Intermolecular Disulfide Bonds Research Articles

Abstract 449: Dimerization of Sortilin Regulates its Trafficking to Extracellular Vesicles and Leads to Secretion of Soluble Sortilin Homodimers, a Potential Novel Biomarker

Objective: Extracellular vesicles (EVs) play a critical role in intercellular communication by transferring microRNAs, lipids and proteins to neighboring cells. Sortilin, a sorting receptor that directs target proteins to the secretary or endocytic compartments of cells, is found in both EVs and cells. In many diseases, including cardiovascular calcification and cancer, sortilin expression levels are atypically high. We previously demonstrated that sortilin promotes vascular calcification via its trafficking of tissue-nonspecific alkaline phosphatase to EVs. Therefore, suppressing trafficking of sortilin to EVs may act as a novel therapy against EV-associated diseases. However, the precise mechanisms of sortilin trafficking remain to be determined. In this study, we hypothesized that dimerization of sortilin regulates its trafficking to EVs. Approach and Results: We found that sortilin forms a homodimer with an intermolecular disulfide bond (IDB) at Cysteine783 (Cys783) residue. Palmitoylation and the IDB formation compete for Cys783. Formation of the IDB led to trafficking of sortilin to EVs by preventing palmitoylation, which further promoted sortilin trafficking to the Golgi apparatus. Moreover, addition of sortilin-derived propeptide decreased sortilin homodimers within EVs, suggesting that the propeptide binding suppresses sortilin trafficking to EVs through prevention of its dimerization. We also found that soluble sortilin secreted by cells overexpressing full-length sortilin forms homodimers with IDBs using various commercially available antibodies against sortilin, thus providing an opportunity for diagnosis of EV-associated diseases by detection of sortilin homodimers in patients. Conclusions: Sortilin is transported to EVs via the formation of homodimers with an IDB, which is endogenously regulated by its own propeptide. Therefore, inhibiting sortilin dimerization may provide a novel therapy against EV-associated diseases, including cardiovascular calcification and cancer. In addition, soluble sortilin homodimer with IDBs could be detected and used as a novel diagnostic biomarker for EV-associated diseases.

Dimerization of the cellular prion protein inhibits propagation of scrapie prions

A central step in the pathogenesis of prion diseases is the conformational transition of the cellular prion protein (PrPC) into the scrapie isoform, denoted PrPSc Studies in transgenic mice have indicated that this conversion requires a direct interaction between PrPC and PrPSc; however, insights into the underlying mechanisms are still missing. Interestingly, only a subfraction of PrPC is converted in scrapie-infected cells, suggesting that not all PrPC species are suitable substrates for the conversion. On the basis of the observation that PrPC can form homodimers under physiological conditions with the internal hydrophobic domain (HD) serving as a putative dimerization domain, we wondered whether PrP dimerization is involved in the formation of neurotoxic and/or infectious PrP conformers. Here, we analyzed the possible impact on dimerization of pathogenic mutations in the HD that induce a spontaneous neurodegenerative disease in transgenic mice. Similarly to wildtype (WT) PrPC, the neurotoxic variant PrP(AV3) formed homodimers as well as heterodimers with WTPrPC Notably, forced PrP dimerization via an intermolecular disulfide bond did not interfere with its maturation and intracellular trafficking. Covalently linked PrP dimers were complex glycosylated, GPI-anchored, and sorted to the outer leaflet of the plasma membrane. However, forced PrPC dimerization completely blocked its conversion into PrPSc in chronically scrapie-infected mouse neuroblastoma cells. Moreover, PrPC dimers had a dominant-negative inhibition effect on the conversion of monomeric PrPC Our findings suggest that PrPC monomers are the major substrates for PrPSc propagation and that it may be possible to halt prion formation by stabilizing PrPC dimers.

