Abstract
Many Gram-negative bacteria have two cytoplasmic thioredoxins, thioredoxin-1 and -2, encoded by the trxA and trxC genes, respectively. Both thioredoxins have the highly conserved WCGPC motif and function as disulfide-bond reductases. However, thioredoxin-2 has unique features: it has an N-terminal motif that binds a zinc ion, and its transcription is under the control of OxyR, which allows it to be up-regulated under oxidative stress. Here, we report the crystal structure of thioredoxin-2 from Rhodobacter capsulatus. The C-terminal region of thioredoxin-2 forms a canonical thioredoxin fold with a central beta-sheet consisting of five strands and four flanking alpha-helices on either side. The N-terminal zinc finger is composed of four short beta-strands (S1-S4) connected by three short loops (L1-L3). The four cysteines are at loops L1 and L3 and form a tetragonal binding site for a zinc ion. The zinc finger is close to the first beta-strand and first alpha-helix of the thioredoxin fold. Nevertheless, the zinc finger may not directly affect the oxidoreductase activity of thioredoxin-2 because the zinc finger is not near the active site of a protomer and because thioredoxin-2 is a monomer in solution. On the basis of structural similarity to the zinc fingers in Npl4 and Vps36, we propose that the N-terminal zinc finger of thioredoxin-2 mediates protein-protein interactions, possibly with its substrates or chaperones.
Highlights
Motif; during the catalytic cycle, the two cysteines alternate between reduced and oxidized forms
The disulfide in thioredoxin must be regenerated by a thioredoxin reductase, which in turn uses the reducing equivalents derived from NADPH [2, 3]
Thioredoxin-2 seems to play a role in the protection against oxidative stress, as the deletion of its gene in Rhodobacter capsulatus results in increased sensitivity to oxidative agents [8, 9]
Summary
Cloning and Protein Expression—The R. capsulatus thioredoxin-2 gene was amplified from genomic DNA by PCR and cloned into pET28aϩ between the NdeI and HindIII sites. The recombinant protein has an N-terminal histidine tag and a thrombin site between the tag and thioredoxin-2. The transformed cells were grown to A600 ϭ 0.7 and induced by the addition of isopropyl -D-thiogalactopyranoside to a final concentration of 0.5 mM. The recombinant thioredoxin-2 protein was in the supernatant and purified by Ni-NTA3 chromatography under native conditions. Thrombin was added to the purified sample at a ratio of 10 units of thrombin to 1 mg of recombinant thioredoxin-2. The cleaved sample was concentrated and further purified by size-exclusion fast protein liquid chromatography in a running buffer of 20 mM HEPES and 0.1 M NaCl, pH 7.4. Crystallization, Data Collection, and Structure Determination—Before trays were set up, dithiothreitol was added to a final concentration of 5 mM, and the protein solution was incubated at room temperature for 1 h.
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