Extra Cysteines Research Articles

1H, 15N and 13C backbone and side-chain assignments of reduced and S-nitrosated C62only mutant of human thioredoxin.

Thioredoxins are ubiquitous and conserved small proteins. The redox-active site is composed of highly conserved Cys32 and Cys35. In higher eukaryotes, thioredoxin evolved to a gain of function in nitrosative control, with 3 extra cysteines, Cys62, Cys69, and Cys73. Human thioredoxin 1 (hTrx) is directly involved in cellular signal transduction through S-nitrosation. The understanding of the mechanism of S-nitrosation is essential. Here we produced a mutant of hTrx containing only Cys62 (C62only). We report the almost full 1H, 15N, and 13C chemical shift assignment of the reduced and S-nitrosated C62only. This study will help to measure the reactivity Cys62 toward S-nitrosants and the stability of S-nitrosated Cys62.

Unusual Cysteine Content in V1 Region of gp120 From an Elite Suppressor That Produces Broadly Neutralizing Antibodies.

Although it is now possible to produce recombinant HIV envelope glycoproteins (Envs) with epitopes recognized by the 5–6 major classes of broadly neutralizing antibodies (bNAbs), these have failed to consistently stimulate the formation of bNAbs in immunized animals or humans. In an effort to identify new immunogens better able to elicit bNAbs, we are studying Envs derived from rare individuals who possess bNAbs and are able to control their infection without the need for anti-retroviral drugs (elite supressors or ES), hypothesizing that in at least some people the antibodies may mediate durable virus control. Because virus evolution in people with the ES only phenotype was reported to be limited, we reasoned the Env proteins recovered from these individuals may more closely resemble the Envs that gave rise to bNAbs compared to the highly diverse viruses isolated from normal progressors. Using a phenotypic assay, we screened 25 controllers and identified two for more detailed investigation. In this study, we examined 20 clade B proviral sequences isolated from an African American woman, who had the rare bNAb/ES phenotype. Phylogenetic analysis of proviral envelope sequences demonstrated low genetic diversity. Envelope proteins were unusual in that most possessed two extra cysteines within an elongated V1 region. In this report, we examine the impact of the extra cysteines on the binding to bNAbs, virus infectivity, and sensitivity to neutralization. These data suggest structural motifs in V1 can affect infectivity, and that rare viruses may be prevented from developing escape.

Characterization of a Novel 4.0-kb Y-type HMW-GS from Eremopyrum distans

A novel 4.0-kb Fy was sequenced and bacterially expressed. This gene, the largest y-type HMW-GS currently reported, is 4,032-bp long and encodes a mature protein with 1,321 amino acid (AA) residues. The 4.0-kb Fy shows novel modifications in all domains. In the N-terminal, it contains only 67 AA residues, as three short peptides are absent. In the repetitive domain, the undecapeptide RYYPSVTSPQQ is completely lost and the dodecapeptide GSYYPGQTSPQQ is partially absent. A novel motif unit, PGQQ, is present in addition to the two standard motif units PGQGQQ and GYYPTSPQQ. Besides, an extra cysteine residue also occurs in the middle of this domain. The large molecular mass of the 4.0-kb Fy is mainly due to the presence of an extra-long repetitive domain with 1,279 AA residues. The novel 4.0-kb Fy gene is of interest in HMW-GS gene evolution as well as to wheat quality improvement with regard to its longest repetitive domain length and extra cysteines residues.

Selectivity filters and cysteine-rich extracellular loops in voltage-gated sodium, calcium, and NALCN channels.

