Disulfide Bonds Play a Critical Role in the Structure and Function of the Receptor-binding Domain of the SARS-CoV-2 Spike Antigen

The current coronavirus pandemic is exerting a tremendously detrimental impact on global health. The Spike proteins of coronaviruses, responsible for cell receptor binding and viral internalization, possess multiple and frequently conserved disulfide bonds raising the question about their role in these proteins. Here, we present a detailed structural and functional investigation of the disulfide bonds of the SARS-CoV-2 Spike receptor-binding domain (RBD). Molecular dynamics simulations of the RBD predict increased flexibility of the surface loops when the four disulfide bonds of the domain are reduced. This flexibility is particularly prominent for the disulfide bond-containing surface loop (residues 456–490) that participates in the formation of the interaction surface with the Spike cell receptor ACE2. In vitro, disulfide bond reducing agents affect the RBD secondary structure, lower its melting temperature from 52 °C to 36–39 °C and decrease its binding affinity to ACE2 by two orders of magnitude at 37 °C. Consistent with these in vitro findings, the reducing agents tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT) were able to inhibit viral replication at low millimolar levels in cell-based assays. Our research demonstrates the mechanism by which the disulfide bonds contribute to the molecular structure of the RBD of the Spike protein, allowing the RBD to execute its viral function.     

Mapping of Disulfide Bonds within the Amino-terminal Extracellular Domain of the Inhibitory Glycine Receptor

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 nAChRα1 ECD, we generated a model of the human GlyRα1 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 GlyRα1 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.     

Intermonomer Hydrogen Bonds Enhance GxxxG-Driven Dimerization of the BNIP3 Transmembrane Domain: Roles for Sequence Context in Helix-Helix Association in Membranes

We determined the sequence dependence of human BNIP3 transmembrane domain dimerization using the biological assay TOXCAT. Mutants in which intermonomer hydrogen bonds between Ser172 and His173 are abolished show moderate interaction, indicating that side-chain hydrogen bonds contribute to dimer stability but are not essential to dimerization. Mutants in which a GxxxG motif composed of Gly180 and Gly184 has been abolished show little or no interaction, demonstrating the critical nature of the GxxxG motif to BNIP3 dimerization. These findings show that side-chain hydrogen bonds can enhance the intrinsic dimerization of a GxxxG motif and that sequence context can control how hydrogen bonds influence helix-helix interactions in membranes. The dimer interface mapped by TOXCAT mutagenesis agrees closely with the interfaces observed in the NMR structure and inferred from mutational analysis of dimerization on SDS-PAGE, showing that the native dimer structure is retained in detergents. We show that TOXCAT and SDS-PAGE give complementary and consistent information about BNIP3 transmembrane domain dimerization: TOXCAT is insensitive to mutations that have modest effects on self-association in detergents but readily discriminates among mutations that completely disrupt detergent-resistant dimerization. The close agreement between conclusions reached from TOXCAT and SDS-PAGE data for BNIP3 suggests that accurate estimates of the relative effects of mutations on native-state protein-protein interactions can be obtained even when the detergent environment is strongly disruptive.     

The MDM2 RING Domain and Central Acidic Domain Play Distinct Roles in MDM2 Protein Homodimerization and MDM2-MDMX Protein Heterodimerization

The oncoprotein murine double minute 2 (MDM2) is an E3 ligase that plays a prominent role in p53 suppression by promoting its polyubiquitination and proteasomal degradation. In its active form, MDM2 forms homodimers as well as heterodimers with the homologous protein murine double minute 4 (MDMX), both of which are thought to occur through their respective C-terminal RING (really interesting new gene) domains. In this study, using multiple MDM2 mutants, we show evidence suggesting that MDM2 homo- and heterodimerization occur through distinct mechanisms because MDM2 RING domain mutations that inhibit MDM2 interaction with MDMX do not affect MDM2 interaction with WT MDM2. Intriguingly, deletion of a portion of the MDM2 central acidic domain selectively inhibits interaction with MDM2 while leaving intact the ability of MDM2 to interact with MDMX and to ubiquitinate p53. Further analysis of an MDM2 C-terminal deletion mutant reveals that the C-terminal residues of MDM2 are required for both MDM2 and MDMX interaction. Collectively, our results suggest a model in which MDM2-MDMX heterodimerization requires the extreme C terminus and proper RING domain structure of MDM2, whereas MDM2 homodimerization requires the extreme C terminus and the central acidic domain of MDM2, suggesting that MDM2 homo- and heterodimers utilize distinct MDM2 domains. Our study is the first to report mutations capable of separating MDM2 homo- and heterodimerization.     

