| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 275, Issue 30, 22743-22749, July 28, 2000
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the National Laboratory of Biomacromolecules, Institute of
Biophysics, Academia Sinica, Beijing 100101, China
Received for publication, March 22, 2000, and in revised form, April 30, 2000
Limited proteolysis of DsbC with trypsin resulted
in a compact and stable C-terminal fragment (residues 66-216), fDsbC,
which retains the active site sequence,
-Cys98-Gly-Tyr-Cys101-, and shows only
minor differences in conformation compared with that of the intact
molecule. The pKa of active site thiol and the
KSS with glutathione are very close to that of DsbC, respectively; however, fDsbC is inactive as an isomerase in
catalyzing the formation of correct disulfide bonds in scrambled RNase
A and denatured and reduced bovine pancreatic trypsin inhibitor and
shows only 13% thiol-protein oxidoreductase activity (TPOR) of DsbC.
In contrast to the dimeric DsbC, fDsbC exists as a monomer and has no
chaperone activity in assisting the reactivation of denatured
D-glyceraldehyde-3-phosphate dehydrogenase. The heterodimer of DsbC with the inactive DsbC carboxymethylated at both active site
thiols shows about 50% TPOR activity of DsbC but no isomerase activity, indicating that the DsbC subunit in the heterodimer displays
full TPOR activity but little, if any, isomerase activity. It is
concluded that the N-terminal sequence (residues 1-65) is essential
for dimer formation and chaperone activity of DsbC. The active sites in
both subunits of the dimeric DsbC appear to be essential for its
isomerase activity.
The Dsb protein family in bacterial periplasm has been recently
characterized to be responsible for the formation of native disulfide
bonds of newly synthesized polypeptides (1). At least six proteins,
DsbA, DsbB, DsbC, DsbD, DsbE, and DsbG, have been identified, and they
work in conjunction with one another (2). DsbC is a soluble protein
catalyzing the rearrangement of disulfide bonds (3) and therefore
considered as the prokaryotic counterpart of the eukaryotic
protein-disulfide isomerase
(PDI)1 (3, 4). PDI has
recently been shown to have not only enzyme activities but also
chaperone activity (5-7). Compared with PDI, DsbC shows lower
isomerase activity but even more marked chaperone activity (8).
As a member of the thioredoxin superfamily, DsbC shares 24% sequence
identity with DsbG (9) but no apparent sequence homology with other
members of the Dsb family or with other members of the thioredoxin
superfamily except for the motif of
Phe-X-X-X-X-Cys-X-X-Cys in the active site (10). DsbC is a homodimer (3), and each of its
23.4-kDa subunits is composed of 216 amino acid residues with four
cysteines, two in the active site sequence of
-Cys98-Gly-Tyr-Cys101- and the other two at
positions 141 and 163, believed to form a disulfide bond with a
structural function (3). It was predicted that the molecule appeared to
consist of C- and N-terminal domains of similar length, with the
C-terminal half recognized as another variation on the thioredoxin
theme (10). The crystal structure of oxidized DsbC shows that the
molecule has a V-shape with each arm for one subunit, and each subunit
consists of a C-terminal thioredoxin-like domain connected by a hinged
linker helix to an N-terminal domain responsible for dimerization (9).
Limited proteolysis has been widely used in protein studies for the
exploration of surface regions, ligand-induced conformational changes,
domain boundaries, and protein unfolding/refolding (11). Based on the
profile of limited digestion by trypsin and V8 protease, the
architecture of PDI appears to be consistent with a model of four
consecutive domains, each with a thioredoxin fold (12).
In this paper, we report the properties of a C-terminal fragment
(residues 66-216) of DsbC generated by limited trypsin digestion. This
fragment, fDsbC, is monomeric in contrast to the dimeric intact
molecule and has a compact structure. Although containing all four
cysteine residues with very similar chemical properties to those of
DsbC, it shows low thiol-protein oxidoreductase (TPOR) activity and
neither isomerase nor chaperone activity. Hybrid experiments suggest
that the dimeric DsbC requires both of its active sites to be intact
for its isomerase activity.
Materials--
8-Anilino-1-naphthalenesulfonic acid (ANS),
bovine serum albumin (98-99% albumin; fraction V),
glyceraldehyde-3-phosphate, guanidine hydrochloride (GdnHCl),
glutathione reductase (yeast, type III), HEPES, imidazole,
NAD+ (97%), bovine pancreatic trypsin inhibitor (BPTI),
trypsin (tosylphenylalanyl chloromethyl ketone-treated), yeast RNA, and
fluorescein isothiocyanate were from Sigma. GSH and NADPH (98%) were
from Roche Molecular Biochemicals. Dithiothreitol (DTT) and molecular
mass markers from Amersham Pharmacia Biotech. Isopropyl
1-thio- Preparation--
The expression plasmid pDsbC containing the
full-length DsbC precursor gene is a generous gift from Dr. Rudi
Glockshuber (Eidgenössische Technische Hochschule,
Hönggerberg, Switzerland). DsbC was prepared according to
Missiakas et al. (13) and Chen et al. (8).
D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from
rabbit muscle (14) was kindly provided by J. Li and N. X. Zhang of
this laboratory. Scrambled RNase A (sRNase) was prepared essentially
according to Hillson et al. (15).
