Two pairs of conserved cysteines are required for the oxidative activity of Ero1p in protein disulfide bond formation in the endoplasmic reticulum

In the major pathway for protein disulfide-bond formation in the endoplasmic reticulum (ER), oxidizing equivalents flow from the conserved ER-membrane protein Ero1p to secretory proteins via protein disulfide isomerase (PDI). Herein, a mutational analysis of the yeast ERO1 gene identifies two pairs of conserved cysteines likely to form redox-active disulfide bonds in Ero1p. Cys100, Cys105, Cys352, and Cys355 of Ero1p are important for oxidative protein folding and for cell viability, whereas Cys90, Cys208, and Cys349 are dispensable for these functions. Substitution of Cys100 with alanine impedes the capture of Ero1p-Pdi1p mixed-disulfide complexes from yeast, and also blocks oxidation of Pdi1p in vivo. Cys352 and Cys355 are required to maintain the fully oxidized redox state of Ero1p, and also play an auxiliary role in thiol-disulfide exchange with Pdi1p. These results suggest a model for the function of Ero1p wherein Cys100 and Cys105 form a redox-active disulfide bond that engages directly in thiol-disulfide exchange with ER oxidoreductases. The Cys352-Cys355 disulfide could then serve to reoxidize the Cys100-Cys105 cysteine pair, possibly through an intramolecular thiol-disulfide exchange reaction.     

Influence of the pK(a) value of the buried, active-site cysteine on the redox properties of thioredoxin-like oxidoreductases

Thioredoxin constitutes the prototype of the thiol-disulfide oxidoreductase family. These enzymes contain an active-site disulfide bridge with the consensus sequence Cys-Xaa-Xaa-Cys. The more N-terminal active-site cysteine is generally a strong nucleophile with an abnormal low pK(a) value. In contrast, the more C-terminal cysteine is buried and only little is known about its effective pK(a) during catalysis of disulfide exchange reactions. Here we have analyzed the pK(a) values of the active-site thiols in wild type thioredoxin and a 400-fold more oxidizing thioredoxin variant by NMR spectroscopy, using selectively (13)C(beta)-Cys-labeled proteins. We find that the effective pK(a) of the buried cysteine (pK(b)) of the variant is increased, while the pK(a) of the more N-terminal cysteine (pK(N)) is decreased relative to the corresponding pK(a) values in the wild type. We propose two empirical models which exclusively require the knowledge of pK(N) to predict the redox properties of thiol-disulfide oxidoreductases with reasonable accuracy.     

Disulfide transfer between two conserved cysteine pairs imparts selectivity to protein oxidation by Ero1

The membrane-associated flavoprotein Ero1p promotes disulfide bond formation in the endoplasmic reticulum (ER) by selectively oxidizing the soluble oxidoreductase protein disulfide isomerase (Pdi1p), which in turn can directly oxidize secretory proteins. Two redox-active disulfide bonds are essential for Ero1p oxidase activity: Cys100-Cys105 and Cys352-Cys355. Genetic and structural data indicate a disulfide bond is transferred from Cys100-Cys105 directly to Pdi1p, whereas a Cys352-Cys355 disulfide bond is used to reoxidize the reduced Cys100-Cys105 pair through an internal thiol-transfer reaction. Electron transfer from Cys352-Cys355 to molecular oxygen, by way of a flavin cofactor, maintains Cys352-Cys355 in an oxidized form. Herein, we identify a mixed disulfide species that confirms the Ero1p intercysteine thiol-transfer relay in vivo and identify Cys105 and Cys352 as the cysteines that mediate thiol-disulfide exchange. Moreover, we describe Ero1p mutants that have the surprising ability to oxidize substrates in the absence of Cys100-Cys105. We show the oxidase activity of these mutants results from structural changes in Ero1p that allow substrates increased access to Cys352-Cys355, which are normally buried beneath the protein surface. The altered activity of these Ero1p mutants toward selected substrates leads us to propose the catalytic mechanism involving transfer between cysteine pairs evolved to impart substrate specificity to Ero1p.     