Interaction of the GntR-family transcription factor Sll1961 with thioredoxin in the cyanobacterium Synechocystis sp. PCC 6803

Changes in the redox state of the photosynthetic electron transport chain act as a signal to trigger acclimation responses to environmental cues and thioredoxin has been suggested to work as a key factor connecting the redox change with transcriptional regulation in the cyanobacterium Synechocystis sp. PCC 6803. We screened for redox-dependent transcription factors interacting with thioredoxin M (TrxM) and isolated the GntR-type transcription factor Sll1961 previously reported to be involved in acclimation responses of the photosynthetic machinery. Biochemical analyses using recombinant Sll1961 proteins of wild type and mutants of three cysteine residues, C124, C229 and C307, revealed that an intramolecular disulfide bond is formed between C229 and C307 under oxidizing conditions and TrxM can reduce it by attacking C307. Sll1961 exists in a dimeric form of about 80 kDa both under reducing and oxidizing conditions. C124 can form an intermolecular disulfide bond but it is not essential for dimerization. Based on these observations, tertiary structure models of the Sll1961 homodimer and the Sll1961-TrxM complex were constructed.

Characterization of low-abundance species in the active pharmaceutical ingredient of CIGB-300: A clinical-grade anticancer synthetic peptide.

CIGB-300 is a first-in-class synthetic peptide-based drug of 25 amino acids currently undergoing clinical trials in cancer patients. It contains an amidated disulfide cyclic undecapeptide fused to the TAT cell-penetrating peptide through a beta-alanine spacer. CIGB-300 inhibits the CK2-mediated phosphorylation leading to apoptosis of tumor cells in vitro, and in vivo in cancer patients. Despite the clinical development of CIGB-300, the characterization of peptide-related impurities present in the active pharmaceutical ingredient has not been reported earlier. In the decision tree of ICHQ3A(R2) guidelines, the daily doses intake, the abundance, and the identity of the peptide-related species are pivotal nodes that define actions to be taken (reporting, identification, and qualification). For this, purity was first assessed by reverse-phase chromatography (>97%) and low-abundance impurities (≤0.27%) were collected and identified by mass spectrometry. Most of the impurities were generated during peptide synthesis, the spontaneous air oxidation of the reduced peptide, and the lyophilization step. The most abundant impurity, with no biological activity, was the full-length peptide containing Met17 transformed into a sulfoxide residue. Interestingly, parallel and antiparallel dimers of CIGB-300 linked by 2 intermolecular disulfide bonds exhibited a higher antiproliferative activity than the CIGB-300 monomer. Likewise, very low abundance trimers and tetramers of CIGB-300 linked by disulfide bonds (≤0.01%) were also detected. Here we describe for the first time the presence of active dimeric species whose feasibility as novel CIGB-300 derived entities merits further investigation.

“Effect of inhibiting protein conformational changes by introducing disulfide bonds in the transmembrane domains of the Hendra virus fusion protein”

Hendra virus (HeV) is a zoonotic pathogen that causes severe respiratory illness and encephalitis. However, no human vaccine or antiviral treatment is currently available. As a member of the Paramyxoviridae family, HeV consists of a non‐segmented negative‐sense RNA genome and is enclosed within a lipid bilayer which contains the surface glycoproteins known as the attachment protein (G) and the fusion protein (F). The G protein plays a role in target cell receptor binding, and the F protein promotes membrane fusion between the viral envelope and the target cell membrane to allow for viral entry and infection. Previous studies have shown that viral fusion proteins undergo conformational changes to drive fusion. For HeV, the F protein associates as a homotrimer, and research from our group suggests that the transmembrane domains (TMDs) stabilize the protein in its pre‐fusion state.1 Based on this research, we hypothesized that modulation of TMD interactions is critical for initiation and completion of the conformational changes that drive membrane fusion. To test this, three HeV F constructs (T483C/V484C, V484C/N485C, N485C/P486C) were generated with double cysteine mutations near the N‐terminal region of the TMD to study the effect of altered conformational flexibility in this region. Initially, the mutants were analyzed for intermolecular disulfide bond formation. Results showed that the mutants primarily associated in a trimeric form under non‐reducing conditions, in contrast to the wild type (WT) protein which was primarily monomeric, suggesting that the mutant TMDs were linked by disulfide bonds. Further experiments showed that all three mutants expressed in cells at levels similar to WT HeV F. However, cleavage of HeV F to its fusion‐active form was reduced for the three mutants, suggesting that the mutants are trafficked more slowly or undergo less proteolytic processing by the endosomal protease cathepsin L compared to WT HeV F. The mutants were then assessed for their ability to promote membrane fusion. Preliminary fusion studies showed that fusion was dramatically reduced for the mutants, suggesting that TMD dissociation is critical for initial conformational changes in HeV F. Future studies will further examine the conformational changes that can occur when dissociation of the TMDs is blocked by the introduction of disulfide bonds.Support or Funding InformationThe authors gratefully acknowledge funding from the NIH/NIAID, grant R01 AI051517.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MI130004, a Novel Antibody-Drug Conjugate Combining Trastuzumab with a Molecule of Marine Origin, Shows Outstanding In Vivo Activity against HER2-Expressing Tumors.