S-Carboxymethylated RNase A (mRNase) was prepared by
overnight incubation of RNase A at 1.8 mM with 130 mM DTT and 6 M GdnHCl in 0.4 M
Tris-HCl buffer (pH 8.0) at room temperature and then modification with
a 10-fold excess of iodoacetic acid in 1.5 M Tris-HCl with
6 M GdnHCl (pH 9) at room temperature for 1 h. The reaction mixture was adjusted to pH 4-4.5 with glacial acetic acid,
dialyzed thoroughly against 0.02 M acetic acid at 4 °C, and lyophilized.
fDsbC was prepared by limited digestion of DsbC with trypsin. DsbC at 1 mg/ml was incubated with trypsin at 0.01 mg/ml in 0.1 M
ammonium bicarbonate buffer (pH 8.1) at 37 °C for 2-3 h. The
reaction mixture was then loaded on a Bio-Scale Q2 column (Bio-Rad) equilibrated with 50 mM Tris-HCl buffer (pH 8.0)
and eluted with a linear gradient of 0-0.2 M NaCl in 20 ml
of the same buffer. The main peak was collected, dialyzed thoroughly against 50 mM NH4HCO3 at 4 °C,
and lyophilized. The condition for trypsin digestion was optimized by
examination of the reaction products on 15% SDS-polyacrylamide gel
electrophoresis (PAGE).
DsbC and fDsbC carboxymethylated at the N-terminal thiol and at both
thiols (mmDsbC and mmfDsbC) in their active site sequence -Cys98-Gly-Tyr-Cys101-, respectively, were
prepared according to Zapun et al. (3) and were determined
to be all devoid of enzyme activity.
Determinations--
Concentrations of proteins were determined
spectrophotometrically at 280 nm with the following absorption
coefficients (A1 cm0.1%): 0.66 for bovine serum albumin, 0.98 for GAPDH (16), and 0.7 for DsbC (3).
The A1 cm0.1% value of fDsbC
was determined to be 0.87 by protein concentration determinations by
the Bradford method (17) using bovine serum albumin as a standard. For
the convenience of comparison, homotetrameric GAPDH and homodimeric
DsbC were considered as protomers in the calculation of molar ratios.
Thiol groups were determined with 5,5-dithiobis (2-nitrobenzoic acid)
in the presence of 6.4 M GdnHCl (18).
The N-terminal amino acid sequence of fDsbC was determined on a
MilliGen/Biosearch model 6600 ProSequencer linked to a Waters high
pressure liquid chromatography system with simultaneous detection at
269 and 313 nm. The mass of fDsbC was determined by electrospray ionization mass spectrometry on a VG Platform spectrometer. The sizes
of native fDsbC and DsbC were estimated by gel filtration on a Superose
12 HR 10/30 column (Amersham Pharmacia Biotech) calibrated with GAPDH,
bovine serum albumin, ovalbumin, chymotrypsinogen, and cytochrome
c as mass markers. Hybridization and nondenaturing PAGE on a
6% gel were performed exactly as described by Zapun et al.
(3) to identify the oligomeric state of DsbC and fDsbC. The ratios of
the protein bands on the gel were scanned by a Bio-Rad DS-670
densitometer. Protein was recovered from the gel by slicing out the
band and cutting into small pieces before soaking in 50 mM
sodium phosphate buffer (pH 7.5) overnight, desalting of the supernatant, and lyophilization.
CD spectra from 200 to 250 nm were determined with a Jasco 500 spectropolarimeter at 25 °C. Fluorescence spectra were measured at
25 °C in a Hitachi F-4010 spectrofluorimeter with excitation wavelengths of 280 and 295 nm for the intrinsic and 373 nm for the ANS
spectra. The sample buffer was 0.1 M potassium phosphate with 2.5 mM EDTA (pH 7.5), and 1 mM DTT was
also used for reduced proteins.
pKa values of the nucleophilic cysteine
(Cys98) were determined by following the increase of
thiolate-specific absorbance at 240 nm with the change of pH at
30 °C in buffer A (10 mM KH2PO4, 10 mM K2HPO4, 10 mM
sodium citrate, 10 mM Tris, 200 mM KCl, and 1 mM EDTA, pH 7.7). DsbC and fDsbC (~200 µM)
were first reduced with 10 mM DTT in 0.1 M
Tris-HCl (pH 8.0) for 10 min at 25 °C, and the excess of DTT was
removed using a Superose 12 HR 10/30 column equilibrated with buffer A. The oxidized proteins treated in the same way without DTT were used as
references. The pH values of 2-ml protein solutions at ~25
µM were measured by an Orion pH meter with stepwise
addition of 200 mM HCl in portions of 25 µl, and the
absorbance values at 280 and 240 nm were recorded on a UV-1601
UV-visible spectrophotometer (SHIMADZU). Since the absorbance of DsbC
at 280 nm was determined to be pH-independent (data not shown),
(A240 red/A280 red)/(A240 oxi/A280 oxi) was used as a measure of the fraction of the Cys98 thiolate
in DsbC and fDsbC. The data were fitted according to the
Henderson-Hasselbach equation (19).
The equilibrium constants for disulfide bond formation in the active
site of fDsbC and DsbC, with glutathione as thiol-disulfide reagent,
were determined according to a method well established by Zapun
et al. (3). The samples were analyzed by reverse phase FPLC
on a PepRPCTM HR 5/5 column (Amersham Pharmacia Biotech) at
room temperature with a gradient of 30-38% (v/v) acetonitrile in 20 ml of 0.1% trifluoroacetic acid.
Activity Assay--
Activities of disulfide isomerase based on
the isomerization of sRNase to the native enzyme and TPOR using the
reduction of insulin with a linkage to glutathione reductase were
assayed according to Lambert and Freedman (20), protein-disulfide
oxidoreductase (PDOR) was determined based on the increases of
fluorescence emission at 519 nm upon the reduction of intercatenary
disulfide bonds of difluoresceinthiocarbamyl-insulin (21). The effects
of DsbC and fDsbC on the disulfide-coupled refolding reaction of fully reduced BPTI were performed basically according to Zapun and Creighton (22). The refolding reaction mixture was incubated at 25 °C for 20 min before the addition of reduced BPTI to initiate the reaction in
order to ensure the redox equilibrium in the active site of DsbC and
fDsbC under the used concentration of GSSG/GSH. The acid-trapped
samples were analyzed by FPLC on a PepRPCTM HR 5/5 reverse
phase column at room temperature with a gradient of 9-36% (v/v)
acetonitrile in 50 ml of 0.1% trifluoroacetic acid.
