(Received for publication, August 25, 1995; and in revised form, November 21, 1995)
From the
Escherichia coli thioredoxin contains two tryptophan
residues (Trp and Trp
) situated close to the
active site disulfide/dithiol. In order to probe the structural and
functional roles of tryptophan in the mechanism of E. coli thioredoxin (Trx), we have replaced Trp
with alanine
using site-directed mutagenesis and expressed the mutant protein W28A
in E. coli. Changes in the behavior of the mutant protein
compared with the wild-type protein have been monitored by a number of
physical and spectroscopic techniques and enzyme assays. As expected,
removal of a tryptophan residue causes profound changes in the
fluorescence spectrum of thioredoxin, particularly for the reduced
protein (Trx-(SH)
), and to a lesser extent for the oxidized
protein (Trx-S
). These results show that the major
contribution to the strongly quenched fluorescence of Trx-S
in both wild-type and mutant proteins is from Trp
,
whereas the higher fluorescence quantum yield of Trx-(SH)
in the wild-type protein is dominated by the emission from
Trp
. The fluorescence, CD, and
H NMR spectra
are all indicative that the mutant protein is fully folded at pH 7 and
room temperature, and, despite the significance of the change, from a
tryptophan in close proximity to the active site to an alanine, the
functions of the protein appear to be largely intact. W28A Trx-S
is a good substrate for thioredoxin reductase, and W28A
Trx-(SH)
is as efficient as wild-type protein in reduction
of insulin disulfides. DNA polymerase activity exhibited by the complex
of phage T7 gene 5 protein and Trx-(SH)
is affected only
marginally by the W28A substitution, consistent with the buried
position of Trp
in the protein. However, the thermodynamic
stability of the molecule appears to have been greatly reduced by the
mutation: guanidine hydrochloride unfolds the protein at a
significantly lower concentration for the mutant than for wild type,
and the thermal stability is reduced by about 10 °C in each case.
The stability of each form of the protein appears to be reduced by the
same amount, an indication that the effect of the mutation is identical
in both forms of the protein. Thus, despite its close proximity to the
active site, the Trp
residue of thioredoxin is not
apparently essential to the electron transfer mechanism, but rather
contributes to the stability of the protein fold in the active site
region.
Thioredoxin (Trx), ()a 12-kDa, heat-stable,
redox-active protein, has been characterized in many functions from a
wide variety of prokaryotic and eukaryotic cells (Holmgren, 1985;
Gleason and Holmgren, 1988; Holmgren, 1989). All thioredoxins contain
an active site cysteine disulfide/dithiol in a conserved sequence
Trp-Cys-Gly-Pro-Cys. The disulfide of oxidized thioredoxin,
Trx-S
, can be reduced to the dithiol by NADPH and a
specific flavoprotein enzyme, thioredoxin reductase, while reduced
thioredoxin, Trx-(SH)
, participates in a number of redox
reactions mostly linked to reduction of protein disulfides. Thioredoxin
has been most extensively studied in Escherichia coli. The
three-dimensional structure of E. coli Trx-S
has
been refined to 1.7 Å by x-ray crystallography (Katti et al., 1990), and high resolution structures of both Trx-S
and Trx-(SH)
have recently been calculated from
multidimensional NMR data (Jeng et al., 1994). The protein
contains 108 amino acid residues, including two tryptophans; one,
Trp
, is conserved throughout known thioredoxin sequences,
while the other, Trp
, is conserved throughout prokaryotes,
being replaced by serine in eukaryotic sequences (Eklund et al., 1991). The active site disulfide is located on a protrusion of the
molecule and is close to the two tryptophans both in the sequence
(Trp
-Ala-Glu-Trp
-Cys
-Gly-Pro-Cys
-)
and in the three-dimensional structure (Katti et al., 1990;
Jeng et al., 1994). The close proximity of the two tryptophans
to the active site provides a probe for detecting structural changes
between the oxidized and reduced molecule, since the tryptophan
fluorescence is affected by the change of oxidation state.
