Crystal structure of the catalytic domain of a human thioredoxin-like
protein
Implications for substrate specificity and a novel regulation mechanism
Jian Jin
1,2
, Xuehui Chen
1
, Yan Zhou
2
, Mark Bartlam
1
, Qing Guo
1
, Yiwei Liu
1
, Yixin Sun
1
, Yu Gao
1,2
,
Sheng Ye
1
, Guangtao Li
2
, Zihe Rao
1
, Boqin Qiang
2
and Jiangang Yuan
2
1

ubiquitously present and evolutionarily conserved from
prokaryotes to higher eukaryotes [1–3]. Thioredoxin was
initially discovered in Escherichia coli as an electron donor
for the essential enzyme ribonucleotide reductase [4] and,
since then, many functions have been assigned to thio-
redoxins not only associated with redox-mediated processes
but also with structural roles. For example, they can also
serve as a reducing agent in sulfate reduction [5,6] and
methionine sulfoxide reduction in E. coli [7]. Moreover,
E. coli thioredoxin-(SH)
2
can act as an essential subunit of
T7 DNA polymerase [8] and is known to function in the
maturation of filamentous bacteriophages M13 and f1
[9,10]. In eukaryotic cells, thioredoxin can facilitate refold-
ing of disulfide-containing proteins [11] and modulate the
activity of some transcription factors such as NF-kB and
AP-1 [12,13]. Other functions include antioxidant action
and the ability to reduce hydrogen peroxide [14], scavenging
of free radicals [15], and protection of cells against oxidative
stress [16].
A recent area of interest is the role of thioredoxin as a
cell growth stimulator and an apoptosis inhibitor, both
in vitro and in vivo. Recombinant human thioredoxin,
when added to minimal culture medium in the absence of
serum, stimulates the proliferation of a number of human
solid tumor cell lines as measured over several days [17].
An adult T cell leukemia-derived factor, which augments
the expression of interleukin-2 receptor and then stimu-
lates T cell growth, was found to be identical to human

Note: a website is available at
(Received 19 November 2001, revised 12 February 2002,
accepted 21 February 2002)
Eur. J. Biochem. 269, 2060–2068 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02844.x
structure consists of a central b sheet that is sandwiched by
a helices. The active site of thioredoxin is localized in a
protrusion of the protein surface [22], and the two cysteine
residues provide the sulfhydryl groups involved in the
thioredoxin-dependent reducing activity. The oxidized form
(thioredoxin-S
2
), where the two cysteine residues are linked
by an intramolecular disulfide bond, is reduced by flavoen-
zyme thioredoxin reductase and NADPH [2]. The reduced
form [thioredoxin-(SH)
2
] contains two thiol groups and can
efficiently catalyze the reduction of many exposed disulfides.
Therefore, thioredoxin can interact with a broad range of
proteins either in electron transport for substrate reduction
or in regulation of activity by a seemingly simple redox
mechanism based on reversible oxidation of two cysteine
thiol groups to a disulfide, accompanied by the transfer of
two electrons and two protons.
Human thioredoxin-like protein (hTRXL, m  32 kDa)
can be regarded as a member of the mammalian
thioredoxin family [27]. The other two members of this
family, thioredoxin-1 (m  12 kDa) and mitochondrial
thioredoxin-2 (m  18 kDa), are much smaller than
hTRXL. Among the three types of thioredoxin proteins,

encer (PerkinElmer).
Northern blot analysis
Total Poly(A)
+
RNA from 13- and 33-week-old human
fetal cerebrum (Biochain) was electrophoresed in a 1%
agarose gel containing 0.66
M
formaldehyde and was
blotted onto a Hybond-N
+
nylon membrane filter (Amer-
sham). The blotted filter and the human adult multiple-
tissue Northern blot membrane (ClonTech) were hybridized
in accordance with manufacturer’s instructions. The probe
is the differentially displayed EST HFBEST12 (GenBank
accession no. U48630) isolated in DDRT-PCR, random-
radiolabeled with [a-
32
P]dCTP.
Cloning procedures, expression and purification
The full-length hTRXL cDNA in pDR2 vector (ClonTech)
was used as a template for PCR to create in-frame
constructs for further cloning. Human thioredoxin full-
length cDNA was also isolated by PCR-amplification using
human fetal brain library (ClonTech) as a tem-plate. PET-
28 vector (Novagen) and PGEX-4T vector (Amersham
Pharmacia Biotech) were used to create histidine-tagged and
GST-fused proteins for bacterial expression.
His-tagged proteins and the GST fusion proteins were

