Functional properties of the protein disulfide oxidoreductaseA member of a novel protein family related to protein disulfide-isomerase Emilia Pedone1, Bin Ren2, Rudolf Ladenstein2, Mose`
Trang 1Functional properties of the protein disulfide oxidoreductase
A member of a novel protein family related to protein disulfide-isomerase
Emilia Pedone1, Bin Ren2, Rudolf Ladenstein2, Mose` Rossi3,4and Simonetta Bartolucci3
1 Istituto di Biostrutture e Bioimmagini, C.N.R., Napoli, Italy; 2 Center for Structural Biochemistry, Karolinska Institutet, Huddinge, Sweden;3Dipartimento di Chimica Biologica, Universita` degli Studi di Napoli Federico II, Napoli, Italy;4Istituto di Biochimica delle Proteine, C.N.R., Napoli, Italy
Protein disulfide oxidoreductases are ubiquitous redox
enzymes that catalyse dithiol–disulfide exchange reactions
with a CXXC sequence motif at their active site A disulfide
oxidoreductase, a highly thermostable protein, was isolated
from Pyrococcus furiosus (PfPDO), which is characterized
by two redox sites (CXXC) and an unusual molecular mass
Its 3D structure at high resolution suggests that it may be
related to the multidomain protein disulfide-isomerase
(PDI), which is currently known only in eukaryotes This
work focuses on the functional characterization of PfPDO
as well as its relation to the eukaryotic PDIs Assays of
oxidative, reductive, and isomerase activities of PfPDO were
performed, which revealed that the archaeal protein not only
has oxidative and reductive activity, but also isomerase
activity On the basis of structural data, two single mutants
(C35S and C146S) and a double mutant (C35S/C146S) of PfPDO were constructed and analyzed to elucidate the specific roles of the two redox sites The results indicate that the CPYC site in the C-terminal half of the protein is fundamental to reductive/oxidative activity, whereas iso-merase activity requires both active sites In comparison with PDI, the ATPase activity was tested for PfPDO, which was found to be cation-dependent with a basic pH optimum and
an optimum temperature of 90C These results and an investigation on genomic sequence databases indicate that PfPDO may be an ancestor of the eukaryotic PDI and belongs to a novel protein disulfide oxidoreductase family Keywords: Archaea; protein disulfide-isomerase; protein disulfide oxidoreductase; Pyrococcus furiosus; redox sites
Protein disulfide oxidoreductases are ubiquitous redox
enzymes that catalyse dithiol–disulfide exchange reactions
These enzymes share a CXXC sequence motif at their active
sites The two cysteines can undergo reversible oxidation–
reduction by shuttling between a dithiol and a disulfide form
in the catalytic process Protein disulfide oxidoreductases
comprise the families of thioredoxin, glutaredoxin, protein
disulfide-isomerase (PDI), and DsbA (disulfide-bond
form-ing) and their homologs Whereas thioredoxin and
glutaredoxin mainly catalyse the reduction of disulfides,
PDI and DsbA catalyse the formation or rearrangement of
disulfide bridges in the protein-folding process
Protein disulfide oxidoreductases have been well studied
in bacteria and eukarya, although to date only a few
archaeal members of this protein family have been isolated,
and therefore very little is known about protein disulfide
oxidoreductases in archaea
A small redox protein with a molecular mass of 12 kDa
was purified from the archaeon Methanobacterium
thermoautotrophicumby McFarlan et al [1] This protein can catalyse the reduction of insulin disulfides and function
as a hydrogen donor for Escherichia coli ribonucleotide reductase The presence of the active-site motif CPYC, which is conserved in all glutaredoxins, suggested that it acts
as a glutaredoxin-like protein Surprisingly, however, the reduced enzyme does not react with either thioredoxin reductase or glutathione differently from other thioredoxins and glutaredoxins [2] In the hyperthermophilic archaeon Methanococcus jannaschii[3], a thioredoxin homologue was identified (Mj0307) [4] that has the sequence CPHC, which had never before been observed in either thioredoxins or glutaredoxins It exhibits biochemical activities similar to thioredoxin, although its structure is more similar to glutaredoxin The observation that a single thioredoxin system is present in M jannaschii and Mb thermoauto-trophicumsuggested that a single thioredoxin-like protein with a glutaredoxin-like structure is enough to maintain redox homeostasis in the archaeal methanogen [5] Guagliardi et al [6] purified a protein disulfide oxido-reductase from the hyperthermophilic archaeon Sulfolobus solfataricus Given its ability to catalyse the reduction of insulin disulfides in the presence of dithiothreitol, the protein was named thioredoxin The monomeric form of the enzyme has an unusual molecular mass of about
26 kDa, compared with that observed in thioredoxin and glutaredoxin (12 kDa)
A homologous protein disulfide oxidoreductase was purified from the hyperthermophilic archaeon Pyrococcus
Correspondence to S Bartolucci, Dipartimento di Chimica Biologica,
Universita` degli Studi di Napoli Federico II, via Mezzocannone 16,
80134 Napoli, Italy Fax: +39 81 2534614, Tel.: +39 81 2534732,
E-mail: bartoluc@unina.it
Abbreviations: PfPDO, protein disulfide oxidoreductase from the
archeon Pyrococcus furiosus; DMS, dimethyl suberimidate; MgATP,
5 m M MgCl 2 , 2 m M ATP; PDI, protein disulfide-isomerase.
