The structure of hepadnaviral core antigens. Identification of free thiols and determination of the disulfide bonding pattern.

A set of wild-type and mutant human, woodchuck, and duck hepatitis viral core proteins have been prepared and used to study the free thiol groups and the disulfide bonding pattern present within the core particle. Human (HBcAg) and woodchuck (WHcAg) core proteins contain 4 cysteine residues, whereas duck (DHcAg) core protein contains a single cysteine residue. Each of the cysteines of HBcAg has been eliminated, either singly or in combinations, by a two-step mutagenesis procedure. All of the proteins were shown to have very similar physical and immunochemical properties. All assemble into essentially identical core particle structures. Therefore disulfide bonds are not essential for core particle formation. No intra-chain disulfide bonds occur. Cys107 is a free thiol buried within the particle structure, whereas Cys48 is present partly as a free sulfhydryl which is exposed at the surface of the particle. Cys61 is always and Cys48 is partly involved in interchain disulfide bonds with the identical residues of another monomer, whereas Cys183 is always involved in a disulfide bond with the Cys183 of a different monomer. WHcAg has the same pattern of bonding, whereas DHcAg lacks any disulfide bonds, and the single free sulfhydryl, Cys153 which is equivalent to Cys107 of HBcAg, is buried.     

Different susceptibility of inter- and intra-chain disulfide bonds to reductive cleavage in native fibronectin and effect of their cleavage on conformation

The two thread-like subunits (Mr approximately equal to 250 000) of the multidomain protein fibronectin are connected by a pair of inter-chain disulfide bridges in their C-terminal regions. In addition each chain contains 29 intra-chain disulfide bonds which are located in 12 type I and 2 type II structural domains in the N-terminal and C-terminal regions of the strands. The 15 to 17 type III domains in the central portion of the strands do not contain disulfide bonds. The susceptibility of inter-chain disulfide bonds to 10mM 1,4-dithiothreitol at pH 7.8 as quantitated by the rate of reductive cleavage of fibronectin into its subunits was found to be only 8-fold larger than that of the intra-chain bonds. Consequently at 90% completion of chain separation 30% of the intra-chain disulfides are also cleaved. The rate of inter-chain disulfide cleavage was found to be identical for fibronectin and a 140-kDa fragment comprising the C-terminal portions of the two subunits. This shows that the relatively high protection of the inter-chain disulfide bonds must originate from interactions between C-terminal domains which are probably also responsible for the V shaped arrangement of the two subunit strands. Changes of circular dichroism and thermal transition profiles for fibronectin and its C-terminal 140-kDa fragment indicated that already partial reduction of the intra-chain disulfide bonds alters the conformations of type I and II domains without affecting the type III domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interchain disulfide bond formation in types I and II procollagen. Evidence for a protein disulfide isomerase catalyzing bond formation

The assembly of reduced pro-alpha chains of type I and type II procollagen into the native triple-helical molecule was examined in vitro in the presence and absence of pure protein disulfide isomerase. The data clearly indicates that protein disulfide isomerase is able to accelerate the formation of native interchain disulfide bonds in these procollagens. It takes about 6 min after disulfide bonding before triple-helical molecules exist, while the time required to produce triple-helical type I procollagen in the presence of protein disulfide isomerase is 9.4 min and that for type II procollagen 17.2 min. These values agree with those obtained for type I and II procollagen in vivo suggesting that protein disulfide isomerase is also an enzyme catalyzing interchain disulfide bond formation in procollagen in vivo. The formation of native disulfide bonds can proceed without any enzyme catalysis but then requires the presence of reduced and oxidized glutathione. Bonding is rather slow in such a case, however, resulting in a delay in the formation of the triple helix.     

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.     

