Chapter 1: Oxidative Folding of Proteins in vivo

The cellular redox state is a crucial mediator of several aspects of life, e.g. growth and apoptosis. It is based on low-molecular-weight thiols such as glutathione (GSH) and protein thiols, providing a reducing or an oxidizing environment. The cytoplasm with its low redox potential favors the reduction of cysteinyl residues, whereas the prokaryotic periplasm supports disulfide-bond formation. In principle thiol-disulfide pairs in proteins have two possible functions. First, disulfides often can contribute to the overall structure and stability of the protein; second, the redox state of the cysteinyl residues can regulate the activity of the protein. The modifications of cysteine residues and thereby the redox state of the particular compartment are controlled by thiol-disulfide oxidoreductases, which mainly belong to the thioredoxin family of proteins.

Escherichia coli thioredoxin 1 (Trx1), the first member of the thioredoxin family of proteins, was discovered more than 40 years ago as an electron donor for ribonucleotide reductase (RNR).^1,2  In all organisms, this enzyme is essential for DNA synthesis during both replication and repair.³  The second member of the Trx family, glutaredoxin (Grx), was discovered as a GSH-dependent electron donor for RNR in an E. coli mutant lacking Trx.⁴  Today, the Trx protein family is first and foremost defined by a structural motif named the Trx fold.⁵  In spite of considerable variation in overall structure, the Trx fold is present in a variety of functionally different proteins:^6,7  thiol-disulfide oxidoreductases, disulfide isomerases, glutathione S-transferases,⁸  thiol-dependent peroxidases⁹  and chloride intracellular channels.¹⁰ 

The Trx fold motif consists of a central four-stranded β-sheet surrounded by three α-helices^11,12  (Figure 1.1.1A). This basic βαβαββα topology can only be found in bacterial glutaredoxins, while thioredoxins contain an additional β-sheet and α-helix at the N-terminus¹³  (Figure 1.1.1B,C). Variations of this motif have also been identified in domains of 723 proteins,¹⁴  e.g. the Trx fold domains of E. coli DsbA, a protein necessary for disulfide-bond formation in the periplasm¹⁵  (Figure 1.1.1D).

Hallmarks of the Trx motif are a cis-proline residue located before β-sheet three and the Cys-X-X-Cys active site motif located on the loop connecting β-sheet one and α-helix one.¹¹  The nature and composition of these two amino acids dramatically affects the standard redox potential of the particular proteins. In E. coli, the strongest reductant, cytosolic Trx (Cys-Gly-Pro-Cys), has a redox potential of ΔE′₀ = −270 mV,¹⁶  the strongest oxidant, DsbA (Cys-Pro-His-Cys), has a redox potential of ΔE′₀ = −122 mV.¹⁷  Mutation of the Cys-Gly-Pro-Cys active site in Trx to the corresponding Cys-Pro-His-Cys active site of DsbA resulted in an increase of its standard midpoint potential to ΔE′₀ = −204 mV.¹⁸  Supportingly, the redox potential of a DsbA mutant harboring the Cys-Gly-Pro-Cys active site of Trx decreased by 92 mV.¹⁹  Several other amino acids outside the active site motif have been determined as important for the redox potential of Trx fold oxidoreductases.^20–22 

