Disulfide bond formation and secretion of Escherichia coli heat-stable enterotoxin II.

The Escherichia coli heat-stable enterotoxin II (STII) is a typical extracellular toxin consisting of 48 amino acid residues, of which 4 are cysteine. There are two disulfide bonds, one between Cys-10 and Cys-48 and one between Cys-21 and Cys-36. We examined the involvement of DsbA in the formation of the disulfide bonds of STII and the role of each in the secretion of STII. A dsbA mutant was transformed with a plasmid harboring the STII gene, and STII was not detected either in the cells or in the culture supernatant. Reducing the level of STII brought about the dsbA mutation restored by introducing the wild-type dsbA gene into the mutant strain. These results showed that DsbA is involved in forming the disulfide bonds of STII and that STII without these disulfide bonds is degraded during secretion. We substituted these four cysteine residues in vivo by oligonucleotide-directed site-specific mutagenesis. The amino acid sequence of the purified STII (C48S) and pulse-chase studies revealed that two intermolecular disulfide bonds must be formed to be efficiently secreted and that cleavage between amino acid residues 14 and 15 is probably the first step in the proteolytic degradation of STII.     

Purification and characterization of Escherichia coli heat-stable enterotoxin II.

Escherichia coli heat-stable enterotoxin II (STII) was purified to homogeneity by successive column chromatographies from the culture supernatant of a strain harboring the plasmid encoding the STII gene. The purified STII evoked a secretory response in the suckling mouse assay and ligated rat intestinal loop assay in the presence of protease inhibitor, but the response was not observed in the absence of the inhibitor. Analyses of the peptide by the Edman degradation method and fast atom bombardment mass spectrometry revealed that purified STII is composed of 48 amino acid residues and that its amino acid sequence was identical to the 48 carboxy-terminal amino acids of STII predicted from the DNA sequence (C. H. Lee, S. L. Mosely, H. W. Moon, S. C. Whipp, C. L. Gyles, and M. So, Infect. Immun. 42:264-268, 1983). STII has four cysteine residues which form two intramolecular disulfide bonds. Two disulfide bonds were determined to be formed between Cys-10-Cys-48 and Cys-21-Cys-36 by analyzing tryptic hydrolysates of STII.     

Maturation of Pseudomonas aeruginosa elastase. Formation of the disulfide bonds

Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme. After propeptide-mediated folding in the periplasm, the proenzyme is autoproteolytically processed, prior to translocation of both the mature enzyme and the propeptide across the outer membrane. The formation of the two disulfide bonds present in the mature enzyme was examined by studying the expression of the wild-type enzyme and of alanine for cysteine mutant derivatives in the authentic host and in dsb mutants of Escherichia coli. It appeared that the two disulfide bonds are formed successively. First, DsbA catalyzes the formation of the disulfide bond between Cys-270 and Cys-297 within the proenzyme. This step is essential for the subsequent autoproteolytic processing to occur. The second disulfide bond between Cys-30 and Cys-57 is formed more slowly and appears to be formed after processing of the proenzyme, and its formation is catalyzed by DsbA as well. This second disulfide bond appeared to be required for the full proteolytic activity of the enzyme and contributes to its stability.     

Maturation pathway of Escherichia coli heat-stable enterotoxin I: requirement of DsbA for disulfide bond formation.

The Escherichia coli heat-stable enterotoxin STp is synthesized as a precursor consisting of pre, pro and mature regions. Mature STp is released into the culture supernatant and is composed of 18-amino-acid resides which contain three intramolecular disulfide bonds. The involvement of DsbA in the formation of the disulfide bonds of STp was examined in this study. A dsbA mutant was transformed with a plasmid harboring the STp gene, and the ST activity was significantly lower than that of the parent strain harboring the same plasmid. Furthermore, purified DsbA induced the conversion of synthetic STp peptide (inactive form) to the active form and increased the ST activity of the culture supernatant derived from the dsbA transformants. These results showed that DsbA directly catalyzes the formation of the disulfide bonds of STp. DsbA is located in periplasmic space, where STp is released as an intermediate form consisting of pro and mature regions. To examine the effect of the pro region on the action of DsbA, we replaced the cysteine residue at position 39 and tested the effect in vivo. The substitution caused a significant decrease of ST activity in the culture supernatant, the accumulation of inactive ST in periplasmic space, and an alteration in the cleavage site of the intermediate of STp. We conclude that Cys-39 is important for recognition by the processing enzymes required for the maturation of STp.     