(Received 19 May 2004, revised 29 June 2004, accepted 8 July 2004)
Trang 2furiosus(PfPDO) [7] PfPDO showed close similarity to the
S solfataricusprotein in molecular mass (25 648 Da) and
dithiothreitol-dependent insulin reduction activity In
addi-tion, both proteins displayed thiol transferase activity by
catalysing the reduction of disulfide bonds in L-cysteine
[7,8] The PfPDO primary structure does not show any
overall sequence similarity to known protein disulfide
oxidoreductases Interestingly, it has two potential active
sites with the conserved CXXC sequence motif A CPYC
sequence is located at the C-terminal half of PfPDO, which
is the conserved active sequence of the glutaredoxin family,
usually located at the N-terminus In addition, a CQYC
sequence, which has never been observed in any other
protein disulfide oxidoreductase, is present at the
N-terminal half of the protein The PfPDO crystal structure
provides some intriguing challenges to the understanding of
the enzyme’s function [9–11] The protein consists of two
homologous units with low sequence identity (18%) Each
unit contains a thioredoxin fold, and the accessibilities of the
two CXXC active sites are rather different The presence of
two homologous units in the same protein resembles the
structure of PDI; in fact, the PDI molecule possesses two
thioredoxin-like domains with two active sites Interestingly,
whereas thioredoxins and glutaredoxins were identified in
both prokaryotes and eukaryotes, DsbA was only found in
prokaryotes PDIs, with multiple thioredoxin/glutaredoxin
domains within a single polypeptide are known in
eukary-otes, and it is likely that the first step in their molecular
evolution was the duplication of an ancestral thioredoxin/
glutaredoxin domain [12] The unusual structural features of
PfPDO suggest that this enzyme probably represents a new
member of the protein disulfide oxidoreductase superfamily
and a new form of isomerase compared with PDI and
DsbA Functional studies of PfPDO are essential to support
this finding, but have not yet been conducted Therefore,
this work focuses on the functional characterization of the
PfPDO protein in an attempt to elucidate its relation with
the eukaryotic multidomain PDI Functional data revealed
that the archaeal protein not only has oxidative and
reductive activity, but also isomerase activity This is the
first example of an archaeal protein characterized with
disulfide isomerase activity
To investigate the specific roles of each PfPDO redox site,
two single mutants (C35S and C146S) were constructed, in
which the N-terminal active-site cysteine residue (Cys35 or
Cys146) was replaced by serine, and a double mutant
(C35S/C146S) All mutants were expressed, purified, and
their activities compared with that of the wild-type protein
To compare the PfPDO with PDI for ATP binding and
hydrolysis, the archaeal protein was also tested for its
ATPase activity
Experimental Procedures
Materials
Bovine insulin, glutathione disulfide (GSSG), glutathione
(GSH), bovine liver PDI, horse liver alcohol
dehydro-genase, bovine pancreas scrambled RNase and all the other
reagents used were from Sigma Molecular-mass standards
for SDS/PAGE were obtained from Pharmacia or
Bio-Rad E coli strain JM101 was purchased from Boehringer
Expression vector pET22(b+), E coli strain BL21(DE3), and CJ236 E coli strain were from AMS Biotechnology (Abingdon, UK) Radioactive materials were obtained from New England Nuclear/Life Science (Boston, MA, USA) 8-Azido-[32P]ATP[aP] was obtained from ICN Deoxynucleotides and restriction and modification enzymes were from Boehringer All materials used for gene amplification were supplied by Stratagene Cloning Systems All synthetic oligonucleotides and the peptide designed by Ruddock et al [13] were from PRIMM (Milan, Italy) Bacterial cultures, plasmid purifications, and transformations were performed as described by Sambrook et al [14]
Construction ofE coli Pf PDO and mutants
Pf PDO(C35S), Pf PDO(C146S) and the double mutant (C35S/C146S)
Isolation of chromosomal DNA from P furiosus was performed as described by Barker [15] From the PfPDO amino-acid sequence from residues 1–7, the following oligonucleotides were designed and used as primers in the PCR gene amplification procedure, using the chromosomal DNA (200 ng) as template: forward primer, 5¢-GGAATT catatgGGATTGATTAGTGACGCTG-3¢, contained a 5¢-NdeI site (indicated in lowercase); reverse primer housed the PfPDO stop codon 3¢ of a unique BamHI (indicated
in lowercase) 5¢-GGAATTcatatgGGATTAGTGACGC TG-3¢ The amplification was performed as described by Saiki [16] for 35 cycles at 45C annealing temperature, on a Perkin–Elmer Cetus Cycler Temp using Pfx polymerase (Stratagene) The amplified DNA fragment (PfPDO), opportunely digested, was inserted into the pET22(b+) plasmid The recombinant clone, designated pET-PfPDO wild-type, represented the expression vector
The mutations Cys35Ser (C35S) and Cys146Ser (C146S) were introduced into the PfPDO DNA by the method of Kunkel [17] The amplified genes, opportunely digested, were ligated to the cloning pET22(b+) plasmid Insertion of the correct mutations was confirmed by DNA sequencing using Sanger’s dideoxy method, with a Sequenase Sequen-cing Kit from Amersham [18]
Expression and purification of recombinantPf PDO mutants
Competent E coli BL21(DE3) cells were transformed with pET-PfPDO wild-type, C35S, C146S, and C35S/C146S, and grown at 37C to different densities in 500 mL terrific-broth medium; isopropyl thio-b-D-galactoside was added to
1 mMfinal concentration, varying the induction time from
2 to 24 h E coli BL21DE3 cells transformed with pET22(b+) represented a negative control Optimized overexpression of all the proteins was obtained by exposing the cells to 1 mM isopropyl thio-b-D-galactoside at a cell density of A600¼ 2.5 for 18 h Cell pellets from 500 mL cultures were resuspended in 5 mL 10 mM Tris/HCl,
