Invariant Cysteine Research Articles

Crystallization and preliminary X-ray analysis of binary and ternary complexes of Haloferax mediterranei glucose dehydrogenase.

Haloferax mediterranei glucose dehydrogenase (EC 1.1.1.47) belongs to the medium-chain alcohol dehydrogenase superfamily and requires zinc for catalysis. In the majority of these family members, the catalytic zinc is tetrahedrally coordinated by the side chains of a cysteine, a histidine, a cysteine or glutamate and a water molecule. In H. mediterranei glucose dehydrogenase, sequence analysis indicates that the zinc coordination is different, with the invariant cysteine replaced by an aspartate residue. In order to analyse the significance of this replacement and to contribute to an understanding of the role of the metal ion in catalysis, a range of binary and ternary complexes of the wild-type and a D38C mutant protein have been crystallized. For most of the complexes, crystals belonging to space group I222 were obtained using sodium/potassium citrate as a precipitant. However, for the binary and non-productive ternary complexes with NADPH/Zn, it was necessary to replace the citrate with 2-methyl-2,4-pentanediol. Despite the radical change in conditions, the crystals thus formed were isomorphous.

Characterization of Disulfide Bonds in Human Nucleoside Triphosphate Diphosphohydrolase 3 (NTPDase3): Implications for NTPDase Structural Modeling

Cell-surface nucleotidases (NTPDases) contain 10 invariant cysteine residues in their extracellular regions. To investigate disulfide structure in human NTPDase3, we made single and double mutants of these 10 cysteines, and analyzed their enzymatic activity, glycosylation pattern, trafficking to the cell membrane, and sensitivity to reduction. The mutants constituted five distinct phenotypes, thus, strongly suggesting disulfide bonds between C92-C116 (first bond), C261-C308 (second bond), C289-C334 (third bond), C347-C353 (fourth bond), and C399-C422 (fifth bond). Due to conservation of the 10 cysteines, the identified five disulfide bonds are likely to exist in all cell-surface NTPDases. The third and fifth bonds are also present in the soluble NTPDases and are critical for processing, trafficking, and enzymatic activity. The fourth bond has minimal effect on processing and function, while the first and second bonds are of intermediate importance. Most of the N-linked glycosylation sites in the wild-type enzyme are processed to complex oligosaccharides, but at least one site is high-mannose or hybrid in structure. Interestingly, disruption of the first disulfide bond resulted in some enzyme that lost sensitivity to endoglycosidase H, suggesting that the first disulfide bond in the wild-type enzyme shields some high-mannose glycans from terminal glycosylation. Comparative modeling by threading and homology modeling of the NTPDase3 sequence revealed a high degree of structural fold similarity with a bacterial exopolyphosphatase (PDB ). The resultant theoretical 3-D model of the extracellular portion of NTPDase3, based on homology with this exopolyphosphatase, is consistent with the assignment of the disulfide bonds occurring in regions of good fold similarity between NTPDase3 and the exopolyphosphatase. The 3-D model obtained for NTPDase3 also suggests the structural basis for the importance of several apyrase conserved regions for the nucleotidase activities of the NTPDases.

Vaccinia Virus H2 Protein Is an Essential Component of a Complex Involved in Virus Entry and Cell-Cell Fusion

The vaccinia virus H2R gene (VACWR 100) is conserved in all sequenced members of the poxvirus family and encodes a protein with a predicted transmembrane domain and four invariant cysteines. A recombinant vaccinia virus, in which expression of the H2 protein is stringently regulated, was unable to replicate without inducer. However, under nonpermissive conditions, all stages of virus morphogenesis appeared normal and extracellular virions were detected at the tips of actin tails. Nevertheless, virus did not spread to neighboring cells nor did syncytia form after low-pH treatment. Purified -H2 and +H2 virions from cells infected in the absence or presence of inducer, respectively, were indistinguishable in microscopic appearance and contained the same complement of major proteins, though only +H2 virions were infectious. The -H2 virions bound to cells, but their cores did not penetrate into the cytoplasm. In addition, exogenously added -H2 virions were unable to mediate the formation of syncytia after low-pH treatment. In contrast, virions lacking the A27 (p14) protein, which was previously considered to have an essential role in fusion, penetrated cells and induced extensive syncytia. The properties of H2, however, are very similar to those recently reported for the A28 protein. Moreover, coimmunoprecipitation experiments indicated an interaction between H2 and A28. Therefore, H2 and A28 are the only proteins presently known to be specifically required for vaccinia virus entry and are likely components of a fusion complex.

