Analysis of disulphide bond connectivity patterns in protein tertiary structure

The analysis of disulphide bond containing proteins in the Protein Data Bank (PDB) revealed that out of 27,209 protein structures analyzed, 12,832 proteins contain at least one intra-chain disulphide bond and 811 proteins contain at least one inter-chain disulphide bond. The intra-chain disulphide bond containing proteins can be grouped into 256 categories based on the number of disulphide bonds and the disulphide bond connectivity patterns (DBCPs) that were generated according to the position of half-cystine residues along the protein chain. The PDB entries corresponding to these 256 categories represent 509 unique SCOP superfamilies. A simple web-based computational tool is made freely available at the website http://www.ccmb.res.in/bioinfo/dsbcp that allows flexible queries to be made on the database in order to retrieve useful information on the disulphide bond containing proteins in the PDB. The database is useful to identify the different SCOP superfamilies associated with a particular disulphide bond connectivity pattern or vice versa. It is possible to define a query based either on a single field or a combination of the following fields, i.e., PDB code, protein name, SCOP superfamily name, number of disulphide bonds, disulphide bond connectivity pattern and the number of amino acid residues in a protein chain and retrieve information that match the criterion. Thereby, the database may be useful to select suitable protein structural templates in order to model the more distantly related protein homologs/analogs using the comparative modeling methods.     

A flavoprotein oxidase defines a new endoplasmic reticulum pathway for biosynthetic disulphide bond formation

Ero1 and Pdi1 are essential elements of the pathway for the formation of disulphide bonds within the endoplasmic reticulum (ER). By screening for alternative oxidation pathways in Saccharomyces cerevisiae, we identified ERV2 as a gene that when overexpressed can restore viability and disulphide bond formation to an ero1-1 mutant strain. ERV2 encodes a luminal ER protein of relative molecular mass 22,000. Purified recombinant Erv2p is a flavoenzyme that can catalyse O2-dependent formation of disulphide bonds. Erv2p transfers oxidizing equivalents to Pdi1p by a dithiol-disulphide exchange reaction, indicating that the Erv2p-dependent pathway for disulphide bond formation closely parallels that of the previously identified Ero1p-dependent pathway.     

The role of glutathione in disulphide bond formation and endoplasmic-reticulum-generated oxidative stress

Glutathione is a ubiquitous molecule found in all parts of the cell where it fulfils a range of functions from detoxification to protection from oxidative damage. It provides the main redox buffer for cells and as such has been implicated in the formation of native disulphide bonds. However, the discovery of the enzyme Ero1 has called into question the exact role of glutathione in this process. In this review, we discuss the arguments for and against a role for glutathione in facilitating disulphide-bond formation and consider its role in protecting the cell from endoplasmic-reticulum-generated oxidative stress.     

A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae

A strain of Vibrio cholerae, which had been engineered to express high levels of the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200-amino-acid exported protein, with a Cys-X-X-Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation in E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active-site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.     

Dynamic nature of disulphide bond formation catalysts revealed by crystal structures of DsbB

In the Escherichia coli system catalysing oxidative protein folding, disulphide bonds are generated by the cooperation of DsbB and ubiquinone and transferred to substrate proteins through DsbA. The structures solved so far for different forms of DsbB lack the Cys104–Cys130 initial‐state disulphide that is directly donated to DsbA. Here, we report the 3.4 Å crystal structure of a DsbB–Fab complex, in which DsbB has this principal disulphide. Its comparison with the updated structure of the DsbB–DsbA complex as well as with the recently reported NMR structure of a DsbB variant having the rearranged Cys41–Cys130 disulphide illuminated conformational transitions of DsbB induced by the binding and release of DsbA. Mutational studies revealed that the membrane‐parallel short α‐helix of DsbB has a key function in physiological electron flow, presumably by controlling the positioning of the Cys130‐containing loop. These findings demonstrate that DsbB has developed the elaborate conformational dynamism to oxidize DsbA for continuous protein disulphide bond formation in the cell.     

