Intrasubunit Disulfide Bridges Research Articles

Arabidopsis seryl-tRNA synthetase: the first crystal structure and novel protein interactor of plant aminoacyl-tRNA synthetase.

The rules of the genetic code are established by aminoacyl-tRNA synthetases (aaRSs) enzymes, which covalently link tRNA with the cognate amino acid. Many aaRSs are involved in diverse cellular processes beyond translation, acting alone, or in complex with other proteins. However, studies of aaRS noncanonical assembly and functions in plants are scarce, as are structural studies of plant aaRSs. Here, we have solved the crystal structure of Arabidopsis thaliana cytosolic seryl-tRNA synthetase (SerRS), which is the first crystallographic structure of a plant aaRS. Arabidopsis SerRS displays structural features typical of canonical SerRSs, except for a unique intrasubunit disulfide bridge. In a yeast two-hybrid screen, we identified BEN1, a protein involved in the metabolism of plant brassinosteroid hormones, as a protein interactor of Arabidopsis SerRS. The SerRS:BEN1 complex is one of the first protein complexes of plant aaRSs discovered so far, and is a rare example of an aaRS interacting with an enzyme involved in primary or secondary metabolism. To pinpoint regions responsible for this interaction, we created truncated variants of SerRS and BEN1, and identified that the interaction interface involves the SerRS globular catalytic domain and the N-terminal extension of BEN1 protein. BEN1 does not have a strong impact on SerRS aminoacylation activity, indicating that the primary function of the complex is not the modification of SerRS canonical activity. Perhaps SerRS performs as yet unknown noncanonical functions mediated by BEN1. These findings indicate that - via SerRS and BEN1 - a link exists between the protein translation and steroid metabolic pathways of the plant cell. DATABASE: Structural data are available in the PDB under the accession number PDB ID 6GIR.

A structural modeling approach for the understanding of initiation and elongation of ALS-linked superoxide dismutase fibrils

Familial amyotrophic lateral sclerosis caused by mutations in copper-zinc superoxide dismutase (SOD1) is characterized by the presence of SOD1-rich inclusions in spinal cords. It has been shown that a reduced intra-subunit disulfide bridge apo-SOD1 can rapidly initiate fibrillation forming an inter-subunits disulfide under mild, physiologically accessible conditions. Once initiated, elongation can proceed via recruitment of either apo or partially metallated disulfide-intact SOD1 and the presence of copper, but not zinc, ions inhibit fibrillation. We propose a structural model, refined through molecular dynamics simulations, that, taking into account these experimental findings, provides a molecular explanation for the initiation and the elongation of SOD1 fibrils in physiological conditions. The model indicates the occurrence of a new dimeric unit, prone to interact one with the other due to the presence of a wide hydrophobic surface and specific electrostatic interactions. The model has dimensions consistent with the SOD1 fibril size observed through electron microscopy and provides a structural basis for the understanding of SOD1 fibrillation.

Homodimeric Chicken Galectin CG-1B (C-14): Crystal Structure and Detection of Unique Redox-Dependent Shape Changes Involving Inter- and Intrasubunit Disulfide Bridges by Gel Filtration, Ultracentrifugation, Site-Directed Mutagenesis, and Peptide Mass Fingerprinting

Intrafamily gene diversification has led to three prototype galectins in chicken [i.e., chicken galectin (CG)-1A, CG-1B, and CG-2] that show distinct expression profiles and developmental regulation. In order to pinpoint structural disparities among them, we determined the crystal structure of CG-1B. Alteration of the position of the Trp ring in the lectin site and the presence of only two ordered water molecules therein, as well as changes in the interface region between the two subunits, set the structure of CG-1B clearly apart from that of CG-1A. Intriguingly, the unique presence of two Cys residues at positions 2 and 7 in the N-terminal region translated into formation of an intersubunit disulfide bridge between the Cys7 residues of the homodimer in the crystal. In solution, oxidation is associated with significant shape changes in the dimeric protein and the additional occurrence of a compacted form with an intrasubunit disulfide bridge between Cys2 and Cys7. The single-site mutant C7S/C7V was not subjected to such changes, supporting the crucial role of Cys7 in redox-dependent shape changes. These results point to the functional significance of the distinctive presence of the two Cys residues in the N-terminal region of CG-1B.

