(Received for publication, September 25, 1995; and in revised form, December 28, 1995)
From the
We have determined the crystal structure of a complex between
the noncompetitive inhibitor (K = 27
µM, K
= 48 µM with respect to oxidized glutathione (GSSG) and K
= 144 µM, K
=
176 µM with respect to NADPH)
6-hydroxy-3-oxo-3H-xanthene-9-propionic acid (XAN) and human
glutathione reductase (hGR). The structure, refined to an R-factor of
0.158 at 2.0 Å resolution, reveals XAN bound in the large cavity
present at the hGR dimer interface where it does not overlap the
glutathione binding site. The inhibitor binding causes extensive local
structural changes that primarily involve amino acid residues from a
30-residue
-helix that lines the cavity and contributes to the
active site of hGR. Despite the lack of physical overlap of XAN with
the GSSG binding site, no GSSG binding is seen in soaks carried out
with high XAN and GSSG concentrations, suggesting that some subtle
interaction between the sites exists. An earlier crystallographic
analysis on the complex between hGR and
3,7-diamino-2,8-dimethyl-5-phenyl-phenazinium chloride (safranin)
showed that safranin bound at this same site. We have found that
safranin also inhibits hGR in a noncompetitive fashion, but it binds
about 16 times less tightly (K
= 453
µM, K
= 586 µM with respect to GSSG) than XAN and does not preclude the binding
of GSSG in the crystal. Although in structure-based drug design
competitive inhibitors are usually targetted, XAN's binding to a
well defined site that is unique to glutathione reductase suggests that
noncompetitive inhibitors could also serve as lead compounds for
structure-based drug design, in particular as components of chimeric
inhibitors.
Malaria is one of the world's most devastating diseases. According to the World Health Organization, 300 million cases of malaria are recorded each year with a fatality exceeding 2 million per annum(1) . The causative agents of malaria are parasitic protozoa of the genus Plasmodium, which infect human erythrocytes and are transmitted by anopheles mosquitoes. Numerous strains of Plasmodium falciparum, the most aggressive species, have developed resistance to currently used drugs; therefore the development of novel, effective chemotherapeutic agents is essential(2) .
Intracellular parasites such as the P.
falciparum require a highly efficient thiol metabolism to protect
themselves from intracellular reactive oxygen species and their
derivatives(3) . This implies that they are highly susceptible
to oxidative stress and that this sensitivity is a promising target for
drug action(4, 5) . The role of oxidative stress as an
important mechanism for the destruction of parasites and tumor cells (6) is particularly well illustrated by many congenital and
acquired factors that generate oxidative stress in human erythrocytes
and offer partial protection against malaria(4) . Central to
defense against intracellular oxidative stress in humans is the
glutathione redox cycle, which involves the enzymes human glutathione
reductase (hGR) ()and glutathione peroxidase. The
flavoenzyme hGR catalyzes the NADPH-dependent recycling of oxidized
glutathione (GSSG) to maintain high levels of reduced glutathione as
shown by the equation below(7) .
Despite the importance of hGR, there is good evidence that hGR is not essential for normal erythrocyte function and that the reduced life-span of hGR-deficient red blood cells is tolerable(8) . As reviewed by Schirmer et al.(2) , hGR is thus a reasonable target for rational drug design. In this direction, various compounds have been reported to inhibit hGR(9, 10, 11) .
In choosing hGR as a target for structure-based drug design, we have considered, in addition to the enzyme's physiological importance, the plethora of structural information that is available; the three-dimensional structure of hGR has been solved and refined to 1.54 Å resolution, and its catalytic mechanism has been well established from crystal structures of the enzyme complexed with its natural substrates GSSG and NADPH, substrate analogues, and various other ligands(12, 13, 14, 15, 16, 17) . Furthermore, the gene for hGR has been cloned and overexpressed in Escherichia coli, which makes possible the study of structure-function relations through the generation of site-directed mutants(18) .
