From the Center for Cellular Switch Protein
Structure, Korea Research Institute of Bioscience and Biotechnology, 52 Euh-eun-dong, Yusong-gu, Daejon 305-806, South Korea and
§ Department of Food and Nutrition, College of Human
Ecology, Institute of Home Economics, Chonnam National University,
Gwangju, 507-757 South Korea
Received for publication, September 18, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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Cellular redox control is often mediated by oxidation
and reduction of cysteine residues in the redox-sensitive proteins, where thioredoxin and glutaredoxin (Grx) play as electron donors for
the oxidized proteins. Despite the importance of protein-protein interactions between the electron donor and acceptor proteins, there
has been no structural information for the interaction of thioredoxin
or Grx with natural target proteins. Here, we present the crystal
structure of a novel Haemophilus influenza peroxiredoxin (Prx) hybrid Prx5 determined at 2.8-Å resolution. The structure reveals that hybrid Prx5 forms a tightly associated tetramer where active sites of Prx and Grx domains of different monomers interact with
each other. The Prx-Grx interface comprises specific charge interactions surrounded by weak interactions, providing insight into
the target recognition mechanism of Grx. The tetrameric structure also
exhibits a flexible active site and alternative Prx-Grx interactions, which appear to facilitate the electron transfer from Grx to Prx domain. Differences of electron donor binding surfaces in Prx proteins
revealed by an analysis based on the structural information explain the
electron donor specificities of various Prx proteins.
Peroxiredoxin (Prx)1 is a
family of proteins that degrade reactive oxygen species in cells
(1-3). Members of the Prx family are implicated in the defense of
cells against oxidative stress as well as in the regulation of
important cellular processes such as transcription, apoptosis, and
cellular signaling (1-3). Prx proteins can be divided into two
subgroups, 2-Cys Prx (1) and 1-Cys Prx (4), depending on the number of
conserved cysteines. 2-Cys Prx proteins have two conserved cysteines,
one in the N-terminal domain and the other in the C-terminal domain.
The N-terminal cysteine directly reacts with reactive oxygen species
substrates, resulting in the formation of cysteine sulfenic acid. The
C-terminal cysteine, which is in the vicinity of the N-terminal
cysteine of the other monomer in the dimeric structure found in most
2-Cys Prx proteins, forms a disulfide bond with the N-terminal cysteine sulfenic acid (5, 6). The disulfide bond is reduced by thioredoxin (Trx) to regenerate enzyme activity (1). In comparison, 1-Cys Prx has
only the N-terminal cysteine, and enzyme activity of 1-Cys Prx usually
does not involve either intermolecular disulfide bond formation or
thioredoxin reduction (4). However, some 1-Cys Prx proteins have an
extra non-conserved cysteine that plays the role of the conserved
C-terminal cysteine in 2-Cys Prx (7). For example, human Prx5, which is
a 1-Cys Prx implicated in tumor necrosis factor Crystal structures for 1-Cys Prx (hORF6 (9) and Prx5 (10)) and 2-Cys
Prx (HBP23 (6), TpxB (11), TryP (12), and AhpC (13)) proteins were
reported. These structures revealed that Prx proteins have a
thioredoxin fold with several insertions between secondary structural
elements of the core thioredoxin fold. All Prx proteins except for
human Prx5 were found as tightly associated dimers ( Recently, novel hybrid Prx proteins with a glutaredoxin (Grx) domain
fused in them were found from several pathogenic bacteria including
Haemophilus influenza (GenBankTM accession
number P44758) (16), Neisseria meningitidis
(GenBankTM accession number CAB94403), and Vibrio
cholerae (GenBankTM accession number AE004330). An
anaerobic sulfur-oxidizing phototroph, Chromatium gracile
also has the hybrid Prx (17). The Prx and Grx domains of these hybrid
proteins have sequence homology with human Prx5 and Escherichia
coli Grx3, respectively (16, 18). The fusion of Grx domain in
these hybrid Prx5 proteins suggests that Grx domain is likely to act as
the electron donor for Prx domain of these proteins (18). H. influenza hybrid Prx5, which is a prototype example of these
hybrid Prx proteins, was first found from a sequence search of the
bacterium's genome and originally named as HI0572 (16). There are
three cysteines in H. influenza hybrid Prx5 that comprise a
total of 241 residues. The first cysteine, Cys-49, corresponds to the
N-terminal cysteine that is absolutely conserved throughout all Prx
proteins. The second and third (Cys-180 and Cys-183) are the two
cysteines in the conserved CXXC motif of Grx domain. There
are no non-conserved cysteines in H. influenza hybrid Prx5.