MacAB is involved in the secretion of Escherichia coli heat-stable enterotoxin II

The heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli is an extracellular peptide toxin that evokes watery diarrhea in the host. Two types of STs, STI and STII, have been found. Both STs are synthesized as precursor proteins and are then converted to the active forms with intramolecular disulfide bonds after being released into the periplasm. The active STs are finally translocated across the outer membrane through a tunnel made by TolC. However, it is unclear how the active STs formed in the periplasm are led to the TolC channel. Several transporters in the inner membrane and their periplasmic accessory proteins are known to combine with TolC and form a tripartite transport system. We therefore expect such transporters to also act as a partner with TolC to export STs from the periplasm to the exterior. In this study, we carried out pulse-chase experiments using E. coli BL21(DE3) mutants in which various transporter genes (acrAB, acrEF, emrAB, emrKY, mdtEF, macAB, and yojHI) had been knocked out and analyzed the secretion of STs in those strains. The results revealed that the extracellular secretion of STII was largely decreased in the macAB mutant and the toxin molecules were accumulated in the periplasm, although the secretion of STI was not affected in any mutant used in this study. The periplasmic stagnation of STII in the macAB mutant was restored by the introduction of pACYC184, containing the macAB gene, into the cell. These results indicate that MacAB, an ATP-binding cassette transporter of MacB and its accessory protein, MacA, participates in the translocation of STII from the periplasm to the exterior. Since it has been reported that MacAB cooperates with TolC, we propose that the MacAB-TolC system captures the periplasmic STII molecules and exports the toxin molecules to the exterior.     

Functional role of two interhelical disulfide bonds in human Cox17 protein from a structural perspective

Human Cox17 is the mitochondrial copper chaperone responsible for supplying copper ions, through the assistance of Sco1, Sco2, and Cox11, to cytochrome c oxidase, the terminal enzyme of the mitochondrial energy-transducing respiratory chain. It consists of a coiled coil-helix-coiled coil-helix domain stabilized by two disulfide bonds and binds one copper(I) ion through a Cys-Cys motif. Here, the structures and the backbone mobilities of two Cox17 mutated forms with only one interhelical disulfide bond have been analyzed. It appears that the inner disulfide bond (formed by Cys-36 and Cys-45) stabilizes interhelical hydrophobic interactions, providing a structure with essentially the same structural dynamic properties of the mature Cox17 state. On the contrary, the external disulfide bond (formed by Cys-26 and Cys-55) generates a conformationally flexible α-helical protein, indicating that it is not able to stabilize interhelical packing contacts, but is important for structurally organizing the copper-binding site region.     

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.     