Evidence That the Streptomyces Developmental Protein WhiD, a Member of the WhiB Family, Binds a [4Fe-4S] Cluster

WhiD is required for the late stages of sporulation in the Gram-positive bacterium Streptomyces coelicolor. WhiD is a member of the WhiB-like family of putative transcription factors that are present throughout the actinomycetes but absent from other organisms. This family of proteins has four near-invariant cysteines, suggesting that these residues might act as ligands for a metal cofactor. Overexpressed WhiD, purified from Escherichia coli, contained substoichiometric amounts of iron and had an absorption spectrum characteristic of a [2Fe-2S] cluster. After Fe-S cluster reconstitution under anaerobic conditions, WhiD contained approximately 4 iron atoms/monomer and similar amounts of sulfide ion and gave an absorption spectrum characteristic of a [4Fe-4S] cluster. Reconstituted WhiD gave no electron paramagnetic resonance signal as prepared but, after reduction with dithionite, gave an electron paramagnetic resonance signal (g approximately 2.06, 1.94) consistent with a one-electron reduction of a [4Fe-4S](2+) cluster to a [4Fe-4S](1+) state with electron spin of S = (1/2). The anaerobically reconstituted [4Fe-4S] cluster was oxygen sensitive. Upon exposure to air, absorption at 410 and 505 nm first increased and then showed a steady decrease with time until the protein was colorless in the near UV/visible region. These changes are consistent with an oxygen-induced change from a [4Fe-4S] to a [2Fe-2S] cluster, followed by complete loss of cluster from the protein. Each of the four conserved cysteine residues, Cys-23, -53, -56, and -62, was essential for WhiD function in vivo.

Conserved extracellular cysteine residues and cytoplasmic loop-loop interplay are required for functionality of the heptahelical MLO protein.

We performed a structure-function analysis of the plasma membrane-localized plant-specific barley (Hordeum vulgare) MLO (powdery-mildew-resistance gene o) protein. Invariant cysteine and proline residues, located either in extracellular loops or transmembrane domains that have been conserved in MLO proteins for more than 400 million years, were found to be essential for MLO functionality and/or stability. Similarly to many metazoan G-protein-coupled receptors known to function as homo- and hetero-oligomers, FRET (fluorescence resonance energy transfer) analysis revealed evidence for in planta MLO dimerization/oligomerization. Domain-swap experiments with closely related wheat and rice as well as diverged Arabidopsis MLO isoforms demonstrated that the identity of the C-terminal cytoplasmic tail contributes to MLO activity. Likewise, analysis of a progressive deletion series revealed that integrity of the C-terminus determines both MLO accumulation and functionality. A series of domain swaps of cytoplasmic loops with the wheat (Triticum aestivum) orthologue, TaMLO-B1, provided strong evidence for co-operative loop-loop interplay either within the protein or between MLO molecules. Our data indicate extensive intramolecular co-evolution of cytoplasmic domains in the evolutionary history of the MLO protein family.

Solution Structure of Cryptdin-4, a Mouse Paneth Cell α-Defensin,

Mammalian defensins are abundant antimicrobial peptides that contribute to host defense. They are characterized by several conserved amino acids, including six invariant cysteine residues which form three intramolecular disulfide bonds and stabilize the tertiary structure. Cryptdin-4 (Crp4), a mouse alpha-defensin with potent in vitro bactericidal activity, has a primary structure distinct from all known alpha-defensins in that its polypeptide backbone uniquely lacks three residues between Cys(IV) and Cys(V). NMR diffusion experiments showed that Crp4 is monomeric in solution, and its three-dimensional solution structure, determined by two-dimensional proton NMR, consists of a triple-stranded antiparallel beta-sheet with the beta-strands joined to each other by a series of tight turns and a beta-hairpin. However, the overall beta-sheet content in Crp4 is lower than that of other alpha-defensin structures, while the shape and orientation of the Crp4 beta-hairpin also differ from those of other alpha-defensin structures. These structural characteristics combined with the high overall cationicity of Crp4 may contribute to its broad bactericidal spectrum and membrane disruptive activity.