In the search for novel payloads to design new antibody-drug conjugates (ADC), marine compounds represent an interesting opportunity given their unique chemical features. PM050489 is a marine compound that binds β-tubulin at a new site and disrupts the microtubule network, hence leading to mitotic aberrations and cell death. PM050489 has been conjugated to trastuzumab via Cys residues through a noncleavable linker, and the resulting ADC, named MI130004, has been studied. Analysis of MI130004 delivered data consistent with the presence of two molecules of PM050489 per antibody molecule, likely bound to both sides of the intermolecular disulfide bond connecting the antibody light and heavy chains. The antitumor activity of MI130004 was analyzed in vitro and in vivo in different cell lines of diverse tumor origin (breast, ovary, and gastric cancer) expressing different levels of HER2. MI130004 showed very high in vitro potency and good selectivity for tumor cells that overexpressed HER2. At the cellular level, MI130004 impaired tubulin polymerization, causing disorganization and disintegration of the microtubule network, which ultimately led to mitotic failure, mirroring the effect of its payload. Treatment with MI130004 in mice carrying histologically diverse tumors expressing HER2 induced a long-lasting antitumor effect with statistically significant inhibition of tumor growth coupled with increases in median survival time compared with vehicle or trastuzumab. These results strongly suggest that MI130004 is endowed with remarkable anticancer activity and confirm the extraordinary potential of marine compounds for the design of new ADCs. Mol Cancer Ther; 17(4); 786-94. ©2018 AACR.

Dimerization of sortilin regulates its trafficking to extracellular vesicles

Extracellular vesicles (EVs) play a critical role in intercellular communication by transferring microRNAs, lipids, and proteins to neighboring cells. Sortilin, a sorting receptor that directs target proteins to the secretory or endocytic compartments of cells, is found in both EVs and cells. In many human diseases, including cancer and cardiovascular disorders, sortilin expression levels are atypically high. To elucidate the relationship between cardiovascular disease, particularly vascular calcification, and sortilin expression levels, we explored the trafficking of sortilin in both the intracellular and extracellular milieu. We previously demonstrated that sortilin promotes vascular calcification via its trafficking of tissue-nonspecific alkaline phosphatase to EVs. Although recent reports have noted that sortilin is regulated by multiple post-translational modifications, the precise mechanisms of sortilin trafficking still need to be determined. Here, we show that sortilin forms homodimers with an intermolecular disulfide bond at the cysteine 783 (Cys783) residue, and because Cys783 can be palmitoylated, it could be shared via palmitoylation and an intermolecular disulfide bond. Formation of this intermolecular disulfide bond leads to trafficking of sortilin to EVs by preventing palmitoylation, which further promotes sortilin trafficking to the Golgi apparatus. Moreover, we found that sortilin-derived propeptide decreased sortilin homodimers within EVs. In conclusion, sortilin is transported to EVs via the formation of homodimers with an intermolecular disulfide bond, which is endogenously regulated by its own propeptide. Therefore, we propose that inhibiting dimerization of sortilin acts as a new therapeutic strategy for the treatment of EV-associated diseases, including vascular calcification and cancer.

