A protein oxidase catalysing disulfide bond formation is localized to the chloroplast thylakoids

In chloroplasts, thiol/disulfide-redox-regulated proteins have been linked to numerous metabolic pathways. However, the biochemical system for disulfide bond formation in chloroplasts remains undetermined. In the present study, we characterized an oxidoreductase, AtVKOR-DsbA, encoded by the gene At4g35760 as a potential disulfide bond oxidant in Arabidopsis. The gene product contains two distinct domains: an integral membrane domain homologous to the catalytic subunit of mammalian vitamin K epoxide reductase (VKOR) and a soluble DsbA-like domain. Transient expression of green fluorescent protein fusion in Arabidopsis protoplasts indicated that AtVKOR-DsbA is located in the chloroplast. The first 45 amino acids from the N-terminus were found to act as a transit peptide targeting the protein to the chloroplast. An immunoblot assay of chloroplast fractions revealed that AtVKOR-DsbA was localized in the thylakoid. A motility complementation assay showed that the full-length of AtVKOR-DsbA, if lacking its transit peptide, could catalyze the formation of disulfide bonds. Among the 10 cysteine residues present in the mature protein, eight cysteines (four in the AtVKOR domain and four in the AtDsbA domain) were found to be essential for promoting disulfide bond formation. The topological arrangement of AtVKOR-DsbA was assayed using alkaline phosphatase sandwich fusions. From these results, we developed a possible topology model of AtVKOR-DsbA in chloroplasts. We propose that the integral membrane domain of AtVKOR-DsbA contains four transmembrane helices, and that both termini and the cysteines involved in catalyzing the formation of disulfide bonds face the oxidative thylakoid lumen. These studies may help to resolve some of the issues surrounding the structure and function of AtVKOR-DsbA in Arabidopsis chloroplasts.     

Reconstitution of a disulfide isomerization system

Isomerization of disulfide bonds is vital for the proper folding of proteins that possess multiple disulfides. In prokaryotes, the catalytic pathway responsible for disulfide isomerization involves thioredoxin, thioredoxin reductase, and the DsbC, DsbG, and DsbD proteins. To be active as isomerases, DsbC and DsbG must be kept reduced. This task is performed by the cytoplasmic membrane protein DsbD. DsbD in turn is reduced by the cytoplasmic thioredoxin and is composed of three domains. The beta domain is membrane-embedded, whereas the alpha and gamma domains are localized to the periplasm. It had been proposed that electrons are transferred within DsbD by a succession of disulfide exchange reactions between the three domains. To test this model using biochemical methods, we purified to homogeneity different polypeptides corresponding to the alpha, beta, gamma, and betagamma domains. Using these domains, we could reconstitute a DsbD activity and, for the first time, reconstitute in vitro the electron transport pathway from NADPH and thioredoxin to DsbC and DsbG. We showed that electrons are transferred from thioredoxin to the beta domain then successively to the gamma domain, the alpha domain, and finally on to DsbC or DsbG. We also determined the redox potential of the gamma domain to be -241 mV, and that of the alpha domain was found to be -229 mV. This shows that the direction of electron flow within DsbD is thermodynamically driven.     

The thioredoxin reductase-thioredoxin system is involved in the entry of tetanus and botulinum neurotoxins in the cytosol of nerve terminals

Tetanus and botulinum neurotoxins cause paralysis by cleaving SNARE proteins within the cytosol of nerve terminals. They are endocytosed inside acidic vesicles and the pH gradient across the membrane drives the translocation of their metalloprotease L domain in the cytosol. This domain is linked to the rest of the molecule by a single interchain disulfide bridge that has to be reduced on the cytosolic side of the membrane to free its enzymatic activity. By using specific inhibitors of the various cytosolic protein disulfides reducing systems, we show here that the NADPH-thioredoxin reductase-thioredoxin redox system is the main responsible for this disulfide reduction. In addition, we indicate auranofin, as a possible basis for the design of novel inhibitors of these neurotoxins.     

An Unconventional Role for Cytoplasmic Disulfide Bonds in Vaccinia Virus Proteins

Previous data have shown that reducing agents disrupt the structure of vaccinia virus (vv). Here, we have analyzed the disulfide bonding of vv proteins in detail. In vv-infected cells cytoplasmically synthesized vv core proteins became disulfide bonded in the newly assembled intracellular mature viruses (IMVs). vv membrane proteins also assembled disulfide bonds, but independent of IMV formation and to a large extent on their cytoplasmic domains. If disulfide bonding was prevented, virus assembly was only partially impaired as shown by electron microscopy as well as a biochemical assay of IMV formation. Under these conditions, however, the membranes around the isolated particles appeared less stable and detached from the underlying core. During the viral infection process the membrane proteins remained disulfide bonded, whereas the core proteins were reduced, concomitant with delivery of the cores into the cytoplasm. Our data show that vv has evolved an unique system for the assembly of cytoplasmic disulfide bonds that are localized both on the exterior and interior parts of the IMV.     