In 1968 E. coli Trx1 was sequenced, revealing the characteristic Cys-Gly-Pro-Cys active site motif²  and seven years later the Trx fold was described for the first time.¹¹  Since then, more than 200 structures of different Trxs were solved including structures of both oxidized and reduced Trxs. These structures are very similar, but reduction induces local conformational changes in the area of the active site (e.g. ref. 23). The two cysteinyl residues in the active site of Trx are utilized to reduce the protein disulfide formed during the catalytic cycle of RNR.^1,3  Today we know Trx as a general disulfide reductase²⁴  reducing disulfide bonds by a ping-pong mechanism²⁵  (Figure 1.1.2). The N-terminal active site thiol of Trxs possesses an unusual low pK_a value,²⁶  whereas the pK_a of the C-terminal active site is higher than that of cysteine in solution.²⁷  The low pK_a of the N-terminal cysteine of the E. coli Trx1 active site was shown to be related to the carboxyl group of Asp 26 and the ε-amino group of Lys 57.²⁷  Hence, the thiol group of the N-terminal active site cysteine is readily deprotonated under physiological conditions. Recently, it was shown by single molecule force-clamp spectroscopy that efficient catalysis requires a reorientation of the substrate disulfide bond.²⁸  This investigation demonstrated that the rate-limiting step of Trx activity is the orientation of the N-terminal active site cysteine of Trx and the two disulfide bridged cysteines of the substrate in a 180° angle. This reorientation provides the condition for the nucleophilic attack of the N-terminal Cys resulting in a covalent intermediate mixed disulfide between the Trx N-terminal active site and one of the substrate's cysteinyl side-chains.²⁶  The C-terminal active site cysteinyl side-chain reduces this disulfide yielding the reduced substrate and a disulfide in the active site of Trx. Subsequently, the disulfide in the active site of Trx is reduced by the dimeric flavo-enzyme thioredoxin reductase (TrxR) at the expense of NADPH (for a more detailed overview, see ref. 12).

E. coli contains two thioredoxins. Trx1, a protein with a molecular mass of 12 kD, and Trx2, a protein of 15.5 kDa, which contains an N-terminal domain of 32 amino acids including two additional Cys-X-X-Cys motifs. These four extra cysteines are able to coordinate zinc.²⁹ 

Glutaredoxins (Grxs) exist in all glutathione (GSH)-containing life forms. As described in Section 1.1.1.1 bacterial Grxs displayed the basic architecture of the Trx fold. Similar to Trxs, the structural comparison of reduced³⁰  and oxidized³¹  E. coli Grx1 revealed more or less identical overall structures but significant changes in and around the active site.³² 

Based on their active site motifs, Grxs can be divided into two major categories: the dithiol Grxs (active site: Cys-Pro-Tyr-Cys) and the monothiol Grxs (active site: Cys-Gly-Phe-Ser). Dithiol Grxs are general thiol-disulfide oxidoreductases reducing some protein disulfides like that in E. coli ribonucleotide reductase with a dithiol mechanism as described for Trxs above (Figure 1.1.2). In addition, Grxs are able to reduce protein-GSH mixed disulfides (de-glutathionylation) utilizing a mechanism that requires only the N-terminal active site residue (monothiol mechanism; for a more detailed overview, see ref. 33). Grxs use GSH as electron donor. The resulting glutathione disulfide (GSSG) is reduced by glutathione reductase (GR) with electrons from NADPH^34,35  (Figure 1.1.2). The molecular mechanism and the functions of monothiol glutaredoxins, which are prevalent inactive in (dithiol) Grx-specific activity assays, are only beginning to emerge (for a recent review see ref. 36).

E. coli contains the three dithiol Grxs 1–3,^37,38  and the monothiol Grx4.³⁹  Grxs 1, 3 and 4 are single domain proteins with molecular masses of 10, 9 and 13 kDa, respectively, whereas Grx2 is a larger protein of 24 kDa. Only the N-terminal part of Grx2 is a Trx-fold domain; the overall structure resembles that of GSH-S-transferases.⁴⁰  Recently, several monothiol Grxs including E. coli Grx4 were described as [FeS] proteins.⁴¹  The cofactors are coordinated by the N-terminal active site cysteine of two Grx monomers and two non-covalently bound molecules of GSH as described before for human Grx2 and poplar GrxC1.^42–44  So far, E. coli Grx4 is the only essential member of the Trx family of proteins in E. coli.³⁹ 

E. coli NrdH (9 kDa) shows similarities to Grxs in its secondary and tertiary structure.⁴⁵  However, it lacks the GSH binding site and is therefore not reduced by GSH. In vitro and in vivo studies identified TrxR as an electron donor of NrdH.^45,46  NrdH was shown to act as electron donor for RNRs.^45,47 

YbbN/Trxsc is a protein of 31 kDa expressed in the cytoplasm of E. coli.⁴⁸  Its N-terminus is homologous to Trxs, but without a Cys-X-X-Cys motif.

