(Received for publication, August 17, 1995; and in revised form, December 26, 1995)
From the
The primary and secondary structure of glutaredoxin-3 (Grx3), a
glutathione-disulfide oxidoreductase from Escherichia coli,
has been determined. The amino acid sequence of Grx3 consists of 82
residues and contains a redox-active motif, Cys-Pro-Tyr-Cys, typical of
the glutaredoxin family. Sequence comparison reveals a homology (33%
identity) to that of glutaredoxin-1 (Grx1) from E. coli as
well as to other members of the thioredoxin superfamily. In addition to
the active site cysteine residues, Grx3 contains one additional
cysteine (Cys) corresponding to one of the two non-active
site (or structural) cysteine residues present in mammalian
glutaredoxins. The sequence-specific
H and
N
nuclear magnetic resonance assignments of reduced Grx3 have been
obtained. From a combined analysis of chemical shifts,
J
coupling constants, sequential
and medium range NOEs, and amide proton exchange rates, the secondary
structure of reduced Grx3 was determined and found to be very similar
to that inferred from amino acid sequence comparison to homologous
proteins. The consequences of the proposed structural similarity to
Grx1 are that Grx3, while possessing a largely intact GSH binding
cleft, would have a very different spatial distribution of charged
residues, most notably surrounding the active site cysteine residues
and occurring in the proposed hydrophobic protein-protein interaction
area. These differences may contribute to the observed very low K
of Grx3 as a reductant of insulin disulfides
or as a hydrogen donor for ribonucleotide reductase. Thus, despite an
identical active site disulfide motif and a similar secondary structure
and tertiary fold, Grx3 and Grx1 display large functional differences
in in vitro protein disulfide oxido-reduction reactions.
In general, glutaredoxins (Grx) ()and thioredoxins
(Trx) are small (9-12 kDa), well characterized proteins capable
of catalyzing thiol-disulfide exchange reactions. Representatives of at
least one of these two protein families have been found in all
organisms studied, indicating that proteins of this type are essential
for cellular functions (for reviews, cf. Gleason and
Holmgren(1988) and Holmgren(1989)). In the cell, glutaredoxins and
thioredoxins differ in the manner they are reduced. Glutaredoxins are
reduced via the ubiquitous tripeptide glutathione (GSH), whereas
thioredoxins are reduced directly by the specific flavoenzyme
thioredoxin reductase. In both cases, reducing equivalents are
ultimately derived from NADPH. In vitro, thioredoxins are
found to be general reductants of a number of different protein
disulfides, whereas glutaredoxins are less capable in this respect, but
are known to readily reduce mixed disulfides between proteins (or low
molecular weight thiol-containing compounds) and GSH (Gravina and
Mieyal, 1993). This activity is measured conventionally using the
-hydroxyethylene disulfide (HED) reduction assay (Holmgren,
1979a). Glutaredoxins can also reduce some specific protein disulfides,
such as the redox-active disulfide of ribonucleotide reductase
(Holmgren, 1979b; Bushweller et al., 1992).
Ribonucleotide
reductases are key enzymes in the biosynthesis of deoxyribonucleotides.
The aerobic classes of ribonucleotide reductases require the presence
of a specific disulfide-reducing agent (e.g. Trx or Grx) for
catalysis (for a recent review, see Reichard(1993)). Trx was thought to
be unique in this role until a viable Escherichia coli mutant
lacking Trx (trxA) was isolated, which eventually led to the
discovery of Grx1 (Holmgren, 1976). The essential roles of Trx and Grx1
were then challenged through the viability of an E. coli trxA,
Grx1 double mutant, A410 (Russel and Holmgren, 1988). From one such
mutant, UC647 (Miranda-Vizuete et al., 1994), two novel E.
coli glutaredoxins were identified, Grx2 and Grx3 (Åslund et al., 1994), and the gene for Grx3 was subsequently cloned. ()Grx3 was found to be an active, albeit inefficient,
hydrogen donor for ribonucleotide reductase in comparison with Grx1.