The closed structure of the MscS mechanosensitive channel. Cross-linking of single cysteine mutants

Mechanosensitive channels must make a large conformational change during the transition from the closed to the open state. The crystal structure of the open form of the Escherichia coli MscS channel was recently solved and depicts a homoheptamer (1). In this study, cross-linking of site-specific cysteine substitutions demonstrates that residues up to 10-33 A apart in the crystal structure readily form disulfide bridges in the closed form and can also be cross-linked by a 10-A linker. Cross-linking between adjacent subunits stabilizes the heptameric form of the channel providing biochemical evidence to support the crystal structure. The data are consistent with the published model (1) in that the membrane domain is highly flexible and that the closed to open transition may involve a significant displacement of transmembrane helices 1 and 2, possibly by as much as 30 A. The data are also consistent with significant flexibility of the cytoplasmic domain.     

Identification of an atypical membrane protein involved in the formation of protein disulfide bonds in oxygenic photosynthetic organisms

The evolution of oxygenic photosynthesis in cyanobacteria nearly three billion years ago provided abundant reducing power and facilitated the elaboration of numerous oxygen-dependent reactions in our biosphere. Cyanobacteria contain an internal thylakoid membrane system, the site of photosynthesis, and a typical Gram-negative envelope membrane system. Like other organisms, the extracytoplasmic space in cyanobacteria houses numerous cysteine-containing proteins. However, the existence of a biochemical system for disulfide bond formation in cyanobacteria remains to be determined. Extracytoplasmic disulfide bond formation in non-photosynthetic organisms is catalyzed by coordinated interaction between two proteins, a disulfide carrier and a disulfide generator. Here we describe a novel gene, SyndsbAB, required for disulfide bond formation in the extracytoplasmic space of cyanobacteria. The SynDsbAB orthologs are present in most cyanobacteria and chloroplasts of higher plants with fully sequenced genomes. The SynDsbAB protein contains two distinct catalytic domains that display significant similarity to proteins involved in disulfide bond formation in Escherichia coli and eukaryotes. Importantly, SyndsbAB complements E. coli strains defective in disulfide bond formation. In addition, the activity of E. coli alkaline phosphatase localized to the periplasm of Synechocystis 6803 is dependent on the function of SynDsbAB. Deletion of SyndsbAB in Synechocystis 6803 causes significant growth impairment under photoautotrophic conditions and results in hyper-sensitivity to dithiothreitol, a reductant, whereas diamide, an oxidant had no effect on the growth of the mutant strains. We conclude that SynDsbAB is a critical protein for disulfide bond formation in oxygenic photosynthetic organisms and required for their optimal photoautotrophic growth.     

The N-terminal domain of PILB from Neisseria meningitidis is a disulfide reductase that can recycle methionine sulfoxide reductases

The PilB protein of the Neisseria genus comprises three domains. Two forms have been recently reported to be produced in vivo. One form, containing the three domains, is secreted from the bacterial cytoplasm to the outer membrane, whereas the second form, which is cytoplasmic, only contains the central and the C-terminal domains. The secreted form was shown to be involved in survival under oxidative conditions. Although previous studies indicated that the central and the C-terminal domains display methionine sulfoxide reductase A and B activities, respectively, no function was described so far for the N-terminal domain. In the present study, the N-terminal domain of the PilB of Neisseria meningitidis was produced as a folded entity, and its biochemical and enzymatic properties have been determined. The data show that the N-terminal domain possesses a disulfide redox-active site with a redox potential in the range of that of thioredoxin. Moreover, the N-terminal domain, either as an isolated form or included in PilB, recycles the oxidized forms of the methionine sulfoxide reductases like thioredoxin. These results, which show that the N-terminal domain exhibits a disulfide reductase activity and probably has a thioredoxin-fold, are discussed in relation to its possible functional role in Neisseria.     

