Acta Cryst. (2009). F65, 651–653doi:10.1107/S1744309109018417
Acta Crystallographica Section F
Cloning, overproduction, purification,
crystallization and preliminary X-ray diffraction
analysis of yeast glutaredoxin Grx5
Yi Wang, Yong-Xing He, Jiang Yu
and Cong-Zhao Zhou*
Hefei National Laboratory for Physical Sciences
at Microscale and School of Life Sciences,
University of Science and Technology of China,
Hefei, Anhui 230026, People’s Republic of
Correspondence e-mail: firstname.lastname@example.org
Received 9 March 2009
Accepted 15 May 2009
Grx5 from the yeast Saccharomyces cerevisiae is a monothiol glutaredoxin that is
involved in iron–sulfur cluster biogenesis. Here, yeast Grx5 was cloned and
overproduced in Escherichia coli. The purified protein was crystallized using the
hanging-drop vapour-diffusion method. Diffraction data for Grx5 were collected
to 1.67 A˚resolution. The crystal of Grx5 belonged to space group R3, with unit-
cell parameters a = b = 85.12, c = 48.95 A˚, ? = ? = 90.00, ? = 120.00?.
Glutaredoxins (Grxs) are small proteins that catalyze the reduction
of protein disulfides or glutathione–protein mixed disulfides using
reduced glutathione (GSH) as the electron donor (Holmgren, 1985,
1989). They belong to the thioredoxin-like protein superfamily and
are involved in a large number of cellular processes (Martin, 1995).
Besides functioning as an antioxidant, yeast glutaredoxin Grx5 plays
an essential role in the biogenesis of iron–sulfur (Fe–S) clusters in
mitochondria (Rodriguez-Manzaneque et al., 2002). Homologues of
yeast Grx5 from other species, including Escherichia coli Grx4,
Chlamydomonas Grx3 and human Grx5, have been reported to
possess similar functions (Rahlfs et al., 2001; Picciocchi et al., 2007;
Zaffagnini et al., 2008). A lack of Grx5 in mitochondria leads to the
shutoff of biosynthesis of the Fe–S cluster, which is used as a cofactor
by several enzymes, such as aconitase and succinate dehydrogenase in
the tricarboxylic acid (TCA) cycle (Rodriguez-Manzaneque et al.,
The monothiol glutaredoxin Grx5 has significant homology to
dithiol glutaredoxins (sequence identity ?25%), mainly in the
carboxyl-terminal region of the molecule (Rodriguez-Manzaneque et
al., 1999; Bellı ´ et al., 2002). In addition to Cys60 at the CGFS site,
Grx5 has an additional cysteine Cys117 in the C-terminal region
(Rodriguez-Manzaneque et al., 1999). This cysteine residue has been
reported to be essential for the antioxidative role of Grx5. In addition
toGrx5, theother two yeast monothiol glutaredoxins, Grx3 and Grx4,
share high sequence homology with Grx5 (sequence identity above
45%) and also play important roles in the Fe–S cluster relevant signal
transduction pathway (Kuma ´novics et al., 2008). However, none of
these three proteins has been investigated from a structural point of
view. Therefore, the crystal structure of Grx5 should provide insight
into its molecular function.
2. Materials and methods
2.1. Cloning and protein expression
The open reading frame of GRX5 lacking the sequence coding for
the N-terminal mitochondrial targeting residues Met1–Tyr29 was
amplified by PCR using the Saccharomyces cerevisiae genomic DNA
as the template and inserted into a pET29a-derived vector between
the NdeI and NotI sites. The protein was overproduced at 310 K in
E. coli Rosetta (DE3) strain using 2?YT culture medium (5 g NaCl,
# 2009 International Union of Crystallography
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16 g bactotryptone and 10 g yeast extract per litre). E. coli Rosetta
(DE3) cells were transformed with this plasmid. To induce expression
of the desired protein, isopropyl ?-d-1-thiogalactopyranoside (IPTG)
was added to a final concentration of 0.2 mM when the OD600was 0.6
and the culture was grown for another 5 h at 310 K. Cells were
harvested by centrifugation at 7330g for 10 min and then resuspended
in lysis buffer (20 mM Tris–HCl pH 7.0). The suspension was soni-
cated and then centrifuged at 29 300g for 25 min at 277 K.
Wang et al.
Acta Cryst. (2009). F65, 651–653
(a) Gel filtration of Grx5 using a HiLoad 16/60 Superdex 75 column. The flow rate was 1 ml min?1. Impurities were contained in peaks I, II and III and target protein was
contained in peak IV. (b) 15% SDS–PAGE analysis of fractions from gel filtration. Lanes 2–10, fractions from the main peak III; lane 1, low-molecular-mass markers (kDa).
(a) Crystals of Grx5. (b) A diffraction image of a Grx5 crystal.
2.2. Protein purification Download full-text
The supernatant was loaded onto an anion-exchange column
(QFF-Sepharose, Amersham Biosciences, Sweden) pre-equilibrated
with buffer containing 20 mM Tris–HCl pH 7.0 and the target protein
was eluted with a linear gradient of NaCl from 0 to 1 M. The protein
was further loaded onto a Superdex75 gel-filtration column (Amer-
sham Biosciences, Sweden) and was eluted with a buffer containing
20 mM Tris–HCl pH 7.0, 100 mM NaCl. The approximate yield of
purified Grx5 was 10 mg per litre of culture. After concentration to
8 mg ml?1, the target protein was added to an equal volume of 100%
glycerol and frozen at 233 K. The protein concentration was
determined by measuring the absorption at 280 nm using a theor-
etical coefficient of 8605 M?1cm?1(http://www.expasy.org/cgi-bin/
2.3. Crystallization and X-ray data collection
Crystals of Grx5 were obtained by the hanging-drop vapour-
diffusion method at 289 K using commercial screens from Hampton
Research. Each drop, consisting of 1 ml 8 mg ml?1protein solution
(20 mM Tris–HCl pH 7.0, 100 mM NaCl, 10 mM DTT) and 1 ml
reservoir solution, was equilibrated against 400 ml reservoir solution.