Several three-dimensional structures of thioredoxins and
glutaredoxins have been determined, the earliest being the crystal
structure of oxidized E. coli thioredoxin (Holmgren et
al., 1975). The first crystal structure of a glutaredoxin was that
of the phage T4 glutaredoxin (Söderberg et
al., 1978). Together, these studies established the now typical
thioredoxin/glutaredoxin polypeptide fold (Eklund et al.,
1984). Subsequent studies by nuclear magnetic resonance (NMR)
spectroscopy have revealed the structures of reduced and oxidized Grx1
(Sodano et al., 1991; Xia et al., 1992), and the
structure of the mixed disulfide between Grx1 and glutathione, Grx1-SG
(Bushweller et al., 1994). In the present work, we have
determined the amino acid sequence of E. coli Grx3 and
compared it to other members of the thioredoxin superfamily. As a first
step toward characterizing the three-dimensional structure of this
newest member of the glutaredoxin family, we have determined the
sequence-specific H and
N NMR assignments and
the secondary structure elements of the reduced form of Grx3 in
solution thereby supporting the proposed sequence alignments and the
close structural similarity of Grx3 to Grx1. Implications of this
structural homology are discussed in light of the observed activities
of Grx3.
Total amino acid compositions were
determined (Pharmacia-LKB Alpha Plus 4151) after hydrolysis of purified
protein or peptide samples for 24 h at 110 °C in evacuated tubes
containing 6 M HCl and 0.5% (w/v) phenol. Amino acid sequence
analysis was performed using Edman degradation on the intact
[C]carboxymethylated protein and the cleavage
fragments using either a Milligen Prosequencer 6600 or an Applied
Biosystems 470A sequencer.
Sequence homology between Grx3 and other glutaredoxins was probed with the aid of the computer program LASERGENE (Dnastar, Inc.) and the programs ALIGN, SSEARCH, and FASTA (Pearson, 1990).
Figure 2: Amino acid sequence alignment of selected glutaredoxins. The amino acid sequences were obtained from the following references: a, human (Padilla et al., 1995); b, yeast 2 (hypothetical Grx-like protein) (Hollenberg et al., 1992); c, phage T4 (Sjöberg and Holmgren, 1972); d, E. coli Grx1 (Höög et al., 1983); e, E. coli Grx3 (this work). Residues identical in all species are shown with a stippled background. Those residues of E. coli Grx1 identified as being involved in the interactions with GSH (Bushweller et al., 1994) are boxed. The proposed secondary structure of Grx3 as determined in this work is indicated with line segments under the Grx3 sequence (see text for details).
Vicinal spin-spin coupling
constants J
were measured from
the two-dimensional
H NOESY spectrum in H
O by
inverse Fourier transformation of the in-phase multiplets (Szyperski et al., 1992). For the identification of slowly exchanging
amide protons, a freshly prepared sample of reduced Grx3 was
lyophilized from 50 mM phosphate buffer, pH 6.8. Exchange was
initiated by addition of
H
O and a single,
homonuclear two-dimensional total correlation spectroscopy spectrum was
recorded after 1 h for a duration of 60 min. Amide protons still
visible in this spectrum are classified as slowly exchanging (k
< 0.02 min
).
Secondary structure analysis using H NMR chemical
shifts was performed according to the technique of Wishart et
al., (1992). Chemical shifts for reduced, unfolded Grx3 have been
determined previously (Nordstrand et al., 1995). Expected
H chemical shifts were calculated from the atomic positions
of the Grx3 model according to the algorithm of Williamson et
al.(1995).
Figure 1: The primary structure of E. coli Grx3 as determined by N-terminal amino acid sequence analysis. The extent of sequential Edman degradation is indicated by line segments under the sequence. N1 refers to analysis of undigested protein. Fragments K4 and K5 are peptides obtained by cleavage with Lys endoprotease. Fragment F1 was obtained after treatment with 70% formic acid for 24 h at 37 °C.
Figure 3:
Contour plot of a two-dimensional N HSQC spectrum recorded of cloned, overexpressed,
N-enriched reduced Grx3 at pH 6.5 and 28 °C. The
sequence-specific residue assignment is indicated next to the peak
using the single-letter code followed by the residue number.