The Disulfide Bonds in Glycoprotein E2 of Hepatitis C Virus Reveal the Tertiary Organization of the Molecule

Hepatitis C virus (HCV), a major cause of chronic liver disease in humans, is the focus of intense research efforts worldwide. Yet structural data on the viral envelope glycoproteins E1 and E2 are scarce, in spite of their essential role in the viral life cycle. To obtain more information, we developed an efficient production system of recombinant E2 ectodomain (E2e), truncated immediately upstream its trans-membrane (TM) region, using Drosophila melanogaster cells. This system yields a majority of monomeric protein, which can be readily separated chromatographically from contaminating disulfide-linked aggregates. The isolated monomeric E2e reacts with a number of conformation-sensitive monoclonal antibodies, binds the soluble CD81 large external loop and efficiently inhibits infection of Huh7.5 cells by infectious HCV particles (HCVcc) in a dose-dependent manner, suggesting that it adopts a native conformation. These properties of E2e led us to experimentally determine the connectivity of its 9 disulfide bonds, which are strictly conserved across HCV genotypes. Furthermore, circular dichroism combined with infrared spectroscopy analyses revealed the secondary structure contents of E2e, indicating in particular about 28% β-sheet, in agreement with the consensus secondary structure predictions. The disulfide connectivity pattern, together with data on the CD81 binding site and reported E2 deletion mutants, enabled the threading of the E2e polypeptide chain onto the structural template of class II fusion proteins of related flavi- and alphaviruses. The resulting model of the tertiary organization of E2 gives key information on the antigenicity determinants of the virus, maps the receptor binding site to the interface of domains I and III, and provides insight into the nature of a putative fusogenic conformational change.     

Deciphering Structural and Functional Roles of Individual Disulfide Bonds of the Mitochondrial Sulfhydryl Oxidase Erv1p