Biochemical and structural characterization of mammalian‐like purine nucleoside phosphorylase from the Archaeon Pyrococcus furiosus

We report here the characterization of the first mammalian-like purine nucleoside phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus (PfPNP). The gene PF0853 encoding PfPNP was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. PfPNP is a homohexamer of 180 kDa which shows a much higher similarity with 5'-deoxy-5'-methylthioadenosine phosphorylase (MTAP) than with purine nucleoside phosphorylase (PNP) family members. Like human PNP, PfPNP shows an absolute specificity for inosine and guanosine. PfPNP shares 50% identity with MTAP from P. furiosus (PfMTAP). The alignment of the protein sequences of PfPNP and PfMTAP indicates that only four residue changes are able to switch the specificity of PfPNP from a 6-oxo to a 6-amino purine nucleoside phosphorylase still maintaining the same overall active site organization. PfPNP is highly thermophilic with an optimum temperature of 120 degrees C and is characterized by extreme thermodynamic stability (T(m), 110 degrees C that increases to 120 degrees C in the presence of 100 mm phosphate), kinetic stability (100% residual activity after 4 h incubation at 100 degrees C), and remarkable SDS-resistance. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. By integrating biochemical methodologies with mass spectrometry we assigned three pairs of intrasubunit disulfide bridges that play a role in the stability of the enzyme against thermal inactivation. The characterization of the thermal properties of the C254S/C256S mutant suggests that the CXC motif in the C-terminal region may also account for the extreme enzyme thermostability.

The Mycobacterial Thioredoxin Peroxidase Can Act as a One-cysteine Peroxiredoxin

Thioredoxin peroxidase (TPx) has been reported to dominate the defense against H(2)O(2), other hydroperoxides, and peroxynitrite at the expense of thioredoxin (Trx) B and C in Mycobacterium tuberculosis (Mt). By homology, the enzyme has been classified as an atypical 2-C-peroxiredoxin (Prx), with Cys(60) as the "peroxidatic" cysteine (C(P)) forming a complex catalytic center with Cys(93) as the "resolving" cysteine (C(R)). Site-directed mutagenesis confirms Cys(60) to be C(P) and Cys(80) to be catalytically irrelevant. Replacing Cys(93) with serine leads to fast inactivation as seen by conventional activity determination, which is associated with oxidation of Cys(60) to a sulfinic acid derivative. However, in comparative stopped-flow analysis, WT-MtTPx and MtTPx C93S reduce peroxynitrite and react with TrxB and -C similarly fast. Reduction of pre-oxidized WT-MtTPx and MtTPx C93S by MtTrxB is demonstrated by monitoring the redox-dependent tryptophan fluorescence of MtTrxB. Furthermore, MtTPx C93S remains stable for 10 min at a morpholinosydnonimine hydrochloride-generated low flux of peroxynitrite and excess MtTrxB in a dihydrorhodamine oxidation model. Liquid chromatography-tandem mass spectrometry analysis revealed disulfide bridges between Cys(60) and Cys(93) and between Cys(60) and Cys(80) in oxidized WT-MtTPx. Reaction of pre-oxidized WT-MtTPx and MtTPx C93S with MtTrxB C34S or MtTrxC C40S yielded dead-end intermediates in which the Trx mutants are preferentially linked via disulfide bonds to Cys(60) and never to Cys(93) of the TPx. It is concluded that neither Cys(80) nor Cys(93) is required for the catalytic cycle of the peroxidase. Instead, MtTPx can react as a 1-C-Prx with Cys(60) being the site of attack for both the oxidizing and the reducing substrate. The role of Cys(93) is likely to conserve the oxidation equivalents of the sulfenic acid state of C(P) as a disulfide bond to prevent overoxidation of Cys(60) under a restricted supply of reducing substrate.

The Crystal Structure of 5′-Deoxy-5′-methylthioadenosine Phosphorylase II from Sulfolobus solfataricus, a Thermophilic Enzyme Stabilized by Intramolecular Disulfide Bonds

The crystal structure of Sulfolobus solfataricus 5'-deoxy-5'-methylthioadenosine phosphorylase II (SsMTAPII) in complex with 5'-deoxy-5'-methylthioadenosine (MTA) and sulfate was determined to 1.45A resolution. The hexameric structure of SsMTAPII is a dimer-of-trimers with one active site per monomer. The oligomeric assembly of the trimer and the monomer topology of SsMTAPII are almost identical with trimeric human 5'-deoxy-5'-methylthioadenosine phosphorylase (hMTAP). SsMTAPII is the first reported hexameric member in the trimeric class of purine nucleoside phosphorylase (PNP) from Archaea. Unlike hMTAP, which is highly specific for MTA, SsMTAPII also accepts adenosine as a substrate. The residues at the active sites of SsMTAPII and hMTAP are almost identical. The broad substrate specificity of SsMTAPII may be due to the flexibility of the C-terminal loop. SsMTAPII is extremely thermoactive and thermostable. The three-dimensional structure of SsMTAPII suggests that the unique dimer-of-trimers quaternary structure, a CXC motif at the C terminus, and two pairs of intrasubunit disulfide bridges may play an important role in its thermal stability.

Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

The extremely heat-stable 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction. 5'-Methylthioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit. Analysis of the fragments obtained after digestion of the protein alkylated without previous reduction identified two intrasubunit disulfide bridges. The enzyme is very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 3.0 M after 22 h incubation. This value decreases to 2.0 M in the presence of 30 mM dithiothreitol, furnishing evidence that disulfide bonds are needed for protein stability. The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays, fluorescence measurements and SDS/PAGE. The finding of multiple disulfide bridges in 5'-methylthioadenosine phosphorylase from Pyrococcus furiosus argues strongly that disulfide bond formation may be a significant molecular strategy for stabilizing intracellular hyperthermophilic proteins.

Complete primary structure of a galactose-specific lectin from the venom of the rattlesnake Crotalus atrox. Homologies with Ca2(+)-dependent-type lectins

The complete primary structure of a galactose-specific lectin contained in the venom of the rattlesnake, Crotalus atrox, was determined. The lectin is composed of two covalently linked, identical subunits, each consisting of 135 amino acid residues. Under physiological conditions the lectin proved to be highly aggregated. The venom lectin contained 9 half-cystines, 8 of which formed four intrasubunit disulfide bridges (Cys3-Cys14, Cys31-Cys131, Cys38-Cys133, and Cys106-Cys123), while Cys86 was involved in an intersubunit disulfide bridge. Because of the high content of disulfide bridges, the intact lectin was extremely resistant to tryptic digestion. The determined amino acid sequence was found to be homologous with those of the so-called carbohydrate recognition domains of Ca2(+)-dependent-type lectins in animal. Among them, 8 amino acid residues (Cys31, Gly69, Trp92, Pro97, Cys106, Asp120, Cys123, and Cys131) were completely conserved. Leu40, Trp67, and Trp81 were also well conserved. The rattlesnake venom lectin showed high hemagglutinating activity. These results, together with the occurrence of similar lectins in crotalid venoms, suggest that these lectins have evolved in order to make the venom a more effective weapon to capture prey animals.

Limited proteolysis of inactive tetrameric chloroplast NADP-Malate dehydrogenase produces active dimers

Carboxy-terminal amino acids of NADP-dependent malate dehydrogenase (EC 1.1.1.82) from pea chloroplasts were removed by treatment with carboxypeptidase Y. This results in the activation of the inactive oxidized enzyme, while activation by light in vivo is thought to occur via reduction of an intrasubunit disulfide bridge. After proteolytic activation the oxidized enzyme had a specific activity of 100 U/mg protein, which is 50% of the maximal activity of the control enzyme in the reduced state. When the truncated enzyme was reduced with dithiothreitol (DTT), the specific activity was further increased to 1200 U/mg. While the native enzyme is composed of four identical subunits of 38,900 Da, the truncated malate dehydrogenase forms dimers composed of two subunits of 38,000 Da. No further change of molecular mass or activity was noticed subsequent to prolonged incubation of native NADP-malate dehydrogenase with carboxypeptidase Y for several days. When the enzyme is denatured by 2 m guanidine-HCl, the proteolytic activation proceeds more rapidly, but only transiently. The truncated enzyme is less accessible to activation by reduced thioredoxin, but the stimulation of activity by DTT alone is more rapid than that of the native enzyme. These results indicate that only a small carboxy-terminal peptide of native NADP-malate dehydrogenase from pea chloroplasts is accessible to proteolytic degradation and that this peptide is involved in the regulation of activity, tetramer formation, and thioredoxin binding. While the pH optimum for catalytic activity of the intact reduced enzyme is at pH 8.0–8.5, it is shifted to more acidic values upon proteolysis of NADP-malate dehydrogenase. At pH values below 8 the reduced truncated enzyme exhibits substrate inhibition by Oxaloacetate.

Isolation and sequence of complementary DNA encoding human extracellular superoxide dismutase.

A complementary DNA (cDNA) clone from a human placenta cDNA library encoding extracellular superoxide dismutase (EC-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) has been isolated and the nucleotide sequence determined. The cDNA has a very high G+C content. EC-SOD is synthesized with a putative 18-amino acid signal peptide, preceding the 222 amino acids in the mature enzyme, indicating that the enzyme is a secretory protein. The first 95 amino acids of the mature enzyme show no sequence homology with other sequenced proteins and there is one possible N-glycosylation site (Asn-89). The amino acid sequence from residues 96-193 shows strong homology (approximately 50%) with the final two-thirds of the sequences of all known eukaryotic CuZn SODs, whereas the homology with the P. leiognathi CuZn SOD is clearly lower. The ligands to Cu and Zn, the cysteines forming the intrasubunit disulfide bridge in the CuZn SODs, and the arginine found in all CuZn SODs in the entrance to the active site can all be identified in EC-SOD. A comparison with bovine CuZn SOD, the three-dimensional structure of which is known, reveals that the homologies occur in the active site and the divergences are in the part constituting the subunit contact area in CuZn SOD. Amino acid sequence 194-222 in the carboxyl-terminal end of EC-SOD is strongly hydrophilic and contains nine amino acids with a positive charge. This sequence probably confers the affinity of EC-SOD for heparin and heparan sulfate. An analysis of the amino acid sequence homologies with CuZn SODs from various species indicates that the EC-SODs may have evolved from the CuZn SODs before the evolution of fungi and plants.