An asterisk following the assignment indicates the cross-peak
is assigned to a side-chain NH.
The core of seven secondary
structural elements within the Grx3 structure is defined in a
straightforward and unambiguous manner from the data summarized in Fig. 4. The three helices identified in Grx3 are labeled
sequentially
(residues 13-24),
(38-47), and
(67-74) in Fig. 4. Many of the expected medium range NOEs could not be
unambiguously assigned due to chemical shift degeneracies or cross-peak
overlap. The four segments of extended structure identified in Grx3 are
labeled sequentially
(residues 3-7),
(28-33),
(53-57), and
(60-62) in Fig. 4. These extended
segments contain numerous interstrand NOE connectivities, enabling
alignment of these four strands into a four-stranded
sheet (Fig. 5). The short fourth strand,
, involving
residues 60-62 is predicted from sequence homologies (Fig. 2) and appears from analysis of interstrand NOEs to
, but the small
J
coupling constant of His 60 indicates a significant deviation
from a regular
-like secondary structure (Fig. 4).
Figure 4:
Survey of NMR-derived structural
parameters characterizing reduced Grx3 in solution at pH 6.8 and 28
°C as a function of the amino acid sequence. The following features
are identified: amide proton exchange rates with solvent water:
, k
< 0.02 min
;
coupling constants
J
(
)
< 6.0 Hz (
) > 7.0 Hz; sequential backbone d
and d
NOE connectivities are classified
as strong, weak, or absent and are represented by the thickness (or
absence) of a bar connecting the residues in question; medium
range NOE connectivities d
(i, i+3) and (i, i+4) are drawn as
line segments connecting the residues contributing to the observed
cross-peak if present. Arrows above the sequence connecting a
proline to the previous residue indicate a sequential connectivity via
either sequential d
or sequential d
NOEs. All NOEs were measured in a
two-dimensional NOESY spectrum recorded with 60-ms mixing
time.
Figure 5:
Diagram showing the alignment of the
four-stranded sheet observed in Grx3 deduced from interstrand NOE
connectivities. The amino acid backbone is represented in stick format with residue numbers indicated above C
positions. Interstrand NOEs are drawn as double-headed arrows connecting protons giving rise to the observed cross-peaks. Slowly
exchanging (k
< 0.02 min
)
amide protons are identified by stippled circles. Hydrogen
bonds suggested by the network of NOEs are drawn as broken lines connecting amide proton and acceptor carbonyl oxygen. The
directionality of individual strands of the
sheet is indicated by bold arrows at the right of the
figure.
While
the sequential and medium range NOEs and J
coupling constants were
successful in identifying the core of the secondary structure elements,
the exact positions of the ends of each of these structures are more
difficult to define. Therefore, the N- and C-terminal extensions of the
helices and
sheets in the three-dimensional structures
could be up to three residues longer than indicated in Fig. 6and 7 and still be consistent with the data presented
here. The experimental determination of the
strand alignment in
Grx3 (Fig. 5) is in full agreement with the independently
obtained sequence alignment (Fig. 2) and justifies both the
position and the length of insertions and deletions in the sequence
alignment with Grx1. Additionally, the conserved cis-proline
(Pro
) present in each of the homologues in Fig. 2is also present in reduced Grx3 as deduced from the
presence of sequential d
and absence of
sequential d
NOEs involving this residue.
Final confirmation of the precise boundaries of the secondary structure
elements awaits the full three-dimensional structure determination
currently in progress.
Figure 6:
Plot
of the differences between the observed H chemical
shifts and the corresponding random coil values,
(H
native) -
(H
random) versus the amino acid sequence of Grx3. Two vertical bars representing differences calculated using (i) average random coil
values taken from Wishart et al.(1992) (black), and
(ii) experimentally determined random coil values for reduced Grx3
taken from Nordstrand et al.(1995) (stippled), are
centered on the amino acid residue position. Regions of secondary
structure identified by analysis of NOEs, J-couplings, and amide proton
exchange rates ( Fig. 4and Fig. 5) are indicated by solid black line segments at the top of the
plot.
