Báo cáo Y học: Crystal structure of the catalytic domain of a human thioredoxin-like protein pdf

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

Laboratory of Structural Biology and the MOE Laboratory of Protein Science, School of Life Science & Engineering, Tsinghua
University, Beijing, China;
2
National Laboratory of Institute of Basic Medical Sciences, Peking Union Medical College and
Chinese Academy of Medical Sciences, National Center of Human Genome Research, Beijing, China
Thioredoxin is a ubiquitous dithiol oxidoreductase found in
many organisms and involved in numerous biochemical
processes. Human thioredoxin-like protein (hTRXL) is
differentially expressed at different development stages of
human fetal cerebrum and belongs to an expanding family of
thioredoxins. We have solved the crystal structure of the
recombinant N-terminal catalytic domain (hTRXL-N) of
hTRXL in its oxidized form at 2.2-A
˚
resolution. Although
this domain shares a similar three-dimensional structure
with human thioredoxin (hTRX), a unique feature of
hTRXL-N is the large number of positively charged residues
distributed around the active site, which has been implicated
in substrate specificity. Furthermore, the hTRXL-N crystal
structure is monomeric while hTRX is dimeric in its four
crystal structures (reduced, oxidized, C73S and C32S/C35S
mutants) reported to date. As dimerization is the key regu-
latory factor in hTRX, the positive charge and lack of dimer
formation of hTRXL-N suggest that it could interact with
the acidic amino-acid rich C-terminal region, thereby sug-
gesting a novel regulation mechanism.
Keywords: dithiol oxidoreductase; hTRXL; crystal structure;
monomeric; N-terminal.
Thioredoxin, a group of redox active proteins, is both

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

thioredoxin [18]. WEHI7.2 cells stably transfected with
human thioredoxin cDNA and displaying increased levels
of cytoplasmic thioredoxin, showed increased growth and
were resistant to drug-induced apoptosis both in vitro and
in vivo [19]. In contrast, redox-inactive mutant thioredoxin
reduces growth and enhances drug-induced apoptosis
when transfected into WEHI7.2 cells [20]. Since the
molecular studies have provided the proof-of-principle
that the thioredoxin system is a rational target for
anticancer drug development, the initial approach was
to develop agents that might selectively inhibit the
thioredoxin system and hence thioredoxin-dependent cell
proliferation [21].
Members of the expanding thioredoxin family are char-
acterized by an amino-acid sequence at the active site, -Cys-
Gly-Pro-Cys-, conserved throughout evolution. Extensive
structural characterization of thioredoxin has been carried
out by both X-ray and NMR methods [22–26]. The globular
Correspondence to Z. Rao, Laboratory of Structural Biology,
School of Life Sciences and Engineering, Tsinghua University,
Beijing, 100084, China.
Fax: + 86 62773145, Tel.: + 86 62771493
E-mail:
Abbreviations: hTRXL, human thioredoxin-like protein; hTRXL-N,
the N-terminal domain of human thioredoxin-like protein; hTRXL-C,
the C-termianal region of human thioredoxin-like protein; hTRXL,
gene of human thioredoxin-like protein; hTRX, human thioredoxin;
EST, expressed sequence tags.
Enzymes: thioredoxin (EC 1.8.4.8); flavoenzyme thioredoxin reductase
(EC 1.6.4.5); thrombin (EC 3.4.21.5).

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,

least is known about hTRXL. Here, we report our work
on the isolation of the gene, hTRXL, the functional
identification of the gene product and the structure
determination of its N-terminal catalytic domain.
MATERIALS AND METHODS
DDRT-PCR and full-length cDNA isolation
Total RNA (2.5 lg) from 13- and 33-week-old human fetal
cerebrum (Biochain) was reverse transcribed by Superscript
II (Gibco-BRL) using a single-base anchored 3¢ primer
(5¢-AAGCTTTTTTTTTTTN-3¢,N¼ C, G, A) each time.
The cDNAs were subsequently amplified by PCR using the
same 3¢ single-base anchored primer and 5¢ arbitrary
primer. A detailed procedure for reverse transcription and
differential display have been described previously [28,29].
The PCR products were electrophoresed on a 6% SDS/
PAGE gel (data not shown). cDNA fragments that showed
differential display were recovered from the dried sequen-
cing gel, reamplified and subcloned into PCRII using the
TA cloning kit (Invitrogen, San Diego, CA, USA). In total,
90 selected expressed sequence tags (ESTs) were cloned and
then sequenced. After database searching, HFBEST12
(GenBank accession no. U48630) was chosen to be used
as a probe labeled with [a-
32
P]dCTP (Amersham) to screen
the human fetal brain kDR2 cDNA library (ClonTech) for
full-length cDNA. Three positive kDR2 phage clones were
isolated and converted to pDR2 plasmid, as described in
ClonTech’s manual. DNA sequencing was performed
according to standard methods on an ABI 377 autosequ-