Cloning and functional expression of invertebrate connexins from Halocynthia pyriformis

Unlike many other ion channels, unrelated gene families encode gap junctions in different animal phyla. Connexin and pannexin genes are found in deuterostomes, while protostomal species use innexin genes. Connexins are often described as vertebrate genes, despite the existence of invertebrate deuterostomes. We have cloned connexin sequences from an invertebrate chordate, Halocynthia pyriformis. Invertebrate connexins shared 25–40% sequence identity with human connexins, had extracellular domains containing six invariant cysteine residues, coding regions that were interrupted by introns, and formed functional channels in vitro. These data show that gap junction channels based on connexins are present in animals that predate vertebrate evolution.

The SufE Sulfur-acceptor Protein Contains a Conserved Core Structure that Mediates Interdomain Interactions in a Variety of Redox Protein Complexes

The isc and suf operons in Escherichia coli represent alternative genetic systems optimized to mediate the essential metabolic process of iron-sulfur cluster (Fe-S) assembly under basal or oxidative-stress conditions, respectively. Some of the proteins in these two operons share strong sequence homology, e.g. the cysteine desulfurases IscS and SufS, and presumably play the same role in the oxygen-sensitive assembly process. However, other proteins in these operons share no significant homology and occur in a mutually exclusive manner in Fe-S assembly operons in other organisms (e.g. IscU and SufE). These latter proteins presumably play distinct roles adapted to the different assembly mechanisms used by the two systems. IscU has three invariant cysteine residues that function as a template for Fe-S assembly while accepting a sulfur atom from IscS. SufE, in contrast, does not function as an Fe-S assembly template but has been suggested to function as a shuttle protein that uses a persulfide linkage to a single invariant cysteine residue to transfer a sulfur atom from SufS to an alternative Fe-S assembly template. Here, we present and analyze the 2.0A crystal structure of E.coli SufE. The structure shows that the persulfide-forming cysteine occurs at the tip of a loop with elevated B-factors, where its side-chain is buried from solvent exposure in a hydrophobic cavity located beneath a highly conserved surface. Despite the lack of sequence homology, the core of SufE shows strong structural similarity to IscU, and the sulfur-acceptor site in SufE coincides with the location of the cysteine residues mediating Fe-S cluster assembly in IscU. Thus, a conserved core structure is implicated in mediating the interactions of both SufE and IscU with the mutually homologous cysteine desulfurase enzymes present in their respective operons. A similar core structure is observed in a domain found in a variety of Fe-S cluster containing flavoenzymes including xanthine dehydrogenase, where it also mediates interdomain interactions. Therefore, the core fold of SufE/IscU has been adapted to mediate interdomain interactions in diverse redox protein systems in the course of evolution.

IscA Mediates Iron Delivery for Assembly of Iron-Sulfur Clusters in IscU under the Limited Accessible Free Iron Conditions

Increasing evidence suggests that IscS, a cysteine desulfurase, provides sulfur for assembly of transient iron-sulfur clusters in IscU. IscU appears to act as a scaffold and eventually transfers the assembled clusters to target proteins. However, the iron donor for the iron-sulfur cluster assembly largely remains elusive. Here we find that Escherichia coli IscU fails to assemble iron-sulfur clusters when the accessible "free" iron in solution is limited by an iron chelator sodium citrate. Remarkably, IscA, an iron-sulfur cluster assembly protein with an iron association constant of 3.0 x 10(19) m(-1), is able to overcome the iron limitation due to sodium citrate and deliver iron for the IscS-mediated iron-sulfur cluster assembly in IscU. Substitution of the invariant cysteine residues Cys-99 or Cys-101 in IscA with serine completely abolishes the iron binding activity of the protein. The IscA mutants that fail to bind iron are unable to mediate iron delivery for the iron-sulfur cluster assembly in IscU under the limited accessible "free" iron conditions. The results suggest that IscA is capable of recruiting intracellular iron and providing iron for the iron-sulfur cluster assembly in IscU in cells in which the accessible "free" iron content is probably restricted.