The refined crystal structure of alpha-cobratoxin from Naja naja siamensis at 2.4-A resolution

The crystal structure of the "long" alpha-neurotoxin alpha-cobratoxin was refined to an R-factor of 19.5% using 3271 x-ray data to 2.4-A resolution. The polypeptide chain forms three loops, I, II, III, knotted together by four disulfide bridges, with the most prominent, loop II, containing another disulfide close to its lower tip. Loop I is stabilized by one beta-turn and two beta-sheet hydrogen bonds; loop II by eight beta-sheet hydrogen bonds, with the tip folded into two distorted right-handed helical turns stabilized by two alpha-helical and two beta-turn hydrogen bonds; and loop III by hydrophobic interactions and one beta-turn. Loop II and one strand of loop III form an antiparallel triple-pleated beta-sheet, and tight anchoring of the Asn63 side chain fixes the tail segment. In the crystal lattice, the alpha-cobratoxin molecules dimerize by beta-sheet formation between strands 53 and 57 of symmetry-related molecules. Because such interactions are found also in a cardiotoxin and alpha-bungarotoxin, this could be of importance for interaction with acetylcholine receptor.     

The susceptibility of disulfide bonds to modification in keratin fibers undergoing tensile stress

Cysteine residues perform a dual role in mammalian hairs. The majority help stabilize the overall assembly of keratins and their associated proteins, but a proportion of inter-molecular disulfide bonds are assumed to be associated with hair mechanical flexibility. Hair cortical microstructure is hierarchical, with a complex macro-molecular organization resulting in arrays of intermediate filaments at a scale of micrometres. Intermolecular disulfide bonds occur within filaments and between them and the surrounding matrix. Wool fibers provide a good model for studying various contributions of differently situated disulfide bonds to fiber mechanics. Within this context, it is not known if all intermolecular disulfide bonds contribute equally, and, if not, then do the disproportionally involved cysteine residues occur at common locations on proteins? In this study, fibers from Romney sheep were subjected to stretching or to their breaking point under wet or dry conditions to detect, through labeling, disulfide bonds that were broken more often than randomly. We found that some cysteines were labeled more often than randomly and that these vary with fiber water content (water disrupts protein-protein hydrogen bonds). Many of the identified cysteine residues were located close to the terminal ends of keratins (head or tail domains) and keratin-associated proteins. Some cysteines in the head and tail domains of type II keratin K85 were labeled in all experimental conditions. When inter-protein hydrogen bonds were disrupted under wet conditions, disulfide labeling occurred in the head domains of type II keratins, likely affecting keratin-keratin-associated protein interactions, and tail domains of the type I keratins, likely affecting keratin-keratin interactions. In contrast, in dry fibers (containing more protein-protein hydrogen bonding), disulfide labeling was also observed in the central domains of affected keratins. This central “rod” region is associated with keratin-keratin interactions between anti-parallel heterodimers in the tetramer of the intermediate filament.     

Disulfide bond assignment of the Ebola virus secreted glycoprotein SGP

The non-structural glycoprotein (SGP) of Ebola virus (EboV) is secreted in large amounts from infected cells as a disulfide-linked homodimer. In this communication, highly purified SGP, derived from Vero E6 cultures infected with the Zaire species of EboV, was used to determine the correct localization of inter- and intrachain disulfide bonds. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis of proteolytic cleavage fragments indicates that all cysteines (six per monomeric unit) form unique disulfide bonds. Monomers of the SGP homodimer are joined in a parallel manner by two intersubunit disulfide bonds formed between paired N-terminal and C-terminal cysteines (C53-C53' and C306-C306'). The remaining cysteines are involved in intrachain disulfide bonding (paired as C108-C135 and C121-C147), which resembles the disulfide bond topology of fibronectin type II domains. The findings presented here provide the foundation for future studies aimed at defining the structural and functional properties of SGP.     

Insulin-like growth factors I and II are unable to form and maintain their native disulfides under in vivo redox conditions

Insulin-like growth factor (IGF) I does not quantitatively form its three native disulfide bonds in the presence of 10 mM reduced and 1 mM oxidized glutathione in vitro [Hober, S. et al. (1992) Biochemistry 31, 1749-1756]. In this paper, we show (i) that both IGF-I and IGF-II are unable to form and maintain their native disulfide bonds at redox conditions that are similar to the situation in the secretory vesicles in vivo and (ii) that the presence of protein disulfide isomerase does not overcome this problem. The results indicate that the previously described thermodynamic disulfide exchange folding problem of IGF-I in vitro is also present in vivo. Speculatively, we suggest that the thermodynamic disulfide exchange properties of IGF-I and II are biologically significant for inactivation of the unbound growth factors by disulfide exchange reactions to generate variants destined for rapid clearance.     