In a recent study on the prediction of ligand binding to proteins by affinity fingerprinting methods(19) , yeast glutathione reductase was used as one of the model protein drug targets. The study identified a number of tight binding inhibitors from a variety of chemical families, yet their modes of action were not investigated. Because yeast glutathione reductase and hGR are homologous (48% overall amino acid sequence identity), we have tested some of the yeast GR inhibitors as inhibitors of hGR and found that they exhibit diverse modes of inhibition. One of them, 6-hydroxy-3-oxo-3H-xanthene-9-propionic acid (XAN), is a noncompetitive inhibitor that binds in a cavity of hGR where the dye 3,7-diamino-2,8-dimethyl-5-phenyl-phenazinium chloride (safranin) had been previously seen to bind(13) . As a result of this finding we have also assessed the inhibition of hGR by safranin, which also behaved as a noncompetitive inhibitor with respect to GSSG, yet its inhibition potency was 16 times weaker than XAN. Here, we report on the inhibition of hGR by these two compounds and on the crystal structure of hGR in complex with XAN at 2.0 Å resolution.
Plasmid-carrying E. coli SG5 cells, which had been grown
for 14 h (A
= 6.5) in 10 liters
of two times concentrated tryptone-yeast extract medium containing 0.1
mg/ml ampicillin, were harvested by centrifugation. The cells were
suspended in lysis buffer (0.1 M Hepes, pH 7.2, 1 mM EDTA, 5 mM
-mercaptoethanol) containing 0.1 mM phenylmethylsulfonyl fluoride and 0.05 mg/ml FAD and were lysed by
French Press (12000 psi) and sonication. The crude extract was
clarified by centrifugation (25,900
g, 1 h, 4 °C).
The supernatant was then adjusted to a 20% ammonium sulfate saturation
by the addition of solid ammonium sulfate and was stirred for 1 h at 4
°C. The resulting precipitate was removed by centrifugation (25,900
g, 1 h, 4 °C), and the supernatant was brought to
an 80% ammonium sulfate saturation and was stirred for 1 h at 4 °C.
The precipitate was then obtained by centrifugation (25,900
g, 1 h, 4 °C) and was subsequently resuspended and
dialyzed in buffer A (20 mM potassium phosphate, 1 mM EDTA, 0.2 M KCl, and 5 mM
-mercaptoethanol,
pH 7.2). The dialysate was applied to a 50-ml column of
2`,5`-ADP-Sepharose equilibrated with buffer A. After extensive washing
with buffer A, the enzyme was eluted with 30 ml of 1 mM NADPH
in buffer A. The enzyme was reoxidized and stabilized by the addition
of 2 mM GSSG. The solution was then dialyzed against buffer B
(20 mM Tris, 1 mM EDTA, 5 mM
-mercaptoethanol, pH 8.5) and applied to a 30-ml column of
DEAE-Sephacel equilibrated with buffer B. After washing with 150 ml of
buffer B, the enzyme was eluted with a linear salt gradient
(0-0.4 M KCl in buffer B). The active fractions were
pooled and dialyzed against buffer B. The dialyzed protein solution was
loaded onto a 10-ml column of Procion Red HE-3B and washed extensively
with buffer B. Pure hGR was eluted with a linear salt gradient of
0-2 M KCl in buffer B. Fractions containing hGR were
pooled, concentrated to 15 mg/ml, and dialyzed against storage buffer
(3% ammonium sulfate, 0.1 M potassium phosphate, pH 7.0).
The final concentration of the enzyme was assessed using
= 11.3 mM
cm
(22) . Enzyme activity was measured
under saturating substrate conditions(23) . The yields per
liter of cell culture were 3-4 mg of electrophoretically
homogeneous (>98% pure) hGR, with an A
/A
ratio of 6.6. The K
for GSSG of 65 µM, and the k
of 12,500 min
agree well
with those reported by Nordhoff et al.(21) for
recombinant hGR and by Worthington and Rosemeyer (24) for the
native enzyme.
Figure 1: Chemical structures of XAN and safranin. Inhibitors are presented with the names used in the text, whereas their full IUPAC names can be found in the abbreviation footnote.
