Invariant Cysteine Research Articles

Identification and characterization of a novel envelope protein in Rana grylio virus

Viral envelope proteins have been proposed to play significant roles in virus infection and assembly. In this study, an envelope protein gene, 53R, was cloned and characterized from Rana grylio virus (RGV), a member of the family Iridoviridae. Database searches found its homologues in all sequenced iridoviruses, and sequence alignment revealed several conserved structural features shared by virus capsid or envelope proteins: a myristoylation site, two predicted transmembrane domains and two invariant cysteine residues. Subsequently, RT-PCR and Western blot detection revealed that the transcripts encoding RGV 53R and the protein itself appeared late during infection of fathead minnow cells and that their appearance was blocked by viral DNA replication inhibitor, indicating that RGV 53R is a late expression gene. Moreover, immunofluorescence localization found an association of 53R with virus factories in RGV-infected cells, and this association was further confirmed by expressing a 53R-GFP fusion protein in pEGFP-N3/53R-transfected cells. Furthermore, detergent extraction and Western blot detection confirmed that RGV 53R was associated with virion membrane. Therefore, the current data suggest that RGV 53R is a novel viral envelope protein and that it may play an important role in virus assembly. This is thought to be the first report on a viral envelope protein that is conserved in all sequenced iridoviruses.

PRGL: A cell wall proline-rich protein containning GASA domain in Gerbera hybrida

PRPs (proline-rich proteins) are a group of cell wall proteins characterized by their proline and hydroproline-rich repetitive peptides. The expression of PRPs in plants is stimulated by wounding and environmental stress. GASA (gibberellic acid stimulated in Arabidopsis) proteins are small peptides sharing a 60 amino acid conserved C-terminal domain containing twelve invariant cysteine residues. Most of GASAs reported are localized to apoplasm or cell wall and their expression was regulated by gibberellins (GAs). It has been reported that, in French bean, these two proteins encoding by two distinct genes formed a two-component chitin-receptor involved in plant-pathogen interactions when plant was infected. We cloned a full-length cDNA of PRGL (proline-rich GASA-like) gene which encodes a protein containing both PRP and GASA-like domains. It is demonstrated that PRGL is a new protein with characteristics of PRP and GASA by analyzing its protein structure and gene expression.

Structures of Prostacyclin Synthase and Its Complexes with Substrate Analog and Inhibitor Reveal a Ligand-specific Heme Conformation Change

Prostacyclin synthase (PGIS) is a cytochrome P450 (P450) enzyme that catalyzes production of prostacyclin from prostaglandin H(2). PGIS is unusual in that it catalyzes an isomerization rather than a monooxygenation, which is typical of P450 enzymes. To understand the structural basis for prostacyclin biosynthesis in greater detail, we have determined the crystal structures of ligand-free, inhibitor (minoxidil)-bound and substrate analog U51605-bound PGIS. These structures demonstrate a stereo-specific substrate binding and suggest features of the enzyme that facilitate isomerization. Unlike most microsomal P450s, where large substrate-induced conformational changes take place at the distal side of the heme, conformational changes in PGIS are observed at the proximal side and in the heme itself. The conserved and extensive heme propionate-protein interactions seen in all other P450s, which are largely absent in the ligand-free PGIS, are recovered upon U51605 binding accompanied by water exclusion from the active site. In contrast, when minoxidil binds, the propionate-protein interactions are not recovered and water molecules are largely retained. These findings suggest that PGIS represents a divergent evolution of the P450 family, in which a heme barrier has evolved to ensure strict binding specificity for prostaglandin H(2), leading to a radical-mediated isomerization with high product fidelity. The U51605-bound structure also provides a view of the substrate entrance and product exit channels.