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

expressed in E. coli strain BL21. His-tagged proteins were
loaded onto a His-Trap column (Novagen) and eluted with
a 5–300 m
M
imidazole gradient at pH 8.0 buffered with
20 m
M
Tris/HCl. GST fusion proteins were bound to
glutathione–Sepharose beads (Amersham Pharmacia Bio-
tech), and were cleaved by incubation with thrombin
protease (Sigma) at 4 °C for 14 h.
Insulin disulfide reduction assay
E. coli thioredoxin (Sigma), His-tagged human thioredoxin
(His-TRX), His-tagged hTRXL, His-tagged hTRXL-N
(residues 1–122) and His-tagged hTRXL-C (residues 105–
289) were compared for the reducing activity of insulin
disulfide bonds as described previously (30). The 600-lL
reaction mixture contained 100 m
M
NaCl/P
i
(pH 7.0),
2m
M
EDTA, 0.13 m
M
bovine insulin (Sigma) and 5 m
M
proteins. A reaction was initiated by adding 1 m
M

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
/

SCALE-
PACK
[32]. Data statistics are given in Table 1. The structure
was solved by molecular replacement with CNS [33] using
the structure of human thioredoxin reduced form (PDB
code: 1ERT) as a search model, then refined smoothly in
alternating steps of automatic adjustment with CNS and
manual adjustment with the program O [34]. The final
model has a final R-factor of 0.222 with a free R-factor of
0.253. Molecular graphics images were generated using a
combination of
BOBSCRIPT
[35],
GRASP
[36],
RASTER
3
D
[37]
and
O
[34].
Ó FEBS 2002 Crystal Structure of hTRXL-N (Eur. J. Biochem. 269) 2061
Data deposition
Coordinates for TRXL-N have been deposited with the
Protein Data Bank (PDB accession no., 1GH2, RCSB
accession no., RCSB001506).
RESULTS AND DISCUSSION
hTRXL is a gene differentially expressed at different
development stages

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
+

) RNA from the
13- and 33-week-old cerebrum demonstrated that the
expression level of hTRXL in the former was distinctly
higher than that in the latter (Fig. 1A). This confirmed that
the results from the DDRT-PCR that hTRXL did have
different expression levels in human cerebrum at different
development stages. Northern blot analysis using mRNA
from multiple adult human tissue showed that the hTRXL
was a ubiquitously expressed gene (Fig. 1B).
Both full-length hTRXL and its N-terminal domain
have the thioredoxin-like reductase activity
To investigate the thioredoxin-like reducing activity of
hTRXL, we expressed recombinant hTRXL and human
thioredoxin as His-tagged forms (His-hTRXL and His-
TRX) in E. coli. Truncated hTRXLs corresponding to the
N-terminal (His-hTRXL-N, residues 1–122) and C-terminal
(His-hTRXL-C, residues 105–289) domains were also
prepared, respectively. The expressed recombinant proteins
were purified by His-Trap column chromatography. In
contrast to previously published work on Txl/TRP32, in
which the full-length proteins did not show any reducing
activity, our experiments showed that both His-hTRXL and
His-hTRXL-N possessed reducing activity for the insulin
disulfide bonds. The former showed the kinetics faster than
His-TRX but slower than E. coli thioredoxin (Sigma), while
the latter showed similar reducing activity to His-TRX
(Fig. 2). hTRXL and hTRXL-N (GST fusion expressed
then cleaved) exhibited the same behavior in insulin disulfide
Table 1. Summary of crystallographic data collection and refinement
statistics.