Denaturation and Renaturation of GAPDH--
Denaturation with
GdnHCl and assisted reactivation of GAPDH upon dilution in the presence
of DsbC and fDsbC were carried out according to Chen et al.
(8). Aggregation during refolding of denatured GAPDH was followed
continuously at 25 °C by 90° light scattering at 488 nm in a
Hitachi F-4010 spectrofluorimeter.
Limited Digestion of DsbC by Trypsin--
As shown in Fig.
1, by digestion of 1 mg/ml DsbC at
37 °C with trypsin at a weight ratio of 0.01, the DsbC band
disappeared completely after 100 min, and after 3 h a fragment of
17 kDa was the only digestion product detectable on the gel. This
component was purified through one-step anion exchange chromatography
and named as fDsbC.
Identification of fDsbC--
The first three amino acid residues
at the N terminus of fDsbC have been determined to be Met-Leu-Leu,
indicating that fDsbC results from the cleavage of DsbC between
Lys65 and Met66 by trypsin (Scheme
I). The mass of fDsbC determined by mass
spectrometry is 16,683 Da, in agreement with the calculated value of
16,687 Da for the fragment of 66-216.
Oligomeric State--
Upon size exclusion chromatography, DsbC was
eluted at the position corresponding to an apparent mass of 58 kDa,
similar to the 67 kDa reported by Zapun et al. (3), and
fDsbC was eluted at 22 kDa, suggesting that the former exists as a
dimer and the latter as a monomer. It should be noted that both of the
apparent mass values are higher than the expected values of 46.9 and
16.7 kDa, respectively and the reduced forms of DsbC and fDsbC, mmDsbC and mmfDsbC, showed exactly the same elution position as those of the
respective oxidized forms, indicating that redox state and thiol
modification do not affect the association properties. Thus, mmDsbC is
a dimer, and mmfDsbC is a monomer.
As shown in Fig. 2, the nondenaturing
PAGE of the hybrid mixtures of DsbC/mmDsbC at all ratios contain three
bands (Fig. 2, lanes 2-6) for the native
protein, the heterodimer, and the modified protein, while the mixture
of fDsbC/mmfDsbC shows only two bands with no heterodimer between the
two homodimers (lane 9). In addition, no band of
heterodimeric hybrid appeared for DsbC/mmfDsbC (lane 11) and DsbC/fDsbC (lane 12). The
above finding indicates the dimeric state of DsbC and the monomeric
state of fDsbC and mmfDsbC, in agreement with the results of gel
filtration.
Cysteine Thiol Groups of fDsbC--
fDsbC freshly prepared, like
DsbC, is in the oxidized form, since no thiol group was detected by
Ellman's assay when it was denatured with 6.4 M
GdnHCl.
DsbC and fDsbC, carboxymethylated at the N-terminal thiol in the active
site sequence -Cys98-Gly-Tyr-Cys101-, show 0.8 and 0.9 thiol groups, respectively, after denaturation, suggesting that
in fDsbC, like in DsbC (8), only the N-terminal thiol group
(Cys98) of the active site was alkylated in the native
conformation, so that only Cys101 can be detected after
denaturation. The other two non-active site thiol groups form a
disulfide bond.
There is no significant change in reactivity of Cys98 in
DsbC caused by removing the N-terminal segment (residues 1-65) as
shown by the pKa values, 4.3 ± 0.2 (n = 3) for fDsbC as compared with 4.1 ± 0.3 (n = 6) for DsbC (Fig.
3). As shown in Fig.
4, the equilibrium constant
KSS for the formation of the accessible disulfide
bond of fDsbC with glutathione is 205 µM, which is close
to the KSS of 273 µM of DsbC (3),
indicating that the disulfide bond of fDsbC at the active site is
nearly as unstable as that of DsbC.
Fluorescence and CD Spectra--
DsbC contains one Trp at position
140 and eight Tyr residues, whereas fDsbC keeps Trp140 but
has only six Tyr residues. The intrinsic fluorescence spectra of fDsbC
with excitation wavelengths at either 280 or 295 nm are similar to that
of the intact molecule in terms of the emission maximum but with
lowered emission intensity (Fig. 5,
A and B). The ANS fluorescence spectra are nearly
the same for DsbC and fDsbC (Fig. 5C). The CD spectrum of
fDsbC is also similar to that of DsbC (Fig. 5D).
For oxidized and reduced fDsbC, like DsbC (3) but unlike DsbA (23),
there is no difference between the intrinsic fluorescence spectra with
excitation wavelength at either 280 or 295 nm, ANS fluorescence
spectra, and CD spectra (data not shown), indicating that the oxidized
and reduced fDsbC have essentially the same fold.
Carboxymethylation at the N-terminal thiol in the active site sequence
-Cys98-Gly-Tyr-Cys101- of DsbC and fDsbC shows
little effect on its intrinsic fluorescence spectra with excitation at
either 280 or 295 nm and ANS fluorescence spectra (data not shown).
However, mmDsbC and mmfDsbC show a 3-nm blue-shifted emission maximum
in their intrinsic fluorescence spectra with excitation at 280 or 295 nm (Fig. 5, A and B). The ANS fluorescence
spectra for mmDsbC and mmfDsbC show a more marked, 15-nm, blue shift in
emission and markedly higher intensity (Fig. 5C). The above
suggest that the alkylation of both thiols in the active site perturbs
the structure of DsbC and fDsbC more markedly than the modification at
the protruding thiol group (Cys98) alone.
