Pathways for protein disulphide bond formation

The folding of many secretory proteins depends upon the formation of disulphide bonds. Recent advances in genetics and cell biology have outlined a core pathway for disulphide bond formation in the endoplasmic reticulum (ER) of eukaryotic cells. In this pathway, oxidizing equivalents flow from the recently identified ER membrane protein Ero1p to secretory proteins via protein disulphide isomerase (PDI). Contrary to prior expectations, oxidation of glutathione in the ER competes with oxidation of protein thiols. Contributions of PDI homologues to the catalysis of oxidative folding will be discussed, as will similarities between eukaryotic and prokaryotic disulphide-bond-forming systems.     

Building bridges: disulphide bond formation in the cell

Disulphides are often vital for the folding and stability of proteins. Dedicated enzymatic systems have been discovered that catalyse the formation of disulphides in the periplasm of prokaryotes. These discoveries provide compelling evidence for the actual catalysis of protein folding in vivo. Disulphide bond formation in Escherichia coli is catalysed by at least three ‘Dsb’ proteins; DsbA, -B and -C. The DsbA protein has an extremely reactive, oxidizing disulphide which it simply donates directly to other proteins. DsbB is required for the reoxidation of DsbA. DsbC is active in disulphide rearrangements and appears to work synergistically with DsbA. The relative rarity of disulphides in cytoplasmic proteins appears to be dependent upon a disulphide-destruction machine. One pivotal cog in this machine is thioredoxin reductase.     

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.     

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.     

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.     

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.     

Catechol as a nucleophilic catalyst of peptide bond formation.

The aminolysis of a mildly activated aminoacid ester, benzyloxycarbonyl-L-phenylalanine cyanomethyl ester, by glycine esters in the presence of catechol has been studied as a model of catalysis by RNA cis-vicinal-diol systems in protein biosynthesis. Catechol accelerated the aminolysis, especially in the presence of bases, probably by nucleophilic catalysis.     

Intramolecular disulphide bond arrangements in nonhomologous proteins

The presence and location of intramolecular disulphide bonds are a key determinant of the structure and function of proteins. Intramolecular disulphide bonds in proteins have previously been analyzed under the assumption that there is no clear relationship between disulphide arrangement and disulphide concentration. To investigate this, a set of sequence nonhomologous protein chains containing one or more intramolecular disulphide bonds was extracted from the Protein Data Bank, and the arrangements of the bonds, Protein Data Bank header, and Structural Characterization of Proteins fold were analyzed as a function of intramolecular disulphide bond concentration. Two populations of intramolecular disulphide bond-containing proteins were identified, with a naturally occurring partition at 25 residues per bond. These populations were named intramolecular disulphide bond-rich and -poor. Benefits of partitioning were illustrated by three results: (1) rich chains most frequently contained three disulphides, explaining the plateaux in extant disulphide frequency distributions; (2) a positive relationship between median chain length and the number of disulphides, only seen when the data were partitioned; and (3) the most common bonding pattern for chains with three disulphide bonds was based on the most common for two, only when the data were partitioned. The two populations had different headers, folds, bond arrangements, and chain lengths. Associations between IDSB concentration, IDSB bonding pattern, loop sizes, SCOP fold, and PDB header were also found. From this, we found that intramolecular disulphide bond-rich and -poor proteins follow different bonding rules, and must be considered separately to generate meaningful models of bond formation.     

Overexpression of the rhodanese PspE, a single cysteine-containing protein, restores disulphide bond formation to an Escherichia coli strain lacking DsbA

Escherichia coli uses the DsbA/DsbB system for introducing disulphide bonds into proteins in the cell envelope. Deleting either dsbA or dsbB or both reduces disulphide bond formation but does not entirely eliminate it. Whether such background disulphide bond forming activity is enzyme-catalysed is not known. To identify possible cellular factors that might contribute to the background activity, we studied the effects of overexpressing endogenous proteins on disulphide bond formation in the periplasm. We find that overexpressing PspE, a periplasmic rhodanese, partially restores substantial disulphide bond formation to a dsbA strain. This activity depends on DsbC, the bacterial disulphide bond isomerase, but not on DsbB. We show that overexpressed PspE is oxidized to the sulphenic acid form and reacts with substrate proteins to form mixed disulphide adducts. DsbC either prevents the formation of these mixed disulphides or resolves these adducts subsequently. In the process, DsbC itself gets oxidized and proceeds to catalyse disulphide bond formation. Although this PspE/DsbC system is not responsible for the background disulphide bond forming activity, we suggest that it might be utilized in other organisms lacking the DsbA/DsbB system.     

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.     

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.     

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.     

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.     

Preparation of protein conjugates via intermolecular disulfide bond formation

Conjugates of two unlike proteins can be prepared via the intermolecular disulfide interchange reaction, namely, protein A containing thiol groups reacts with protein B containing 4-dithiopyridyl groups to yield a conjugate with the release of 4-thiopyridone. Thiol groups can be introduced into proteins upon amidination with methyl 3-mercaptopropionimidate ester or 2-iminothiolane, and 4-dithiopyridyl groups can be introduced into proteins with these same reagents in the presence of 4,4'-dithiodipyridine. 2-Iminothiolane is stable on storage in contrast to the known lability of imidate esters; therefore 2-iminothiolane is a more convenient reagent for the modification of protein than are the imidate esters. All the reactions can be carried out easily under mild conditions in good yields. Conjugates of bovine plasma albumin with itself, ribonuclease, or a copolymer of D-glutamic acid and D-lysine and of sheep antibody and horseradish peroxidase were prepared with modified proteins containing an average of 1 to 5 thiol or dithiopyridyl groups per mol. These conjugates formed mainly dimers, trimers, and tetramers. The peroxidase labeled antibody retained more than 80% of its enzymatic and antigenic binding activities.