pH 8.4, and crude extracts were prepared by disrupting the cells with 20 min pulses at 20 Hz (Sonicator Ultrasonic liquid processor; Heat System Ultrasonics Inc., Farming-dale, NY, USA) and ultracentrifugation at 160 000 g for
30 min Recombinant wild-type protein and its mutants
Trang 3were purified in a similar way The crude extracts were
subjected to heat treatment at 80C and then centrifuged at
5000 g at 4C for 15 min, removing almost 70% of the
mesophilic host proteins The crude extracts were applied to
a 2.6 cm· 60 cm column (HiLoad Superdex 75;
Pharma-cia) connected to an FPLC system (PharmaPharma-cia) and eluted
with 10 mMTris/HCl (pH 8.4)/0.2MNaCl at a flow rate of
2 mLÆmin)1 The active fractions were pooled,
concentra-ted, and extensively dialysed against 10 mM Tris/HCl,
pH 8.4 They were then loaded on an anion-exchange
Mono Q column in 10 mMTris/HCl, pH 8.4, connected to
an FPLC system (Pharmacia), and eluted with a linear
gradient (0/0.3M NaCl) in 30 min at a flow rate of
0.5 mLÆmin)1 A single peak was observed on RP-HPLC
and a single protein band on SDS/PAGE
Analytical methods for protein characterization
Protein concentration was determined using BSA as the
standard [19] The molar absorption coefficient, obtained
by the method used by the Schepertz laboratory (http://
paris.chem.yale.edu), was 19 724M )1Æcm)1
Protein homogeneity was assessed by SDS/PAGE
[12.5% (w/v) gels] using the silver staining procedure of
Rabilloud et al [20] In addition, proteins were analysed by
nondenaturing electrophoresis [12.5% (w/v) polyacrylamide
slab gel]
The molecular mass of the proteins was estimated using
electrospray mass spectra recorded on a Bio-Q triple
quadrupole instrument (Micromass) Samples were
dis-solved in 1% (v/v) acetic acid/50% (v/v) acetonitrile and
injected into the ion source at a flow rate of 10 mLÆmin)1
using a Phoenix syringe pump Spectra were collected and
elaborated using MASSLYNX software provided by the
manufacturer Calibration of the mass spectrometer was
performed with horse heart myoglobin (16 951.5 Da)
UV-CD spectra in 10 mM sodium phosphate, pH 7.0,
using a 1-mm path-length cell at 185–260 nm at 25C, were
recorded on a Jasco J-710 spectropolarimeter equipped with
a Peltier thermostatic cell holder (Jasco, model PTC-343)
for all the proteins
Counting integral numbers of residues by chemical
modification
The procedure of Hollecker & Creighton [21] was used to
detect the different exposure of the cysteine residues All the
proteins (PfPDO and mutants at a final concentration of
200 mM) were incubated in a final volume of 1 mL for
30 min at 37C in 10 mMTris/HCl (pH 8.0)/10 mMEDTA
(pH 7.0) in native, reduced (10 mM dithiothreitol), and
reduced and denatured (10 mMdithiothreitol and 8Murea)
conditions Successively in a final volume of 10 lL, five
different solutions containing 0.25Miodoacetate (in 0.25M
Tris/HCl, pH 8.0, and 0.25MKOH) and 0.25M
iodoacet-amide (in 0.25M Tris/HCl, pH 8.0) were prepared in the
following ratios: 0 : 1 (250 mM); (250 mM) 1 : 0; 1 : 1 (each
125 mM); (187.5 mM) 3 : 1 (62.5 mM); (225 mM) 9 : 1
(25 mM) At the end of the incubation, 40 lL of the mixture
was added to each of the five solutions; these were then left
to react on ice for 5 min The reaction mixtures were
analysed by nondenaturing electrophoresis [12.5% (w/v)
polyacrylamide slab gel] The ladder or control is repre-sented by a mixture of 10 mL taken from each of the five reaction mixtures The method consists of adding various iodoacetamide and iodoacetate ratios to portions of the protein to generate a complete spectrum of protein mole-cules with 0, 1, or 4 acidic carboxymethyl groups, where 4 is the integral number of cysteine residues Protein in which all thiol groups were blocked with iodoacetate, if well exposed, migrated more slowly than that blocked with iodoacetamide, because of the acidic carboxymethyl groups Cross-linking with dimethyl suberimidate (DMS)
Following the procedure of Davies & Stark [22], 10 lg PfPDO was incubated for 2 h at room temperature with different quantities of DMS (1 : 1, 1 : 2.5, 1 : 5, 1 : 10) to determine the best protein to DMS ratio Molecular mass and yield were checked by SDS/PAGE [12.5% (w/v) polyacrylamide gel]
Assay of enzyme activities Insulin reductase activity.Reductase activity was assayed
by Holmgren’s turbidimetric method [23] with a few modifications The catalytic reduction of insulin disulfide bonds was measured at 30C Protein was added in 1 mL
100 mM sodium phosphate buffer, pH 7.0, containing
2 mM EDTA and 1 mg bovine insulin A control cuvette contained only buffer and insulin The reaction was started
by the addition of 2 mM dithiothreitol to both cuvettes Increasing turbidity from precipitation of the insulin B chain was recorded at 650 nm The stock solution of insulin (10 mgÆmL)1) was prepared according to the Holmgren protocol
Oxidation activity.The disulfide bond-forming activity of the proteins was monitored using the synthetic decapep-tide NRCSQGSCWN containing two cysteine residues at position 3 and 8 designed by Ruddock et al [13] The peptide contains a fluorescent group (tryptophan) on one side of one cysteine residue and a protonated group (arginine) on the other side of the second cysteine residue, and the two cysteine residues are separated by a flexible linker region The linker is long enough to permit the formation of an unstrained disulfide bond, and the peptide is small and water soluble Oxidation of this dithiol peptide to the disulfide state is accompanied by a change in tryptophan fluorescence emission intensity In fact, on oxidation, the fluorescent group and the protonated group are brought close together, and quenching on the fluorophore occurs where arginine is the charged quencher Fluorescence quenching was used
