An intramolecular disulfide bond is required for the thermostability of methyl parathion hydrolase, OPHC2

OPHC2, a methyl parathion hydrolase (MPH) from Pseudomonas pseudoalcaligenes C2-1 (CGMCC 1150), can degrade a wide range of organophosphate pesticides. Compared with other MPHs, OPHC2 exhibits high thermostability. Its thermostability mechanism, however, remains unknown. In the present study, sequence analysis demonstrated that two cysteines (Cys110 and Cys146) exist in OPHC2, but not in other MPHs. The three-dimensional structural model of OPHC2 performed by computer-assisted homology modelling revealed a potential stacking network with residues Cys110 and Cys146, which probably formed an intramolecular disulfide bond. Furthermore, both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and thiol-titration analyses indicated that OPHC2 contains a disulfide bond. Substitution of the disulfide bond-forming cysteines with alanine, leucine or methionine residues substantially decreased the thermostability of OPHC2, suggesting that disulfide bond formation affects conformational stability. These results, combined with three-dimensional structural modelling, demonstrated that the formation of a C110-C146 disulfide bond may stabilise the conformation of OPHC2, contributing to its thermostability.     

Disulfide bond in Pseudomonas aeruginosa lipase stabilizes the structure but is not required for interaction with its foldase

Pseudomonas aeruginosa secretes a 29-kDa lipase which is dependent for folding on the presence of the lipase-specific foldase Lif. The lipase contains two cysteine residues which form an intramolecular disulfide bond. Variant lipases with either one or both cysteines replaced by serines showed severely reduced levels of extracellular lipase activity, indicating the importance of the disulfide bond for secretion of lipase through the outer membrane. Wild-type and variant lipase genes fused to the signal sequence of pectate lyase from Erwinia carotovora were expressed in Escherichia coli, denatured by treatment with urea, and subsequently refolded in vitro. Enzymatically active lipase was obtained irrespective of the presence or absence of the disulfide bond, suggesting that the disulfide bond is required neither for correct folding nor for the interaction with the lipase-specific foldase. However, cysteine-to-serine variants were more readily denatured by treatment at elevated temperatures and more susceptible to proteolytic degradation by cell lysates of P. aeruginosa. These results indicate a stabilizing function of the disulfide bond for the active conformation of lipase. This conclusion was supported by the finding that the disulfide bond function could partly be substituted by a salt bridge constructed by changing the two cysteine residues to arginine and aspartate, respectively.     

Efficient approach to synthesis of two-chain asymmetric cysteine analogs of receptor-binding region of transforming growth factor-alpha.

The putative receptor-binding region of human transforming growth factor-alpha (TGF alpha) has been shown to be contributed by two fragments: an A-chain (residue 12-18) and a 17-residue carboxyl fragment (residue 34-50) that includes a disulfide-containing C-loop (residue 34-43). An approach to the synthesis of two-chain analogs containing an intermolecular disulfide linked A-chain and the 17-residue carboxyl fragment (C-fragment) possessing receptor-binding activity is described. The synthesis was achieved by the solid-phase method using the Boc-benzyl protecting group strategy. The single Cys of the A-chain was activated as a mixed disulfide with 2-thiopyridine to form the intermolecular disulfide bond with Cys41 or Cys46 of the C-fragment on the resin support. Prior to this reaction, the acetamido (Acm) protecting group of Cys41 or Cys46 was removed by Hg(OAc)2 on the resin support. The peptide and side chain protecting groups including the S-methylbenzyl moiety of the Cys34 and Cys43 were concomitantly cleaved by high HF. The intramolecular disulfide with two unprotected Cys was formed in the presence of an intermolecular disulfide. This intramolecular disulfide bond formation was usually not feasible under the traditionally-held scheme at basic pH since disulfide interchange would occur faster than intramolecular oxidation. To prevent the disulfide interchange, a new method was devised. The intramolecular disulfide bond oxidation was mediated by dimethylsulfoxide at an acidic pH, at which the disulfide interchange reaction was suppressed. The desired product was obtained with a 60-70% yield.(ABSTRACT TRUNCATED AT 250 WORDS)     

