From the
Division of Biological Chemistry and Molecular Microbiology, School of
Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom,
Macromolecular Crystallography Group, European
Synchrotron Radiation Facility (ESRF), BP 220, F-38043 Grenoble Cedex 9,
France, the
European Molecular Biology
Laboratory Grenoble outstation, 6 rue Jules Horowitz, BP 156, F-38042 Grenoble
Cedex 9, France, and the ¶Institut de Biochimie
et Génétique Cellulaire, UMR CNRS 5095, Université Victor
Segalen Bordeaux II, 1 Rue Camille Saint-Saëns, 33077 Bordeaux Cedex,
France
Received for publication, February 21, 2003
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ABSTRACT |
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INTRODUCTION |
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The first crystal structure of tryparedoxin, the protein from Crithidia
fasciculata (CfTryX1), revealed a compact globular molecule
classed in the same fold family as the functional homologue thioredoxin (TrX,
see Fig. 2a)
(5). The TrX fold is based on a
twisted five-stranded central -sheet with two helices on either side
(6,
7), but although classed in the
same family, TryX is distinct from TrX in several respects
(5). The parasite protein is
significantly larger,
16 kDa as compared with 12 kDa for TrX, and carries
additional elements of secondary structure, in particular a
-hairpin at
the N terminus. The relationship of secondary structure with the amino acid
sequence is so different for TryX and TrX that it is meaningless to compare
the overall sequences. A noteworthy similarity is, however, the presence of a
redox-active disulfide at the N terminus of an
-helix. In TrX, this
disulfide is contained in the motif Trp-Cys-Gly/Ala-Pro-Cys, whereas in TryX,
the motif is Trp-Cys-Pro-Pro-Cys. A least-squares fit of the central
-strands of TrX and TryX align these motifs on top of each other
(5).
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We now report structures for a second isoform of C. fasciculata TryX (CfTryX2) and provide brief comparisons with CfTryX1 and structures of the disulfide form and chemically reduced CfTryX2 recently published (9). Unable to crystallize TryX as a homogeneous free dithiol form, we sought to generate this structure by photoreduction of the redox-active disulfide of CfTryX1 using the intense x-ray beam available from an undulator beamline at the European Synchrotron Radiation Facility (ESRF). As a control for this novel experiment and to study the effect of removing the disulfide linkage, Cys-43 was mutated to alanine, and the structure (CfTryX-C43A) was determined to 1.30-Å resolution. We also determined the structure of Trypanosoma brucei tryparedoxin (TbTryX) at 2.3-Å resolution, which presents an active site significantly different from any other TryX structure. Analysis of the interactions between TbTryX and a symmetry-related molecule suggests structural alterations that may be relevant to the interaction between TryX and the partner peroxidase, TryP. Based on crystal structures of a chloroplast TrX (10), one of its redox partners, fructose-1,6-bisphosphate phosphatase (11), and our own sequence analyses, we propose that the conformational lability of the tryptophan lid may contribute to specific redox events.
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MATERIALS AND METHODS |
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All recombinant proteins were expressed in Escherichia coli strain BL21 (DE3). Expression and purification protocols followed those published by Alphey et al. (13) and involved the use of metal ion affinity chromatography to exploit the presence of the N-terminal histidine tag, which was introduced by using the pET-15b vector and which was subsequently removed by cleavage with thrombin (Amersham Biosciences). Protein concentration was determined spectrophotometrically at 280 nm using a theoretical extinction coefficient of 38030 M1 cm1 (14), and purity was evaluated using SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
Crystallization, Data Collection, and Data
ProcessingCrystals were grown using the hanging drop vapor
diffusion setup, and diffraction data were processed, reduced, and scaled
using the HKL (15) and CCP4
suite of programs (see Table I)
(16). Crystals of
CfTryX1 and CfTryX-C43A were obtained using the published
conditions (13). Two
tetragonal crystal forms (A and B) of CfTryX2 were obtained. Form A
presented as rods and appeared in drops containing 10 mg
ml1 protein, 15% w/v polyethylene glycol 8000, 30
mM sodium cacodylate, pH 6.5, 5 mM dithiothreitol, and
60 mM ammonium sulfate. These crystals display space group
P42212 with unit cell dimensions of a =
b = 111.7, c = 56.5 Å and are isomorphous to samples
studied by Hofmann et al.