Erv1p is a FAD-dependent sulfhydryl oxidase of the mitochondrial intermembrane space. It contains three conserved disulfide bonds arranged in two CXXC motifs and one CX₁₆C motif. Experimental evidence for the specific roles of the individual disulfide bonds is lacking. In this study, structural and functional roles of the disulfides were dissected systematically using a wide range of biochemical and biophysical methods. Three double cysteine mutants with each pair of cysteines mutated to serines were generated. All of the mutants were purified with the normal FAD binding properties as the wild type Erv1p, showing that none of the three disulfides are essential for FAD binding. Thermal denaturation and trypsin digestion studies showed that the CX₁₆C disulfide plays an important role in stabilizing the folding of Erv1p. To understand the functional role of each disulfide, small molecules and the physiological substrate protein Mia40 were used as electron donors in oxygen consumption assays. We show that both CXXC disulfides are required for Erv1 oxidase activity. The active site disulfide is well protected thus requires the shuttle disulfide for its function. Although both mutants of the CXXC motifs were individually inactive, Erv1p activity was partially recovered by mixing these two mutants together, and the recovery was rapid. Thus, we provided the first experimental evidence of electron transfer between the shuttle and active site disulfides of Erv1p, and we propose that both intersubunit and intermolecular electron transfer can occur.Disulfide bonds play very important roles in the structure and function of many proteins by stabilizing protein folding and/or acting as thiol/disulfide redox switches. The process of disulfide formation is catalyzed by dedicated enzymes in vivo (1–4). Erv1p is a FAD-dependent sulfhydryl oxidase located in the Saccharomyces cerevisiae mitochondrial intermembrane space (4–6). It is an essential component of the redox regulated Mia40/Erv1 import and assembly pathway used by many of the cysteine-containing intermembrane space proteins, such as members of the “small Tim” and Cox17 families (7–10). Upon import of a Cys-reduced substrate, Mia40 interacts with the substrate via intermolecular disulfide bond and shuttles a disulfide to its substrate. Although oxidized Mia40 promotes disulfide bond formation in the substrates, Erv1p functions in catalyzing reoxidation of the reduced Mia40 and/or release of the substrate (11–13).The common features for the FAD-dependent sulfhydryl oxidases are that the enzymes can catalyze the electron transfer from substrate molecules (e.g. protein thiols) through the noncovalent bound FAD cofactor to molecular oxygen or oxidized cytochrome c (14). The sulfhydryl oxidases can be divided into three groups: Ero1 enzymes, multidomain quiesin sulfhydryl oxidases, and single domain Erv (essential for respiration and vegetative growth)/ALR proteins. The yeast Ero1p and the mammalian homologues (Ero1α and Ero1β) are large flavoenzymes present in the ER with at least five disulfide bonds, but only two of the disulfide bonds are conserved. The conserved cysteines are essential for the catalytic activity of Ero1p forming the active site CXXC and shuttle disulfide CX₄C, respectively (15, 16). Furthermore, nonconserved disulfide bonds have been shown recently to be important in regulating the activity of both yeast and mammalian Ero1 (17–19). The second group of oxidases, the multidomain quiesin sulfhydryl oxidases, have important functions in higher eukaryotes (14, 20). Quiesin sulfhydryl oxidases consist of an Erv/ALR module fused to one or more thioredoxin-like domains with two conserved CXXC motifs in the Erv/ALR module. Quiesin sulfhydryl oxidase enzymes are found in many subcellular and extracellular locations, but not in mitochondria. Instead, single domain Erv/ARL enzymes of the third group are found in the 7mitochondria of many eukaryotic cells (21). Erv1p belongs to this single domain Erv/ARL family, which includes the human mitochondrial ARL, plant AtErv1, and yeast Erv2p of the ER lumen.The Erv/ARL enzymes are characterized by a highly conserved central catalytic core of ∼100 amino acids, which includes an active site CXXC motif (Cys¹³⁰–Cys¹³³ for Erv1p), CX₁₆C disulfide bond (Cys¹⁵⁹–Cys¹⁷⁶ for Erv1p), and residues involved in FAD binding (Fig. 1A). Based on the partial crystal structure data of Erv2p (22) and AtErv1 (23), the catalytic core of Erv proteins contains a four-helix bundle forming the noncovalent FAD-binding site with the active site CXXC in close proximity to the isoalloxazine ring of FAD. In addition, the long range CX₁₆C disulfide bond of the Erv proteins brings the short fifth helix to the four-helix bundle in proximity to the adenine ring of FAD (Fig. 1A). Thus, the CX₁₆C disulfide bond is proposed to play a structural role in stabilizing the FAD binding and/or protein folding, but direct experimental evidence to verify the roles is lacking. Apart from the catalytic core, the other parts of the proteins seem flexible and unfolded. Importantly, all members of the Erv/ALR family have at least an additional disulfide bond located in the nonconserved N- or C-terminal region to the catalytic core (Fig. 1B), which is hypothesized as a shuttle disulfide based on the partial crystal structure of Erv2 (22). The hypothesized shuttle disulfide of Erv2p CXC and AtErv1 CX₄C are located in the C terminus, but Erv1p (Cys³⁰–Cys³³) and ALR have a CXXC shuttle disulfide located N-terminal to the catalytic core. Furthermore, structural and chemical data have suggested that Erv/ARL enzymes form homodimer or oligomers in the presence or absence of intermolecular disulfide bonds (5, 23, 24).Open in a separate windowFIGURE 1.Structure and conserved Cys motifs of Erv/ALR enzymes. A, modeled structures of the conserved central catalytic core domain of Erv1p dimer based on the crystal structure data of AtErv1 (Protein Data Bank accession number 2HJ3, residues 73–173, the helix 1 starts with residue 75). The helices of the four-helix bundle and the short fifth helix are labeled from 1 to 5. The two disulfides are shown as yellow spheres, and the cofactor FAD is in red. The Cys¹³⁰–Cys¹³³ is the redox active disulfide located closely to the isoalloxazine ring of FAD. The N and C termini were labeled as N and C, respectively. The structure was generated using Pymol program. B, schematic of the primary structure of yeast, plant, and human sulfhydryl oxidase with the conserved Cys motifs. The conserved central catalytic core regions are shown as black bars, and the nonconserved regions are in gray.Yeast mitochondrial Erv1p contains a total of six Cys residues forming three pairs of disulfide bonds (residues 30–33, 130–133, and 159–176) as described above. Previous studies with single Cys mutants showed that although all three disulfide bonds are essential for Erv1p function in vivo, only Cys¹³⁰–Cys¹³³ disulfide is required for the oxidase activity of Erv1p in vitro (24). The conclusion that only Cys¹³⁰–Cys¹³³ disulfide is required for Erv1p oxidase activity in vitro was based on a study using the artificial substrate DTT² as the electron donor. Abnormal color changes were observed for some of the single Cys mutants of Erv1p in the previous study that were probably caused by protein misfolding or formation of non-native disulfides because of the presence of a redox active but unpaired Cys. It is clear that Cys¹³⁰–Cys¹³³ is the active site disulfide; however, experimental evidence for the role of Cys³⁰–Cys³³ disulfide is lacking, and the specific role played by the unique CX₁₆C motif of Erv proteins is unknown.In this study, we dissected the structural and functional roles of all three individual disulfides of Erv1p systematically. To avoid misfolding via unpaired Cys, three double Cys mutants of Erv1p were generated with each of the disulfides mutated to serines. All three mutants were successfully purified with the normal FAD binding properties of the wild type (WT) Erv1p. Various biophysical and biochemical methods were used to study the folding and oxidase activity of the WT and Erv1p mutants. Both artificial and the natural substrate (Mia40) of Erv1p were used as electron donors to understand the functional mechanism of Erv1p. Our results show that both the first (Cys³⁰–Cys³³) and second (Cys¹³⁰–Cys¹³³) disulfides are essential for Erv1 oxidase activity in vitro. Although none of the three disulfides are essential for FAD binding, the third disulfide (Cys¹⁵⁹–Cys¹⁷⁶) plays an important role in stabilizing the folding of Erv1p. More importantly, this study provided direct experimental evidence to show that Cys³⁰–Cys³³ functionally acts as a shuttle disulfide passing electrons to the active site Cys¹³⁰–Cys¹³³ disulfide. Moreover, the electron transfer seems to occur through both intersubunit and intermolecular interactions.     