A Unique DNA Binding Domain Converts T-Cell Factors into Strong Wnt Effectors

Wnt regulation of gene expression requires binding of LEF/T-cell factor (LEF/TCF) transcription factors to Wnt response elements (WREs) and recruitment of the activator beta-catenin. There are significant differences in the abilities of LEF/TCF family members to regulate Wnt target genes. For example, alternatively spliced isoforms of TCF-1 and TCF-4 with a C-terminal "E" tail are uniquely potent in their activation of LEF1 and CDX1. Here we report that the mechanism responsible for this unique activity is an auxiliary 30-amino-acid DNA interaction motif referred to here as the "cysteine clamp" (or C-clamp). The C-clamp contains invariant cysteine, aromatic, and basic residues, and surface plasmon resonance (SPR) studies with recombinant C-clamp protein showed that it binds double-stranded DNA but not single-stranded DNA or RNA (equilibrium dissociation constant = 16 nM). CASTing (Cyclic Amplification and Selection of Targets) experiments were used to test whether this motif influences WRE recognition. Full-length LEF-1, TCF-1E, and TCF-1E with a mutated C-clamp all bind nearly identical WREs (TYYCTTTGATSTT), showing that the C-clamp does not alter WRE specificity. However, a GC element downstream of the WRE (RCCG) is enriched in wild-type TCF-1E binding sites but not in mutant TCF-1E binding sites. We conclude that the C-clamp is a sequence-specific DNA binding motif. C-clamp mutations destroy the ability of beta-catenin to regulate the LEF1 promoter, and they severely impair the ability of TCF-1 to regulate growth in colon cancer cells. Thus, E-tail isoforms of TCFs utilize two DNA binding activities to access a subset of Wnt targets important for cell growth.

Functional Characterization of Mia40p, the Central Component of the Disulfide Relay System of the Mitochondrial Intermembrane Space

Mia40p and Erv1p are components of a translocation pathway for the import of cysteine-rich proteins into the intermembrane space of mitochondria. We have characterized the redox behavior of Mia40p and reconstituted the disulfide transfer system of Mia40p by using recombinant functional C-terminal fragment of Mia40p, Mia40C, and Erv1p. Oxidized Mia40p contains three intramolecular disulfide bonds. One disulfide bond connects the first two cysteine residues in the CPC motif. The second and the third bonds belong to the twin CX(9)C motif and bridge the cysteine residues of two CX(9)C segments. In contrast to the stabilizing disulfide bonds of the twin CX(9)C motif, the first disulfide bond was easily accessible to reducing agents. Partially reduced Mia40C generated by opening of this bond as well as fully reduced Mia40C were oxidized by Erv1p in vitro. In the course of this reaction, mixed disulfides of Mia40C and Erv1p were formed. Reoxidation of fully reduced Mia40C required the presence of the first two cysteine residues in Mia40C. However, efficient reoxidation of a Mia40C variant containing only the cysteine residues of the twin CX(9)C motif was observed when in addition to Erv1p low amounts of wild type Mia40C were present. In the reconstituted system the thiol oxidase Erv1p was sufficient to transfer disulfide bonds to Mia40C, which then could oxidize the variant of Mia40C. In summary, we reconstituted a disulfide relay system consisting of Mia40C and Erv1p.

Expression of the recombinant bacterial outer surface protein A in tobacco chloroplasts leads to thylakoid localization and loss of photosynthesis

Bacterial lipoproteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. A prominent example is the outer surface protein A (OspA) of Borrelia burgdorferi, which has been efficiently used as a vaccine for the prevention of Lyme disease. In a previous study, OspA could be produced in tobacco chloroplasts in a lipidated and immunogenic form. To further explore the potential of chloroplasts for the production of bacterial lipoproteins, the role of the N-terminal leader sequence was investigated. The amount of recombinant OspA could be increased up to ten-fold by the variation of the insertion site in the chloroplast genome. Analysis of OspA mutants revealed that replacement of the invariant cysteine residue as well as deletion of the leader sequence abolishes palmitolyation of OspA. Also, decoration of OspA with an N-terminal eukaryotic lipidation motif does not lead to palmitoylation in chloroplasts. Strikingly, the bacterial signal peptide of OspA efficiently targets the protein to thylakoids, and causes a mutant phenotype. Plants accumulating OspA at 10% total soluble protein could not grow without exogenously supplied sugars and rapidly died after transfer to soil under greenhouse conditions. The plants were found to be strongly affected in photosystem II, as revealed by the analyses of temporal and spatial dynamics of photosynthetic activity by chlorophyll fluorescence imaging. Thus, overexpression of OspA in chloroplasts is limited by its concentration-dependent interference with essential functions of chloroplastic membranes required for primary metabolism.