Enzymatic Activities--
Like DsbC, fDsbC catalyzes the reduction
of insulin in its TPOR activity but with a considerably lower activity,
13.5 ± 1.4% of that of DsbC (n = 3). As shown in
the Table I for the kinetic constants of
insulin reduction catalyzed by fDsbC and DsbC, the Km for fDsbC is 2.58-fold that for DsbC, and the
kcat of fDsbC is 12.4% that of DsbC. Thus the
factor of kcat/Km of fDsbC is
only 4.8% that of DsbC. However, fDsbC shows no isomerase activity
using sRNase as a substrate compared with DsbC with an isomerase
activity of 590 units/g. The kinetics of disappearance of the fully
reduced unfolded BPTI and the appearance of the native three
disulfide-bonded BPTI catalyzed by DsbC and fDsbC was compared in order
to provide more information about the enzyme activity of fDsbC (Fig.
6). In the absence of any catalyst, the
half-time for the disappearance of the reduced BPTI, about 4.2 min, was decreased to 3.2 min by fDsbC and further to 2 min by DsbC. This indicated that fDsbC catalyzes the oxidation of the thiols in reduced
BPTI like DsbC but at a lower rate. However, in contrast to DsbC, which
catalyzed 50% of BPTI to be reactivated in 13 min, fDsbC had
little effect on the formation of native BPTI just like the case
without any catalyst. The above finding indicates that fDsbC showed no
isomerase activity as well with reduced BPTI as a substrate.
Enzymatic Activities of Hybrid Mixtures of DsbC/mmDsbC--
Table
II shows the relative intensity of the
three bands for the native protein, the heterodimer, and the inactive
modified protein, the TPOR activity, and the isomerase activity of the hybrid mixtures with different ratios of DsbC/mmDsbC in Fig. 2. The
native species, DsbC, is fully active, and the modified mmDsbC is
inactive. It is noted that the experimentally determined TPOR activities of hybrid mixtures agree well with the sums of the relative
intensity of the native and the half-intensity of the heterodimer
bands. The isomerase activities experimentally determined are very
close to the corresponding values of the relative band intensity of the
native species. The above finding suggests that the heterodimer of
DsbC/mmDsbC shows little (if any) isomerase activity but almost 50%
TPOR activity of DsbC; i.e. the DsbC subunit in the
heterodimer displays full TPOR activity but little (if any) isomerase
activity. The isomerase activities of DsbC have been determined in the
absence and presence of the same concentration of mmDsbC and found to
be identical (530 units/g). This eliminated the possibility of
unproductive binding of the substrate by mmDsbC that might contribute
to the large decrease of the isomerase activity of the hybrid mixtures
of DsbC and mmDsbC.
Effects of fDsbC on Reactivation and Aggregation of Denatured
GAPDH--
Reactivation of denatured GAPDH was used to examine the
chaperone activity of fDsbC independent to its thiol-protein
oxidoreductase activity (5). As shown in Fig.
7A, reactivation of GAPDH in the presence of DsbC increases from 6 to 30% as the molar ratio of
DsbC to GAPDH increases to 25. However, fDsbC at the same range of
molar ratios shows no effect on the reactivation of the denatured GAPDH. In the presence of a 20-fold molar excess of DsbC, the aggregation of denatured GAPDH during refolding decreases significantly in both rate and extent, but fDsbC at the same concentration has no
effect (Fig. 7B).
Effects of Nonnative RNase A on PDOR Activities of DsbC and
fDsbC--
The PDOR assay recently developed using a fluorescent
derivative of insulin as the substrate (21) was employed for measuring the effects of peptides on the reductase activity of fDsbC for its
higher sensitivity than the conventional TPOR assay. As shown in Fig.
8A, sRNase, as a big misfolded
peptide and a good substrate for DsbC, inhibits the PDOR activity of
both DsbC and fDsbC. However, mRNase at higher concentrations inhibits
the PDOR activity of DsbC but has no effect on the activity of fDsbC at
the same concentrations (Fig. 8B).
Compact Structure of fDsbC--
Although there are 21 potential
sites for trypsin cleavage in DsbC, as shown in Scheme I, the protein
is only cleaved at a limited number of sites under the employed
conditions with the site between Lys65 and
Met66, one of the most liable to be cleaved. All of the 16 sites within the fDsbC fragment seem to be unaffected during the first
3 h of digestion at 37 °C even with 1:10 (weight ratio) trypsin
or prolonged digestion time of 20 h with 1:100 trypsin at
37 °C.
The resistance toward trypsin proteolysis of the C-terminal fragment
suggests a compact structure of this part of the DsbC molecule. The
peptide segment around the nick site between Lys65 and
Met66 appears to be a flexible and exposed loop or a mobile
linker region between domains (11). The nick site is indeed located within the movable hinged linker
The N-terminal domain, consisting of six-stranded anti-parallel
fDsbC Is Monomeric in Contrast to the Dimeric DsbC--
By size
exclusion chromatography and nondenaturing electrophoresis of hybrid
mixtures, it is concluded that fDsbC is monomeric. The fact that fDsbC
retains the general conformation as in the intact molecule led us to
suggest that the N-terminal fragment of 65 residues is responsible for
the dimerization of DsbC subunits, and this is now confirmed by the
crystal structure (9). The two subunits of DsbC form a V-shaped
molecule with each arm consisting of two separate domains connected by
a hinged linker
In nondenaturing PAGE of a hybrid mixture of DsbC/mmDsbC at a ratio of
1:1, the relative proportion of the native protein, the heterodimer,
and the inactive modified protein is not 1:2:1 as would be expected
from random association of the subunits but actually approximately
1:1:1. It is interesting to find that a rerun of nondenaturing PAGE of
the recovered heterodimeric protein of DsbC/mmDsbC from the gel shows
again three bands with a ratio of approximately 1:1:1 (data not shown).