Peroxiredoxins (reviewed in ref. 49), a ubiquitous family of thiol-dependent proteins reducing peroxides like hydrogen peroxide or peroxinitrite, contain Trx-fold domains. In E. coli, four of these peroxiredoxins are present. The primary scavenger of endogenous hydrogen peroxide is alkyl hydroperoxide peroxidase C (AhpC),⁵⁰  which is reduced by AhpF. Thiol peroxidase (Tpx), the gene product of btuE, a GSH peroxidase homolog, and bacterioferritin comigratory protein (BCP) are reduced by Trx1, or at least are Trx dependent.^51,52 

GSH-S-transferases, as described for E. coli Grx2, contain a Trx domain. The E. coli genome encodes for eight of these proteins transferring GSH to electrophilic compounds and thereby detoxifying them.⁵³ 

In the periplasm, oxidative folding takes place performed by proteins of the Dsb family (reviewed in ref. 54 and Chapter 1.2). DsbA introduces disulfides into substrates, and the protein disulfide isomerases DsbC and DsbG reduce incorrect formed disulfide bonds. All these proteins are Trx-fold oxidoreductases.^15,55,56  The eukaryotic counterparts to E. coli Dsbc/DsbG, for instance human and yeast PDIs (protein disulfide isomerases), are Trx-fold proteins as well.^57,58 

As in the chapters above we will focus on E. coli as the best investigated prokaryotic organism. Here, numerous functions have been described for Trxs and Grxs, both as electron donors as well as regulators of cellular function in response to oxidative stress (Figure 1.1.3).

Within the framework of this chapter, it is important to note that a number of functions described for these proteins are independent from their oxidoreductase activity, for instance E. coli Trx as subunit of the T7 DNA polymerase complex,⁵⁹  or its activity as molecular (co-)chaperone (ref. 60, see 1.1.3.).

Trxs and Grxs keep a reduced environment in the cytoplasm by reducing protein disulfides and protein-GSH mixed disulfides.

By modulating the redox state of protein thiols Trxs and Grxs can function as regulators of transcription factors. OxyR is such a factor activating transcription of several E. coli genes encoding proteins defending against hydrogen peroxide induced oxidative stress, e.g. catalase 1, AhpCF, GSH reductase, Grx1 and Trx2.⁶¹  The formed disulfide, which activates OxyR, is most likely in vivo reduced by Grx1.⁶¹  The activity of the transcription factor SoxR is regulated by a [2Fe2S] cluster. If the cluster is oxidized, the transcription of proteins involved in defense against oxidative stress is activated.⁶²  In vitro, the reconstitution of the [FeS] cluster of SoxR is promoted by the Trx system. In addition, SoxR inactivation is inhibited in E. coli mutants lacking TrxR and GR.⁶³ 

Trx and Grx have also been described as electron donors for antioxidant enzymes, for instance, peroxiredoxins as described in Section 1.1.1.4.

Methionine sulfoxides that may form during oxidative stress by reactive oxygen species are reduced by methionine sulfoxide reductases (Msrs).⁶⁴  In E. coli six Msrs are present,⁶⁵  which are most likely using Trx1 as the electron donor in vivo.⁶⁶  Since increased expression of Grx1 and Trx2 in an E. coli mutant lacking Trx1 allows growth using methionine sulfoxide as the sole source of methionine, these two oxidoreductants are also potential electron donors for Msrs in vivo.⁶⁶ 

Trx and Grx were first described as electron donors for RNR.^1,4,67  RNRs catalyze the reduction of nucleotides to deoxynucleotides and are thus essential for DNA synthesis.^68,69  E. coli cells are equipped with three RNR enzymes. While RNR1a and RNRIII are essential for aerobic and anaerobic metabolism, respectively, RNR1b is not essential.^69,70  The rate-limiting step during enzymatic turn-over of ribonucleotides by RNR1a is the reduction of a disulfide performed by Trxs or Grxs.^1,4,37,71,72  In E. coli, Grx1 may be the primary electron donor for RNR1a in vivo.^73,74  Trx1 may be involved in activation of RNRIII⁷⁵  via reduction of a disulfide.⁷⁶ 

