The Structure of Human Thioredoxin Reductase 1 Provides Insights into C-terminal Rearrangements During Catalysis

Abstract

Human thioredoxin reductase (hTrxR) is a homodimeric flavoprotein crucially involved in the regulation of cellular redox reactions, growth and differentiation. The enzyme contains a selenocysteine residue at its C-terminal active site that is essential for catalysis. This redox center is located on a flexible arm, solvent-exposed and reactive towards electrophilic inhibitors, thus representing a target for antitumor drug development. During catalysis reducing equivalents are transferred from the cofactor NADPH to FAD, then to the N-terminal active site cysteine residues and from there to the flexible C-terminal part of the other subunit to be finally delivered to a variety of second substrates at the molecule's surface. Here we report the first crystal structure of hTrxR1 (Sec→Cys) in complex with FAD and NADP⁺ at a resolution of 2.8 Å. From the crystals three different conformations of the carboxy-terminal arm could be deduced. The predicted movement of the arm is facilitated by the concerted action of the three side-chain residues of N418, N419 and W407, which act as a guiding bar for the C-terminal sliding process. As supported by previous kinetic data, the three visualized conformations might reflect different stages in enzymatic catalysis. Comparison with other disulfide reductases including human glutathione reductase revealed specific inhibitor binding sites in the intersubunit cavity of hTrxR that can be exploited for structure-based inhibitor development.

Introduction

Mammalian thioredoxin reductases (TrxRs) are members of the pyridine nucleotide disulfide reductase family of dimeric flavoenzymes with high homology to glutathione reductases. TrxR (EC 1.8.1.9) reduces a broad range of substrates including the direct reduction of low M_r compounds like lipoamide, hydrogen peroxide and alloxane as well as direct reduction of proteins like disulfide isomerase and NK-lysine and reduction of plasma glutathione peroxidase via reduced thioredoxin. Its main function, however, is the reduction of the 12 kDa disulfide protein thioredoxin (Trx) to its dithiol-containing form:1., 2., 3., 4.

$$\text{NADPH}+{\mathrm{H}}^{+}+{\text{Trx−S}}_2\rightleftharpoons {\text{NADP}}^{+}+{\text{Trx−(SH)}}_2\text{.}$$

Apart from its “classical” function as an electron shuttle for ribonucleotide reductase, Trx modulates the activity of transcription factors, supports protein biosynthesis and folding, regulates enzyme activities, serves as an anti-oxidant and can act extracellularly as an autocrine growth factor.2., 5. The central functions of the thioredoxin system make it an attractive target for anti-tumor drug development.4., 6.

In mammals, three types of TrxRs have been characterized: the cytosolic form hTrxR1⁷ and the mitochondrial hTrxR28., 9. which share 52% sequence homology, as well as the thioredoxin glutathione reductase (TGR), a hybrid enzyme possessing an additional glutaredoxin domain.10., 11. TGR occurs predominantly in the microsomal fraction of testis tissue. Human TrxR1 is transcribed by a house-keeping-type promoter and is regulated post-transcriptionally. Heterogeneity of the 5′ untranslated region in mammalian TrxR mRNA results from alternative first exon splicing.12., 13.

Mammalian TrxRs contain selenocysteine (Sec, U) as the penultimate amino acid.14., 15. The species-specific usage of the selenocysteine insertion sequence (SECIS) element makes a heterologous expression of the mammalian enzyme difficult, a challenge that has been tackled by different groups.16., 17., 18. Various approaches including site-directed mutagenesis,19., 20., 21. selenium depletion²² and selective digestion or alkylation23., 24. proved that the selenocysteine is essential for the catalytic mechanism of human TrxR1. After binding of NADPH, electrons are transported from NADPH via FAD to the N-terminal active site disulfide and from there to the C-terminal redox center of the other subunit, which finally reduces the substrate.15., 20., 25. The first three-dimensional structure of a mammalian TrxR1, namely a Sec498Cys rat TrxR mutant, was solved by Sandalova et al.²⁶ The protein was found to be similar to GR including the three domain structure of a monomer as well as FAD and NADPH binding residues. Interestingly, most residues directly interacting with GSSG in GR are conserved in rat TrxR although the enzyme does not turn over GSSG.²⁶ Also for hTrxR1 in its truncated form, which was considered to resemble GR mechanistically, this phenomenon was observed and attributed to electrostatic repulsion rather than to steric hindrance effected by the C-terminal extension.²⁷ Recently, the structure of mouse TrxR2 was reported, differing in some key active site residues from the rat TrxR1 structure.²⁸ Conformational changes were observed upon NADPH binding and an active site tyrosine residue was found to be responsible for optimal placement of the nicotinamide moiety. Variations in the flexible C-terminal part of mouse TrxR2 suggested differences in activities of cytosolic and mitochondrial TrxR.²⁸

On the basis of its redox properties, the selenocysteine in hTrxR has been reported to contribute to redox signaling and, specifically, to mediating responses to oxidative stress.29., 30. Cell death can be rapidly induced by selenium-compromised TrxR1 but not by the fully active selenoenzyme.³¹ In accordance with the emerging importance of the selenium-containing C-terminal extension of hTrxR, it was postulated that these extensions of proteins represent a general mechanism of evolution of new protein function.³²

Here we report the first three-dimensional structure of human TrxR1; NADP⁺ is bound. We describe three distinct conformations of the carboxy-terminal amino acid chain, which reflect different stages in the catalytic cycle of the enzyme. Comparison of hTrxR1 with other disulfide reductases gives important information on potential specific inhibitor binding sites.