The Disulfide Bond Cys255-Cys279 in the Immunoglobulin-Like Domain of Anthrax Toxin Receptor 2 Is Required for Membrane Insertion of Anthrax Protective Antigen Pore

Anthrax toxin receptors act as molecular clamps or switches that control anthrax toxin entry, pH-dependent pore formation, and translocation of enzymatic moieties across the endosomal membranes. We previously reported that reduction of the disulfide bonds in the immunoglobulin-like (Ig) domain of the anthrax toxin receptor 2 (ANTXR2) inhibited the function of the protective antigen (PA) pore. In the present study, the disulfide linkage in the Ig domain was identified as Cys255-Cys279 and Cys230-Cys315. Specific disulfide bond deletion mutants were achieved by replacing Cys residues with Ala residues. Deletion of the disulfide bond C255-C279, but not C230-C315, inhibited the PA pore-induced release of the fluorescence dyes from the liposomes, suggesting that C255-C279 is essential for PA pore function. Furthermore, we found that deletion of C255-C279 did not affect PA prepore-to-pore conversion, but inhibited PA pore membrane insertion by trapping the PA membrane-inserting loops in proteinaceous hydrophobic pockets. Fluorescence spectra of Trp59, a residue adjacent to the PA-binding motif in von Willebrand factor A (VWA) domain of ANTXR2, showed that deletion of C255-C279 resulted in a significant conformational change on the receptor ectodomain. The disulfide deletion-induced conformational change on the VWA domain was further confirmed by single-particle 3D reconstruction of the negatively stained PA-receptor heptameric complexes. Together, the biochemical and structural data obtained in this study provides a mechanistic insight into the role of the receptor disulfide bond C255-C279 in anthrax toxin action. Manipulation of the redox states of the receptor, specifically targeting to C255-C279, may become a novel strategy to treat anthrax.     

Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes

Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys(119)-Cys(299)) and a unique N-linked glycosylation site (Asn(309)) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr(78)) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved.     

A reporter platform for the monitoring of in vivo conformational changes in AcrB

AcrB is an inner membrane protein in Escherichia coli that is a component of a triplex AcrA-AcrB-TolC (AcrAB-TolC) multidrug efflux pump. The AcrAB-TolC complex and its homologues are highly conserved among Gram-negative bacteria and are major players in conferring multidrug resistance (MDR) in many pathogens. In this study we developed a disulfide trapping method that may reveal AcrB conformational changes under the native condition in the cell membrane. Specifically, we created seven disulfide bond pairs in the periplasmic domain of AcrB, which can be used as probes to determine local conformational changes. We have developed a rigorous protocol to measure the extent of disulfide bond formation in membrane proteins under the native condition. The rigorousness of the method was verified through examining the extent of disulfide bond formation in Cys pairs separated by different distances. The blocking-reducing-labeling scheme combined with fluorescence labeling made the current method convenient, reliable, and quantitative.     

Membrane immunoglobulins are stabilized by interchain disulfide bonds occurring within the extracellular membrane-proximal domain

Membrane-bound immunoglobulins have, in addition to the transmembrane and cytoplasmic portions, an extracellular membrane-proximal domain (EMPD), absent in the secretory forms. EMPDs of immunoglobulin isotypes alpha, gamma, and epsilon contain cysteines whose role has so far not been elucidated. Using a genetic strategy, we investigated the ability of these cysteines to form disulfide bridges. Shortened versions of human membrane immunoglobulins, depleted of cysteines known to form intermolecular disulfide bonds, were constructed and expressed on the surface of a B-cell line. The resulting membrane proteins contain a single chain fragment of variable regions (scFv) linked to the dimerizing domain from the immunoglobulin heavy chains (CH3 for alpha and gamma or CH4 for epsilon isotypes), followed by the corresponding EMPD and the transmembrane and cytoplasmic domains. The two functional membrane versions of the epsilon chain, containing the short and long EMPD, were analyzed. Our results show that the single cysteine within alpha1L and gamma1 EMPD and the short version of epsilon EMPD form an interchain disulfide bond. Conversely, the cysteine resident in the epsilon transmembrane domain remains unreacted. epsilon-long EMPD contains four cysteines; two are involved in interchain bonds while the remaining two are likely forming an intrachain bridge. Expression of a full-length membrane epsilon heavy chain mutant, in which Cys(121) and Cys(209) within domain CH2 (involved in interchain bridges) were mutated to alanines, confirmed that, within the complete IgE, EMPD cysteines form interchain disulfide bonds. In conclusion, we unveil evidence for additional covalent stabilization of membrane-bound immunoglobulins.     

