The disulfide bond isomerase DsbC is activated by an immunoglobulin-fold thiol oxidoreductase: crystal structure of the DsbC-DsbDalpha complex

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.     

Mutations of the membrane-bound disulfide reductase DsbD that block electron transfer steps from cytoplasm to periplasm in Escherichia coli

The cytoplasmic membrane protein DsbD keeps the periplasmic disulfide isomerase DsbC reduced, using the cytoplasmic reducing power of thioredoxin. DsbD contains three domains, each containing two reactive cysteines. One membrane-embedded domain, DsbDbeta, transfers electrons from thioredoxin to the carboxy-terminal thioredoxin-like periplasmic domain DsbDgamma. To evaluate the role of conserved amino acid residues in DsbDbeta in the electron transfer process, we substituted alanines for each of 19 conserved amino acid residues and assessed the in vivo redox states of DsbC and DsbD. The mutant DsbDs of 11 mutants which caused defects in DsbC reduction showed relatively oxidized redox states. To analyze the redox state of each DsbD domain, we constructed a thrombin-cleavable DsbD (DsbDTH) from which we could generate all three domains as separate polypeptide chains by thrombin treatment in vitro. We divided the mutants with strong defects into two classes. The first mutant class consists of mutant DsbDbeta proteins that cannot receive electrons from cytoplasmic thioredoxin, resulting in a DsbD that has all six of its cysteines disulfide bonded. The second mutant class represents proteins in which the transfer of electrons from DsbDbeta to DsbDgamma appears to be blocked. This class includes the mutant with the most clear-cut defect, P284A. We relate the properties of the mutants to the positions of the amino acids in the structure of DsbD and discuss mechanisms that would interfere with the electron transfer process.     

High-resolution structures of Escherichia coli cDsbD in different redox states: A combined crystallographic, biochemical and computational study

Escherichia coli DsbD transports electrons from cytoplasmic thioredoxin to periplasmic target proteins. DsbD is composed of an N-terminal (nDsbD) and a C-terminal (cDsbD) periplasmic domain, connected by a central transmembrane domain. Each domain possesses two cysteine residues essential for electron transport. The transport proceeds via disulfide exchange reactions from cytoplasmic thioredoxin to the central transmembrane domain and via cDsbD to nDsbD, which then reduces the periplasmic target proteins. We determined four high-resolution structures of cDsbD: oxidized (1.65 A resolution), chemically reduced (1.3 A), photo-reduced (1.1 A) and chemically reduced at pH increased from 4.6 to 7. The latter structure was refined at 0.99 A resolution, the highest achieved so far for a thioredoxin superfamily member. The data reveal unprecedented structural details of cDsbD, demonstrating that the domain is very rigid and undergoes hardly any conformational change upon disulfide reduction or interaction with nDsbD. In full agreement with the crystallographic results, guanidinium chloride-induced unfolding and refolding experiments indicate that oxidized and reduced cDsbD are equally stable. We confirmed the structural rigidity of cDsbD by molecular dynamics simulations. A remarkable feature of cDsbD is the pKa of 9.3 for the active site Cys461: this value, determined using two different experimental methods, surprisingly was around 2.5 units higher than expected on the basis of the redox potential. Additionally, taking advantage of the very high quality of the cDsbD structures, we carried out pKa calculations, which gave results in agreement with the experimental findings. In conclusion, our wide-scope analysis of cDsbD, encompassing atomic-resolution crystallography, computational chemistry and biophysical measurements, highlighted two so far unrecognized key aspects of this domain: its unusual redox properties and extreme rigidity. Both are likely to be correlated to the role of cDsbD as a covalently linked electron shuttle between the membrane domain and the N-terminal periplasmic domain of DsbD.     

Thermodynamic aspects of DsbD-mediated electron transport

DsbD from Escherichia coli transports electrons from cytoplasmic thioredoxin across the inner membrane to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of three domains: a periplasmic N-terminal domain, a central transmembrane domain (tmDsbD) and a periplasmic C-terminal domain. Each domain contains two essential cysteine residues that are required for electron transport. In contrast to the quinone reductase DsbB, HPLC analysis of the methanol/hexane extracts of purified DsbD revealed no presence of quinones, suggesting that the tmDsbD interacts with thioredoxin and the periplasmic C-terminal domain exclusively via disulfide exchange. We also demonstrate that a DsbD variant containing only the redox-active cysteine pair C163 and C285 in tmDsbD, reconstituted into liposomes, has a redox potential of − 0.246 V. The results show that all steps in the DsbD-mediated electron flow are thermodynamically favorable.     