Antimicrobial and Pore-forming Peptides of Free-living and Potentially Highly Pathogenic Naegleria fowleri Are Released from the Same Precursor Molecule

The pore-forming polypeptides of Naegleria fowleri, naegleriapores A and B, are processed from separate multipeptide precursor structures. According to their transcripts, each precursor molecule appears to contain additional naegleriapore-like polypeptides, all of which share a structural motif of six invariant cysteine residues within their amino acid sequence. To identify the putative pronaegleriapore-derived peptides at the protein level, amoebic extracts were screened for small cysteine-rich polypeptides by fluorescently labeling their cysteine residues. Three novel naegleriapore isoforms derived from the precursor molecule of naegleriapore B were identified. Two of the isoforms were purified to homogeneity and tested for their biological activity. The pore-forming activity of the novel peptides was remarkably lower than that of the originally isolated naegleriapores, but both peptides killed bacteria by permeabilizing their cytoplasmic membranes. Collectively, these results indicate that naegleriapore isoforms with antibacterial and pore-forming activity are proteolytically released from the same precursor protein, presumably to generate a phylogenetically ancient complementary antimicrobial arsenal.

Functional Analysis of a Divergent System II Protein, Ccs1, Involved in c-Type Cytochrome Biogenesis

The Ccs1 gene, encoding a highly divergent novel component of a system II type c-type cytochrome biogenesis pathway, is encoded by the previously defined CCS1 locus in Chlamydomonas reinhardtii. phoA and lacZalpha bacterial topological reporters were used to deduce a topological model of the Synechocystis sp. 6803 Ccs1 homologue, CcsB. CcsB, and therefore by analogy Ccs1, possesses a large soluble lumenal domain at its C terminus that is tethered in the thylakoid membrane by three closely spaced transmembrane domains in the N-terminal portion of the protein. Molecular analysis of ccs1 alleles reveals that the entire C-terminal soluble domain is essential for Ccs1 function and that a stromal loop appears to be important in vivo, at least for maintenance of Ccs1. Site-directed mutational analysis reveals that a single histidine (His(274)) within the last transmembrane domain, preceding the large lumenal domain, is required for c-type cytochrome assembly, whereas an invariant cysteine residue (Cys(199)) is shown to be non-essential. Ccs1 is proposed to interact with other Ccs components based on its reduced accumulation in ccs2, ccs3, ccs4, and ccsA strains.

SseA, a 3-mercaptopyruvate sulfurtransferase from Escherichia coli: crystallization and preliminary crystallographic data.

SseA, the translation product of the Escherichia coli sseA gene, is a 31 kDa protein endowed with 3-mercaptopyruvate:cyanide sulfurtransferase activity in vitro. As such, SseA is the prototype of a sulfurtransferase subfamily distinguished from the better known rhodanese sulfurtransferases, which display thiosulfate:cyanide sulfurtransferase activity. The physiological role of the two homologous enzyme families, whose catalytic activity is centred on a reactive invariant cysteine, is a matter of debate. In this framework, the forthcoming crystal structure analysis of SseA will be based on the tetragonal crystal form (space group P4(1) or P4(3)) reported here, with unit-cell parameters a = b = 150.2, c = 37.9 A.

Structural and functional characterization of the Pseudomonas hydroperoxide resistance protein Ohr.

Bacteria have developed complex strategies to detoxify and repair damage caused by reactive oxygen species. These compounds, produced during bacterial aerobic respiration as well as by the host immune system cells as a defense mechanism against the pathogenic microorganisms, have the ability to damage nucleic acids, proteins and phospholipid membranes. Here we describe the crystal structure of Pseudomonas aeruginosa Ohr, a member of a recently discovered family of organic hydroperoxide resistance proteins. Ohr is a tightly folded homodimer, with a novel alpha/beta fold, and contains two active sites located at the monomer interface on opposite sides of the molecule. Using in vitro assays, we demonstrate that Ohr functions directly as a hydroperoxide reductase, converting both inorganic and organic hydroperoxides to less toxic metabolites. Site-directed mutagenesis confirms that the two conserved cysteines in each active site are essential for catalytic activity. We propose that the Ohr catalytic mechanism is similar to that of the structurally unrelated peroxiredoxins, directly utilizing highly reactive cysteine thiol groups to elicit hydroperoxide reduction.