Periplasmic disulphide bond formation is essential for cellulase secretion by the plant pathogen Erwinia chrysanthemi

Secretion to the cell exterior of cellulase EGZ and of at least six pectinases enables the Gram-negative Erwinia chrysanthemi to cause severe plant disease. The C-terminal cellulose-binding domain (CBD) of EGZ was found to contain a disulphide bond which forms, in the periplasm, between residues Cys-325 and Cys-382. Dithiothreitol (DTT)-treatment of native EGZ showed that the disulphide bond was dispensable, both for catalysis and cellulose binding. Adding DTT to E. chrysanthemi cultures led to immediate arrest of secretion of EGZ which accumulated in the periplasm where the CBD was eventually proteolysed. Site-directed mutagenesis that affected Cys residues involved in disulphide bond formation resulted in molecules that were catalytically active and able to bind to cellulose but were no longer secreted, Instead they accumulated in the periplasm. Interestingly, the region around EGZ Cys-325 is conserved in two pectinases secreted by the same pathway as EGZ. We conclude that the conserved Cys, and possibly adjacent residues, bear essential information for EGZ to be secreted and that periplasmic disulphide bond formation is an obligatory step which provides a pre-folded functional form of EGZ with secretion competence.     

The requirement for DsbA in pullulanase secretion is independent of disulphide bond formation in the enzyme

Results from previous studies have suggested that an intramolecular disulphide bond in the exoprotein pullulanase is needed for its recognition and transport across the outer membrane. This interpretation of the data is shown here to be incorrect: pullulanase devoid of all potential disulphide bonds is secreted with apparently the same efficiency as the wild-type protein. Furthermore, the periplasmic disulphide bond, oxidoreductase DsbA, previously shown to catalyse the formation of a disulphide bond in pullulanase and to decrease its transit time in the periplasm, is shown here to be required for the rapid secretion of pullulanase devoid of disulphide bonds. Several possible explanations for the role of DsbA in pullulanase secretion are discussed.     

How thioredoxin can reduce a buried disulphide bond

We present a study of the interaction between thioredoxin and the model enzyme pI258 arsenate reductase (ArsC) from Staphylococcus aureus. ArsC catalyses the reduction of arsenate to arsenite. Three redox active cysteine residues (Cys10, Cys82 and Cys89) are involved. After a single catalytic arsenate reduction event, oxidized ArsC exposes a disulphide bridge between Cys82 and Cys89 on a looped-out redox helix. Thioredoxin converts oxidized ArsC back towards its initial reduced state. In the absence of a reducing environment, the active-site P-loop of ArsC is blocked by the formation of a second disulphide bridge (Cys10-Cys15). While fully reduced ArsC can be recovered by exposing this double oxidized ArsC to thioredoxin, the P-loop disulphide bridge is itself inaccessible to thioredoxin. To reduce this buried Cys10-Cys15 disulphide-bridge in double oxidized ArsC, an intra-molecular Cys10-Cys82 disulphide switch connects the thioredoxin mediated inter-protein thiol-disulphide transfer to the buried disulphide. In the initial step of the reduction mechanism, thioredoxin appears to be selective for oxidized ArsC that requires the redox helix to be looped out for its interaction. The formation of a buried disulphide bridge in the active-site might function as protection against irreversible oxidation of the nucleophilic cysteine, a characteristic that has also been observed in the structurally similar low molecular weight tyrosine phosphatase.     

Thiol and disulphide contents of hen ovalbumin. C-Terminal sequence and location of disulphide bond

1. The thiol and disulphide contents of hen ovalbumin were investigated by p-chloromercuribenzoate titration, by determination of cysteic acid content after performic acid oxidation, by measurement of uptake of radioactive iodoacetic acid, and by assay of S-aminoethylcysteine after reaction with ethyleneimine. All results showed that ovalbumin had 6 half-cystine residues. Experiments with and without reducing agents demonstrated that there were 4 thiol groups and 1 disulphide bond. 2. A peptide containing equimolar amounts of S-carboxymethyl-cysteine, serine, valine and proline, but no lysine or arginine, was obtained by radioactive labelling of the cysteine residues with iodo[¹⁴C]acetic acid followed by electrophoretic and chromatographic separation of tryptic digests. It was concluded that the C-terminal sequence of ovalbumin is -Cys-Val-Ser-Pro. 3. The location of the disulphide bond was studied by using a double-labelling technique. It was shown that one end of the disulphide was located in this C-terminal peptide.     

