The disulfide bond formation (DSB) system: so much more than a housekeeper.

The Structure of a Two-Disulfide Intermediate Assists in Elucidating the Oxidative Folding Pathway of a Cyclic Cystine Knot Protein

Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX9C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX9C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel and a member of the superfamily of cysteine loop (Cys-loop) neurotransmitter receptors, which also comprises the nicotinic acetylcholine receptor (nAChR). Within the extracellular domain (ECD), the eponymous Cys-loop harbors two conserved cysteines, assumed to be linked by a superfamily-specific disulfide bond. The GlyR ECD carries three additional cysteine residues, two are predicted to form a second, GlyR-specific bond. The configuration of none of the cysteines of GlyR, however, had been determined directly. Based on a crystal structure of the nAChRalpha1 ECD, we generated a model of the human GlyRalpha1 where close proximity of the respective cysteines was consistent with the formation of both the Cys-loop and the GlyR-specific disulfide bonds. To identify native disulfide bonds, the GlyRalpha1 ECD was heterologously expressed and refolded under oxidative conditions. By matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we detected tryptic fragments of the ECD indicative of disulfide bond formation for both pairs of cysteines, as proposed by modeling. The identity of tryptic fragments was confirmed using chemical modification of cysteine and lysine residues. As evident from circular dichroism spectroscopy, mutagenesis of single cysteines did not impair refolding of the ECD in vitro, whereas it led to partial or complete intracellular retention and consequently to a loss of function of full-length GlyR subunits in human embryonic kidney 293 cells. Our results indicate that the GlyR ECD forms both a Cys-loop and a GlyR-specific disulfide bond. In addition, cysteine residues appear to be important for protein maturation in vivo.

The formation of disulfides within proteins entering the secretory pathway is catalyzed by the protein disulfide isomerase family of endoplasmic reticulum localized oxidoreductases. One such enzyme, ERp57, is thought to catalyze the isomerization of non-native disulfide bonds formed in glycoproteins with unstructured disulfide-rich domains. Here we investigated the mechanism underlying ERp57 specificity toward glycoprotein substrates and the interdependence of ERp57 and the calnexin cycle for their correct folding. Our results clearly show that ERp57 must be physically associated with the calnexin cycle to catalyze isomerization reactions with most of its substrates. In addition, some glycoproteins only require ERp57 for correct disulfide formation if they enter the calnexin cycle. Hence, the specificity of ER oxidoreductases is not only determined by the physical association of enzyme and substrate but also by accessory factors, such as calnexin and calreticulin in the case of ERp57. These conclusions suggest that the calnexin cycle has evolved with a specialized oxidoreductase to facilitate native disulfide formation in complex glycoproteins.

We have studied the effects of polysaccharide and protein crowding agents on the refolding of oxidized and reduced hen lysozyme in order to test the prediction that association constants of interacting macromolecules in living cells are greatly increased by macromolecular crowding relative to their values in dilute solutions. We demonstrate that whereas refolding of oxidized lysozyme is hardly affected by crowding, correct refolding of the reduced protein is essentially abolished due to aggregation at high concentrations of crowding agents. The results show that the protein folding catalyst protein disulfide isomerase is particularly effective in preventing lysozyme aggregation under crowded conditions, suggesting that crowding enhances its chaperone activity. Our findings suggest that the effects of macromolecular crowding could have major implications for our understanding of how protein folding occurs inside cells.

