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James C A Bardwell

Publications and source records attributed to James C A Bardwell.

At least 19 recordsLinked to original sources
PubMed2006

The origami of thioredoxin-like folds.

Origami is the Japanese art of folding a piece of paper into complex shapes and forms. Much like origami of paper, Nature has used conserved protein folds to engineer proteins for a particular task. An example of a protein family, which has been used by Nature numerous times, is the thioredoxin superfamily. Proteins in the thioredoxin superfamily are all structured with a beta-sheet core surrounded with alpha-helices, and most contain a canonical CXXC motif. The remarkable feature of these proteins is that the link between them is the fold; however, their reactivity is different for each member due to small variations in this general fold as well as their active site. This review attempts to unravel the minute differences within this protein family, and it also demonstrates the ingenuity of Nature to use a conserved fold to generate a diverse collection of proteins to perform a number of different biochemical tasks.

Amino Acid Motifs↗
PubMed2006

Evidence for conformational changes within DsbD: possible role for membrane-embedded proline residues.

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.

Amino Acid Sequence↗
PubMed2005

Copper stress causes an in vivo requirement for the Escherichia coli disulfide isomerase DsbC.

In Escherichia coli, the periplasmic disulfide oxidoreductase DsbA is thought to be a powerful but nonspecific oxidant, joining cysteines together the moment they enter the periplasm. DsbC, the primary disulfide isomerase, likely resolves incorrect disulfides. Given the reliance of protein function on correct disulfide bonds, it is surprising that no phenotype has been established for null mutations in dsbC. Here we demonstrate that mutations in the entire DsbC disulfide isomerization pathway cause an increased sensitivity to the redox-active metal copper. We find that copper catalyzes periplasmic disulfide bond formation under aerobic conditions and that copper catalyzes the formation of disulfide-bonded oligomers in vitro, which DsbC can resolve. Our data suggest that the copper sensitivity of dsbC- strains arises from the inability of the cell to rearrange copper-catalyzed non-native disulfides in the absence of functional DsbC. Absence of functional DsbA augments the deleterious effects of copper on a dsbC- strain, even though the dsbA- single mutant is unaffected by copper. This may indicate that DsbA successfully competes with copper and forms disulfide bonds more accurately than copper does. These findings lead us to a model in which DsbA may be significantly more accurate in disulfide oxidation than previously thought, and in which the primary role of DsbC may be to rearrange incorrect disulfide bonds that are formed during certain oxidative stresses.

Adaptation, Physiological↗
PubMed2005

The crystal structure of TrxA(CACA): Insights into the formation of a [2Fe-2S] iron-sulfur cluster in an Escherichia coli thioredoxin mutant.

Escherichia coli thioredoxin is a small monomeric protein that reduces disulfide bonds in cytoplasmic proteins. Two cysteine residues present in a conserved CGPC motif are essential for this activity. Recently, we identified mutations of this motif that changed thioredoxin into a homodimer bridged by a [2Fe-2S] iron-sulfur cluster. When exported to the periplasm, these thioredoxin mutants could restore disulfide bond formation in strains lacking the entire periplasmic oxidative pathway. Essential for the assembly of the iron-sulfur was an additional cysteine that replaced the proline at position three of the CGPC motif. We solved the crystalline structure at 2.3 Angstroms for one of these variants, TrxA(CACA). The mutant protein crystallized as a dimer in which the iron-sulfur cluster is replaced by two intermolecular disulfide bonds. The catalytic site, which forms the dimer interface, crystallized in two different conformations. In one of them, the replacement of the CGPC motif by CACA has a dramatic effect on the structure and causes the unraveling of an extended alpha-helix. In both conformations, the second cysteine residue of the CACA motif is surface-exposed, which contrasts with wildtype thioredoxin where the second cysteine of the CXXC motif is buried. This exposure of a pair of vicinal cysteine residues apparently allows thioredoxin to acquire an iron-sulfur cofactor at its active site, and thus a new activity and mechanism of action.

Binding Sites↗
PubMed2005

Mutational analysis of the disulfide catalysts DsbA and DsbB.