Solution structure and backbone dynamics of the cysteine 103 to serine mutant of the N-terminal domain of DsbD from Neisseria meningitidis

The DsbD protein is essential for electron transfer from the cytoplasm to the periplasm of Gram-negative bacteria. Its N-terminal domain dispatches electrons coming from cytoplasmic thioredoxin (Trx), via its central transmembrane and C-terminal domains, to its periplasmic partners: DsbC, DsbE/CcmG, and DsbG. Previous structural studies described the latter proteins as Trx-like folds possessing a characteristic C-X-X-C motif able to generate a disulfide bond upon oxidation. The Escherichia coli nDsbD displays an immunoglobulin-like fold in which two cysteine residues (Cys103 and Cys109) allow a disulfide bond exchange with its biological partners.We have determined the structure in solution and the backbone dynamics of the C103S mutant of the N-terminal domain of DsbD from Neisseria meningitidis. Our results highlight significant structural changes concerning the beta-sheets and the local topology of the active site compared with the oxidized form of the E. coli nDsbD. The structure reveals a "cap loop" covering the active site, similar to the oxidized E. coli nDsbD X-ray structure. However, regions featuring enhanced mobility were observed both near to and distant from the active site, revealing a capacity of structural adjustments in the active site and in putative interaction areas with nDsbD biological partners. Results are discussed in terms of functional consequences.     

Structural basis and kinetics of DsbD-dependent cytochrome c maturation

DsbD from Escherichia coli transports two electrons from cytoplasmic thioredoxin to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of an N-terminal periplasmic domain (nDsbD), a C-terminal periplasmic domain, and a central transmembrane domain. Each domain possesses two cysteines required for electron transport. Herein, we demonstrate fast (3.9 x 10(5) M(-1)s(-1)) and direct disulfide exchange between nDsbD and CcmG, a highly specific disulfide reductase essential for cytochrome c maturation. We determined the crystal structure of the disulfide-linked complex between nDsbD and the soluble part of CcmG at 1.94 A resolution. In contrast to the other two known complexes of nDsbD with target proteins, the N-terminal segment of nDsbD contributes to specific recognition of CcmG. This and other features, like the possibility of using an additional interaction surface, constitute the structural basis for the adaptability of nDsbD to different protein substrates.     

Kinetics of the intramolecular disulfide exchange between the periplasmic domains of DsbD

DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of a periplasmic, N-terminal domain (nDsbD), a central transmembrane domain and a periplasmic, C-terminal domain (cDsbD). Each of these domains contains two essential cysteine residues that are required for intermolecular disulfide exchange between DsbD and substrates, and intramolecular disulfide exchange between the three DsbD domains. In order to determine the rate of intramolecular electron transfer from cDsbD to nDsbD, we constructed a redox-sensitive tryptophan variant of cDsbD (cDsbD(W)) that shows an approximately threefold increase in fluorescence upon reduction and has the same redox potential and reactivity as wild-type cDsbD. cDsbD(W) was then used for the construction of fusion proteins with nDsbD and cDsbD(W), connected via flexible linkers of different length. Using the DsbD substrate DsbC, which can only be reduced by nDsbD and does not react with cDsbD, we could directly measure the intramolecular electron transfer from cDsnD(W) to nDsbB in the fusion proteins. We show that the intramolecular disulfide exchange is significantly faster than the reaction between isolated nDsbD and cDsbD. Nevertheless, the effective concentration of 0.2 mM of the domains in the fusions is comaparably low. The rate of 23 s(-1) for the intramolecular disulfide exchange in the fusions was independent of the linker length and may represent the upper limit for the substrate turnover of full-length DsbD.     

Transfer of electrons across the cytoplasmic membrane by DsbD, a membrane protein involved in thiol-disulphide exchange and protein folding in the bacterial periplasm

Reduction of non-native protein disulphides in the periplasm of Escherichia coli is catalysed by three enzymes, DsbC, DsbG and DsbE, each of which harbours a catalytic Cys-X-X-Cys dithiol motif. This dithiol motif requires continuous reduction for activity. Genetic evidence suggests that the source of periplasmic reducing power resides within the cytoplasm, provided by thioredoxin (trxA) and thioredoxin reductase (trxB). Cytoplasmic electrons donated by thioredoxin are thought to be transferred into the periplasm via the DsbD membrane protein. To understand the molecular nature of electron transfer, we have analysed the membrane topology of DsbD. DsbD is exported by an N-terminal signal peptide. The N- and C-terminal domains are positioned in the periplasmic space, connected by eight transmembrane segments. Electron transfer was shown to require five cysteine sulphydryl of DsbD. Trans complementation of mutant DsbD molecules revealed intermolecular electron transfer. We discuss a model whereby the membrane-embedded disulphides of DsbD accept electrons from cytoplasmic thioredoxin and transfer them to the C-terminal periplasmic dithiol motif of DsbD.     