How nature discriminates sodium from calcium ions in eukaryotic channels has been difficult to resolve because they contain four homologous, but markedly different repeat domains. We glean clues from analyzing the changing pore region in sodium, calcium and NALCN channels, from single-cell eukaryotes to mammals. Alternative splicing in invertebrate homologs provides insights into different structural features underlying calcium and sodium selectivity. NALCN generates alternative ion selectivity with splicing that changes the high field strength (HFS) site at the narrowest level of the hourglass shaped pore where the selectivity filter is located. Alternative splicing creates NALCN isoforms, in which the HFS site has a ring of glutamates contributed by all four repeat domains (EEEE), or three glutamates and a lysine residue in the third (EEKE) or second (EKEE) position. Alternative splicing provides sodium and/or calcium selectivity in T-type channels with extracellular loops between S5 and P-helices (S5P) of different lengths that contain three or five cysteines. All eukaryotic channels have a set of eight core cysteines in extracellular regions, but the T-type channels have an infusion of 4–12 extra cysteines in extracellular regions. The pattern of conservation suggests a possible pairing of long loops in Domains I and III, which are bridged with core cysteines in NALCN, Cav, and Nav channels, and pairing of shorter loops in Domains II and IV in T-type channel through disulfide bonds involving T-type specific cysteines. Extracellular turrets of increasing lengths in potassium channels (Kir2.2, hERG, and K2P1) contribute to a changing landscape above the pore selectivity filter that can limit drug access and serve as an ion pre-filter before ions reach the pore selectivity filter below. Pairing of extended loops likely contributes to the large extracellular appendage as seen in single particle electron cryo-microscopy images of the eel Nav1 channel.

Enhanced oxygen tolerance of hydrogenase from Klebsiella oxytoca HP1 by Gly–Cys exchanges nearby Fe–S clusters as biocatalysts in biofuel cells or hydrogen production

Hydrogenase is the key point of H2-based biotechnology. However, the O2-sensitivity largely hinders its applications in biofuel cells and biological H2 production. Therefore, substantial breakthrough on understanding the molecular basis of O2-sensitivity and developing more O2-tolerant hydrogenases are urgently required. In this study, we found adding extra cysteines to the vicinity of the proximal Fe–S cluster to the NiFe active centre could largely enhance oxygen tolerance of hydrogen-evolving hydrogenase 3 from Klebsiella oxytoca HP1 (KoHyd3), through homologous sequence comparison and site-directed mutagenesis. Ratio of aerobic hydrogen yield to anaerobic hydrogen yield (RHH) of Gly47Cys (Gly47 was replaced with Cys47), Gly50Cys, Gly113Cys, Gly120Cys and Gly50Cys–Gly120Cys (double exchange) were increased by 46.99%, 42.15%, 59.19%, 44.74% and 78.72%, respectively, comparing with that of wild type. Moreover, TiO2-KoHyd3 (Gly47Cys, Gly50Cys, Gly113Cys, Gly120Cys and Gly50Cys–Gly120Cys) particles acted well in UV light-driven H2 production from water. These results revealed that extra cysteines nearby Fe–S clusters had significant effects on oxygen tolerance of KoHyd3. It also provided a promising way to produce O2-tolerant hydrogenase as biocatalysts in biofuel cells or H2 production by photolysis of water.

Influenza Virus M2e with Additional Cysteine Residues Shows Enhanced Immunogenicity and Protection Against Lethal Virus Challenge

The amino acid sequence of the extracellular domain of matrix protein 2 (M2e) is conserved among all subtypes of influenza A viruses. Therefore, the M2e peptide can be considered as a target antigen for the development of a universal influenza vaccine. We evaluated the effects of adding cysteine residues to a peptide of amino acids 2-24 of M2e. Mice immunized with some of these peptides containing one, two, three, four, or five extra cysteines displayed enhanced antibody titers to M2e. In addition, immunization with a peptide containing three extra cysteines, along with an aluminum adjuvant, protected mice more effectively against a lethal influenza virus challenge than the original M2e peptide. These results indicated that an M2e peptide containing additional cysteine residues could be a universal influenza vaccine candidate even without the addition of strong adjuvants.