We determined the crystal structure of H. influenza hybrid
Prx5 (hyPrx5) to understand the functional role of fusion between Prx
and electron donor proteins as well as electron donor specificity. The
tetrameric structure of hyPrx5 reveals detailed information on the
conformation of catalytic sites that account for the
glutathione-dependent peroxidase activity of hyPrx5. The
tetramerization is mediated by interconnecting Prx and Grx domains of
the protein, suggesting that the fusion is essential for the
tetramerization. Most importantly, Prx and Grx domains of different
monomers form significant interaction with each other to allow the
intermolecular electron transfer between the two domains. The Prx-Grx
interaction found in the hyPrx5 tetramer reveals the atomic-level
features of the Grx surface used for the interaction with an intact
protein target. Thus, the tetrameric structure of hyPrx5 provides a
framework in understanding the mechanism of target recognition by Grx
as well as activities of the Prx-Grx hybrid proteins.
Crystallization and Structure Determination--
HyPrx5 was
crystallized at room temperature by the vapor diffusion method. For the
crystallization, 2 µl of reservoir solution (1.6 M
ammonium sulfate, 0.2 M sodium acetate, 0.1 M
Tris-HCl (pH 8.0)) and 2 µl of protein solution (10 mg/ml) were mixed
and equilibrated against the reservoir. Tetragonal crystals appeared within 2 or 3 days and grew to the their full size of 0.5 × 0.3 × 0.3 mm after 1 week. The crystals belong to the
P43212 space group with cell constants of
a = b = 72.85 Å, c = 229.63 Å. There are two molecules of hyPrx5 in the asymmetric unit.
The multi-wavelength anomalous diffraction (MAD) (19) data were
collected at the Photon Factory beamline 18B by using a crystal grown
from selenomethionyl-substituted protein. Data collected at three
wavelengths (peak, edge, and remote) were integrated and scaled with
the programs MOSFLM (20) and SCALA in the CCP4 suite (21).
Heavy atom search by using the program SHAKE-AND-BAKE (22) located 12 of 14 selenium sites, and heavy atom parameters were refined by using
the program SHARP (23). Phases were optimized with solvent-flattening
and 2-fold NCS averaging by using the program DM (21). A summary of
data collection, phasing, and refinement statistics is shown in Table
I. The model was built by using the
program O (24) and refined with the program CNS (25). The randomly
selected 5% of the data were set aside for the
Rfree calculation. Refinement included 2-fold
NCS restraints, an overall anisotropic B factor, and bulk solvent
correction. The data set collected at the edge wavelength ( Analytical Ultracentrifugation--
Sedimentation equilibrium
analysis (31) was performed with the Beckman Optima XL-A and An-60Ti
analytical rotor at 20 °C. The air-oxidized hyPrx5 protein (~0.5
mg/ml) was prepared in 20 mM Tris-HCl (pH 7.5) and 100 mM NaCl. For the preparation of the reduced hyPrx5 sample,
1 mM dithiothreitol (about 80-fold molar excess for the
reduction of disulfide bond in hyPrx5) was supplemented in the hyPrx5
solution, and the sample was preincubated for 1 h before the
experiment. The sample absorbance was monitored at 280 nm during
centrifugation at 8,000 rpm until equilibrium was reached (about
24 h). Data were fitted using the equation corresponding to IDEAL1
model implemented in Beckman data analysis software, Aobs = A0exp[((1 Overall Structure--
The refined structure of hyPrx5 reveals two
discrete domains, peroxiredoxin (residues 3-162) and glutaredoxin
(residues 171-241) domains with a connecting loop (residues 163-170)
(Fig. 1a). No direct
interactions are found between Prx and Grx domains in the monomeric
structure. There are two monomers of hyPrx5 in the asymmetric unit of
crystals, and the two monomers have different domain arrangements enabled by the flexible linker between Prx and Grx domains (Fig. 1b). The dimer in the asymmetric unit associates with
another dimer generated by a crystallographic 2-fold symmetry operation of the first dimer, resulting in a tightly associated tetramer (Fig. 1,
c and d). The tetramer has a donut shape with the
outer and inner diameters of about 85 and 18 Å, respectively (Fig.