(9). Form B crystals displayed
a bipyramidal morphology and grew from solutions of
10 mg
ml1 protein, 500 mM sodium citrate, 30
mM sodium HEPES, pH 7.5, 5 mM dithiothreitol. They are
in space group P41212 with unit cell dimensions of
a = b = 114.3, c = 102.0 Å. Single crystals
of both forms were cryo-protected by soaking in crystallization mother liquor
containing either 15% (form A) or 10% (form B) of glycerol prior to transfer
in a nitrogen gas stream at 170 °C. A single crystal of form A was
used on the ESRF bending magnet beamline BM14, and data collection was carried
out at
= 0.977 Å to dmin = 1.5 Å with
an MarCCD133 detector in a single sweep totaling 90o of oscillation
in 0.5o steps. For form B, a single crystal was mounted on the ESRF
undulator beamline ID14-EH2, and data were collected at
= 0.933
Å using an ADSC QUANTUM4 detector. Despite the relatively large size of
the crystal used for data collection (
250 x 150 x 150
µm3), diffraction maxima were only visible to
2.2-Å
resolution, and these were only apparent after a relatively long exposure time
of 45 s/0.5o oscillation. Radiation damage was evident after 75
images, and the crystal was translated such that a fresh section was exposed
to the x-ray beam and a further 44 0.5o images were collected. Both
batches of data were processed and scaled together yielding a data set
complete to 2.35 Å.
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For the time course experiment on CfTryX1 and the analysis of
CfTryX-C43A, crystals were cryo-protected with 40% polyethylene
glycol monomethyl ether 2000 and then flash-cooled at 170 °C. Data
were collected on ID14-EH2 ( = 0.933 Å) using the STRATEGY
program (17) to determine the
angular range for collection. For the time course experiment, a series of data
sets were measured over the same oscillation range. Data sets A and B were
consecutive and measured to a resolution of 1.5 Å. An intermediate
exposure of 780 s, which corresponds to the total exposure time for
measurement of a data set, was made while rotating the crystal, although no
data were actually recorded. Data set C was then measured, and it was noted
that the sample now only diffracted to 1.7-Å resolution. The radiation
damage to the crystal after data set CfTryX-C was judged too great to
warrant further useful data collection. The three data sets and the models
derived from each are labeled CfTryX-A, -B, and -C, respectively.
Clumps of small monoclinic plate-like crystals of TbTryX grew over
a period of weeks in drops made by mixing a solution of 10 mg
ml1 protein, 50 mM HEPES, pH 7.5, with
the reservoir solution of 30% polyethylene glycol 4000, 100 mM
sodium acetate, pH 4.6, 200 mM ammonium acetate. The crystals
display space group P21 with unit cell dimensions of a =
30.6, b = 31.5, c = 56.9 Å, b = 93.4°. The
asymmetric unit comprises a monomer with 30% solvent content and
Vm of 1.8 Å3
Da1. A small fragment (
200 x 50
x 10 µm3) was removed from a clump of crystals and passed
through a cryo-protectant consisting of reservoir solution adjusted to include
20% 2-methyl-2,4-pentanediol, and then transferred into a stream of nitrogen
gas at 170 °C. Data were measured to 2.3-Å resolution on a
Rigaku rotating anode (copper K
= 1.5418 Å)-Raxis IV
image plate system.
Structure Solution and RefinementThe initial phases for
both CfTryX2 structures were obtained using the molecular replacement
(MR) technique as implemented in the CNS software package
(18) with data in the
resolution ranges 153 Å for form A and 204 Å for
form B. The structure of CfTryX1 (Protein Data Bank code 1QK8
[PDB]
)
(5) stripped of all solvent
molecules was used as the search model. After this procedure, it was clear
that both crystal forms contain two molecules/asymmetric unit, which results
in calculated Matthews coefficients (Vm)
(19) of 2.3 and 4.4
Å3 Da1 for forms A and B,
respectively. The unit cell of form A has a much lower bulk solvent volume,
46%, than the 72% observed for form B, and this helps to explain the different
diffraction limits of the two forms. For crystal form A, the initial MR phases
obtained were extended to the resolution limit of the data set, 1.5 Å,
using a combination of non-crystallographic symmetry averaging, solvent
flattening, and histogram matching as implemented in the program DM
(20) after first calculating
reliable A-weighted figures-of-merit (FOM)
(21) for the MR phase set. The
resulting electron density map (Fobs,
DM, FOMDM)
was of excellent quality, and a model was constructed using the program
ARP/wARP (22). Refinement was
then carried out using CNS interspersed with rounds of rebuilding in QUANTA
(Accelrys) during which solvent molecules were included. To complete the
refinement, a final round was performed using the program REFMAC
(23) in which the two sulfur
atoms in the active site were refined with anisotropic temperature factors.