as the basis for monitoring the disulfide bond-forming activity of PfPDO
Spectrofluorimetric analysis.The assay was performed in McIlvaine buffer (0.2M disodium hydrogen phosphate/ 0.1M citric acid, pH 7.0) with 2 mM GSH, 0.5 mM
GSSG and 5 lM PfPDO The reaction mixture was placed in a fluorescence cuvette with a final assay volume of 1 mL After mixing, the cuvette was placed
in a thermostatically controlled Perkin–Elmer LS50B
Trang 4spectrofluorimeter for 1 min to allow thermal
equilibra-tion of the soluequilibra-tion to 50C Next, 5 lM substrate
peptide was added, mixed, and the change in fluorescence
intensity (excitation 295 nm, emission 350 nm, slits
10/10 nm) was monitored over an appropriate time
(15 min) As a control, the same experiment was carried
out in the absence of any protein; no decrease in
fluorescence intensity was observed [13]
HPLC analysis Alternatively the oxidation activity was
measured by HPLC analysis (Varian) The reduced and
oxidized forms of the peptide have different retention times
and are eluted separately on reverse-phase chromatography
[L Birolo and A Tosco (1999) personal communication]
The peptide was eluted in a single peak and stored at)20 C
in the elution buffer (30% acetonitrile in 0.1%
trifluoro-acetic acid; v/v/v) at a concentration of 1.05 mM The
peptide concentration was determined
spectrophotometi-cally using an absorption coefficient of 5600M )1Æcm)1at
278 nm The oxidized state of the peptide was generated by
incubating the peptide at a concentration of 50 lMin 0.2M
Tris/HCl, pH 8.4, at 20C for 15 h The reduced state was
generated by incubating the peptide in McIlvaine buffer
(0.2M disodium hydrogen phosphate/0.1M citric acid,
pH 7.0) at a final concentration of 50 lM and 1 mM
dithiothreitol in a final volume of 50 lL
The assay mixture contained 5 lM reduced peptide,
100 mMGSH (stock solution 60.1 mgÆmL)1), 25 mMGSSG
(stock solution 30.7 mgÆmL)1) and the protein PfPDO (final
concentration 10, 50, 100, 150 or 200 lM) The mixture was
incubated at different temperatures (50C, 60 C or 70 C)
for different times in the presence of different concentrations
of the protein After incubation, the mixture was loaded on
the HPLC reversed-phase Vydac C18 column equilibrated
in buffer A [0.1% (v/v) trifluoroacetic acid in water]
Chromatography was carried out with a linear gradient
0–100% buffer B (95% acetonitrile, 0.07% trifluoroacetic
acid; v/v/v) in buffer A at a flow rate of 1 mLÆmin)1for
35 min
Re-activation of scrambled RNase.The isomerase activity
was assayed by Lambert’s method Re-activation of
scrambled RNase was monitored after incubation of
PfPDO in 50 mM sodium phosphate, pH 7.5, in a total
volume of 0.9 mL, with 10 lL dithiothreitol (1 mMstock
solution, final concentration 10 lM) for 2 min at 30C [24]
A 0.1 mL portion of scrambled RNase (Sigma;
0.5 mgÆmL)1 in 10 mM acetic acid, final concentration
4 lM) was added, and at different times after this addition
10 lL samples were withdrawn and assayed for RNase
activity Each sample was added to an assay mixture of
1 mL 0.5 mgÆmL)1 RNA in 50 mM Tris/HCl, pH 7.5
RNase activity on yeast RNA was assayed by the method
outlined by Kunitz [25] with some modifications, and under
conditions in which the decrease in A300was linear for at
least 3–4 min Yeast RNA was dissolved in water, and the
pH was kept neutral by performing the assay in 50 mMTris/
HCl, pH 7.5 The positive control was re-activation of
scrambled RNase catalysed by PDI (bovine liver; Sigma)
Nonenzymatic reactivation of scrambled RNase was
cor-rected for by using the same mixture without the addition of
any of the proteins
Detection of ATP binding by CD
CD measurements were performed in a Jasco J-720 spectropolarimeter in 20 mM Tris/HCl (pH 7.5)/5 mM
MgCl2at 25C Each sample was scanned five times, noise reduction was applied, and baseline buffer spectra were subtracted from sample spectra before molar ellipticities were calculated To obtain spectra in the near-UV region (250–320 nm), the cell path length was 1 cm and the protein concentration 1 mgÆmL)1 The CD spectra were evaluated
at 260 nm
Cross-linking ofPf PDO with 8-Azido-[32
P]ATP[aP]
To analyze the ability of PfPDO to cross-link to 8-azido-ATP, 3 mg protein was incubated in the presence of 2 mCi 8-azido[32P]ATP[aP] for 30 min in 50 mMTris/HCl, pH 8.0
or 10 mMGly/NaOH, pH 10.0, containing 2 mMEDTA,
1 mMdithiothreitol and 5 mMMgCl2at 60 or 70 C To induce cross-linking, samples were exposed for 10 min to
UV irradiation and then resolved by SDS/PAGE in 12% polyacrilamide gel and visualized by radioautography on a Fuji medical X-ray film The same procedure was used for incubation of PfPDO at pH 10.0 at 70C in the presence of
an increasing concentration of unlabeled ATP (50 lMand
1 mM) [26]
Fluorescence measurements Samples of PfPDO (100 lg) were incubated for 10 min at
70C, 80 C, or 90 C in the presence of MgATP, and then loaded on a Superdex 75 HiLoad column (Amersham Pharmacia Biotech; 1· 30 cm; eluent 10 mM Tris/HCl,
pH 7.5, 0.2MNaCl; flow rate 0.3 mLÆmin)1) to remove the nucleotide excess The protein samples recovered from the columns and a sample of native PfPDO were analyzed for fluorescence at 3 lMfinal protein concentration (excitation wavelength 280 nm; emission recorded between 310 and
410 nm) using a Perkin–Elmer LS50B spectrofluorimeter at
25C [27]
Assay of ATPase activity
A colorimetric assay was routinely used to measure ATPase activity following the method of Lanzetta et al [28] To
100 lL of sample (water and 10 mg protein) was added
800 lL of green malachite/ammonium molybdate in 1M
