Tautomeric state and pKa of the phosphorylated active site histidine in the N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system

The phosphorylated form of the N-terminal domain of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been investigated by one-bond and long-range 1H-15N correlation spectroscopy. The active site His 189 is phosphorylated at the Nepsilon2 position and has a pKa of 7.3, which is one pH unit higher than that of unphosphorylated His 189. Because the neutral form of unphosphorylated His 189 is in the Ndelta1-H tautomer, and its Nepsilon2 atom is solvent inaccessible and accepts a hydrogen bond from the hydroxyl group of Thr 168, both protonation and phosphorylation of His 189 must be accompanied by a change in the side-chain conformation of His 189, specifically from a chi(2) angle in the g+ conformer in the unphosphorylated state to the g- conformer in the phosphorylated state.     

Regulation of the raffinose permease of Escherichia coli by the glucose-specific enzyme IIA of the phosphoenolpyruvate:sugar phosphotransferase system

In enteric bacteria, chromosomally encoded permeases specific for lactose, maltose, and melibiose are allosterically regulated by the glucose-specific enzyme IIA of the phosphotransferase system. We here demonstrate that the plasmid-encoded raffinose permease of enteric bacteria is similarly subject to this type of inhibition.     

Backbone NMR assignments and secondary structure of the N-terminal domain of DnaB helicase from E. coli

A case of adult onset myopathy who showed a peculiar sleep-related respiratory disorder (SRRD) is reported. She recovered from respiratory failure after tracheostomy and/or with the aid of the respirator used only during the night. Sleep study without the use of respirator revealed that her sleep was highly fragmented by frequent arousal responses due to inspiratory effort but not by apnea or hypopnea. To our knowledge this type of SRRD has not been described.     

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.     

Mutational analysis of the Escherichia coli K-12 TolA N-terminal region and characterization of its TolQ-interacting domain by genetic suppression

The Tol-Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity. They form two complexes in the cell envelope. Transmembrane domains of TolQ, TolR, and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. The N-terminal transmembrane domain of TolA anchors the protein to the cytoplasmic membrane and interacts with TolQ and TolR. Extensive mutagenesis of the N-terminal part of TolA was carried out to characterize the residues involved in such processes. Mutations affecting the function of TolA resulted in a lack or an alteration in TolA-TolQ or TolR-TolA interactions but did not affect the formation of TolQ-TolR complexes. Our results confirmed the importance of residues serine 18 and histidine 22, which are part of an SHLS motif highly conserved in the TolA and the related TonB proteins from different organisms. Genetic suppression experiments were performed to restore the functional activity of some tolA mutants. The suppressor mutations all affected the first transmembrane helix of TolQ. These results confirmed the essential role of the transmembrane domain of TolA in triggering interactions with TolQ and TolR.     

Identification of peptides inhibiting enzyme I of the bacterial phosphotransferase system using combinatorial cellulose-bound peptide libraries

The effect of hyperbaric oxygen (HBO) treatment on regeneration of the rat sciatic nerve was studied. The sciatic nerve was crushed with a pair of pliers and the animals were either left untreated or subjected to a series of 45-min exposures to 100% O2 at 3.3 atm absolute pressure at 0, 4, and 8 h postoperatively and then every 8 h. Regeneration was evaluated using the pinch-reflex test at 3, 4, or 5 days following surgery and with neurofilament staining at 4 days. The regeneration distances at all time points were significantly longer in animals exposed to hyperbaric oxygen treatment independent of the evaluation procedure. A short initial period of the same HBO treatment schedule, with no more treatments after 25 h, appeared as effective as when treatments were maintained being given every 8 h until evaluation. We conclude that HBO treatment stimulates axonal outgrowth following a nerve crush lesion.     

Mutational analysis of invariant arginines in the IIAB(Man) subunit of the Escherichia coli phosphotransferase system