Roles of the intramolecular disulfide bridge in MotX and MotY, the specific proteins for sodium-driven motors in Vibrio spp

The proteins PomA, PomB, MotX, and MotY are essential for the motor function of Na+-driven flagella in Vibrio spp. Both MotY and MotX have the two cysteine residues (one of which is in a conserved tetrapeptide [CQLV]) that are inferred to form an intramolecular disulfide bond. The cysteine mutants of MotY prevented the formation of an intramolecular disulfide bond, which is presumably important for protein stability. Disruption of the disulfide bridge in MotX by site-directed mutagenesis resulted in increased instability, which did not, however, affect the motility of the cells. These lines of evidence suggest that the intramolecular disulfide bonds are involved in the stability of both proteins, but only MotY requires the intramolecular bridge for proper function.     

Using intramolecular disulfide bonds in tau protein to deduce structural features of aggregation-resistant conformations

Because tau aggregation likely plays a role in a number of neurodegenerative diseases, understanding the processes that affect tau aggregation is of considerable importance. One factor that has been shown to influence the aggregation propensity is the oxidation state of the protein itself. Tau protein, which contains two naturally occurring cysteine residues, can form both intermolecular disulfide bonds and intramolecular disulfide bonds. Several studies suggest that intermolecular disulfide bonds can promote tau aggregation in vitro. By contrast, although there are data to suggest that intramolecular disulfide bond formation retards tau aggregation in vitro, the precise mechanism underlying this observation remains unclear. While it has been hypothesized that a single intramolecular disulfide bond in tau leads to compact conformations that cannot form extended structure consistent with tau fibrils, there are few data to support this conjecture. In the present study we generate oxidized forms of the truncation mutant, K18, which contains all four microtubule binding repeats, and isolate the monomeric fraction, which corresponds to K18 monomers that have a single intramolecular disulfide bond. We study the aggregation propensity of the oxidized monomeric fraction and relate these data to an atomistic model of the K18 unfolded ensemble. Our results argue that the main effect of intramolecular disulfide bond formation is to preferentially stabilize conformers within the unfolded ensemble that place the aggregation-prone tau subsequences, PHF6* and PHF6, in conformations that are inconsistent with the formation of cross-β-structure. These data further our understanding of the precise structural features that retard tau aggregation.     

Intramolecular disulfide bond is a critical check point determining degradative fates of ATP-binding cassette (ABC) transporter ABCG2 protein

Human ABCG2 belongs to the ATP-binding cassette (ABC) transporter family and plays an important role in various biological reactions, such as xenobiotic elimination and homeostasis of protoporphyrin. We previously reported that ABCG2 exists in the plasma membrane as a homodimer bound via a disulfide bond at Cys-603. In the present study, we examined the importance of an intramolecular disulfide bond for stability of the ABCG2 protein. Substitution of either Cys-592 or Cys-608 located in the extracellular loop to glycine resulted in a significant decrease in protein levels of ABCG2 when expressed in Flp-In-293 cells. Interestingly, the protein levels of those ABCG2 variants were remarkably enhanced by treatment with the proteasome inhibitor MG132. Concomitantly, increases in ubiquitinated forms of those variant proteins were detected by immunoprecipitation. In contrast, neither the protein level nor the ubiquitinated state of the ABCG2 wild-type (WT) was affected by MG132 treatment. Ubiquitin-mediated protein degradation is suggested to be involved in degradation of misfolded ABCG2 proteins lacking the intramolecular disulfide bond. On the other hand, the protein level of ABCG2 WT increased more than 4-fold when cells were treated with bafilomycin A(1), which inhibits lysosomal degradation, whereas the C592G or C608G variant was little affected by the same treatment. These results strongly suggest that two distinct pathways exist for protein degradation of ABCG2 WT and mutants lacking the intramolecular disulfide bond. Namely, the WT ABCG2 is degraded in lysosomes, and the misfolded ABCG2 lacking intramolecular disulfide bond undergoes ubiquitin-mediated protein degradation in proteasomes.     

Connexon integrity is maintained by non-covalent bonds: intramolecular disulfide bonds link the extracellular domains in rat connexin-43.