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.

Expression and characterization of recombinant rattusin, an α-defensin-related peptide with a homodimeric scaffold formed by intermolecular disulfide exchanges

Rattusin is an α-defensin-related peptide isolated from the small intestine of rats. The primary sequence of linear rattusin is composed of 31 amino acids containing five cysteines with a unique spacing pattern. It forms a homodimeric scaffold in which the primary structure occurs in an antiparallel fashion formed by five intermolecular disulfide (SS) bonds. Rattusin is a highly potent antibiotic, which not only exhibits broad-spectrum antimicrobial activity, but also maintains its antimicrobial activity at physiological salt concentrations. Therefore, to develop new antibiotics based on rattusin, structural and functional studies of rattusin should be performed. For this purpose, large amounts of linear rattusin precursor must be obtained through appropriate preparation methods. Therefore, we established a mass production technique for linear rattusin by using recombinant protein expression and purification procedures. We verified that structure and activity of the recombinant rattusin are identical to the chemically synthesized rattusin. The described method for producing recombinant rattusin provides a high yield of rattusin, which can be used to study the biochemical and functional properties of rattusin and for the development of rattusin-based peptide antibiotics.

An inter-subunit disulfide bond of artemin acts as a redox switch for its chaperone-like activity.

Encysted embryos of Artemia are among the most stress-resistant eukaryotes partly due to the massive amount of a cysteine-rich protein termed artemin. High number of cysteine residues in artemin and their intramolecular spatial positions motivated us to investigate the role of the cysteine residues in the chaperone-like activity of artemin. According to the result of Ellman's assay, there are nine free thiols (seven buried and two exposed) and one disulfide bond per monomer of artemin. Subsequent theoretical analysis of the predicted 3D structure of artemin confirmed the data obtained by the spectroscopic study. Native and reduced/modified forms of artemin were also compared with respect to their efficiency in chaperoning activity, tertiary structure, and stability. Since the alkylation and reduction of artemin diminished its chaperone activity, it appears that its chaperoning potential depends on the formation of intermolecular disulfide bond and the presence of cysteine residues. Comparative fluorescence studies on the structure and stability of the native and reduced protein revealed some differences between them. Due to the redox-dependent functional switching of artemin from the less to more active form, it can be finally suggested as a redox-dependent chaperone.

Multiple role of 3-mercaptopyruvate sulfurtransferase: antioxidative function, H2 S and polysulfide production and possible SOx production.

Rat 3-mercaptopyruvate sulfurtransferase (MPST) is a 32 808Da simple protein. Cys247 is a catalytic site, and Cys154 and Cys263 are on the enzyme surface. MPST is found in all tissues, particularly in the kidneys, although the localization of its activity differs in each tissue. In this review, four functions of MPST are reviewed: (i) antioxidative function: Cys247 is redox-sensitive and serves as a redox-sensing switch. It is oxidized to cysteine sulfenate, which has a low redox potential, upon which the enzyme is inactivated. Then, reduced thioredoxin (Trx) with a reducing system (Trx reductase and NADPH) reduces the sulfenate to restore activity; meanwhile, Cys154 and Cys263 form an intermolecular disulfide bond, which serves as another redox-sensing switch. Consequently, Trx specifically cleaves the intermolecular disulfide bond by converting it from the inactive form (dimer) to the active form (monomer). (ii) Hydrogen sulfide and polysulfide production: hydrogen sulfide is produced via reduction of the persulfurated sulfur-acceptor substrate by reduced Trx or Trx with a reducing system; as an alternative process, stable polysulfurated or persulfurated Cys247 as a reaction intermediate is reduced by Trx with a reducing system to release hydrogen sulfide and polysulfides. (iii) Possible sulfur oxide production: sulfur oxides (SO, SO2 and SO3 ) can be produced in the redox cycle of sulfane sulfur formed at the catalytic site Cys247 (Cys-SO- , Cys-SO2- and Cys-SO3- ) as reaction intermediates and released by reduced Trx or Trx with a reducing system. (iv) Possible anxiolytic-like effects: MPST-knockout mice exhibited anxiolytic-like effects.

