Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates

Abstract

Thioredoxin functions in nearly all organisms as the major thiol-disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin has been reported to contribute to a wide variety of physiological functions by interacting with specific sets of substrates in different cell types. To investigate the function of the essential thioredoxin A (TrxA) in the low-GC Gram-positive bacterium Bacillus subtilis, we purified wild-type TrxA and three mutant TrxA proteins that lack either one or both of the two cysteine residues in the CxxC active site. The pure proteins were used for substrate-binding studies known as "mixed disulfide fishing" in which covalent disulfide-bonded reaction intermediates can be visualized. An unprecedented finding is that both active-site cysteine residues can form mixed disulfides with substrate proteins when the other active-site cysteine is absent, but only the N-terminal active-site cysteine forms stable interactions. A second novelty is that both single-cysteine mutant TrxA proteins form stable homodimers due to thiol oxidation of the remaining active-site cysteine residue. To investigate whether these dimers resemble mixed enzyme-substrate disulfides, the structure of the most abundant dimer, C32S, was characterized by X-ray crystallography. This yielded a high-resolution (1.5A) X-ray crystallographic structure of a thioredoxin homodimer from a low-GC Gram-positive bacterium. The C32S TrxA dimer can be regarded as a mixed disulfide reaction intermediate of thioredoxin, which reveals the diversity of thioredoxin/substrate-binding modes.

Fig. 1

Mixed disulfide fishing. Mixed disulfide fishing was performed with the highly pure His6-tagged BsTrxA with the wild-type active site or with active-site-specific mutations, and cytoplasmic proteins from the TrxA-depleted B. subtilis WB800 ItrxA strain. (a) To show that the BsTrxA proteins used for mixed disulfide fishing were highly pure, 0.1 μg of each BsTrxA protein variant was loaded on SDS-PAGE. Upon electrophoresis, the gel was silver-stained. WT, BsTrxA with wild-type active site; C29S, C29S single-mutant BsTrxA; C32S, C32S single-mutant BsTrxA; C29S–C33S, C29S–C32S double-mutant BsTrxA. (b) To visualize possible stable interactions between BsTrxA and its substrates, 2 μg of each BsTrxA protein variant was mixed with 50 μl of cytoplasmic protein extract. After 5 min of incubation, proteins were separated on a nonreducing gel, and BsTrxA–substrate complexes were visualized by Western blotting, with antibodies raised against B. subtilis TrxA. Background: The cytoplasmic extract mock-treated with 2 μl of water instead of BsTrxA protein reveals a few nonspecific cross-reactions of the BsTrxA antibody. (c) Immunodetection of purified BsTrxA–substrate complexes. For purification of the BsTrxA–substrate bound complexes, the C-terminal His6-tag of the pure BsTrxA proteins was used. Pure BsTrxA with the wild type or a mutant active site was mixed with cytoplasmic protein extracts as indicated for (b). Subsequently, magnetic beads precharged with nickel were added, and the His6-tag of the BsTrxA proteins was allowed to bind to the nickel of the magnetic beads for 10 min. A magnet was then used to collect the beads with bound BsTrxA. After the beads had been washed nine times, the BsTrxA proteins were eluted from the beads with a buffer containing imidazole. The eluted proteins were separated by nonreducing SDS-PAGE, and the BsTrxA–substrate bound complexes were visualized by Western blotting with antibodies against BsTrxA.

Fig. 2

Redox states of BsTrxA monomers and dimers. (a) Purified His6-tagged BsTrxA proteins were separated by capillary electrophoresis using a 2100 Bioanalyzer (Agilent Technologies). WT, BsTrxA with wild-type active site; C29S, C29S single-mutant BsTrxA; C32S, C32S single-mutant BsTrxA; C29S–C32S, C29S–C32S double-mutant BsTrxA. To monitor the presence of free thiols in the purified BsTrxA proteins, samples were incubated in the presence or in the absence of 0.3 mM AMS (lanes marked + AMS or − AMS). To test whether the C29S and C32S dimers are formed by disulfide bonding, these proteins were incubated with 10 mM DTT (lanes marked + DTT). DTT was absent from all other samples. The image of the Bioanalyzer chromatogram was generated using the 2100 Expert Software package (Agilent Technologies). (b) C32S BsTrxA protein (∼ 2.5 μg) was reduced with increasing concentrations of DTT (shown on top of the panel) and separated by capillary electrophoresis as described for (a). The dimer-to-monomer ratios (Dimer [%]) are shown at the bottom of the panel.