Assays were carried out in 1 ml of the following solution: 0.2 M KCl, 0.1 M potassium phosphate, pH 7.0, 1 mM EDTA, 0.1 mM NADPH, 30-1000 µM GSSG, 0.6 nM hGR, and 35 nM bovine serum albumin. The added bovine serum albumin stabilized hGR, which otherwise rapidly lost activity at the low concentration used in the assay(22) . For the inhibition studies, inhibitors were added to the above mixture at the desired concentrations, whereas all other parameters stayed the same. In the case of XAN, where the inhibitor was dissolved in 0.1 M Tris at pH 8, control assays were performed in the presence of Tris. For each inhibitor we used three different concentrations. The inhibitor concentration was kept constant, and activity measurements were made at 30-1000 µM GSSG. For XAN we have also carried out activity measurements by keeping a saturating 1 mM concentration of GSSG and taking measurements at 5-100 µM NADPH. The reaction was initiated by the addition of NADPH once all other reagents were present in the assay mixture.
The
inhibition constants, K and K
, for noncompetitive inhibitors were estimated
by fitting the data to the following equation using the FORTRAN
programs of Cleland(25) .
where [S] is the concentration of the variable
substrate, [I] is the concentration of the inhibitor, K represents the binding of inhibitor to free
enzyme, and K
represents binding to the
enzyme-substrate complex. For pure noncompetitive inhibition K
= K
, and for pure
competitive inhibition K
= infinity.
The model for XAN was built using the ChemNote utility of
QUANTA (Molecular Simulations, Inc.) and minimized using CHARMm
(Molecular Simulations, Inc.). Binding of XAN to the enzyme was
observed in difference electron density maps using coefficients (F - F
) and
calculated phases from the refinement of the native structure
(
). Initially, data set XAN-1 at 2.3 Å
resolution (Table 1) was used. Difference electron density maps
showed incomplete density for the tricyclic xanthene moiety of the
molecule, but after one round of positional refinement using the native
hGR structure after removal of water molecules in the XAN binding site,
the density improved. XAN was subsequently fitted, and manual changes
to the protein model were made using the program CHAIN(30) .
Further cycles of conventional positional refinement against data
between 10 and 2.3 Å resolution, and manual fitting yielded a
structure with an R-factor of 0.143. At this point data were collected
from crystals equilibrated in artificial mother liquor containing XAN
and GSSG (data set XAN-2, Table 1). Because binding of GSSG was
not observed, data sets XAN-1 and XAN-2 were merged to generate a more
complete 2.0 Å resolution data set (Table 1). Several
further rounds of manual refitting and positional refinement resulted
in our final model of the complex with an R-factor of 0.158 for all
data between 10 and 2.0 Å resolution.
Kinetic studies showed that XAN inhibition of hGR is
noncompetitive with respect to GSSG, with K = 27 µM and K
=
48 µM (see Fig. 2). Additional experiments at
saturating concentrations of GSSG and varying concentrations of NADPH
revealed that XAN is also noncompetitive to NADPH with K
= 144 µM and K
= 176 µM (data not shown).
This mode of inhibition suggests that XAN binds reversibly both to the
free enzyme and the enzyme-substrate complex at a site distinct from
that of the natural substrate(s). Furthermore, assays done in the
absence of GSSG showed that XAN itself does not act as an electron
acceptor. The observed noncompetitive inhibition of XAN shows that the
diverse compounds identified by the affinity fingerprinting approach to
drug design (19) may bind to various sites on the enzyme. Given
this extra degree of freedom, the predictive success of this study
makes it all the more noteworthy.
Figure 2:
Kinetic results for the inhibition of hGR
by XAN and safranin. Lineweaver-Burk plots showing the effect of XAN (A) and safranin (B) on hGR activity at different
GSSG concentrations (30-1000 µM) and a saturating
100 µM NADPH concentration. The concentrations of XAN used
were 10 (), 20 (+), and 30 µM (
). For
safranin the concentrations tested were 100 (
), 200 (+), and
300 µM (
). The data points marked as (
)
correspond to control measurements in the absence of
inhibitor.
The catalytic mechanism of hGR is
a bi-bi ping-pong mechanism with NADPH reacting first to produce the
reduced enzyme (EH), which in turn binds and reduces
GSSG(14, 15) :
Because there are more than just the E and ES forms of the
enzyme present, the K values need not be exactly
equated with single dissociation constants. Nevertheless, two factors
suggest that there is an element of XAN inhibition that is competitive
with GSSG. First is the 5-fold poorer inhibition by XAN in the presence
of saturating GSSG (K
= 144
µM) compared with the presence of saturating NADPH (K
= 27 µM), and second is
the 1.8-fold difference in K
versus
K
(48 µMversus 27
µM) with respect to GSSG. Both observations suggest that
GSSG-bound forms of the enzyme bind to XAN more weakly, which directly
implies that XAN bound forms of the enzyme bind GSSG more weakly.