HCl, followed by mixing After 1 min, 100 lL 34% citrate was added and mixed This solution was read immediately
at 660 nm
In an alternative assay, the ATPase activity of PfPDO was assayed in mixtures containing 2 mM ATP, 15 lCi [32P]ATP[aP], 5 mM MgCl2 and 10 lg pure protein in
50 mM Tris/HCl, pH 7.5 (150 lL final volume) After a
5 min incubation at 70C, a 25 lL aliquot was withdrawn and added to 0.5 mL of a suspension containing 50 mM
HCl, 5 mMH3PO4and 7% activated charcoal The mixture was then centrifuged at 4000 g for 20 min The radioactivity
of the supernatant was determined in a 100 lL aliquot using
a liquid-scintillation counter (Beckman) In rate calcula-tions, spontaneous ATP hydrolysis in the absence of PfPDO was corrected for [29]
Trang 5Production of wild-type and mutantPf PDO
To determine the redox state and the accessibility of the
cysteine residues of the PfPDO redox sites, electrophoretic
analysis was performed, as described by Hollecker et al
[21], on the protein treated under different conditions
(native, reduced, and reduced and denatured) (Table 1) By
comparing the results obtained from the different gels, it
was possible to confirm the crystallographic data that the
most reactive cysteine was Cys146, as this was observed at
the lowest ratio of iodoacetate to iodoacetamide This was
followed by Cys149, Cys35, and Cys38, which was the last
to react and the least accessible residue [21]
To investigate the role of the putative redox sites of
PfPDO, three mutants were constructed (C35S, C146S, and
C35S/C146S) by mutagenizing the most exposed cysteines
of each of the redox sites: specifically, Cys35 at the
N-terminal site and Cys146 at the C-terminal site were
replaced by serine [30]
PfPDO and mutants were expressed in E coli
BL21(DE3) Overexpression of all the proteins was
obtained by exposing the cells to 1 mM isopropyl
thio-b-D-galactoside at a cell density of A¼ 2.5 To optimize the
production of the recombinant proteins, transformed cells
were exposed to the inducer for 2–24 h; maximum
expres-sion was obtained after 18 h of induction
The crude extract of E coli was subjected to one thermal
precipitation step at 80C for 20 min to remove almost
70% of the mesophilic host proteins During the
purifica-tion procedure, the proteins were assayed after reducpurifica-tion of
protein disulfides on insulin as substrate; when the
inter-chain disulfide bridges are reduced between inter-chains A and B
of the insulin, the turbidity of the solution increases because
of precipitation of the free B chain [23] After gel-filtration
chromatography and anion-exchange chromatography, a
single peak was observed on RP-HPLC, and a single band
on SDS/PAGE The protein yield from 1 L of culture was
40 mg for all the recombinant proteins
The molecular mass of the proteins was analysed by
electrospray mass spectroscopy The measured mass of
PfPDO was 25 648 ± 0.5 Da The measured mass of C35S
and C146S was 25 628 ± 0.5 Da, and that of C35S/C146S
was 25 613 ± 0.4 Da Thus, the difference in mass was in
perfect agreement with the mutations introduced
To see if the mutations introduced had an effect on the
structure of the protein, far-UV CD spectra were recorded
for all the proteins The spectra were very similar, showing that all the proteins are completely folded and indicating that the mutations did not result in any obvious change in overall structure
Characterization of the activities of wild-type and mutantPf PDO
PfPDO reduces insulin disulfide in the presence of dithio-threitol at 30C The analysis was performed in the presence of increasing concentrations of the pure proteins,
as well as in their absence (the spontaneous precipitation reaction), because dithiothreitol is the reducing agent that recycles the oxidized protein (Fig 1) The activity was assayed at 1.2 lM for all the proteins Both the wild-type PfPDO and the mutant C35S were active in the insulin reductase assay [23], whereas the activity of the mutant C146S and the double mutant was similar to the control This shows that the active site in the C-terminal half (CPYC) is responsible for the reductase activity
In the presence of 5 lMPfPDO, oxidation of the dithiol peptide designed by Ruddock was observed at neutral pH
by the spectrofluorimetric assay (Fig 2A) Separation of the oxidized and reduced forms of the peptide by HPLC allowed quantification of the oxidative activity as a ratio between the areas of oxidized/reduced peptide The assays were performed at a concentration of 100 lM protein, at different times and different temperatures [50C, 60 C, and 70C (data not shown)] The best conditions were
Table 1 Exposed cysteine residues by the Hollecker method [21] The proteins in native, reduced (10 m M dithiothreitol), and reduced and denatured (10 m M dithiothreitol and 8 M urea) conditions were treated with different amounts of iodoacetate/iodoacetamide [1 : 1 (each 250 m M ), 1 : 3, 1 : 9 ratios of neutral to acidic reagents] and separated by SDS/PAGE The appearance of a band in the different conditions used (native, reduced, reduced and denatured) on the different proteins (PfPDO and mutants) is evidence of the exposure of that cysteine residue nd, Not determined.
Iodoacetate/iodoacetamide 1 : 1 3 : 1 9 : 1 1 : 1 3 : 1 9 : 1 1 : 1 3 : 1 9 : 1 PfPDO wild-type – – – C146 C146 C146/149 C146 C146/149 C146/149/35 C35S mutant – – – C146 C146 C146/149 C146 C146/149 C146/149/38
C35S/C146S mutant nd nd nd C149 C149 C149/38 C149 C149 C149/38
Fig 1 Assay of reductase activity by measuring the reduction of bovine insulin disulfides The dithiothreitol-dependent reduction of bovine insulin disulfides was carried out as described in Experimental proce-dures in the absence [control (–––)] or presence of 1.2 l M PfPDO wild-type (ÆÆÆÆ), PfPDO (C35S) (- - - -), or PfPDO (C146S) and PfPDO (C35S)/(C146S) (-Æ-Æ).