In order to test the validity of the proposed amino acid
sequence homology of Grx3 with other members of the thioredoxin
superfamily (Fig. 2) and to gain further insight into the
secondary structure, we have analyzed the H chemical
shifts of reduced Grx3 (Table 2) using the procedure of Wishart et al.(1992). Fig. 6displays a plot of the difference
between the H
chemical shift measured for reduced Grx3
and the H
chemical shift reported for the same residue
type in a ``random coil'' conformation,
(H
) -
(H
), at each position in the
amino acid sequence. Regions in which the observed H
chemical shifts are significantly (>0.1 ppm) shifted to lower
field (negative differences) identify
helices and regions in
which the observed H
chemical shifts are shifted to
higher field (positive differences) identify
strands (Wishart et al., 1992). With the exception of the short fourth strand
of the
sheet (residues 60-62), each secondary structure
element proposed by the sequence alignment to Grx1 (Fig. 2) is
also indicated from this analysis of Grx3 (Fig. 6). Recently,
the complete NMR resonance assignment of the reduced, unfolded Grx3 was
determined (Nordstrand et al., 1995). When used in place of
the average ``random coil'' values of H
chemical shifts proposed by Wishart et al.(1992), the
experimentally determined H
chemical shifts for Grx3
produce a very similar pattern (Fig. 6). Interestingly, an
additional, short helix containing residues 78-82 is predicted
when using the experimental random coil H
chemical
shifts, but not by the average shift values of Wishart et
al.(1992). The presence of this helical segment is further
supported by the NMR parameters summarized in Fig. 4. The
overall good correlation of the predictions from analysis of the
H
chemical shifts (Fig. 6) with independently
determined secondary structural elements ( Fig. 4and Fig. 5) further supports the secondary structure outlined in Fig. 4.
Having established a similar secondary structure
content and location for Grx3 and Grx1, we were next interested whether
the tertiary fold of Grx3 could be confirmed by similar means using the
conformational information contained in the H NMR
chemical shifts. Exploiting the sequence homology of Grx3 with Grx1,
for which three NMR structures exist (Sodano et al., 1991; Xia et al., 1992; Bushweller et al., 1994), we have
constructed a three-dimensional model of oxidized Grx3, Grx3 model. The
model contains the same basic secondary structure elements as found in
Grx1 in the same relative orientation (Fig. 7). The secondary
structure analysis of Grx3 model reveals a four-stranded
sheet
containing residues 2-7 (
), 27-33
(
), 53-57 (
), and 60-62
(
) and three
helices containing residues
11-24 (
), 39-49 (
), and
64-75 (
). There is thus overall good agreement
between the secondary structures present in Grx3 model and the
experimentally determined secondary structure of Grx3 (Fig. 4Fig. 5Fig. 6). Apart from the minor
displacement of helix 2 (Fig. 7, top), Grx3 model is
very similar to the NMR solution structure of Grx1 upon which it is
based. The truncated first helix of Grx3, suggested by both the
sequence comparison to Grx1 and the secondary structure determination
by NMR, contains one less helical turn at the C terminus than Grx1 (Fig. 1, Fig. 4, and Fig. 7) providing a shortened
link to the second
strand. Analysis of Grx3 model reveals that
the side chains of Grx3 are also well accommodated in the Grx1
structure with minimal backbone distortions.
Figure 7:
Comparison of the structure of Grx3 model (light) with the mean NMR solution conformer of oxidized Grx1
calculated from the solution structures determined by Xia et
al.(1992) (dark). The two proteins are displayed backbone
cartoons using the program Ribbons (Carson, 1991) following
superposition of backbone atoms of shared regular secondary structure
elements (see text for details). Selected amino acid positions of Grx3
model are numbered at the position of
C.