After our crystallographic studies (see below) showed that XAN bound
in a site known to bind safranin (13) (Fig. 1), we
tested the inhibitory effects of safranin. Safranin also behaved as a
noncompetitive inhibitor with respect to GSSG (Fig. 2) but with
markedly less potency than XAN, having K =
453 µM and K
= 586 µM with respect to GSSG.
To characterize the mode of binding and the inhibition mechanism of XAN and safranin, we have analyzed crystal structures of hGR soaked with XAN alone, GSSG alone, a combination of XAN and GSSG, and a combination of safranin and GSSG (Table 1).
XAN binds at the crystallographic 2-fold axis in the large cavity at
the dimer interface of hGR with a stoichiometry of one XAN molecule/hGR
dimer. The cavity has been previously identified as one that binds
safranin and menadione and to a lesser degree lipoate,
-
-GSSG, and S-(2, 4-dinitrophenyl)-glutathione, but
whether it has a biological function is still unknown. The cavity
connects to both GSSG binding sites through two channels that also
serve as mediators between the bulk solvent and the
cavity(13) . Structural refinement using the conventional
positional refinement routines in X-PLOR yielded our final model for
the hGR-XAN complex at 2.0 Å resolution, which has an R-factor
= 0.158. Fig. 3A shows the final electron
density in the region of the bound XAN inhibitor. The electron density
for the xanthene portion of XAN is somewhat irregular, suggesting
mobility of XAN at its binding site (Fig. 3B). This is
supported by the fact that the B-factors for all atoms in XAN range
between 35 and 40 Å
. We have modelled and refined our
structure with XAN exhibiting planarity in its xanthene moiety in order
to be consistent with the nomenclature and chemical structure of the
compound (Fig. 1) and with previously determined crystal
structures of compounds that also have a xanthene
group(31, 32) . The region that accommodates XAN in
the hGR-XAN complex exhibits high structural disorder with B-factors in
the 40-60 Å
range in the native hGR structure.
This effect is particularly pronounced for amino acid residues
Phe
and Met
, for which the possibility that
they exist in alternate conformations has been suggested(12) .
Upon binding of XAN, however, these residues, along with His
and His
reorient substantially (Fig. 4A) and become quite rigid with B-factors
dropping to near 15 Å
.
Figure 3:
A, quality of the electron density maps.
The final 2F - F
;
electron density
after refinement of the hGR-XAN complex at 2.0 Å resolution is
shown. The map is contoured at 1.0
. Shown is the
region of the XAN binding site including the refined model for the
inhibitor molecule. The electron density for the carboxylate group of
the inhibitor is lower because each position of the carboxylate is only
present at half occupancy, whereas the remaining atoms of the inhibitor
overlap with their symmetry mates, effectively bringing them to full
occupancy. At the same time, however, it is worth noting the remarkable
ordering of the residues involved in the binding of XAN, which is
manifested in the low B-factors (main chain and side chain atoms). In
the unliganded structure of hGR, this region exhibits high disorder
with B-factors in the 40-60 Å
range. B,
electron-
electron interactions between XAN and
Phe
. Shown is an excerpt from the 2F
- F
;
electron density after refinement of the hGR-XAN complex at 2.0
Å resolution shown in A. We have modelled XAN with its
xanthene ring being planar based on the fact that the structures of
fluorescein and erythrosine B ethanolate (which also have a xanthene
moiety) also exhibit planarity of their
tricycle(30, 31) . The irregular electron density for
the tricycle is consistent with the relatively high B-factors of the
atoms in the xanthene moiety (35-40 Å
) and with
some slight puckering of the xanthene.