1c). When viewed from the side, the tetramer appears as a
cylinder of 65 Å height (Fig. 1d).
Prx domain of hyPrx5 has a typical thioredoxin fold, which comprises
the central Tetrameric Association--
The tetramerization of hyPrx5 is
achieved mainly by two strong subunit contacts, Prx-Prx (monomers A-B
and C-D) and Grx-Grx (monomers A-D and B-C) contacts (Fig. 1,
c and d). In addition to these contacts, the
Prx-Grx interaction, which has important implications in the peroxidase
mechanism of hyPrx5, is also involved in the tetramerization (see
below). To verify the tetramerization of hyPrx5 in solution we
determined the molecular weight of hyPrx5 by analytical
ultracentrifugation (Fig. 3). In oxidized and
reduced conditions, the molecular masses were estimated to be 117.0 and 115.1 kDa, respectively. These values are close to the expected tetrameric molecular mass of hyPrx5 (~108 kDa), indicating that hyPrx5 forms a tetramer in solution and the oligomeric state is not
dependent on redox conditions. Dynamic light scattering and gel
filtration experiments showed similar results (data not shown). The
redox-independent tetramerization of hyPrx5 should be important for its
biological function since the electron transfer reaction between the
Prx and Grx domains occur in the tetrameric interface, and the
tetramerization maintains the optimal geometry between the Prx and Grx
domains fused by a flexible linker (see below).
The Prx-Prx contact in hyPrx5 is completely different from the dimeric
interface in other Prx proteins (6, 9-12). The dimeric contact between
two Prx domains of hyPrx5 is formed by the face perpendicular to the
direction of strands in the central sheet, whereas that of other Prx
proteins makes use of the face parallel to the direction of strands.
The Prx-Prx contact of hyPrx5 involves residues in loops
The Grx-Grx interface of hyPrx5 is formed by donating helix Active Sites--
HyPrx5 has two redox-active sites; one in the
Prx domain (Cys-49) is used to directly react with reactive oxygen
species substrates, and the other in the Grx domain (Cys-180 and
Cys-183 in the CXXC motif) reduces the oxidized active site
cysteine in Prx domain to regenerate peroxidase activity of the enzyme.
The most striking thing in the tetrameric structure of hyPrx5 is that
active sites of the Prx and Grx domains from different monomers come
close to each other in the tetrameric state (Figs. 1c and
4a), whereas the two active sites in the same monomer are
placed distantly. The C
Oxidized Grx proteins are reduced by reduced glutathione (GSH) via the
Grx-SG mixed disulfide intermediate. The structure of E. coli Grx3 in complex with GSH (Grx3-SG) (33) showed that GSH binds
to a concave surface near the CXXC motif. In the complex structure, the main chain atoms of the Cys residue in GSH form hydrogen
bonds with those of Val-52 in Grx3 (corresponding to Val-220 in hyPrx5
(Fig. 2)), resulting in an antiparallel intermolecular
Two crystallographically independent molecules in the asymmetric unit
of hyPrx5 crystals exhibit different environments for the reactive
cysteine (Cys-49) that also affect the Prx-Grx interface in the
tetrameric structure (see below). In one hyPrx5 molecule (monomer A),
the reactive cysteine is located at the start of helix
In the other hyPrx5 molecule (monomer B) of the asymmetric unit, the
region of active site cysteine (Cys-49) is unwound from helix Prx-Grx Interaction--
In the hyPrx5 tetramer, the surface near
the CXXC motif of Grx domain, which is at one end of the
central
Although Prx domain of hyPrx5 accepts electrons from Grx domain, human
Prx5 with strong homology with hyPrx5 uses only Trx for the electron
donor (7). Examination of the molecular surface of human Prx5 provides
an explanation for the specificity of electron donors (Fig.