The above result indicates a lower affinity to form heterodimers
compared with that of homodimers and an equilibrium between the
heterodimer and the homodimers. According to the crystal structure, the
dimerization is essentially via the hydrogen bonds between the
Enzyme and Chaperone Activities--
fDsbC, as a monomer, shows no
isomerase activity in catalyzing the formation of native molecules
either from sRNase or from reduced BPTI but retains 13.5% TPOR
activity and part of the oxidase activity of homodimeric DsbC.
It is suggested that the combination of the two basic structural
features, i.e. a large uncharged surface (40 × 40 × 25 Å) consisting of the cleft of the V for substrate binding and a
thiolate active site, account for its isomerase activity. The broad
uncharged cleft in DsbC molecule is sufficient to allow the binding of
target protein and may be involved in both the chaperone and isomerase activities (9). Moreover, the hinged linker helix may provide sufficient flexibility to allow the binding of different sized substrates (9).
Since fDsbC has an intact active site with unchanged chemical
properties (the protruded Cys98, pKa,
and redox constant KSS) and a slightly perturbed
molecular conformation, the absence of the large uncharged surface
within the V-shaped dimeric molecule resulting from the removal of the
N-terminal dimerization domains very likely leads to the absence of
isomerase activity. In this connection, the Km of
fDsbC for insulin is 2.58-fold higher than that of DsbC, indicating a
poorer substrate binding with fDsbC than with DsbC. The fact that the
DsbC monomer in the heterodimer of DsbC/mmDsbC shows no isomerase
activity but full TPOR activity of DsbC suggests that both of the
active sites of dimeric DsbC may be necessary for its isomerase
activity and that the monomeric state is sufficient for its TPOR
activity. Our present data provide an experimental demonstration of the
speculation by Zapun et al. (3) that in the catalyzed
isomerization of mispaired disulfides of a scrambled protein with no
free thiols, the simultaneous break of the another disulfide may be
needed, so that a different disulfide can be formed via disulfide
interchange. Similar to PDI, the two active sites with one in each
domain of DsbC are obviously favorable and probably necessary to
catalyze the isomerization of scrambled substrate. The catalyzed
reduction of a disulfide only needs one active site of the enzyme to
function. In this respect, it is speculated that in the recycling of
DsbC in cells the reduction of the disulfide in each domain of DsbC by
DsbD may be independent. In the determination of reaction kinetics of
DsbC with glutathione, Zapun et al. (3) proposed that the
two active sites of DsbC function independently, since no indication of
interactions between the two monomers of the DsbC dimer was found.
Meanwhile, the sufficient area for substrate binding is also necessary
for TPOR and oxidase activities, since fDsbC lacking the N-terminal
domain has much lower activities. The possibility is not excluded that
the conformational change in modified subunit could be responsible for
the activities of the heterodimer. It is also to be recalled that in
contrast to the dimeric DsbC and PDI with substantial isomerase
activity, thioredoxin is monomeric and shows little isomerase activity
(24). DsbA is also monomeric but has an additional helical insert,
which, together with the thioredoxin domain, provides a much more
extended hydrophobic surface than that of thioredoxin for substrate
binding, and therefore DsbA shows low isomerase activity of about 5%
of that PDI (25) and 20% of DsbC (3). In this connection, it is known
that the active sites of some enzymes are shared between different
subunits (26, 27). For DsbA and the monomeric domain a of PDI with 14%
isomerase activity (28) of PDI, their small size may allow two enzyme
molecules to attack two disulfide bonds of a substrate simultaneously.
Since the chaperone and PDOR activities of DsbC can be inhibited by
sRNase and mRNase at high concentrations but not by small peptides, it
has been suggested that DsbC has an extended surface for peptide
binding so that only a relatively large unfolded peptide is able to
compete with the substrate or the target folding intermediate for
binding to DsbC (8). fDsbC is no longer active as a chaperone on the
reactivation and prevention of aggregation of denatured GAPDH, and its
PDOR activity is not inhibited by mRNase, suggesting that fDsbC has
lost the ability for large peptide binding and consequently the
activity as a chaperone. This has now also been supported by the
three-dimensional structure (9). In contrast to mRNase, sRNase
decreases PDOR activity of DsbC and fDsbC at very low concentrations,
since DTT present in the determination of PDOR activity may reduce the
disulfide of sRNase and lead to disulfide exchange with DsbC and fDsbC,
and results in the inhibition of the PDOR activity. Many chaperones
have ring structures to form a peptide binding site at the end of the
ring contributed by each subunit (29); e.g. GroEL has two
seven-member rings, the eukaryotic cytosolic chaperonin, CCT, and
archaebacteria thermosome have two eight-member rings, and
archaebacteria TF55 has two rings of nine subunits. In contrast, in
other chaperones, like dimeric PDI (30) and DsbC, the multidomain
structure seems to contribute to form an extended site for substrate binding.
We sincerely thank Dr. Rudi Glockshuber for
the generous gift of the plasmid pDsbC, Dr. Peter Metcalf for sending
the proof for Ref. 9 prior to publication, and Dr. Xiaohong Qian for mass determination. We also gratefully acknowledge Professor C. L. Tsou for continuous encouragement for this work.
*
This work was supported by Chinese Ministry of Science and
Technology Grant G1999075608 and China Natural Science Foundation Grants 39870177 and 39990600.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.
Published, JBC Papers in Press, May 1, 2000, DOI 10.1074/jbc.M002406200
The abbreviations used are:
PDI, protein-disulfide isomerase;
fDsbC, C-terminal fragment (residues
66-216) of DsbC;
TPOR, thiol-protein oxidoreductase;
ANS, 8-anilino-1-naphthalenesulfonic acid;
GdnHCl, guanidine hydrochloride;
BPTI, bovine pancreatic trypsin inhibitor;
DTT, dithiothreitol;
GAPDH, D-glyceraldehyde-3-phosphate dehydrogenase;
sRNase, RNase A
with scrambled disulfide bonds;
mRNase, S-carboxymethylated
RNase A;
PAGE, polyacrylamide gel electrophoresis;
mmDsbC and mmfDsbC, DsbC and fDsbC, respectively, carboxymethylated at both thiols in their
active sites;
PDOR, protein-disulfide oxidoreductase;
FPLC, fast
protein liquid chromatography.
