Crystal structure of fully oxidized human thioredoxin

Accepted Manuscript Crystal structure of fully oxidized human thioredoxin Jungwon Hwang, Loi T. Nguyen, Young Ho Jeon, Chan Yong Lee, Myung Hee Kim PII:

S0006-291X(15)30703-8

DOI:

10.1016/j.bbrc.2015.10.003

Reference:

YBBRC 34687

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 30 September 2015 Accepted Date: 1 October 2015

Please cite this article as: J. Hwang, L.T. Nguyen, Y.H. Jeon, C.Y. Lee, M.H. Kim, Crystal structure of fully oxidized human thioredoxin, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.10.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Crystal structure of fully oxidized human thioredoxin

Myung Hee Kima,* a

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Jungwon Hwanga,1, Loi T. Nguyena,b,1, Young Ho Jeonc, Chan Yong Leeb,

Infection and Immunity Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Korea

College of Pharmacy, Korea University, Sejong 339-770, Korea

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Department of Biochemistry, Chungnam National University, Daejeon 305-764, Korea

*Corresponding author. Fax: +82 42 879 8595.

These authors contributed equally to this work.

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E-mail address: [email protected] (M. H. Kim)

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b

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In addition to the active cysteines located at positions 32 and 35 in humans, mammalian cytosolic thioredoxin (TRX) possesses additional conserved cysteine residues at positions 62, 69, and 73. These non-canonical cysteine residues, that are distinct from prokaryotic TRX and also

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not found in mammalian mitochondrial TRX, have been implicated in biological functions

regulating signal transduction pathways via their post-translational modifications. Here, we

describe for the first time the structure of a fully oxidized TRX. The structure shows a non-active

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Cys62-Cys69 disulfide bond in addition to the active Cys32-Cys35 disulfide. The non-active disulfide switches the α3-helix of TRX, composed of residues Cys62 to Glu70, to a bulging loop

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and dramatically changes the environment of the TRX residues involved in the interaction with its reductase and other cellular substrates. This structural modification may have implications for a number of potential functions of TRX including the regulation of redox-dependent signaling

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pathways.

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Keywords: Thioredoxin, Oxidoreductase, Redox, X-ray structure

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1. Introduction Human cytosolic thioredoxin (TRX), a ubiquitous 12 kDa protein, is essential for life.

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Through its oxidoreductase activities involving its active site C-G-P-C motif and NADPHdependent selenoenzyme thioredoxin reductase (TrxR), TRX plays critical roles in maintaining an appropriately reducing cytosol for various cellular processes [1]. The active site motif of TRX

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is strictly conserved from bacteria to humans [2], with the two active cysteines (at positions 32 and 35 in humans) undergoing a dithiol-disulfide exchange reaction.

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Even though all TRX proteins show a similar structure comprising a central core of a fivestranded β-sheet and four peripheral α-helices, mammalian cytosolic TRX possesses additional conserved cysteine residues (located at positions 62, 69, and 73 in humans) that are distinct from prokaryotic TRX and also not found in mammalian mitochondrial TRX [3]. These non-canonical

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cysteine residues have been implicated in the regulation of signal transduction pathways via post-translational modifications including glutathionylation [4], S-nitrosylation [5], and thiol oxidation [6, 7]. A glutathionylated TRX via Cys73 (corresponding to Cys72 in ref 4) was

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identified in oxidative-stressed lymphocytes, with this modification regulating TRX enzymatic activity in a reversible manner [4]. An S-nitrosylation detected at Cys69 has been implicated in

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the maintenance of the redox regulatory activity and the anti-apoptotic function of TRX in endothelial cells under basal conditions [8]. Another S-nitrosylation of TRX via Cys73 was found to be associated with the reversible and specific transfer of a nitrosothiol onto the caspase 3 Cys163 residue in vitro, with no significant S-nitrosylation observed for Cys62 and Cys69 [5]. The transnitrosation reaction between TRX Cys73 and caspase 3 Cys163 suggested an involvement in the regulation of apoptosis. 3

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Fully oxidized mammalian TRX was considered to alter its reduction rate by TrxR more slowly than TRX oxidized only at the active site, suggesting that the oxidation of the non-active site cysteines may change the rate of reduction and control a redox mechanism of TRX function

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[6, 7]. Consequently, a proteomic study analyzing a second disulfide bond formed between Cys62 and Cys69 demonstrated its considerable effect on TRX activity under oxidizing conditions [9].