Figure 4:
A, overlay of XAN-free and XAN-bound
structures of hGR at the dimer interface. The XAN-bound structure is
shown in solid lines. Note the extensive structural changes
that take place upon binding of XAN to the free enzyme. The shifts are
supported by the strong positive and negative electron density peaks in
difference electron density maps (F - F
;
), as well as by the
quality of the final 2F
- F
;
electron density
map (Fig. 3A). Interesting and rather unique is the
head-on confrontation of the C
1 atoms of His
and
His
with an interatomic distance of 3.1 Å. Despite
the pronounced rearrangements in this part of the structure, no
significant changes can be observed elsewhere. B, stereo view
of the hydrogen bonding interactions at the XAN binding site. The
identity of each amino acid residue is indicated by the one-letter
amino acid code and the hGR residue number. Hydrogen bonds are
represented by thin dashed lines, and the distance between
hydrogen bond donor and acceptor pairs is shown (in
Å).
An additional structural
change involves Cys and Cys
, which form an
intersubunit disulfide bridge in the native hGR structure. In the
hGR-XAN complex the electron density for the disulfide bridge decreases
and elongates (Fig. 3A), indicating increased mobility
of this group (along with residues 88-94) and possibly an opening
of the disulfide in a fraction of the molecules. In the liganded
structure, residues 88-94 have the highest B-factors in the
structure and make up the only region whose B-factors are higher in the
XAN-bound structure than in the XAN-free state.
The observed
conformational changes result in a hydrogen bond network involving the
inhibitor and neighboring amino acid residues (Fig. 4B). In the native hGR structure, N1 of
His
makes a short (2.6 Å) hydrogen bond with the
N
1 of His
. In the XAN-bound structure the two
residues move apart (Fig. 4A) so that each one can form
a hydrogen bond with the oxygen on the carboxylate group of the
inhibitor. His
and His
make additional
hydrogen bonds with the reoriented Cys
and
Cys
, which further supports a partially open conformation
for the disulfide bridge between Cys
and
Cys
. His
is now able to form a new hydrogen
bond (2.8 Å) with Tyr
. A hydrogen bond between
Tyr
and Asp
, which is present in unliganded
hGR, is maintained in the hGR-XAN complex despite the fact that both
these residues have shifted slightly. The tricyclic moiety participates
in the hydrogen bonding network through interactions of its oxygens at
positions 15 and 16 with Water
, Asn
and
their symmetry-related partners. Moreover, the central oxygen at
position 10 of the xanthene makes a rather long hydrogen bond to Water
. Nonpolar interactions between hGR and XAN are made
primarily by Phe
and Phe
, which align almost
parallel to and about 4 Å away from the plane of the
inhibitor's tricycle forming a ``sandwich'' type of
interaction (Fig. 3B).
Surprisingly, the binding site is in a region that is not well conserved between human and yeast GR. Among the residues involved in the binding site (Fig. 4B), none is conserved. Although we still think it is likely that XAN binds at an equivalent site in yeast GR, this need not be so, and even if it does many details of the binding have to be different. In this light we must view the similarity of the inhibition constants of XAN for yeast GR and hGR as fortuitous.
To characterize the effects of XAN binding on GSSG binding, hGR crystals were soaked in artificial mother liquor containing both compounds (XAN-2, Table 1). In this soak only XAN was seen to bind, and a control soak with all components of XAN-2 but inhibitor showed that GSSG bound at high occupancy, thus eliminating the possibility that the buffer of the soak hampers binding of GSSG. Interestingly, when we attempted to obtain a double complex containing safranin and GSSG (SAF-1, Table 1), we observed binding of both compounds. The lack of GSSG binding in the XAN-2 soak surprised us because the kinetics had only suggested that XAN was slightly competitive with respect to GSSG (see above). One possible explanation is that under the conditions of the crystalline enzyme (e.g.. high ionic strength), the XAN-hGR interaction changes so that the binding of XAN directly interferes with GSSG binding. However, the results for safranin make this hypothesis less attractive. Another possibility is that the binding of XAN does indeed decrease the binding affinity of GSSG but that this is not visible in the kinetics because of the complexity of the two-substrate hGR reaction(33) . We note in this regard that for similar reasons the aldose reductase inhibitor zopolrestat behaves kinetically as a noncompetitive inhibitor but is crystallographically seen to block the aldose binding site(34) .