The N-terminal Sequence (Residues 1-65) Is Essential for
Dimerization, Activities, and Peptide Binding of Escherichia
coli DsbC*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactoside was from Serva;
5,5-dithiobis(2-nitrobenzoic acid) was from Fluka; and iodoacetic acid
was from Merck. All other chemicals were local products of analytical grade.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (31K):
[in a new window]
Fig. 1.
SDS-PAGE (15%) profile of the products of
DsbC by limited proteolysis with trypsin. Lane
1, DsbC. DsbC at 1 mg/ml was incubated with trypsin of 0.01 mg/ml at 37 °C for 50 min (lane 2), 100 min
(lane 3), 150 min (lane 4),
and 200 min (lane 5). Lane
6, purified fDsbC; lane 7, mass
markers.
View larger version (25K):
[in a new window]
Scheme 1.
Potential sites for trypsin attack in the
DsbC sequence. All Lys and Arg residues in the DsbC sequence (10)
are shown in boldface type. The active
98CGYC101 is
double-underlined, and Cys141 and
Cys163 are underlined. The only Trp residue
(Trp140) is dotted. The hinged linker of
residues 62-77 between the N- and C-terminal domains (9) is
framed. The arrow from the top
indicates the cleavage site of DsbC by trypsin to obtain the fDsbC
fragment. The N-terminal three amino acid residues (MLL) of fDsbC are
marked with asterisks. The arrows from the
bottom show the other detected cleavage sites of DsbC.
View larger version (13K):
[in a new window]
Fig. 2.
Nondenaturing PAGE (6%) of hybrid
mixtures. The experiments were carried out according to Zapun
et al. (3). Lane 1, mmDsbC;
lanes 2, 3, 4,
5, and 6, hybrids of DsbC/mmDsbC at ratios of 3:1, 2:1, 1:1,
1:2, and 1:3, respectively; lane 7, DsbC;
lane 8, mmfDsbC; lane 9,
hybrid of fDsbC/mmfDsbC at a ratio of 1:1; lane
10, fDsbC; lane 11, hybrid of
DsbC/mmfDsbC; lane 12, hybrid of
DsbC/fDsbC.
View larger version (17K):
[in a new window]
Fig. 3.
Determination of pKa
values of the active site thiol, Cys98, in DsbC and
fDsbC. The increase of the specific absorption of thiolate ion at
240 nm relative to the thiol group was used to determine the
pKa of Cys98 in DsbC ( 
) and fDsbC
(
) by pH titration. The experiments were carried out as described
under "Experimental Procedures." The oxidized proteins were used as
a reference.
(A240 red/A280 red)/(A240 oxi/A280 oxi)
was used as a measure of the fraction of the Cys98
thiolate. The pKa values were calculated by fitting
the data according to the Henderson-Hasselbach equation.
View larger version (30K):
[in a new window]
Fig. 4.
Measurement of the thiol-disulfide
equilibrium of the active site disulfide bond of fDsbC and DsbC with
glutathione. DsbC (A and B) and fDsbC
(C and D) at a monomer concentration of about 6 µM were incubated with various mixtures containing either
of two concentrations of GSSG and varying amounts of GSH in 0.1 M Tris-HCl (pH 7.5), 0.2 M KCl, and 1 mM EDTA at 25 °C for 20 min. The equilibrium reaction
mixtures were acid-quenched and analyzed by reverse phase FPLC.
A and C, relative proportions of reduced forms
( , ) and oxidized forms (
, 
) at varying concentrations of
GSSG and GSH. Filled and open symbols
were for mixtures that contained 2 and 1 mM GSSG,
respectively. B and D, the plot of reduced
form/oxidized form versus [GSH]2/[GSSG]
should be linear and gives the value of KSS. The
solid lines are the results of linear fits to the
data.
View larger version (39K):
[in a new window]
Fig. 5.
Conformational analysis. Intrinsic
fluorescence spectra with excitation at 280 nm (A) and at
295 nm (B) are shown. C, ANS fluorescence
spectra; D, CD spectra. The protein concentrations were 15 µM in A and B, 11.5 µM in C, 18 µM (fDsbC), and 12.8 µM (DsbC) in D. Curve 1,
DsbC; curve 2, fDsbC; curve
3, mmDsbC; curve 4, mmfDsbC.
Kinetic parameters for the reduction of insulin catalyzed by DsbC
and fDsbC
View larger version (25K):
[in a new window]
Fig. 6.
Effects of fDsbC and DsbC on the refolding of
reduced BPTI. Reactions were initiated by the addition of fully
reduced BPTI to a final concentration of 10 µM into 0.1 M Tris buffer (pH 7.5) with 0.2 M KCl, 1 mM EDTA, 0.5 mM GSSG, and 2 mM GSH
in the absence ( 
, 
) or presence of 8 µM DsbC (,
) or 8 µM fDsbC (, ) at 25 °C. The aliquot
was withdrawn for acidification at various times as indicated. The
native (open symbols) and the reduced BPTI
(solid symbols) were separated and quantified by
reverse phase FPLC.
Relative intensity of protein bands in nondenaturing PAGE and the
isomerase and TPOR activities of the hybrid mixture of DsbC/mmDsbC
at different ratios
View larger version (19K):
[in a new window]
Fig. 7.