Small‐molecule reductants inhibit multicatalytic activity of AA‐NADase from Agkistrodon acutus venom by reducing the disulfide‐bonds and Cu(II) of enzyme

AA‐NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA‐NADase contains Cu(II) and has disulfide‐bond linkages between two peptide chains. The effects of the reduction of the disulfide‐bonds and Cu(II) in AA‐NADase by small‐molecule reductants on its NADase and ADPase activities have been investigated by polyacrylamide gel electrophoresis, high performance liquid chromatography, electron paramagnetic resonance spectroscopy and isothermal titration calorimetry. The results show that AA‐NADase has six disulfide‐bonds and fifteen free cysteine residues. L‐ascorbate inhibits AA‐NADase on both NADase and ADPase activities through the reduction of Cu(II) in AA‐NADase to Cu(I), while other reductants, dithiothreitol, glutathione and tris(2‐carboxyethyl)phosphine inhibit both NADase and ADPase activities through the reduction of Cu(II) to Cu(I) and the cleavage of disulfide‐bonds in AA‐NADase. Apo‐AA‐NADase can recover its NADase and ADPase activities in the presence of 1 mM Zn(II). However, apo‐AA‐NADase does not recover any NADase or ADPase activity in the presence of 1 mM Zn(II) and 2 mM TCEP. The multicatalytic activity relies on both disulfide‐bonds and Cu(II), while Cu(I) can not activate the enzyme activities. AA‐NADase is probably only active as a dimer. The inhibition curves for both ADPase and NADase activities by each reductant share a similar trend, suggesting both ADPase and NADase activities probably occur at the same site. In addition, we also find that glutathione and L‐ascorbate are endogenous inhibitors to the multicatalytic activity of AA‐NADase. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 141–149, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Conformational analysis of two cyclic disulfide peptides

Complete nmr and CD studies of two cyclic tetrapeptides with disulfide bonds, Ac-L-Pen-L-Pro-D-Val-L-Cys-NH2 (1) and Ac-L-Cys-L-Pro-D-Val-L-Cys-NH2 (2) bonds have been carried out in different solvents to investigate the formation and stabilization of beta-turn structures and to determine the stereochemistry of the disulfide linkage. Both peptides have three-dimensional structures with a type II beta-turn, as derived from quantitative nuclear Overhauser effect data. The combined use of CD and nmr indicates that the dihedral angle of the disulfide bridge is different in the two peptides, although the chirality is maintained.     

Role of disulfide bonds in modulating internal motions of proteins to tune their function: molecular dynamics simulation of scorpion toxin Lqh III

A series of 1-ns MD simulations were performed on the scorpion toxin Lqh III in native and disulfide bond broken states. The removal of disulfide bonds has caused hydrogen bond network alteration in the five-residue turn, the long loop, the alpha-helix, the loop connecting strands II and III, and the C-terminal region. In addition and more importantly, it has influenced the amplitude of the fluctuations of five-residue turn, loops, and C-terminal region with a minor effect on the fluctuations of the cysteines in the broken bond sites. These findings suggest that disulfide bonds are not the most important factors in rigidifying their own locations, while they have more important effects at a global scale. Furthermore, our results reveal that disulfide bonds have considerable influence on the functionally important essential modes of motions and the correlations between the motions of the binding site residues. Therefore, we can conclude that disulfide bonds have a crucial role in modulating the function via adjusting the dynamics of scorpion toxin molecules. Although this conclusion cannot be generalized to all peptides and proteins, it demonstrates the importance of more investigations on this aspect of disulfide bond efficacy.     

鸭β-防御素5基因的分离、鉴定及其生物学作用

为克隆与表达鸭β-防御素5 (AvBD5)基因及测定其生物学特性,采用RT-PCR方法从鸭肺脏组织中扩增到鸭AvBD5,将测得的序列与已发现的禽β-防御素和部分哺乳类动物β-防御素的氨 基酸序列构建进化树进行同源性分析.结果显示鸭AvBD5 cDNA大小为201 bp,编码66个氨基酸残基,内含6个位置保守的半胱...     