On the role of the cis-proline residue in the active site of DsbA

In addition to the Cys-Xaa-Xaa-Cys motif at position 30-33, DsbA, the essential catalyst for disulfide bond formation in the bacterial periplasm shares with other oxidoreductases of the thioredoxin family a cis-proline in proximity of the active site residues. In the variant DsbA(P151A), this residue has been changed to an alanine, an almost isosteric residue which is not disposed to adopt the cis conformation. The substitution strongly destabilized the structure of DsbA, as determined by the decrease in the free energy of folding. The pKa of the thiol of Cys30 was only marginally decreased. Although in vivo the variant appeared to be correctly oxidized, it exhibited an activity less than half that of the wild-type enzyme with respect to the folding of alkaline phosphatase, used as a reporter of the disulfide bond formation in the periplasm. DsbA(P151A) crystallized in a different crystal form from the wild-type protein, in space group P2(1) with six molecules in the asymmetric unit. Its X-ray structure was determined to 2.8 A resolution. The most significant conformational changes occurred at the active site. The loop 149-152 adopted a new backbone conformation with Ala151 in a trans conformation. This rearrangement resulted in the loss of van der Waals interactions between this loop and the disulfide bond. His32 from the Cys-Xaa-Xaa-Cys sequence presented in four out of six molecules in the asymmetric unit a gauche conformation not observed in the wild-type protein. The X-ray structure and folding studies on DsbA(P151A) were consistent with the cis-proline playing a major role in the stabilization of the protein. A role for the positioning of the substrate is discussed. These important properties for the enzyme function might explain the conservation of this residue in DsbA and related proteins possessing the thioredoxin fold.     

Site-directed mutagenesis of cysteine residues in the pro region of the transforming growth factor beta 1 precursor. Expression and characterization of mutant proteins

Three cysteine residues are located in the pro region of the transforming growth factor beta 1 (TGF-beta 1) precursor at amino acid positions 33, 223, and 225. Previous studies (Gentry, L. E., Lioubin, M. N., Purchio, A. F., and Marquardt, H. (1988) Mol. Cell. Biol. 8, 4162-4168) with purified recombinant TGF-beta 1 (rTGF-beta 1) precursor produced by Chinese hamster ovary (CHO) cells revealed that Cys-33 can form a disulfide bond with at least 1 cysteine residue in mature TGF-beta 1, contributing to the formation of a 90-110-kDa protein. We now show that Cys-223 and Cys-225 form interchain disulfide bonds. Site-directed mutagenesis was used to change these Cys codons to Ser codons, and mutant constructs were transfected into COS cells. Analysis of recombinant proteins by immunoblotting showed that by substituting Cys-33 the 90-110-kDa protein is not formed, and thus, more mature dimer (24 kDa) is obtained, corresponding to a 3- to 5-fold increase in biological activity. Substitution of Cys-223 and/or Cys-225 resulted in near wild-type levels of mature TGF-beta 1. Furthermore, cells transfected with plasmid coding for Ser at positions 223 and 225 expressed only monomeric precursor proteins and released bioactive TGF-beta 1 that did not require acid activation, suggesting that dimerization of the precursor pro region may be necessary for latency.     

DsbB is required for the pathogenesis process of Xanthomonas campestris pv. campestris

The DsbA/DsbB oxidation pathway is one of the two pathways that catalyze disulfide bond formation of proteins in the periplasm of gram-negative bacteria. It has been demonstrated that DsbA is essential for multiple virulence factors of several animal bacterial pathogens. In this article, we present genetic evidence to show that the open reading frame XC_3314 encodes a DsbB protein that is involved in disulfide bond formation in periplasm of Xanthomonas campestris pv. campestris, the causative agent of crucifer black rot disease. The dsbB mutant of X. campestris pv. campestris exhibited attenuation in virulence, hypersensitive response, cell motility, and bacterial growth in planta. Furthermore, mutation in the dsbB gene resulted in ineffective type II and type III secretion systems as well as flagellar assembly. These findings reveal that DsbB is required for the pathogenesis process of X. campestris pv. campestris.     

Extracellular secretion of Escherichia coli heat-stable enterotoxin I across the outer membrane.