Novel Disulfide Bond-Mediated Dimerization of the CARD Domain Was Revealed by the Crystal Structure of CARMA1 CARD

CARMA1, BCL10 and MALT1 form a large molecular complex known as the CARMA1 signalosome during lymphocyte activation. Lymphocyte activation via the CARMA1 signalosome is critical to immune response and linked to many immune diseases. Despite the important role of the CARMA1 signalosome during lymphocyte activation and proliferation, limited structural information is available. Here, we report the dimeric structure of CARMA1 CARD at a resolution of 3.2 Å. Interestingly, although CARMA1 CARD has a canonical six helical-bundles structural fold similar to other CARDs, CARMA1 CARD shows the first homo-dimeric structure of CARD formed by a disulfide bond and reveals a possible biologically important homo-dimerization mechanism.     

Conformational Dynamics and Structural Plasticity Play Critical Roles in the Ubiquitin Recognition of a UIM Domain

Ubiquitin-interacting motifs (UIMs) are an important class of protein domains that interact with ubiquitin or ubiquitin-like proteins. These approximately 20-residue-long domains are found in a variety of ubiquitin receptor proteins and serve as recognition modules towards intracellular targets, which may be individual ubiquitin subunits or polyubiquitin chains attached to a variety of proteins. Previous structural studies of interactions between UIMs and ubiquitin have shown that UIMs adopt an extended structure of a single α-helix, containing a hydrophobic surface with a conserved sequence pattern that interacts with key hydrophobic residues on ubiquitin. In light of this large body of structural studies, details regarding the presence and the roles of structural dynamics and plasticity are surprisingly lacking. In order to better understand the structural basis of ubiquitin-UIM recognition, we have characterized changes in the structure and dynamics of ubiquitin upon binding of a UIM domain from the yeast Vps27 protein. The solution structure of a ubiquitin-UIM fusion protein designed to study these interactions is reported here and found to consist of a well-defined ubiquitin core and a bipartite UIM helix. Moreover, we have studied the plasticity of the docking interface, as well as global changes in ubiquitin due to UIM binding at the picoseconds-to-nanoseconds and microseconds-to-milliseconds protein motions by nuclear magnetic resonance relaxation. Changes in generalized-order parameters of amide groups show a distinct trend towards increased structural rigidity at the UIM-ubiquitin interface relative to values determined in unbound ubiquitin. Analysis of ¹⁵N Carr-Purcell-Meiboom-Gill relaxation dispersion measurements suggests the presence of two types of motions: one directly related to the UIM-binding interface and the other induced to distal parts of the protein. This study demonstrates a case where localized interactions among protein domains have global effects on protein motions at timescales ranging from picoseconds to milliseconds.     