Vibrio spp. secrete proaerolysin as a folded dimer without the need for disulphide bond formation

Proaerolysin is an extracellular dimeric protein that is secreted across the inner and outer membranes of Aeromonas spp. in separate steps. To investigate the role of protein folding in the second step, one or more cysteine residues were introduced and the mutant proaerolysins were expressed in Aeromonas hydrophila and Aeromonas salmonicida , as well as Vibrio cholerae . Replacing Met-41 with Cys resulted in expression of a protein that could form a dimer in which the monomers were linked together by a disulphide bridge. A double mutant was also made, in which Gly-202 and Ile-445 were replaced with cysteine in order to allow the formation of an intrachain disulphide bridge when the molecule was correctly folded. The M41C covalent dimer and G202C/I445C proaerolysin with the new intrachain bridge were both easily detected inside the bacteria, and they later appeared in the culture supernatants. Small amounts of incorrectly folded proaerolysin were also observed in the cells, but they were not secreted. We conclude that proaerolysin folds and dimerizes before being released from the cell, and that correct folding is a requirement for secretion to occur. The proton ionophore CCCP reduced release of the folded proteins. Unoxidized protein was secreted by cells grown in β-mercaptoethanol and by a dsbA mutant of V. cholerae , indicating that disulphide bond formation may not be essential for release.     

The relationship between disulphide bond formation, processing and secretion of lipo-β-lactamase in yeast

The hybrid prokaryotic lipo-beta-lactamase mature and precursor proteins spontaneously form an intramolecular disulphide bond when oxidized in vitro. When expressed in Saccharomyces cerevisiae (in vivo) the lipo-beta-lactamase precursor is in a reduced form whereas the majority of the mature protein is oxidized. The results indicate that in yeast, the lipo-beta-lactamase precursor is first processed (the signal peptide is removed) and then oxidized to form a disulphide bond in the mature protein. Reduced-mature lipo-beta-lactamase was found to reach the yeast periplasm and the process depends on endoplasmic reticulum (ER) entry even though the protein is not oxidized. This result is remarkable since in eukaryotes, disulphide bond formation occurs in the ER. Oxidized mature lipo-beta-lactamase can also be released from the sphaeroplast into the yeast periplasm. Mutant lipo-beta-lactamase genes in which cysteine residue 131 was changed to either tyrosine or threonine, were efficiently processed and secreted in yeast, which is consistent with the finding that reduced-mature non-mutant lipo-beta-lactamase can be secreted. We discuss the possibility that the folding mechanism of lipo-beta-lactamase in vitro may be fundamentally different from the process in the eukaryotic system of S. cerevisiae.     

Incomplete protein disulphide bond conformation and decreased protein expression result from high cell growth during heterologous protein expression in Pichia pastoris

Previous report has shown that the expression of recombinant human consensus interferon-α mutant (cIFN) in Pichia pastoris in bioreactor is limited with respect to the incorrectly folded cIFN with incomplete disulfide bond, which lead to the degradation and aggregation of cIFN. In this study, the origin of incorrectly folded cIFN is firstly studied. Fed-batch fermentation in bioreactor shows that the incorrectly folded cIFN is formed intramolecularly and secreted to the extracellular environment. Further chemostat cultures indicate that the specific growth rate is the critical factor for the production of incorrect cIFN. In addition, cell shows reduced expression level of cIFN at high specific growth rate. We also demonstrate that the incorrectly folded cIFN could form aggregates intracellularly and these aggregates are non-covalent forms. Taken together, these results suggest that the efficient heterologous expression of cIFN is limited by high cell growth that is unique from expression limitations seen for soluble proteins. A balance has to be found between the increase for high efficient expression of heterologous proteins and requirement of the high cell growth during the expression of recombinant proteins in P. pastoris.     

A new Escherichia coli gene, dsbG, encodes a periplasmic protein involved in disulphide bond formation, required for recycling DsbA/DsbB and DsbC redox proteins

We have identified and functionally characterized a new Escherichia coli gene, dsbG , whose product is involved in disulphide bond formation in the periplasm. The dsbG gene was cloned from a multicopy plasmid library lacking the dsbB redox protein-encoding gene. Multicopy dsbG -carrying clones were selected, since they allowed E. coli to grow at lethal concentrations of dithiothreitol. In a complementary genetic approach, point mutations were independently obtained and mapped to the dsbG gene. Such mutations led simultaneously to a dithiothreitol-sensitive phenotype and an increased σ^E-dependent heat shock response, which reflects the presence of misfolded proteins in the extracytoplasm. In agreement with these observations, dsbG mutants were shown to accumulate reduced forms of a variety of disulphide bond-containing proteins in the periplasm. This DsbG defect could be rescued by addition to the growth medium of either oxidized dithiothreitol or cystine, or by overexpression of the dsbA or dsbB genes. DsbG is synthesized as a precursor form of 27.5 kDa and processed to a 25.7 kDa mature species located in the periplasm. DsbG was overproduced, purified to homogeneity and shown to have redox properties of thiol–disulphide oxidoreductases in vitro . Replacement of the first Cys residue of the predicted active site, Phe–(Xaa)₄–Cys–Pro–Tyr–Cys by Ala, completely inactivated DsbG protein function. Taken together, all our results demonstrate that DsbG acts in vivo as an efficient thiol–disulphide oxidase. In addition, dsbG is the first member of the dsb family for which null mutations are conditionally lethal and can be propagated only if supplemented with oxidants in the growth medium. We propose that the main role of DsbG is to maintain the proper redox balance between the DsbA/DsbB and DsbC systems.     