The assembly of the β-barrel proteins present in the outer membrane (OM) of Gram-negative bacteria is poorly characterized. After translocation across the inner membrane, unfolded β-barrel proteins are escorted across the periplasm by chaperones that reside within this compartment. Two partially redundant chaperones, SurA and Skp, are considered to transport the bulk mass of β-barrel proteins. We found that the periplasmic disulfide isomerase DsbC cooperates with SurA and the thiol oxidase DsbA in the folding of the essential β-barrel protein LptD. LptD inserts lipopolysaccharides in the OM. It is also the only β-barrel protein with more than two cysteine residues. We found that surAdsbC mutants, but not skpdsbC mutants, exhibit a synthetic phenotype. They have a decreased OM integrity, which is due to the lack of the isomerase activity of DsbC. We also isolated DsbC in a mixed disulfide complex with LptD. As such, LptD is identified as the first substrate of DsbC that is localized in the OM. Thus, electrons flowing from the cytoplasmic thioredoxin system maintain the integrity of the OM by assisting the folding of one of the most important β-barrel proteins.

Transglutaminase 2 (TG2) in the extracellular matrix is largely inactive but is transiently activated upon certain types of inflammation and cell injury. The enzymatic activity of extracellular TG2 thus appears to be tightly regulated. As TG2 is known to be sensitive to changes in the redox environment, inactivation through oxidation presents a plausible mechanism. Using mass spectrometry, we have identified a redox-sensitive cysteine triad consisting of Cys(230), Cys(370), and Cys(371) that is involved in oxidative inactivation of TG2. Within this triad, Cys(370) was found to participate in disulfide bonds with both Cys(230) and its neighbor, Cys(371). Notably, Ca(2+) was found to protect against formation of these disulfide bonds. To investigate the role of each cysteine residue, we created alanine mutants and found that Cys(230) appears to promote oxidation and inactivation of TG2 by facilitating formation of Cys(370)-Cys(371) through formation of the Cys(230)-Cys(370) disulfide bond. Although vicinal disulfide pairs are found in several transglutaminase isoforms, Cys(230) is unique for TG2, suggesting that this residue acts as an isoform-specific redox sensor. Our findings suggest that oxidation is likely to influence the amount of active TG2 present in the extracellular environment.

The hydroxamate siderophore receptor FhuA is a TonB-dependent outer membrane protein of Escherichia coli composed of a C-terminal 22-stranded beta-barrel occluded by an N-terminal globular cork domain. During siderophore transport into the periplasm, the FhuA cork domain has been proposed to undergo conformational changes that allow transport through the barrel lumen; alternatively, the cork may be completely displaced from the barrel. To probe such changes, site-directed cysteine mutants in the cork domain (L109C and Q112C) and in the barrel domain (S356C and M383C) were created within the putative siderophore transport pathway. Molecular modeling predicted that the double cysteine mutants L109C/S356C and Q112C/M383C would form disulfide bonds, thereby tethering the cork and barrel domains. The double cysteine FhuA mutants were denatured under nonreducing conditions and fluorescently labeled with thiol-specific Oregon Green maleimide. Subsequent SDS-PAGE analysis revealed two distinct species: FhuA containing a disulfide bond and FhuA with free sulfhydryl groups. To address the role of the putative siderophore transport pathway and to evaluate possible rearrangements of the cork domain during ferricrocin transport, disulfide bond formation was enhanced by an oxidative catalyst. Cells containing double cysteine FhuA mutants that were subjected to oxidation during ferricrocin transport exhibited disulfide bond formation to near completion. After disulfide tethering of the cork to the barrel, ferricrocin transport was equivalent to transport by untreated cells. These results demonstrate that blocking the putative siderophore transport pathway does not abrogate ferricrocin uptake. We propose that, during siderophore transport through FhuA, the cork domain remains within the barrel rather than being displaced.

Members of the protein-disulfide isomerase superfamily catalyze the formation of intra- and intermolecular disulfide bonds, a rate-limiting step of protein folding in the endoplasmic reticulum (ER). Here we compared maturation of one obligate and two facultative calnexin substrates in cells with and without ERp57, the calnexin-associated, glycoprotein-specific oxidoreductase. ERp57 deletion did not prevent the formation of disulfide bonds during co-translational translocation of nascent glycopolypeptides in the ER. It affected, however, the post-translational phases of oxidative influenza virus hemagglutinin (HA) folding, resulting in significant loss of folding efficiency for this obligate calnexin substrate. Without ERp57, HA also showed reduced capacity to recover from an artificially induced aberrant conformation, thus revealing a crucial role of ERp57 during post-translational reshuffling to the native set of HA disulfides. ERp57 deletion did not affect maturation of the model facultative calnexin substrates E1 and p62 (and of most cellular proteins, as shown by lack of induction of ER stress). ERp72 was identified as one of the ER-resident oxidoreductases associating with the orphan ERp57 substrates to maintain their folding competence.