Role of Intermolecular Disulfide Bonds of the Organic Solvent-Stable PST-01 Protease in Its Organic Solvent Stability

The PST-01 protease is secreted by the organic solvent-tolerant microorganism Pseudomonas aeruginosa PST-01 and is stable in the presence of various organic solvents. Therefore, the PST-01 strain and the PST-01 protease are very useful for fermentation and reactions in the presence of organic solvents, respectively. The organic solvent-stable PST-01 protease has two disulfide bonds (between Cys-30 and Cys-58 and between Cys-270 and Cys-297) in its molecule. Mutant PST-01 proteases in which one or both of the disulfide bonds were deleted were constructed by site-directed mutagenesis, and the effect of the disulfide bonds on the activity and the various stabilities was investigated. The disulfide bond between Cys-270 and Cys-297 in the PST-01 protease was found to be essential for its activity. The disulfide bond between Cys-30 and Cys-58 played an important role in the organic solvent stability of the PST-01 protease.     

Novel Pectate Lyase Genes of Heterodera glycines Play Key Roles in the Early Stage of Parasitism

Pectate lyases are known to play a key role in pectin degradation by catalyzing the random cleavage of internal polymer linkages (endo-pectinases). In this paper, four novel cDNAs, designated Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7, that encode pectate lyases were cloned and characterized from the soybean cyst nematode, Heterodera glycines. The predicted protein sequences of HG-PEL-3, HG-PEL-4 and HG-PEL-6 differed significantly in both their amino acid sequences and their genomic structures from other pectate lyases of H. glycines (HG-PEL-1, HG-PEL-2 and HG-PEL-7). A phylogenetic study revealed that the pectate lyase proteins of H. glycines are clustered into distinct clades and have distinct numbers and positioning of introns, which suggests that the pectate lyase genes of H. glycines may have evolved from at least two ancestral genes. A Southern blot analysis revealed that multiple Hg-pel-6-like genes were present in the H. glycines genome. In situ hybridization showed that four novel pectate lyases (Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7) were actively transcribed in the subventral esophageal gland cells. A semi-quantitative RT-PCR assay supported the finding that the expression of these genes was strong in the egg, pre-parasitic second-stage juvenile (J2) and early parasitic J2 stages and that it declined in further developmental stages of the nematode. This expression pattern suggests that these proteins play a role in the migratory phase of the nematode life cycle. Knocking down Hg-pel-6 using in vitro RNA interference resulted in a 46.9% reduction of the number of nematodes that invaded the plants and a 61.5% suppression of the development of H. glycines females within roots compared to the GFP-dsRNA control. Plant host-derived RNAi induced the silencing of the Hg-pel-6gene, which significantly reduced the nematode infection levels at 7 Days post inoculation (dpi). Similarly, this procedure reduced the number of female adults at 40 dpi, which suggests the important roles of this gene in the early stages of parasitism. Our combined data suggest that two types of pectate lyases are present in the H. glycines genome and may have different roles during infection.     

The Sinorhizobium meliloti LpxXL and AcpXL Proteins Play Important Roles in Bacteroid Development within Alfalfa