Rat heart connexin-43 (RCx43) has been isolated using a modified procedure that is rapid and can be used with fresh or frozen hearts. When RCx43 is isolated in the presence of the alkylating reagent iodoacetamide, no intermolecular disulfide bonds are found. However, the alkylated RCx43 does have at least one intramolecular disulfide bond. By using site directed antibodies and proteolytic cleavage the location of the intramolecular disulfide bonding is shown between the two extracellular loops of RCx43.     

A Primary Role for Disulfide Formation in the Productive Folding of Prokaryotic Cu,Zn-superoxide Dismutase

Enzymatic activation of Cu,Zn-superoxide dismutase (SOD1) requires not only binding of a catalytic copper ion but also formation of an intramolecular disulfide bond. Indeed, the disulfide bond is completely conserved among all species possessing SOD1; however, it remains obscure how disulfide formation controls the enzymatic activity of SOD1. Here, we show that disulfide formation is a primary event in the folding process of prokaryotic SOD1 (SodC) localized to the periplasmic space. Escherichia coli SodC was found to attain β-sheet structure upon formation of the disulfide bond, whereas disulfide-reduced SodC assumed little secondary structure even in the presence of copper and zinc ions. Moreover, reduction of the disulfide bond made SodC highly susceptible to proteolytic degradation. We thus propose that the thiol-disulfide status in SodC controls the intracellular stability of this antioxidant enzyme and that the oxidizing environment of the periplasm is required for the enzymatic activation of SodC.     

Mechanism of the electron transfer catalyst DsbB from Escherichia coli

The membrane protein DsbB from Escherichia coli is essential for disulfide bond formation and catalyses the oxidation of the periplasmic dithiol oxidase DsbA by ubiquinone. DsbB contains two catalytic disulfide bonds, Cys41-Cys44 and Cys104-Cys130. We show that DsbB directly oxidizes one molar equivalent of DsbA in the absence of ubiquinone via disulfide exchange with the 104-130 disulfide bond, with a rate constant of 2.7 x 10 M(-1) x s(-1). This reaction occurs although the 104-130 disulfide is less oxidizing than the catalytic disulfide bond of DsbA (E(o)' = -186 and -122 mV, respectively). This is because the 41-44 disulfide, which is only accessible to ubiquinone but not to DsbA, is the most oxidizing disulfide bond in a protein described so far, with a redox potential of -69 mV. Rapid intramolecular disulfide exchange in partially reduced DsbB converts the enzyme into a state in which Cys41 and Cys44 are reduced and thus accessible for reoxidation by ubiquinone. This demonstrates that the high catalytic efficiency of DsbB results from the extreme intrinsic oxidative force of the enzyme.     

A redox-sensitive loop regulates plasminogen activator inhibitor type 2 (PAI-2) polymerization

Plasminogen activator inhibitor type 2 (PAI-2) is the only wild-type serpin that polymerizes spontaneously under physiological conditions. We show that PAI-2 loses its ability to polymerize following reduction of thiol groups, suggesting that an intramolecular disulfide bond is essential for the polymerization. A novel disulfide bond was identified between C79 (in the CD-loop) and C161 (at the bottom of helix F). Substitution mutants in which this disulfide bond was broken did not polymerize. Reactive center loop peptide insertion experiments and binding of bis-ANS to hydrophobic cavities indicate that the C79-C161 disulfide bond stabilizes PAI-2 in a polymerogenic conformation with an open A-beta-sheet. Elimination of this disulfide bond causes A-beta-sheet closure and abrogates the polymerization. The finding that cytosolic PAI-2 is mostly monomeric, whereas PAI-2 in the secretory pathway is prone to polymerize, suggests that the redox status of the cell could regulate PAI-2 polymerization. Taken together, our data suggest that the CD-loop functions as a redox-sensitive switch that converts PAI-2 between an active stable monomeric and a polymerogenic conformation, which is prone to form inactive polymers.     