For crystal form B, a similar protocol to that described for form A was used
to extend the MR phases to the diffraction limit of the data set. A model was
built manually with QUANTA, and refinement carried out in a similar manner to
that for form A.
The CfTryX-A, -B, and -C structures are isomorphous with the disulfide form of CfTryX1 (5), which provided the starting model for refinements using REFMAC. Following rigid body refinement, additional rounds of positional and B-factor refinement combined with graphics fitting (O) (24) were carried out. Water molecules were added using ARP/wARP. Once the R-factor and R-free had dropped from about 40 to 25%, anisotropic B-factor refinement was introduced.
The structure of TbTryX was solved by MR (AMoRe) (25) using a poly-Ala structure of CfTryX1 as the search model. A clear solution was obtained that, after rigid body refinement, gave an R-factor of 48% and a correlation coefficient of 0.56 for data in the range of 302.3-Å resolution. Density modification (DM) improved the electron density map that was then used for model building. Simulated annealing molecular dynamics (to reduce model bias), least-squares refinement with CNS, together with the placement of water molecules completed the analysis.
Approximately 5% of each data set was set aside to provide an R-free to monitor the progress of all refinements (26), whereas PROCHECK (27) and OOPS (28) were used to assess model geometry. Further experimental details are provided (see Table I) and in the Protein Data Bank depositions.
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RESULTS AND DISCUSSION |
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The Second Tryparedoxin Isoform of C. fasciculata
(CfTryX2)The structure of CfTryX2 was determined
independently in two crystal forms, each of which presents two
molecules/asymmetric unit. A pairwise least-squares superposition of all
C atoms for the four molecules gave root mean square deviation values
that ranged from 0.2 to 0.5 Å, and the results are similar whether we
used a MR protocol or the anomalous dispersion from sulfur atoms
(29) to provide the initial
phase information. When comparing our MR-derived structures with those of
CfTryX2 determined by Hofmann et al.
(9), least-squares
superposition values of between 0.2 and 0.6 Å were observed. These
values indicate close agreement of the second isoform structures irrespective
of how or where they were determined or the redox state of the protein (see
below). The structures reported here confirm that, when compared with the
structure of CfTryX1, the helices
1 and
2 are closer to
each other in the structure of CfTryX2, allowing the formation of a
hydrogen bonding network around the less solvent-exposed sulfur atom in the
active site SS bridge
(9). Both crystal forms of
CfTryX2 were grown from solutions containing dithiothreitol. The
electron and difference density maps were suggestive of a time and space
average of SS bridge oxidized and reduced states. The refined SS
distances are 2.9 and 2.8 Å for the two molecules in form A and 3.2 and
3.0 Å for the two molecules in form B.
Radiation-induced Cleavage of the Redox-active Disulfide The exposure of protein crystals to an intense x-ray source changes the properties of the sample (3033). These changes, which include an increase in unit cell volume, a decreased resolution to which diffraction data can be observed, increased mosaicity, and an increased Wilson B-factor, are indicative of general radiation damage. It has also been noted that although atomic B-factor values increase for successive data sets, the change is not equally distributed for all atoms but rather occurs at glutamate, aspartate, and cysteine residues. In the latter case, it appears that disulfide breakage contributes significantly to this increase in B-factors. In general, only one cysteine in a disulfide actually moves during bond breakage, whereas its partner remains well fixed (31). Weik et al. (32), in a study of x-ray-induced damage to Torpedo californica acetyl-cholinesterase observed that active site residues are among the most radiation-sensitive of residues and suggested, in a similar fashion to disulfide bonds, that these groups constituted "weak links" in protein structures. Our study on CfTryX1 targeted a redox-active disulfide, which should constitute an even weaker link than a structural disulfide. This is indeed the case since the radiation dose required to break this SS bond is much less than that reported for structural SS links in lysozyme for example (31).
During the time course experiment, from CfTryX-A to
CfTryX-C (Table I), we
noted general symptoms of radiation damage to the sample; resolution
decreased, the B factors for consecutive data sets increased, the unit cell
volumes increased from 136,800 to 136,930 to 137,340 Å3, and
the mean fractional isomorphous differences also increased from 0.08 (A and B)
to 0.12 (A and C). In the disulfide form of CfTryX1, the
Cys-40Cys-43 SS
distance is 2.2 Å. In
CfTryX-A, the distance between the two S
atoms has increased
to 2.5 Å. This is most likely due to partial reduction or damage caused
by the high x-ray intensity of the undulator beamline. In CfTryX-B,
the S
-S
distance is 2.8 Å, and in CfTryX-C, it
has increased to 3.0 Å (Fig.