5c). The surface of human Prx5 equivalent to the Grx
interaction surface of the hyPrx5 Prx domain is mostly hydrophobic, and
there are no negatively charged surface patches for the interaction with Grx. The hydrophobic surface is likely to interact favorably with
the hydrophobic surface of Trx (Fig. 5d) that is located at
the equivalent place on the Prx interaction surface of the hyPrx5 Grx
domain. The same hydrophobic surface of Trx is also used in the
interaction with Trx reductase (37) that donates electrons to Trx.
Recently, another member of Prx with high homology with human Prx5,
poplar Prx, was reported to exhibit dual specificity for electron
donors (18). That is, poplar Prx accepts electrons from both Grx and
Trx. In poplar Prx, the negatively charged residues (Glu-149 and
Glu-156 of poplar Prx for Asp-148 and Asp-156 of hyPrx5, respectively)
playing an important role in the interaction with Grx are conserved
(Fig. 2). In addition, the electron donor binding surface of poplar Prx
has a Trx-favoring character, too. In poplar Prx, Ser-51 of the hyPrx5
Prx domain is replaced with a hydrophobic residue, Leu-53, which is
likely to enable poplar Prx interact with Trx as well as Grx (Fig.
2).
The tetrameric structure of hyPrx5 reveals for the first time the
interaction of Grx with its natural target protein (the hyPrx5 Prx
domain). In the interaction between Prx and Grx domains of hyPrx5, the
major interaction force is derived from two charge interactions. Other
parts of the interaction are made of weak hydrogen bonds and van der
Waals interactions, indicating that the interaction between Grx and Prx
domains is less specific than the usual protein-protein interactions.
Previously, the complex structure of human Trx with peptide fragments
of target proteins, NF
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signaling (7) and
apoptosis regulation (8), uses thioredoxin as the electron donor, and
the disulfide bond in human Prx5 is formed between the N-terminal
conserved cysteine and the extra non-conserved cysteine (7).
2)
or decamers ((
2)5) utilizing the dimer as a
basic unit. 2-Cys Prx proteins exhibit the structural transition between the dimeric and decameric states depending on environmental status such as redox states, ionic strength, and pH (11, 13-15). For
example, the Salmonella typhimurium AhpC, a 2-Cys Prx,
strongly favors the decameric structure in the reduced state, whereas
the oxidized enzyme exists as a mixture of lower order oligomeric assemblies (13). Crystal lattices of human Prx5 (10) do not show the
same dimer found in other Prx proteins, although the possibility of
dimerization was reported previously (7).
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= 0.9795), which was nearly complete, was used in the refinement. The
stereochemical analysis using the program PROCHECK (26) showed that two
residues are in generously allowed regions, and there is no residue in disallowed regions. The final model contains residues 3-239 in molecule A, residues 3-241 in molecule B, and 4 sulfate ions. The
R-value of the final model is 23.9%
(Rfree = 28.3%) at 2.8-Å resolution. The
figures were prepared by using the programs GRASP (27), RIBBONS (28),
MOLSCRIPT (29), and BOBSCRIPT (30).
Crystallographic data
s
)
2/2RT)Mr(r2
r02)] + E, where
s is the partial specific volume of macromolecule (0.736 ml/g),
is the solvent density (1.002 g/ml),
is the angular
velocity of the rotor, R is the universal gas constant, T is the absolute temperature, A0 is
the absorbance at reference at radius r0,
r0 is the reference radius,
Mr is the gram molecular weight of the
macromolecule, and E is the base-line offset. The program
SigmaPlot v5.0 (SPSS Inc.) was used for the data fitting.