Sequence-specific interaction of nascent antiterminator RNA with the zinc-finger motif of Escherichia coli RNA polymerase.

The N-terminal Zn-finger motif of the beta' subunit of RNA polymerase contains two pairs of invariant cysteines flanking a moderately well-conserved segment of 13 amino acids that is rich in basic residues. Previous work showed that replacement of certain Zn-finger residues prevented transcription antitermination in response to phage HK022 put sites. Nascent put RNA binds to and modifies transcribing polymerase, so that it becomes resistant to termination. To characterize the Zn finger further, we replaced each of the basic residues with alanine and determined the effects of the substitutions on termination, antitermination and cell viability. All the mutants were defective in put-mediated antitermination. The severity of the defect depended on the mutant and on the sequence of the upstream stem-loop of put RNA. Some, but not all, mutants distinguished between put variants that differed in this region. This suggests that the Zn-finger motif interacts directly and specifically with put RNA. All the mutants in the basic residues complemented a temperature-sensitive beta' mutant for cell growth at a non-permissive temperature, and those mutant enzymes that were tested transcribed and terminated normally in vitro on a template that lacked a put site.

Influence of amino acid numbers between two ligand cysteines of zinc finger proteins on affinity and specificity of DNA binding

The C 2H 2-type zinc finger motif has the consensus sequence X 2–Cys–X 2,4–Cys–X 12–His–X 3–5–His and two or four amino acid residues between two ligand cysteines are well conserved. To evaluate this conservation, we prepared six mutant peptides derived from the three-zinc-finger domain of Sp1 whose C-terminal finger has two amino acid residues between the two invariant cysteines. Their circular dichroism spectra suggest that these mutants have an ordered secondary structure comparable with that of the wild type. The insertion mutants (X=3–5) bind to DNA with the somewhat lower affinity than the wild type (X=2). On the other hand, the DNA binding affinity of the deletion mutants (X=0 and 1) is significantly reduced. In particular, the extent of the reduction in two mutants with Cys–Cys and Cys–Pro–Cys is remarkable. The methylation interference analyses demonstrate that this decreased DNA binding affinity is due to lowering of the extent of the base contacts by the central and C-terminal fingers. This information reveals not only a clue to evolution of the zinc finger but also the possibility of the existence of the zinc finger with altered number of amino acid residues between the two ligand cysteines.

Redox Regulation of Cdc25C

The Cdc25 family of dual specific phosphatases are critical components of cell cycle progression and checkpoint control. Certain stresses such as ultraviolet light stimulate the rapid and selective destruction of Cdc25A protein through a Chk1 protein kinase-dependent pathway. We demonstrate that in contrast to cellular stresses previously examined, hydrogen peroxide exposure affects Cdc25C but not Cdc25A levels. Pharmacological inhibition of Chk1 activity or a mutant of Cdc25C that lacks the Chk1 phosphorylation site still undergoes degradation in response to oxidants. We also demonstrate that in vitro hydrogen peroxide stimulates an intramolecular disulfide bond between the active site cysteine at position 377 and another invariant cysteine at position 330. The in vivo stability of Cdc25C is substantially reduced by the mutation of either of these two cysteine residues. In contrast, a double (C2) mutant of both cysteine 330 and cysteine 377 results in a protein that is more stable than wild type Cdc25C and is resistant to oxidative stress-induced degradation. In addition, the C2 mutant, which is unable to form an intramolecular disulfide bond, has reduced binding to 14-3-3 in vitro and in vivo. These results suggest that oxidative stress may induce cell cycle arrest in part through the degradation of Cdc25C.

Structure, function, and inhibition of chemokines.

Chemokines are the largest family of cytokines in human immunophysiology. These proteins are defined by four invariant cysteines and are categorized based on the sequence around the first two cysteines, which leads to two major and two minor subfamilies. Chemokines function by activating specific G protein-coupled receptors, which results in, among other functions, the migration of inflammatory and noninflammatory cells to the appropriate tissues or compartments within tissues. Some of these proteins and receptors have been implicated or shown to be involved in inflammation, autoimmune diseases, and infection by HIV-1. The three-dimensional structure of each monomer is virtually identical, but the quaternary structure of chemokines is different for each subfamily. Structure-function studies reveal several regions of chemokines to be involved in function, with the N-terminal region playing a dominant role. A number of proteins and small-molecule antagonists have been identified that inhibit chemokine activities. In this review, we discuss aspects of the structure, function, and inhibition of chemokines.