The mannose transporter of bacterial phosphotransferase system mediates uptake of mannose, glucose, and related hexoses by a mechanism that couples translocation with phosphorylation of the substrate. It consists of the transmembrane IIC(Man)-IID(Man) complex and the cytoplasmic IIAB(Man) subunit. IIAB(Man) has two flexibly linked domains, IIA(Man) and IIB(Man), each containing a phosphorylation site (His-10 and His-175). Phosphoryl groups are transferred from the phosphoryl carrier protein phospho-HPr to His-10, hence to His-175 and finally to the 6' OH of the transported hexose. Phosphate-binding sites and phosphate-catalytic sites frequently contain arginines, which by their guanidino group can stabilize phosphate through hydrogen bonding and electrostatic interactions. IIB(Man) contains five arginines which are invariant in the homologous IIB subunits of Escherichia coli, Klebsiella pneumoniae and Bacillus subtilis. The IIA domains have no conserved arginines. The five arginines were replaced by Lys or Gln one at a time, and the mutants were analyzed for transport and phosphorylation activity. All five IIB mutants can still be phosphorylated at His-175 by the IIA domain. R172Q is completely inactive with respect to glucose phosphotransferase (phosphoryltransfer from His-175 to the 6' OH of Glc) and hexose transport activity. R168Q has no hexose transport and strongly reduced phosphotransferase activity. R204K has no transport but almost normal phosphotransferase activity. R304Q has only slightly reduced transport activity. R190K behaves like wild-type IIAB(Man). Arg-168, Arg-172, and Arg-304 are part of the hydrogen bonding network on the surface of IIB, which contains the active site His-175 and the interface with the IIA domain (Schauder, S., Nunn, R.S., Lanz, R., Erni, B. and Schirmer, T. (1998) J. Mol. Biol. 276, 591-602) (Protein Data Bank accession code 1BLE). Arg-204 is at the putative interface between IIB(Man) and the IIC(Man)-IID(Man) complex.     

NMR structure of Escherichia coli glutaredoxin 3-glutathione mixed disulfide complex: implications for the enzymatic mechanism

Glutaredoxins (Grxs) catalyze reversible oxidation/reduction of protein disulfide groups and glutathione-containing mixed disulfide groups via an active site Grx-glutathione mixed disulfide (Grx-SG) intermediate. The NMR solution structure of the Escherichia coli Grx3 mixed disulfide with glutathione (Grx3-SG) was determined using a C14S mutant which traps this intermediate in the redox reaction. The structure contains a thioredoxin fold, with a well-defined binding site for glutathione which involves two intermolecular backbone-backbone hydrogen bonds forming an antiparallel intermolecular beta-bridge between the protein and glutathione. The solution structure of E. coli Grx3-SG also suggests a binding site for a second glutathione in the reduction of the Grx3-SG intermediate, which is consistent with the specificity of reduction observed in Grxs. Molecular details of the structure in relation to the stability of the intermediate and the activity of Grx3 as a reductant of glutathione mixed disulfide groups are discussed. A comparison of glutathione binding in Grx3-SG and ligand binding in other members of the thioredoxin superfamily is presented, which illustrates the highly conserved intermolecular interactions in this protein family.     

Regulation of glycerol metabolism in Enterococcus faecalis by phosphoenolpyruvate-dependent phosphorylation of glycerol kinase catalyzed by enzyme I and HPr of the phosphotransferase system

Using a polyclonal antibody against glycerol kinase from Enterococcus faecalis, we could demonstrate that glycerol kinase is inducible by growth on glycerol-containing medium and that during growth on glycerol the enzyme is mainly phosphorylated. Glucose and other sugars metabolized via the Embden-Meyerhof pathway strongly repressed the synthesis of glycerol kinase, while if glycerol was also present during growth, low activity, reflecting partial induction and the presence of mainly unphosphorylated, less active enzyme, was found. With gluconate, which is also a substrate of the phosphotransferase system, repression of glycerol kinase was less severe, but the enzyme was mainly present in the less active, unphosphorylated form. Effects of growth on different carbon sources on glycerol uptake are also reported.     

Multinuclear NMR resonance assignments and the secondary structure of Escherichia coli thioesterase/protease I: a member of a new subclass of lipolytic enzymes

Escherichia coli thioesterase/protease I is a 183 amino acid protein with a molecular mass of 20,500. This protein belongs to a new subclass of lipolytic enzymes of the serine protease superfamily, but with a new GDSLS consensus motif, of which no structure has yet been determined. The protein forms a tetramer at pH values above 6.5 and exists as a monomer at lower pH values. Both monomer and tetramer are catalytically active. From analysis of a set of heteronuclear multidimensional NMR spectra with uniform and specific amino acid labeled protein samples, we have obtained near-complete resonance assignments of the backbone 1H, 13C and 15N nuclei (BMRB databank accession number 4060). The secondary structure of E. coli thioesterase/protease I was further deduced from the consensus chemical shift indices, backbone short- and medium-range NOEs, and amide proton exchange rates. The protein was found to consist of four beta-strands and seven alpha-helices, arranged in alternate order. The four beta-strands were shown to form a parallel beta-sheet. The topological arrangement of the beta-strands of -1x, +2x, +1x appears to resemble that of the core region of the alpha beta hydrolase superfamily, typically found in common lipases and esterases. However, substantial differences, such as the number of beta-strands and the location of the catalytic triad residues, make it difficult to give a definitive classification of the structure of E. coli thioesterase/protease I at present.     