Free-living Sinorhizobium meliloti lpxXL and acpXL mutants lack lipid A very-long-chain fatty acids (VLCFAs) and have reduced competitiveness in alfalfa. We demonstrate that LpxXL and AcpXL play important but distinct roles in bacteroid development and that LpxXL is essential for the modification of S. meliloti bacteroid lipid A with VLCFAs.Sinorhizobium meliloti and Brucella abortus form chronic intracellular infections within legumes and mammalian hosts, respectively (3, 20), and their BacA proteins play essential roles in these processes (8, 12). The precise function(s) of the BacA proteins has not been resolved, but free-living S. meliloti and B. abortus mutants lacking BacA have increased resistance to the glycopeptide bleomycin (9, 12) and there are ∼50% decreases in their lipid A very-long-chain fatty acid (VLCFA) contents (4, 7). It has also been determined that the increased resistance of an S. meliloti bacA null mutant to bleomycin and a truncated eukaryotic peptide, Bac7_1-16, is independent of its lipid A VLCFA alteration (6, 15). Together, these findings support a model in which BacA could have multiple nonoverlapping functions which lead to lipid A VLCFA modification and peptide uptake. The fact that two symbiotically defective S. meliloti BacA site-directed mutants (Q193G and R389G) (13) show defects in BacA-mediated lipid A VLCFA modification (4) but are still capable of peptide uptake (15) suggests that the S. meliloti lipid A VLCFA modification could play a key role in the symbiosis of this organism with alfalfa.Since the mechanism by which BacA leads to the lipid A VLCFA modification has not been resolved (4), S. meliloti mutants were constructed with mutations in the lpxXL and acpXL genes, which encode a lipid A VLCFA acyl transferase and a VLCFA acyl carrier protein directly involved in the biosynthesis of VLCFA-modified lipid A (5, 23). The S. meliloti lpxXL and acpXL mutants completely lack the lipid A VLCFA modification in their free-living states, but, unlike the S. meliloti bacA null mutant, these mutants can still form a successful symbiosis with alfalfa (5, 8, 23). However, the fact that the S. meliloti acpXL and lpxXL mutants are substantially less competitive in the alfalfa symbiosis than the parent strain (5, 23) indicates that the AcpXL and LpxXL proteins play important roles in at least one of the stages of the alfalfa symbiosis. Although the free-living S. meliloti acpXL and lpxXL mutants completely lack the lipid A VLCFA, they produce different species of lipid A (5). For example, in the absence of AcpXL, S. meliloti is able to modify lipid A with either C16:0 or C18:0 in the position normally modified with the VLCFA in the parent strain lipid A. This process is LpxXL dependent, as it does not occur in either an S. meliloti lpxXL single mutant or an S. meliloti acpXL lpxXL double mutant. In addition, since a Rhizobium leguminosarum acpXL mutant completely lacks the lipid A VLCFA modification in its free-living state but its lipid A is partially modified with the VLCFA to ∼58% of the amount in the parent strain lipid A during passage through peas (25), it is also possible that the S. meliloti acpXL mutant and possibly the S. meliloti lpxXL mutant undergo further lipid A changes during the interaction with alfalfa.In this study, we found that LpxXL and AcpXL play important but distinct roles in S. meliloti bacteroid development during alfalfa symbiosis. Additionally, we demonstrated that there is a minor host-induced AcpXL-independent mechanism by which S. meliloti bacteroid lipopolysaccharide (LPS) can be modified with the VLCFA. In contrast, we found that the LpxXL protein plays an essential role in the modification of S. meliloti bacteroids with VLCFAs.     

B Cells Play Key Roles in Th2-Type Airway Immune Responses in Mice Exposed to Natural Airborne Allergens

Humans are frequently exposed to various airborne allergens. In addition to producing antibodies, B cells participate in immune responses via various mechanisms. The roles of B cells in allergic airway inflammation and asthma have been controversial. We examined the functional importance of B cells in a mouse model of asthma, in which mice were exposed repeatedly to common airborne allergens. Naïve wild-type BALB/c mice or B cell-deficient JH^−/− mice were exposed intranasally to a cocktail of allergen extracts, including Alternaria, Aspergillus, and house dust mite, every other day for two weeks. Ovalbumin was included in the cocktail to monitor the T cell immune response. Airway inflammation, lung pathology, and airway reactivity were analyzed. The airway exposure of naïve wild type mice to airborne allergens induced robust eosinophilic airway inflammation, increased the levels of Th2 cytokines and chemokines in the lung, and increased the reactivity to inhaled methacholine. These pathological changes and immune responses were attenuated in B cell-deficient JH^−/− mice. The allergen-induced expansion of CD4⁺ T cells was impaired in the lungs and draining lymph nodes of JH^−/− mice. Furthermore, lymphocytes from JH^−/− mice failed to produce Th2 cytokines in response to ovalbumin re-stimulation in vitro. Our results suggest that B cells are required for the optimal development of Th2-type immune responses and airway inflammation when exposed to common airborne allergens. The therapeutic targeting of B cells may be beneficial to treat asthma in certain patients.     

Disulfide Linkages and a Three-Dimensional Structure Model of the Extracellular Ligand-binding Domain of Guanylyl Cyclase C

Guanylyl cyclase C (GC-C) is a single-transmembrane receptor that is specifically activated by endogenous ligands, including guanylin, and the exogenous ligand, heat-stable enterotoxin. Using combined HPLC separation and MS analysis techniques the positions of the disulfide linkages in the extracellular ligand-binding domain (ECD) of GC-C were determined to be between Cys⁷–Cys⁹⁴, Cys⁷²–Cys⁷⁷, Cys¹⁰¹–Cys¹²⁸ and Cys¹⁷⁹–Cys²²⁶. Furthermore, a three-dimensional structural model of the ECD was constructed by homology modeling, using the structure of the ECD of GC-A as a template (van den Akker et al., 2000, Nature, 406: 101–104) and the information of the disulfide linkages. Although the GC-C model was similar to the known structure of GC-A, importantly its ligand-binding site appears to be located on the quite different region from that in GC-A.     