H NMR
chemical shifts of oxidized Grx3 were calculated from the
three-dimensional coordinates of Grx3 model using the algorithm of
Williamson et al.(1995), which considers contributions from
ring current, magnetic anisotropy, and electric field shifts. The
resulting calculated H
chemical shifts for Grx3
correlate well with those observed for Grx3 in solution. The standard
deviation (S.D.) of calculated to observed H
chemical
shifts is 0.44 ppm. The accuracy of NMR solution structures with S.D.
values around 0.3 ppm has been compared to x-ray crystal structures of
2.0-Å resolution (Williamson et al., 1995). S.D. values
of 0.45 ppm are not without precedent among experimentally determined
NMR solution and x-ray crystal structures (Williamson et al.,
1995). Some discrepancies are expected from the fact that the
experimental chemical shifts were obtained from reduced Grx3 whereas
the model was built of the oxidized form. Thus, while a S.D. value of
0.44 ppm is, in these terms, clearly indicative of a structure of lower
quality than a 2-Å resolution crystal structure, this value is
sufficiently small to support the tertiary fold of Grx3 as presented in
Grx3 model.
Figure 8:
Comparison of Grx3 (), Grx1 (
),
and Grx1 (C14S) (
) as reductants of insulin disulfides. See text
for details.
Since the structural differences between Grx3 and Grx1 involve differences in charge distribution around the active site cysteine residues (see below), the importance of these charges should decrease with increasing ionic strength. The activity of Grx3 versus that of Grx1 with ribonucleotide reductase was found to increase upon addition of sodium chloride or ammonium sulfate (data not shown). Since ribonucleotide reductase is known to be inhibited by these salts, the assays were performed with sodium acetate, which has no inhibitory effect on ribonucleotide reductase (Brown et al., 1969). The activity of Grx3 with ribonucleotide reductase increased by 100% when 800 mM sodium acetate was added (Fig. 9). Ribonucleotide reductase was not inhibited, and Grx1 was only slightly inhibited under these conditions. It is interesting to note that the sequence of the last 23 residues of ribonucleotide reductase subunit R1 (EDAQDDLVPSIQDDGCESGACKI) consists of numerous charged residues. This segment of R1 also contains the cysteine residues that are reduced by glutaredoxin or thioredoxin (Mao et al., 1987; Åberg et al., 1989). Electrostatic interactions are thus likely to be important for the interaction of glutaredoxin to this region of ribonucleotide reductase. The results presented here (Fig. 9) supports the hypothesis that electrostatic interactions play an important role in the diminished activity of Grx3 compared to Grx1 with ribonucleotide reductase.
Figure 9:
Ribonucleotide reductase activity using
different hydrogen donors as a function of ionic strength. Hydrogen
donors included 25 mM DTT(), 1 mM Grx1 (
),
and 0.35 mM Grx3 (
).
A number of similarities and differences in
the charged amino acids which map near the active site cysteines
(Cys and Cys
) are noticed. Some differences
are conservative as, for example, Lys
in the turn preceding
the active site cysteine residues, which has arginine as a counterpart
in Grx1. However, the negative charge of Glu9 in Grx3 has no
counterpart in Grx1. Likewise, there is no counterpart for Glu
of Grx1, just before the conserved cis-proline (Pro
in Grx3) and Asp
in Grx3 (Thr
in Grx1).
It is also possible that the numerous amino acid differences observed
in the loop containing the second helix (residues 32-53 in Grx3)
are important determinants contributing to the low reactivity of Grx3
with ribonucleotide reductase compared to Grx1 (Fig. 9).
Overall, based on the amino acid sequence, Grx3 is expected to be more
basic than Grx1 with calculated pI values of 7.1 and 5.2, respectively.
Similar to mammalian glutaredoxins (Fig. 2), Grx3 contains a
cysteine (Cys) residue after the two conserved glycine
residues in the bend before the last helix. This residue corresponds to
Tyr
of Grx1. The function of this single cysteine residue
is not known. From the position of Cys
, in the first turn
of the third helix, one might expect this thiol to have an increased
reactivity caused by the localized partial positive charge of the helix
dipole (Hol et al., 1978). In addition, mammalian
glutaredoxins have a second, non-active site, cysteine located three
residues before the cysteine residue corresponding to Cys
in Grx3, whereas in prokaryotic glutaredoxins, there is a
conserved histidine residue at this position (Fig. 2). In
mammalian thioredoxins, an analogous internal cysteine pair can be
found similar to those in mammalian glutaredoxins. A regulatory
function of this cysteine pair in thioredoxins has been suggested (Ren et al., 1993). We have observed dimers of Grx3 following
SDS-polyacrylamide gel electrophoresis under non-reducing conditions
(Åslund et al., 1994), presumably due to intermolecular
disulfide bond formation.