Evaluating the antioxidant effects of human hair protein extracts

The intrinsically high cysteine content in human hair keratins and keratin associated proteins confer hair its outstanding mechanical strength through the formation of strong intermolecular disulfide bonds. In addition, these proteins offer the potential to be exploited as potent antioxidants. This report presents our findings on the antioxidant effects of human hair protein extracts and their consequent protective role against oxidative stress in human dermal fibroblast (HDF) cultures. Protein extracts were obtained from human hair using sodium sulfide as the reducing agent, and characterized using SDS-PAGE, Western blotting, MALDI-ToF mass spectrometry and amino acid analysis. Cysteine was found to account for 11.2 mol % in the extracted fractions. By measuring 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging activity, the hair protein fractions were shown to possess significant antioxidant ability (IC50 = 16.22 μM). As a supplement in cell culture media, the extracts protected HDFs from H2O2 induced oxidative stress, which was demonstrated by the maintenance of cell viability and reduced reactive oxygen species production. Besides offering mechanical support as a scaffolding material, the unique antioxidizing ability of human hair protein extracts may also be exploited in biomedical applications.

Functional consequences of piceatannol binding to glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the key redox-sensitive proteins whose activity is largely affected by oxidative modifications at its highly reactive cysteine residue in the enzyme’s active site (Cys149). Prolonged exposure to oxidative stress may cause, inter alia, the formation of intermolecular disulfide bonds leading to accumulation of GAPDH aggregates and ultimately to cell death. Recently these anomalies have been linked with the pathogenesis of Alzheimer’s disease. Novel evidences indicate that low molecular compounds may be effective inhibitors potentially preventing the GAPDH translocation to the nucleus, and inhibiting or slowing down its aggregation and oligomerization. Therefore, we decided to establish the ability of naturally occurring compound, piceatannol, to interact with GAPDH and to reveal its effect on functional properties and selected parameters of the dehydrogenase structure. The obtained data revealed that piceatannol binds to GAPDH. The ITC analysis indicated that one molecule of the tetrameric enzyme may bind up to 8 molecules of polyphenol (7.3 ± 0.9). Potential binding sites of piceatannol to the GAPDH molecule were analyzed using the Ligand Fit algorithm. Conducted analysis detected 11 ligand binding positions. We indicated that piceatannol decreases GAPDH activity. Detailed analysis allowed us to presume that this effect is due to piceatannol ability to assemble a covalent binding with nucleophilic cysteine residue (Cys149) which is directly involved in the catalytic reaction. Consequently, our studies strongly indicate that piceatannol would be an exceptional inhibitor thanks to its ability to break the aforementioned pathologic disulfide linkage, and therefore to inhibit GAPDH aggregation. We demonstrated that by binding with GAPDH piceatannol blocks cysteine residue and counteracts its oxidative modifications, that induce oligomerization and GAPDH aggregation.

Quantitative analyses of the equilibria among DNA complexes of a blue-light-regulated bZIP module, Photozipper.