The ABP-120 C-end region from Entamoeba histolytica interacts with sulfatide, a new lipid target

EhABP-120 is the first filamin identified in the parasitic protozoan Entamoeba histolytica. Filamins are a family of cross-linking actin-binding proteins that promote a dynamic orthogonal web. They have been reported to interact directly with more than 30 cellular proteins and some phosphoinositides. The biochemical consequences of these interactions may have either positive or negative effects on the cross-linking function and also form a link between the cytoskeleton and plasma membrane. In this study, the EhABP-120 carboxy-terminal domain (END) was biochemically characterized. This domain was able to associate to 3-sulfate galactosyl ceramide, a new lipid target for a member of the filamin family. Also, the END domain was able to dimerize “in vitro.” Molecular modeling analysis showed that the dimeric region is stabilized by a disulfide bond. Electrostatic and docking studies suggest that an electropositive concave pocket at the dimeric END domain interacts simultaneously with several sulfogalactose moieties of the sulfatide.     

Structural Characteristics of the Redox-sensing Coiled Coil in the Voltage-gated H+ Channel

Oxidation is an important biochemical defense mechanism, but it also elicits toxicity; therefore, oxidation must be under strict control. In phagocytotic events in neutrophils, the voltage-gated H⁺ (Hv) channel is a key regulator of the production of reactive oxygen species against invading bacteria. The cytoplasmic domain of the Hv channel forms a dimeric coiled coil underpinning a dimerized functional unit. Importantly, in the alignment of the coiled-coil core, a conserved cysteine residue forms a potential intersubunit disulfide bond. In this study, we solved the crystal structures of the coiled-coil domain in reduced, oxidized, and mutated (Cys → Ser) states. The crystal structures indicate that a pair of Cys residues forms an intersubunit disulfide bond dependent on the redox conditions. CD spectroscopy revealed that the disulfide bond increases the thermal stability of the coiled-coil protein. We also reveal that two thiol modifier molecules are able to bind to Cys in a redox-dependent manner without disruption of the dimeric coiled-coil assembly. Thus, the biochemical properties of the cytoplasmic coiled-coil domain in the Hv channel depend on the redox condition, which may play a role in redox sensing in the phagosome.     

Two membrane juxtaposed signaling modules in ANF-RGC are interlocked

Atrial natriuretic factor (ANF) receptor guanylate cyclase ANF-RGC is a single transmembrane spanning modular protein. Juxtaposed to each side of the transmembrane module is a Cys423-Cys432 disulfide ANF signaling module motif and the ATP-regulated transduction module (ARM) motif. The signaling module motif is conserved in nearly all membrane guanylate cyclases and is believed to be critical in the signaling activities of all membrane guanylate cyclases. The present study with the model system of the olfactory membrane guanylate cyclase shows that this concept is not valid. Furthermore, the study shows that in ANF-GC the signaling motif works through the ARM domain. A new signaling model is proposed where in its natural state the disulfide structural motif represses the ARM domain activity, which, in turn, represses the catalytic module activity of ANF-RGC. ANF signaling relieves the disulfide structural motif restraint on the ARM inhibition and stimulates the catalytic module of the cyclase.     

The channel domain of colicin A is inhibited by its immunity protein through direct interaction in the Escherichia coli inner membrane.

A bacterial signal sequence was fused to the colicin A pore-forming domain: the exported pore-forming domain was highly cytotoxic. We thus introduced a cysteine-residue pair in the fusion protein which has been shown to form a disulfide bond in the natural colicin A pore-forming domain between alpha-helices 5 and 6. Formation of the disulfide bond prevented the cytotoxic activity of the fusion protein, presumably by preventing the membrane insertion of helices 5 and 6. However, the cytotoxicity of the disulfide-linked pore-forming domain was reactivated by adding dithiothreitol into the culture medium. We were then able to co-produce the immunity protein with the disulfide linked pore-forming domain, by using a co-immunoprecipitation procedure, in order to show that they interact. We showed both proteins to be co-localized in the Escherichia coli inner membrane and subsequently co-immunoprecipitated them. The interaction required a functional immunity protein. The immunity protein also interacted with a mutant form of the pore-forming domain carrying a mutation located in the voltage-gated region: this mutant was devoid of pore-forming activity but still inserted into the membrane. Our results indicate that the immunity protein interacts with the membrane-anchored channel domain; the interaction requires a functional membrane-inserted immunity protein but does not require the channel to be in the open state.     

Labile disulfide bonds are common at the leucocyte cell surface

Redox conditions change in events such as immune and platelet activation, and during viral infection, but the biochemical consequences are not well characterized. There is evidence that some disulfide bonds in membrane proteins are labile while others that are probably structurally important are not exposed at the protein surface. We have developed a proteomic/mass spectrometry method to screen for and identify non-structural, redox-labile disulfide bonds in leucocyte cell-surface proteins. These labile disulfide bonds are common, with several classes of proteins being identified and around 30 membrane proteins regularly identified under different reducing conditions including using enzymes such as thioredoxin. The proteins identified include integrins, receptors, transporters and cell-cell recognition proteins. In many cases, at least one cysteine residue was identified by mass spectrometry as being modified by the reduction process. In some cases, functional changes are predicted (e.g. in integrins and cytokine receptors) but the scale of molecular changes in membrane proteins observed suggests that widespread effects are likely on many different types of proteins including enzymes, adhesion proteins and transporters. The results imply that membrane protein activity is being modulated by a 'redox regulator' mechanism.     

