The high-resolution three-dimensional solution structures of the oxidized and reduced states of human thioredoxin

Abstract

Background Thioredoxin is a ubiquitous protein and is involved in a variety of fundamental biological functions. Its active site is conserved and has two redox active cysteines in the sequence Trp-Cys-Gly-Pro-Cys. No structures of the oxidized and reduced states from the same species have been determined at high resolution under the same conditions and using the same methods. Hence, any detailed comparison of the two oxidation states has been previously precluded.

Results The reduced and oxidized states of the (C62A, C69A, C73A) mutant of human thioredoxin have been investigated by multidimensional heteronuclear NMR. Structures for both states were determined on the basis of approximately 28 experimental restraints per residue, and the resulting precision of the two structures is very high. Consequently, subtle differences between the oxidized and reduced states can be reliably assessed and evaluated. Small differences, particularly within and around the active site can be discerned.

Conclusions Overall, the structures of the reduced and oxidized states of the (C62A, C69A, C73A) mutant of human thioredoxin are very similar (with a backbone atomic root mean square difference of about 0.9 å) and the packing of side chains within the protein core is nearly identical. The conformational change between oxidized and reduced human thioredoxin is very small and localized to areas in spatial proximity to the redox active cysteines. These subtle structural differences, in addition to the restriction of conformational freedom within the active site upon oxidation, may be important for the different activities of thioredoxin involving a variety of target proteins.

Introduction

Thioredoxin is a ubiquitous dithiol oxidoreductase found in many organisms and involved in numerous biochemical processes [1], [2]. Although the most widely studied thioredoxin is from Escherichia coli , thioredoxins from other bacteria, bacteriophages, plants, and mammals have also been isolated and sequenced, and have been found to exhibit between 25 and 70 % sequence identity with E. coli thioredoxin [3]. Thioredoxin activity is related to the thiol–disulfide redox chemistry of two cysteine residues found in the conserved active site sequence Trp-Cys-Gly-Pro-Cys which is present in all species [1], [2]. Thioredoxin acts as a hydrogen donor for reductive enzymes such as ribonucleotide reductase [4], [5]; functions as a general reductant for disulfides in proteins including insulin, oxytocin, and fibrinogen [1]; and serves as a regulatory factor for enzymes or receptors in photosynthetic systems [6]. Specific activities associated with human thioredoxin include the induction of interleukin-2 receptor expression [7], autocrine growth factor properties [8], and the stimulation of the DNA-binding activity of the transcription factor NF-κ B [9], [10].

The mechanism of the redox-related functions of E. coli thioredoxin has been proposed to involve nucleophilic attack by the more reactive Cys32 thiolate anion on a disulfide-containing substrate, producing a mixed-disulfide intermediate, which is then attacked by the thiolate of Cys35 to yield a reduced substrate and free, oxidized thioredoxin [11]. A number of experimental measurements including fluorescence [12], NMR [13], [14], [15], hydrogen exchange [16], specific volume and adiabatic compressibility [17] have indicated that there are subtle conformational differences between the oxidized and reduced states of thioredoxin, the nature of which have not yet been characterized.

E. coli thioredoxin also has several non-redox related functions. For example, it constitutes an essential subunit in the phage T7 DNA polymerase and is required in the assembly of filamentous phages [18], [19]. These activities are associated with the reduced protein, and it has been established that the redox cycle is not required since mutants of thioredoxin with one or both active site cysteines replaced by serine or alanine are also active [19]. Subtle structural and dynamic differences between the reduced and oxidized forms have been invoked to explain these functional changes [20]. Mutagenesis studies indicate that the conserved active site region plays an important role in interacting with other proteins for both redox and non-redox related functions of thioredoxin [18], [19], [20], [21], [22], [23], [24]. In contrast to our extensive knowledge about E. coli thioredoxin, little is known about the functional activities of human thioredoxin beyond its general redox properties.

The three-dimensional structures of a number of different thioredoxins have been determined, with the aim of shedding light on the catalytic mechanism of thioredoxin. These include the structure of oxidized E. coli thioredoxin by X-ray crystallography [25], [26], and the solution structures of reduced E. coli [27] and human [28] thioredoxins determined by two- dimensional NMR. The E. coli and human proteins exhibit very similar three-dimensional folds despite the large variation in amino acid sequence (25 % sequence identity with some deletions and insertions). However, none of the structures were determined at high resolution under the same conditions and using the same methods for both the reduced and oxidized states of thioredoxin from the same species. Hence, any detailed comparisons of the oxidized and reduced states is precluded, particularly as the structural differences are clearly very subtle in nature. Moreover, the precision and accuracy of the two previous NMR structure determinations of reduced E. coli [27] and human [28] thioredoxins would be insufficient to permit the detection of minor changes between the reduced and oxidized states.

Clearly, a detailed structural comparison between reduced and oxidized forms of human thioredoxin will be of considerable importance for understanding the functional properties of human thioredoxin and of thioredoxins in general. The comparison may also shed light on the structural role of the reduced state of thioredoxin for its non-redox related function.

In this paper we present high-resolution three-dimensional structures of the reduced and oxidized states of the triple Cys → Ala mutant (C62A, C69A, C73A) of human thioredoxin in solution using multidimensional heteronuclear NMR spectroscopy. This mutant, rather than the wild-type protein, was chosen for study in order to circumvent any problems arising from intermolecular disulfide bond formation by the three free non-active site cysteine residues upon oxidation. Previous studies have shown that this mutant has virtually the same activity as the wild-type protein and that the structures of the mutant and wild-type proteins are essentially indistinguishable [29]. The present structures have been determined on the basis of about 28 experimental restraints per residue. As a result, the precision with which the coordinates have been determined is very high, namely 0.18–0.19 å for the backbone atoms, 0.58–0.60 å for all atoms, and 0.30–0.31 å for all atoms of residues which do not exhibit conformational disorder in solution. This represents an approximately 50 % increase in the number of nuclear Overhauser enhancement (NOE) derived interproton distance restraints, a 120 % increase in the backbone precision and a 70 % increase in the precison of ordered side chains relative to our previous structure determination of wild-type reduced human thioredoxin [28]. As a result, subtle differences between the oxidized and reduced states can be observed with great confidence. We show that while the global fold is extremely similar between the reduced and oxidized forms of human thioredoxin, there exist some important localized conformational differences particularly in the active site region.