Thioredoxin 2, an oxidative stress-induced protein, contains a high affinity zinc binding site

Two thioredoxins have been described in Escherichia coli, TrxA and Trx2. Both thioredoxins are capable of reducing disulfide bonds using a conserved pair of cysteine residues present in a WCGPC motif. A number of unique structural and regulatory features distinguish the Trx2 subfamily from the much larger TrxA family. The Trx2 subfamily has an additional N-terminal domain of +/-30 residues, which contains two additional conserved CXXC motifs. Moreover, the gene coding for Trx2 is under control of the oxidative stress transcription factor OxyR in E. coli. This suggests that Trx2 may play a role in the cellular defense against oxidative stress. We show here that Trx2 contains zinc in a 1:1 stoichiometry, making it the first identified zinc-binding thioredoxin. The zinc atom is coordinated by the four cysteines of the two N-terminal CXXC motifs. The zinc center of Trx2 binds zinc with a very high affinity (K-a of > 10(18) M-1). We show that in vitro oxidation of the zinc binding cysteines by H2O2 releases the zinc and induces a conformational change. The zinc-free protein conserves its reductase activity. Altogether, our results suggest that the zinc center might play the role of a redox switch, changing a yet to be identified activity

Trigger factor both holds and folds its client proteins

ATP-independent chaperones like trigger factor are generally assumed to play passive roles in protein folding by acting as holding chaperones. Here we show that trigger factor plays a more active role. Consistent with a role as an aggregation inhibiting chaperone, we find that trigger factor rapidly binds to partially folded glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and prevents it from non-productive self-association by shielding oligomeric interfaces. In the traditional view of holding chaperone action, trigger factor would then be expected to transfer its client to a chaperone foldase system for complete folding. Unexpectedly, we noticed that GAPDH folds into a monomeric but otherwise rather native-like intermediate state while trigger factor-bound. Upon release from trigger factor, the mostly folded monomeric GAPDH rapidly self-associates into its native tetramer and acquires enzymatic activity without needing additional folding factors. The mechanism we propose here for trigger factor bridges the holding and folding activities of chaperone function

Crystallization of Dsba, An Escherichia-Coli Protein Required for Disulfide Bond Formation Invivo

DsbA is a 21 kDa protein that facilitates disulphide bond formation and is required for the correct folding and stability of a number of exported proteins in Escherichia coli. Crystals of oxidized DsbA have been obtained from polyethylene glycol 8000 (20 to 25% 0·1 M-cacodylate buffer (pH 6·5) and 1% 2-mehtyl-2,4-pentanediol. Oxidation of the protein is critical for reproducibly obtaining high quality crystals. The resulting crystals diffract to 2 Å and belong to the monoclinic space group C 2 with cel dimensions a=117·5 Å, b =65·0 Å, c76·3 Å, β=126·3° with two molecules in the asymmetric unit.No Full Tex

Hsp15: a ribosome-associated heat shock protein

We are analyzing highly conserved heat shock genes of unknown or unclear function with the aim of determining their cellular role. Hsp15 has previously been shown to be an abundant nucleic acid-binding protein whose synthesis is induced massively at the RNA level upon temperature upshift. We have now identified that the in vivo target of Hsp15 action is the free 50S ribosomal subunit. Hsp15 binds with very high affinity (K(D) <5 nM) to this subunit, but only when 50S is free, not when it is part of the 70S ribosome. In addition, the binding of Hsp15 appears to correlate with a specific state of the mature, free 50S subunit, which contains bound nascent chain. This provides the first evidence for a so far unrecognized abortive event in translation. Hsp15 is suggested to be involved in the recycling of free 50S subunits that still carry a nascent chain. This gives Hsp15 a very different functional role from all other heat shock proteins and points to a new aspect of translation