Bacteria are able to satisfy their need for reduced sulfur by assimilation of inorganic sulfate. Reduction of sulfate (SO$42−$) to sulfide (S²⁻) requires eight electrons and takes place in two steps. First, sulfate is activated to adenylylsulfate (APS) by ATP sulfurylase and then to phoshoadenylylsulfate (PAPS) by APS kinase and subsequently reduced to sulfite (SO$32−$) by PAPS reductase. Secondly, sulfite is reduced to sulfide using six electrons provided by NADPH (reviewed in ref. 139). Trx1 and Grx1 were identified as electron donors for APS kinase⁷⁷  and PAPS reductase in E. coli.^78–81 

Arsenate reductase (ArsC) detoxifies arsenate to arsenite with electrons provided by E. coli Grxs 1, 2 and 3.⁸²  The substrate for Grxs is a glutathionylated arsenate intermediate.

[FeS] clusters, found in all life forms, can undergo reversible redox reactions, determine protein structure, act as catalytic centers and as sensitive sensors of iron and various oxygen species.^83,84  The biosynthesis of these cofactors is therefore essential for catalytic function of several enzymes. During biosynthesis sulfide and iron are delivered to a scaffold protein, which coordinates the newly synthesized [FeS] cluster before transferring it to target proteins. In E. coli, two independent systems for [FeS] cluster biosynthesis are present: the Isc (iron sulfur cluster) and the Suf (mobilization of sulfur) systems.^85,86  The Trx system was shown to mediate iron binding of IscA⁸⁷  and SufA,⁸⁸  proteins that have been described as alternative scaffold proteins,⁸⁹  or as potential iron donors for the formation of [FeS] clusters in the scaffold IscU.^90,91  Monothiol Grxs including E. coli Grx4 are crucially involved in iron–sulfur cluster biosynthesis and regulation of iron homeostasis.^92–94 

Trxs are by far the most well known members of the Trx family. The investigations on these proteins, as well as Grxs, provided general insights in functions and mechanisms of all members of the Trx family. Even though most of these initial general experiments were done in vitro, they provided important concepts for the in vivo situation. The different ways in which Trxs and Grxs can participate in prokaryotic but also eukaryotic protein folding are the subject of this chapter.

Trxs and Grxs has been described as regulators of many proteins involved in folding via their redox activities.^95,96 

In prokaryotes, Trx and Grx catalyze the reduction of protein disulfides in E. coli Hsp33.⁹⁷  This reduction led to the formation of Zn coordinating inactive monomers. Under conditions of oxidative stress, Hsp33 forms dimers protecting unfolded proteins against aggregation (see also Chapter 1.8).

Yeast Ssp2⁹⁸  and human Hdj2,⁹⁹  two redox-regulated co-chaperones, are reduced by Trxs.

As described in Section 1.1.1.4 peroxiredoxins interact with the Trx system. Some eukaryotic peroxiredoxins were characterized as chaperones. H₂O₂-induced chaperone activity of yeast and human Prxs 2 requires the active site cysteinyl side-chains and the Trx system as cofactor.^100,101  In concert with yeast Prx2, the Trx system protects ribosomes against stress-induced aggregation.¹⁰² 

Electrons provided by Trx1 are also essential for the correct oxidative folding of proteins in the E. coli periplasm (Figure 1.1.4, black), where the two protein disulfide isomerases DsbC and DsbG reduce incorrect formed disulfide bonds for further isomerization.⁵⁴  A constant electron supply is guaranteed by the membrane protein DsbD, which itself is reduced by cytoplasmic Trx.¹⁰³ 