Solution NMR structure and backbone dynamics of the major cold-shock protein (CspA) from Escherichia coli: evidence for conformational dynamics in the single-stranded RNA-binding site

The major cold-shock protein (CspA) from Escherichia coli is a single-stranded nucleic acid-binding protein that is produced in response to cold stress. We have previously reported its overall chain fold as determined by NMR spectroscopy [Newkirk, K., Feng, W., Jiang, W., Tejero, R., Emerson, S. D., Inouye, M., and Montelione, G. T. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5114-5118]. Here we describe the complete analysis of 1H, 13C, and 15N resonance assignments for CspA, together with a refined solution NMR structure based on 699 conformational constraints and an analysis of backbone dynamics based on 15N relaxation rate measurements. An extensive set of triple-resonance NMR experiments for obtaining the backbone and side chain resonance assignments were carried out on uniformly 13C- and 15N-enriched CspA. Using a subset of these triple-resonance experiments, the computer program AUTOASSIGN provided automatic analysis of sequence-specific backbone N, Calpha, C', HN, Halpha, and side chain Cbeta resonance assignments. The remaining 1H, 13C, and 15N resonance assignments for CspA were then obtained by manual analysis of additional NMR spectra. Dihedral angle constraints and stereospecific methylene Hbeta resonance assignments were determined using a new conformational grid search program, HYPER, and used together with longer-range constraints as input for three-dimensional structure calculations. The resulting solution NMR structure of CspA is a well-defined five-stranded beta-barrel with surface-exposed aromatic groups that form a single-stranded nucleic acid-binding site. Backbone dynamics of CspA have also been characterized by 15N T1, T2, and heteronuclear 15N-1H NOE measurements and analyzed using the extended Lipari-Szabo formalism. These dynamic measurements indicate a molecular rotational correlation time taum of 4.88 +/- 0.04 ns and provide evidence for fast time scale (taue < 500 ps) dynamics in surface loops and motions on the microsecond to millisecond time scale within the proposed nucleic acid-binding epitope.     

Secondary structure of the DNA-binding domain of the c-Myb oncoprotein in solution. A multidimensional double and triple heteronuclear NMR study

Euglycemic hyperinsulinemia evokes both sympathetic activation and vasodilation in skeletal muscle, but the mechanism remains unknown. To determine whether insulin per se or insulin-induced stimulation of carbohydrate metabolism is the main excitatory stimulus, we performed, in six healthy lean subjects, simultaneous microneurographic recordings of muscle sympathetic nerve activity, plethysmographic measurements of calf blood flow, and calorimetric determinations of carbohydrate oxidation rate. Measurements were made during 2 h of: (a) insulin/glucose infusion (hyperinsulinemic [6 pmol/kg per min] euglycemic clamp), (b) exogenous glucose infusion at a rate matched to that attained during protocol a, and (c) exogenous fructose infusion at the same rate as for glucose infusion in protocol b. For a comparable rise in carbohydrate oxidation, insulin/glucose infusion that resulted in twofold greater increases in plasma insulin concentrations than did glucose infusion alone, evoked twofold greater increases in both muscle sympathetic nerve activity and calf blood flow. Fructose infusion, which increased carbohydrate oxidation comparably, but had only a minor effect on insulinemia, did not stimulate either muscle sympathetic nerve activity or calf blood flow. These observations suggest that in humans hyperinsulinemia per se, rather than insulin-induced stimulation of carbohydrate metabolism, is the main mechanism that triggers both sympathetic activation and vasodilation in skeletal muscle.     

Proton NMR of Escherichia coli sulfite reductase: the unligated hemeprotein subunit

