers of 398 residues each of 418 amino
acids of the chemical sequence and 6 inhibitor molecules. Atomic
coordinates and structure factors for both the unliganded and complexed
CM-mGST-
structures have been deposited with the Brookhaven Protein
Data Bank.
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RESULTS |
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Structure of the Cys-47-carboxymethylated Enzyme--
The global
fold of the unliganded CM-mGST P1-1, excluding residues 34-51, is
similar to that of the native enzyme when complexed with glutathione
adducts. The rms deviation in the C positions
is 0.33 Å between molecule A of the present structure and molecule A
in the S-(p-nitrobenzyl)glutathione complex
structure. Other comparisons show similar values. The rms
difference between monomers in the present structure is 0.107 Å.
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Structure of the Cys-47-carboxymethylated Enzyme Complexed with
S-(p-Nitrobenzyl)glutathione--
The global fold of the CM-mGST P1-1
in the complexed enzyme is again similar to the native complexed
enzyme. For example, the rms deviation in the
C positions, excluding residues 38-48, is 0.37 Å between molecule A of the present structure and molecule A in the
S-(p-nitrobenzyl)glutathione complex structure
(12). The S-(p-nitrobenzyl)glutathione is present
at the active site (Fig. 1). When the structure of CM-mGST P1-1 (Fig.
3B) is compared with the structure obtained in the presence
of S-(p-nitrobenzyl)glutathione, it is obvious
that the disordered helix B region is partially restructured on binding ligand. For example, residues Ile-35, Asp-36, Thr-37, and Trp-38 are
now in well defined electron density. Cys-47, Leu-48, Tyr-49, Gly-50,
and Gln-51 are also in density.
Kinetic Analysis of the Cys-47-carboxymethylated
Enzyme--
During our initial purification of the carboxymethylated
protein we confirmed the report of Tamai et al. (21) that
the thiol-modified enzyme (CM-mGST P1-1) does not bind to
S-hexylglutathione-Sepharose or to glutathione-Sepharose.
However, we observed very low levels of
1-chloro-2,4-dinitrobenzene-conjugating activity in the wash-through fraction and, in preliminary work, could find no evidence for contamination with unmodified protein (29) consistent with the hypothesis that the modified enzyme retained some residual activity. By
monitoring the wash through material from the affinity column at far
higher concentrations of glutathione (20-100 mM) than we would normally use (1 mM), it is possible to detect
significant 1-chloro-2,4-dinitrobenzene-conjugating activity in the
nonbound fraction. Detailed kinetic studies indicate that the CM
reaction increases the Km for glutathione from 150 µM to approximately 100 mM (Fig.
6). We have confirmed this apparent
decrease in affinity for GSH by monitoring the
GSH-dependent quenching of protein fluorescence when values
for KDGSH for the native and
CM-modified protein are 150 µM and 80 mM, respectively. The Km for the second substrate
1-chloro-2,4-dinitrobenzene is hardly affected (1 mM native
enzyme and 2 mM for the CM-modified enzyme). The turnover
number for the CM-modified enzyme is 30 s1, which is
close to the value reported by Phillips (38) for the native enzyme of
51 s
1. It thus appears that the CM modification simply
increases the Km for glutathione by almost 3 orders
of magnitude but that under saturating conditions the enzyme turns over
almost as efficiently as the native enzyme. This kinetic data is
entirely consistent with the partial destructuring of the glutathione
binding site but with no rearrangement around the catalytic tyrosine. Interestingly the addition of a hydrophobic moiety
(p-nitrobenzyl) to the glutathione sulfur atom results in
tight binding to CM-mGST-
that is similar (38 µM) to
the value obtained for the native enzyme (12 µM). Again
this is in accord with the structural data where the hydrophobic moiety
interacts with the H-site, and for the CM-mGST-
enzyme the
disordered helix B region is partially restructured around the
glutathione element.
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DISCUSSION |
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Cys-47 was believed to be an essential catalytic residue because
of its high reactivity and because its chemical modification affects
the enzyme activity. Surprisingly the first Pi class GST structure
solved (8) showed that this residue was removed about 12 Å from the
active site. The structure of the Cys-47-carboxymethylated mGST P1-1
proves that the effect of the chemical modification is a loss of
electron density in the helix B and 310B region, a
17-residue stretch including important binding site residues. The loss
of electron density may be due to high thermal motions or oscillations
between a number of conformers. The side chain of Cys-47 is buried by
the side chains of Leu-43, Thr-46, Lys-53, and Tyr-63 in the native
complexes and is essentially inaccessible to solvent (see also Ref.
18). In addition the stability of the
B-310B
substructure must be marginal with exposed hydrophobic residues like
Leu-42. The addition of a carboxymethyl group at the S
atom of Cys-47 would cause severe contacts with the surrounding side
chains, in particular with Leu-43, and prevent the correct folding of
this area.
However, our kinetic studies demonstrate that the carboxymethylated
enzyme is still active with a kcat comparable
with that of the native protein. Simply the carboxymethylation of
Cys-47 increases the Km for glutathione by 3 orders
of magnitude. Under saturating conditions the modified enzyme turns
over almost as efficiently as the native one. In agreement with this,
the complex of CM-mGST- with
S-(p-nitrobenzyl)glutathione proves that a
product analog can bind to the modified enzyme and that this binding
partially organizes the active site except for Lys-44. Inhibitor
binding results in the organization of 6-10 residues out of 17, but
the ligand binding is unable to fully organize the area, most probably
because of the steric hindrance of CM-Cys-47 with Leu-43.