An unusual mode of iron–sulfur-cluster coordination in a teleost glutaredoxin

Glutaredoxins that contain a Cys-X-X-Cys active site motif are glutathione-dependent thiol-disulfide oxidoreductases. Vertebrate glutaredoxin 2 is characterized by two extra cysteines that form an intra-molecular disulfide bridge. Zebrafish glutaredoxin 2 contains four additional cysteines that are conserved within the infraclass of bony fish (teleosts). Here, we present a biochemical and biophysical characterization of zebrafish glutaredoxin 2, focusing on iron–sulfur-cluster coordination. The coordination of [2Fe2S]2+-clusters in monomers of this protein was revealed by both absorption and Mössbauer spectroscopy as well as size exclusion chromatography. All other holo-glutaredoxins represent [FeS]-cluster bridged dimers using two molecules of non-covalently bound glutathione and the N-terminal active site cysteines as ligands. These cysteine residues were not required for [FeS]-cluster coordination in zebrafish glutaredoxin 2. A crystal structure of the teleost protein revealed high structural similarity to its human homologue. The two vertebrate-specific cysteines as well as two of the teleost-specific cysteines are positioned within a radius of 7Å near the C-terminus suggesting a potential role in [FeS]-cluster coordination. Indeed, mutated proteins lacking these teleost-specific cysteines lost the ability to bind the cofactor. Hence, the apparent mode of [FeS]-cluster coordination in zebrafish glutaredoxin 2 could be different from all yet described [FeS]-glutaredoxins.

Molecular cloning and characterization of alboaggregin D, a novel platelet activating protein, from Green pit viper (Cryptelytrops albolabris) venom

Viper venoms are abundant sources of proteins affecting hemostasis. This study aimed to clone and purify a high-molecular-weight C-type lectin-like protein (snaclec) from Green pit viper (Cryptelytrops albolabris) venom, as well as to characterize its effects on human platelets.Based on the partial sequences from the C. albolabris venom gland library, we cloned full-length cDNAs encoding the snaclec subunits using 5′RACE and 3′RACE methods. The cDNA sequence of the α subunit contained 477 base pairs (bp) that were translated into 23 amino acid residue signal peptide and a 135-residue mature protein. The cDNA sequence of the β subunit contained 447bp that were translated into 23-residue signal peptide and a 125-residue mature protein. Compared with known sequences of dimeric snaclecs, these peptides contained extra cysteines that probably formed a high-order multimer. In parallel, a snaclec was isolated from C. albolabris crude venom using gel filtration followed by ion-exchange chromatography. The purified C. albolabris snaclec on SDS-PAGE showed the apparent molecular mass of 120kDa under native condition and 2 bands of 14 and 17 kD under reduced condition suggesting a tetramer of heterodimers (αβ)4. Liquid chromatography-tandem mass spectrometry analysis of the peptides found perfect matches with the conceptually translated sequences from the cDNA library. This protein was unique from any other snaclecs previously purified from C. albolabris and named alboaggregin D. It induced human platelet aggregation in the absence of any cofactor with the EC50 of 0.25nM and caused tyrosine phosphorylation in human platelets. Antibodies against either platelet glycoprotein (GP) Ib or GPVI could inhibit alboaggregin D-induced platelet aggregation. This snaclec may be useful for dissecting the mechanisms of platelet activation.

General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold

Nanobodies, single-domain antigen-binding fragments of camelid-specific heavy-chain only antibodies offer special advantages in therapy over classic antibody fragments because of their smaller size, robustness, and preference to target unique epitopes. A Nanobody differs from a human heavy chain variable domain in about ten amino acids spread all over its surface, four hallmark Nanobody-specific amino acids in the framework-2 region (positions 42, 49, 50, and 52), and a longer third antigen-binding loop (H3) folding over this area. For therapeutic applications the camelid-specific amino acid sequences in the framework have to be mutated to their human heavy chain variable domain equivalent, i.e. humanized. We performed this humanization exercise with Nanobodies of the subfamily that represents close to 80% of all dromedary-derived Nanobodies and investigated the effects on antigen affinity, solubility, expression yield, and stability. It is demonstrated that the humanization of Nanobody-specific residues outside framework-2 are neutral to the Nanobody properties. Surprisingly, the Glu-49 --> Gly and Arg-50 --> Leu humanization of hallmark amino acids generates a single domain that is more stable though probably less soluble. The other framework-2 substitutions, Phe-42 --> Val and Gly/Ala-52 --> Trp, are detrimental for antigen affinity, due to a repositioning of the H3 loop as shown by their crystal structures. These insights were used to identify a soluble, stable, well expressed universal humanized Nanobody scaffold that allows grafts of antigen-binding loops from other Nanobodies with transfer of the antigen specificity and affinity.

