Oxidative Folding of Proteins in vivo

CHAPTER 1.1

Thioredoxins and the Regulation of Redox Conditions in Prokaryotes

CARSTEN BERNDT AND ARNE HOLMGREN

The Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden

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.

1.1.1 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). In all organisms, this enzyme is essential for DNA synthesis during both replication and repair. 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. Today, the Trx protein family is first and foremost defined by a structural motif named the Trx fold. In spite of considerable variation in overall structure, the Trx fold is present in a variety of functionally different proteins: thiol-disulfide oxidoreductases, disulfide isomerases, glutathione S-transferases, thiol-dependent peroxidases and chloride intracellular channels.

1.1.1.1 The Thioredoxin Fold

The Trx fold motif consists of a central four-stranded β-sheet surrounded by three α-helices (Figure 1.1.1A). This basic βαβα ββα topology can only be found in bacterial glutaredoxins, while thioredoxins contain an additional β-sheet and α-helix at the N-terminus (Figure 1.1.1B,C). Variations of this motif have also been identified in domains of 723 proteins, 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. 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, the strongest oxidant, DsbA (Cys-Pro-His-Cys), has a redox potential of ΔE'0 = — 122 mV. 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. Supportingly, the redox potential of a DsbA mutant harboring the Cys-Gly-Pro-Cys active site of Trx decreased by 92 mV. Several other amino acids outside the active site motif have been determined as important for the redox potential of Trx fold oxidoreductases.

1.1.1.2 Thioredoxins and the Thioredoxin System

In 1968 E. coli Trxl 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. 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.' Today we know Trx as a general disulfide reductase reducing disulfide bonds by a ping-pong mechanism (Figure 1.1.2). The N-terminal active site thiol of Trxs possesses an unusual low pKa value, whereas the pKa of the C-terminal active site is higher than that of cysteine in solution. The low pKa of the N-terminal cysteine of the E. coli Trxl active site was shown to be related to the carboxyl group of Asp 26 and the ε-amino group of Lys 57. 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. 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. 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. Trxl, 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.

1.1.1.3 Glutaredoxins and the Glutaredoxin System

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 reduced and oxidized E. coli Grx1 revealed more or less identical overall structures but significant changes in and around the active site.

Based on their active site...