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

working
(%) 22.2 (6026 reflections)
R
free
(%) 25.3 (337 reflections)
No. of nonhydrogen atoms
Protein atoms 816
Solvent 44
Rmds deviation from ideal values
Bond length (A
˚
) 0.02
Bond angle (°) 1.97
Average B-factor (A
˚
2
)
Protein atoms 24.0
Solvent molecules 37.3
a
Numbers in parentheses are the corresponding numbers for the
highest resolution shell (2.30–2.22 A
˚
).
Fig. 1. Expression pattern of the hTRXL transcript. Differential
expression of hTRXL in human fetal cerebrum of 33- and 13-weeks-
old. Human adult tissue Poly(A
+
) RNA Northern blot (ClonTech).
The

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

Asn109 to Gly122 are not visible in the electron density
map. The numbering convention used for hTRXL through-
out starts from the N-terminal methionine, which is different
from hTRX (1ERT), offsetting by 2.
The hTRXL-N molecule contains a typical thioredoxin
fold, consisting of two large folding units: one babab and
another bba (Fig. 3A). Although the amino-acid sequence
of hTRXL-N shows relatively low identity with that of
thioredoxin from different species, the three-dimensional
structure is similar (Table 2). Distinct differences occur
primarily in the four peripheral ahelices of different
molecules, while the hydrophobic core consisting of b
sheets shows little difference with other TRX (Fig. 3B).
Active site
The location of the active site in all of the known
thioredoxin structures is identical. It includes the end of
b-2, two to three linking amino acids and the beginning of
a-2 (Fig. 3A). It is evident that hTRXL-N (as well as other
thioredoxins including hTRX) is distinct from most com-
mon enzymes, whose active site is usually located in a deep
cleft. This is because in TRX, the active site is located
on a pronounced protrusion of the molecular surface
(Fig. 3A,B), demonstrating that the thioredoxin family
proteins are apt at interacting with larger molecules. This
would agree with its role in various redox reactions with
disulfide containing proteins in vivo and in vitro, as reported
previously [4,12,13,40–42].
Despite low sequence identity, dissimilar crystal forms and
dissimilar intermolecular contacts near the active site in the
crystal, the conformation of the active site (-Cys-Gly-Pro-

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

change between oxidized and reduced hTRXL-N would be
very small and localized in the vicinity of the redox active
cysteines, in agreement with the conclusions obtained from
structural information on both human and E. coli thio-
redoxins, based on both crystallography and NMR [22–26].
Nevertheless, the subtle structural differences between
hTRXL-N and hTRX may be important for the different
activities of thioredoxin involving a variety of target proteins.
However, a remarkable feature of hTRXL-N protein is
the large number of positively charged residues distributed
around the active site. As shown in Fig. 5, in hTRXL,
Lys28, Arg32, Arg39 and His62 replace residues Asp26,
Thr30, Met37 and Asp60 that are highly conserved within
the thioredoxins of mammals and chick. This suggests that
the reaction site of the possible substrates may be rich in
negatively charged residues. Another possibility is that the
four positively charged residues might play an important
role in the interaction with the C-terminal region since the
latter carries a large number of acidic amino acids.
Substrate specificity
Alhough it is difficult to identify the true physiological
partners of hTRXL, it was reported that this protein is not a
substrate for thioredoxin reductase in the insulin assay,
unlike human TRX and thioredoxins in other species
[38,39]. The substituted residues around the active site may
suggest different ligand specificity for hTRXL-N. A mul-
tiple alignment of 83 samples of thioredoxins and thio-
redoxin related proteins from archebacteria to human was
performed (data shown only includes thioredoxins from
mammals and chick). Ninety-six percent of residues are

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

conserved throughout evolution.
Fig. 3. hTRXL-N structure. (A) Overall structure of N-terminal domain. Residues involved in the active site are depicted as ball and stick.
Cysteines are coloured in yellow (Cys34 and Cys37); Near the active site, positively charged residues are coloured in blue (Lys28, Arg32, Arg39 and
His62); other residues are coloured in grey (Met31, Gly33, Gly35 and Pro36); The disulfide bond between Cys34 and Cys37 of active site is coloured
in orange. The figure was drawn using
BOBSCRIPT
(44). (B) Backbone superpositions of the six structures of hTRXL-N and other thioredoxin related
proteins or domains. 1GH2 (crystal structure of hTRXL-N, 2–108), 2TRX (crystal structure of E. coli thioredoxin, 1–108), 1ERU and 1ERT
(crystal structure of oxidized and reduced human thioredoxin, 1–105), 1DBY (NMR structure of thioredoxin in Chlamydomonas reinhardtii, 1–107),
1MEK (NMR structure of thioredoxin domain of protein-disulfide isomerase, 1–120) are coloured in cyan, blue, red, green, magenta, and yellow,
respectively. For detailed rmsd values, see Table 2. Superposition calculation was performed using
SHP
program [49].
2064 J. Jin et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Instead of the large imidazole side chain of Trp31 in
human TRX (Fig. 6B), which lies both in the active site and
in the dimer interface of human TRX, Gly33 takes its place
in hTRXL-N. This substitution may contribute to the
inability of hTRXL-N to react with thioredoxin reductase.
The role of this Trp residue in E. coli thioredoxin has been
studied by site-directed mutagenesis: the apparent K
m
value
of thioredoxin reductase with thioredoxin (TRX) as its
substrate was increased twofold for the mutant TRX
W31A, as compared with the wild type thioredoxin while
K
cat
value remained the same. This results in a 50%
reduction in catalytic efficiency (K

