Crystal structure of DsbDγ reveals the mechanism of redox potential shift and substrate specificity<sup>1</sup>

Kim, Jae-Hoon; Kim, Seung Jun; Jeong, Dae Gwin; Son, Jeong Hee; Ryu, Seong Eon

doi:10.1016/s0014-5793(03)00434-4

Cited by 51 publications

(52 citation statements)

References 38 publications

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“…Although cleavage of ␤ takes place efficiently, no band corresponding to that of alkylated cleaved ␤ was observed (Fig. 5, compare lane 4 with lanes [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. This result indicates that ␣␤, exhibiting its periplasmic face outside on in right-side-out vesicles, is subject to cleavage by TEV protease but does not have either cysteine exposed on this face of the membrane.…”

Section: Both Catalytic Cysteines Of Dsbd␤ Embedded In the Membrane Omentioning

confidence: 97%

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Role and location of the unusual redox-active cysteines in the hydrophobic domain of the transmembrane electron transporter DsbD

Katzen

Beckwith

2003

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Both Catalytic Cysteines Of Dsbd␤ Embedded In the Membrane Omentioning

confidence: 97%

“…DsbD is composed of three domains: an N-terminal immunoglobulin-like domain (␣), a central hydrophobic core (␤) with eight transmembrane segments (TM segments), and a Cterminal periplasmic domain (␥), which appears to be a member of the thioredoxin superfamily (12)(13)(14)(15)(16)(17) (Fig. 1A).…”

mentioning

confidence: 99%

Role and location of the unusual redox-active cysteines in the hydrophobic domain of the transmembrane electron transporter DsbD

Katzen

Beckwith

2003

Proc. Natl. Acad. Sci. U.S.A.

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“…In vivo and in vitro experiments suggest that electrons are transferred via a succession of disulfide bond exchange reactions, from thioredoxin to ␤, then to ␥, then to ␣, and finally to DsbC/DsbG (3,11,18). The crystal structures of both periplasmic domains ␣ and ␥ have been solved, and there is a significant amount of biochemical information available for these two domains (7,12,18). In contrast, much less is known about the membrane domain ␤.…”

mentioning

confidence: 99%

Evidence for Conformational Changes within DsbD: Possible Role for Membrane-Embedded Proline Residues

Hiniker

Vertommen²,

Bardwell

et al. 2006

J Bacteriol

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The mechanism by which DsbD transports electrons across the cytoplasmic membrane is unknown. Here we provide evidence that DsbD's conformation depends on its oxidation state. Our data also suggest that four highly conserved prolines surrounding DsbD's membrane-embedded catalytic cysteines may have an important functional role, possibly conferring conformational flexibility to DsbD.Many periplasmic and secreted proteins contain disulfide bonds that are required to stabilize the protein's structure. In Escherichia coli, the well-studied DsbA-DsbB pathway catalyzes formation of disulfides in substrate proteins (2). A second, less studied, pathway performs disulfide bond rearrangement. In this pathway, the inner membrane protein DsbD maintains periplasmic disulfide isomerases DsbC and DsbG in the reduced and active form (8,14). This is necessary for DsbC and DsbG to attack incorrect disulfides and catalyze disulfide rearrangements. Genetic studies have shown that DsbD receives its electrons from cytoplasmic thioredoxin and transfers these electrons across the inner membrane to DsbC/DsbG (17). DsbD therefore connects the periplasmic isomerization pathway to the reductive power of the cytoplasm by transferring electrons from the cytoplasm to the periplasm and, correspondingly, disulfide bonds from the periplasm to the cytoplasm.DsbD is a 59-kDa protein with three domains. Two of these domains (␣ and ␥) are periplasmic while the third, ␤, is located in the inner membrane. Each domain contains a conserved pair of cysteine residues, which are essential for activity (19). In vivo and in vitro experiments suggest that electrons are transferred via a succession of disulfide bond exchange reactions, from thioredoxin to ␤, then to ␥, then to ␣, and finally to DsbC/DsbG (3,11,18). The crystal structures of both periplasmic domains ␣ and ␥ have been solved, and there is a significant amount of biochemical information available for these two domains (7,12,18). In contrast, much less is known about the membrane domain ␤. In particular, the mechanism by which the ␤ domain transports disulfides across the inner membrane remains unresolved. In fact, the question of how disulfides get across membranes has not to our knowledge been solved for any system. There are examples of systems that transport electrons across membranes, including the malateaspartate and glycerophosphate shuttles. In these systems, electrons are carried by metabolites that are transported from one compartment of the cell to the other. However, DsbD is thought to transport electrons without using a metabolite or a cofactor. If true, this makes DsbD unique among known electron transport proteins. We therefore wanted to examine this longstanding mystery of how reducing equivalents get across membranes.Conformational changes. If DsbD transports disulfides without using a cofactor, major conformational changes are likely to take place to allow the membrane-embedded cysteine residues to be alternatively exposed to the cytoplasm and to the periplasm. To test this hypoth...