Despite our relatively detailed crystallographic
results pertaining to the interactions XAN makes with the enzyme, the
inhibition mechanism remains unclear. Based on the observed
noncompetitive inhibition, we expected to see structural changes that
might affect placement of active site residues or alter the mode of
GSSG binding without hindering the affinity of GSSG to the active site.
In this way catalysis could be impaired in a manner that could not be
overcome by high concentrations of GSSG. In the hGR-XAN complex, XAN is
located about 20 Å from the redox-active disulfide of hGR and is
about 10 Å from the nearest atom of GSSG in its expected binding
mode. This indicates that the observed inhibition is not due to a
direct interaction between the inhibitor and the active site of the
enzyme (Fig. 5). We note that most residues changing
conformation or directly interacting with XAN (His,
Phe
, Met
, Asp
, and
His
) reside on the long
-helix that contains the
active site disulfide. Despite this fact, no significant movements of
atoms in or near the active site disulfide can be observed. The only
structural effects that can be seen near the active site are small
scale main chain shifts close to the GSSG binding site and in regions
that directly interact with GSSG. The most prominent of these is a
0.3-Å shift of Met
whose main chain nitrogen
donates a hydrogen bond to the
-Glu-II-OT of GSSG in the hGR-GSSG
complex (14) . Although it would be surprising, it is
conceivable that these small conformational changes coupled with the
observed decrease in the mobility of the crystalline enzyme could
affect binding of GSSG and catalysis. Studies that have been carried
out using crystalline ribonuclease A as a model have suggested that
enzyme flexibility is required for catalytic function and that a
decrease in enzyme flexibility can hamper the activity of the
enzyme(35) . Some evidence against this hypothesis is provided
by safranin, which also inhibits hGR but does not produce extensive
conformational changes either in the hGR-safranin complex (13) or in our hGR-safranin-GSSG structure. Phe
and Phe
are the only residues that are markedly
affected by the binding of safranin. An alternative explanation for our
results is that the crystallographically seen binding site is a passive
one not responsible for inhibition and that the true inhibitory site
has not been observed in the crystals. Although this is possible, we
think it is unlikely because the XAN binding site is adjacent to the
GSSG binding site and is only seen to be highly occupied for compounds
that show this type of inhibition. Although we cannot presently detail
the mechanism by which XAN and safranin inhibit hGR, we know of no
other noncovalent, noncompetitive inhibitors that do not occupy the
active site and for which the structural mechanism of inhibition is
known. In this light, further investigation of the mechanism of
inhibition of these inhibitors should be valuable. We expect that
calorimetric analyses, and fast reaction kinetics will shed more light
on the inhibition mechanism of XAN and safranin.
Figure 5:
Stereo diagram showing the location of
XAN's binding site relative to the overall hGR structure. The hGR
dimer is characterized by crystallographic 2-fold symmetry. The
inhibitor binds at this 2-fold symmetry axis, and it has to be noted
that although both symmetry mates of XAN are shown here, only one XAN
molecule binds hGR at a time. The binding site is about 22 Å away
from either GSSG binding sites, which are indicated here by the active
site catalytic cysteines Cys, Cys
,
Cys
, and Cys
, which in the oxidized form of
the enzyme form disulfide bridges. These amino acid residues reside on
the two symmetry related
-helices that largely account for the
binding of XAN.
The residues lining the XAN binding pocket in hGR are not well conserved among homologs of hGR, so although compounds binding at this site are not competitive inhibitors, they are reasonable for structure-based drug design. In this regard, earlier results by Becker et al.(9) and more recent work by Kirsch et al.(36) have focussed on flavin derivatives that are hGR inhibitors and are expected to bind at the same dimer interface cavity that XAN and safranin do. In the case of XAN we see possibilities to rationally improve the potency of the compound. The enzyme-inhibitor interactions are tight in many regions, but there is room to make more extensive interactions through the placement of substituents of appropriate length at positions 3, 4, 5, and 6 of the tricyclic moiety of XAN. Furthermore, a replacement of XAN's carboxylate group with amide or hydroxyl groups might provide more optimal interactions with the surrounding environment, and at the same time it would also test the importance of the negatively charged tail of XAN for inhibition and binding.
The atomic coordinates and structure factors (codes 1XAN and R1XANSF) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.