dithio-
threitol, and the A
650
was immediately recorded at room
temperature. Measurements were performed using 1-min
recordings and the nonenzymatic reduction of insulin by
dithiothreitol was recorded in a control cuvette without
thioredoxin.
Crystallization and data collection
The hTRXL-N crystals were grown by hanging-drop
vapor-diffusion in ammonium sulfate system. Native
data for TRXL-N was collected in house using a Rigaku
rotating anode X-ray source and a MAR345 image plate to
2.22 A
˚
(31).
Structure determination
The crystals belong to space group C
2
with the unit cell
dimensions of a ¼ 87.5 A
˚
, b ¼ 48.5 A
˚
, c ¼ 29.8 A
˚
,
b ¼ 99.59°. The data were processed with
DENZO
/

mRNA extracted from human fetal brain tissues at different
developmental stages (13- and 33-week-old cerebrum) was
used for DDRT-PCR and the isolated EST (GenBank,
accession no. U48630) with different expression patterns in
these two stages was cloned into pBlue-Script vector and
sequenced. cDNA library screening was performed using
the EST obtained as a probe labeled by a-
32
Pandthe
screening resulted in isolation of a novel, full length cDNA
clone, hTRXL (human thioredoxin-like protein, GenBank
accession no. AF051896). hTRXL is 1230-bp in length and
contains an 867-bp ORF, which encodes for a protein with
289 amino acids and a calculated molecular mass of
32 kDa. A search of the nonredundant protein sequence
database was performed using the
BLAST
program. Besides
sharing the same sequence with Txl/TRP32 [38,39], the 105
residue N-terminal domain shared 42% identity and 55%
similarity to human thioredoxin and contained the con-
served active site sequence CGPC (Cys-Gly-Pro-Cys). The
C-terminal 184 amino acids of hTRXL, which is rich in
acidic amino acids, had no similarity to any proteins in the
public databases. The full-length cDNA isolated and cloned
by the method of DDRT-PCR and cDNA library screening
is identical to the previously published Txl/TRP32 sequence
[38,39].
Northern blot analysis using poly (A
+

A. Data statistics
Resolution (A
˚
) 100–2.2 A
˚
Space group C
2
Unit cell (A
˚
, °) a ¼ 87.5
b ¼ 48.5
c ¼ 29.8
b ¼ 99.59
R
merge
(%) 0.089 (0.316)
a
No. of reflections 6710 (624)
a
Completeness (%) 99.8 (98.7)
a
I/I
(I)
8.4
B. Refinement statistics
Resolution (A
˚
) 15–2.2 A
˚
R

32
P-labeled probe is the EST obtained from DDRT-PCR (Gen-
Bank accession no. U48630) and the control used is b-actin cDNA
(ClonTech).
2062 J. Jin et al. (Eur. J. Biochem. 269) Ó FEBS 2002
reduction assay (data not shown). As expected, the His-
hTRXL-C failed to reduce insulin, demonstrating that the
N-terminal region is responsible for the dithio-reducing
enzymatic activity and the C-terminal region has little direct
effect on the activity of the enzyme. The function of this
unique C-terminal domain remains unknown.
Overall structure
Crystals of the catalytic domain of hTRXL (hTRXL-N)
were obtained from ammonium sulfate by hanging-drop
vapor-diffusion method [31]. The crystals diffracted beyond
2.2-A
˚
resolution. The structure was determined by molecu-
lar replacement with CNS [33] using the crystal structure of
human thioredoxin in its reduced form as a search model
(PDB ID: 1ERT). The structure was refined to a crystallo-
graphic R-factor of 0.222 at 2.2-A
˚
resolution (Table 1). The
overall structure is very similar to hTRX (rmsd 0.83 A
˚
)with
the main difference being that hTRXL-N crystallized as a
monomer while the hTRX crystallized as a disulfide-linked
dimer. The N-terminal methionine and the C-terminus from

Cys-) of the hTRXL-N determined in the present study is
very close to those of human and E. coli thioredoxin. In
addition to the disulfide-bond between the two cysteine
residues, three pairs of hydrogen bonds are formed in the
active site of hTRXL-N (Fig. 4), accounting for the
compactness and stability of the active site. The H-bond
length between the carbonyl oxygen of Cys34 and the amide
nitrogen of Leu38 is 2.99 A
˚
in this structure, as compared
with 3.21 A
˚
in the oxidized form of hTRX (1ERU) and
3.49 A
˚
in the reduced form of hTRX (1ERT), respectively.
Cys37 is stabilized by an S–O hydrogen bond with the
hydroxyl of Thr76 (bond length 3.3 A
˚
), which is not present
in 1ERU and 1ERT due to a substitution of Thr76 for
Met74. The carbonyl oxygen of Gly35 forms a well-aligned
H-bond to the amide nitrogen of Arg39 with a length of
2.87 A
˚
, in comparison with the corresponding H-bond in
1ERU and 1ERT (both 3.00 A
˚
), which suggests the a helix
appears more compact in our structure. The conformational