3). Similar results were obtained in the structure of
CfTryX2 in the presence of 2-mercaptoethanol where the
S
-S
distances for the two copies in the asymmetric unit are 3.0
and 3.4 Å (9).
Minor Structural Perturbation Results from Disulfide
BreakageIn contrast to previous observations of radiation-induced
damage to structural disulfides
(31), we do not see
deterioration in electron density for the S atoms of Cys-40 or Cys-43
(Fig. 3). As the disulfide
breaks, the Cys-40 side chain moves toward solvent, and the flanking residues
Trp-39 and Pro-41 move slightly up and out (not shown). Structures of TrX in
the reduced form have been determined (Ref.
10 and references therein) and
also CfTryX2 (9), and
similar observations have been made.
In addition to breaking the disulfide using synchrotron radiation, it was anticipated that the C43A mutant would allow Cys-40 to adopt a position similar to that occupied when the protein is reduced. The mutant structure correlates well with CfTryX-C (Figs. 4 and 5). The C43A mutation also produced small shifts within the active site involving Ser-36 and Tyr-80. In CfTryX1, the side chain of Ser-36 is held in position through interactions with the immobile Cys-43, but in the mutant, Ala-43 no longer has a stabilizing effect on Ser-36. The side chain of Ser-36 adopts a different position, forming a hydrogen bond with the hydroxyl of Tyr-80, which has moved some 1.2 Å from the native structure to stabilize the new arrangement (Fig. 4).
The overall conclusion from the radiation-induced disulfide breakage and
mutant structure analyses is that the active site of TryX appears to be
relatively unperturbed by the redox state. Minor structural changes occur that
serve to make Cys-40 S slightly more accessible to react with the
cognate partners. This is similar to what has been observed in structures of
the dithiol form of TrX by itself
(10) or in complex with
thioredoxin reductase (34).
Capitani et al. (10)
studied the variation in oxidation state of the disulfide in a chloroplast
TrX, but in contrast to our study, they first measured data on the reduced
form of TrX, and then over a period of almost 2 days, using an in-house x-ray
source and crystals at 4 °C, were able to isolate a data set that
indicated that the disulfide had reformed without any large perturbation to
the active site.
Structure of TbTryX and a Model for the Interaction with
TryPThe high degree of sequence conservation (Figs.
2b and
5) and similar biophysical
properties (35,
36) of TbTryX as
compared with the C. fasciculata tryparedoxins suggest that the
three-dimensional structures should be similar. An overlap of
CfTryX1, CfTryX2, and TbTryX highlights the
structural homology of tryparedoxins (Fig.
5). The root mean square deviation for 139 C atoms in
common between TbTryX and CfTryX1 is 0.8 Å. The
largest differences are observed in the N-terminal region, in particular at
the turn between
1 and
2. This is on the opposite side of the
molecule from the redoxactive site.
The similarities extend beyond the overlay of C atoms to the
residues that constitute the hydrophobic core of the protein. Thirteen
aromatic residues in CfTryX1 (tyrosines 34, 54, and 80, tryptophans
70 and 86, phenylalanines 32, 35, 46, 53, 63, 77, 91, 104) are strictly
conserved in the three tryparedoxins (Fig.
2b). In addition, phenylalanines at positions 33, 57, 67,
and 81 of CfTryX1 are replaced by Leu-33, His-57, Leu-67, and Tyr-81
of TbTryX. The residues that cluster around the redoxactive site are
also highly conserved between CfTryX and TbTryX. Indeed
those residues that were first implicated in CfTryX1 binding
trypanothione (5), namely
Trp-39, Pro-41, Pro-42, Arg-44, Trp-70, Asp-71, Glu-72, Lys-83, Ile-109,
Pro-110, and Arg-128, are strictly conserved. Hofmann et al.
(9) were able to confirm that
the last three residues did in fact interact with the ligand in a mutant
CfTryX2 glutathionylspermidine complex. This suggests that similar
molecular features determine the association with the TryX redox partners in
both Crithidia and Trypanosoma.
An overlay of residues that comprise the active site of TbTryX and
CfTryX1 reveals Trp-39 in a different position in TbTryX
than in the CfTryX structures
(Fig. 6). In CfTryX1,
Trp-39 is placed over the redox disulfide, and N1 donates a hydrogen
bond to the carbonyl of Trp-70
(5). In TbTryX, Trp-39
adopts a different rotamer and is flipped out at the surface of the protein
with N
1 forming a hydrogen bond with Asp-76 O
2 from a
symmetry-related molecule (not shown). In the TbTryX crystal
structure, a symmetry-related molecule is positioned such that the
Val-59-Ala-60-Lys-61 segment is placed in the cleft at the redox-active site.