Crystal of Grx5 grew to dimensions of 0.7 ? 0.8 ? 0.6 mm within one
week using a reservoir containing 1.6 M ammonium sulfate, 0.1 M
Tris–HCl pH 8.0. A mixture of 30% glycerol with the reservoir
solution described above was used as a cryogenic liquor. The X-ray
diffraction data were collected at 100 K in a liquid-nitrogen gas
stream using a Rigaku MM007 X-ray generator (? = 1.54178 A˚) with
a MAR Research 345 image-plate detector at the School of Life
Sciences, University of Science and Technology of China (USTC,
Hefei, People’s Republic of China). 137 frames were collected with 1?
oscillation and 10 min exposure per frame at a crystal-to-detector
distance of 120 mm. The diffraction data were indexed and integrated
using the program MOSFLM 7.0.4 (Leslie, 1992) and scaled using
SCALA (Evans, 1993).
3. Results and discussion
The gel-filtration method was used as the final purification step and
the purity of the target protein was further checked by SDS–PAGE
(Fig. 1). Crystals of Grx5 appeared in drops obtained using a reser-
voir containing 1.6 M ammonium sulfate, 0.1 M Tris–HCl pH 8.0
(Fig. 2a). A diffraction image for Grx5 is shown in Fig. 2(b). The data-
collection statistics are given in Table 1. The crystal of Grx5 belonged
to spacegroup R3, with unit-cell parameters a =b =85.12,c=48.95 A˚,
? = ? = 90.00, ? = 120.00?. Assuming a Matthews coefficient of
2.63 A˚3Da?1and a solvent content of 53.18%, the asymmetric unit
contains one molecule (Matthews, 1968). The structure solution has
been obtained using the molecular-replacement method with the
program MOLREP (Vagin & Teplyakov, 1997), using E. coli Grx4
(PDB code 1yka, sequence identity 38%) as the search model, and
confirmed the presence of a single Grx5 molecule in the asymmetric
unit (Fladvad et al., 2005). Structure refinement is in progress.
This work was funded by grant 30470366 from the Chinese
National Natural Science Foundation and projects 2006CB910202
and 2006CB806501 of the Ministry of Science and Technology of
Bellı ´, G., Polaina, J., Tamarit, J., de la Torre, M. A., Rodrı ´guez-Manzaneque,
M. T., Ros, J. & Herrero, E. (2002). J. Biol. Chem. 277, 37590–37596.
Evans, P. R. (1993). Proceedings of the CCP4 Study Weekend. Data Collection
and Processing, edited by L. Sawyer, N. Isaacs & S. Bailey, pp. 114–122.
Warrington: Daresbury Laboratory.
Evans, P. (2006). Acta Cryst. D62, 72–82.
Fladvad, M., Bellanda, M., Fernandes, A. P., Mammi, S., Vlamis-Gardikas, A.,
Holmgren, A. & Sunnerhagen, M. (2005). J. Biol. Chem. 280, 24553–24561.
Holmgren, A. (1985). Methods Enzymol. 113, 525–540.
Holmgren, A. (1989). J. Biol. Chem. 264, 13963–13966.
Kuma ´novics, A., Chen, O. S., Li, L., Bagley, D., Adkins, E. M., Lin, H., Dingra,
N. N., Outten, C. E., Keller, G., Winge, D., Ward, D. M. & Kaplan, J. (2008).
J. Biol. Chem. 283, 10276–10286.
Leslie,A.G.W. (1992).JntCCP4/ESF–EACBMNewsl. ProteinCrystallogr.26.
Martin, J. L. (1995). Structure, 3, 245–250.
Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497.
Picciocchi, A., Saguez, C., Boussac, A., Cassier-Chauvat, C. & Chauvat, F.
(2007). Biochemistry, 46, 15018–15026.
Rahlfs, S., Fischer, M. & Becker, K. (2001). J. Biol. Chem. 276, 37133–37140.
Rodriguez-Manzaneque, M. T., Ros, J., Cabiscol, E., Sorribas, A. & Herrero, E.
(1999). Mol. Cell. Biol. 19, 8180–8190.
Rodriguez-Manzaneque, M. T., Tamarit, J., Bellı ´, G., Ros, J. & Herrero, E.
(2002). Mol. Biol. Cell, 13, 1109–1121.
Vagin, A. & Teplyakov, A. (1997). J. Appl. Cryst. 30, 1022–1025.
Zaffagnini, M., Michelet, L., Massot, V., Trost, P. & Lemaire, S. D. (2008). J.
Biol. Chem. 283, 8868–8876.
Acta Cryst. (2009). F65, 651–653Wang et al.
Data-collection and processing statistics.
Values in parentheses are for the highest resolution shell.
Unit-cell parameters (A˚,?)
a = b = 85.12, c = 48.95,
? = ? = 90.00, ? = 120.00
Resolution range (A˚)
Total No. of observations
No. of unique reflections
† Calculated according to Evans (2006).