Several proteomic approaches identified molecular chaperones as new targets of the Trx and Grx systems: human Trx1 was identified as interaction partner of 14-3-3 ζ proteins during interphase and mitosis in HeLa cells.¹⁰⁴  Synergistically with α-crystallin, the human Trx system can recover inactivated GR in human aged clear and cataract lenses.¹⁰⁵  Cyclophilin, a peptidyl-prolyl cis-trans isomerase essential for protein folding^106,107  is activated via reduction by spinach Trx-m.¹⁰⁸  Cyclophilin was also found in investigations aiming at the identification of new targets of A. thaliana cytosolic Trx-h3 and spinach plastidic Trx-m.^109,110  Using poplar Grx-C4 as bait Rouhier et al. identified a 14-3-3 protein (At5g6543), Hsp60, Hsp70 and cyclophilin as interacting proteins in extracts of different plants.¹¹¹  The following human chaperones were identified as targets for reversible glutathionylation: Hsp10,¹¹²  Hsp60,^113,114  Hsp70,^112–115  Hsp90,¹¹³  Hsc70,^113,115  14-3-3 proteins,¹¹³  cyclophilin A,^112,114,115  and Cox17.¹¹⁶  As mentioned in Section 1.1.1.3 Grxs are highly specific for protein-GSH mixed disulfides, which suggests that all the above-listed chaperone-GSH mixed disulfides are likely targets for Grxs in vivo. Indeed, it was shown that glutathionylated human Hsc70 is a substrate for Grx1, and that reduction of the mixed disulfide decreased chaperone activity.¹¹⁷ 

Trxs and Grxs are not only regulators of proteins involved in folding, they are also able to act directly in oxidative folding or as chaperones.

As described above (Section 1.1.1.2), a recent paper dealing with the characterization of the reaction mechanism of E. coli Trx1 using single molecule force-clamp spectroscopy suggests that a local refolding of the substrate is part of the catalytic activity.²⁸  Hence, chaperone activity is coupled to rate enhancements in the order of four to five orders of magnitude compared to small reductants like dithiothreitol in reduction of protein disulfides.

E. coli Trx1 fusion was shown to increase the levels of soluble proteins heterologously expressed in E. coli, for instance numerous mammalian proteins,^118–120  Clostridium tetani fragment C of tetanus toxin¹²¹  or the single-chain variable fragment of antibodies.¹²²  Several plasmids were constructed to express Trx fusion proteins to avoid inclusion body formation during recombinant expression of proteins in E. coli.^118,119  The induction of correct folding was independent of Trx's redox activity since fusion with a Trx mutant harboring an Ala-Gly-Pro-Ala active site was as efficient as the wild type Trx fusion partner.¹²²  The fused Trx, covalently linked to the protein of interest, may act as a molecular chaperone preventing precipitation and aggregation of the fused partners until these reach a stable folding state.^118,119 

Direct activity as molecular chaperone was demonstrated in vitro by Richarme and coworkers. E. coli Trx1 and the Trx homolog YbbN/Trxsc were able to refold citrate synthase and α-glucosidase with an efficiency comparable to those of chaperones like DnaK and different heat shock proteins.^48,60  As observed for the chaperone activity of fused Trx neither the redox state of the Trxs nor the active site cysteines or other amino acids important for redox function are required for chaperone activity measured in vitro.^60,123  Corroboratively, Trx1 can stimulate the refolding of MglB, a protein without cysteines.⁶⁰  Unlike molecular chaperones, Trx and YbbN/Trxsc do not preferentially bind unfolded proteins and do not protect citrate synthase against thermal degradation.^48,60 

It is not only E. coli proteins that have been described as chaperones since also Trx1, but not Trx2 from Helicobacter pylori, promoted the renaturation of arginase.¹²³ 

Eukaryotic mitochondrial monothiol Grx5 and several other monothiol Grxs including E. coli Grx4 have been implied in iron–sulfur cluster biosynthesis.^92–94  Depletion of Grx5 led to increased iron levels and decreased enzymatic function of iron-sulfur proteins such as aconitase and succinate dehydrogenase.⁹²  The transfer of [FeS] clusters from scaffold to target protein (see Section 1.1.2.2.) is dependent on a functional HscA/HscB chaperone system.^85,124  Grx5 deletion in yeast resulted in accumulation of iron–sulfur clusters on the scaffold protein.⁹³  This phenotype can be rescued by over-expression of the HscA-type chaperone Ssq1⁹²  indicating that Grx5 may act in concert with the HscA/Ssq1 HscB/Jac1 chaperone couple in [FeS] cluster biosynthesis.

Protein disulfide isomerases catalyze the isomerization of disulfide-bond formation in the oxidative environments of the prokaryotic periplasm and the eukaryotic endoplasmatic reticulum (overviews in refs. 54 and 125).