Endoplasmic reticulum oxidation 1 (ERO1) is a conserved eukaryotic flavin adenine nucleotide-containing enzyme that promotes disulfide bond formation by accepting electrons from reduced protein disulfide isomerase (PDI) and passing them on to molecular oxygen. Although disulfide bond formation is an essential process, recent experiments suggest a surprisingly broad tolerance to genetic manipulations that attenuate the rate of disulfide bond formation and that a hyperoxidizing ER may place stressed cells at a disadvantage. In this study, we report on the development of a high throughput in vitro assay for mammalian ERO1alpha activity and its application to identify small molecule inhibitors. The inhibitor EN460 (IC(50), 1.9 mum) interacts selectively with the reduced, active form of ERO1alpha and prevents its reoxidation. Despite rapid and promiscuous reactivity with thiolates, EN460 exhibits selectivity for ERO1. This selectivity is explained by the rapid reversibility of the reaction of EN460 with unstructured thiols, in contrast to the formation of a stable bond with ERO1alpha followed by displacement of bound flavin adenine dinucleotide from the active site of the enzyme. Modest concentrations of EN460 and a functionally related inhibitor, QM295, promote signaling in the unfolded protein response and precondition cells against severe ER stress. Together, these observations point to the feasibility of targeting the enzymatic activity of ERO1alpha with small molecule inhibitors.

A three-disulfide intermediate, des-[65-72] RNase A, lacking the disulfide bond between Cys65 and Cys72, is formed in one of the rate-determining steps of the oxidative regeneration pathways of bovine pancreatic ribonuclease A (RNase A). An analog of this intermediate, [C65S, C72S] RNase A, has been characterized in terms of structure and thermodynamic stability. Triple-resonance NMR data were analyzed using an automated assignment program, AUTOASSIGN. Nearly all backbone 1H, 13C, and 15N resonances and most side-chain 13C(beta) resonances of both wild-type (wt) and [C65S, C72S] RNase A were assigned unambiguously. Analysis of NOE, 13C(alpha) chemical shift, and 3J(H(N)-H(alpha)) scalar coupling data indicates that the regular backbone structure of the major form of [C65S, C72S] RNase A is very similar to that of the major form of wt RNase A, although small structural differences are indicated in the mutation site and in spatially adjacent beta-sheet structures comprising the hydrophobic core. Thermodynamic analysis demonstrates that [C65S, C72S] RNase A (Tm of 38.5 degrees C) is significantly less stable than wt RNase A (Tm of 55.5 degrees C) at pH 4.6. Although the structural comparison of wt RNase A and this analog of an oxidative folding intermediate indicates only localized effects around the Cys65 and Cys72 sites, these thermodynamic measurements indicate that formation of the fourth disulfide bond, Cys65-Cys72, on this oxidative folding pathway results in global stabilization of the native chain fold. This conclusion is supported by comparisons of amide 1H/2H exchange rates which are significantly faster throughout the entire structure of [C65S, C72S] RNase A than in wt RNase A. More generally, our study indicates that the C65-C72 disulfide bond of RNase A contributes significantly in stabilizing the structure of the hydrophobic core of the native protein. Formation of this disulfide bond in the final step of this oxidative folding pathway provides significant stabilization of the native-like structure that is present in the corresponding three-disulfide folding intermediate.