A mechanistic investigation of the thiol-disulfide exchange step in the reductive dehalogenation catalyzed by tetrachlorohydroquinone dehalogenase

Tetrachlorohydroquinone dehalogenase catalyzes the reductive dehalogenation of tetrachloro- and trichlorohydroquinone to give 2,6-dichlorohydroquinone in the pathway for degradation of pentachlorophenol by Sphingobium chlorophenolicum. Previous work has suggested that this enzyme may have originated from a glutathione-dependent double bond isomerase such as maleylacetoacetate isomerase or maleylpyruvate isomerase. While some of the elementary steps in these two reactions may be similar, the final step in the dehalogenation reaction, a thiol-disulfide exchange reaction that removes glutathione covalently bound to Cys13, certainly has no counterpart in the isomerization reaction. The thiol-disulfide exchange reaction does not appear to have been evolutionarily optimized. There is little specificity for the thiol; many thiols react at high rates. TCHQ dehalogenase binds the glutathione involved in the thiol-disulfide exchange reaction very poorly and does not alter its pK(a) in order to improve its nucleophilicity. Remarkably, single-turnover kinetic studies show that the enzyme catalyzes this step by approximately 10000-fold. This high reactivity requires an as yet unidentified protonated group in the active site.     

Roles of two conserved cysteine residues in the activation of human adenovirus proteinase.

The roles of two conserved cysteine residues involved in the activation of the adenovirus proteinase (AVP) were investigated. AVP requires two cofactors for maximal activity, the 11-amino acid peptide pVIc (GVQSLKRRRCF) and the viral DNA. In the AVP-pVIc crystal structure, conserved Cys104 of AVP has formed a disulfide bond with conserved Cys10 of pVIc. In this work, pVIc formed a homodimer via disulfide bond formation with a second-order rate constant of 0.12 M(-1) s(-1), and half of the homodimer could covalently bind to AVP via thiol-disulfide exchange. Alternatively, monomeric pVIc could form a disulfide bond with AVP via oxidation. Regardless of the mechanism by which AVP becomes covalently bound to pVIc, the kinetic constants for substrate hydrolysis were the same. The equilibrium dissociation constant, K(d), for the reversible binding of pVIc to AVP was 4.4 microM. The K(d) for the binding of the mutant C10A-pVIc was at least 100-fold higher. Surprisingly, the K(d) for the binding of the C10A-pVIc mutant to AVP decreased at least 60-fold, to 6.93 microM, in the presence of 12mer ssDNA. Furthermore, once the mutant C10A-pVIc was bound to an AVP-DNA complex, the macroscopic kinetic constants for substrate hydrolysis were the same as those exhibited by wild-type pVIc. Although the cysteine in pVIc is important in the binding of pVIc to AVP, formation of a disulfide bond between pVIc and AVP was not required for maximal stimulation of enzyme activity by pVIc.     

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.     

Nonnative interactions between cysteines direct productive assembly of P22 tailspike protein

Nonnative disulfide bond formation can play a critical role in the assembly of disulfide bonded proteins. During the folding and assembly of the P22 tailspike protein, nonnative disulfide bonds form both in vivo and in vitro. However, the mechanism and identity of cysteine disulfide pairs remains elusive, particularly for P22 tailspike, which contains no disulfide bonds in its native, functional form. Understanding the interactions between cysteine residues is important for developing a mechanistic model for the role of nonnative cysteines in P22 tailspike assembly. Prior in vivo studies have suggested that cysteines 496, 613, and 635 are the most likely site for sulfhydryl reactivity. Here we demonstrate that these three cysteines are critical for efficient assembly of tailspike trimers, and that interactions between cysteine pairs lead to productive assembly of native tailspike.     

Cysteine reactivity and thiol-disulfide interchange pathways in AhpF and AhpC of the bacterial alkyl hydroperoxide reductase system

AhpC and AhpF from Salmonella typhimurium undergo a series of electron transfers to catalyze the pyridine nucleotide-dependent reduction of hydroperoxide substrates. AhpC, the peroxide-reducing (peroxiredoxin) component of this alkyl hydroperoxidase system, is an important scavenger of endogenous hydrogen peroxide in bacteria and acts through a reactive, peroxidatic cysteine, Cys46, and a second cysteine, Cys165, that forms an active site disulfide bond. AhpF, a separate disulfide reductase protein, regenerates AhpC every catalytic cycle via electrons from NADH which are transferred to AhpC through a tightly bound flavin and two disulfide centers, Cys345-Cys348 and Cys129-Cys132, through putative large domain movements. In order to assess cysteine reactivity and interdomain interactions in both proteins, a comprehensive set of single and double cysteine mutants (replacing cysteine with serine) of both proteins were prepared. Based on 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and AhpC reactivity with multiple mutants of AhpF, the thiolate of Cys129 in the N-terminal domain of AhpF initiates attack on Cys165 of the intersubunit disulfide bond within AhpC for electron transfer between proteins. Cys348 of AhpF has also been identified as the nucleophile attacking the Cys129 sulfur of the N-terminal disulfide bond to initiate electron transfer between these two redox centers. These findings support the modular architecture of AhpF and its need for domain rotations for function, and emphasize the importance of Cys165 in the reductive reactivation of AhpC. In addition, two new constructs have been generated, an AhpF-AhpC complex and a "twisted" form of AhpF, in which redox centers are locked together by stable disulfide bonds which mimic catalytic intermediates.     

