[Show abstract][Hide abstract] ABSTRACT: The central hydrophobic domain of the membrane protein DsbD catalyzes the transfer of electrons from the cytoplasm to the periplasm of Escherichia coli. Two cysteine residues embedded in transmembrane segments are essential for this process. Our results, based on cysteine alkylation and site-directed proteolysis, provide strong evidence that these residues are capable of forming an intramolecular disulfide bond. Also, by using a combination of two complementary genetic approaches, we show that both cysteines appear to be solvent-exposed to the cytoplasmic side of the inner membrane. These data are inconsistent with earlier topological models that place these residues on opposite sides of the membrane and permit the formulation of alternate hypotheses for the mechanism of this unusual transmembrane electron transfer.
Proceedings of the National Academy of Sciences 10/2003; 100(18):10471-6. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Disulfide bonds formed between pairs of cysteines are important features of the structure of many proteins. Elaborate electron transfer pathways have evolved Escherichia coli to promote the formation of these covalent bonds and to ensure that the correct pairs of cysteines are joined in the final folded protein. These transfers of electrons consist, in the main, of cascades of disulfide bond formation or reduction steps between a series of proteins (DsbA, DsbB, DsbC, and DsbD). A surprising variety of mechanisms and protein structures are involved in carrying out these steps.
Annual Review of Biochemistry 02/2003; 72:111-35. · 26.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Escherichia coli disulfide bond isomerase DsbC rearranges incorrect disulfide bonds during oxidative protein folding. It is specifically activated by the periplasmic N-terminal domain (DsbDalpha) of the transmembrane electron transporter DsbD. An intermediate of the electron transport reaction was trapped, yielding a covalent DsbC-DsbDalpha complex. The 2.3 A crystal structure of the complex shows for the first time the specific interactions between two thiol oxidoreductases. DsbDalpha is a novel thiol oxidoreductase with the active site cysteines embedded in an immunoglobulin fold. It binds into the central cleft of the V-shaped DsbC dimer, which assumes a closed conformation on complex formation. Comparison of the complex with oxidized DsbDalpha reveals major conformational changes in a cap structure that regulates the accessibility of the DsbDalpha active site. Our results explain how DsbC is selectively activated by DsbD using electrons derived from the cytoplasm.
The EMBO Journal 10/2002; 21(18):4774-84. · 10.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Modular organization of proteins has been postulated as a widely used strategy for protein evolution. The multidomain transmembrane protein DsbD catalyzes the transfer of electrons from the cytoplasm to the periplasm of Escherichia coli. Most bacterial species do not have DsbD, but instead their genomes encode a much smaller protein, CcdA, which resembles the central hydrophobic domain of DsbD. We used reciprocal heterologous complementation assays between E.coli and Rhodobacter capsulatus to show that, despite their differences in size and structure, DsbD and CcdA are functional homologs. While DsbD transfers reducing potential to periplasmic protein disulfide bond isomerases and to the cytochrome c thioreduction pathway, CcdA appears to be involved only in cytochrome c biogenesis. Our findings strongly suggest that, by the acquisition of additional thiol-redox active domains, DsbD expanded its substrate specificity.
The EMBO Journal 09/2002; 21(15):3960-9. · 10.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The correct formation of disulfide bonds in the periplasm of Escherichia coli involves Dsb proteins, including two related periplasmic disulfide-bond isomerases, DsbC and DsbG. DsbD is a membrane protein required to maintain the functional oxidation state of DsbC and DsbG. In this work, purified proteins were used to investigate the interaction between DsbD and DsbC. A 131-residue N-terminal fragment of DsbD (DsbDalpha) was expressed and purified and shown to form a functional folded domain. Gel filtration results indicate that DsbDalpha is monomeric. DsbDalpha was shown to interact directly with and to reduce the DsbC dimer, thus increasing the isomerase activity of DsbC. The DsbC-DsbDalpha complex was characterized, and formation of the complex was shown to require the N-terminal dimerization domain of DsbC. These results demonstrate that DsbD interacts directly with full-length DsbC and imply that no other periplasmic components are required to maintain DsbC in the functional reduced state.
Proceedings of the National Academy of Sciences 09/2001; 98(17):9551-6. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cytoplasmic membrane protein DsbD transfers electrons from the cytoplasm to the periplasm of E. coli, where its reducing power is used to maintain cysteines in certain proteins in the reduced state. We split DsbD into three structural domains, each containing two essential cysteines. Remarkably, when coexpressed, these truncated proteins restore DsbD function. Utilizing this three piece system, we were able to determine a pathway of the electrons through DsbD. Our findings strongly suggest that the pathway is based on a series of multistep redox reactions that include direct interactions between thioredoxin and DsbD, and between DsbD and its periplasmic substrates. A thioredoxin-fold domain in DsbD appears to have the novel role of intramolecular electron shuttle.
[Show abstract][Hide abstract] ABSTRACT: The active-site cysteines of the Escherichia coli periplasmic protein disulfide bond isomerase (DsbC) are kept reduced by the cytoplasmic membrane protein, DsbD. DsbD, in turn, is reduced by cytoplasmic thioredoxin, indicating that DsbD transfers disulfidereducing potential from the cytoplasm to the periplasm. To understand the mechanism of this unusual mode of electron transfer, we have undertaken a genetic analysis of DsbD. In the process, we discovered that the previously suggested start site for the DsbD protein is incorrect. Our results permit the formulation of a model of DsbD membrane topology. Also, we show that six cysteines of DsbD conserved among DsbD homologs are essential for the reduction of DsbC, DsbG and for a reductive pathway leading to c-type cytochrome assembly in the periplasm. Our findings suggest a testable model for the DsbD-dependent transfer of electrons across the membrane, involving a cascade of disulfide bond reduction steps.
The EMBO Journal 12/1999; 18(21):5963-71. · 10.75 Impact Factor