The interrelationship between the acquisition of ordered structure, prolyl isomerization, and the formation of the disulfide bonds in assisted protein folding was investigated by using a variant of ribonuclease T1 (C2S/C10N-RNase T1) with a single disulfide bond and two cis-prolyl bonds as a model protein. The thiol-disulfide oxidoreductase DsbA served as the oxidant for forming the disulfide bond and prolyl isomerase A as the catalyst of prolyl isomerization. Both enzymes are from the periplasm of Escherichia coli. Reduced C2S/C10N-RNase T1 is unfolded in 0 M NaCl, but native-like folded in > or = 2 M NaCl. Oxidation of 5 microM C2S/C10N-RNase T1 by 8 microM DsbA (at pH 7.0, 25 degrees C) is very rapid with a t1/2 of about 10 s (the second-order rate constant is 7 x 10(3) s-1 M-1), irrespective of whether the reduced molecules are unfolded or folded. When they are folded, the product of oxidation is the native protein. When they are denatured, first the disulfide bond is formed in the unfolded protein chains and then the native structure is acquired. This slow reaction is limited in rate by prolyl isomerization and catalyzed by prolyl isomerase. The efficiency of this catalysis is strongly decreased by the presence of the disulfide bond. Apparently, the rank order of chain folding, prolyl isomerization, and disulfide bond formation can vary in the oxidative folding of ribonuclease T1. Such a degeneracy could generally be an advantage for protein folding both in vitro and in vivo.

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.

Mycobacteria, including Mycobacterium tuberculosis-the etiological agent of tuberculosis-possess a unique and impermeable cell envelope that is critical for survival and antibiotic resistance. The assembly and maintenance of this envelope depend on properly folded proteins, yet the role of disulfide bond formation in these processes remains poorly understood. Mycobacteria rely on two membrane enzymes, disulfide bond formation protein A (DsbA) and vitamin K epoxide reductase (VKOR), for introducing disulfide bonds into exported proteins. In silico studies predict that ~64% of exported proteins contain even numbers of cysteine residues and thence disulfide bonding; nevertheless, substrates of the DsbA-VKOR pathway remain largely unknown. Here, we demonstrate that DsbA and VKOR introduce disulfide bonds into substrate proteins and identify several essential proteins that depend on oxidative folding in the mycobacterial cell envelope. Using bioinformatics and cysteine profiling proteomics, we uncover numerous exported proteins that require disulfide bonds for stability. Cysteine derivatization in whole cells confirms that key proteins, including LamA (MmpS3), PstP, LpqW, and EmbB, rely on disulfide bonds for proper function. Furthermore, chemical inhibition of VKOR phenocopies vkor deletion, thus highlighting its essential role in maintaining mycomembrane integrity. These findings address a critical gap in understanding mycobacterial cell envelope biogenesis and underscore the DsbA-VKOR system as a promising target for disrupting cell envelope homeostasis in drug-resistant Mycobacteria.IMPORTANCEThis work addresses a major deficiency in understanding mycobacterial cell envelope processes and highlights the biological and clinical implications of oxidative protein folding in mycobacteria. This process, marked by the formation of disulfide bonds, is essential for the stability of exported proteins. While disulfide bond formation studies in Gram-negative bacteria suggested a similar role in mycobacteria, the underlying consequences of disulfide bonds remained unclear. Thus, we began investigating the diverse physiological functions dependent on disulfide bonds in Mycobacteria using a combination of bioinformatics, proteomics, and genetic and biochemical approaches. We identified hundreds of proteins affected by oxidative protein folding and validated essential substrates of this process. We show that disulfide bonds are not only crucial for the stability and function of key mycobacterial proteins but also represent a novel therapeutic target against antimicrobial resistance. Our findings underscore the potential of targeting disulfide bond formation to disrupt mycomembrane assembly, opening new avenues for antimycobacterial drug development.

Influence of Protein Conformation on Disulfide Bond Formation in the Oxidative Folding of Ribonuclease T 1