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“…3B). It allows helix ␣2Ј to change its orientation relative to the central ␤-sheet and allows helix ␣2 to adopt a longer linear segment (44).…”

Section: Resultsmentioning

confidence: 99%

Insight into Disulfide Bond Catalysis in Chlamydia from the Structure and Function of DsbH, a Novel Oxidoreductase

Mac

Hacht

Hung

et al. 2008

Journal of Biological Chemistry

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The Chlamydia family of human pathogens uses outer envelope proteins that are highly cross-linked by disulfide bonds but nevertheless keeps an unusually high number of unpaired cysteines in its secreted proteins. To gain insight into chlamydial disulfide bond catalysis, the structure, function, and substrate interaction of a novel periplasmic oxidoreductase, termed DsbH, were determined. The structure of DsbH, its redox potential of ؊269 mV, and its functional properties are similar to thioredoxin and the C-terminal domain of DsbD, i.e. characteristic of a disulfide reductase. As compared with these proteins, the two central residues of the DsbH catalytic motif (CMWC) shield the catalytic disulfide bond and are selectively perturbed by a peptide ligand. This shows that these oxidoreductase family characteristic residues are not only important in determining the redox potential of the catalytic disulfide bond but also in influencing substrate interactions. For DsbH, three functional roles are conceivable; that is, reducing intermolecular disulfides between proteins and small molecules, keeping a specific subset of exported proteins reduced, or maintaining the periplasm of Chlamydia in a generally reducing state.Chlamydia are obligate intracellular eubacteria that are phylogenetically distant from other bacterial divisions (1). Virtually every human being will be infected with Chlamydia pneumoniae in their lifetime, and this organism accounts for ϳ10% of pneumonia cases and 5% of bronchitis cases in the United States (2, 3). Acute infections are usually mild in immunocompetent hosts, but severe pneumonias are observed in immunocompromised patients. Chronic C. pneumoniae infections have been associated with arterial disease (4), where C. pneumoniae act to continuously stimulate inflammation and thereby contribute to stroke and coronary heart disease. Chlamydia have an unique developmental cycle in which the extracellular infectious form, the elementary body, is metabolically inactive and highly resistant to lysis. This is thought to be due in large part to a complex of outer envelope proteins that are highly crosslinked by disulfide bonds (5). Disulfide cross-linked envelope proteins appear to substitute for peptidoglycan in the elementary body (6). This is in contrast to the intracellular, non-infectious but metabolically active reticulate body which employs peptidoglycan (7), like other Gram-negative bacteria. The reliance of the elementary body on disulfide bond-linked proteins suggests that interference with chlamydial disulfide bond catalysis offers a potential avenue for treating chlamydial infections in addition to antibiotics administration. Despite the extensive use of disulfide cross-linked envelope proteins, Chlamydia exhibit an unusually high number of unpaired cysteines in their secreted proteins compared with Escherichia coli. Is the machinery for periplasmic disulfide bond catalysis in Chlamydia different from E. coli?Functional gene assignment of the C. pneumoniae TW183 genome predicts 5 periplasmic...

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Crystal structure of DsbDγ reveals the mechanism of redox potential shift and substrate specificity¹

Cited by 51 publications

References 38 publications

Role and location of the unusual redox-active cysteines in the hydrophobic domain of the transmembrane electron transporter DsbD

Role and location of the unusual redox-active cysteines in the hydrophobic domain of the transmembrane electron transporter DsbD

Evidence for Conformational Changes within DsbD: Possible Role for Membrane-Embedded Proline Residues

Insight into Disulfide Bond Catalysis in Chlamydia from the Structure and Function of DsbH, a Novel Oxidoreductase

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Crystal structure of DsbDγ reveals the mechanism of redox potential shift and substrate specificity1

Cited by 51 publications

References 38 publications

Role and location of the unusual redox-active cysteines in the hydrophobic domain of the transmembrane electron transporter DsbD

Role and location of the unusual redox-active cysteines in the hydrophobic domain of the transmembrane electron transporter DsbD

Evidence for Conformational Changes within DsbD: Possible Role for Membrane-Embedded Proline Residues

Insight into Disulfide Bond Catalysis in Chlamydia from the Structure and Function of DsbH, a Novel Oxidoreductase

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Crystal structure of DsbDγ reveals the mechanism of redox potential shift and substrate specificity¹