Crystal structures of reduced and oxidized DsbA: investigation of domain motion and thiolate stabilization

BACKGROUND: The redox proteins that incorporate a thioredoxin fold have diverse properties and functions. The bacterial protein-folding factor DsbA is the most oxidizing of the thioredoxin family. DsbA catalyzes disulfide-bond formation during the folding of secreted proteins. The extremely oxidizing nature of DsbA has been proposed to result from either domain motion or stabilizing active-site interactions in the reduced form. In the domain motion model, hinge bending between the two domains of DsbA occurs as a result of redox-related conformational changes. RESULTS: We have determined the crystal structures of reduced and oxidized DsbA in the same crystal form and at the same pH (5.6). The crystal structure of a lower pH form of oxidized DsbA has also been determined (pH 5.0). These new crystal structures of DsbA, and the previously determined structure of oxidized DsbA at pH 6.5, provide the foundation for analysis of structural changes that occur upon reduction of the active-site disulfide bond. CONCLUSIONS: The structures of reduced and oxidized DsbA reveal that hinge bending motions do occur between the two domains. These motions are independent of redox state, however, and therefore do not contribute to the energetic differences between the two redox states. Instead, the observed domain motion is proposed to be a consequence of substrate binding. Furthermore, DsbA's highly oxidizing nature is a result of hydrogen bond, electrostatic and helix-dipole interactions that favour the thiolate over the disulfide at the active site.     

Three-dimensional solution structure of Callinectes sapidus metallothionein-1 determined by homonuclear and heteronuclear magnetic resonance spectroscopy

Metallothionein is a cysteine-rich metal-binding protein whose biosynthesis is closely regulated by the level of exposure of an organism to zinc, copper, cadmium, and other metal salts. The metallothionein from Callinectes sapidus is known to bind six divalent metal ions in two separate metal-binding clusters. Heteronuclear 1H-113Cd and homonuclear 1H-1H NMR correlation experiments have been used to establish that the two clusters reside in two distinct protein domains. The three-dimensional solution structure of the metallothionein has been determined using the distance and angle constraints derived from these two-dimensional NMR data sets and a distance geometry/simulated annealing protocol. There are no interdomain short distance (< or = 4.5 A) constraints observed in this protein, enabling the calculation of structures for the N-terminal, beta domain and the C-terminal, alpha domain separately. A total of 18 structures were obtained for each domain. The structures are based on a total of 364 experimental NMR restraints consisting of 277 approximate interproton distance restraints, 12 chi 1 and 51 phi angular restraints, and 24 metal-to-cysteine connectivities obtained from 1H-113Cd correlation experiments. The only element of regular secondary structure in either of the two domains is a short segment of helix in the C-terminal alpha domain between Lys42 and Thr48. The folding of the polypeptide backbone chain in each domain, however, gives rise to several type I beta turns. There are no type II beta turns.     

Interactions of reduced and oxidized nicotinamide mononucleotide with wild-type and alphaD195E mutant proton-pumping nicotinamide nucleotide transhydrogenases from Escherichia coli

The interaction of reduced nicotinamide mononucleotide (NMNH), constituting one half of NADH, with the wild-type and alphaD195E proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli was investigated. Reduction of thio-NADP+ by NMNH was catalysed at approximately 30% of the rate with NADH. Other activities including proton pumping and the cyclic reduction of 3'-acetyl-pyridine-NAD+ by NMNH in the presence of NADP+ were more strongly inhibited. The alphaD195 residue is assumed to interact with the 2'-OH moiety of the adenosine-5'-phosphate, i.e., the second nucleotide of NADH. Mutation of this residue to alphaD195E resulted in a 90% decrease in activity with NMNH as well as NADH as substrate, suggesting that it produced global structural changes of the NAD(H) binding site. The results suggest that the NMN moiety of NADH is a substrate of transhydrogenase, and that the adenine nucleotide is not required for catalysis or proton pumping.     