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The structures of human TRX determined to date involve its oxidized [3], reduced [3], and Snitrosylated [10] forms. Among these, the oxidized TRX structures contain disulfide bonds

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formed between active residues Cys32 and Cys35, and dimerization Cys73 and Cys73` (the residue belonging to the other molecule is indicated by a prime). A fully oxidized TRX structure containing disulfide bond between the non-active residues Cys62 and Cys69 has been missing. Such a structure would allow a more complete molecular understanding of the redox-related

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regulation of TRX.

Recently, we reported the molecular mechanism of the negative regulation of TRX by the TRX-interacting protein (TXNIP), the only known endogenous inhibitor of TRX [11, 12].

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During this study, we found a crystal in a crystallization screening plate containing the Nterminal TXNIP domain and TRX complex. Unexpectedly, the crystal was of TRX only, and its

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structure revealed a fully oxidized conformation containing disulfide bonds between Cys32 and Cys35, Cys62 and Cys69, and Cys73 and Cys73`. Here, we report the molecular details of the fully oxidized TRX structure and its biological implications in the regulation of TRX function.

2. Materials and methods

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2.1. DNA cloning, protein expression, and purification The plasmid co-expressing TRX and the N-terminal domain of TXNIP (residues 3–156) was constructed using a two-promoter vector system as described in a previous study [11].

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Expression of the complex was induced in E. coli Rosetta GamiTM (DE3) by treatment with 0.5 mM isopropyl β-D-thiogalactopyranoside at 21°C for 40 h. The cells were then harvested by centrifugation at 5,000 g at 4°C for 15 min. The cell pellets were re-suspended in cooled buffer

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A (50 mM Tris-HCl, pH 8.0 and 300 mM NaCl), and the cell suspension was ultrasonicated. Cell lysates were centrifuged at 25,000 g at 4°C for 1 h. The supernatant containing hexahistidine-

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tagged N-terminal domain of TXNIP and TRX was loaded onto a Ni-NTA agarose column that was pre-equilibrated with cooled buffer A. The column was intensively washed with buffer A and eluted with 250 mM imidazole. The hexahistidine tag was then removed from the N-terminal TXNIP domain by incubation with rTEV protease (Invitrogen, Carlsbad, CA, USA) at 4°C for

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16 h, followed by size exclusion and second Ni-NTA column chromatographies. The purified complex protein was dialyzed against 50 mM Tris-HCl, pH 7.0, concentrated to 12.1 mg/ml, and

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stored at -80°C until use.

2.2. Crystallization, data collection, and structure determination

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Crystallization was carried out using the sitting drop vapor diffusion technique. The purified complex protein was crystallized in 2.0 M ammonium citrate and 0.1 M Bis-Tris propane, pH 7.0 at 21°C. A crystal was observed after about 6 months of setting up the crystallization. The crystal was cryoprotected in the crystallization solution containing 23% glycerol and flash-cooled in liquid nitrogen to 100 K. Diffraction data were collected at a resolution of 1.4 Å at 5C beamline of the Pohang Accelerator Laboratory (PAL, Pohang, Korea), and processed using the HKL2000 5

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package [13]. The crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 53.204, b = 61.231, and c = 64.839 Å. Considering the unit-cell volume (211218.2 Å3), only one TRX molecule was expected in the asymmetric unit. Accordingly, the

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crystal structure was determined by molecular replacement method with the program MOLREP [14] using TRX (PDB ID 2HSH) as the search template [10]. The structure was revised using COOT [15] and refined with REFMAC5 [16]. The crystallographic data are summarized in

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3. Results and discussion

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Table 1.

