Oxidized and reduced forms of Human Thioredoxin 2
1
A. Smeets, C. Evrard, B. Knoops and J.-P. Declercq
Unit of Structural Chemistry (CSTR), Université Catholique de Louvain, Place L. Pasteur 1, B-1348 Louvain-la1
Neuve, Belgium; Laboratory of Cell Biology, Université Catholique de Louvain, Place Croix du Sud 5, B-1348
Louvain-la-Neuve, Belgium
Thioredoxins are a class of small redox proteins found in most organisms. They are characterized by a
highly conserved active site in a so-called thioredoxin fold that contains two cysteine residues able to
alternate through oxidized and reduced states [1]. This redox activity is assisted by the flavoenzyme
thioredoxin reductase and NADPH. Two forms of human thioredoxin have been cloned, thioredoxin 1
(TXN-1) and thioredoxin 2 (TXN-2). The structure of TXN-1 has been largely studied whereas no
structural data were available for TXN-2 until now. The actif site of TXN-1 contains the conserved
Trp-Cys-Gly-Pro-Cys-Lys motif as well as several additional cysteines which are not observed in the
sequence of TXN-2. The conserved active site is also present in TXN-2 which shows a mitochondrial
import sequence on its N-terminal end. In TXN-1, a disulfure bridge mediated dimerization occurs
leading to a regulatory homo-dimer.
We have solved and refined three isomorphous structures of TXN2. The space group is P1, with a =
49 Å , b = 49 Å , c = 79 Å , α = 88°, β = 83°, γ = 79°. Each structure contains 6 independent
molecules in the asymmetric unit, labelled A to F. In the first structure, despite the crystallization in
presence of DTT, we observe respectively the following distances between the Sγ atoms of Cys31
and Cys34, the two catalytic residues: 2.69, 2.87, 3.15, 3.62, 3.02, 3.15 Å. These values are to be
compared to the typical distance observed in a disulfide bridge, 2.05 Å and to the sum of the Van
der Waals radii of two sulfur atoms, 3.6 Å, which should correspond to the minimum distance
when the disulfide bridge is not present. With the exception of the molecule D which is certainly
reduced, all the other molecules show
distances intermediate between the
values expected in the reduced form
and the oxidized form. It must be
concluded that in the crystal, some
molecules are reduced while the
other ones are oxidised and that the
sulfur atoms appear in some average
electron density. Since in the reduced
form, the two sulfur atoms are not
too far away from each other, it was
supposed
that
oxidation
and
reduction could take place in already
grown crystals. We have indeed
succeeded to prepare the completely
oxidized and the completely reduced
crystal forms by soaking crystals in
solutions
containing
hydrogen
peroxide
(1
mM)
and
tris(hydroxymethyl)phosphine
(10
mM) respectively. The resulting Sγ-Sγ
distances are: 2.07, 2.12, 2.08, 2.08,
2.08, 2.11 Å in the oxidized form and
3.63, 3.52, 3.63, 3.73, 3.71, 3.58 Å in
the reduced form.
Figure 1 Ribbon diagram of one of the oxidized molecules. The beginning and the end of the secondary
structural elements are labelled.
The resolutions achieved are 2.0 Å, 1.8 Å and 2.0 Å, respectively in the initial, oxidized and reduced
forms of TXN2, with the corresponding Rfactor(Rfree) = 0.20(0.26), 0.17(0.23) and 0.22(0.28). Figure 1
shows one of the oxidized molecules with the formation of the intramolecular disulfide bond
between Cys31 and Cys34. The structures of the oxidized and reduced forms are very similar. Only
the orientation of the side chain of Cys31 is affected by the transition between the reduced and the
oxidized state.
The formation of dimers observed in TXN-1 [2], via the formation of an intermolecular disulfide
bridge is also observed in TXN-2 (Figure 2a), in spite of the absence of the additional Cys residue
forming the disulfide bond in TXN-1.
b)
a)
Direction y - 1
Direction x + 1
F
A
F’
D
D’
A
E’
C’
D
F
B
E
C
B
Figure 2:
C
E
a) Three dimers (A-B, C-D and E-F) present in the triclinic unit-cell of TXN-2.
b) Association (thin lines) of the dimers (thick line) to form infinite two-dimensional polymers
In the 2formation of the three dimers (A-B, C-D, E-F), the buried surface contact per monomer is about
480 Å . In the same unit-cell, we also observe
close contacts between molecules A-D, A-F, B-C and BE, with buried surfaces close to 400 Å2. Applying lattice translations reported in figure 2b, molecule
A is also in close contact with molecules D' and F', and molecule B with molecules C' and E', with
buried surfaces per monomer comprised between 450 Å2 and 550 Å2. These contacts thus give rise
to the formation of two-dimensional polymers which were not observed in TXN-1.
References
[1] A. Holmgren, Structure 3, 239 (1995)
[2] A. Weischel, J.R. Gasdaska, G. Powis & R.W. Montfort, Structure 4, 735 (1996)