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

J Mol Biol. 2008 Jun 6;379(3):520-34. doi: 10.1016/j.jmb.2008.03.077. Epub 2008 Apr 10.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus subtilis / chemistry
  • Bacillus subtilis / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Binding Sites
  • Crystallography, X-Ray
  • Cysteine / chemistry
  • Dimerization
  • Disulfides / chemistry*
  • Humans
  • Hydrogen Bonding
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation*
  • Oxidation-Reduction
  • Protein Structure, Quaternary*
  • Thioredoxins / chemistry*
  • Thioredoxins / genetics*
  • Thioredoxins / metabolism

Substances

  • Bacterial Proteins
  • Disulfides
  • Thioredoxins
  • Cysteine

Associated data

  • PDB/2VOC