Defensins: endogenous antibiotic peptides from human leukocytes.

A variety of endogenous antimicrobial peptides equip mammals, amphibians, insects and plants to defend themselves against microbial pathogens. Defensins are small peptides of mammalian cells that contain 29-35 amino acid residues, including six invariant cysteines that form three intramolecular disulphide bonds. They are produced by the sequential proteolysis of precursors that contain approximately 95 amino acids and are synthesized by several types of cells, especially the bone marrow precursors of blood neutrophils. Defensins constitute 5-7% of the total protein in human neutrophils and are present in high concentration in the azurophil granules and phagocytic vacuoles. The production of defensins by human neutrophils amounts to approximately 10 mg/kg body weight per day. In certain mammalian species lung macrophages and specialized epithelial (Paneth) cells in the small intestine also produce defensins. Defensins are complexly folded, amphipathic, rich in antiparallel beta-sheet but devoid of alpha-helical domains. Their unusually broad antimicrobial spectrum encompasses Gram-positive and Gram-negative bacteria, many fungi, mycobacteria, spirochetes and several enveloped viruses. The antimicrobial properties of defensins result from their insertion into target cell membranes and the formation of voltage-sensitive channels. Given their abundance and broad spectrum of antimicrobial activity, defensins may contribute substantially to innate resistance to infection.

Functional Modulation of the Geminivirus AL2 Transcription Factor and Silencing Suppressor by Self-Interaction

The DNA genomes of geminiviruses have a limited coding capacity that is compensated for by the production of small multifunctional proteins. The AL2 protein encoded by members of the genus Begomovirus (e.g., Tomato golden mosaic virus) is a transcriptional activator, a silencing suppressor, and a suppressor of a basal defense. The related L2 protein of Beet curly top virus (genus Curtovirus) shares the pathogenicity functions of AL2 but lacks transcriptional activation activity. It is known that AL2 and L2 can suppress local silencing by interacting with adenosine kinase (ADK) and can suppress basal defense by interacting with SNF1 kinase. However, how the activities of these viral proteins are regulated remains an unanswered question. Here, we provide some answers by demonstrating that AL2, but not L2, interacts with itself. The zinc finger-like motif (CCHC) is required but is not sufficient for AL2 self-interaction. Alanine substitutions for the invariant cysteine residues that comprise the motif abolish self-interaction or cause aberrant subnuclear localization but do not abolish interaction with ADK and SNF1. Using bimolecular fluorescence complementation, we show that AL2:AL2 complexes accumulate primarily in the nucleus, whereas AL2:ADK and L2:ADK complexes accumulate mainly in the cytoplasm. Further, the cysteine residue mutations impair the ability of AL2 to activate the coat protein promoter but do not affect local silencing suppression. Thus, AL2 self-interaction correlates with nuclear localization and efficient activation of transcription, whereas AL2 and L2 monomers can suppress local silencing by interacting with ADK in the cytoplasm.