The residues involved in binding of
glutathione have been identified in the NMR solution structure of the
mixed disulfide Grx1-SG (Bushweller et al., 1994) and are
enclosed in boxes in Fig. 2. The interaction of GSH
with Grx3 is likely to be very similar to that with Grx1, with the GSH
molecule binding in a cleft on the surface of Grx3. The cysteine thiol
of GSH would interact with Cys of Grx3 and the conserved
Tyr
would form one side of the binding site. A tyrosine or
a phenylalanine residue is always found at this position in
glutaredoxins (Nikkola et al., 1991). The conformation of the
conserved cis-proline at position 53 should allow for main-chain
hydrogen bonding between glutathione and the protein as it does for
Grx1. The preceding residues (Thr
and Val
)
are also conserved in Grx3. The amino group of the
-glutamyl of
GSH can interact with the
-hydroxyl of Thr
and a
-carboxyl group of one of the aspartic acid residues following
Cys
. The first of these two aspartic acid residues is at a
position where a serine or threonine is positioned in other
glutaredoxins (Fig. 2). It has been shown for T4 glutaredoxin
that an Asp
Ser substitution at this position
increases the activity of this glutaredoxin with GSH (Nikkola et
al., 1991), indicating that the charge distribution in this area
is indeed important for GSH binding. Despite the two aspartic acid
residues following Cys
in Grx3, the turnover of GSH is
comparatively high, as measured by the HED assay. On a molar basis,
Grx3 has a 50% higher activity than Grx1 in the HED assay (Åslund et al., 1994). The Asp-Asp motif of Grx3 is also found in an
analogous position in the yeast DNA sequence corresponding to a
hypothetical glutaredoxin (Yeast 2, Fig. 2). Most
intriguing is the role of Cys
. This residue could hydrogen
bond with the carboxyl of the
-glutamyl of GSH. Its location in
the GSH binding cleft suggests, by analogy, that Grx3 may be a good
model for GSH-protein interaction in mammalian glutaredoxins and
furthermore presents the possibility of regulation through
intermolecular disulfide bond formation.
Despite a similar
three-dimensional structure, Grx3 was found to have only a fraction of
the capacity of Grx1 as a disulfide reductant despite an identical
active site sequence (CPYC). The conformations of oxidized and reduced
Grx1 have been shown by NMR to be very similar. The similar appearance
of the circular dichroism spectra of oxidized and reduced Grx3
indicates little conformational change between the two forms. ()One possible explanation for the different reactivities of
Grx1 and Grx3 is the observed differences in the distribution of
charged residues in and around the proposed surface for interaction
with other proteins. The increased activity of Grx3 in the
ribonucleotide reductase assay as a function of ionic strength supports
this point. An increase in redox potential of Grx3 compared to the
other hydrogen donors to ribonucleotide reductase (e.g. Trx
and Grx1) is also a possible explanation for the reduced activity. The
effect of disulfide bonds on the conformational stability of globular
proteins must necessarily reflect the reciprocal effect of the protein
conformation on the stability of the disulfide bonds (Creighton, 1986).
This linkage relationship, when applied to the oxidoreductases,
proposes that differences in conformational stability between the
oxidized and reduced forms of these proteins should effect the
stability of the active site disulfide bond and hence affect the redox
potential in a predictable manner. This relationship has been verified
experimentally for the E. coli proteins Trx (Lin and Kim,
1989) and DsbA (Zapun et al., 1993; Wunderlich et
al., 1993), where the oxidized form of the physiologically
reducing Trx was found to be more stable than the reduced form whereas
in the physiologically oxidizing DsbA, it is the reduced form that is
more stable. In the framework of Grx3, an increased redox potential
(less negative) could be achieved by increasing the stability of the
reduced with respect to the oxidized form. This relatively
straightforward mechanism could turn out to be a general phenomenon by
which the process of evolution controls the gross redox function of
these proteins.