In vitro and in vivo redox states of the Escherichia coli periplasmic oxidoreductases DsbA and DsbC

DsbC is a periplasmic protein of Escherichia coli that was previously identified by a genetic selection that rescued sensitivity to dithiothreitol in Tn10 mutagenized cells. The Erwinia chrysanthemi dsbC gene was identified in a previous genetic screen to restore motility in a dsbA null strain. In order to analyze the biochemical role of E. coli DsbC, the protein was overexpressed, purified, and compared with DsbA in terms of disulfide isomerization, thiol oxidation, and in vivo redox state. In vitro, DsbC and DsbA have an equivalent kcat for disulfide isomerization with the model substrate, misfolded insulin-like growth factor-1. However, DsbA is a more effective oxidant than DsbC of protein dithiols. In vivo, DsbA is found exclusively in the oxidized state in wild-type strains grown in rich media. On the other hand, in vivo DsbC has one pair of cysteines oxidized and one pair reduced. DsbD is required to maintain this reduced pair of cysteines, confirming previous genetic results. A dsbC deletion strain showed decreases in the production of some, but not all, heterologous proteins containing multiple disulfide bonds. Notably, those proteins affected by the dsbC deletion do not have the cysteines paired consecutively.     

Q-band resonance Raman spectra of oxidized and reduced mitochondrial bc1 complexes

Recently published crystallographic studies of mitochondrial bc1 complexes have stimulated renewed interest in the active site architecture of these important integral membrane proteins. We present resonance Raman spectra obtained via variable excitation within the heme Q-band from samples poised in several different net redox states. Appropriate subtraction and polarization analysis allows the vibrational behavior of the individual heme bL,bH, and c1 sites to be assessed. The spectra of the b hemes are particularly noteworthy. They exhibit evidence for a protonation equilibrium involving heme axial ligands and reveal a marked structural heterogeneity at the heme bH site that most likely involves nonplanar distortions of the macrocycle. The possible implications of these findings for heme functionality are discussed.     

Functional and metabolic evaluation of the athlete's heart by magnetic resonance imaging and dobutamine stress magnetic resonance spectroscopy

BACKGROUND: The question of whether training-induced left ventricular hypertrophy in athletes is a physiological rather than a pathophysiological phenomenon remains unresolved. The purpose of the present study was to detect any abnormalities in cardiac function in hypertrophic hearts of elite cyclists and to examine the response of myocardial high-energy phosphate metabolism to high workloads induced by atropine-dobutamine stress. METHODS AND RESULTS: We studied 21 elite cyclists and 12 healthy control subjects. Left ventricular mass, volume, and function were determined by cine MRI. Myocardial high-energy phosphates were examined by 31P magnetic resonance spectroscopy. There were no significant differences between cyclists and control subjects for left ventricular ejection fraction (59+/-5% versus 61+/-4%), left ventricular cardiac index (3.4+/-0.4 versus 3.4+/-0.4 L x min(-1) x m[-2]), peak early filling rate (562+/-93 versus 535+/-81 mL/s), peak atrial filling rate (315+/-93 versus 333+/-65 mL/s), ratio of early and atrial filling volumes (3.0+/-1.0 versus 2.6+/-0.6), mean acceleration gradient of early filling (5.2+/-1.4 versus 5.8+/-1.9 L/s2), mean deceleration gradient of early filling(-3.1 +/- 0.9 versus -3.2 +/- 0.7 L/s2), mean acceleration gradient of atrial filling (3.6+/-1.8 versus 4.5+/-1.7 L/s2), and atrial filling fraction (0.23+/-0.06 versus 0.26+/-0.04, respectively). Cyclists and control subjects showed similar decreases in the ratio of myocardial phosphocreatine to ATP measured with 31P magnetic resonance spectroscopy during atropine-dobutamine stress (1.41+/-0.20 versus 1.41+/-0.18 at rest to 1.21+/-0.20 versus 1.16+/-0.13 during stress, both P=NS). CONCLUSIONS: Left ventricular hypertrophy in cyclists is not associated with significant abnormalities of cardiac function or metabolism as assessed by MRI and spectroscopy. These observations suggest that training-induced left ventricular hypertrophy in cyclists is predominantly a physiological phenomenon.     