Tryptophan fluorescence is highly quenched in Trx-S. A
3.5-fold increase in fluorescence is observed upon reduction. This is
thought to be due mainly to the removal of the fluorescence quenching
provided by the disulfide bond (Holmgren, 1972), as well as to slight
shifts in the positions of the tryptophan rings accompanying a local
conformational change which may include a change in hydrogen bonding
interactions of the tryptophan ring (Dyson et al., 1990; Jeng et al., 1994). A number of studies, including chemical
modification of Trp
(Holmgren, 1981), comparison of yeast
and calf thymus thioredoxins (Mérola et al., 1989), and studies of several Trp
mutants (Krause and
Holmgren, 1991), indicated that this increase in fluorescence is due to
Trp
. Oxidized Trp
mutant proteins have a very
low tryptophan fluorescence emission from the remaining
Trp
, but reduction results in a large (up to 11-fold)
increase of fluorescence intensity (Krause and Holmgren, 1991). The
Trp
aromatic ring is in close proximity to the disulfide
in wt Trx-S
(Katti et al., 1990; Jeng et al., 1994), and changes in NMR chemical shifts upon reduction indicate
that its position differs slightly in Trx-(SH)
(Dyson et al., 1989). Characterization of mutant thioredoxin with
Trp
replaced by a nonaromatic residue can be used to
determine the role of this residue in the changes of fluorescence
emission intensity, as well as to elucidate any involvement in the
oxidoreduction mechanism of thioredoxin. Also, since Trp
is a conserved residue in prokaryotes, it might be expected to be
of structural importance. In this study, we have chosen to examine the
mutant where Trp
is substituted by the smallest
hydrophobic residue, alanine. While other substitutions, such as the
insertion of serine, would be of some interest, since the mammalian
thioredoxins have serine at this position, we considered that the
disruption of structure caused by the insertion of a hydrophilic side
chain deep into the hydrophobic pocket behind the active site might
have a number of effects that could not be analyzed with any certainty.
The W28A mutation is the simplest change, with fewest competing
effects.
The present study also examines the role of Trp in bacteriophage systems. The reduced form of E. coli thioredoxin is essential for an assembly of filamentous phages f1
and M13 (Russel and Model, 1985) and for phage T7 DNA replication (Mark
and Richardson, 1976). T7 DNA polymerase consists of the T7
phage-encoded gene 5 protein (g5p) and reduced E. coli thioredoxin. Isolated g5p has a high single-stranded exonuclease
activity, but only a very low 5`-3` polymerase and 3`-5`
double-stranded exonuclease activities. Addition of Trx-(SH)
to g5p in vitro reconstitutes highly processive DNA
polymerase and double-stranded exonuclease activities. To date, little
is known about how thioredoxin acts to give T7 DNA polymerase high
processivity. The role of Trx in T7 DNA polymerase is apparently
different from its well known redox functions and is probably linked to
a structural interaction which stabilizes the binding of g5p to a
primer-template (Tabor et al., 1987; Slaby and Holmgren,
1989). Site-directed mutagenesis of E. coli thioredoxin with
subsequent DNA polymerase assay has been successfully used for probing
the participation of specific amino acid residues in the binding
interaction with the gene 5 protein (Huber et al., 1986;
Krause and Holmgren, 1991; Krause et al., 1991).
The mutagenesis host strain E. coli CU9276 (hsdR17, mcrAB, recA1, (lac-proAB), (F`traD36,
proAB
, lacI
Z
M15) was supplied by Dr.
E. Holmgren, Pharmacia BioScience Center, Stockholm. E. coli strain JF521 (
(lac-pro-AB),thi, supE, metE46, srl300::
Tn10trx A2(7004), recA, (F`traD36, proAB+,
lacI
Z
M15)) and plasmids pUC118 and pUC118-trxA were generous gifts from Dr. J. Fuchs, Department of Biochemistry,
Gortner Laboratory, St. Paul, MN.