The carboxymethylation of Cys-47 even though affecting residues implied in the substrate binding does not affect residues close to the glutathione sulfur site like Tyr-7 or Tyr-108, which might be somehow involved in catalysis (17, 39-41). Neither does it affect the water structure of the pocket where W1, W2, and W3 (Fig. 5) maintain their positions, although extra water molecules appear to fill the space freed by the absence of the substrate. Again this agrees with the turnover capability of the CM-modified enzyme because the appropriate environment around the glutathione thiol or thiolate group is preserved.
Furthermore in the unliganded CM-mGST P1-1 we do not find any evidence
that Tyr-7 is in the tyrosinate form. There is no positively charged
residue to stabilize a Tyr-O ion in the vicinity for
CM-mGST P1-1, and this is also the case for the native enzyme complexed
with glutathione adducts. On the contrary the hydroxyl group of Tyr-7
seems to be protonated, accepting a hydrogen bond from the main chain
NH group of Arg-13 and giving a hydrogen bond to a well defined water
molecule, W0, that occupies the thiol position in the absence of
glutathione (Fig. 5, A and B). W0 is obviously
displaced when the substrate glutathione binds, and the H-bond acceptor
would then be the glutathione thiol(ate). Our results do not support
the hypothesis of a tyrosinate residue acting as a general base and
accepting a proton from the glutathione thiol. A similar conclusion has
been reported for the human Alpha class GST A1-1 (16). Its role must
be just stabilizing the thiolate by hydrogen bonding and positioning it
in the right orientation for the nucleophilic attack on the hydrophobic
substrate. Because there is strong evidence that the glutathione
molecule is in the thiolate form when bound to the enzyme, the most
feasible hypothesis is that one of the water molecules at the active
site accepts the proton (42). For GST A1-1 Widersten et al.
(43) have suggested that a water molecule could relay the thiol proton
to the
-carboxylate group of glutathione.
The present work does not completely solve the question of whether
substrate binding induces conformational changes in native mGST P1-1.
In this respect there are two possible scenarios: (i) The first
scenario is that unliganded native mGST P1-1 has a disorder similar to
the one observed in the free CM-mGST P1-1 enzyme. This disorder affects
residues that are involved in binding of glutathione but are not
directly implicated in catalysis. The binding of the glutathione
substrate would then organize the B-310B area. The H-subsite would be substantially the same in the free enzyme, except
for Ile-35, and, therefore, the hydrophobic substrate would readily
bind. Ile-35 would close the lipophilic crevice at one side, once the
substrate enters. The difficulties in crystallizing the native enzyme
in the absence of GSH or GS conjugates seem to indicate some
flexibility or disorder, although this argument does not extend to the
free CM-mGST P1-1, which was indeed crystallized in spite of having a
17-residue portion of the polypeptide chain disordered. (ii) The
alternative scenario consists of a native mGST P1-1 perfectly
structured in its free form. The structure of Schistosoma
japonica GST, which is different from the mammalian enzymes but
related to the Mu class, shows that the active site is unchanged when
comparing the ligand-free and the ligand-bound enzyme (15). In the
class an unaltered G-subsite is observed in the absence of ligand,
although the distinct C-terminal
-helix of this class, which covers
the H-subsite when the hydrophobic substrate is bound, appears to be
disordered in the unliganded form (16).
For mGST P1-1, with the present data, neither of the two scenarios can be ruled out. However, we note that neither of them implies any rearrangement of residues in the surroundings of Tyr-7 or Tyr-108. Only water molecules that fill the ligand-free active site, some of them H-bonded to these two tyrosine residues, would be expelled by the binding of the substrates.
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ACKNOWLEDGEMENTS |
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We thank Dusan Turk for setting up the NCS protocol in MAIN, Isabel García-Sáez for help and discussion, and David Sheehan for helpful comments.
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FOOTNOTES |
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* This work was supported by Ministerio de Educación y Ciencia Grant PB95-0224 and by funds from the Center de Referència en Biotecnologia (Generalitat de Catalunya). Data collection at the European Molecular Biology Laboratory Outstation at the Deutsches Elektronensynchrotron was supported by the European Union Large Installations Project CHGECT93-0040.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 structure factors (codes 1BAY and R1BAYSF) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
¶ Supported by a Wellcome Toxicology Studentship.
To whom correspondence should be addressed. Tel.:
34-3-4006149; Fax: 34-3-2045904; E-mail: mcccri{at}cid.csic.es.
1 The abbreviations used are: GST, glutathione S-transferase; GSH, glutathione; GS-conjugate, glutathione S-conjugate; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid; mGST, mouse GST; NCS, noncrystallographic symmetry; CM, carboxymethylation; PEG, polyethylene glycol.
2 A. Párraga, I. García-Sáez, S. B. Walsh, T. J. Mantle, and M. Coll, submitted for publication.
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REFERENCES |
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