Role of Dimerization in the Catalytic Properties of the Escherichia coli Disulfide Isomerase DsbC

The bacterial protein-disulfide isomerase DsbC is a homodimeric V-shaped enzyme that consists of a dimerization domain, two α-helical linkers, and two opposing thioredoxin fold catalytic domains. The functional significance of the two catalytic domains of DsbC is not well understood yet. We have engineered heterodimer-like DsbC derivatives covalently linked via (Gly₃-Ser) flexible linkers. We either inactivated one of the catalytic sites (CGYC), or entirely removed one of the catalytic domains while maintaining the putative binding area intact. Variants having a single active catalytic site display significant levels of isomerase activity. Furthermore, mDsbC[H45D]-dim[D53H], a DsbC variant lacking an entire catalytic domain but with an intact dimerization domain, also showed isomerase activity, albeit at lower levels. In addition, the absence of the catalytic domain allowed this protein to catalyze in vivo oxidation. Our results reveal that two catalytic domains in DsbC are not essential for disulfide bond isomerization and that a determining feature in isomerization is the availability of a substrate binding domain.Disulfide bonds are critical for the proper folding and structural stability of many exocytoplasmic proteins. The Dsb family of thiol:disulfide oxidoreductase enzymes catalyzes oxidative protein folding in the periplasm of Escherichia coli by means of two independent pathways (1–3). In the DsbA-DsbB oxidation pathway, DsbA, a very strong oxidant, catalyzes the formation of disulfide bonds on newly translocated proteins (4). The DsbA disulfide is rapidly recycled by DsbB, a membrane protein that transfers electrons from DsbA onto quinones (5–7). In the DsbC-DsbD isomerization pathway, non-native disulfides are reduced or rearranged by DsbC. DsbC is maintained in a reduced, catalytically active state via the transfer of electrons from the inner membrane protein DsbD that in turn accepts electrons from thioredoxin 1 and ultimately from NADPH (via thioredoxin reductase) within the cytoplasm (8, 9). Large kinetic barriers keep the oxidation and isomerization pathways isolated, preventing the establishment of a futile cycle of electron transfer. Accordingly, reactions between enzymes of the two pathways, for example the oxidation of DsbC by DsbB or the reduction of DsbA by DsbD, are 10³–10⁷-fold slower than the physiologically relevant DsbA-DsbB and DsbC-DsbD reactions (10). Nonetheless, the kinetic barrier between DsbB and DsbC can be breached by introducing mutations that result in structural changes in DsbC (11, 12).DsbC is a homodimer with each monomer comprising an N-terminal dimerization domain and a C-terminal thioredoxin-like catalytic domain fused by an α-helical linker. The crystal structure of DsbC reveals that the two monomers come together to form a V-shaped protein. The inner surface of the resulting cleft is patched with uncharged and hydrophobic residues suggesting an important role in the binding of substrate proteins. The active sites comprising the sequence Cys⁹⁸-Gly⁹⁹-Tyr¹⁰⁰-Cys¹⁰¹ in each of the monomeric subunits are located in the arms of the “V” facing each other (13). Isomerization involves an attack onto a substrate disulfide by Cys⁹⁸ resulting in the formation of a mixed disulfide, which then is resolved by either another cysteine from the substrate or by Cys¹⁰¹ from DsbC (14, 15). Besides its isomerase activity, DsbC is also known to display chaperone activity preventing protein aggregation during refolding (16). In E. coli, disulfide bond isomerization is the limiting step in the oxidative folding of many heterologous proteins that contain multiple cysteines. Overexpression of DsbC has been shown to enhance the yield of proteins such as human nerve growth factor, human tissue plasminogen activator (tPA)² and immunoglobulins (17–19).DsbC is topologically analogous to the eukaryotic protein-disulfide isomerase (PDI). The structural similarities between the two enzymes may have resulted from convergent evolution by thioredoxin-like domain replication in the case of PDI and domain recruitment in DsbC (20, 21). PDI comprises two thioredoxin-like catalytic domains (a and a′) separated by two non-catalytic domains (b and b′), in addition to a c domain (22). In PDI, the catalytic domains are different and functionally nonequivalent (23). Substrate binding is mediated primarily by the b′ domain; the two catalytic domains, a and a′, can catalyze oxidation of small model peptides indicating that they must also have low substrate binding affinity (24).The DsbC monomer is essentially devoid of RNase A isomerase activity (25). Sun and Wang (44) reported that DsbC with one catalytic site impaired by carboxymethylation is also essentially inactive but, in separate studies, Zapun et al. (26) did not detect cooperativity between the two catalytic sites indicating that they function independently of each other (26). Moreover, unlike PDI, the significance of the putative peptide binding cleft of DsbC on disulfide isomerization has not been ascertained. However, while DsbA or TrxA with a PDI active site dipeptide (CGHC) display very little isomerase activity in vitro and in vivo (27–29), we recently showed that upon fusion to a dimerization region that provides a putative substrate binding surface (the E. coli peptidyl proline isomerase FkpA) they acquire the ability to assist the folding of periplasmically expressed multidisulfide heterologous proteins (30).In the present work, we engineered heterodimer-like covalently linked DsbC derivatives in which one of the catalytic sites has been inactivated (Fig. 1A) or one of the catalytic domains has been entirely removed while maintaining the intact peptide binding cleft (which is normally formed by association of the N-terminal domains of the two monomers) (Fig. 3A). We show that DsbC forced monomers with one functional active site, or with one thioredoxin domain only, display significant isomerization activity. Interestingly, the latter variant is partially reduced in vivo indicating that the presence of both thioredoxin domains is important for the avoidance of protein oxidation by DsbB.Open in a separate windowFIGURE 1.A, protein structure of DsbC, and molecular models of mDsbC-mDsbC and the single active site covalently linked mutants. Dimerization domains are shown in gray, thioredoxin domains in black, and the active sites in white. B, gel filtration FPLC of DsbC and linked variants. Purified proteins were run on a Superdex^TM 200 column in PBS, 10% glycerol buffer.Open in a separate windowFIGURE 3.A, molecular model of mDsbC-dim. Dimerization domains are shown in gray, thioredoxin domain in black, and catalytic site in white. B, gel filtration FPLC of mDsbC-dim as compared with DsbC. Purified proteins were run on a Superdex^TM 200 column in PBS, 10% glycerol buffer. C, MALS measurement of the molar masses of the components of mDsbC-dim together with their hydrodynamic radii. The data show monomeric, dimeric, and tetrameric states. The relative concentrations were determined by the refractive index differences.     