The arrangement of intra- and intermolecular disulfide bonds in the carboxyterminal, non-collagenous aggregation and cross-linking domain of basement-membrane type IV collagen

The hexameric complex of globular domains of type IV collagen was isolated after collagenase digestion of human placenta and the different monomers and dimers present were chromatographically separated. The ratio of alpha 1(IV)NC1 to alpha 2(IV)NC1 was 2:1. About 50% of the NC1 domains were connected to dimers. Predominantly alpha 1-alpha 1 dimers were found. Only 12% were alpha 2-alpha 2 dimers and no alpha 1-alpha 2 dimers could be detected. The majority (88%) of the intermolecular bonds was found to be disulfide bridges. The remainder could not be cleaved by reduction. To elucidate the arrangement of the disulfide bonds, the unreduced alpha 1(IV)NC1 monomers were treated with cyanogen bromide, the disulfide-bridged peptides isolated and characterized by Edman degradation. Each of the two homologous subdomains within a monomer is stabilized by an identical set of three disulfide bonds. In subdomain I, cysteines at positions 20 and 53 are connected with the C-terminal cysteine pair 108 and 111. Thus formed, the disulfide knot stabilizes two interconnected loops of 32 and 54 residues, respectively. A smaller loop of five residues occurs due to a disulfide bond between the cysteines 65 and 71. A similar disulfide arrangement is indicated for subdomain II which is separated from subdomain I by a segment of 20 amino acid residues. The same arrangement of disulfide bonds has been strongly suggested for the alpha 2(IV)NC1 monomer by the isolation and characterization of its disulfide-bridged tryptic fragments. Similar investigations on the dimeric alpha 1(IV)NC1 domain established the arrangement of the intermolecular disulfide bonds. They are formed by a complete disulfide exchange between corresponding disulfide knots of two monomeric NC1 domains.     

Structural and functional relationships in two families of beta-1,4-glycanases.

CenA and Cex are beta-1,4-glycanases produced by the cellulolytic bacterium Cellulomonas fimi. Both enzymes are composed of two domains and contain six Cys residues. Two disulfide bonds were assigned in both enzymes by peptide analysis of the isolated catalytic domains. A further disulfide bond was deduced in both cellulose-binding domains from the absence of free thiols under denaturing conditions. Corresponding Cys residues are conserved in eight of nine other known C. fimi-type cellulose-binding domains. CenA and Cex belong to families B and F, respectively, in the classification of beta-1,4-glucanases and beta-1,4-xylanases based on similarities in catalytic domain primary structure. Disulfide bonds in the CenA catalytic domain correspond to the two disulfide bonds in the catalytic domain of Trichoderma reesei cellobiohydrolase II (family B) which stabilize loops forming the active-site tunnel. Sequence alignment indicates the probable occurrence of disulfides at equivalent positions in the two other family B enzymes. Partial resequencing of the gene encoding Streptomyces KSM-9 beta-1,4-glucanase CasA (family B) revealed five errors in the original nucleotide sequence analysis. The corrected amino acid sequence contains an Asp residue corresponding to the proposed proton donor in hydrolysis catalysed by cellobiohydrolase II. Cys residues which form disulfide bonds in the Cex catalytic domain are conserved in XynZ of Clostridium thermocellum and Xyn of Cryptococcus albidus but not in the other eight known family F enzymes. Like other members of its family, Cex catalyses xylan hydrolysis. The catalytic efficiency (kcat/Km) for hydrolysis of the heterosidic bond of p-nitrophenyl-beta-D-xylobioside is 14,385 min-1.mM-1 at 25 degrees C; the corresponding kcat/Km for p-nitrophenyl-beta-D-cellobioside hydrolysis is 296 min-1.mM-1.     