The effects of DsbC and fDsbC on the
reactivation (A) and aggregation (B)
during refolding of denatured GAPDH. The refolding was initiated
by a 50-fold dilution of GdnHCl-denatured GAPDH to 2.8 µM
in 0.1 M phosphate buffer (pH 7.5) containing 5 mM DTT, 2.5 mM EDTA, and DsbC ( ) or fDsbC
() at different molar ratios to GAPDH as indicated. The reactivation
mixture was first kept at 4 °C for 30 min and then for additional
3 h at 25 °C before an aliquot containing 2 µg of GAPDH was
taken for activity assay at 25 °C. Aggregation was followed by light
scattering at 488 nm after dilution of GAPDH to 2.8 µM in
the absence (dashed line) and presence of DsbC
(solid curve 1) or fDsbC
(solid curve 2) at a ratio to GAPDH of
20.
View larger version (19K):
[in a new window]
Fig. 8.
Effects of nonnative RNase A on the PDOR
activity of DsbC and fDsbC. The reaction mixture contained 0.078 µM difluoresceinthiocarbamyl-insulin, 0.2 mM
DTT, 2.5 mM EDTA, 0.51 µM DsbC ( ) or 3.2 µM fDsbC (), and different concentrations of sRNase
(A) or mRNase (B) as indicated. The reaction was
initiated by the addition of DTT and measured by following the emission
intensity at 519 nm with excitation at 495 nm. 1 unit of PDOR is the
amount of enzyme that transforms 1 µmol of
difluoresceinthiocarbamyl-insulin per min. The data with
error bars are expressed as mean ± S.D.
(n = 2).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix, which connects the
N-terminal domain (residues 1-61) and the C-terminal domain (residues
78-216) with a thioredoxin fold (9) and is most accessible for trypsin attack. The fragment fDsbC is thus just the C-terminal thioredoxin domain with 12 residues in the linker at the N terminus.
-sheets (9), is obviously much less compact than the C-terminal region for its greater susceptibility to digestion, since in the first
50 min of digestion two additional weak bands with a mass of about 21 kDa (cleaved between Lys28 and Thr29) and 19 kDa (cleaved between Lys44 and His45) appeared
in addition to the band of the intact molecules and the main band with
a mass of 17 kDa (cleaved between Lys65 and
Met66) and is digested to small pieces during trypsin treatment.
-helix (see Scheme
II). The N-terminal domains (residues
1-61) from each monomer form a pair of six-stranded anti-parallel
-sheets that form the dimer interface at the base of the V shaped
molecule. The N-terminal domain is thus named the dimerization domain.
It becomes very clear that removal of the dimerization domain results
in fDsbC as two separate monomeric C-terminal thioredoxin domains.
View larger version (40K):
[in a new window]
Scheme 2.
Schematic presentation of the domain
structure of the DsbC molecule. DsbC is a V-shaped dimeric
molecule with each arm of the V for one subunit. Each subunit consists
of a C-terminal thioredoxin-like domain (C) and an
N-terminal domain (N) responsible for dimerization. The two
domains are connected by a hinged linker helix. The arrow
represents the cleavage site of DsbC by trypsin. Thiols at active sites
are shown as balls. The dimer interface consists essentially
of hydrogen bonds between the two N domains. There is a broad uncharged
cleft between the two domains.
-sheets of the N-terminal domains and the two C-terminal thioredoxin
domains with the active sites are normally 38 Å apart across the
cleft. In addition, the linker helix is sufficiently long that the
N-terminal association domain and the C-terminal catalytic domain
within a monomer also have minimal interaction. It is hard to
understand why the modification at the active site of one subunit
affects the dimerization with a normal subunit unless the moderate
perturbed conformation of the modified subunit is responsible. We also
found that the heterodimer appeared from a mixture of DsbC and mmDsbC
under nondenaturing conditions, indicating that native DsbC and mmDsbC
also exist in equilibrium of dissociation and association (data not
shown). However, the process under nondenaturing conditions is very
slow and takes about 24 h to reach equilibrium.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: National Laboratory of
Biomacromolecules, Inst. of Biophysics, Academia Sinica, 15 Datun Rd.,
Beijing 100101, China. Tel.: 86-10-64888502; Fax: 86-10-64872026;
E-mail: chihwang@sun5.ibp.ac.cn.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Missiakas, D.,
and Raina, S.
(1997)
J. Bacteriol.
179,
2465-2471
2.
Raina, S.,
and Missiakas, D.
(1997)
Annu. Rev. Microbiol.
51,
179-202
3.
Zapun, A.,
Missiakas, D.,
Raina, S.,
and Creighton, T. E.
(1995)
Biochemistry
34,
5075-5089
4.
Sone, M.,
Akiyama, Y.,
and Ito, K.
(1997)
J. Biol. Chem.
272,
10349-10352
5.
Cai, H.,
Wang, C. C.,
and Tsou, C. L.
(1994)
J. Biol. Chem.
269,
24550-24552
6.
Song, J. L.,
and Wang, C. C.
(1995)
Eur. J. Biochem.
231,
312-316
7.
Yao, Y.,
Zhou, Y. C.,
and Wang, C. C.
(1997)
EMBO J.
16,
651-658
8.
Chen, J.,
Song, J. L.,
Zhang, S.,
Wang, Y.,
Cui, D. F.,
and Wang, C. C.
(1999)
J. Biol. Chem.
274,
19601-19605
9.
McCarthy, A. A.,
Haebel, P. W.,
Torronen, A.,
Rybin, V.,
Baker, E. N.,
and Metcalf, P.
(2000)
Nat. Struct. Biol.
7,
196-199
10.
Frishman, D.
(1996)
Biochem. Biophys. Res. Commun.
219,
686-689
11.
Hubbard, S. J.
(1998)
Biochim. Biophys. Acta
1382,
191-206
12.
Freedman, R. B.,
Gane, P. J.,
Hawkins, H. C.,
Hlodan, R.,
Mclaughlin, S. H.,
and Parry, J. W. L.
(1998)
Biol. Chem.
379,
321-328
13.
Missiakas, D.,
Georgopoulos, C.,
and Raina, S.