Trx1, when exported to the periplasm, is able to complement E. coli strains deficient in the periplasmatic thiol oxidase DsbA at concentrations that allow efficient re-oxidation of Trx by DsbB¹²⁶  (Figure 1.1.4, green). Mutated Trx1 variants mimicking active sites of other Trx-fold oxidoreductases resulting in a higher redox potentials (see Section 1.1.1.1) were more efficient in complementation.^126,127  Beyond that, some Trx1 mutations were able to perform disulfide-bond formation pathway independent of DsbA and DsbB.¹²⁸  The active site mutants of E. coli Trx1 were created by random mutagenesis and the most efficient mutants contained the active sites Cys-Ala-Cys-Cys and Cys-Ala-Cys-Ala. These Trx mutants coordinated an [2Fe2S] cluster that was essential for the catalysis of oxidative protein folding (Figure 1.1.4, blue).

As described in Section 1.1.2.1. Trxs and Grxs usually maintain a reducing environment in the cytosol by reduction of disulfide bonds. But when the Trx system is impaired, for instance by inhibition of TrxR, disulfide-bond formation can occur in the cytoplasm of E. coli.¹²⁹  This effect is enhanced if, in addition to the Trx system, the Grx system is disturbed as well.¹³⁰  It was shown that the resulting disulfide bridge formations in the cytoplasm are attributed not only to the lack of reductase activity of Trxs 1 and 2 and Grx1, but also to the accumulation of oxidized redoxins that serve as oxidants and catalysts of disulfide-bond formation⁶⁶  (Figure 1.1.4, red).

In vitro both E. coli Trx1 and Grx1 are able to refold ribonuclease A (RNase A). Oxidized and a mixture of oxidized and reduced Trx efficiently catalyzes the refolding of reduced, denatured RNase, or oxidized, scrambled RNase, respectively.¹³¹  As shown by kinetic analyses Grx1 only needs the N-terminal active site cysteine for RNase A refolding.¹³²  The further investigation of the mechanism showed that Grx1 acted by the monothiol mechanism (Figure 1.1.2) and that ribonuclease-GSH mixed disulfide was the substrate.¹³³  The investigation of an in vitro model of the mechanism by which PDI catalyzes formation of disulfide bonds in the presence of GSSG indicated that PDI primarily catalyzes formation and breakage of GSH-protein mixed disulfides.¹³⁴  E. coli Grx1 displayed synergistic activity together with PDI when the redox potential of the GSH redox buffer was low enough to reduce the active site disulfide in Grx,¹³³  but was also able to refold RNase A without PDI.^135,136  Therefore, GSH mixed disulfides were believed to be important folding intermediates in vivo. Today it seems that in eukaryotic systems GSH is needed as a redox balancing system, providing reducing equivalents for the reduction of PDIs, and for the protection against reactive oxygen species, which are formed during oxidation of PDIs via Ero1 using oxygen as the ultimate electron acceptor (reviewed in ref. 137). In E. coli GSH is present in the periplasm but not essential for disulfide-bond formation or isomeration.¹³⁸ 

Over the last 40 years research has shown the essential roles of thiol-disulfide oxidoreductases of the Trx family of proteins in regulating the cellular redox conditions. In prokaryotes these proteins are crucial for both reduction of protein thiols in cytoplasm and oxidation of protein thiols in periplasm. The numerous interaction partners of Trx-fold proteins identified so far indicate the potential of this structural motif as protein binding scaffold promising more interaction partners detected by future research. Protein interaction is of course a condition for donating or accepting electrons but also for folding activity. Therefore it is not surprising that – in addition to regulation of different proteins involved in folding – Trxs and Grxs possess protein folding activity (Figure 1.1.5). In vivo an essential chaperone activity of Trx and Grx for the correct folding of a specific substrate is not yet described, but the in vitro investigations prepared the ground for the insights of the enzymatic mechanisms of proteins like DsbA or PDI.

This work was supported by the Deutsche Forschungsgemeinschaft, Karolinska Institutet, the Swedish Cancer Society, the Swedish Research Council Medicine and the Knut and Alice Wallenberg Foundation.