Aureochrome1 is a blue-light-receptor protein identified in a stramenopile alga, Vaucheria frigida. Photozipper (PZ) is an N-terminally truncated, monomeric, V. frigida aureochrome1 fragment containing a basic leucine zipper (bZIP) domain and a light–oxygen–voltage (LOV)-sensing domain. PZ dimerizes upon photoexcitation and consequently increases its affinity for the target sequence. In the present study, to understand the equilibria among DNA complexes of PZ, DNA binding by PZ and mutational variants was quantitatively investigated by electrophoretic-mobility-shift assay and fluorescence-correlation spectroscopy in the dark and light states. DNA binding by PZ was sequence-specific and light-dependent. The half-maximal effective concentration of PZ for binding to the target DNA sequence was ~40 nM in the light, which was >10-fold less than the value in the dark. By contrast, the dimeric PZ-S2C variant (with intermolecular disulfide bonds) had higher affinity for the target sequence, with dissociation constants of ~4 nM, irrespective of the light conditions. Substitutions of Glu159 and Lys164 in the leucine zipper region decreased the affinity of PZ for the target sequence, especially in the light, suggesting that these residues form inter-helical salt bridges between leucine zipper regions, stabilizing the dimer–DNA complex. Our quantitative analyses of the equilibria in PZ–DNA-complex formation suggest that the blue-light-induced dimerization of LOV domains and coiled-coil formation by leucine zipper regions are the primary determinants of the affinity of PZ for the target sequence.

Lrpap1 as a specific marker of proximal pronephric kidney tubuli in Xenopus laevis embryos.

LRPAP1, also known as receptor associated protein (RAP) is a small protein of 40 kDa associated with six of the seven members of the evolutionary conserved family of LDL receptors. Numerous studies showed that LRPAP1 has a dual function, initially as a chaperone insuring proper formation of intermolecular disulfide bonds during biogenesis of low density lipoprotein (LDL) receptors and later as an escort protein during trafficking through the endoplasmic reticulum and the early Golgi compartment, preventing premature interaction of receptor and ligand. Because of the general influence of LRPAP1 protein on lipid metabolism, we analyzed the temporal and spatial expression of the Xenopus laevis ortholog of lrpap1. Here, we show that lrpap1 was expressed in the developing neural system, the eye and ear anlagen, the branchial arches, the developing skin and the pronephric kidney. The very high expression level of lrpap1 specifically in the proximal tubules of the developing pronephros establishes this gene as a novel marker for the analysis of pronephros formation.

CGG repeat DNA assisted dimerization of CGG/CGG binding molecule through intermolecular disulfide formation.

A new DNA binding small molecule, NCD-CC is reported. NCD-CC has a NCD domain, which recognizes the G-G mismatch in a CGG/CGG triad, and a cysteinylcystein (CC) moiety. Dimerization of NCD-CC through intermolecular disulfide bond formation was accelerated in the presence of CGG repeat DNA.

Spectroscopic and calorimetric characterization of spermine oxidase and its association forms.

Spermine oxidase (SMOX) is a flavin-containing enzyme that oxidizes spermine to produce spermidine, 3-aminopropanaldehyde, and hydrogen peroxide. SMOX has been shown to play key roles in inflammation and carcinogenesis; indeed, it is differentially expressed in several human cancer types. Our previous investigation has revealed that SMOX purified after heterologous expression in Escherichia coli actually consists of monomers, covalent homodimers, and other higher-order forms. All association forms oxidize spermine and, after treatment with dithiothreitol, revert to SMOX monomer. Here, we report a detailed investigation on the thermal denaturation of SMOX and its association forms in native and reducing conditions. By combining spectroscopic methods (circular dichroism, fluorescence) and thermal methods (differential scanning calorimetry), we provide new insights into the structure, the transformation, and the stability of SMOX. While the crystal structure of this protein is not available yet, experimental results are interpreted also on the basis of a novel SMOX structural model, obtained in silico exploiting the recently solved acetylspermine oxidase crystal structure. We conclude that while at least one specific intermolecular disulfide bond links two SMOX molecules to form the homodimer, the thermal denaturation profiles can be justified by the presence of at least one intramolecular disulfide bond, which also plays a critical role in the stabilization of the overall three-dimensional SMOX structure, and in particular of its flavin adenine dinucleotide-containing active site.

Restoration of glycoprotein Erns dimerization via pseudoreversion partially restores virulence of classical swine fever virus.