Escherichia coli heat-stable enterotoxin Ip (STIp) is an extracellular toxin consisting of 18 amino acid residues that is synthesized as a precursor of pre (amino acid residues 1 to 19), pro (amino acid residues 20 to 54), and mature (amino acid residues 55 to 72) regions. The precursor synthesized in the cytoplasm is translocated across the inner membrane by the general export pathway consisting of Sec proteins. The pre region functions as a leader peptide and is cleaved during translocation. However, it remains unknown how the resulting peptide (pro-mature peptide) translocates across the outer membrane. In this study, we investigated the structure of the STIp that passes through the outer membrane to determine how it translocates through the outer membrane. The results showed that the pro region is cleaved in the periplasmic space. The generated peptide becomes the mature form of STIp, which happens to have disulfide bonds, which then passes through the outer membrane. We also showed that STIp with a carboxy-terminal peptide consisting of 3 amino acid residues passes through the outer membrane, whereas STIp with a peptide composed of 37 residues does not. Amino acid analysis of mutant STIp purified from culture supernatant revealed that the peptide composed of 37 amino acid residues was cleaved into fragments of 5 amino acid residues. In addition, analyses of STIps with a mutation at the cysteine residue and the dsbA mutant strain revealed that the formation of an intramolecular disulfide bond within STIp is not absolutely required for the mature region of STIp to pass through the outer membrane.     

大肠杆菌DsbA蛋白的氧化还原性质和构象变化

DsbA蛋白是大肠杆菌周质空间内的巯基 /二硫键氧化酶 ,主要催化底物蛋白质二硫键的形成。利用定点突变结合色氨酸类似物标记技术 ,研究了DsbA蛋白的氧化还原性质和构象变化。结果显示 :(1 )DsbA蛋白的还原态比氧化态的结构更加稳定 ,说明DsbA的强氧化性来源于氧化态构象的紧张状态 ;(2 )DsbA氧化和还原态间特殊的荧光变化主要来源于Trp76在不同状态间微观环境的差异 ;(3 )色氨酸类似物标记不会对DsbA蛋白的结构和功能产生明显的影响 ,利用1 9F NMR进一步证实了DsbA氧化还原状态间的构象变化 ,而且这种变化主要影响Trp76的局部环境 ,而对Trp1 2 6的局部环境没有太大的影响     

大肠杆菌二硫键形成蛋白A（DsbA）研究进展

二硫键形成蛋白A(DisulfidebondformationproteinA,DsbA)是存在于大肠杆菌周质胞腔内的一种参与新生蛋白质折叠过程中催化二硫键形成的折叠酶。综述了DsbA三维结构、进化过程、协助蛋白质体内外复性方面的研究进展。DsbA比硫氧还原蛋白具有更强的氧化性,其强氧化性来自于Cys30残基异常低的pKa值和不稳定的氧化型结构,通过定点突变的研究表明了Cys30残基是DsbA活性中心最关键的氨基酸残基之一。DsbA不论在体内与目标蛋白融合表达还是在体外以折叠酶形式添加,都能有效地催化蛋白质的折叠复性,同时DsbA还具有部分分子伴侣的活性。     

Preferential binding of an unfolded protein to DsbA.

The oxidoreductase DsbA from the periplasm of escherichia coli introduces disulfide bonds into proteins at an extremely high rate. During oxidation, a mixed disulfide is formed between DsbA and the folding protein chain, and this covalent intermediate reacts very rapidly either to form the oxidized protein or to revert back to oxidized DsbA. To investigate its properties, a stable form of the intermediate was produced by reacting the C33A variant of DsbA with a variant of RNase T1. We find that in this stable mixed disulfide the conformational stability of the substrate protein is decreased by 5 kJ/mol, whereas the conformational stability of DsbA is increased by 5 kJ/mol. This reciprocal effect suggests strongly that DsbA interacts with the unfolded substrate protein not only by the covalent disulfide bond, but also by preferential non-covalent interactions. The existence of a polypeptide binding site explains why DsbA oxidizes protein substrates much more rapidly than small thiol compounds. Such a very fast reaction is probably important for protein folding in the periplasm, because the accessibility of the thiol groups for DsbA can decrease rapidly when newly exported polypeptide chains begin to fold.     

Complementation of DsbA deficiency with secreted thioredoxin variants reveals the crucial role of an efficient dithiol oxidant for catalyzed protein folding in the bacterial periplasm.