29] Redox regulation of protein tyrosine phosphatases by hydrogen peroxide: Detecting sulfenic acid intermediates and examining reversible inactivation

Publisher Summary This chapter describes a mechanism of redox reduction for protein tyrosine phosphatases (PTPs) in which the transient intracellular production of hydrogen peroxide, as a result of growth factor/mitogen stimulation of cell surface receptors, leads to the rapid inactivation of PTPs by the modification of their invariant catalytic cysteine thiolate (R–S) to an inactive sulfenic acid (R–SOH). The chapter describes methods to test if specific PTPs can be regulated by reversible inactivation with hydrogen peroxide. These techniques include a continuous and end point spectrophotometric assay for the inactivation of PTPs by hydrogen peroxide, a method to examine the reversibility of the hydrogen peroxide inactivation of PTPs by monitoring the reactivation kinetics with thiol compounds, three separate techniques to verify that catalytic cysteine thiolate is the target for hydrogen peroxide inactivation, a chemical modification technique to trap the cysteine sulfenic acid intermediate, and a method to examine if growth factor treatment of cells results in a reversible inactivation of the PTP of interest.

Differential Regulation of the Orphan Nuclear ReceptorSmall Heterodimer Partner (SHP) Gene Promoter by Orphan Nuclear Receptor ERR Isoforms

The orphan nuclear receptor small heterodimer partner (SHP; NR0B2) interacts with a wide array of nuclear receptors and represses their transcriptional activity. SHP expression is regulated by several other members of the nuclear receptor superfamily, including the orphan receptors SF-1 and LRH-1, and the bile acid receptor FXR. We have found that the SHP promoter is also activated by the estrogen receptor-related receptor gamma (ERRgamma) but not the related ERRalpha and ERRbeta isoforms. SHP and ERRgamma mRNAs are coexpressed in several tissues, including pancreas, kidney, and heart, confirming the potential relevance of this transactivation. ERRgamma transactivation is dependent on only one of five previously characterized DNA-binding sites for SF-1, and this element differs from previously reported ERR response elements. However, treatment with the histone deacetylase inhibitor trichostatin A significantly increased ERRalpha and ERRbeta activity on this element indicating that the lack of activity of ERRalpha and -beta may depend on their association with co-repressor in vivo. Furthermore, using protease sensitivity assays on DNA bound receptors it was demonstrated that DNA sequence of different response elements may cause allosteric modulation of ERR proteins, which in turn may be responsible for the differential activities of these receptors on different response elements. SHP inhibits ERRgamma transactivation and physically interacts with all three members of ERR subfamily, as demonstrated by both yeast two-hybrid and biochemical assays. As with other SHP targets, this interaction is dependent on the AF-2 coactivator-binding site of ERRgamma and the previously described N-terminal receptor interaction domain of SHP. Several recently described SHP mutations associated with moderate obesity in humans block the inhibition of ERRgamma activity. Overall, these results identify a new autoregulatory loop controlling SHP gene expression and significantly extend the potential functional roles of the three ERRs.

The hairpin structure of the (6)F1(1)F2(2)F2 fragment from human fibronectin enhances gelatin binding.

The solution structure of the (6)F1(1)F2(2)F2 fragment from the gelatin-binding region of fibronectin has been determined (Protein Data Bank entry codes 1e88 and 1e8b). The structure reveals an extensive hydrophobic interface between the non-contiguous (6)F1 and (2)F2 modules. The buried surface area between (6)F1 and (2)F2 ( approximately 870 A(2)) is the largest intermodule interface seen in fibronectin to date. The dissection of (6)F1(1)F2(2)F2 into the (6)F1(1)F2 pair and (2)F2 results in near-complete loss of gelatin-binding activity. The hairpin topology of (6)F1(1)F2(2)F2 may facilitate intramolecular contact between the matrix assembly regions flanking the gelatin-binding domain. This is the first high-resolution study to reveal a compact, globular arrangement of modules in fibronectin. This arrangement is not consistent with the view that fibronectin is simply a linear 'string of beads'.