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

map contoured at 1r at the hTRXL-N active site at 2.2 A
˚
resolution. Hydrogen bonds are represented as dotted
lines.
Fig. 5. Multiple alignments of thioredoxin
homologs. Analignmentofthesheep
(SWISSPROT accession no. P50413), Macaca
mulatta (accession no. P29451), rat (accession
no. P11232), mouse (accession no. P10639),
chicken (accession no. P08629), human (Homo
sapiens, accession No. P10599) and rabbit
(accession no. P08628) thioredoxin is depicted.
The14 residues conserved in the dimer inter-
face are indicated with asterisks.
Ó FEBS 2002 Crystal Structure of hTRXL-N (Eur. J. Biochem. 269) 2065
substrates in the hTRX–NFjB and hTRX–Ref1 complexes
were wrapped around the protrusion of the reactive Cys32
in a crescent-shaped groove. However, the orientation of the
Ref1 peptide is opposite to that of the target peptide from
NFjB. The ability of hTRX to recognize peptides in
opposite orientations indicates that this redox protein has
succeeded in balancing specificity in substrate recognition
with requirement for access to a variety of substrates. In this
way, hTRX and perhaps thioredoxins from other species
might have the potential to target a wide range of proteins
within the cell. A comparison of corresponding hydropho-
bic surfaces of hTRXL-N (1GH2) and the substrate-binding
surface of hTRX (1CQH) reveals that a similar groove can
also be found in the hTRXL surface. To deduce the
molecular basis of the possible substrate specificity we

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

take place in this area. Human TRX is believed to function
as a monomer in redox reactions, but the active site is largely
blocked by dimer formation. Hence it has been proposed
that dimer formation may play a role in regulating human
thioredoxin [44,45]. The changes in the corresponding
region of the dimer interface in hTRXL-N imply that
different regulatory mechanisms may occur in hTRXL. It
can be hypothesized that the unique hTRXL C-terminal
region may have a similar role in regulation as in the dimer
formation.
Finally, we have not been able to crystallize the full-length
protein. In order to gain some structural information and
function clues of the unique C-terminal region, we used fold
recognition and modeling to establish a model structure.
Secondary structure prediction was performed using
JPRED
program [46,47]. The predicted secondary structure in the
C-terminal region was shown to be relatively low, and this
may partly explain why it was difficult to crystallize the
hTRXL full-length protein and its C-terminal region. Fold
recognition was performed using the
FORESST
program [48],
Fig. 6. Space-filling model of the disabled
dimer interface with H-bond formation residues
substituted in hTRXL-N, and compared with its
corresponding dimer formation surface in
hTRX monomer (reduced) and coloured by
residue type: aliphatic (white), positive (blue),
negative (red), cysteine (yellow), polar (purple)

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

with the N-terminal domain, the mechanism of regulating
the catalytic activity may be similar to that of the dimer. In
other word, the active site would be physically blocked and
would have to ÔdissociateÕ to achieve active conformation by
exposure of the active site. However, we can not exclude the
possibility that this site functions as a recruiting factor or
signal sequence leading the N-terminal thioredoxin-like
domain to approach certain substrates. A study to find the
possible substrates in the electron transport chain is
currently underway.
ACKNOWLEDGEMENTS
Z. R. was supported by the following grants: NSFC no. 39870174 and
no. 39970155; project Ô863Õ, No. 2001AA233011; project Ô973Õ,
No. G1999075602, No. G1999011902 and no. 1998051105. J. Y. was
supported by the National Natural Sciences Foundation of China
(39830070), the National High Technology Research and Development
Program (Z19-02-02-01) and the National Program for Key Basic
Research Projects (G1998051002).
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