Unique disulfide bond structures found in ST8Sia IV polysialyltransferase are required for its activity

NCAM polysialylation plays a critical role in neuronal development and regeneration. Polysialylation of the neural cell adhesion molecule (NCAM) is catalyzed by two polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST), which contain sialylmotifs L and S conserved in all members of the sialyltransferases. The members of the ST8Sia gene family, including ST8Sia II and ST8Sia IV are unique in having three cysteines in sialylmotif L, one cysteine in sialylmotif S, and one cysteine at the COOH terminus. However, structural information, including how disulfide bonds are formed, has not been determined for any of the sialyltransferases. To obtain insight into the structure/function of ST8Sia IV, we expressed human ST8Sia IV in insect cells, Trichoplusia ni, and found that the enzyme produced in the insect cells catalyzes NCAM polysialylation, although it cannot polysialylate itself ("autopolysialylation"). We also found that ST8Sia IV does not form a dimer through disulfide bonds. By using the same enzyme preparation and performing mass spectrometric analysis, we found that the first cysteine in sialylmotif L and the cysteine in sialylmotif S form a disulfide bridge, whereas the second cysteine in sialylmotif L and the cysteine at the COOH terminus form a second disulfide bridge. Site-directed mutagenesis demonstrated that mutation at cysteine residues involved in the disulfide bridges completely inactivated the enzyme. Moreover, changes in the position of the COOH-terminal cysteine abolished its activity. By contrast, the addition of green fluorescence protein at the COOH terminus of ST8Sia IV did not render the enzyme inactive. These results combined indicate that the sterical structure formed by intramolecular disulfide bonds, which bring the sialylmotifs and the COOH terminus within close proximity, is critical for the catalytic activity of ST8Sia IV.     

The vitelline envelope of eggs from the giant keyhole limpet Megathura crenulata. II. Products formed by lysis with sperm enzymes and dithiothreitol.

The egg vitelline envelope of the marine invertebrate, Megathura crenulata, was lyzed either by sperm lysins A, B, C or by dithiothreitol. In each case the lysis mixture consisted of two major fractions, I and II, that could be separated by hydroxylapatite chromatography and had different electrophoretic mobilities on cellulose acetate strips. The amino acid, amino sugar, and neutral sugar compositions of fractions I and II were similar and resembled that of the intact vitelline envelope. Fractions I and II of each lysis mixture emerged in the exclusion volume of a Sepharose 6B column. A vitelline envelope fragment enzymatically formed by lysin was further degraded by dithiothreitol to form smaller fragments. A model of the vitelline envelope of the Megathura crenulata egg is suggested whereby the envelope is composed of polypeptide chains cross-linked by disulfide bonds and built to a large extent of closely spaced threonine residues. Most of the threonine residues are linked to carbohydrate units. Dithiothreitol dissolves the envelope by reducing disulfide bonds, whereas lysins most likely dissolve the envelope by degrading polypeptide chains.     

Reversible inactivation of AT(2) angiotensin II receptor from cysteine-disulfide bond exchange

Dithiothreitol (DTT) treatment of angiotensin II (Ang II) type 2 (AT(2)) receptor potentiates ligand binding, but the underlying mechanism is not known. Two disulfide bonds proposed in the extracellular domain were examined in this report. Based on the analysis of ligand affinity of cysteine (Cys, C) to alanine (Ala, A) substitution mutants, we provide evidence that Cys(35)-Cys(290) and Cys(117)-Cys(195) disulfide bonds are formed in the wild-type AT(2) receptor. Disruption of the highly conserved Cys(117)-Cys(195) disulfide bond linking the second and third extracellular segments leads to inactivation of the receptor. The Cys(35)-Cys(290) bond is highly sensitive to DTT. Its breakage results in an increased binding affinity for both Ang II and the AT(2) receptor-specific antagonist PD123319. Surprisingly, in the single Cys mutants, C35A and C290A, a labile population of receptors is produced which can be re-folded to high-affinity state by DTT treatment. These results suggest that the free -SH group of Cys(35) or Cys(290) competes with the disulfide bond formation between Cys(117) and Cys(195). This Cys-disulfide bond exchange results in production of the inactive population of the mutant receptors through formation of a non-native disulfide bond.     