3.1. Structure of the fully oxidized human TRX

Although our original aim to solve the structure of the N-terminal domain of TXNIP

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complexed with TRX failed due to the transient nature of the interaction between the two proteins [11], we were fortunate to be able to determine the structure of the fully oxidized TRX. The fully oxidized TRX is comprised of a central core with a five-stranded β-sheet flanked

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by three α-helices (Fig. 1A). This is in contrast to the β-sheet flanked by four α-helices seen in all reported TRX structures. The active site disulfide bond formed between Cys32 and Cys35 and its

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surrounding environment in the fully oxidized structure are identical to the previously published form of the TRX structure (PDB ID 2HSH) in which only the active site is oxidized [10] (Fig. 1B, left panel). The most remarkable structural feature of the fully oxidized TRX is the second disulfide bond, formed between residues Cys62 and Cys69 (Fig. 1B, right panel). This disulfide bond formation completely disrupts the third α-helix comprising residues Cys62 to Glu70, and instead forms a bulging loop. In this conformation, the hydrophobic residues Val65 and Ala66, 6

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which in the helix were primarily involved in maintaining the overall stability of the structure through formation of hydrophobic interactions with neighboring residues (Lys8, Phe11, Phe27, Val57, Val59, Cys62, and Val71), become fully exposed to solvent (Fig. 1B and C). The loop

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formed by the second disulfide bond is further stabilized by the interactions of the Ala66 backbone with the Lys8 side-chain amino group and the Glu68 side-chain carboxyl group with the Gln12 side-chain amide group (data not shown).

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During refinement of the structure, extra electron density was observed near the second disulfide bond and assigned to a benzoic acid, perhaps originating from metabolites of culture

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medium or trace elements of crystallization reagents (Fig. 2A). The benzoic acid stabilizes the loop through interactions of its carboxyl group with Gln63 and Glu68 backbone amides and the Ser67 hydroxyl group (Fig. 2A). Its aromatic ring makes hydrophobic interactions with residues Ile5, Lys8, Phe11, Val57, Asp61, and Cys62 (Fig. 2A).

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While only a single TRX molecule was observed in the asymmetric unit, crystallographic symmetry analysis revealed dimerization of TRX with symmetry mates via an intermolecular disulfide bond formed between Cys73 and Cys73` (Fig. 2B). The dimer conformation is identical

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to previously reported TRX dimers mediated by Cys73 residues [3].

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3.2. Biological implications of the fully oxidized TRX TrxR reduces fully oxidized mammalian TRX much more slowly than TRX oxidized only at the active site [6, 7, 17]. This suggests that oxidation of the non-active site thiols may control the rate of reduction by TrxR and regulate TRX function in redox signaling or oxidative stress. Furthermore, proteomic analysis showed that TRX may form a disulfide bond between Cys62 and Cys69 [9]. Disulfide formation inhibited the re-activation of TRX by TrxR in vitro, 7

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indicating that oxidation of the non-active site thiols may provide a structural switch affecting TRX function [9]. Recently, the structure of TRX in complex with TrxR revealed the electron transport of TrxR

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to its substrate TRX taking place via an intermolecular disulfide formed between the catalytic residues TRX Cys32 and TrxR SeCys498`. The active site residue in TrxR, SeCys498, was mutated to Cys498 in the structure to trap the reaction intermediate showing the electron

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transport via the intermolecular disulfide formation (PDB ID 3QFA, Fig. 3A) [18]. The structure illustrates the critical involvement of TRX residues 59 to 74 in the interaction with TrxR. These

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residues form a pocket in which Trp114, located on a σ-helix in TrxR, is buried (Fig. 3A). Among the critical residues, the α3-helix of TRX comprised of Cys62 to Cys69 plays an important role for the interaction, not only through formation of the pocket for TrxR Trp114, but through additional stabilization via a salt bridge with TrxR (Fig. 3A). Mutation of Trp114 in

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TrxR resulted in no detectable activity towards TRX(C73S) [18]. While the TrxR Trp114 side chain indole sits on the TRX pocket, the TRX Trp31 indole fits inside a pocket formed by TRX residues Ala29, Thr30, and Met74, TrxR residues Asn107, Gly110, Ser111, and Trp114, and the

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intermolecular disulfide between TRX Cys32 and TrxR Cys498` (Fig. 3A). The high resolution structure of the fully oxidized TRX allows the rationalization of the