The valine side chain fills the site, which in CfTryX is occupied by
the Trp-39 lid, apparently forcing the tryptophan to adopt a different
conformation. The alanine methyl group points toward the redox-active
disulphide, and the lysine side chain is directed away from the disulfide.
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This alternative conformation of the tryptophan lid has been noted previously in TrX and attributed to crystal packing effects, in the case of a mutant E. coli TrX (37), or due to sequence effects in a chloroplast TrX (10). The adjustment serves to expose the N-terminal cysteine of the redox-active disulfide; therefore, we decided to investigate whether such conformational pliability is relevant to the function of TryX or TrX when they interact with their cognate peroxidases and other proteins. We first considered TryP, a 2Cys [PDB] -peroxiredoxin, well characterized biochemically (3) and for which a crystal structure of the reduced form has been determined (2).
All peroxiredoxins carry an essential N-terminal cysteine, often in a
tetrapeptide Val-Cys-Pro-Thr motif. The 2Cys
[PDB]
-peroxiredoxins have, in addition,
a conserved C-terminal cysteine in a Val-Cys-Pro tripeptide motif, which
interacts with the TryX Trp-Cys-Pro-Pro-Cys redox center. Reduced TryX
interacts with oxidized TryP, but the only structure available for TryP is
that of the reduced form (2).
However, the structure of the homologous and oxidized form of the
2Cys
[PDB]
-peroxiredoxin HBP23 has been determined
(38). Since TryP and HPB23
share almost 60% sequence identity and are closely related in
three-dimensional structure
(2), we superimposed the
C-terminal Val-Cys-Pro motif of one monomer of the dimeric HBP23 onto the
symmetry-related Val-Ala-Lys tripeptide of TbTryX. The C fit
was with an root mean square deviation of 0.3 Å. Although this can only
be a crude model, we note that the side chain of HBP23 Val-172 adopts a
different orientation as compared with the TbTryX Val-59' but
that HBP23 Cys-173 is turned directly toward the redox-active Cys-40 of the
tryparedoxin (Fig. 6b)
with the S
atoms 3.3 Å apart. The model suggests that when TryX
associates with TryP, a repositioning of the tryptophan lid might occur in
conjunction with other molecular features such as the electrostatic
interactions discussed by Hofmann et al.
(3) to facilitate interaction
of the redox components.
If the combination of a pliable tryptophan and a valine-cysteine dipeptide
is indeed important for the tryparedoxinperoxidase interaction, we reasoned
that it might also contribute to thioredoxin-protein associations. Thioredoxin
peroxidases are homologous to TryP, and the Val-Cys-Pro motif is strictly
conserved (3), which would be
consistent with our hypothesis. Also, the truncated form of chloroplast TrX
shows the tryptophan lid in the open conformation, and we note that one
partner for this TrX is chloroplast FBPase, for which a structure is available
(11). Chloroplast TrX
regulates the activity of FBPase by reduction of the disulfide formed between
Cys-153 and Cys-173 (pea FBPase numbering). Cys-153 occurs in the sequence
Val-Cys-Gln-Pro-Gly located on a flexible loop, whereas Cys-173 occurs in an
-helix. A search of the EXPASY data base (ca.expasy.org) indicated that
the pentapeptide segment, with valine preceding the redox-active cysteine, is
strictly conserved in chloroplast FBPase. The observations hint at a role for
a Val-Cys combination to interact with a conformationally labile tryptophan to
assist TryX and TrX pass on their reducing equivalents. Definitive proof would
require a structure of the functional complexes, and we are trying to obtain
this for TryX-TryP.
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FOOTNOTES |
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* This work was supported by a Wellcome Trust senior research fellowship (to
W. N. H.) and program grant (to A. H. F.), a Biotechnology and Biological
Sciences Research Council (BBSRC) studentship (to M. S. A.), and a BBSRC Sir
David Phillips Research Fellowship (to C. S. B.). The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|| To whom correspondence should be addressed. Tel.: 44-1382-345745; Fax: 44-1382-345764; E-mail: w.n.hunter{at}dundee.ac.uk.
1 The abbreviations used are: TryX, tryparedoxin; TryR, trypanothione
reductase; TryP, tryparedoxin peroxidase; TrX, thioredoxin; MR, molecular
replacement; ESRF, European Synchrotron Radiation Facility; FBPase,
fructose-1,6-bisphosphatase; T[SH]2, the reduced form of
trypanothione disulfide; Tb, T. brucei; Cf, C.
fasciculata.
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ACKNOWLEDGMENTS |
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REFERENCES |
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