FASTDXL: A Generalized Screen to Trap Disulfide-Stabilized Complexes for Use in Structural Studies

Structural studies of macromolecular complexes have produced extraordinary insights into a wide variety of biological processes. Unfortunately, as structural biologists pursue larger and more challenging assemblies, weakly stable and/or nonspecific interactions can become significant roadblocks to structure determination. We have developed a rapid and effective pool-based screen, termed FASTDXL (focused array screening technique for disulfide X-linking), to produce and identify disulfide-stabilized protein-nucleic acid assemblies. A significant strength of FASTDXL is that it can take advantage of prior structural knowledge about molecular interactions, but does not necessarily rely upon it. A detailed application of the approach to the difficult problem of trapping a bacterial primase-ssDNA complex is described, validating the method as a route toward obtaining diffracting crystals suitable for structure determination.

Suppression of hepatitis B viral gene expression by protein-tyrosine phosphatase PTPN3

Protein-tyrosine phosphatase PTPN3 is a membrane-associated non-receptor protein-tyrosine phosphatase. PTPN3 contains a N-terminal FERM domain, a middle PDZ domain, and a C-terminal phosphatase domain. Upon co-expression of PTPN3, the level of human hepatitis B viral (HBV) RNAs, 3.5 kb, 2.4/2.1 kb, and 0.7 kb transcribed from a replicating HBV expression plasmid is significantly reduced in human hepatoma HuH-7 cells. When the expression of endogenous PTPN3 protein is diminished by specific small interfering RNA, the expression of HBV genes is enhanced, indicating that the endogenous PTPN3 indeed plays a suppressive role on HBV gene expression. PTPN3 can interact with HBV core protein. The interaction is mediated via the PDZ domain of PTPN3 and the carboxyl-terminal last four amino acids of core. Either deletion of PDZ domain of PTPN3 or substitution of PDZ ligand in core has no effect on PTPN3-mediated suppression. These results clearly show that the interaction of PTPN3 with core is not required for PTPN3 suppressive effect. Mutation of (359)serine and (835)serine of 14-3-3beta binding sites to alanine, which slightly reduces the interaction with 14-3-3beta, does not influence the PTPN3 effect. In contrast, mutation of the invariant (842)cysteine residue in phosphatase domain to serine, which makes the phosphatase activity inactive, does not change its subcellular localization and interaction with core or 14-3-3beta, but completely abolishes PTPN3-mediated suppression. Furthermore, deletion of FERM domain does not affect the phosphatase activity or interaction with 14-3-3beta, but changes the subcellular localization from cytoskeleton-membrane interface to cytoplasm and nucleus, abolishes binding to core, and diminishes the PTPN3 effect on HBV gene expression. Taken together, these results demonstrate that the phosphatase activity and FERM domain of PTPN3 are essential for its suppression of HBV gene expression.

Molecular characterisation and expression of CD4 in two distantly related marsupials: The gray short-tailed opossum ( Monodelphis domestica) and tammar wallaby ( Macropus eugenii)

The gene and corresponding cDNA for CD4 in the gray short-tailed opossum, Monodelphis domestica, and the cDNA sequence for CD4 in the tammar wallaby, Macropus eugenii, have been characterised. The opossum CD4 homolog reveals conserved synteny, preserved genomic organisation and analogous structural arrangement to human and mouse CD4. Opossum and tammar CD4 exhibit typical eutherian CD4 features including the highly conserved p56 lck binding motif in the cytoplasmic region and the invariant cysteine residues in extracellular domains 1 and 4. Interestingly, the marsupial CD4 sequences substitute a tryptophan for the first cysteine in domain 2 negating the formation of a disulphide bond as seen in other eutherian CD4 sequences except human and mouse. Overall the marsupial CD4 sequences share amino acid identity of 59% to each other and 37–41% with eutherian mammals. However, in contrast to eutherian homologs, the marsupial CD4 sequences were found to be truncated at the terminal end of the cytoplasmic tail. This is the first report confirming the presence of CD4 in a marsupial and describing its key features.