Role of individual cysteine residues and disulfide bonds in the structure and function of Aspergillus ribonucleolytic toxin restrictocin.

Restrictocin, produced by the fungus Aspergillus restrictus, belongs to the group of ribonucleolytic toxins called ribotoxins. It specifically cleaves a single phosphodiester bond in a conserved stem and loop structure in the 28S rRNA of large ribosomal subunit and potently inhibits eukaryotic protein synthesis. Restrictocin contains 149 amino acid residues and includes four cysteines at positions 5, 75, 131, and 147. These cysteine residues are involved in the formation of two disulfide bonds, one between Cys 5 and Cys 147 and another between Cys 75 and Cys 131. In the current study, all four cysteine residues were changed to alanine individually and in different combinations by site-directed mutagenesis so as to remove one or both the disulfides. The mutants were expressed and purified from Escherichia coli. Removal of any cysteine or any one of the disulfide bonds individually did not affect the ability of the toxin to specifically cleave the 28S rRNA or to inhibit protein synthesis in vitro. However, the toxin without both disulfide bonds completely lost both ribonucleolytic and protein synthesis inhibition activities. The active mutants, containing only one disulfide bond, exhibited relatively high susceptibility to trypsin digestion. Thus, none of the four cysteine residues is directly involved in restrictocin catalysis; however, the presence of any one of the two disulfide bonds is absolutely essential and sufficient to maintain the enzymatically active conformation of restrictocin. For maintenance of the unique stability displayed by the native toxin, both disulfide bonds are required.     

Randomization of the entire active-site helix alpha 1 of the thiol-disulfide oxidoreductase DsbA from Escherichia coli

DsbA from Escherichia coli is the most oxidizing member of the thiol-disulfide oxidoreductase family (E(o)' = -122 mV) and is required for efficient disulfide bond formation in the periplasm. The reactivity of the catalytic disulfide bond (Cys(30)-Pro(31)-His(32)-Cys(33)) is primarily due to an extremely low pK(a) value (3.4) of Cys(30), which is stabilized by the partial positive dipole charge of the active-site helix alpha1 (residues 30-37). We have randomized all non-cysteine residues of helix alpha1 (residues 31, 32, and 34-37) and found that two-thirds of the resulting variants complement DsbA deficiency in a dsbA deletion strain. Sequencing of 98 variants revealed a large number of non-conservative replacements in active variants, even at well conserved positions. This indicates that tertiary structure context strongly determines alpha-helical secondary structure formation of the randomized sequence. A subset of active and inactive variants was further characterized. All these variants were more reducing than wild type DsbA, but the redox potentials of active variants did not drop below -210 mV. All inactive variants had redox potentials lower than -210 mV, although some of the inactive proteins were still re-oxidized by DsbB. This demonstrates that efficient oxidation of substrate polypeptides is the crucial property of DsbA in vivo.     

Probing the dark state tertiary structure in the cytoplasmic domain of rhodopsin: proximities between amino acids deduced from spontaneous disulfide bond formation between Cys316 and engineered cysteines in cytoplasmic loop 1