Active-site properties of the oxidized and reduced C-terminal domain of DsbD obtained by NMR spectroscopy

The periplasmic C-terminal domain of the Escherichia coli DsbD protein (cDsbD) has a thioredoxin fold. The two cysteine residues in the CXXC motif serve as the reductant for the disulfide bond of the N-terminal domain which can in turn act as a reductant for various periplasmic partners. The resulting disulfide bond in cDsbD is reduced via an unknown mechanism by the transmembrane helical domain of the protein. We show by NMR analysis of (13)C, (15)N-labelled cDsbD that the protein is rigid, is stable to extremes of pH and undergoes only localized conformational changes in the vicinity of the CXXC motif, and in adjacent regions of secondary structure, upon undergoing the reduced/oxidized transition. pK(a) values have been determined, using 2D NMR, for the N-terminal cysteine of the CXXC motif, Cys461, as well as for other active-site residues. It is demonstrated using site-directed mutagenesis that the negative charges of the side-chains of Asp455 and Glu468 in the active site contribute to the unusually high pK(a) value, 10.5, of Cys461. This value is higher than expected from knowledge of the reduction potential of cDsbD. In a double mutant of cDsbD, D455N/E468Q, the pK(a) value of Cys461 is lowered to 8.6, a value close to that expected for an unperturbed cysteine residue. The pK(a) value of the second cysteine in wild-type cDsbD, Cys464, is significantly higher than the maximum pH value that was studied (pH 12.2).     

Structural basis and kinetics of inter- and intramolecular disulfide exchange in the redox catalyst DsbD

DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to the periplasmic disulfide isomerase DsbC. DsbD contains two periplasmically oriented domains at the N- and C-terminus (nDsbD and cDsbD) that are connected by a central transmembrane (TM) domain. Each domain contains a pair of cysteines that are essential for catalysis. Here, we show that Cys109 and Cys461 form a transient interdomain disulfide bond between nDsbD and cDsbD in the reaction cycle of DsbD. We solved the crystal structure of this catalytic intermediate at 2.85 A resolution, which revealed large relative domain movements in DsbD as a consequence of a strong overlap between the surface areas of nDsbD that interact with DsbC and cDsbD. In addition, we have measured the kinetics of all functional and nonfunctional disulfide exchange reactions between redox-active, periplasmic proteins and protein domains from the oxidative DsbA/B and the reductive DsbC/D pathway. We show that both pathways are separated by large kinetic barriers for nonfunctional disulfide exchange between components from different pathways.     

Thiol-disulfide exchange in an immunoglobulin-like fold: structure of the N-terminal domain of DsbD

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.     

Six conserved cysteines of the membrane protein DsbD are required for the transfer of electrons from the cytoplasm to the periplasm of Escherichia coli.

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.     

1H, 13C and 15N resonance assignments for the oxidized and reduced states of the N-terminal domain of DsbD from Escherichia coli

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 (1)H, (13)C and (15)N 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.     

Reconstitution of a disulfide isomerization system

Isomerization of disulfide bonds is vital for the proper folding of proteins that possess multiple disulfides. In prokaryotes, the catalytic pathway responsible for disulfide isomerization involves thioredoxin, thioredoxin reductase, and the DsbC, DsbG, and DsbD proteins. To be active as isomerases, DsbC and DsbG must be kept reduced. This task is performed by the cytoplasmic membrane protein DsbD. DsbD in turn is reduced by the cytoplasmic thioredoxin and is composed of three domains. The beta domain is membrane-embedded, whereas the alpha and gamma domains are localized to the periplasm. It had been proposed that electrons are transferred within DsbD by a succession of disulfide exchange reactions between the three domains. To test this model using biochemical methods, we purified to homogeneity different polypeptides corresponding to the alpha, beta, gamma, and betagamma domains. Using these domains, we could reconstitute a DsbD activity and, for the first time, reconstitute in vitro the electron transport pathway from NADPH and thioredoxin to DsbC and DsbG. We showed that electrons are transferred from thioredoxin to the beta domain then successively to the gamma domain, the alpha domain, and finally on to DsbC or DsbG. We also determined the redox potential of the gamma domain to be -241 mV, and that of the alpha domain was found to be -229 mV. This shows that the direction of electron flow within DsbD is thermodynamically driven.     