(1994)
EMBO J.
13,
2013-2020
14.
Liang, S. J.,
Lin, Y. Z.,
Zhou, J. M.,
Tsou, C. L.,
Wu, P.,
and Zhou, Z.
(1990)
Biochim. Biophys. Acta
1038,
240-246
15.
Hillson, D. A.,
Lambert, N.,
and Freedman, R. B.
(1984)
Methods Enzymol.
107,
281-294
16.
Scheek, R. M.,
and Slater, E. C.
(1982)
Methods Enzymol.
89,
305-308
17.
Bradford, M. M.
(1976)
Anal. Biochem.
72,
248-254
18.
Riddles, P. W.,
Blakeley, R. L.,
and Zerner, B.
(1983)
Methods Enzymol.
91,
49-60
19.
Huber-Wunderlich, M.,
and Glockshuber, R.
(1998)
Folding Design
3,
161-171
20.
Lambert, N.,
and Freedman, R. B.
(1983)
Biochem. J.
213,
225-234
21.
Heuck, A. P.,
and Wolosiuk, R. A.
(1997)
Anal. Biochem.
248,
94-101
22.
Zapun, A.,
and Creighton, T. E.
(1994)
Biochemistry
33,
5202-5211
23.
Wunderlich, M.,
and Glockshuber, R.
(1993)
Protein Sci.
2,
717-726
24.
Hawkins, H. C.,
Blackburn, E. C.,
and Freedman, R. B.
(1991)
Biochem. J.
275,
349-353
25.
Zheng, W. D.,
Quan, H.,
Song, J. L.,
Yang, S. L.,
and Wang, C. C.
(1997)
Arch. Biochim. Biophys.
337,
326-331
26.
Giroux, E.,
Williams, M. K.,
and Kantrowitz, E. R.
(1994)
J. Biol. Chem.
269,
31404-31409
27.
Grant, G. A.,
Kim, S. J.,
Xu, X. L.,
and Hu, Z.
(1999)
J. Biol. Chem.
274,
5357-5361
28.
Darby, N. J.,
and Creighton, T. E.
(1995)
Biochemistry
34,
11725-11735
29.
Ranson, N. A.,
White, H. E.,
and Saibil, H. R.
(1998)
Biochem. J.
333,
233-242
30.
Klappa, P.,
Ruddock, L. W.,
Darby, N. J.,
and Freedman, R. B.
(1998)
EMBO J.
17,
927-935
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  G. Tian, F.-X. Kober, U. Lewandrowski, A. Sickmann, W. J. Lennarz, and H. Schindelin The Catalytic Activity of Protein-disulfide Isomerase Requires a Conformationally Flexible Molecule J. Biol. Chem., November 28, 2008; 283(48): 33630 - 33640. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Arredondo, L. Segatori, H. F. Gilbert, and G. Georgiou De Novo Design and Evolution of Artificial Disulfide Isomerase Enzymes Analogous to the Bacterial DsbC J. Biol. Chem., November 14, 2008; 283(46): 31469 - 31476. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Messens, J.-F. Collet, K. Van Belle, E. Brosens, R. Loris, and L. Wyns The Oxidase DsbA Folds a Protein with a Nonconsecutive Disulfide J. Biol. Chem., October 26, 2007; 282(43): 31302 - 31307. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Quan, I. Schneider, J. Pan, A. Von Hacht, and J. C. A. Bardwell The CXXC Motif Is More than a Redox Rheostat J. Biol. Chem., September 28, 2007; 282(39): 28823 - 28833. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Segatori, L. Murphy, S. Arredondo, H. Kadokura, H. Gilbert, J. Beckwith, and G. Georgiou Conserved Role of the Linker {alpha}-Helix of the Bacterial Disulfide Isomerase DsbC in the Avoidance of Misoxidation by DsbB J. Biol. Chem., February 24, 2006; 281(8): 4911 - 4919. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-y. Shi, X.-g. Hong, and C.-c. Wang The C-terminal (331-376) Sequence of Escherichia coli DnaJ Is Essential for Dimerization and Chaperone Activity: A SMALL ANGLE X-RAY SCATTERING STUDY IN SOLUTION J. Biol. Chem., June 17, 2005; 280(24): 22761 - 22768. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Moutevelis and J. Warwicker Prediction of pKa and redox properties in the thioredoxin superfamily Protein Sci., October 22, 2004; 13(10): 2744 - 2752. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Barnewitz, C. Guo, M. Sevvana, Q. Ma, G. M. Sheldrick, H.-D. Soling, and D. M. Ferrari Mapping of a Substrate Binding Site in the Protein Disulfide Isomerase-related Chaperone Wind Based on Protein Function and Crystal Structure J. Biol. Chem., September 17, 2004; 279(38): 39829 - 39837. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Segatori, P. J. Paukstelis, H. F. Gilbert, and G. Georgiou Engineered DsbC chimeras catalyze both protein oxidation and disulfide-bond isomerization in Escherichia coli: Reconciling two competing pathways PNAS, July 6, 2004; 101(27): 10018 - 10023. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Zhao, Y. Peng, S.-f. Hao, Z.-h. Zeng, and C.-c. Wang Dimerization by Domain Hybridization Bestows Chaperone and Isomerase Activities J. Biol. Chem., October 31, 2003; 278(44): 43292 - 43298. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-F. Collet, J. Riemer, M. W. Bader, and J. C. A. Bardwell Reconstitution of a Disulfide Isomerization System J. Biol. Chem., July 19, 2002; 277(30): 26886 - 26892. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Goldstone, P. W. Haebel, F. Katzen, M. W. Bader, J. C. A. Bardwell, J. Beckwith, and P. Metcalf DsbC activation by the N-terminal domain of DsbD PNAS, August 1, 2001; (2001) 171315498. [Abstract] [Full Text] [PDF]
|
|
|
|
< |