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Fig. 1.
Monomeric and tetrameric structures.
a, the monomeric structure of hyPrx5 with secondary
structural elements. The redox active cysteines (Cys-49 in Prx domain
and Cys-180 and Cys-183 in Grx domain) were drawn in a
ball and stick representation. Cys-180 and
Cys-183 are disulfide-bonded. The boundaries for secondary structural
elements are 1 (13-18),
2 (21-26),
3 (35-41),
4
(71-76),
5 (96-99),
6 (112-114),
7 (122-124),
8
(127-132),
9 (135-141),
10 (172-176),
11 (198-201),
12
(222-225),
13 (228-230),
1 (27-31),
2 (47-66),
3
(80-89),
4 (104-108),
5 (155-162),
6 (181-193),
7
(208-214), and
8 (233-238). b, superposition of two
hyPrx5 monomers. C
-carbon traces of two monomers in the asymmetric
unit are presented as superposed by using C
-carbon atoms of Prx
domain. Monomers A and B are colored black and
red, respectively. c, the tetrameric structure of
hyPrx5 seen from above the tetramer. Each monomer with a different
color is labeled as A, B, C, and
D, counterclockwise. C
positions of the redox active
cysteines in Prx and Grx domains are indicated as pink and
yellow spheres, respectively. d, the tetrameric
structure of hyPrx5 seen from the side with the point of view
perpendicular to that of a.
-sheet with four strands (
3,
4,
8, and
9)
and three helices (
2,
4, and
5) flanking the sheet (Figs. 1a and 2). In addition to the
thioredoxin fold, Prx domain of hyPrx5 has several insertions, (i)
N-terminal two antiparallel
strands (
1 and
2) followed by a
short
-helix (
1), (ii) helix
3 and strand
5 between strand
4 and helix
4, (iii) two short antiparallel
-strands (strands
6 and
7) between helix
4 and strand
8. A search for
homologous structures by using the DALI server (32) confirmed that the
structure of Prx domain of hyPrx5 is similar to that of other Prx
proteins. Of these, the structure of human Prx5 (10) is most similar to
that of the hyPrx5 Prx domain. A total of 151 of 160 C
atoms in the
hyPrx5 Prx domain can be aligned with the corresponding atoms of human
Prx5 with an root mean square deviation of 1.50 Å. The structure of
the hyPrx5 Grx domain is very similar to that of Grx3 (33), comprising a central
sheet with four strands (
10,
11,
12, and
13)
flanked by three helices (
6,
7, and
8) (Figs. 1a
and 2). The root mean square value of C
-carbon alignment between the
hyPrx5 Grx domain and Grx3 is 1.32 Å for 66 of 69 residues in the
hyPrx5 Grx domain.
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Fig. 2.
Sequence alignment of hyPrx5 and related
proteins. Sequences of four representative hyPrx5 proteins from
different species (H. influenza, Neisseria
meningitis, V. cholerae, and C. gracile),
poplar Prx, human Prx5, and E. coli Grx3 are aligned using
the program CLUSTALW (41). Secondary structural elements of
H. influenza hyPrx5 are indicated above the
aligned sequences. The Prx and Grx domains are colored blue
and red, respectively. The linker region between the two
domains is indicated as a green line. Residues with 100%
identity are colored yellow, and those with a conservation
value above 7.0 defined in the program ALSCRIPT (42) are colored
cyan. In the assignment of conservation values, the Prx and
Grx domains were treated separately. The redox-active cysteines
(Cys-49, Cys-180, and Cys-183) are indicated as inverted
triangles.
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Fig. 3.