A dark state tertiary structure in the cytoplasmic domain of rhodopsin is presumed to be the key to the restriction of binding of transducin and rhodopsin kinase to rhodopsin. Upon light-activation, this tertiary structure undergoes a conformational change to form a new structure, which is recognized by the above proteins and signal transduction is initiated. In this and the following paper in this issue [Cai, K., Klein-Seetharaman, J., Altenbach, C., Hubbell, W. L., and Khorana, H. G. (2001) Biochemistry 40, 12479-12485], we probe the dark state cytoplasmic domain structure in rhodopsin by investigating proximity between amino acids in different regions of the cytoplasmic face. The approach uses engineered pairs of cysteines at predetermined positions, which are tested for spontaneous formation of disulfide bonds between them, indicative of proximity between the original amino acids. Focusing here on proximity between the native cysteine at position 316 and engineered cysteines at amino acid positions 55-75 in the cytoplasmic sequence connecting helices I-II, disulfide bond formation was studied under strictly defined conditions and plotted as a function of the position of the variable cysteines. An absolute maximum was observed for position 65 with two additional relative maxima for cysteines at positions 61 and 68. The observed disulfide bond formation rates correlate well with proximity of these residues found in the crystal structure of rhodopsin in the dark. Modeling of the engineered cysteines in the crystal structure indicates that small but significant motions are required for productive disulfide bond formation. During these motions, secondary structure elements are retained as indicated by the lack of disulfide bond formation in cysteines that do not face toward Cys316 in the crystal structure model. Such motions may be important in light-induced conformational changes.     

Mutating the four extracellular cysteines in the chemokine receptor CCR6 reveals their differing roles in receptor trafficking,ligand binding,and signaling

CCR6 is the receptor for the chemokine MIP-3 alpha/CCL20. Almost all chemokine receptors contain cysteine residues in the N-terminal domain and in the first, second, and third extracellular loops. In this report, we have studied the importance of all cysteine residues in the CCR6 sequence using site-directed mutagenesis and biochemical techniques. Like all G protein-coupled receptors, mutating disulfide bond-forming cysteines in the first (Cys118) and second (Cys197) extracellular loops in CCR6 led to complete elimination of receptor activity, which for CCR6 was also associated with the accumulation of the receptor intracellularly. Although two additional cysteines in the N-terminal region and the third extracellular loop, which are present in almost all chemokine receptors, are presumed to form a disulfide bond, this has not been demonstrated experimentally for any of these receptors. We found that mutating the cysteines in the N-terminal domain (Cys36) and the third extracellular loop (Cys288) neither significantly affected receptor surface expression nor completely abolished receptor function. Importantly, contrary to several previous reports, we demonstrated directly that instead of forming a disulfide bond, the N-terminal cysteine (Cys36) and the third extracellular loop cysteine (Cys288) contain free SH groups. The cysteine residues (Cys36 and Cys288), rather than forming a disulfide bond, may be important per se. We propose that CCR6 forms only a disulfide bond between the first (Cys118) and second (Cys197) extracellular loops, which confines a helical bundle together with the N-terminus adjacent to the third extracellular loop, creating the structural organization critical for ligand binding and therefore for receptor signaling.     

Analyses of Native Disulfide Bond Formations in the Early Stage of the Folding Process in Mutant Lysozymes Where the Long-Range Interactions in the Denatured State Were Modulated

In order to clarify whether modulation of long-range interactions in the denatured state affect native disulfide bond (SS bond) formations of hen egg white lysozyme (HEL) containing a pair of cysteine residues, we examined the extent of SS bond formation among 12 variants containing a pair of cysteines. The loss of clusters 5 and 6 in the denatured state affected the formation of Cys30-Cys115 and Cys6-Cys127 respectively.     

Analyses of native disulfide bond formations in the early stage of the folding process in mutant lysozymes where the long-range interactions in the denatured state were modulated

In order to clarify whether modulation of long-range interactions in the denatured state affect native disulfide bond (SS bond) formations of hen egg white lysozyme (HEL) containing a pair of cysteine residues, we examined the extent of SS bond formation among 12 variants containing a pair of cysteines. The loss of clusters 5 and 6 in the denatured state affected the formation of Cys30-Cys115 and Cys6-Cys127 respectively.     

Thioredoxin-dependent enzymatic activation of mercaptopyruvate sulfurtransferase. An intersubunit disulfide bond serves as a redox switch for activation