Crystal structure of a heterodimer of phospholipase A2 from Naja naja sagittifera at 2.3 Å resolution reveals the presence of a new PLA2‐like protein with a novel cys 32–Cys 49 disulphide bridge with a bound sugar at the substrate‐binding site

The crystal structure of the phospholipase A2 (PLA2) heterodimer from Naja naja sagittifera reveals the presence of a new PLA2-like protein with eight disulphide bridges. The heterodimer is formed between a commonly observed group I PLA2 having seven characteristic disulfide bonds and a novel PLA2-like protein (Cys-PLA2) containing two extra cysteines at two highly conserved sites (positions 32 and 49) of structural and functional importance. The crystals of the heterodimer belong to tetragonal space group P41212 with cell dimensions, a = b = 77.7 A and c = 68.4 A corresponding to a solvent content of 33%, which is one of the lowest values observed so far in the PLA2 crystals. The structure has been solved with molecular replacement method and refined to a final R value of 21.6% [Rfree = 25.6%]. The electron density revealed the presence of cysteines 32 and 49 that are covalently linked to give rise to an eighth disulphide bridge in the PLA2-like monomer. A non-protein high-quality electron density was also observed at the substrate-binding site in the PLA2-like protein that has been interpreted as N-acetylglucosamine. The overall tertiary folds of the two monomers are similar having all features of PLA2-type folding. A zinc ion is detected at the interface of the heterodimer with fivefold coordination while another zinc ion was found on the surface of Cys-PLA2 with sixfold coordination. The conformations of the calcium-binding loops of both monomers are significantly different from each other as well as from those in other group I PLA2s. The N-acetylglucosamine molecule is favorably placed in the substrate-binding site of Cys-PLA2 and forms five hydrogen bonds and several van der Waals interactions with protein atoms, thus indicating a strong affinity. It also provides clue of the possible mechanism of sugar recognition by PLA2 and PLA2-like proteins. The formation of heterodimer seems to have been induced by zinc ion.

Cysteine 116 participates in intermolecular bonding of the human VEGF 121 homodimer

VEGF 121, the 121-amino acid form of vascular endothelial growth factor is a homodimer with nine cysteine residues per monomer. While three intramolecular and two intermolecular disulfide bonds have been mapped, the state of the ninth cysteine, Cys116, is not known. In this study, we determined that human VEGF 121 contains a third interchain disulfide bond between Cys116 of each monomer. We also isolated a VEGF 121 variant with two extra cysteines bound to each Cys116. No evidence was found for the exsistence of Cys116 in the reduced state. In fact, selective reduction of the Cys116 interchain disulfide bond yielded an unstable VEGF 121 molecule, which reoxidized quickly. Biological activities of VEGF 121 Cys116 variants were assessed. The oxidative state of Cys116 has no effect on binding or proliferation activities but may be important for overall stability of the molecule.