A Natural Variant of the Heme-Binding Signature (R441C) Resulting in Complete Loss of Function of CYP2D6

A new variant allele CYP2D6*62 (g.4044C>T; R441C) of the drug-metabolizing cytochrome P450 (P450) CYP2D6 was identified in a person with reduced sparteine oxidation phenotype, which was unexpected based on a genetic CYP2D6*1A/*41 background. The recombinantly expressed variant protein had no activity toward propafenone as a result of missing heme incorporation. Sequence alignment revealed that the positively charged R441 residue is part of the heme-binding signature but not strictly conserved among all the P450s. A compilation of described P450 monooxygenase variants revealed that other enzymes can functionally tolerate even nonconservative amino acid changes at the corresponding position (i.e., the invariant cysteine 2). This suggests that heme binding in mammalian P450s depends not only on the integrity of the heme-binding signature but also on other family- and subfamily-specific sequence determinants.

Crystal Structure of Helicobacter pylori Formamidase AmiF Reveals a Cysteine-Glutamate-Lysine Catalytic Triad

Helicobacter pylori AmiF formamidase that hydrolyzes formamide to produce formic acid and ammonia belongs to a member of the nitrilase superfamily. The crystal structure of AmiF was solved to 1.75A resolution using single-wavelength anomalous dispersion methods. The structure consists of a homohexamer related by 3-fold symmetry in which each subunit has an alpha-beta-beta-alpha four-layer architecture characteristic of the nitrilase superfamily. One exterior alpha layer faces the solvent, whereas the other one associates with that of the neighbor subunit, forming a tight alpha-beta-beta-alpha-alpha-beta-beta-alpha dimer. The apo and liganded crystal structures of an inactive mutant C166S were also determined to 2.50 and 2.30 A, respectively. These structures reveal a small formamide-binding pocket that includes Cys(166), Glu(60), and Lys(133) catalytic residues, in which Cys(166) acts as a nucleophile. Analysis of the liganded AmiF and N-carbamoyl d-amino acid amidohydrolase binding pockets reveals a common Cys-Glu-Lys triad, another conserved glutamate, and different subsets of ligand-binding residues. Molecular dynamic simulations show that the conserved triad has minimal fluctuations, catalyzing the hydrolysis of a specific nitrile or amide in the nitrilase superfamily efficiently.

Crystal Structure of the Serine Protease Domain of Prophenoloxidase Activating Factor-I

A family of serine proteases (SPs) mediates the proteolytic cascades of embryonic development and immune response in invertebrates. These proteases, called easter-type SPs, consist of clip and chymotrypsin-like SP domains. The SP domain of easter-type proteases differs from those of typical SPs in its primary structure. Herein, we report the first crystal structure of the SP domain of easter-type proteases, presented as that of prophenoloxidase activating factor (PPAF)-I in zymogen form. This structure reveals several important structural features including a bound calcium ion, an additional loop with a unique disulfide linkage, a canyon-like deep active site, and an exposed activation loop. We subsequently show the role of the bound calcium and the proteolytic susceptibility of the activation loop, which occurs in a clip domain-independent manner. Based on biochemical study in the presence of heparin, we suggest that PPAF-III, highly homologous to PPAF-I, contains a surface patch that is responsible for enhancing the catalytic activity through interaction with a nonsubstrate region of a target protein. These results provide insights into an activation mechanism of easter-type proteases in proteolytic cascades, in comparison with the well studied blood coagulation enzymes in mammals.

Mapping essential domains of Mycobacterium smegmatis WhmD: insights into WhiB structure and function.