Rat 3-mercaptopyruvate sulfurtransferase (MST) contains three exposed cysteines as follows: a catalytic site cysteine, Cys(247), in the active site and Cys(154) and Cys(263) on the surface of MST. The corresponding cysteine to Cys(263) is conserved in mammalian MSTs, and Cys(154) is a unique cysteine. MST has monomer-dimer equilibrium with the assistance of oxidants and reductants. The monomer to dimer ratio is maintained at approximately 92:8 in 0.2 m potassium phosphate buffer containing no reductants under air-saturated conditions; the dimer might be symmetrical via an intersubunit disulfide bond between Cys(154) and Cys(154) and between Cys(263) and Cys(263), or asymmetrical via an intersubunit disulfide bond between Cys(154) and Cys(263). Escherichia coli reduced thioredoxin (Trx) cleaved the intersubunit disulfide bond to activate MST to 2.3- and 4.9-fold the levels of activation of dithiothreitol (DTT)-treated and DTT-untreated MST, respectively. Rat Trx also activated MST. On the other hand, reduced glutathione did not affect MST activity. E. coli C35S Trx, in which Cys(35) was replaced with Ser, formed some adducts with MST and activated MST after treatment with DTT. Thus, Cys(32) of E. coli Trx reacted with the redox-active cysteines, Cys(154) and Cys(263), by forming an intersubunit disulfide bond and a sulfenyl Cys(247). A consecutively formed disulfide bond between Trx and MST must be cleaved for the activation. E. coli C32S Trx, however, did not activate MST. Reduced Trx turns on a redox switch for the enzymatic activation of MST, which contributes to the maintenance of cellular redox homeostasis.     

Computational and mutational analysis of human glutaredoxin (thioltransferase): probing the molecular basis of the low pKa of cysteine 22 and its role in catalysis

Human glutaredoxin (GRx), also known as thioltransferase, is a 12 kDa thiol-disulfide oxidoreductase that is highly selective for reduction of glutathione-containing mixed disulfides. The apparent pK(a) for the active site Cys22 residue is approximately 3.5. Previously we observed that the catalytic enhancement by glutaredoxin could be ascribed fully to the difference between the pK(a) of its Cys22 thiol moiety and the pK(a) of the product thiol, each acting as a leaving group in the enzymatic and nonenzymatic reactions, respectively [Srinivasan et al. (1997), Biochemistry 36, 3199-3206]. Continuum electrostatic calculations suggest that the low pK(a) of Cys22 results primarily from stabilization of the thiolate anion by a specific ion-pairing with the positively charged Lys19 residue, although hydrogen bonding interactions with Thr21 also appear to contribute. Variants of Lys19 were considered to further assess the predicted role of Lys19 on the pK(a) of Cys22. The variants K19Q and K19L were generated by molecular modeling, and the pK(a) value for Cys22 was calculated for each variant. For K19Q, the predicted Cys22 pK(a) is 7.3, while the predicted value is 8.3 for K19L. The effects of the mutations on the interaction energy between the adducted glutathionyl moiety and GRx were roughly estimated from the van der Waals and electrostatic energies between the glutathionyl moiety and proximal protein residues in a mixed disulfide adduct of GRx and glutathione, i.e., the GRx-SSG intermediate. The values for the K19 mutants differed by only a small amount compared to those for the wild type enzyme intermediate. Together, the computational analysis predicted that the mutant enzymes would have markedly reduced catalytic rates while retaining the glutathionyl specificity displayed by the wild type enzyme. Accordingly, we constructed and characterized the K19L and K19Q mutants of two forms of the GRx enzyme. Each of the mutants retained glutathionyl specificity as predicted and displayed diminution in activity, but the decreases in activity were not to the extent predicted by the theoretical calculations. Changes in the respective Cys22-thiol pK(a) values of the mutant enzymes, as shown by pH profiles for iodoacetamide inactivation of the respective enzymes, clearly revealed that the K19-C22 ion pair cannot fully account for the low pK(a) of the Cys22 thiol. Additional contributions to stabilization of the Cys22 thiolate are likely donated by Thr21 and the N-terminal partial positive charge of the neighboring alpha-helix.     

Stereoselective disulfide formation stabilizes the local peptide conformation in nisin mimics

Nisin is a polymacrocyclic peptide antimicrobial with high activity against Gram-positive bacteria. Lanthionine and methyllanthionine bridges, closing the macrocycles, are stabilized by thioether bonds, formed between cysteines and dehydrated serine or threonine. The role of polypeptide backbone conformation in the formation of macrocycles A and B within cysteine mutants of nisin residues 1?12 is investigated here by molecular dynamics simulations. Enantiomeric combinational space of Cys3 and Cys7 and of Cys8 and Cys11 is examined for the preference of disulfide bond formation over helical turn formation within this region. A clear preference for spontaneous disulfide formation and closure of rings 3,7 and 8,11 is demonstrated for the D-Cys3, D-Cys7, L-Cys8, L-Cys11 nisin homologue, while interlinked rings A and B are obtained through disulfide bridges between L-Cys3 and D-Cys8 and between D-Cys7 and D-Cys11. This study offers a simple designer approach to solid phase synthesis of macrocyclic peptides and lantibiotic analogues.     