Truncation of NH2-terminal Amino Acid Residues Increases Agonistic Potency of Leukotactin-1 on CC Chemokine Receptors 1 and 3

Leukotactin-1 (Lkn-1) is a human CC chemokine that binds to both CC chemokine receptor 1 (CCR1) and CCR3. Structurally, Lkn-1 is distinct from other human CC chemokines in that it has long amino acid residues preceding the first cysteine at the NH(2) terminus, and contains two extra cysteines. NH(2)-terminal amino acids of Lkn-1 were deleted serially, and the effects of each deletion were investigated. In CCR1-expressing cells, serial deletion up to 20 amino acids (Delta20) did not change the calcium flux-inducing activity significantly. Deletion of 24 amino acids (Delta24), however, increased the agonistic potency approximately 100-fold. Deletion of 27 or 28 amino acids also increased the agonistic potency to the same level shown by Delta24. Deletion of 29 amino acids, however, abolished the agonistic activity almost completely showing that at least 3 amino acid residues preceding the first cysteine at the NH(2) terminus are essential for the biological activity of Lkn-1. Loss of agonistic activity was due to impaired binding to CCR1. In CCR3-expressing cells, Delta24 was the only form of Lkn-1 mutants that revealed increased agonistic potency. Our results indicate that posttranslational modification is a potential mechanism for the regulation of biological activity of Lkn-1.

Characterization of Free α- and β-Chains of Recombinant Macrophage-Stimulating Protein

Human serum macrophage-stimulating protein (MSP) induces motile activity of murine resident peritoneal macrophages and is a growth and motility factor for epithelial cells. It belongs to the plasminogen-related family of kringle proteins, and is secreted as a single-chain, 78-kDa, biologically inactive pro-MSP. Proteolytic cleavage of pro-MSP at a single site yields active MSP, a disulfide-linked αβ-chain heterodimer. However cleavage of recombinant pro-MSP yielded not only the disulfide-linked heterodimer, but also free α- and β-chains, indicating that some of the recombinant molecules lacked an αβ-chain disulfide. We purified the free chains for characterization. The β-chain of MSP has three extra cysteines, Cys527, Cys562, and Cys672, which are not found in the plasminogen β-chain. Disulfide bond analysis showed a Cys527–Cys562, but also a Cys588–Cys672. Coopting Cys588by Cys672prevented the expected formation of a disulfide between α-chain Cys468and β-chain Cys588. Concomitant studies determined structures of oligosaccharides at the three Asn-linked glycosylation sites of MSP. The oligosaccharides at the three Asn loci are heterogeneous; 11 different sugars were identified, all being sialylated fucosyl biantennary structures. We also located the pro-MSP signal peptide cleavage site at Gly18-Gln19and the scissile bond for formation of mature MSP at Arg483–Val484.

Ligand binding and covalent structure of an oxygen-binding heme protein from Rhodobacter sphaeroides, a representative of a new structural family of c-type cytochromes.

The amino acid sequence of an oxygen-binding heme protein (SHP) from Rhodobacter sphaeroides has been determined. The cysteines, which bind the single heme group in the 112-residue protein, are located at positions 43 and 46. SHP is similar in size to the large membrane-bound form of the class I cytochrome c5 of Azotobacter vinelandii (116 residues) and in the location of the heme binding site at positions 48 and 51. Two extra cysteines in SHP (residues 89 and 97) are located in positions similar to those of cytochrome c5 (residues 98 and 101) and form a disulfide bridge in both proteins. In total, four regions of alpha-helix are predicted, covering 46% of the protein, which is comparable to that in other small cytochromes. SHP is thus distantly related to small class I c-type cytochromes but is representative of a distinct family by virtue of its high-spin nature, the lack of a strong sixth ligand, and its capacity to bind oxygen. Potentially, the most important characteristic of SHP is its ability to transiently bind oxygen during autoxidation, which occurs with a half-life of 3 min with a 4-fold excess of O2. SHP also binds carbon monoxide, azide, and cyanide. The kinetics of reduction by free flavins indicate that SHP is less reactive than other class I cytochromes c and that the heme is less accessible to solvent. There is localized positive charge (+3) at the site of reduction of SHP, although the overall protein charge is -2. This may account in part for the ability of SHP to bind anions.