Fig. 3

Overall fold and the dimer–interface interactions of the C32S BsTrxA dimer. (a) Overall fold of the C32S BsTrxA dimer. The disulfide-bonded C32S BsTrxA chains are shown in yellow (chain A) and in blue (chain B). The C29–C29 disulfide bond between the chains is shown in orange. (b) Stereo diagram of the active site of the C32S BsTrxA dimer with an electrostatic surface shown for chain A. The C32S active-site residues Trp28, Cys29, Gly30, and Pro31 are shown. Carbon atoms are shown in yellow in chain A and in blue in chain B. Nitrogen and oxygen atoms are in dark blue and red, respectively. The disulfide bond atoms are in orange, and hydrogen bonds are shown in green. (c) The shallow hydrophobic binding sites for the Trp28 residues on the opposite chains of the dimer in the C32S mutant of BsTrxA. The color coding is the same as in (b), and the view is an approximately 90° rotation relative to (b).

Fig. 4

Comparison of the B. subtilis TrxA homodimer and BsTrxA–ArsC complex structures. The B. subtilis TrxA homodimer structure is shown as a cartoon with chains A and B in yellow and blue, respectively. The disulfide bridge and the Ser32 are shown in orange, as in Fig. 3. The BsTrxA–ArsC complex structure is shown in purple, with the disulfide bridge and residue Ser82 (from the C82S mutation in ArsC) shown in red.

Fig. 5

Comparison of protein/peptide binding modes in the BsTrxA C32S dimer and other thioredoxin peptide and protein complexes. (a) BsTrxA was superimposed on the thioredoxin from other complexes using PyMOL. The superimposed structures, the surface of chain A of BsTrxA, and the peptide or protein chain of the bound molecule are shown. The disulfides and large hydrophobic residues that contribute to binding are also shown. The N- and C-termini of the bound peptides and proteins are indicated with the letters N and C. The proteins shown in (a) are as follows: the BsTrxA dimer with residues 26–38 of chain B shown as cartoons and the side chains of Trp28 and Cys29 shown as sticks (this study; dark blue); the NMR structures of human thioredoxin complexed with substrate peptides from Ref-1 (1CQH; red) or NF-κB (1MDJ; green) with the side chains of residues Cys65 and Tyr67 of the Ref-1 peptide and of residues Trp60 and Cys62 of the NF-κB peptide; the NMR structure of the B. subtilis TrxA and ArsC (2IPT; purple) with residues Cys89 and Met91; and the X-ray structure of the complex between barley thioredoxin and the α-amylase serine proteinase inhibitor (2IWT; orange) with residues Trp147 and Cys148. (b) Comparison of the BsTrxA (dark blue) dimer interface with the binding of thioredoxin reductase by E. coli thioredoxin (1F6M; yellow) and the X-ray structures of the complexes between spinach chloroplast thioredoxins Trx-f and Trx-m and ferrodoxin–thioredoxin reductase (2PU9 and 2PUK; in cyan and magenta, respectively). Trp28 is shown for the TrxA structure (dark blue), and the E. coli thioredoxin reductase structure shows Phe142 (yellow).

Fig. 6

Ser32 interactions in the BsTrxA C32S structure. (a) The loop from residue 26–32 in chain A of the BsTrxA C32 structure is shown with the hydrogen bonds to the Ser32 residue. C29 from chain B is also shown, and the disulfide bond is shown in orange. Oxygen and nitrogen atoms are in red and blue, respectively. The carbon atoms of chains A and B are in blue and yellow, respectively. Hydrogen bonds are shown in green. (b) The environment surrounding S32 is shown. The catalytic loops with residues 25–33 and 72 are shown as sticks, and other parts of the structure are shown as cartoons. The colors are the same as in (a).