Role of the intramolecular disulfide bond in FlgI, the flagellar P-ring component of Escherichia coli

The P ring of the bacterial flagellar motor consists of multiple copies of FlgI, a periplasmic protein. The intramolecular disulfide bond in FlgI has previously been reported to be essential for P-ring assembly in Escherichia coli, because the P ring was not assembled in a dsbB strain that was defective for disulfide bond formation in periplasmic proteins. We, however, found that the two Cys residues of FlgI are not conserved in other bacterial species. We then assessed the role of this intramolecular disulfide bond in FlgI. A Cys-eliminated FlgI derivative formed a P ring that complemented the flagellation defect of our DeltaflgI strain when it was overproduced, suggesting that disulfide bond formation in FlgI is not absolutely required for P-ring assembly. The levels of the mature forms of the FlgI derivatives were significantly lower than that of wild-type FlgI, although the precursor protein levels were unchanged. Moreover, the FlgI derivatives were more susceptible to degradation than wild-type FlgI. Overproduction of FlgI suppressed the motility defect of DeltadsbB cells. Additionally, the low level of FlgI observed in the DeltadsbB strain increased in the presence of l-cystine, an oxidative agent. We propose that intramolecular disulfide bond formation facilitates the rapid folding of the FlgI monomer to protect against degradation in the periplasmic space, thereby allowing its efficient self-assembly into the P ring.     

Disulfide bond within mu-calpain active site inhibits activity and autolysis

Oxidative processes have the ability to influence micro-calpain activity. In the present study the influence of oxidation on activity and autolysis of micro-calpain was examined. Furthermore, LC-MS/MS analysis was employed to identify and characterize protein modifications caused by oxidation. The results revealed that the activity of micro-calpain is diminished by oxidation with H(2)O(2) in a reversible manner involving cysteine and that the rate of autolysis of micro-calpain concomitantly slowed. The LC-MS/MS analysis of the oxidized micro-calpain revealed that the amino acid residues 105-133 contained a disulfide bond between Cys(108) and Cys(115). The finding that the active site cysteine in micro-calpain is able to form a disulfide bond has, to our knowledge, not been reported before. This could be part of a unique oxidation mechanism for micro-calpain. The results also showed that the formation of the disulfide bond is limited in the control (no oxidant added), and further limited in a concentration-dependent manner when beta-mercaptoethanol is added. However, the disulfide bond is still present to some extent in all conditions indicating that the active site cysteine is potentially highly susceptible to the formation of this intramolecular disulfide bond.     

Oxidative Inhibition of Protein Phosphatase 2A Activity: Role of Catalytic Subunit Disulfides

A molecular basis for the inhibition of brain protein phosphatase 2A (PP2A) activity by oxidative stress was examined in a high-speed supernatant (HSS) fraction from rat cerebral cortex. PP2A activity was subject to substantial disulfide reducing agent-reversible inhibition in the HSS fraction. Results of gel electrophoresis support the conclusions that inhibition of PP2A activity was associated with the both the disulfide cross-linking of the catalytic subunit (PP2A_C) of the enzyme to other brain proteins and with the formation of an apparent novel intramolecular disulfide bond in PP2A_C. Additional findings that the vicinal dithiol cross-linking reagent phenylarsine oxide (PAO) produced a potent dithiothreitol-reversible inhibition of PP2A activity suggest that the cross-linking of PP2A_C vicinal thiols to form an intramolecular disulfide bond may be sufficient to inhibit PP2A activity under oxidative stress. We propose that the dithiol–disulfide equilibrium of a vicinal thiol pair of PP2A_C may confer redox sensitivity on cellular PP2A.     