Analytical ultracentrifugation. The
oxidized (a) and reduced (b) hyPrx5 samples were
centrifuged at 8000 rpm with successive measurements of absorbance
(Abs) at 280 mm until equilibrium. The absorbance data were
fit with curves for molecular masses of 117.0 and 115.1 kDa for the
oxidized and reduced samples, respectively. Both values are close to
the expected molecular weight of the hyPrx5 tetramer. The oxidized
hyPrx5 sample was loaded into the first sample-channel (channel range,
5.8-6.1 cm from the center of rotation), whereas the reduced hyPrx5
sample was loaded into the second channel (channel range, 6.4-6.7 cm
from the center of rotation).
3-
2
(residues 45-47),
4-
3 (residues 81-82),
5-
4 (residues
101-102), and
6-
7 (residues 118-121). In addition, residues
Trp-22, Asp-79, Ala-85, and Arg-123, whose side chains are near the
loop, regions play an important role in the dimerization. The
interface, which buries 10.5% (826 Å2) of the total
surface area of Prx domain, is highly complementary and involves mainly
hydrophobic interactions with a salt bridge (Arg-A123-Asp-B79). The
same dimeric association is found also in the structure of human Prx5
that is highly homologous to Prx domain of hyPrx5. Even though the
report (10) describing the structure of human Prx5 did not mention
dimeric contact, the crystal lattice assembled by using the reported
structure shows the same dimeric association. Conservation of the
dimeric contact in the two homologous proteins indicates that the
dimeric association may play an important role in the function of
human Prx5 as in hyPrx5. Previous biochemical studies also suggested
the dimerization of human Prx5 in solution (7).
7 of
each monomer to the dimeric interface, resulting in a five-layered
sandwich structure. Helix
7 of the first monomer is
placed above the central
sheet of the monomer and interacts with
the central
sheet of the next monomer. There are also strong
interactions between the two helices. The Grx-Grx interface, which is
predominantly made of hydrophobic interactions, includes residues in
helix
7 (residues 208-210 and 212-214) and loop
11-
7
(residues 205-207). The interface buries 716 Å2 that
corresponds to 17.0% of the total surface area of Grx domain. Helix
7 is conserved in other Grx proteins, suggesting a possibility of
dimerization in other Grx proteins, too. Consistent with the possibility, an NMR study of Grx1 including sedimentation equilibrium measurements (34) showed that the protein had monomer/dimer equilibrium
in its oxidized state, whereas the reduced form was monomeric.
distance between Cys-A49 of monomer A and
Cys-D180 of monomer D is 15.08 Å, and there is no intervening
structure between the two residues. Although the distance is not close
enough for forming a disulfide bond, we propose that the local
conformation change (see below) could easily bring the two cysteines
into the proximity. For example, the C
distance between the
disulfide bond partner cysteines in reduced state of human Prx5 is
13.84 Å, and a local conformation change is thought to enable the
disulfide bond formation (10). In comparison, the distance between
Cys-A49 and Cys-A180 of the same monomer A is 32.08 Å, which is too
far for a disulfide bond formation without a major change in the
tetrameric association.
-bridge (33).
When we superimposed the structure of the hyPrx5 Grx domain with that
of Grx3 in complex with GSH, the GSH-interacting region of Grx3 was
very well aligned with the corresponding region of the hyPrx5 Grx
domain (data not shown), indicating that GSH is likely to bind to the
hyPrx5 Grx domain involving the formation of intermolecular
-bridge
as it does to Grx3. In the similar superposition carried out with the
tetrameric hyPrx5, we found that the location of GSH bound to Grx3
corresponds to the intermolecular cleft in the hyPrx5 tetramer between
Grx domain of molecule D and Prx domain of molecule A (Fig.
4a), suggesting that GSH is able
to reduce the oxidized Grx of hyPrx5 without affecting the tetrameric
geometry. Thus, the tetramerization of hyPrx5, which is possible by the
fusion of Prx and Grx domains, seems to play an important role in
increasing the antioxidant enzyme efficiency by forming optimal
geometry for both peroxidase and reduction reactions, which is
necessary for the pathogenic bacterium H. influenza in
defending itself against the antimicrobial defense system of host
phagocytes.
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Fig. 4.