In prokaryotes, disulfides are generated by the DsbA-DsbB system. DsbB functions to generate disulfides by quinone reduction. These disulfides are passed to the DsbA protein and then to folding proteins. To investigate the DsbA-DsbB catalytic system, we performed an in vivo selection for chromosomal dsbA and dsbB mutants. We rediscovered many residues previously shown to be important for the activity of these proteins. In addition, we obtained one novel DsbA mutant (M153R) and four novel DsbB mutants (L43P, H91Y, R133C, and L146R). We also mutated residues that are highly conserved within the DsbB family in an effort to identify residues important for DsbB function. We found classes of mutants that specifically affect the apparent K(m) of DsbB for either DsbA or quinones, suggesting that quinone and DsbA may interact with different regions of the DsbB protein. Our results are consistent with the interpretation that the residues Q33 and Y46 of DsbB interact with DsbA, Q95 and R48 interact with quinones, and that residue M153 of DsbA interacts with DsbB. All of these interactions could be due to direct amino acid interactions or could be indirect through, for instance, their effect on protein structure. In addition, we find that the DsbB H91Y mutant severely affects the k(cat) of the reaction between DsbA and DsbB and that the DsbB L43P mutant is inactive, suggesting that both L43 and H91 are important for the activity of DsbB. These experiments help to better define the residues important for the function of these two protein-folding catalysts.

Amino Acid Substitution↗
PubMed2004

Catalysis of disulfide bond formation and isomerization in the Escherichia coli periplasm.

Disulfide bond formation is a catalyzed process in vivo. In prokaryotes, the oxidation of cysteine pairs is achieved by the transfer of disulfides from the highly oxidizing DsbA/DsbB catalytic machinery to substrate proteins. The oxidizing power utilized by this system comes from the membrane-embedded electron transport system, which utilizes molecular oxygen as a final oxidant. Proofreading of disulfide bond formation is performed by the DsbC/DsbD system, which has the ability to rearrange non-native disulfides to their native configuration. These disulfide isomerization reactions are sustained by a constant supply of reducing power provided by the cytoplasmic thioredoxin system, utilizing NADPH as the ultimate electron source.

Catalysis↗
PubMed2004

An engineered pathway for the formation of protein disulfide bonds.

We have engineered a pathway for the formation of disulfide bonds. By imposing evolutionary pressure, we isolated mutations that changed thioredoxin, which is a monomeric disulfide reductase, into a [2Fe-2S] bridged dimer capable of catalyzing O2-dependent sulfhydryl oxidation in vitro. Expression of the mutant protein in Escherichia coli with oxidizing cytoplasm and secretion via the Tat pathway restored disulfide bond formation in strains that lacked the complete periplasmic oxidative machinery (DsbA and DsbB). The evolution of [2Fe-2S] thioredoxin illustrates how mutations within an existing scaffold can add a cofactor and markedly change protein function.

Amino Acid Motifs↗
PubMed2004

Snapshots of DsbA in action: detection of proteins in the process of oxidative folding.

DsbA, a thioredoxin superfamily member, introduces disulfide bonds into newly translocated proteins. This process is thought to occur via formation of mixed disulfide complexes between DsbA and its substrates. However, these complexes are difficult to detect, probably because of their short-lived nature. Here we show that it is possible to detect such covalent intermediates in vivo by a mutation in DsbA that alters cis proline-151. Further, this mutant allowed us to identify substrates of DsbA. Alteration of the cis proline, highly conserved among thioredoxin superfamily members, may be useful for the detection of substrates and intermediate complexes in other systems.

Amino Acid Motifs↗
PubMed2004

In vivo substrate specificity of periplasmic disulfide oxidoreductases.

In Escherichia coli, a family of periplasmic disulfide oxidoreductases catalyzes correct disulfide bond formation in periplasmic and secreted proteins. Despite the importance of native disulfide bonds in the folding and function of many proteins, a systematic investigation of the in vivo substrates of E. coli periplasmic disulfide oxidoreductases, including the well characterized oxidase DsbA, has not yet been performed. We combined a modified osmotic shock periplasmic extract and two-dimensional gel electrophoresis to identify substrates of the periplasmic oxidoreductases DsbA, DsbC, and DsbG. We found 10 cysteine-containing periplasmic proteins that are substrates of the disulfide oxidase DsbA, including PhoA and FlgI, previously established DsbA substrates. This technique did not detect any in vivo substrates of DsbG, but did identify two substrates of DsbC, RNase I and MepA. We confirmed that RNase I is a substrate of DsbC both in vivo and in vitro. This is the first time that DsbC has been shown to affect the in vivo function of a native E. coli protein, and the results strongly suggest that DsbC acts as a disulfide isomerase in vivo. We also demonstrate that DsbC, but not DsbG, is critical for the in vivo activity of RNase I, indicating that DsbC and DsbG do not function identically in vivo. The absence of substrates for DsbG suggests either that the in vivo substrate specificity of DsbG is more limited than that of DsbC or that DsbG is not active under the growth conditions tested. Our work represents one of the first times the in vivo substrate specificity of a folding catalyst system has been systematically investigated. Because our methodology is based on the simple assumption that the absence of a folding catalyst should cause its substrates to be present at decreased steady-state levels, this technique should be useful in analyzing the substrate specificity of any folding catalyst or chaperone for which mutations are available.