Identification and molecular characterization of insulin-like growth factor binding proteins (IGFBP-1, -2, -3, -4, -5 and -6)

Six different insulin-like growth factor binding proteins (IGFBPs) have been identified by molecular cloning of their cDNAs from rat and human tissues and designated as IGFBP-1, -2, -3, -4, -5 and -6. The total number of amino acid residues for the mature rat BPs ranges from 201 for IGFBP-6 to 270 for IGFBP-2, while the human homologs range from 216 for IGFBP-6 to 289 for IGFBP-2. Except for IGFBP-6, all rat and human IGFBPs contain 18 homologous cysteines; twelve are located at the N-terminal and span approximately one-third of the total amino acid sequence, while the remaining six are distributed at the C-terminal and span the last one-third of the protein sequence. Both rat and human IGFBP-4 possess two extra cysteines at the mid-region of the molecule. By contrast, rat and human IGFBP-6 contain only 14 and 16 cysteines, respectively. Absence of the two and four cysteines in the N-terminal region in the human and rat IGFBP-6 resulted in the deletion of the invariant Gly-Cys-Gly-Cys-Cys sequence which is present in all the other five IGFBPs. Both rat and human IGFBP-3 possess multiple N-linked glycosylation sites at the mid-region of the molecule, which accounts for their apparent molecular size being larger than the calculated molecular weight, based on the amino acid sequence. One potential N-linked glycosylation site is located at the mid-region of rat and human IGFBP-4, whereas only human but not rat IGFBP-6 possesses one N-linked glycosylation site at the extreme C-terminal of the molecule. An RGD sequence is found in the C-terminal of IGFBP-1 and -2. In this short review, updated information on the structural identification and molecular cloning of the six IGFBPs will be presented. In addition, the potential regulation of the BPs at the transcriptional and translational levels will be discussed.

Molecular Cloning of the cDNAs Encoding a Novel Insulin-Like Growth Factor-Binding Protein from Rat and Human

cDNA clones encoding a novel insulin-like growth factor-binding protein (IGFBP) purified from rat serum and human bone cell-conditioned medium have been isolated from rat liver and human placenta, liver, and ovary cDNA libraries. The deduced amino acid sequences of the cDNAs revealed a mature polypeptide consisting of 233 amino acids for the rat, while the human structure contains an additional four-amino acid sequence in the middle region of the molecule. This protein, now proposed to be named IGFBP-4, contains two extra cysteines compared with the previously characterized IGFBP-1, -2, and -3, but the alignment of the remaining 18 cysteines is conserved across the four IGFBPs. Amino acid sequence comparison among the four binding proteins within the rat species demonstrated that both the amino- and carboxy-terminal one thirds of the molecules are highly conserved, while the middle one third region, where no cysteines are present except for the two that exist in IGFBP-4, is the most divergent. The overall sequence homology among the four rat IGFBPs is very similar (53-59%), suggesting that their individual genes diverged from a single ancestral gene at about the same evolutionary time point. Northern analysis of the IGFBP-4 mRNA in rat tissue demonstrated that transcription of the IGFBP-4 gene is highly active in the liver, although a single 2.6-kilobase IGFBP-4 mRNA band was detectable in all tissues examined, including adrenal, testis, spleen, heart, lung, kidney, liver, stomach, hypothalamus, and brain cortex.

Sequence of carbonic anhydrase II cDNA from chick retina.

Sequences of three cDNA clones for carbonic anhydrase II (CA-II) from chick retina are presented. The longest cDNA clone encodes all but the first three amino acids of CA-II, and the encoded sequence generally agrees with published fragments of CA-II sequence from chick red blood cells. It is 70% identical to human CA-II; the active-site residues are conserved, but the chick protein has six extra cysteines. There is a long 3'-untranslated region which contains a second open reading frame, but this is not conserved. There appears to be a single CA-II gene in the chick. Some anomalies in cDNA synthesis and in Bal31 deletion are noted.