NMR assignments, secondary structure and hydration of oxidized Escherichia coli flavodoxin

Recombinant flavodoxin from Escherichia coli was uniformly enriched with 15N and 13C isotopes and its oxidized form in aqueous solution investigated by three-dimensional NMR spectroscopy. Nearly complete 1H, 15N and 13C resonance assignments were obtained. The secondary structure was determined from chemical shift, NOE and 3J(HNH alpha) coupling constant data. Like homologous long-chain flavodoxins, E. coli flavodoxin contains a five-stranded parallel beta-sheet and five helices. The beta-strands were found to comprise the residues 3-8, 29-34, 48-56, 80-89, 114-116 and 141-145. The helices comprise residues 12-25, 40-45, 62-73, 98-108 and 152-166. The FMN-binding site was determined by intermolecular NOEs and low-field shifted amide proton resonances induced by the phosphoester group of the cofactor. The data are in good agreement with a previously predicted model of E. coli flavodoxin [Havel, T. F. (1993) Mol. Sim. 10, 175-210]. The analysis of of water-flavodoxin NOEs revealed the presence of two, possibly three, buried hydration water molecules which are located at sites, where homologous flavodoxins from Anacystis nidulans and Anabena 7120 contain conserved hydration water molecules. One of these water molecules mediates hydrogen bonds between the protein backbone and the ribityl chain of the FMN cofactor.     

Differences in the magnesium dependences of the class I and class II aminoacyl-tRNA synthetases from Escherichia coli

The magnesium dependences of the ATP/PPi exchange and tRNA aminoacylation of reactions were measured for six aminoacyl-tRNA synthetases (isoleucyl-, tyrosyl- and arginyl-tRNA synthetases from class I, and histidyl-, lysyl- and phenylalanyl-tRNA synthetases from class II). The measured values were subjected to best-fit analyses using sum square error calculations between the data and the calculated curves in order to find the mode of participation of the Mg2+ and to optimize the sets of the kinetic constants. The following four dependences were observed: the class II synthetases require three Mg2+ for the activation reaction (including the one in MgATP), but the class I synthetases require only one Mg2+ (in MgATP); in class II synthetases both MgPPi and Mg2PPi participate in the pyrophosphorolysis of the aminoacyl adenylate. Arginyl-tRNA synthetase from class I also shows a better fit if also Mg2PPi reacts, but in the isoleucyl- and tyrosyl-tRNA synthetases only MgPPi but not Mg2PPi is used in the pyrophosphorolysis. Different synthetases have different requirements for the tRNA-bound Mg2+ and spermidine, independent of the enzyme class. 1-4 Mg2+ or spermidines are required in the best fit models. At the end of the reaction in all the synthetases analysed the dissociation of Mg2+ from the product aminoacyl-tRNA essentially enhances the subsequent dissociation of the aminoacyl-tRNA from the enzyme. The binding of ATP to the E. aminoacyl-tRNA complex also speeds up the dissociation of the aminoacyl-tRNA from most of these enzymes.     

Evolutionary relationships among pathogenic and nonpathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies

Within the species Escherichia coli, there are commensal strains and a variety of pathogenic strains, including enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and urinary tract infection (UTI) strains. The pathogenic strains are identified by serotype and by possession of specific virulence determinants (toxins and adhesions, etc.) encoded by either monocistronic genes, plasmids, or pathogenicity islands. Although there are studies on the relationships between selected pathogenic strains, the relatedness among the majority of the pathogenic forms to each other, to commensal E. coli, and to the genus Shigella (which has often been suggested to be part of E. coli) has not been determined. We used multilocus enzyme electrophoresis (MLEE) at 10 enzyme loci and the sequence of the mdh housekeeping gene to study the genetic relationships of pathogenic E. coli strains (including Shigella clones), namely, 5 EPEC strains (serotypes O111 and O55), 3 EHEC strains (serotype O157), 6 ETEC strains (serotypes O78, O159, and O148), 5 EIEC strains (serotypes O124, O28, and O112), and 13 Shigella strains representing clones Flexneri, Dysenteriae, Boydii, and Sonnei, to commensal E. coli strains. Both the MLEE and mdh sequence trees reveal that EPEC, EHEC, ETEC, EIEC, and UTI strains are distributed among the ECOR set groups, with no overall clustering of EPEC, ETEC, EIEC, or UTI strains. The genus Shigella is shown to comprise a group of closely related pathogenic E. coli strains. Six pathogenic strains, i.e., M502 (EIEC; O112ac:NM), M503 (EPEC; O111:H12), M526 (ETEC; O159:H4), M522 (EPEC; O111ac:H12), M524 (ETEC; O78:H11), and M506 (ETEC; O78:H11), were found to have mdh sequences identical to those of five ECOR group A strains (ECOR5, ECOR10, ECOR14, ECOR6, and K-12). All 11 strains are closely related by MLEE. The results indicate that pathogenic strains of E. coli do not have a single evolutionary origin within E. coli but have arisen many times. The results also suggest the possibility that any E. coli strain acquiring the appropriate virulence factors may give rise to a pathogenic form.     

Solution structure of the recombinant oxidized rabbit uteroglobin using homonuclear and heteronuclear multidimensional NMR

Rabbit uteroglobin (rab-UG) is a 16-kDa homodimeric secretory protein with potent anti-inflammatory/immunomodulatory properties. Its physiological role is still unclear, although it was observed that several small hydrophobic molecules bind to the oxidized and the reduced uteroglobin. It is suggested that the formation and/or disruption of the two disulphide bridges not only regulates this binding itself, but also the affinity to the ligand. The determination of the solution structure has been started with the assignment of 1H, 15N and 13C resonances of the oxidized rabbit uteroglobin, based on several two-dimensional and three-dimensional homonuclear and heteronuclear double and triple resonance experiments. The assignment was possible with the overproduction of the wild-type as well as of uniformly 15N-labeled and 15N/13C-labeled samples of the recombinant protein. A complete assignment of 1H, 15N and 13C resonances, the secondary-structure elements and the tertiary structure in solution is presented. The tertiary solution structure was found to be in good agreement with the previously determined crystal structure of rab-UG and with the solution structure of human uteroglobin (h-UG). h-UG and rab-UG are extremely stable proteins within a wide range of pH and temperatures. Some of the binding characteristics of ligands of rab-UG and a mutant with all cysteine residues exchanged to serine residues are discussed.     

The design of spin probes for electron magnetic resonance spectroscopy and imaging

The problems that have to be addressed in designing suitable spin probes are manifold. In this article it will be shown that some the properties the probes need to possess are (i) a simple EMR spectrum (in some types of imaging or oximetry a single line is desirable), (ii) sharp lines with no unresolved splittings, to provide maximum sensitivity to detection, (iii) chemical, metabolic and thermal stability, (iv) easy possible synthesis to permit substitution with isotopes, prosthetic groups or groups to control the partition coefficients in lipid/water systems, and (v) electronic anisotropy, if rotational correlation times are to be determined, for example for the measurement of local viscosity.     

Nuclear magnetic resonance spectroscopy: methodology and applications to the study of asphyxia neonatorum

Cerebral metabolism has been extensively studied by magnetic resonance spectroscopy (MRS). MRS allows the study of neonates brain maturation as well as the onset and the evolution of brain injury. The use of phosphorous spectroscopy allows the quantification of phosphorylated metabolites. Thus, the measurement of the relative concentrations of creatine-phosphate and inorganic-phosphate is a prognostic factor of the outcome of a neonate after birth asphyxia. Absolute concentrations have more recently been studied and seem to be more significant. Proton MRS gives access to brain metabolites such as choline, lactate, N-acetyl aspartate and taurine. Its use is more recent than the phosphorous spectroscopy but first results already show its potential in neonatology.     