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molecular mechanism of TRX function mediated by its non-active site disulfide. The α3-helix of TRX is unquestionably essential for stable interaction with TrxR (Fig. 3A). Disruption of the α3helix of TRX by the non-active site disulfide bond formation dramatically changes the environment of TRX residues 59 to 74 (Fig. 1). Such an altered environment would disturb the interaction of TRX with TrxR. More specifically, the interactions of TrxR Arg121 with TRX Ser67 and Glu70, and TrxR Glu122 with TRX Lys72, that seem to be critical for the stable 8

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electron transport of TrxR to TRX (Fig. 3A), would be impacted by formation of the Cys62Cys69 disulfide because this disulfide significantly changes the location of residues Ser67, Glu70, and Lys72 (Fig. 3B). The bulging loop itself formed by non-active site disulfide of TRX

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may present an obstacle to the optimal structural interactions with the α-helix (H) and a β-strand (S) of TrxR (Fig. 3B). Overall, these results suggest that the second disulfide bond may be one of the modifications of TRX that modulate its biological activity by controlling the interaction of

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TRX with TrxR.

The TRX and TrxR system is one of the major antioxidant systems involved in maintaining

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the reduced state of many cellular substrates, particularly several transcription factors, such as activator protein 1 (AP-1) [19] and NF-κB [20, 21]. Reduction of cysteine residues in transcription factors increases DNA binding activity and induces expression of target genes [19, 21], which may lead to a diverse range of effects in cellular physiology. It has been demonstrated

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that the interaction between TRX and TrxR is sensitive to the presence of the second disulfide, which causes biphasic reduction of the two disulfide bonds in TRX and decreases the reduction rate of the active site of TRX by TrxR [9]. Interestingly, while the active site is reduced by TrxR,

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the non-active site disulfide is a substrate for reduction by the active site of TRX [9]. These findings suggest that, in addition to its influence on the TrxR reducing activity towards TRX, the

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second disulfide of TRX may affect the interaction and reduction rates of TRX with other cellular substrates. Because the α3-helix and the loop between α3-helix and strand β4 of TRX affect the interaction with cellular substrates such as NF-κB [20] (Fig. 3C), disruption of the α3helix resulting from formation of the second disulfide bond may lead to delays in the reduction rates of these cellular substrates (Fig. 3D).

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TRX forms homodimers mediated by an intermolecular Cys73-Cys73` disulfide bond in close proximity to the α3-helix, a conformation that may lead to the active site becoming inaccessible to TrxR [7]. As mentioned above, the disulfide between Cys62 and Cys69 caused

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the biphasic reduction of the two disulfide bonds of TRX and decreased the reduction rate of the active site of TRX by TrxR [9]. However, a TRX mutant (C62S/C69S) that was unable to form the second disulfide bond, under conditions that still allowed Cys73-mediated homodimer

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formation, remained active as a substrate for TrxR [7, 22], suggesting that TRX dimerization may be not involved in the early lag phase of reduction by TrxR [9].

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In conclusion, the switch of the third α-helix into a bulge loop via the reversible oxidation of the non-active cysteine residues in TRX suggests a number of potential functional implications, specifically the regulation of reduction rate of TRX by TrxR and recognition of many cellular substrates, which may affect diverse signaling pathways and physiology. Thus, mammalian TRX

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may respond to and regulate the cellular environment via more sophisticated and complex modifications than those described to date, using all of its five conserved cysteine residues,

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Cys32, Cys35, Cys62, Cys69, and Cys73.

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Acknowledgments

We thank the beamline staff at Pohang Light Source (beamline 5C), Korea for their help with data collection. This work was supported by the National Research Foundation of Korea (NRF) grant (NRF-2010-0029767 and no. 2014R1A2A1A01005971) funded by the Korea government (MSIP) and the KRIBB Initiative program (KGM4541521).

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Conflict of interest

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The authors have no financial conflicts of interest.