Influence of Disulfide Bonds on the Induction of Helical Conformation in Proteins

The effect(s) of TFE (2,2,2-trifluoroethanol) on three different conformational states (native, denatured, and carboxymethylated) of CTX III and RNase A has been examined. Contrary to the general belief, the results of the present study reveal that TFE can induce helical conformation in a protein which has no sequence propensity to form a helix. It is found that the helix induction in TFE is intricately related to the destabilization of the tertiary structural conformation in proteins. More importantly, the disulfide bonds in proteins are found to have significant influence on the TFE-mediated helix induction. The results obtained in this study strongly suggest that information pertaining to the influence of disulfide bonds on helix induction need to be considered to improve the accuracy of secondary structure prediction algorithms.     

The Role of Intermolecular Disulfide Bonds in Stabilizing the Structure of Peroxiredoxins

The comparative characterization of thermal stability of human peroxiredoxins 1–6 (Prx1–Prx6) has been performed by physicochemical and biochemical methods and the role of disulfide bonds in stabilizing their structure has been shown. Prx1 and Prx2 among the tested peroxiredoxins exhibit the highest peroxidase activity and thermal stability. Prx1 and Prx2 are more than 2 times more active on average with H₂O₂ and tert-butyl hydroperoxide as substrates compared to other peroxiredoxins and retain at least 50% activity after 30 min heating at a temperature of 64°C, which is more than 10°C higher compared to Prx3–Prx6. The reduction of the disulfide bonds between Prx1 and Prx2 leads to a decrease of their thermal stability, comparable to the thermal stability of Prx3–Prx6, which confirms the important role of the intermolecular S–S bonds in stabilizing the structure of these proteins.