Snapshots of DsbA in Action: Detection of Proteins in the Process of Oxidative Folding

101

Dsb proteins control the formation and rearrangement of disulfide bonds during the folding of secreted and membrane proteins in bacteria. DsbG, a member of this family, has disulfide bond isomerase and chaperone activity. Here, we present two crystal structures of DsbG at 1.7-and 2.0-Å resolution that are meant to represent the reduced and oxidized forms, respectively. The oxidized structure, however, reveals a mixture of both redox forms, suggesting that oxidized DsbG is less stable than the reduced form. This trait would contribute to DsbG isomerase activity, which requires that the active-site Cys residues are kept reduced, regardless of the highly oxidative environment of the periplasm. We propose that a Thr residue that is conserved in the cis-Pro loop of DsbG and DsbC but not found in other Dsb proteins could play a role in this process. Also, the structure of DsbG reveals an unanticipated and surprising feature that may help define its specific role in oxidative protein folding. Thus, the dimensions and surface features of DsbG show a very large and charged binding surface that is consistent with interaction with globular protein substrates having charged surfaces. This finding suggests that, rather than catalyzing disulfide rearrangement in unfolded substrates, DsbG may preferentially act later in the folding process to catalyze disulfide rearrangement in folded or partially folded proteins.A key step in the protein folding process is the formation of disulfide bonds between Cys residues. Organisms ranging from bacteria to humans have developed systems to control this oxidative process (1). The Dsb family of proteins catalyzes disulfide bond formation in bacteria through two distinct pathways, an oxidative and a reducing͞isomerase pathway (2, 3). The DsbA-DsbB, or oxidative, pathway (4-6) introduces disulfide bonds into newly translocated proteins, but it can result in nonnative disulfide bonds. The DsbC͞DsbG-DsbD, or isomerase, pathway (7-9) catalyzes the rearrangement of incorrect disulfide bonds, allowing proteins to fold correctly.Despite important advances in this field, the mechanism of disulfide bond isomerization is poorly understood. Furthermore, it is not clear why two isomerases, DsbC and DsbG, are encoded in bacteria. The two proteins are related distantly, sharing only 24% sequence identity, and DsbG expresses at lower levels than DsbC. In addition, DsbG exhibits a more narrow substrate specificity than DsbC. Thus, it does not catalyze the classic redox protein reaction (insulin reduction), and unlike DsbC, it does not catalyze oxidative refolding of RNase (10).We undertook structural studies of DsbG to shed light on its function and to identify reasons why two isomerases are encoded. The results provide evidence that, as with DsbA (11), the oxidized forms of DsbG and DsbC are less stable than their reduced forms, and they indicate that the two disulfide isomerases may recognize different protein targets. MethodsDiffraction Data Measurement. Native and selenomethionine (SeMet)-labeled DsbG w...

Section: Discussionmentioning

confidence: 97%

Crystal structures of the DsbG disulfide isomerase reveal an unstable disulfide

Heras

Edeling

Schirra

et al. 2004

101

“…The recent interest in redox-regulated proteins fueled by the increasing recognition of their important physiological roles however, led to the development of several global thiol trapping techniques (10)(11)(12)(13). These methods, albeit capable of detecting proteins with redox-sensitive cysteines in lysates and intact cells, often lack the ability to directly identify the proteins or cysteines involved, and more importantly, to quantify the extent of oxidative thiol modifications.…”

mentioning

confidence: 99%

Quantifying changes in the thiol redox proteome upon oxidative stress in vivo

Leichert

Gehrke

Gudiseva

et al. 2008

486

542

Antimicrobial levels of reactive oxygen species (ROS) are produced by the mammalian host defense to kill invading bacteria and limit bacterial colonization. One main in vivo target of ROS is the thiol group of proteins. We have developed a quantitative thiol trapping technique termed OxICAT to identify physiologically important target proteins of hydrogen peroxide (H 2O2) and hypochlorite (NaOCl) stress in vivo. OxICAT allows the precise quantification of oxidative thiol modifications in hundreds of different proteins in a single experiment. It also identifies the affected proteins and defines their redox-sensitive cysteine(s). Using this technique, we identified a group of Escherichia coli proteins with significantly (30 -90%) oxidatively modified thiol groups, which appear to be specifically sensitive to either H 2O2 or NaOCl stress. These results indicate that individual oxidants target distinct proteins in vivo. Conditionally essential E. coli genes encode one-third of redoxsensitive proteins, a finding that might explain the bacteriostatic effect of oxidative stress treatment. We identified a select group of redox-regulated proteins, which protect E. coli against oxidative stress conditions. These experiments illustrate that OxICAT, which can be used in a variety of different cell types and organisms, is a powerful tool to identify, quantify, and monitor oxidative thiol modifications in vivo.chaperone ͉ proteomics ͉ thiol modification

“…Furthermore, a mutant form of DsbA that traps substrates in vivo has been shown to interact directly with LptD (30). However, the number and connectivity of disulfide bonds in LptD is unknown.…”

mentioning

confidence: 99%

Nonconsecutive disulfide bond formation in an essential integral outer membrane protein

Ruiz

Chng

Hiniker

et al. 2010

102

174

The Gram-negative bacterial envelope is bounded by two membranes. Disulfide bond formation and isomerization in this oxidizing environment are catalyzed by DsbA and DsbC, respectively. It remains unknown when and how the Dsb proteins participate in the biogenesis of outer membrane proteins, which are transported across the cell envelope after their synthesis. The Escherichia coli protein LptD is an integral outer membrane protein that forms an essential complex with the lipoprotein LptE. We show that oxidation of LptD is not required for the formation of the LptD/E complex but it is essential for function. Remarkably, none of the cysteines in LptD are essential because either of two nonconsecutive disulfide bonds suffices for function. Oxidation of LptD, which is efficiently catalyzed by DsbA, does not involve the isomerase DsbC, but it requires LptE. Thus, oxidation is completed only after LptD interacts with LptE, an interaction that occurs at the outer membrane and seems necessary for LptD folding.β-barrel protein | lipoprotein | protein folding