Trang 650C for 3 h A linear relation between activity and
concentration was detected for all the proteins (Fig 2B)
Wild-type PfPDO and C35S were able to oxidize the
peptide with maximum activity at a concentration of 150
and 200 lM, respectively C146S had residual oxidative
activity, but the double mutant was completely inactive,
demonstrating the predominant role of the redox site at the
C-terminus in the oxidative activity
The action of PfPDO in catalysing interchange of
intramolecular disulfides in scrambled RNase results in
restoration of the native disulfide pairing and the
concom-itant return of RNase activity Thus, the isomerase activity
of PfPDO was assayed by a time-course incubation during
which aliquots were removed and RNase activity with RNA
was measured Re-activation of scrambled RNase was
performed with all the proteins Only the wild-type protein
was able to refold the scrambled RNase (Fig 3), indicating
that isomerase activity requires the participation of both
N-terminal and C-terminal active sites Refolding of the
scrambled RNase in the presence of PDI was used as a
positive control, and the absence of the recovery of the
RNase activity in the presence of the thioredoxin from Alicyclobacillus acidocaldarius was used as a negative control The refolding of the scrambled RNase in the presence of PfPDO seems to be less efficient when using PDI However, the temperature of the assay, which is limited by the stability of the protein substrate RNase, is very far from the optimal growth temperature of the hyperthermopilic micro-organism
Characterization of wild-typePf PDO
A detailed study of PfPDO structure highlighted certain putative ATP-binding sites (the presence of P-loops, a common motif in ATP-binding proteins), the primary structure of which consists of a glycine-rich sequence followed by a conserved lysine and a serine or a theonine [31] In particular, PfPDO has the sequences, GKDFG(88– 94), GLPAG(97–101), GKGKILG(167–173), which resemble, with some deviations, the glycine-rich motif, GXXGXG, of the ATPase domains of the eukaryotic chaperone hsp90 [32], the type II DNA topoisomerases, and MutL DNA mismatch-repair proteins [33] The hypothet-ical nucleotide-binding sites are presumably located in loops between b3 and b4, a4 and b4, and a6 and b6 To study the role played by the putative binding of ATP in the conformation of PfPDO, a spectrofluorimetric analysis was performed The presence of Trp184 enabled us to perform intrinsic fluorescence experiments The tryptophan emission spectrum of native PfPDO displayed a maximum around k¼ 345 (data not shown) A PfPDO sample incubated in the presence of hydrolysable ATP (MgATP) gave a similar spectrum to that of the native protein
Far-UV CD spectra in the presence and absence of ATP (data not shown) gave the same results as the spectrofluorimetric analysis, i.e no change in the conformation of the protein in the presence of the nucleotide These experiments indicate that the binding and/or hydrolysis of ATP do not have any effect on the conformation of PfPDO, possibly because of the localization of the amino-acid residues involved in ATP binding in exposed regions Near-UV CD spectroscopy was performed, which provides information on the environment
of aromatic residues in folded proteins The aromatic CD
Fig 2 Assay of oxidative activity by measuring the formation of the
disulfide bridge in the peptide NRCSQGSCWN (A)
Spectrophoto-metric method The disulfide bond-forming activity of PfPDO was
monitored using the synthetic decapeptide NRCSQGSCWN
Oxida-tion of this dithiol peptide to the disulfide state is accompanied by a
change in tryptophan fluorescence emission intensity (a) Control, the
same assay performed without the protein; (b) PfPDO; (c) PfPDO
(C35S); (d) PfPDO (C146S) (B) HPLC The disulfide bond-forming
activity of PfPDO is monitored using the synthetic decapeptide
NRCSQGSCWN and the oxidation of this dithiol peptide to the
di-sulfide state is accompanied by a change in time of retention on a
Vydac C18 Oxidative activity is expressed as a ratio between the peak
of oxidized and reduced peptide The assay was performed at 50 C,
with an incubation time of 210 min, at increasing concentration of
PfPDO wild-type (r), PfPDO (C35S) (j); PfPDO (C146S) (m);
PfPDO (C35S)/(C146S) and control (d).
Fig 3 Assay of isomerase activity of PfPDO by measuring re-activa-tion of scrambled RNase The recovery of RNase activity as a funcre-activa-tion
of time is presented after preincubation with PDI (m); PfPDO wild-type (r); PfPDO (C35S) and PfPDO (C146S) and PfPDO (C35S)/ (C146S) (j); control (d) RNase activity with RNA was measured.
Trang 7spectra of PfPDO in the absence and presence of ATP (up
to 324 mM) are shown in Fig 4 The ellipticity of the protein
was positive between 255 and 300 nm A signal around
279 nm can be assigned to tyrosine residues, and the major
intensity at 268 nm and 261.5 nm can be attributed to the
numerous phenylalanine residues (12 of them) After ATP
was added, the signal attributed to tryptophan and tyrosine
residues does not seem to have been affected, whereas the
signal attributed to phenylalanine residues changed
consid-erably
Interestingly, close to the P-loop domain, there is a
phenylalanine residue at position 91 In addition, our
spectra indicate that other aromatic residues are in close
proximity to the ATP-binding domain The CD data
indicate ATP binding with co-operativity and a Kd of
230 lM
The ATP binding to PfPDO was confirmed by
cross-linking to 8-azido-ATP after UV irradiation (Fig 5A,B)
The data show ATP binding for PfPDO Alcohol
dehy-drogenase (horse liver; Sigma) was used as a negative
control because it is known not to bind ATP, even though it
contains a putative nucleotide-binding site The alcohol
dehydrogenase did not show any affinity for the ATP
analog, suggesting that the binding to PfPDO was specific
under the conditions used It has been reported that some
non-ATP-binding proteins (for example, BSA) bind
8-azido-ATP in a nonspecific way However, in these cases,
the bound analog could not be displaced by the unlabeled
nucleotide [34] In this work, photoaffinity labeling of
PfPDO with 8-azido-[32P]ATP[aP] was decreased by the
presence of unlabeled ATP, indicating that ATP and the
analog 8-azido-ATP recognize the same binding site
The ATPase activity of PfPDO was demonstrated The
hydrolysis of ATP was linear for up to 30 min at every
temperature examined with the colorimetric and radioactive
assays used (see Experimental Procedures) The hydrolysis
of ATP by PfPDO required the presence of bivalent metal
ions, Mg2+giving the highest rate (Fig 6A) compared with
the activity observed in the absence of ions When assayed in the pH range 4.0–10.0, PfPDO catalyzed hydrolysis of ATP with a maximum around basic values (Fig 6B) Assays performed in the temperature range 30) 90 C showed that,
at 90C, PfPDO is still fully able to hydrolyse ATP (Fig 6C) The rate of spontaneous ATP hydrolysis was followed in the same range of temperature and pH Freshly purified PfPDO hydrolysed ATP with a Vmaxof 127.5 nmol
PireleasedÆmin)1Æmg)1(Mg2+, pH 10.0, 90C)
The ability of PfPDO to bind and hydrolyse ATP is another property that links this protein with the multifunc-tional PDI, as this feature has been observed in the eukaryotic protein [35]
In addition, as PfPDO exists as a dimer in the crystal form and PDI is a dimer in its 3D structure, we analysed the dimerization of PfPDO by gel filtration and in the presence
of the cross-linking agent DMS In all the conditions tested, the presence of the dimer was never observed It was observed only in the presence of the cross-linking reagent DMS In particular, a ratio of PfPDO to DMS of 1 : 2.5 proved to be optimal (Fig 7)
Discussion
Insufficient information is available on protein disulfide oxidoreductases from archaea to define their physiological function(s) with any certainty Disulfide bonds are now known to occur in many thermophilic and intracellular archaeal proteins, and this observation highlights the importance of the glutaredoxin/thioredoxin system in these micro-organisms
Hyperthermophiles are generally capable of growing under extreme conditions such as low pH, high pressure, and high salt concentration Most of these organisms are anaerobes, have extraordinarily heat-stable proteins, and use ingenious strategies for stabilizing nucleic acids and other macromolecules in vivo [36]
Fig 4 Measurement of K d for ATP by CD Near-UV CD spectra
recorded in 20 m M Tris/HCl (pH 7.5)/5 m M MgCl 2 and in the
pres-ence of increasing concentrations of ATP (0–324 m M ) The inset shows
normalized CD variation at 260 nm vs increasing [ATP]
concentra-tion CD values at 260 nm were normalized and elaborated using the
programme Microsoft Excel 2000 The curve for the determination of
the K d for ATP was obtained using the program KALEIDA GRAPH 3.0.