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Figure legends

Fig. 1. Overall structure of fully oxidized human TRX. (A) Cylinder representation of a

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representative human TRX (PDB ID 2HSH). The structure of an active site oxidized TRX (PDB ID 2HSH, left panel) in gray is compared with the structure of the fully oxidized TRX in yellow

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(right panel). The α3-helix and its corresponding region in fully oxidized TRX are displayed in red. Each β-strand and α-helix is labeled. (B) Effects of Cys62-Cys69 disulfide bond formation on the structure of TRX. The α3-helix that maintains the overall structural stability of TRX through hydrophobic interactions (PDB ID 2HSH, left panel) is replaced by a bulging loop

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induced by formation of the Cys62-Cys69 disulfide (right panel). (C) 2Fo-Fc electron density map contoured at 1.0 σ of the fully oxidized TRX showing the non-active disulfide bond.

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Fig. 2. Structural highlights of the fully oxidized human TRX. (A) A benzoic acid stabilizing the

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bulging loop formed by the non-active disulfide bond. The Fo − Fc map was calculated before the inclusion of benzoic acid and is contoured at 3.5 σ. (B) The structure of the fully oxidized human TRX showing the active disulfide bond between Cys32 and Cys35, non-active disulfide bond between Cys62 and Cys69, and intermolecular disulfide bond between Cys73 and Cys73`. The 2Fo-Fc electron density map contoured at 1.0 σ of the intermolecular disulfide bond between Cys73 and Cys73` is shown. 14

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Fig. 3. Biological implications of the structural modification caused by formation of the Cys62Cys67 disulfide. (A) The structure of the TRX and TrxR complex (PDB ID 3QFA) showing the

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electron transport of TrxR to TRX via an intermolecular disulfide between the catalytic residues TRX Cys32 and TrxR Cys498`. The interaction residues between TrxR (green) and TRX (gray) critical for the stable electron transport are shown. The residue belonging to the other molecule

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of TrxR is primed and displayed in cyan. H, σ-helix; S, β-strand. (B) Effects of Cys62-Cys69 disulfide on the interaction between TRX and TrxR. Formation of this disulfide significantly

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alters the location of the TRX residues (yellow) involved in TrxR (green) interactions. H, σhelix; S, β-strand. (C) The α3-helix of TRX is critical for the interaction with its cellular substrate NF-κB. The TRX residues (gray) involved in the interaction with NF-κB (green) are displayed. The residues Cys62, Cys69, and Met74 in TRX are substituted with Ala62, Ala69,

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and Thr74, respectively, in the structure of the TRX and NF-κB complex (PDB ID 1MDJ). (D) Formation of the non-active disulfide bond of TRX (yellow) may preclude the interaction with

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NF-κB (green) and lead to delayed reduction of this cellular substrate.

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Table 1. Data collection and refinement statistics. Data collection Space group Cell dimensions (Å) A B C

53.20 61.23 64.84 90 1.00 50-1.40 (1.45-1.40) 114,689 20,475 5.6 (3.3) 96.9 (83.7) 11.2 (49.2) 17.88 (1.91)

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105 aa 112 1 benzoic acid, 1 glycerol, 1 chloride ion 15.8/20.9

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Resolution (Å) Model composition Protein Waters Ligands Rworkb/Rfreec (%) R.m.s. deviations Bond lengths (Å) Bond angles (°) PDB code

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α, β, γ Wavelength Resolution (Å) No. of total reflections No. of unique reflections Redundancy Completeness (%) Rsym (%)a I/σ(I) Refinement

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I222

0.027 2.248 5DQY

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The numbers in parentheses describe the relevant value for the highest resolution shell. a Rsym =∑ |Ii-| / ∑I where Ii is the intensity of the i-th observation and is the mean intensity of the reflections. b Rwork = ∑||Fobs| – |Fcalc|| / ∑|Fobs| where Fcalc and Fobs are the calculated and observed structure factor amplitude, respectively. c Rfree = ∑||Fobs| – |Fcalc|| / ∑|Fobs| where all reflections belong to a test set of randomly selected data.

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Highlights

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The structure of fully oxidized human thioredoxin (TRX) shows a non-active disulfide


Non-active disulfide switches the α3-helix of TRX to a bulging loop.


The structural modification of TRX via a disulfide suggests a mechanism for the

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regulation of TRX functions in cellular redox responses.