The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.The thioredoxin fold is the core scaffold of numerous proteins that control disulfide redox activity in the cell (1-3). These redox proteins share very little sequence homology, but all of them incorporate the four-stranded ␤-sheet, three flanking ␣-helices, and the redox-active CXXC motif of the TRX 5 fold (Fig. 1A). The archetype of the family is thioredoxin (4), a disulfide reductase that maintains a reducing cytosolic environment. Other TRX fold redox proteins include the Dsb proteins (1), which regulate the formation of disulfide bonds in prokaryotes, and protein-disulfide isomerase (5), which catalyzes the oxidation and shuffling of disulfides in the endoplasmic reticulum of eukaryotic cells.This wide range of redox activities of TRX fold proteins is thought to be a consequence of modifications to the common scaffold, which result in different redox properties. Thus, the redox potential of Escherichia coli thioredoxin is very reducing, at Ϫ271 mV (6, 7), whereas that of the oxidizing periplasmic protein E. coli DsbA is Ϫ120 mV (8). Thioredoxin fold proteins that participate in a wide range of thiol disulfide exchange reactions, such as the eukaryotic protein-disulfide isomerases, have intermediate redox potentials (around Ϫ160 mV (9)).Thioredoxin-related proteins provide an attractive model for the study of how protein function is dictated by sequence and three-dimensional structure; this is because their functions are, in part, determined by their redox properties, which in turn, are easy to quantify. For example, mutations in thioredoxin that make its redox potential more oxidative complement null mutations in the oxidase DsbA (10, 11). A detailed understanding of how thioredoxin fold sequence affects redox properties provides an excellent opportunity to relate sequence and function. Previous work has focused on the role of the CXXC "redox rheostat" active site in determining the properties of thioredoxin-related proteins (3,12,13). Experiments that exchange the X-X dipeptide of one thiol-disulfide oxidoreductase with that of another generally result in an oxidoreductase with a redox potential partially shifted in the direction of the oxidoreductase protein that served as the source of the dipepti...

In Gram-negative bacteria, the introduction of disulfide bonds into folding proteins occurs in the periplasm and is catalyzed by donation of an energetically unstable disulfide from DsbA, which is subsequently re-oxidized through interaction with DsbB. Gram-positive bacteria lack a classic periplasm but nonetheless encode Dsb-like proteins. Staphylococcus aureus encodes just one Dsb protein, a DsbA, and no DsbB. Here we report the crystal structure of S. aureus DsbA (SaDsbA), which incorporates a thioredoxin fold with an inserted helical domain, like its Escherichia coli counterpart EcDsbA, but it lacks the characteristic hydrophobic patch and has a truncated binding groove near the active site. These findings suggest that SaDsbA has a different substrate specificity than EcDsbA. Thermodynamic studies indicate that the oxidized and reduced forms of SaDsbA are energetically equivalent, in contrast to the energetically unstable disulfide form of EcDsbA. Further, the partial complementation of EcDsbA by SaDsbA is independent of EcDsbB and biochemical assays show that SaDsbA does not interact with EcDsbB. The identical stabilities of oxidized and reduced SaDsbA may facilitate direct re-oxidation of the protein by extracellular oxidants, without the need for DsbB.The formation of native disulfide bonds through air oxidation of cysteine pairs is a slow reaction and organisms ranging from bacteria to humans encode enzymatic systems to catalyze the process. In eukaryotes, oxidative folding in the endoplasmic reticulum is primarily catalyzed by protein-disulfide isomerases that are reoxidized by Ero1p/Erv2p proteins (reviewed in Ref. 1). In Escherichia coli, dithiol oxidation takes place in the periplasm through the action of the Dsb 3 (Disulfide bond) family of proteins. Dsb proteins form two distinct pathways, the DsbA-DsbB (oxidative) pathway that introduces disulfides indiscriminately, and the DsbC/DsbG-DsbD (isomerization) pathway that shuffles incorrect disulfides (2, 3).Probably the best-studied Dsb protein is EcDsbA, the primary disulfide catalyst in E. coli (reviewed in Refs. 2, 4). This promiscuously oxidizing protein is a 21-kDa monomer containing a CPHC active site in a thioredoxin (TRX) fold with an inserted helical domain of ϳ80 residues. Upon catalyzing disulfide bond formation in substrate proteins, reduced EcDsbA relies on EcDsbB, a quinone reductase, to recover its catalytically active, and higher energy, oxidized form (5

The alpha-proteobacterium Wolbachia pipientis is a highly successful intracellular endosymbiont of invertebrates that manipulates its host's reproductive biology to facilitate its own maternal transmission. The fastidious nature of Wolbachia and the lack of genetic transformation have hampered analysis of the molecular basis of these manipulations. Structure determination of key Wolbachia proteins will enable the development of inhibitors for chemical genetics studies. Wolbachia encodes a homologue (alpha-DsbA1) of the Escherichia coli dithiol oxidase enzyme EcDsbA, essential for the oxidative folding of many exported proteins. We found that the active-site cysteine pair of Wolbachia alpha-DsbA1 has the most reducing redox potential of any characterized DsbA. In addition, Wolbachia alpha-DsbA1 possesses a second disulfide that is highly conserved in alpha-proteobacterial DsbAs but not in other DsbAs. The alpha-DsbA1 structure lacks the characteristic hydrophobic features of EcDsbA, and the protein neither complements EcDsbA deletion mutants in E. coli nor interacts with EcDsbB, the redox partner of EcDsbA. The surface characteristics and redox profile of alpha-DsbA1 indicate that it probably plays a specialized oxidative folding role with a narrow substrate specificity. This first report of a Wolbachia protein structure provides the basis for future chemical genetics studies.

In Escherichia coli, interactions between the replication initiation protein DnaA, the ␤ subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. DNA replication consists of three sequential steps: initiation, elongation, and termination. In Escherichia coli the initiation of a new round of chromosome replication occurs when the initiation protein, DnaA, binds to a 9-mer DnaA box within the chromosomal origin, oriC (reviewed in references 34 and 41). In vitro studies have shown that the binding of DnaA to oriC in the presence of the DNA structural protein HU or IHF (14,16,40) stimulates opening of the DNA duplex by melting the AT-rich 13-mer region in oriC (4,9,26). The unwound region then provides an entry site for the DnaB-DnaC helicase, which expands the region of single-stranded DNA. DnaG primase, single-stranded DNA-binding protein, DNA polymerase III holoenzyme (Pol III), and other proteins required for the replication fork formation are then recruited, and bidirectional DNA synthesis is initiated (13).In normal growth, cells replicate their DNA once before cell division and in E. coli there are at least three different mechanisms that block the occurrence of multiple initiations. The