Anadara ovalis hemoglobins: Distinct dissociation and ligand binding characteristics

1. 1. The red cells of the arcid clam Anadara ovalis contain two electrophoretically distinct hemoglobins: Hb Major (Hb Ma) and Hb Minor (Hb Mi). The major component consists of two electrophoretically indistinguishable tetramers each composed of two heterodimers; the minor hemoblogin is a homodimer whose subunits are different from the tetramer. 2. 2. Functionally, Hb Ma has a higher P 50, exhibits a concentration dependent oxygen affinity, has significant ligand cooperativity ( n = 2.0), lacks a Bohr effect and is unaffected by ATP. 3. 3. HB Mi has a P 50 which is lower and independent of hemoglobin concentration, shows appreciable cooperativity ( n = 1.4) and exhibits no heterotropic effects. 4. 4. Both Hb Ma and Mi are resistant to dissociation in the presence of 1.0M NaI, NaCl and guanidine-HCl but dissociate to monomers when converted to the aquamet but not the cyanmet derivative. 5. 5. The dissociation is completely inhibited by mercaptoethanol. 6. 6. The large number of reactive - SH groups (10–13 per tetramer) suggests that the monomerization is mediated by intra-subunit disulfide bridge formation.

Profile of the disulfide bonds in acetylcholinesterase.

The inter- and intrasubunit disulfide bridges for the 11 S form of acetylcholinesterase isolated from Torpedo californica have been identified. Localized within the basal lamina of the synapse, the dimensionally asymmetric forms of acetylcholinesterase contain either two (13 S) or three (17 S) sets of catalytic subunits linked to collagenous and noncollagenous structural subunits. Limited proteolysis of these molecules yields a tetramer of catalytic subunits that sediments at 11 S. Each catalytic subunit contains 8 cysteine residues which were identified following tryptic digestion of the reduced, 14C-carboxymethylated protein. The tryptic peptides were purified by gel filtration followed by reverse-phase high performance liquid chromatography (HPLC) and then sequenced. The disulfide bonding profile was determined by treating the native, nonreduced 11 S form of acetylcholinesterase with a fluorescent, sulfhydryl-specific reagent, monobromobimane, prior to tryptic digestion. Peptides again were resolved by gel filtration and reverse-phase HPLC. One fluorescent cysteine-containing peptide was identified, indicating that a single sulfhydryl residue, Cys231, was present in its reduced form. Three pairs of disulfide-bonded peptides were identified. These were localized in the polypeptide chain based on the cDNA-deduced sequence of the protein and were identified as Cys67-Cys94, Cys254-Cys265, and Cys402-Cys521. Hence, three loops are found in the secondary structure. Cys572, located in the carboxyl-terminal tryptic peptide, was disulfide-bonded to an identical peptide and most likely forms an intersubunit cross-link. Since the 6 cysteine residues in acetylcholinesterase that are involved in intrachain disulfide bonds are conserved in the sequence of the homologous protein thyroglobulin, it is likely that both proteins share a common folding pattern in their respective tertiary structures. Cys231 and the carboxyl-terminal cysteine residue Cys572 are not conserved in thyroglobulin.

The molecular weight of rabbit muscle aldolase and the properties of the subunits in acid solution

Re-examination of the weight-average molecular weight of rabbit muscle aldolase in 0.15 m NaCl by the high-speed sedimentation equilibrium and field relaxation methods gave values of 160,000 ± 1500 and 162,500, respectively. When the protein was allowed to dissociate in an acidic solution (pH 2.0, μ = 0.06) the weight-average molecular weight was estimated as 40,000 ± 2000. The results are consistent with a structure composed of four polypeptide chains. Extensive polydispersity was observed after the protein was dialyzed in acidic buffer for more than 3 days in the cold. The thermodynamic parameters calculated for the breakdown of carboxymethylated aldolase under this condition at two temperatures suggested that the subunits are degraded in acid, with the cleavage of one or more acid-labile peptide bonds. O-Phenanthroline oxidized aldolase, containing approximately four disulfide bonds per protein molecule, showed evidence of degradation in acid only after treatment with mercaptoethanol. Intra-subunit disulfide bridges in the oxidized enzyme appear to hold the fragments together.