The thiol/disulfide oxidoreductase DsbA is the strongest oxidant of the thioredoxin superfamily and is required for efficient disulfide bond formation in the periplasm of Escherichia coli. To determine the importance of the redox potential of the final oxidant in periplasmic protein folding, we have investigated the ability of the most reducing thiol/disulfide oxidoreductase, E.coli thioredoxin, of complementing DsbA deficiency when secreted to the periplasm. In addition, we secreted thioredoxin variants with increased redox potentials as well as the catalytic a-domain of human protein disulfide isomerase (PDI) to the periplasm. While secreted wild-type thioredoxin and the most reducing thioredoxin variant could not replace DsbA, all more oxidizing thioredoxin variants as well as the PDI a-domain could complement DsbA deficiency in a DsbB-dependent manner. There is an excellent agreement between the activity of the secreted thioredoxin variants in vivo and their ability to oxidize polypeptides fast and quantitatively in vitro. We conclude that the redox potential of the direct oxidant of folding proteins and in particular its reactivity towards reduced polypeptides are crucial for efficient oxidative protein folding in the bacterial periplasm.     

Fluorescence quenching in the DsbA protein from Escherichia coli: complete picture of the excited-state energy pathway and evidence for the reshuffling dynamics of the microstates of tryptophan

The disulfide oxidoreductase DsbA is a strong oxidant of protein thiols and is required for efficient disulfide bond formation in the bacterial periplasm. DsbA contains two tryptophans: W76 and W126. The fluorescence of W76 changes upon reduction of the disulfide bridge, as analyzed previously (Hennecke et al., Biochemistry 1997;36:6391-6400). The fluorescence of W126 is highly quenched. The only two potential side chain quenchers are Q74 and N127, and these were replaced by alanine, resulting in a threefold increase in fluorescence intensity. The fluorescence intensity increase is not due to the removal of dynamic quenchers but to an increase in the population with the longest lifetime. In this report, the possibility of a change in the conformation of W126 is investigated theoretically by using molecular mechanics and dynamic simulations and experimentally by using a reaction with N-bromosuccinimide. This reacts preferably with the most exposed microstate of tryptophan, which is responsible for the longest lifetime. The simulations and the experimental results reveal that the amino acid replacements allow W126 to increase the population of its antiperpendicular conformation. The selectivity of the N-bromosuccinimide reaction allows the visualization of the reshuffling kinetics at exhausting reagent concentration. To the authors' knowledge, this is the first time that the kinetics of Trp population reshuffling have been measured.     

High-level expression and secretion of a lysine-containing analog of Escherichia coli heat-stable enterotoxin.

The mechanism of action of the heat-stable enterotoxin STa secreted from enterotoxigenic forms of Escherichia coli has remained elusive, in part due to a tedious, low-yield purification procedure. We report here a method for obtaining large amounts of a biologically active lysine-containing analog of STa. Initial attempts to express the toxin using an expression vector that did not encode a signal sequence resulted in no biologically active material being recovered either from lysed cells or as a secretory product. However, use of the secretion vector pJAL36, which contains the STII enterotoxin signal sequence, allowed large amounts of an STa derivative containing the additional sequence Ser-Thr-Lys at the amino terminus of the mature enterotoxin to be readily purified from culture supernatants. This enterotoxin analog, known as KSTa-1, was equal in biological and receptor binding activity to the native toxin STa. The lysine residue present in KSTa-1 promises to be useful as a reactive amino acid that is readily derivatized to allow coupling of the enterotoxin to supports for affinity chromatography and antigenic conjugates. Additionally, the insertion of the lysine residue carboxy terminal to the Ser-Thr sequence adds a reversible "handle" to the toxin sequence in that the Ser-Thr-Lys segment can be removed by treatment with trypsin, releasing the native form of STa.     