Environment of the redox active site.
a, Prx-Grx interaction and glutathione binding in the hyPrx5
tetramer. Glutathione model (shown in the figure as a ball
and stick diagram) was created by superposing the complex
structure of Grx3-glutathione (PDB code 3GRX) on the hyPrx5 Grx domain.
Surfaces of Prx (monomer A) and Grx (monomer D) domains are colored
red and gold, respectively. Redox active
cysteines (Cys-49 and Cys-180) and residues involved in the Prx-Grx
interaction are labeled. b, stereo view of the
2Fo Fc electron density map.
The electron density map around the Prx active site of hyPrx5 is
presented as superimposed with the refined model. The map was contoured
at a 0.9
level. c, stereo view of the Prx active site of
hyPrx5. The side chains of residues (Arg-126 and Thr-46) contributing
to the reactivity of the sulfur atom of Cys-49 were drawn as a
ball-and-stick representation. Distances between
the interacting atoms are shown in the figure.
2 as in other
Prx proteins (3). The positively charged side chain of Arg-126 makes
good interaction with the sulfur atom of Cys-49 (distance, 3.04 Å)
(Fig. 4, b and c), which stabilizes the thiolate
form of Cys-49 as in other Prx proteins (3). In addition, the sulfur
atom forms a hydrogen bond with the side chain of Thr-46 (distance,
3.09 Å) (Fig. 4, b and c) that is also strongly
implicated in the stabilization of the thiolate form (3). These strong
charge and hydrogen bond interactions of the active site sulfur atom
should contribute to increasing nucleophilic reactivity of the sulfur
atom of hyPrx5.
2, and
the active site Cys-49 becomes a part of loop
3-
2. The loop makes
no significant interactions with other parts of the tetramer,
indicating that the region may be flexible in solution. The unwound
structure would have an important role in the formation of the mixed
disulfide bond formation. Because of the unwinding, the C
distance
between Cys-B49 and Cys-C180 becomes 11.63 Å, indicating that the
conformation change and flexibility of the redox loop containing
Cys-B49 may bring the cysteine into the distance needed for the
disulfide bond formation with Cys-C180. Consistent with the observation
in the hyPrx5 structure, the helix unwinding near the active site
cysteine of 2-Cys Prx proteins was proposed earlier for the mechanism
of the disulfide bond formation between two distant cysteines in TpxB
(11). The flexibility of the redox active loop in the
redox-dependent transcription factor, OxyR, also plays a
role in the disulfide bond formation between two distant cysteines
(35).
sheet, interacts with the active site region of Prx domain
(Fig. 5, a and b).
Because of different domain organizations of two monomers in the
asymmetric unit of hyPrx5 crystals (Fig. 1b), the tetramer
has two different types of Prx-Grx association. The interaction surface
of Grx domain, which comprises two prominent positive-charged regions
(Fig. 5b), forms charge interactions with the Prx domain.
Although the Prx interaction surface of Grx domain is the same in both
interactions, there are two different Grx interaction surfaces of Prx
domain (Fig. 5a). In monomers A(Prx)-D(Grx) interaction
(equivalent to monomers C(Prx)-B(Grx) interaction),
Arg-D212 and Lys-D177 form charge interactions with the
Asp-A154-Asp-A156 patch and Asp-A148, whereas in monomers
B(Prx)-C(Grx) interaction (equivalent to monomers D(Prx)-A(Grx)
interaction) the same residues of Grx domain (Arg-C212 and Lys-C177)
interact with the Asp-B89-Glu-B90 patch and Glu-B59. In A(Prx)-D(Grx)
interactions, Ile-D208 that protrudes from the surface fits into a
shallow pocket made by Phe-A150. The A(Prx)-D(Grx) and B(Prx)-C(Grx)
interactions bury 255 and 222 Å2 of intermolecular
surfaces, respectively. Interestingly, the two different interaction
surfaces are correlated with conformation of the redox active site of
Prx domain. That is, in A(Prx)-D(Grx) interaction, the Prx active site
(monomer A) is well arranged for the catalytic activity (Fig. 4,
b and c), whereas in the B(Prx)-C(Grx) interaction, the Prx active site (monomer B) is unwound and flexible (see "Active Sites"), suggesting that A(Prx)-D(Grx)
interaction may be the form of the initial peroxidase reaction, and
B(Prx)-C(Grx) interaction may be used for the reduction of oxidized Prx
domain by the Grx domain of the neighboring monomer. Thus, the Prx-Grx interaction in the hyPrx5 tetramer is likely to alternate between the
two states in a coordinate fashion during the peroxidase and reduction
reactions, maintaining overall geometry of the tetramer. The possible
flip-flop mechanism of hyPrx5 indicates that hyPrx5 may be a new
example of multimeric enzymes with half-of-the-sites reactivity, where
only half of multiple active sites are catalytically active at a time
(36).