Fig. 2. Reductase activity of thioredoxin proteins. E. coli thioredoxin,
His-hTRXL (full-length), His-hTRXL-N, His-hTRXL-C and His-
hTRX (5 l
M
each) were assayed for their ability to reduce the disulfide
bonds of insulin as described previously [42]. The incubation mixtures
contained, in a final volume of 600 lL: 100 m
M
NaCl/P
i
(pH 7.0),
2m
M
EDTA, 0.13 m
M
bovine insulin (Sigma) and 1 m
M
dithiothrei-
tol. Only dithiothreitol without thioredoxin served as control. The
absorbance at 650 nm is plotted against time.
Ó FEBS 2002 Crystal Structure of hTRXL-N (Eur. J. Biochem. 269) 2063
conserved within the thioredoxins of mammals and chick.
In contrast, many of them are substituted in hTRXL-N
(Fig. 5) and this may lead to divergence in substrate
specificity. As expected, many residues in the four a helixes
and loops on the molecular surface were found to be
substituted while the residues in the five b sheets of the
internal hydrophobic core are generally conserved. The
most noticeable substitutions are Lys28, Met31, Arg32,
Gly33, Leu38, Arg39, His62 and Thr76, which are highly

cat
/K
m
value) of the
mutant [43]. So it can be deduced that a similar effect takes
place when a Gly33 in hTRXL replaces the equivalent
Trp31 in hTRX. Similarly, flexible long side chains of
Lys28, Met31, Arg32, Leu38, Arg39 and His62 substituted
in the areas in spatial proximity to the active site are also
likely to contribute to substrate interaction, leading to
divergence in substrate specificity.
The NMR structures of human thioredoxin complexed
with its target peptides from NFjB and Ref1, respectively,
were reported several years ago [25,26]. The peptide
Table 2. Sequence identity and rmsd deviations of five representative structures compared with hTRXL-N (1GH2).
Protein structure PDB ID Sequence identity rmsd
Reduced human thioredoxin, 1–105 1ERT 42% 0.80 A
˚
Oxidized human thioredoxin, 1–105 1ERU 42% 0.83 A
˚
E. coli thioredoxin, 1–108 2TRX 26% 0.97 A
˚
Thioredoxin in chlamydomonas reinhardtii, 1–107 1DBY 19% 1.10 A
˚
Thioredoxin domain of protein-disulfide isomerase, 1–120 1MEK 18% 1.33 A
˚
Fig. 4. Stereoviews of the 2F
o
) F
c

compared the residues in the crescent-shaped groove in
hTRX with those in the corresponding region in hTRXL-N.
In contrast to the 42% sequence identity to hTRX in the
whole of hTRXL-N, the assumed substrate-binding region
( 20 residues) shows a sequence identity of about 68.5%,
and therefore suggests the similarity in the manner of
binding. Despite this similarity, hTRXL is perhaps more
inclined to bind proteins whose binding sites are negatively
charged as there are four positively charged substitutions
distributed around the active site as mentioned above.
The monomeric structure of hTRXL-N
hTRXL-N is monomeric in its crystal structure determined
in the present work, while human thioredoxin (TRX) is
dimeric in the four crystal structures reported to date
(reduced, oxidized, C73S and C32S/C35S). The dimer
interface of TRX consists of three components: an
1100 A
˚
2
hydrophobic patch, five hydrogen bonds and the
Cys73–Cys73 disulfide bond [23]. The substitution of these
hydrogen bond forming residues in hTRXL-N may account
for the formation of a monomer, instead of a dimer in the
case of TRX. Furthermore, the loss of intermolecular
disulfide-bonds and the disbandment of the hydrophobic
patch may also obstruct the dimer formation (Fig. 6).
The14 residues in the dimer interface are highly conserved
among the thioredoxins of eight vertebrate species (Fig. 5),
yet 10 of these 14 residues are substituted in hTRXL-N,
suggesting that important structural and functional changes

and alcohol (cyan). Note that the numbering
for hTRXL-N is larger by 2 than that in
hTRX for the corresponding equivalent resi-
dues.
Fig. 7. Molecular surface comparison between
hTRX and hTRXL-N. Molecular surface
representations of hTRX (A) and hTRXL-N
(B) around the active surface in the same
orientation were produced using GRASP.
Electrostatic surface potentials are contoured
from )30(red)to30(blue)k
b
TÆe
)1
.The
ellipses highlight the position of active site in
hTRX and hTRXL-N, respectively.
2066 J. Jin et al. (Eur. J. Biochem. 269) Ó FEBS 2002
the top solutions were classified from the SCOP database
webserver and they all belonged to the Ôall-betaÕ family of
proteins. Most of the top solutions share the immunoglo-
bulin-like fold and the model was constructed according to
the structure template of transthyretin (PDB code 1ETA)
with the highest Z-score based on the sequence alignment
from fold recognition.
As shown in Fig. 7, the molecular surface around the
active site of hTRXL-N (1GH2) is very different compared
with that of hTRX (1ERU). The former is more positive (or
much less negative) than the latter. As the C-terminal region
is rich in acidic amino acids, if it does have some interaction

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