Sequential observations of brain edema with proton magnetic resonance imaging and spectroscopy

The purpose of this study was to assess the relationship between morphological and metabolic changes in brain edema using proton magnetic resonance systems. The serial changes during the first 24 hours in the cold-injury trauma rat brain model were investigated by proton magnetic resonance imaging (1H MRI) and high-resolution proton MR spectroscopy (1H MRS). We also analyzed the efficacy of AVS 1,2-bis (nicotinamide)-propane which can scavenge free radicals to the edema in this experiment. The edema was developing extensively via the corpus callosum in ipsi- and contralateral hemispheres as shown by gradually increased signal intensity on 1H MRI. 1H MRS initially showed accumulation of acetate and lactate, and transient increasing of glutamine. After 24 hours, the increased glutamine decreased below the control, alanine increased, and N-acetyl asparatate decreased with the edema development. AVS-treatment significantly suppressed edema development, increases of lactate and alanine and decreases of N-acetyl asparatate. We suggest that the cold-induced lesion contains anaerobic glycolysis deterioration and results in severe brain tissue breakdown. AVS is proved valuable for the treatment of this edema lesion. Clinical 1H MRS showed prolonged lactate elevation and significant decreases of other metabolites in human ischemic stroke edema. In peritumoral edema, decreased N-acetyl asparatate gradually improved, and slightly elevated lactate disappeared after tumor removal. 1H MRS feasibly characterizes the ischemic and peritumoral edema and makes a quantitative analysis in human brain metabolism. We believe the combined 1H MRI and MRS study is a practical method to monitor the brain conditions and will make it easy and possible to find new therapeutic agents to some brain disorders.     

A flavodoxin that is required for enzyme activation: the structure of oxidized flavodoxin from Escherichia coli at 1.8 A resolution

In Escherichia coli, flavodoxin is the physiological electron donor for the reductive activation of the enzymes pyruvate formate-lyase, anaerobic ribonucleotide reductase, and B12-dependent methionine synthase. As a basis for studies of the interactions of flavodoxin with methionine synthase, crystal structures of orthorhombic and trigonal forms of oxidized recombinant flavodoxin from E. coli have been determined. The orthorhombic form (space group P2(1)2(1)2(1), a = 126.4, b = 41.10, c = 69.15 A, with two molecules per asymmetric unit) was solved initially by molecular replacement at a resolution of 3.0 A, using coordinates from the structure of the flavodoxin from Synechococcus PCC 7942 (Anacystis nidulans). Data extending to 1.8-A resolution were collected at 140 K and the structure was refined to an Rwork of 0.196 and an Rfree of 0.250 for reflections with I > 0. The final model contains 3,224 non-hydrogen atoms per asymmetric unit, including 62 flavin mononucleotide (FMN) atoms, 354 water molecules, four calcium ions, four sodium ions, two chloride ions, and two Bis-Tris buffer molecules. The structure of the protein in the trigonal form (space group P312, a = 78.83, c = 52.07 A) was solved by molecular replacement using the coordinates from the orthorhombic structure, and was refined with all data from 10.0 to 2.6 A (R = 0.191; Rfree = 0.249). The sequence Tyr 58-Tyr 59, in a bend near the FMN, has so far been found only in the flavodoxins from E. coli and Haemophilus influenzae, and may be important in interactions of flavodoxin with its partners in activation reactions. The tyrosine residues in this bend are influenced by intermolecular contacts and adopt different orientations in the two crystal forms. Structural comparisons with flavodoxins from Synechococcus PCC 7942 and Anaebaena PCC 7120 suggest other residues that may also be critical for recognition by methionine synthase.     