Identification of human vesicle monoamine transporter (VMAT2) lumenal cysteines that form an intramolecular disulfide bond

The vesicle monoamine transporter (VMAT2) concentrates monoamine neurotransmitter into synaptic vesicles. To obtain structural information regarding this large membrane protein by analysis of disulfide bonds and other intramolecular cross-links, we engineered a strategic thrombin cleavage site into deglycosylated, HA-tagged human VMAT2. Insertion of this protease site did not disrupt ligand binding or serotonin transport. Thrombin cleavage at an engineered site in the predicted cytoplasmic loop between transmembrane (TM) domains 6 and 7 (loop 6/7) was rapid and quantitative in the absence of any detergent. The loop 6/7 thrombin site allowed assessment of an intramolecular disulfide bond between the N- and C-terminal halves of the transporter. Consistent with this hypothesis, after quantitative loop 6/7 thrombin cleavage, in the absence of reducing agent, VMAT2 migrated on SDS-polyacrylamide gels as a full-length transporter. Addition of dithiothreitol resulted in complete conversion from full-length to thrombin-cleaved size, demonstrating a DTT-reversible covalent bond. The identity of the disulfide-bound cysteine pair was suggested by the observation that replacement of Cys 126 or Cys 333 with serine both reduced [(3)H]serotonin transport. Replacement of either Cys 126 or Cys 333 was found to eliminate the DTT-reversible intramolecular covalent bond. We conclude that human VMAT2 Cys 126 in loop 1/2 and Cys 333 in loop 7/8 form a disulfide bond which contributes to efficient monoamine transport.     

Unpaired cysteine-54 interferes with the ability of an engineered disulfide to stabilize T4 lysozyme

We have introduced an intramolecular disulfide bond into T4 lysozyme and have shown this molecule to be significantly more stable than the wild-type molecule to irreversible thermal inactivation [Perry, L.J., & Wetzel, R. (1984) Science (Washington, D.C.) 226, 555-557]. Wild-type T4 lysozyme contains two free cysteines, at positions 54 and 97, and no disulfide bonds. By directed mutagenesis of the cloned T4 lysozyme gene, we replaced Ile-3 with Cys. Oxidation in vitro generated an intramolecular disulfide bond; proteolytic mapping showed this bond to connect Cys-3 to Cys-97. While this molecule exhibited substantially more stability against thermal inactivation than wild type, its stability was further enhanced by additional modification with thiol-specific reagents. This and other evidence suggest that at basic pH and elevated temperatures Cys-54 is involved in intermolecular thiol/disulfide interchange with the engineered disulfide, leading to inactive oligomers. Mutagenic replacement of Cys-54 with Thr or Val in the disulfide-cross-linked variant generated lysozymes exhibiting greatly enhanced stability toward irreversible thermal inactivation.     

2,2'-Bispyridyl disulfide rapidly induces intramolecular disulfide bonds in peptides.

A linear peptide containing two reduced cysteine residues can be rapidly converted to its oxidized cyclic form containing an intramolecular disulfide bond by adding an excess of 2,2'-bispyridyl disulfide (2,2'-dipyridyl disulfide or 2,2'-dithiodipyridine) to conventional buffer solutions. The reactants and products are easily separated by reverse-phase chromatography. This reaction will find wide application in forming intramolecular disulfide bonds because of its selectivity for free sulfhydryl groups, quickness, safety, and applicability under acidic conditions.     

The multisubunit structure of synaptophysin. Relationship between disulfide bonding and homo-oligomerization

Synaptophysin, a major membrane protein of synaptic vesicles, contains four transmembrane regions and two intravesicular loops. Synaptophysin monomers associate into homopolymers that have the potential to form channels in the synaptic vesicle membrane. Here we show that in native synaptophysin, homopolymers are linked by noncovalent forces. The molecule contains unstable intramolecular disulfide bonds that undergo disulfide exchange during solubilization, thereby covalently cross-linking neighboring synaptophysin molecules. The locations of the intramolecular disulfide bonds in synaptophysin were determined, revealing that each of the two intravesicular loops of synaptophysin is circularized by a single disulfide bond. Cross-linking of synaptophysin by disulfide bonds can be triggered in synaptic vesicles and in intact cells by a cycle of reduction and oxidation, suggesting that native synaptophysin is a homomultimer in situ. In addition, chemical cross-linking of native synaptophysin demonstrates that a low molecular weight protein is specifically associated with synaptophysin complexes and is lost upon reduction of the intramolecular disulfide bonds. These data suggest that native synaptophysin forms a noncovalent homomultimeric complex whose structure and interaction with other proteins are dependent on the integrity of its intramolecular disulfide bonds and phospholipid environment.