Fig 5 ATP-binding capacity of PfPDO Cross-linking of PfPDO with 8-azido-[ 32 P]ATP[aP]: 3 lg PfPDO was incubated with 2 mCi 8-azido-[ 32
P]ATP[aP] for 30 min at pH 8.0 and pH 10.0 at 60 and 70 C To induce cross-linking, samples were exposed for 10 min to UV irradi-ation and then resolved by SDS/PAGE in 12% polyacrylamide gel and visualized by radioautography (A) Lanes 1 and 2, pH 8.0 at 60 C and
70 C; lanes 3 and 4, pH 10.0 at 60 C and 70 C (B) The same procedure was used by incubating PfPDO at pH 10.0 at 70 C in the presence of increasing concentrations of unlabeled ATP Lane 1, 0 m M
unlabeled ATP; lane 2, 50 m M unlabeled ATP; lane 3, 1 m M unlabeled ATP.
Trang 8Recently, from the resolution of the whole genome
sequences of various hyperthermophilic archaea, it is clear
that these hyperthermophiles have proteins endowed with
thioredoxin/glutaredoxin motifs, suggesting the ubiquity of
this system in nature
The protein from P furiosus described here may provide
an important contribution to our understanding of the
function of these proteins in hyperthermophilic archaea and
bacteria In fact, PfPDO is able to catalyse the oxidation of
dithiols, as well as the reduction and rearrangement of
disulfides In the presence of glutathione, up to 70C,
PfPDO catalyses the formation of a disulfide bond between
the two cysteines of the peptide, an activity similar to that
observed for DsbA at 25C [13] At 30 C, PfPDO is able
to catalyse the reduction of insulin disulfides in the presence
of dithiothreitol Disulfide rearrangement was also observed
at a similar temperature using RNase with scrambled
disulfides as substrate
Using the two single mutants (C35S and C146S) and the double mutant (C35S/C146S), we have demonstrated that the C-terminal site (CPYC), which is common to all the glutaredoxins, determines the reductive activity This result
is in agreement with crystallographic data, which suggest a reductive nature for the C-unit The lower capacity of the N-unit to reduce disulfide bridges may be due to intrinsic factors, such as a higher redox potential and major conformational tension of the disulfide, but it may also depend on external factors such as steric impediments caused by a closed conformation of the active site in the N-unit As regards the oxidative activity, the two units also display differences in their functional properties, with the site at the C-termus always predominant, the mutant with a nonmutagenized site at the N-terminus showing very low activity at 50C Higher temperatures, closer to the physiological temperature at which the micro-organism
P furiosuslives, may be necessary to obtain more kinetic energy and allow an open conformation at the site Alternatively, a different substrate may be required because
of the polar nature of the amino acids close to the active site
On the other hand, both sites are necessary for the disulfide isomerase activity In fact, only wild-type PfPDO was able
to refold scrambled RNase This is in agreement with a functional model of PDI in which the domains function synergistically [37,38] The emerging model of PDI compri-ses four structural domains, a, b, b¢ and a¢, plus a linker region between b¢ and a¢ and a C-terminal acidic extension
In this model of PDI function, individual domains with specialized roles contribute to different activities to enable the catalysis of complex isomerizations in substantially folded protein substrates Mutations at the first cysteine of the active site in either the N-terminal or C-terminal thioredoxin domain inhibits the capacity of PDI to catalyse thiol–disulfide exchange reactions in vitro, reducing enzy-matic activity to negligible levels In fact, the redox/ isomerase activities of PDI, as in thioredoxin, are due to the reactivity of the N-terminal Cys residue in two
Fig 6 ATPase activity of PfPDO The assays were performed under
standard conditions (see Experimental Procedures) except for the ions
at 5 m M (A) The activity assayed under standard conditions (Mg2+at
90 C, pH 7.5) was 67.3 nmol P i releasedÆmin)1Æmg)1, which was taken
as 100% Activity was assayed at different pH values [50 m M sodium
acetate for pH 4.0–5.5 (m); 50 m M sodium phosphate for pH 6.0–7.0
(j); 50 m M Tris/HCl for pH 7.5–8.4 (e); 50 m M glycine/NaOH for
pH 9.0–10.5 (d)] (B) and temperatures (c) The activity assayed under
standard conditions was 127.5 nmol P i releasedÆmin)1Æmg)1(Mg 2+ ,
pH 10.0, 90 C), which was taken as 100% Data are means from at
least three independent experiments.
Fig 7 Cross-linking of PfPDO with DMS After 2 h of incubation at room temperature in the presence of the cross-linking agent DMS, the samples were loaded on an SDS/12.5% polyacrylamide gel Lane 1, PfPDPfPD/DMS in a ratio 1 : 2.5; lane 2, control, PfPDO with no DMS; lane 3, markers of molecular mass.