Selective Removal of Individual Disulfide Bonds within a Potato Type II Serine Proteinase Inhibitor from Nicotiana alata Reveals Differential Stabilization of the Reactive-Site Loop

The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) belongs to the potato type II family also known as the PinII family of proteinase inhibitors, one of the major families of canonical proteinase inhibitors. T1 contains four disulfide bonds, two of which (C4-C41 and C8-C37) stabilize the reactive-site loop. To investigate the influence of these two disulfide bonds on the structure and function of potato II inhibitors, we constructed two variants of T1, C4A/C41A-T1 and C8A/C37A-T1, in which these two disulfide bonds were individually removed and replaced by alanine residues. Trypsin inhibition assays show that wild-type T1 has a K_i of < 5 nM, C4A/C41A-T1 has a weaker K_i of ∼ 350 nM, and the potency of the C8A/C37A variant is further decreased to a K_i of ∼ 1.8 μM. To assess the influence of the disulfide bonds on the structure of T1, we determined the structure and dynamics of both disulfide variants by NMR spectroscopy. The structure of C4A/C41A-T1 and the amplitude of intrinsic flexibility in the reactive-site loop resemble that of the wild-type protein closely, despite the lack of the C4-C41 disulfide bond, whereas the timescale of motions is markedly decreased. The rescue of the structure despite loss of a disulfide bond is due to a previously unrecognized network of interactions, which stabilizes the structure of the reactive-site loop in the region of the missing disulfide bond, while allowing intrinsic motions on a fast (picosecond-nanosecond) timescale. In contrast, no comparable interactions are present around the C8-C37 disulfide bond. Consequently, the reactive-site loop becomes disordered and highly flexible in the structure of C8A/C37A-T1, making it unable to bind to trypsin. Thus, the reactive-site loop of T1 is stabilized differently by the C8-C37 and C4-C41 disulfide bonds. The C8-C37 disulfide bond is essential for the inhibitory activity of T1, whereas the C4-C41 disulfide bond is not as critical for maintaining the three-dimensional structure and function of the molecule but is responsible for maintaining flexibility of the reactive-site loop on a microsecond-nanosecond timescale.     

Gamma-interferon-inducible lysosomal thiol reductase (GILT). Maturation, activity, and mechanism of action

We recently identified a gamma-interferon-inducible lysosomal thiol reductase (GILT), constitutively expressed in antigen-presenting cells, that catalyzes disulfide bond reduction both in vitro and in vivo and is optimally active at acidic pH. GILT is synthesized as a 35-kDa precursor, and following delivery to major histocompatibility complex (MHC) class II-containing compartments (MIICs), is processed to the mature 30-kDa form via cleavage of N- and C-terminal propeptides. The generation of MHC class II epitopes requires both protein denaturation and reduction of intra- and inter-chain disulfide bonds prior to proteolysis. GILT may be important in disulfide bond reduction of proteins delivered to MIICs and consequently in antigen processing. In this report we show that, like its mature form, precursor GILT reduces disulfide bonds with an acidic pH optimum, suggesting that it may also be involved in disulfide bond reduction in the endocytic pathway. We also show that processing of precursor GILT can be mediated by multiple lysosomal proteases and provide evidence that the mechanism of action of GILT resembles that of other thiol oxidoreductases.     

The nonconsecutive disulfide bond of Escherichia coli phytase (AppA) renders it dependent on the protein-disulfide isomerase, DsbC

The formation of protein disulfide bonds in the Escherichia coli periplasm by the enzyme DsbA is an inaccurate process. Many eukaryotic proteins with nonconsecutive disulfide bonds expressed in E. coli require an additional protein for proper folding, the disulfide bond isomerase DsbC. Here we report studies on a native E. coli periplasmic acid phosphatase, phytase (AppA), which contains three consecutive and one nonconsecutive disulfide bonds. We show that AppA requires DsbC for its folding. However, the activity of an AppA mutant lacking its nonconsecutive disulfide bond is DsbC-independent. An AppA homolog, Agp, a periplasmic acid phosphatase with similar structure, lacks the nonconsecutive disulfide bond but has the three consecutive disulfide bonds found in AppA. The consecutively disulfide-bonded Agp is not dependent on DsbC but is rendered dependent by engineering into it the conserved nonconsecutive disulfide bond of AppA. Taken together, these results provide support for the proposal that proteins with nonconsecutive disulfide bonds require DsbC for full activity and that disulfide bonds are formed predominantly during translocation across the cytoplasmic membrane.