Thiol−Disulfide Exchange in an Immunoglobulin-like Fold: Structure of the N-Terminal Domain of DsbD † , ‡

Department of Biochemistry & Molecular Biology, University of Chicago, Chicago, Illinois, United States
Biochemistry (Impact Factor: 3.02). 07/2002; 41(22):6920-7. DOI: 10.1021/bi016038l
Source: PubMed


Escherichia coli DsbD transports electrons across the plasma membrane, a pathway that leads to the reduction of protein disulfide bonds. Three secreted thioredoxin-like factors, DsbC, DsbE, and DsbG, reduce protein disulfide bonds whereby an active site C-X-X-C motif is oxidized to generate a disulfide bond. DsbD catalyzes the reduction of the disulfide of DsbC, DsbE, and DsbG but not of the thioredoxin-like oxidant DsbA. The reduction of DsbC, DsbE, and DsbG occurs by transport of electrons from cytoplasmic thioredoxin to the C-terminal thioredoxin-like domain of DsbD (DsbD(C)). The N-terminal domain of DsbD, DsbD(N), acts as a versatile adaptor in electron transport and is capable of forming disulfides with oxidized DsbC, DsbE, or DsbG as well as with reduced DsbD(C). Isolated DsbD(N) is functional in electron transport in vitro. Crystallized DsbD(N) assumes an immunoglobulin-like fold that encompasses two active site cysteines, C103 and C109, forming a disulfide bond between beta-strands. The disulfide of DsbD(N) is shielded from the environment and capped by a phenylalanine (F70). A model is discussed whereby the immunoglobulin fold of DsbD(N) may provide for the discriminating interaction with thioredoxin-like factors, thereby triggering movement of the phenylalanine cap followed by disulfide rearrangement.

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Available from: Celia W Goulding, Dec 17, 2013
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    • "The cysteine pair of this domain (C103 and C109) is located in an unusual CX 5 C motif and is shielded from solvent exposure by the socalled ''cap-loop'' residues (residues 66–72) which adopt a more open conformation upon complex formation with the partner proteins (Stirnimann et al. 2006). X-ray structures for oxidized and reduced nDsbD (Goulding et al. 2002; Haebel et al. 2002; Mavridou et al. 2011) as well as for its covalent mixed disulfide complexes with partner proteins (cDsbD, cDsbD and CcmG) (Haebel et al. 2002; Rozhkova et al. 2004; Stirnimann et al. 2005) have been determined. We have shown in previous studies that NMR can provide important insights into the control of the specificity and reactivity of cDsbD that cannot be obtained from X-ray structures alone (Bushell et al. 2002; Mavridou et al. 2007, 2009, 2011). "
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    ABSTRACT: Viability and pathogenicity of Gram-negative bacteria is linked to the cytochrome c maturation and the oxidative protein folding systems in the periplasm. The transmembrane reductant conductor DsbD is a unique protein which provides the necessary reducing power to both systems through thiol-disulfide exchange reactions in a complex network of protein–protein interactions. The N-terminal domain of DsbD (nDsbD) is the delivery point of the reducing power originating from cytoplasmic thioredoxin to a variety of periplasmic partners. Here we report 1H, 13C and 15N assignments for resonances of nDsbD in its oxidized and reduced states. These assignments provide the starting point for detailed investigations of the interactions of nDsbD with its protein partners.
    Biomolecular NMR Assignments 10/2011; 6(2). DOI:10.1007/s12104-011-9347-9 · 0.76 Impact Factor
    • "A, proposed pathway of electron flow from thioredoxin (TrxA) in the cytoplasm, via the three domains of DsbD, to the cytochrome c maturation (Ccm) and disulfide bond isomerization pathways in the periplasm is shown. The crystal structure of nDsbD is from Protein Data Bank code 1L6P (8), cDsbD from Protein Data Bank code 1UC7 (11), and the nDsbD-cDsbD complex from Protein Data Bank code 1VRS (12). The cyan boxes indicate the thrombin cleavage sites introduced into full-length DsbD to allow detection of the nDsbD-cDsbD complex following its formation in vivo. "
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    ABSTRACT: The bacterial protein DsbD transfers reductant from the cytoplasm to the otherwise oxidizing environment of the periplasm. This reducing power is required for several essential pathways, including disulfide bond formation and cytochrome c maturation. DsbD includes a transmembrane domain (tmDsbD) flanked by two globular periplasmic domains (nDsbD/cDsbD); each contains a cysteine pair involved in electron transfer via a disulfide exchange cascade. The final step in the cascade involves reduction of the Cys(103)-Cys(109) disulfide of nDsbD by Cys(461) of cDsbD. Here we show that a complex between the globular periplasmic domains is trapped in vivo only when both are linked by tmDsbD. We have found previously ( Mavridou, D. A., Stevens, J. M., Ferguson, S. J., & Redfield, C. (2007) J. Mol. Biol. 370, 643-658 ) that the attacking cysteine (Cys(461)) in isolated cDsbD has a high pK(a) value (10.5) that makes this thiol relatively unreactive toward the target disulfide in nDsbD. Here we show using NMR that active-site pK(a) values change significantly when cDsbD forms a complex with nDsbD. This modulation of pK(a) values is critical for the specificity and function of cDsbD. Uncomplexed cDsbD is a poor nucleophile, allowing it to avoid nonspecific reoxidation; however, in complex with nDsbD, the nucleophilicity of cDsbD increases permitting reductant transfer. The observation of significant changes in active-site pK(a) values upon complex formation has wider implications for understanding reactivity in thiol:disulfide oxidoreductases.
    Journal of Biological Chemistry 12/2008; 284(5):3219-26. DOI:10.1074/jbc.M805963200 · 4.57 Impact Factor
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    • "All six of these cysteines are required for DsbD activity (Figure 1). The N-terminal periplasmic domain, DsbDa, is the domain that directly reduces DsbC (Katzen and Beckwith, 2000; Goulding et al, 2002; Haebel et al, 2002). DsbDa is then itself reduced by the C-terminal periplasmic domain, DsbDg, a thioredoxin-like polypeptide (Kim et al, 2003). "
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    ABSTRACT: The membrane-embedded domain of the unusual electron transporter DsbD (DsbDbeta) uses two redox-active cysteines to catalyze electron transfer between thioredoxin-fold polypeptides on opposite sides of the bacterial cytoplasmic membrane. How the electrons are transferred across the membrane is unknown. Here, we show that DsbDbeta displays an inherent functional and structural symmetry: first, the two cysteines of DsbDbeta can be alkylated from both the cytoplasm and the periplasm. Second, when the two cysteines are disulfide-bonded, cysteine scanning shows that the C-terminal halves of the cysteine-containing transmembrane segments 1 and 4 are exposed to the aqueous environment while the N-terminal halves are not. Third, proline residues located pseudo-symmetrically around the two cysteines are required for redox activity and accessibility of the cysteines. Fourth, mixed disulfide complexes, apparent intermediates in the electron transfer process, are detected between DsbDbeta and thioredoxin molecules on each side of the membrane. We propose a model where the two redox-active cysteines are located at the center of the membrane, accessible on both sides of the membrane to the thioredoxin proteins.
    The EMBO Journal 09/2007; 26(15):3509-20. DOI:10.1038/sj.emboj.7601799 · 10.43 Impact Factor
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