View larger version (49K):
[in a new window]
Fig. 5.
Interaction surfaces implicated in the
Prx-Grx or Prx-Trx interaction. Electrostatic potential surfaces
of the hyPrx5 Prx domain, the hyPrx5 Grx domain, human Prx5 (PDB code
1HD2), and human Trx (PDB code 1ERU) were calculated by using the
program GRASP (27). Positive and negative charges are represented as
blue and red, respectively. a, the
surface of the hyPrx5 Prx domain. Residues involved in the Prx-Grx
contact in the hyPrx5 tetramer are labeled and surrounded by a
yellow lines. The alternative interaction surface (see
"Prx-Grx Interaction") is indicated with a green
line. b, the surface of the hyPrx5 Grx domain. Residues
involved in the Prx-Grx contact of in the hyPrx5 tetramer are labeled
and surrounded by a yellow line. c, the surface
of human Prx5. Residues of human Prx5 corresponding to those
participating in the Prx-Grx contact of hyPrx5 are labeled and
surrounded by a yellow line. The point of view in the figure
is the same as in a. The orientation was determined by
superposing the two structures (the hyPrx5 Prx domain and human Prx5).
Thr-48, Ser-51, Phe-150, Asp-154, and Asp-156 of the hyPrx5 Prx domain
correspond to Gly-46, Lys-49, Leu-149, Leu-153, and Pro-155 of human
Prx5, respectively (for the sequence alignment, see Fig. 2).
d, the surface of human Trx. Residues of human Trx involved
in the Trx-Trx reductase contact (PDB code 1F6M) are labeled and
surrounded by a yellow line. The point of view in the figure
is the same as in b. The orientation was determined by
superposing the two structures (the hyPrx5 Grx domain and human Trx).
The C trace of the NF
B peptide bound to human Trx (Ref. 38, PDB
code 1MDI) was drawn as a cyan tube.
B (38) and Ref-1 (39), revealed that the
target peptides bind a crescent-shaped groove near the reactive
cysteine of Trx (Fig. 5d). The peptide interaction surface
corresponds to the upper part of the Prx interaction surface of the
hyPrx5 Grx domain. However, the peptide interaction could not predict
the complete surface of Trx for the interaction with three-dimensional
target proteins. In contrast, the Prx interaction surface of the hyPrx5 Grx domain provides information on the surface needed to interact with
an intact protein target. The nature of Prx-Grx interaction involving
weak interactions together with the pivotal charge interactions seems
to allow Grx to interact with diverse target proteins having variations
of the surface.
![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. N. Sakabe and the staffs of the Photon Factory beamline BL18B for help with data collection.
![]() |
FOOTNOTES |
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* This work was supported by a National Creative Research Initiatives grant from the Ministry of Science and Technology, Korea.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.
The atomic coordinates and the structure factors (code 1NM3) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ To whom correspondence should be addressed. E-mail: ryuse@mail.kribb.re.kr.
Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M209553200
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ABBREVIATIONS |
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The abbreviations used are: Prx, peroxiredoxin; Trx, thioredoxin; Grx, glutaredoxin; hyPrx5, hybrid Prx5.
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