Evidence for activation of tissue factor by an allosteric disulfide bond

Tissue Factor (TF) is the mammalian plasma membrane cofactor responsible for initiation of blood coagulation. Binding of blood coagulation factor VIIa to TF activates the serine proteinase zymogens factors IX and X by limited proteolysis leading to the formation of a thrombin and fibrin meshwork that stabilizes the thrombus. TF on the plasma membrane of cells resides mostly in a cryptic configuration, which rapidly transforms into an active configuration in response to certain stimuli. The extracellular part of TF consists of two fibronectin type III domains. The disulfide bond in the membrane proximal domain (Cys186-Cys209) is atypical for domains of this type in that it links adjacent strands in the same beta sheet, what we have called an allosteric bond. Ablation of the allosteric disulfide by mutating both cysteine residues severely impairs procoagulant activity. The thiol-alkylating agents N-ethylmaleimide and methyl methanethiolsulfonate block TF activation by ionomycin, while the thiol-oxidizing agent HgCl2 and dithiol cross-linkers promote activation. TF activation could not be explained by exposure of phosphatidylserine on the outer leaflet of the plasma membrane. Cryptic TF contained unpaired cysteine thiols that were depleted upon activation, and de-encryption was associated with a change in the conformation of the membrane-proximal domain. These findings imply that the Cys186-Cys209 disulfide bond is reduced in the cryptic form of TF and that activation involves formation of the disulfide. It is likely that formation of this disulfide bond changes the conformation of the domain that facilitates productive binding of factors IX and X.     

Channel domain of colicin A modifies the dimeric organization of its immunity protein

Proteins conferring immunity against pore-forming colicins are localized in the Escherichia coli inner membrane. Their protective effects are mediated by direct interaction with the C-terminal domain of their cognate colicins. Cai, the immunity protein protecting E. coli against colicin A, contains four cysteine residues. We report cysteine cross-linking experiments showing that Cai forms homodimers. Cai contains four transmembrane segments (TMSs), and dimerization occurs via the third TMS. Furthermore, we observe the formation of intramolecular disulfide bonds that connect TMS2 with either TMS1 or TMS3. Co-expression of Cai with its target, the colicin A pore-forming domain (pfColA), in the inner membrane prevents the formation of intermolecular and intramolecular disulfide bonds, indicating that pfColA interacts with the dimer of Cai and modifies its conformation. Finally, we show that when Cai is locked by disulfide bonds, it is no longer able to protect cells against exogenous added colicin A.     

Defining the native disulfide topology in the somatomedin B domain of human vitronectin

The N-terminal 44 amino acid residues of the human plasma glycoprotein vitronectin, known as the somatomedin B (SMB) domain, mediates the interaction between vitronectin and plasminogen activator inhibitor 1 (PAI-1) in a variety of important biological processes. Despite the functional importance of the Cys-rich SMB domain, how its four disulfide bridges are arranged in the molecule remains highly controversial, as evidenced by three different disulfide connectivities reported by several laboratories. Using native chemical ligation and orthogonal protection of selected Cys residues, we chemically synthesized all three topological analogs of SMB with predefined disulfide connectivities corresponding to those previously published. In addition, we oxidatively folded a fully reduced SMB in aqueous solution, and prepared, by CNBr cleavage, the N-terminal segment of 51 amino acid residues of intact vitronectin purified from human blood. Proteolysis coupled with mass spectrometric analysis and functional characterization using a surface plasmon resonance based vitronectin-PAI-1-SMB competition assay allowed us to conclude that 1) only the Cys(5)-Cys(21), Cys(9)-Cys(39), Cys(19)-Cys(32), and Cys(25)-Cys(31) connectivity is present in native vitronectin; 2) only the native disulfide connectivity is functional; and 3) the native disulfide pairings can be readily formed during spontaneous (oxidative) folding of the SMB domain in vitro. Our results unequivocally define the native disulfide topology in the SMB domain of human vitronectin, providing biochemical as well as functional support to the structural findings on a recombinant SMB domain by Read and colleagues (Zhou, A., Huntington, J. A., Pannu, N. S., Carrell, R. W., and Read, R. J. (2003) Nat. Struct. Biol. 10, 541-544).