A growing body of evidence suggests that the WhiB-like proteins exclusive to the GC-rich actinomycete genera play significant roles in pathogenesis and cell division. Each of these proteins contains four invariant cysteine residues and a conserved helix-turn-helix motif. whmD, the Mycobacterium smegmatis homologue of Streptomyces coelicolor whiB, is essential in M. smegmatis, and the conditionally complemented mutant M. smegmatis 628-53 undergoes filamentation under nonpermissive conditions. To identify residues critical to WhmD function, we developed a cotransformation-based assay to screen for alleles that complement the filamentation phenotype of M. smegmatis 628-53 following inducer withdrawal. Mycobacterium tuberculosis whiB2 and S. coelicolor whiB complemented the defect in M. smegmatis 628-53, indicating that these genes are true functional orthologues of whmD. Deletion analysis suggested that the N-terminal 67 and C-terminal 12 amino acid residues are dispensable for activity. Site-directed mutagenesis indicated that three of the four conserved cysteine residues (C90, C93, and C99) and a conserved aspartate (D71) are essential. Mutations in a predicted loop glycine (G111) and an unstructured leucine (L116) were poorly tolerated. The region essential for WhmD activity encompasses 6 of the 10 residues conserved in all seven M. tuberculosis WhiBs, as well as in most members of the WhiB family identified thus far. WhmD structure was found to be sensitive to the presence of a reducing agent, suggesting that the cysteine residues are involved in coordinating a metal ion. Iron-specific staining strongly suggested that WhmD contains a bound iron atom. With this information, we have now begun to comprehend the functional significance of the conserved sequence and structural elements in this novel family of proteins.

Two Cathelicidin Genes Are Present in both Rainbow Trout ( Oncorhynchus mykiss ) and Atlantic Salmon ( Salmo salar )

Further to the previous finding of the rainbow trout rtCATH_1 gene, this paper describes three more cathelicidin genes found in salmonids: two in Atlantic salmon, named asCATH_1 and asCATH_2, and one in rainbow trout, named rtCATH_2. All the three new salmonid cathelicidin genes share the common characteristics of mammalian cathelicidin genes, such as consisting of four exons and possessing a highly conserved preproregion and four invariant cysteines clustered in the C-terminal region of the cathelin-like domain. The asCATH_1 gene is homologous to the rainbow trout rtCATH_1 gene, in that it possesses three repeat motifs of TGGGGGTGGC in exon IV and two cysteine residues in the predicted mature peptide, while the asCATH_2 gene and rtCATH_2 gene are homologues of each other, with 96% nucleotide identity. Salmonid cathelicidins possess the same elastase-sensitive residue, threonine, as hagfish cathelicidins and the rabbit CAP18 molecule. The cleavage site of the four salmonid cathelicidins is within a conserved amino acid motif of QKIRTRR, which is at the beginning of the sequence encoded by exon IV. Two 36-residue peptides corresponding to the core part of rtCATH_1 and rtCATH_2 were chemically synthesized and shown to exhibit potent antimicrobial activity. rtCATH_2 was expressed constitutively in gill, head kidney, intestine, skin and spleen, while the expression of rtCATH_1 was inducible in gill, head kidney, and spleen after bacterial challenge. Four cathelicidin genes have now been characterized in salmonids and two were identified in hagfish, confirming that cathelicidin genes evolved early and are likely present in all vertebrates.

Entry of Vaccinia Virus and Cell-Cell Fusion Require a Highly Conserved Cysteine-Rich Membrane Protein Encoded by the A16L Gene

The vaccinia virus A16L open reading frame encodes a 378-amino-acid protein with a predicted C-terminal transmembrane domain and 20 invariant cysteine residues that is conserved in all sequenced members of the poxvirus family. The A16 protein was expressed late in infection and incorporated into intracellular virus particles with the N-terminal segment of the protein exposed on the surface. The cysteine residues were disulfide bonded via the poxvirus cytoplasmic redox system. Unsuccessful attempts to isolate a mutant virus with the A16L gene deleted suggested that the protein is essential for replication. To study the role of the A16 protein, we made a recombinant vaccinia virus that has the Escherichia coli lac operator system regulating transcription of the A16L gene. In the absence of inducer, A16 synthesis was repressed and plaque size and virus yield were greatly reduced. Nevertheless, virus morphogenesis occurred and normal-looking intracellular and extracellular virus particles formed. Purified virions made in the presence and absence of inducer were indistinguishable, though the latter had 60- to 100-fold-lower specific infectivity. A16-deficient virions bound to cells, but their cores did not penetrate into the cytoplasm. Furthermore, A16-deficient virions were unable to induce low-pH-triggered syncytium formation. The phenotype of the inducible A16L mutant was similar to those of mutants in which synthesis of the A21, A28, H2, or L5 membrane protein was repressed, indicating that at least five conserved viral proteins are required for entry of poxviruses into cells as well as for cell-cell fusion.