Kinetics of disulfide exchange reactions of monomer and dimer loops of cysteine-valine-cysteine peptides

Rate constants have been determined in 3 M guanidine hydrochloride for disulfide exchange reactions between glutathione and two synthetic peptides containing a cysteine-valine-cysteine region. Equilibrium experiments demonstrate the absence of noncovalent peptide aggregation in this solvent. Procedures are given for separating seven different components in quenched reactions, including the fully reduced cysteine cluster, the monomeric disulfide loop, parallel and antiparallel dimer loops, and the three monomers containing one or two mixed disulfides with glutathione. Intramolecular rate constants for (1) formation of a sterically strained monomer loop, (2) transfer of glutathione between the two cysteines on the same peptide chain, and (3) formation of unstrained dimer loops correspond to a series of processes forming rings of increasing size. In one sequence, these rate constants are 3, 6, and about 21 s-1, respectively. The larger loops are formed more easily. In the other sequence, rate constants for formation and opening of monomer loops are accelerated 180- and 1300-fold, respectively, relative to analogous reactions in a peptide containing eight residues between the two cysteines. This gives a 7-fold smaller equilibrium constant for ring closure in the cysteine cluster. Dimer formation occurs by a mechanism utilizing the accelerated opening of monomer loops. Results provide information assisting efforts to develop strategies for directing disulfide pairing in novel protein structures. Results also help define factors contributing to formation of undesired oligomers during efforts to refold cysteine-containing proteins obtained by bacterial expression of mammalian genes.     

The Venus's-flytrap and cysteine-rich domains of the human Ca2+ receptor are not linked by disulfide bonds

The extracellular N-terminal domain of the human Ca(2+) receptor (hCaR) consists of a Venus's-flytrap (VFT) domain and a cysteine-rich (Cys-rich) domain. We have shown earlier that the Cys-rich domain is critical for signal transmission from the VFT domain to the seven-transmembrane domain. The VFT domain contains 10 cysteines: two of them (Cys(129) and Cys(131)) were identified as involved in intermolecular disulfide bonds necessary for homodimerization, and six others (Cys(60)-Cys(101), Cys(358)-Cys(395), and Cys(437)-Cys(449)) are predicted to form three intramolecular disulfide bonds. The Cys-rich domain contains nine cysteines, the involvement of which in disulfide bond formation has not been defined. In this work, we asked whether the remaining cysteines in the hCaR VFT, namely Cys(236) and Cys(482), form disulfide bond(s) with cysteines in the Cys-rich domain. We constructed mutant hCaRs with a unique tobacco etch virus (TEV) protease recognition site inserted between the VFT domain and the Cys-rich domain. These mutant hCaRs remain fully functional compared with the wild type hCaR. After TEV protease digestion of the mutant hCaR proteins, dimers of the VFT were identified on Western blot under nonreducing conditions. We concluded that there is no disulfide bond between the VFT and the Cys-rich domains in the hCaR.     

Respiratory chain strongly oxidizes the CXXC motif of DsbB in the Escherichia coli disulfide bond formation pathway

Escherichia coli DsbB has four essential cysteine residues, among which Cys41 and Cys44 form a CXXC redox active site motif and the Cys104-Cys130 disulfide bond oxidizes the active site cysteines of DsbA, the disulfide bond formation factor in the periplasm. Functional respiratory chain is required for the cell to keep DsbA oxidized. In this study, we characterized the roles of essential cysteines of DsbB in the coupling with the respiratory chain. Cys104 was found to form the inactive complex with DsbA under respiration-defective conditions. While DsbB, under normal aerobic conditions, is in the oxidized state, having two intramolecular disulfide bonds, oxidation of Cys104 and Cys130 requires the presence of Cys41-Cys44. Remarkably, the Cys41-Cys44 disulfide bond is refractory to reduction by a high concentration of dithiothreitol, unless the membrane is solubilized with a detergent. This reductant resistance requires both the respiratory function and oxygen, since Cys41-Cys44 became sensitive to the reducing agent when membrane was prepared from quinone- or heme-depleted cells or when a membrane sample was deaerated. Thus, the Cys41-Val-Leu-Cys44 motif of DsbB is kept both strongly oxidized and strongly oxidizing when DsbB is integrated into the membrane with the normal set of respiratory components.