Trang 9thioredoxin-like boxes (Cys-Gly-His-Cys) within the a and
a¢ domains of the protein [39] Although the two domains
do not possess equivalent catalytic activities or
substrate-binding affinities, they can function independently from
each other
PfPDO resembles eukaryotic PDI, as it has two
thio-redoxin-like motifs In PDI, the thiothio-redoxin-like regions are
separated from each other in the primary structure, whereas
in PfPDO they are connected directly In this work, only the
first cysteine of each redox site was mutated to investigate
the effect on the function of the protein, demonstrating that
the active site at the C-terminus is basic for oxidative and
reductive activities and that the two units do not seem to be
functionally independent, considering that only the
wild-type enzyme is able to refold scrambled RNase Unlike PDI,
which is a homodimer of two 57 kDa subunits, PfPDO
seems to be a monomer, dimerization only occurring in the
presence of the cross-linking agent DMS
The ability of PfPDO to bind and hydrolyse ATP
supports its relationship to PDI [40] In fact, an
ATP-binding site and ATPase activity related to its chaperone
role have been reported in PDI [41] Whereas PDI binds
ATP with a Kdof 9.66 lM, PfPDO binds ATP with a Kdof
230 lM PfPDO is a hyperthermostable protein, and the
studies of its functional and catalytic properties are limited
by the temperature at which its activities are studied Such temperatures are usually far below the physiological tem-perature (70–103C) at which P furiosus lives The ATPase activity does not seem to be linked to the isomerase or redox activities, as in the presence of ATP no differences in the activities are observed This is in full agreement with a report that the site of phosphorylation, and thus probably the ATPase active site, lies somewhere within the central domain of the PDI [42], and that this site is far away from the redox active sites in the sequence Furthermore, the measurements of the rates of PDI-catalysed refolding of scrambled RNase A, in the absence or presence of ATP, show that ATP has little or no effect on this activity Interestingly, comparison of the genomes of archaea and bacteria showed the existence of a group of redox proteins with a similar molecular mass to PfPDO Clearly, all these proteins also contain two active sites, although they were often initially assigned as hypothetical thioredoxins and glutaredoxins [43–52] The presence of the redox site, CQYC, at the N-terminus of protein disulfide oxidoreduc-tase in P furiosus, P abyssi, and P horikoshii, and also in the more distant S solfataricus, further confirm the import-ance of this site for protein function (Fig 8) It is worth noting that amino-acid residues that are probably involved
in putative ATP binding, such as Gly88, Gly97, Pro99,
Fig 8 Comparison of the amino-acid sequences of different protein disulfide oxidoreductases The sequences were from the following sources: Pf,
P furiosus; Ph, P horikoshii; Pa, P abissi; Ss, S solfataricus; St, S tokodaii; Ap, Aeropyrum pernix; Ta, Thermoplasma acidophilum; Tv, Ther-moplasma volcanium; Fa, Ferroplasma acidarmanus; Tm, Thermotoga maritima; Aa, Aquifex aeolicus; Tt, Thermoanaerobacter tengcongensis The residues identical with the sequence of PfPDO in at least 90% of the sequences are indicated in bold The underlined residues indicate the active sites.
Trang 10Gly167 and Gly170, are well conserved, indicating their
importance The genomes of the hyperthemophilic bacteria
Aquifex aeolicus, Thermotoga maritima and
Thermoanae-robacter tengcongensisdo not encode a protein related to
bacterial DsbA and no DsbA-like protein in Archaea were
found, suggesting that PfPDO-like proteins represent a new
family characteristic of extremophiles (like DsbA in bacteria
and PDI in eukarya) It should be noted that we found
PfPDO-like proteins only in thermophilic bacteria, i.e
Aquifex aeolicus, Thermotoga maritima and
Thermoanae-robacter tengcongensis A preferential horizontal gene
transfer has been noticed between archaea and
hyperther-mophilic bacteria, such as Aquifex and Thermotoga; in fact
their proteins show greater similarity to archaeal than to
bacterial homologs [53] The reality of horizontal gene flow
from archaea to thermophilic bacteria becomes even more
tangible on examination of the proteins encoded in the
genome of Thermoanaerobacter tengcongensis which
con-tains more archaeal genes than appear in other bacteria
The exclusive presence of PfPDO-like proteins in
extremophiles may suggest that they have a special role
in the adaptation to extreme conditions The P horikoshii
genome also contains a glutaredoxin-homolog gene (88%
identity with the glutaredoxin from P furiosus) [54] This
protein is the first glutaredoxin-homolog protein that
directly mediates electron transfer from a thioredoxin
reductase-like flavoprotein to protein disulfide in archaea
The redox-active sequence motifs CPYC and CQYC
suggest that P horikoshii redox protein (PhRP) belongs
to the same family as PfPDO PhRP has insulin-reducing
activity Site-directed mutagenesis studies revealed that the
active site of the redox protein corresponds to a CPYC
sequence located in the middle of the sequence, as in
PfPDO As regards PhRP activities, the disulfide
forma-tion and its rearrangement were not detected when
reduced or scrambled RNases were used as substrates
at 25C However, the possibility that CQYC may play
some role and that PhRP has PDI-like activity in vivo at
the optimum growth temperature of P horikoshii cannot
be excluded
The various functions of PfPDO make it an interesting
model system for clarifying the long-standing debate on the
content of cysteine residues and disulfide in thermophilic
proteins Disulfide bonds have only rarely been found in
intracellular proteins The pattern is consistent with a
chemically reducing environment inside the cells and with a
PDI role in the endoplasmic reticulum However, recent
experiments and new calculations based on genomic data of
archaea provide striking contradictions to this pattern
Recent results indicate that the intracellular proteins of
certain hyperthermophilic archaea, especially some
cren-archaea such as Pyrobaculum aerophilum and Aeropyrum
pernix, are rich in disulfide bonds [55] This finding points to
the role of disulfide bonds in stabilizing many thermostable
proteins and suggests new chemical environments inside
these microbes
Acknowledgements
We thank Dr Raffaele Cannio and Dr Enrico Bucci for stimulating
discussions This work was supported by grants from MIUR (PRIN
2002).
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