Transmembrane electron transfer by the membrane protein DsbD occurs via a disulfide bond cascade

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.     

Evolutionary domain fusion expanded the substrate specificity of the transmembrane electron transporter DsbD

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.     

Laboratory evolution of Escherichia coli thioredoxin for enhanced catalysis of protein oxidation in the periplasm reveals a phylogenetically conserved substrate specificity determinant

Thioredoxin exported into the Escherichia coli periplasm catalyzes the oxidation of protein thiols in a DsbB-dependent function. However, the oxidative activity of periplasmic thioredoxin is insufficient to render dsbA(-) cells susceptible to infection by M13, a phenotype that is critically dependent on disulfide bond formation in the cell envelope. We sought to examine the molecular determinants that are required in order to convert thioredoxin from a reductant into an efficient periplasmic oxidant. A genetic screen for mutations in thioredoxin that render dsbA(-) cells sensitive to infection by M13 led to the isolation of a single amino acid substitution, G74S. In vivo the TrxA(G74S) mutant exhibited enhanced catalytic activity in the oxidation of alkaline phosphatase but was unable to oxidize FlgI and restore cell motility. In vitro studies revealed that the G74S substitution does not affect the redox potential of the thioredoxin-active site or its kinetics of oxidation by DsbB. Thus, the gain of function afforded by G74S stems in part from its altered substrate specificity, which also rendered the protein more resistant to reduction by DsbD/DsbC in the periplasm.     

Crystal structures of E. coli CcmG and its mutants reveal key roles of the N-terminal beta-sheet and the fingerprint region

CcmG, also designated DsbE, functions as a periplasmic protein thiol:disulfide oxidoreductase and is required for cytochrome c maturation. Here we report the crystal structures of Escherichia coli CcmG and its two mutants, P144A and the N-terminal fifty seven-residue deletion mutant, and two additional deletion mutants were studied by circular dichroism. Structural comparison of E. coli CcmG with its deletion mutants reveals that the N-terminal beta-sheet is essential for maintaining the folding topology and consequently maintaining the active-site structure of CcmG. Pro144 and Glu145 are key residues of the fingerprint region of CcmG. Pro144 is in cis-configuration, and it makes van der Waals interactions with the active-site disulfide Cys80-Cys83 and forms a C--H...O hydrogen bond with Thr82, helping stabilize the active-site structure. Glu145 forms a salt-bridge and hydrogen-bond network with other residues of the fingerprint region and with Arg158, further stabilizing the active-site structure. The cis-configuration of Pro144 makes the backbone nitrogen and oxygen of Ala143 exposed to solvent, favorable for interacting with binding partners. The key role of cis-Pro144 is verified by the P144A mutant, which contains trans-Ala144 and displays redox property changes. Structural comparison of E. coli CcmG with the recently reported structure of CcmG in complex with the N-terminal domain of DsbD reveals that Tyr141 undergoes conformational changes upon binding DsbD. A cis-proline located at the N-terminus of the first beta-strand of the betabetaalpha motif of the thioredoxin-like domain is a conserved structural feature of the thioredoxin superfamily.     

An Extended Active-site Motif Controls the Reactivity of the Thioredoxin Fold

Proteins belonging to the thioredoxin (Trx) superfamily are abundant in all organisms. They share the same structural features, arranged in a seemingly simple fold, but they perform a multitude of functions in oxidative protein folding and electron transfer pathways. We use the C-terminal domain of the unique transmembrane reductant conductor DsbD as a model for an in-depth analysis of the factors controlling the reactivity of the Trx fold. We employ NMR spectroscopy, x-ray crystallography, mutagenesis, in vivo functional experiments applied to DsbD, and a comparative sequence analysis of Trx-fold proteins to determine the effect of residues in the vicinity of the active site on the ionization of the key nucleophilic cysteine of the -CXXC- motif. We show that the function and reactivity of Trx-fold proteins depend critically on the electrostatic features imposed by an extended active-site motif.