The classical swine fever virus (CSFV) represents one of the most important pathogens of swine. The CSFV glycoprotein Erns is an essential structural protein and an important virulence factor. The latter is dependent on the RNase activity of this envelope protein and, most likely, its secretion from the infected cell. A further important feature with regard to its function as a virulence factor is the formation of disulfide-linked Erns homodimers that are found in virus-infected cells and virions. Mutant CSFV lacking cysteine (Cys) 171, the residue responsible for intermolecular disulfide bond formation, were found to be attenuated in pigs (Tews BA, Schürmann EM, Meyers G. J Virol 2009;83:4823-4834). In the course of an animal experiment with such a dimerization-negative CSFV mutant, viruses were reisolated from pigs that contained a mutation of serine (Ser) 209 to Cys. This mutation restored the ability to form disulphide-linked Erns homodimers. In transient expression studies Erns mutants carrying the S209C change were found to form homodimers with about wt efficiency. Also the secretion level of the mutated proteins was equivalent to that of wt Erns. Virus mutants containing the Cys171Ser/Ser209Cys configuration exhibited wt growth rates and increased virulence when compared with the Cys171Ser mutant. These results provide further support for the connection between CSFV virulence and Erns dimerization.

Activating mutations in quorum-sensing regulator Rgg2 and its conformational flexibility in the absence of an intermolecular disulfide bond

Rap/Rgg/NprR/PlcR/PrgX (RRNPP) quorum-sensing systems use extracellular peptide pheromones that are detected by cytoplasmic receptors to regulate gene expression in firmicute bacteria. Rgg-type receptors are allosterically regulated through direct pheromone binding to control transcriptional activity; however, the receptor activation mechanism remains poorly understood. Previous work has identified a disulfide bond between Cys-45 residues within the homodimer interface of Rgg2 from Streptococcus dysgalactiae (Rgg2Sd). Here, we compared two Rgg2Sd(C45S) X-ray crystal structures with that of wild-type Rgg2Sd and found that in the absence of the intermolecular disulfide, the Rgg2Sd dimer interface is destabilized and Rgg2Sd can adopt multiple conformations. One conformation closely resembled the "disulfide-locked" Rgg2Sd secondary and tertiary structures, but another displayed more extensive rigid-body shifts as well as dramatic secondary structure changes. In parallel experiments, a genetic screen was used to identify mutations in rgg2 of Streptococcus pyogenes (rgg2Sp ) that conferred pheromone-independent transcriptional activation of an Rgg2-stimulated promoter. Eight mutations yielding constitutive Rgg2 activity, designated Rgg2Sp*, were identified, and five of them clustered in or near an Rgg2 region that underwent conformational changes in one of the Rgg2Sd(C45S) crystal structures. The Rgg2Sp* mutations increased Rgg2Sp sensitivity to pheromone and pheromone variants while displaying decreased sensitivity to the Rgg2 antagonist cyclosporine A. We propose that Rgg2Sp* mutations invoke shifts in free-energy bias to favor the active state of the protein. Finally, we present evidence for an electrostatic interaction between an N-terminal Asp of the pheromone and Arg-153 within the proposed pheromone-binding pocket of Rgg2Sp.

Solution structure and interaction with copper in vitro and in living cells of the first BIR domain of XIAP

The X-chromosome linked inhibitor of apoptosis (XIAP) is a multidomain metalloprotein involved in caspase inhibition and in copper homeostasis. It contains three zinc-binding baculoviral IAP repeats (BIR) domains, which are responsible for caspase interaction. Recently, it has been suggested that the BIR domains can bind copper, however high resolution data on such interaction is missing. Here we characterize by NMR the structural properties of BIR1 in solution, and the effects of its interaction with copper both in vitro and in physiological environments. BIR1 is dimeric in solution, consistent with the X-ray structure. Cysteine 12, located in the unfolded N-terminal region, has a remarkably low redox potential, and is prone to oxidation even in reducing physiological environments. Interaction of BIR1 with copper(II) results in the oxidation of cysteine 12, with the formation of either an intermolecular disulfide bond between two BIR1 molecules or a mixed disulfide bond with glutathione, whereas the zinc binding site is not affected by the interaction.