M13-vector DNA,
-
S-dATP, and other molecular biology materials were
purchased from Boehringer Mannheim, Pharmacia Biotech Inc., and
Amersham Corp. DNA sequencing was done by the dideoxy method using T7
DNA polymerase kit and equipment from Pharmacia Biotech Inc.
Most of the DNA work was done according to standard procedures (Sambrook et al., 1989). Screening for mutants was performed directly by sequencing M13 DNA purified by the method of Vieira and Messing(1987).
Insulin was used to determine the protein
disulfide reductase activity of thioredoxin coupled to NADPH oxidation
via thioredoxin reductase (Holmgren, 1979). The reactions, containing
0.5-1.5 µM thioredoxin, 0.4 mM NADPH, 160
µM insulin, 2 mM EDTA in 0.1 M phosphate
buffer, pH 7.0, were started by addition of 200 nM thioredoxin
reductase. The rate of NADPH oxidation was calculated from the decrease
in absorbance at 340 nm, using a molar extinction coefficient for NADPH
of 6,200 Mcm
.
To determine the amino acid composition, protein samples were lyophilized and hydrolyzed with 6 M HCl, 0.5% phenol for 24 h at 110 °C in vacuo and analysis was performed with Beckman 121M amino acid analyzer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was run on 8-12% gradient gels using the Phast System from Pharmacia.
The host E. coli strain JF521
lacking the chromosomal wild-type trxA gene was used for
expression of the pUC118-trxA-encoded W28A thioredoxin. The
expression was inducible by
isopropyl--D-thiogalactopyranoside, since pUC118 contains
the lac promoter just in front of the trxA gene. The
insertion of the fragment into the pUC118 polylinker region introduces
a TAA stop codon between lacZ and trxA, thus
excluding a protein fusion (Krause and Holmgren, 1991).
Figure 1:
Portion of a 500 MHz H 2Q
NMR spectrum showing cross-peaks between the NH and C
H
protons of the reduced form of the W28A mutant of thioredoxin. Positive
and negative contour levels are plotted without distinction. Selected
resonance assignments are shown.
Figure 2:
Stimulation of DNA polymerase activity of
T7 gene 5 protein by thioredoxin. Reaction mixtures contained 3 nM g5p and increasing concentrations of wt Trx-(SH) (
) or W28A Trx-(SH)
(
).
Figure 3:
Tryptophan fluorescence spectra of wt Trx,
W28A Trx, and W31A Trx. All spectra were recorded with 1 µM concentrations of the proteins at pH 7.0. The reduction was
accomplished with 1 µM DTT. , wt Trx-S
;
, wt Trx-(SH)
;
, W28A Trx-S
;
, W28A Trx-(SH)
;
, W31A Trx-S
;
, W31A Trx-(SH)
.
Figure 4:
A and B, tryptophan fluorescence
spectra of wt Trx (A) and W28A Trx (B) in guanidine
hydrochloride. , native Trx-S
;
, native
Trx-(SH)
;
, Trx-S
in 1 M GdnHCl;
, Trx-(SH)
in 1 M GdnHCl;
, Trx-S
in 5 M GdnHCl;
,
Trx-(SH)
in 5 M GdnHCl. C, denaturation
of thioredoxins by GdnHCl: variation in tryptophan fluorescence
emission at 350 nm with increasing concentrations of GdnHCl for wt
Trx-S
(
), wt Trx-(SH)
(
), W28A
Trx-S
(
), and W28A Trx-(SH)
(
).
The
ellipticity at 222 nm as a function of GdnHCl concentration is shown in Fig. 5for wt and W28A thioredoxin. For both proteins, the
oxidized form is significantly more stable than the reduced form to
GdnHCl denaturation, consistent with the greater thermal stability of
Trx-S (see below). Interestingly, the concentration of
GdnHCl at which half of the protein is denatured differs by exactly the
same amount between the two forms of W28A Trx compared with wt,
indicating that changing Trp
does not affect the
relationship between the structures of the two oxidation states. This
observation is entirely consistent with the results of the enzymatic
assays, which indicate no significant difference in activity for the
mutant.
