Crystal structures of the DsbG disulfide isomerase reveal an unstable disulfide

Abstract

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...

Laboratory evolution of one disulfide isomerase to resemble another

“…However, there are many structural differences between DsbC and DsbG. In particular, the putative substrate-binding cleft of DsbG is much larger (60 Å ϫ 31 Å ϫ 31 Å) than that of DsbC (40 Å ϫ 25 Å ϫ 25 Å), perhaps indicating that DsbG's substrates are larger than those of DsbC (14). Addi-tionally, comparison of DsbG's surface with that of DsbC reveals many differences; specifically, DsbG contains a significant number of charged surface patches, in contrast to DsbC's largely hydrophobic and uncharged surface.…”

“…Structural studies show that DsbC and DsbG have superficially similar structures: both are V-shaped homodimers consisting of two thioredoxin domains connected by a linker helix to an N-terminal dimerization domain (14)(15)(16). However, there are many structural differences between DsbC and DsbG.…”

Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis

“…For example, the oxidant EcDsbA has CXHC and the isomerase EcDsbC has CXYC. Furthermore, the residue preceding the highly conserved cis-Pro loop of the thioredoxin fold is a valine in DsbAs and a threonine in DsbCs [32]. Surprisingly, the sequence of -DsbA1 incorporates features of both oxidases and isomerases; the active site motif is CYHC and the residue preceding the cis-Pro loop is a threonine rather than a valine.…”

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“…It suggests the two conserved cysteines Cys119 and Cys122 were crucial to DsbG disulphide isomerization activity. In contrast, the DsbG of E. coli is unable to catalyse the oxidative refolding of RNaseA in vitro 6,12 .…”

“…The thioredoxin-like proteins share no overall sequence homology, yet they all contain a CXXC motif and a cis-Pro loop. The Thr in the cis-Pro loop may favour the reduced form of the protein by interaction with the sulphur of the first Cys in the active site 12 . The active site of the putative DsbG (AEF0162) (Cys-Ile-Tyr-Cys) has an Ile residue.…”