Ryanodine Receptor Type 1 (RyR1) Mutations C4958S and C4961S Reveal Excitation-coupled Calcium Entry (ECCE) Is Independent of Sarcoplasmic Reticulum Store Depletion

Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.

Thioredoxin Reductase System Mediates Iron Binding in IscA and Iron Delivery for the Iron-Sulfur Cluster Assembly in IscU*

IscA is a key member of the iron-sulfur cluster assembly machinery found in bacteria and eukaryotes. Previously, IscA was characterized as an alternative iron-sulfur cluster assembly scaffold, as purified IscA can host transient iron-sulfur clusters. However, recent studies indicated that IscA is an iron-binding protein that can provide iron for the iron-sulfur cluster assembly in a proposed scaffold IscU (Ding H., Clark, R. J., and Ding, B. (2004) J. Biol. Chem. 279, 37499-37504). To further elucidate the roles of IscA in the biogenesis of iron-sulfur clusters, we reevaluate the iron binding activity of IscA under physiologically relevant conditions. The results indicate that in the presence of the thioredoxin reductase system, Escherichia coli IscA binds iron with an iron association constant of 2.0 x 10(19) M(-1) in vitro. Whereas all three components (thioredoxin 1, thioredoxin reductase and NADPH) in the thioredoxin reductase system are essential for mediating the iron binding in IscA, only catalytic amounts of thioredoxin 1 and thioredoxin reductase are required. In contrast, IscU fails to bind iron in the presence of the thioredoxin reductase system, suggesting that the iron binding in IscA is specific. Nevertheless, the thioredoxin reductase system can promote the iron-sulfur cluster assembly in IscU in the presence of the iron-loaded IscA, cysteine desulfurase (IscS), and L-cysteine, demonstrating a physiologically relevant system for the biogenesis of iron-sulfur clusters. The results provide additional evidence for the hypothesis that IscA is capable of recruiting intracellular "free" iron and delivering the iron for the iron-sulfur cluster assembly in IscU.

Vaccinia Virus A21 Virion Membrane Protein Is Required for Cell Entry and Fusion

We provide the initial characterization of the product of the vaccinia virus A21L (VACWR140) gene and demonstrate that it is required for cell entry and low pH-triggered membrane fusion. The A21L open reading frame, which is conserved in all sequenced members of the poxvirus family, encodes a protein of 117 amino acids with an N-terminal hydrophobic domain and four invariant cysteines. Expression of the A21 protein occurred at late times of infection and was dependent on viral DNA replication. The A21 protein contained two intramolecular disulfide bonds, the formation of which required the vaccinia virus-encoded cytoplasmic redox pathway, and was localized on the surface of the lipoprotein membrane of intracellular mature virions. A conditional lethal mutant, in which A21L gene expression was regulated by isopropyl-beta-d-thiogalactopyranoside, was constructed. In the absence of inducer, cell-to-cell spread of virus did not occur, despite the formation of morphologically normal intracellular virions and extracellular virions with actin tails. Purified virions lacking A21 were able to bind to cells, but cores did not penetrate into the cytoplasm and synthesize viral RNA. In addition, virions lacking A21 were unable to mediate low pH-triggered cell-cell fusion. The A21 protein, like the A28 and H2 proteins, is an essential component of the poxvirus entry/fusion apparatus for both intracellular and extracellular virus particles.