Figure 5:
Guanidine HCl unfolding of wt and W28A
thioredoxin measured by circular dichroism at pH 7.0. The fraction
denatured was determined from the percent ellipticity change relative
to the ellipticity difference between fully native (without GdnHCl) and
fully denatured (5 M GdnHCl) thioredoxin at 222 nm. , wt
Trx-S
;
, wt Trx-(SH)
;
, W28A
Trx-S
;
, W28A
Trx-(SH)
.
However, it is clear from Fig. 5that the stability
of W28A in either oxidation state is significantly decreased relative
to the wild-type protein. The GdnHCl concentration for half of the
protein to be denatured is decreased from 2.38 M to 1.73 M for Trx-S and from 1.60 M to 1.00 M for Trx-(SH)
. A similar plot (not shown) for the
fluorescence intensity results shown in Fig. 3is more complex
due to the complexity of the fluorescence changes, including changes in
emission wavelength, upon reduction of either wt or W28A mutant Trx,
but still shows the same overall behavior. These results indicate that
although the protein is correctly folded under native conditions, as
indicated by the similarity of the mutant and wt NMR spectra (Fig. 1), the stability of the W28A mutant protein has been
significantly decreased by the change of tryptophan for alanine at
position 28.
The thermal stability of the oxidized and reduced forms
of the mutant and wild-type thioredoxins was estimated by following the
ellipticity at 222 nm as a function of temperature (data not shown).
The transition temperatures of the mutant proteins are significantly
lowered from those of the wild-type proteins (80 °C compared with
88 °C for Trx-S and 70 °C compared with 80 °C
for Trx-(SH)
). Once again, the relative difference between
the two oxidation states has been preserved, consistent with the
functional similarity of the mutant to the wild-type and with the
behavior of the proteins toward denaturation in GdnHCl.
The two tryptophan residues are highly conserved in
thioredoxins (Eklund et al., 1991). Trp is
conserved throughout prokaryotic and eukaryotic proteins and its
replacement with alanine causes significant changes in function (Krause
and Holmgren, 1991). Consistent with this, Trp
is situated
in an unusual position on the surface of the molecule (Katti et
al., 1990; Jeng et al., 1994). By contrast, Trp
is conserved only in prokaryotic thioredoxins (Eklund et al., 1991) and is largely buried in the hydrophobic core of the
molecule (Jeng et al., 1994). The residue that replaces
Trp
in eukaryotes is serine, which is not even remotely
homologous to tryptophan; this argues that tryptophan at this position
is probably not necessary for the function of thioredoxin. Its
conservation among such a large and varied group as the prokaryotes
argues that it is necessary for some other reason, possibly for
stabilizing local structure in the vicinity of the active site cysteine
residues.
We have shown that the replacement of Trp with alanine results in no significant change in the functional
properties of thioredoxin. The NMR, fluorescence, and CD spectra of the
mutant protein under native conditions all indicate that the protein is
correctly folded, consistent with its correct function both in assays
that require reduction or oxidation of the cysteines and in assays such
as the T7 DNA polymerase reaction that measure tightness and
specificity of binding. However, both fluorescence and CD measurements
as a function of added guanidine hydrochloride and temperature clearly
show that the stability of the W28A protein is significantly decreased
relative to wild type. Furthermore, the effects of the mutation on the
stability of the protein are the same for both Trx-S
and
Trx-(SH)
, consistent with the integrity of function in this
mutant. It is perhaps somewhat surprising that such a significant
mutation as the replacement of a tryptophan by an alanine in close
proximity to the active site should have as little effect on the
function of the protein as it does in this case. These results are an
indication of the robust nature of thioredoxin and of its stability of
function toward a wide variety of mutational challenges.