Controlling pore assembly of staphylococcal gamma-haemolysin by low temperature and by disulphide bond formation in double-cysteine LukF mutants

Staphylococcal LukF and Hlg2 are water-soluble monomers of gamma-haemolysin that assemble into oligomeric pores on the erythrocyte membranes. Here, we have created double-cysteine LukF mutants, in which single disulphide bonds connect either the prestem domain and the cap domain (V12C-T136C, Cap-Stem), or two beta-strands within the prestem domain (T117C-T136C, Stem-Stem) to control pore assembly of gamma-haemolysin at intermediate stages. The disulphide-trapped mutants were inactive in erythrocyte lysis, but gained full haemolytic activity if the disulphide bonds were reduced. The disulphide bonds blocked neither the membrane binding ability nor the intermediate prepore oligomerization, but efficiently inhibited the transition from prepores to pores. The prepores of Cap-Stem were dissociated into monomers in 1% SDS. In contrast, the prepores of Stem-Stem were stable in SDS and had ring-shaped structures similar to those of wild-type LukF, as observed by transmission electron microscopy. The transition of both mutants from prepores to pores could even be achieved by reducing disulphide bonds at low temperature (2 degrees C), whereas prepore oligomerization was effectively inhibited by low temperature. Finally, real-time transition of Stem-Stem from prepores to pores on ghost cells, visualized using a Ca2+-sensitive fluorescent indicator (Rhod2), was shown by the sequential appearance of fluorescence spots, indicating pore-opening events. Taken together, these data indicate that the prepores are legitimate intermediates during gamma-haemolysin pore assembly, and that conformational changes around residues 117 and 136 of the prestem domain are essential for pore formation, but not for membrane binding or prepore oligomerization. We propose a mechanism for gamma-haemolysin pore assembly based on the demonstrated intermediates.     

Disulphide bond formation in food protein aggregation and gelation

In this short review we discuss the role of cysteine residues and cystine bridges for the functional aggregation of food proteins. We evaluate how formation and cleavage of disulphide bonds proceeds at a molecular level, and how inter- and intramolecular disulfide bonds can be detected and modified. The differences between heat-, high-pressure-, and denaturant-induced unfolding and aggregation are discussed. The effect of disulphide bonding between aggregates of proteins and protein mixtures on the functional macroscopic properties of space filling networks in protein gels is briefly presented.     

A protein oxidase catalysing disulfide bond formation is localized to the chloroplast thylakoids

In chloroplasts, thiol/disulfide-redox-regulated proteins have been linked to numerous metabolic pathways. However, the biochemical system for disulfide bond formation in chloroplasts remains undetermined. In the present study, we characterized an oxidoreductase, AtVKOR-DsbA, encoded by the gene At4g35760 as a potential disulfide bond oxidant in Arabidopsis. The gene product contains two distinct domains: an integral membrane domain homologous to the catalytic subunit of mammalian vitamin K epoxide reductase (VKOR) and a soluble DsbA-like domain. Transient expression of green fluorescent protein fusion in Arabidopsis protoplasts indicated that AtVKOR-DsbA is located in the chloroplast. The first 45 amino acids from the N-terminus were found to act as a transit peptide targeting the protein to the chloroplast. An immunoblot assay of chloroplast fractions revealed that AtVKOR-DsbA was localized in the thylakoid. A motility complementation assay showed that the full-length of AtVKOR-DsbA, if lacking its transit peptide, could catalyze the formation of disulfide bonds. Among the 10 cysteine residues present in the mature protein, eight cysteines (four in the AtVKOR domain and four in the AtDsbA domain) were found to be essential for promoting disulfide bond formation. The topological arrangement of AtVKOR-DsbA was assayed using alkaline phosphatase sandwich fusions. From these results, we developed a possible topology model of AtVKOR-DsbA in chloroplasts. We propose that the integral membrane domain of AtVKOR-DsbA contains four transmembrane helices, and that both termini and the cysteines involved in catalyzing the formation of disulfide bonds face the oxidative thylakoid lumen. These studies may help to resolve some of the issues surrounding the structure and function of AtVKOR-DsbA in Arabidopsis chloroplasts.