Conserved role of the linker alpha-helix of the bacterial disulfide isomerase DsbC in the avoidance of misoxidation by DsbB

In the bacterial periplasm the co-existence of a catalyst of disulfide bond formation (DsbA) that is maintained in an oxidized state and of a reduced enzyme that catalyzes the rearrangement of mispaired cysteine residues (DsbC) is important for the folding of proteins containing multiple disulfide bonds. The kinetic partitioning of the DsbA/DsbB and DsbC/DsbD pathways partly depends on the ability of DsbB to oxidize DsbA at rates >1000 times greater than DsbC. We show that the resistance of DsbC to oxidation by DsbB is abolished by deletions of one or more amino acids within the alpha-helix that connects the N-terminal dimerization domain with the C-terminal thioredoxin domain. As a result, mutant DsbC carrying alpha-helix deletions could catalyze disulfide bond formation and complemented the phenotypes of dsbA cells. Examination of DsbC homologues from Haemophilus influenzae, Pseudomonas aeruginosa, Erwinia chrysanthemi, Yersinia pseudotuberculosis, Vibrio cholerae (30-70% sequence identity with the Escherichia coli enzyme) revealed that the mechanism responsible for avoiding oxidation by DsbB is a general property of DsbC family enzymes. In addition we found that deletions in the linker region reduced, but did not abolish, the ability of DsbC to assist the formation of active vtPA and phytase in vivo, in a DsbD-dependent manner, revealing that interactions between DsbD and DsbC are also conserved.     

Mutations in the C-terminal part of insulin-like growth factor (IGF)-binding protein-1 result in dimer formation and loss of IGF binding capacity.

In an attempt to define domains in insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) that are involved in IGF binding, we subjected the carboxyl end of the coding region of IGFBP-1 cDNA to mutagenesis. Mutant cDNAs were isolated, characterized by sequencing, and cloned in an expression vector under control of the simian virus-40 (SV40) early promoter. The constructs were transfected into COS-1 cells, and the mutant proteins, secreted into the culture medium, were analyzed for IGF binding by ligand blotting. The results obtained show that deletion of the C-terminal 20 amino acids or introduction of frame-shifts in this region resulted in loss of IGF binding and for some mutants in the formation of dimeric IGFBP-1 molecules. These dimers are probably formed when cysteine-226 (Cys-226) is missing, and its putative partner is able to form intermolecular disulfide bonds. Site-directed mutagenesis demonstrated that most of the introduced point mutations in the C-terminal region did not affect IGF binding. Only mutation of Cys-226 to tyrosine completely abolished IGF binding, as did the introduction of a negatively charged amino acid in the vicinity of this residue. Again, dimers were observed, supporting that Cys-226 is essential for the conformation of IGFBP-1. In addition, our data suggest that an IGF-binding domain may be located in the vicinity of the intramolecular disulfide bond formed by Cys-226 and its putative partner.     

Effect of sequences of the active-site dipeptides of DsbA and DsbC on in vivo folding of multidisulfide proteins in Escherichia coli

We have examined the role of the active-site CXXC central dipeptides of DsbA and DsbC in disulfide bond formation and isomerization in the Escherichia coli periplasm. DsbA active-site mutants with a wide range of redox potentials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsbA promoter by integration of the respective alleles into the bacterial chromosome. The dsbA alleles gave significant differences in the yield of active murine urokinase, a protein containing 12 disulfides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA. No direct correlation between the in vitro redox potential of dsbA variants and the urokinase yield was observed. These results suggest that the active-site CXXC motif of DsbA can play an important role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential. However, under aerobic conditions, there was no significant difference among the DsbA mutants with respect to phenotypes depending on the oxidation of proteins with few disulfide bonds. The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined. A library of DsbC expression plasmids with the active-site dipeptide randomized was screened for mutants that have increased disulfide isomerization activity. A number of DsbC mutants that showed enhanced expression of a variant of human tissue plasminogen activator as well as mouse urokinase were obtained. These DsbC mutants overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse. Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial periplasmic space.