31P magnetic resonance spectroscopy in the frontal lobe of major depressed patients

Most research with 31P-magnetic resonance spectroscopy (31P-MRS) in affective disorders has been done in the field of bipolar disturbances. Reduced frontal and temporal lobe phosphomonoester (PME) concentrations were measured in the euthymic state, whereas increased values were found in the depressed state. In bipolar-II patients reduced phosphocreatine (PCr) concentrations were reported in the euthymic, depressed, and manic state. The aim of the present study was to explore whether PME and PCr were also altered in the frontal lobe of major depressed, unipolar patients. Therefore, we used 31P-MRS to investigate the relative phospholipid and high-energy phosphate concentrations in the frontal lobe of 14 unipolar patients, mostly medicated, and 8 age-matched controls. We found increased PME and decreased ATP values. Other 31P-MRS parameters were not different in both groups. Phosphomonoester percentages correlated negatively with the degree of depression. Thus, the main alterations found in bipolar depressed patients could also be demonstrated in unipolar depressed patients. The results are discussed with regard to disturbed phospholipid and intracellular high-energy phosphate metabolism in depressed patients.     

One- and two-dimensional NMR characterization of oxidized and reduced cytochrome c' from Rhodocyclus gelatinosus

1D and 2D NMR spectra of both the reduced and oxidized forms of cytochrome c' from Rhodocyclus gelatinosus have been recorded. The analysis of the pH dependence of the 1H NMR spectrum of the ferric form has been performed, and two main ionizing groups have been identified. By comparison of the pH dependence of the available spectra of cytochromes c', an ambiguity remaining from previous studies on related cytochromes c' has been solved. By means of 2D spectra, an assignment of all the paramagnetically shifted signals is proposed both for the ferrous and for the ferric forms.     

Ultrastructural aspects of the interaction between Escherichia coli and cultured epithelial cells

Microorganisms from two Escherichia coli serotypes were allowed to interact with cultured epithelial cells and the interaction process was followed from 30 to 360 min. Destruction of the microvilli on the epithelial monolayers as well as of the cells themselves was observed only with 0111:H2 serotype.     

In vivo neurochemistry of the brain in schizophrenia as revealed by magnetic resonance spectroscopy

Magnetic resonance spectroscopy (MRS), an application of the methods of nuclear magnetic resonance (NMR), is a functional imaging modality that provides a view of localized biochemistry in vivo. A number of studies applying MRS to the neurochemistry of schizophrenia have been reported, which encompass a range of patient populations, states of medication, anatomic regions, nuclear species, and MRS techniques. A brief review of the history and methodology of NMR and MRS is presented. Comparison is made of MRS capabilities with other functional imaging modalities. Aspects of the neurochemistry of schizophrenia relevant to MRS studies are reviewed, as are the reported MRS studies involving patients with schizophrenia. Areas of consistent findings include decreased phosphomonoesters and increased phosphodiesters in frontal lobes, and decreases in the putative neuronal cell marker, N-acetylaspartate, in temporal lobes. Studies of neurotransmitters such as glutamate, gamma-aminobutyric acid, and glutamine have generated inconsistent results. New insights into alterations in neurochemistry in schizophrenia have been provided by MRS. Studies of neurotransmitters have future potential with improvements in field strength and in spectral editing techniques. MRS has the potential to measure brain medication levels and simultaneous effects on neurochemistry. MRS may assist in characterizing high-risk populations, and ultimately guide medication use.     

Quantitative proton magnetic resonance spectroscopy of the basal ganglia in patients with affective disorders

We have retrospectively reviewed 53 cases (62 hips) with a diagnosis of slipped capital wedge epiphysis. After admission to our Hospital, a skin longitudinal traction was applied for 2 weeks, All patients were treated afterward with pinning in situ without manipulation in the operating room. Group A (31 hips) consisted of patients treated with smooth K wires and group B (31 hips) of patients treated with cannulated screws. We found a high incidence of pin penetration in group A (27 hips), whereas there was just one case in group B. Physical closure was considered when 75% of proximal growth plate disappeared in the frog lateral view and both groups showed similar values (7 months). Chondrolysis was observed in just three cases in group A, and one case had an avascular necrosis. Few complications were observed compared with the high rate of pin penetration, and we suggest that preoperative traction may be a relevant factor contributing to the low incidence of avascular necrosis (1.6%).