Cysteine↗
PubMed2004

Disulfide relays between and within proteins: the Ero1p structure.

The essential flavoenzyme Ero1p both creates de novo disulfide bonds and transfers these disulfides to the folding catalyst protein disulfide isomerase (PDI). The recently solved crystal structure of Ero1p, in combination with previous biochemical, genetic and structural data, provides insight into the mechanism by which Ero1p accomplishes these tasks. A comparison of Ero1p with the smaller flavoenzyme Erv2p highlights important structural elements that are shared by these flavin adenine dinucleotide (FAD)-binding sulfhydryl oxidases and suggests some general themes that might be common to proteins that generate disulfide bonds.

Crystallography, X-Ray↗
PubMed2003

Disulfide bond formation involves a quinhydrone-type charge-transfer complex.

The chemistry of disulfide exchange in biological systems is well studied. However, the detailed mechanism of how oxidizing equivalents are derived to form disulfide bonds in proteins is not clear. In prokaryotic organisms, it is known that DsbB delivers oxidizing equivalents through DsbA to secreted proteins. DsbB becomes reoxidized by reducing quinones that are part of the membrane-bound electron-transfer chains. It is this quinone reductase activity that links disulfide bond formation to the electron transport system. We show here that purified DsbB contains the spectral signal of a quinhydrone, a charge-transfer complex consisting of a hydroquinone and a quinone in a stacked configuration. We conclude that disulfide bond formation involves a stacked hydroquinone-benzoquinone pair that can be trapped on DsbB as a quinhydrone charge-transfer complex. Quinhydrones are known to be redox-active and are commonly used as redox standards, but, to our knowledge, have never before been directly observed in biological systems. We also show kinetically that this quinhydrone-type charge-transfer complex undergoes redox reactions consistent with its being an intermediate in the reaction mechanism of DsbB. We propose a simple model for the action of DsbB where a quinhydrone-like complex plays a crucial role as a reaction intermediate.

Bacterial Proteins↗
PubMed2003

Thioredoxin 2, an oxidative stress-induced protein, contains a high affinity zinc binding site.

Two thioredoxins have been described in Escherichia coli, TrxA and Trx2. Both thioredoxins are capable of reducing disulfide bonds using a conserved pair of cysteine residues present in a WCGPC motif. A number of unique structural and regulatory features distinguish the Trx2 subfamily from the much larger TrxA family. The Trx2 subfamily has an additional N-terminal domain of +/- 30 residues, which contains two additional conserved CXXC motifs. Moreover, the gene coding for Trx2 is under control of the oxidative stress transcription factor OxyR in E. coli. This suggests that Trx2 may play a role in the cellular defense against oxidative stress. We show here that Trx2 contains zinc in a 1:1 stoichiometry, making it the first identified zinc-binding thioredoxin. The zinc atom is coordinated by the four cysteines of the two N-terminal CXXC motifs. The zinc center of Trx2 binds zinc with a very high affinity (Ka of >1018 m-1). We show that in vitro oxidation of the zinc binding cysteines by H2O2 releases the zinc and induces a conformational change. The zinc-free protein conserves its reductase activity. Altogether, our results suggest that the zinc center might play the role of a redox switch, changing a yet to be identified activity.

Amino Acid Motifs↗
PubMed2002

DsbB catalyzes disulfide bond formation de novo.

DsbA and DsbB are responsible for disulfide bond formation. DsbA is the direct donor of disulfides, and DsbB oxidizes DsbA. DsbB has the unique ability to generate disulfides by quinone reduction. It is thought that DsbB oxidizes DsbA via thiol disulfide exchange. In this mechanism, a disulfide is formed across the N-terminal pair of cysteines (Cys-41/Cys-44) in DsbB by quinone reduction. This disulfide is then transferred on to the second pair of cysteine residues in DsbB (Cys-104/Cys-130) and then finally transferred to DsbA. We have shown here the redox potential of the two disulfides in DsbB are -271 and -284 mV, respectively, and considerably less oxidizing than the disulfide of DsbA at -120 mV. In addition, we have found the Cys-104/Cys-130 disulfide of DsbB to actually be a substrate for DsbA in vitro. These findings indicate that the disulfides in DsbB are unsuitable to function as the oxidant of DsbA. Furthermore, we have shown that mutants in DsbB that lack either pair or all of its cysteines are also capable of oxidizing DsbA. These unexpected findings raise the possibility that the oxidation of DsbA by DsbB does not occur via thiol disulfide exchange as is widely assumed but rather, directly via quinone reduction.

Amino Acid Sequence↗
PubMed2002

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.

Catalysis↗