The isolated hemeprotein subunit of sulfite reductase (SiR-HP) from Escherichia coli consists of a high spin ferric isobacteriochlorin (siroheme) coupled to a diamagnetic [4Fe-4S]2+ cluster. When supplied with an artificial electron donor, such as methyl viologen cation radical, SiR-HP can catalyze the six electron reductions of sulfite to sulfide and nitrite to ammonia. Thus, the hemeprotein subunit appears to represent the minimal protein structure required for multielectron reductase activity. Proton magnetic resonance spectra are reported for the first time on unligated SiR-HP at 300 MHz in all three redox states. The NMR spectrum of high spin ferric siroheme at pH 6.0 was obtained for the purpose of comparing its spectrum with that of oxidized SiR-HP. On the basis of line widths, T1 measurements, and 1D NOE experiments, preliminary assignments have been made for the oxidized enzyme in solution. The pH profile of oxidized SiR-HP is unusual in that a single resonance shows a 9 ppm shift over a range of only 3 pH units with an apparent pK = 6.7 +/- 0.2. Resonances arising from the beta-CH2 protons of cluster cysteines have been assigned using deuterium substitution for all redox states. One beta-CH2 resonance has been tentatively assigned to the bridging cysteine on the basis of chemical shift, T1, line width, and the presence of NOEs to protons from the siroheme ring. The observed pattern of hyperfine shifts can be used as a probe to measure the degree of coupling between siroheme and cluster in solution. The cluster iron sites of the resting (oxidized) enzyme are found to possess both positive and negative spin density which is in good agreement with Mossbauer results on frozen enzyme. The NMR spectrum of the 1-electron reduced form of SiR-HP is consistent with an intermediate spin (S = 1) siroheme. Intermediate spin Fe(II) hemes have only been previously observed in 4-coordinate model compounds. However, the amount of electron density transferred to the cluster, as measured by the isotropic shift of beta-CH2 resonances, is comparable to that present in the fully oxidized enzyme despite diminution of the total amount of unpaired spin density available. Addition of a second electron to SiR-HP, besides generating a reduced S = 1/2 cluster with both upfield and downfield shifted cysteine resonances, converts siroheme to the high spin (S = 2) ferrous state.(ABSTRACT TRUNCATED AT 400 WORDS)     

NMR analysis of the N-terminal SRCR domain of human CD5: engineering of a glycoprotein for superior characteristics in NMR experiments

Despite its obscure and short effect, plasma exchange (PE) remains a mainstay in the treatment of liver disease. However, the question still remains as to whether or not PE suppresses the regeneration of the liver because PE deprives patients of hepatotrophic factors. The effect of PE, which could be a total blood exchange (TBE) in a syngeneic setting, on liver regeneration following a 68% partial hepatectomy (PH) was investigated in rats. In Group 1, 20 ml of blood from normal rats was infused while native blood was removed at 6 and 12 h after PH. In Group 2, 20 ml of blood obtained from PH rats at the same time points was infused. The regeneration rate, labeling index of proliferating cell nuclear antigen (PCNA), and plasma hepatocyte growth factor (HGF) level were determined, and standard liver function tests performed at 24, 48, and 72 h. Although all liver function tests improved in Group 1 at 24 and 48 h, the regeneration rate was significantly impaired. Similarly, the PCNA labeling index was significantly lower in Group 1 than that in Group 2. The plasma HGF level was significantly reduced in Group 1 (6 h blood out versus blood in: 1.1+/-0.5 vs. 0.1+/-0.1 ng/ml, p < 0.05). TBE with normal blood following PH suppressed the early stage of liver regeneration, in part, because of the reduction of HGF even though the blood was purified.     

Solution structure of the His12 --> Cys mutant of the N-terminal zinc binding domain of HIV-1 integrase complexed to cadmium

The solution structure of His12 --> Cys mutant of the N-terminal zinc binding domain (residues 1-55; IN(1-55)) of HIV-1 integrase complexed to cadmium has been solved by multidimensional heteronuclear NMR spectroscopy. The overall structure is very similar to that of the wild-type N-terminal domain complexed to zinc. In contrast to the wild-type domain, however, which exists in two interconverting conformational states arising from different modes of coordination of the two histidine side chains to the metal, the cadmium complex of the His12 --> Cys mutant exists in only a single form at low pH. The conformation of the polypeptide chain encompassing residues 10-18 is intermediate between the two forms of the wild-type complex.     

The crystal structure of Escherichia coli purine nucleoside phosphorylase: a comparison with the human enzyme reveals a conserved topology

BACKGROUND: Purine nucleoside phosphorylase (PNP) from Escherichia coli is a hexameric enzyme that catalyzes the reversible phosphorolysis of 6-amino and 6-oxopurine (2'-deoxy)ribonucleosides to the free base and (2'-deoxy)ribose-1-phosphate. In contrast, human and bovine PNPs are trimeric and accept only 6-oxopurine nucleosides as substrates. The difference in the specificities of these two enzymes has been utilized in gene therapy treatments in which certain prodrugs are cleaved by E. coli PNP but not the human enzyme. The trimeric and hexameric PNPs show no similarity in amino acid sequence, even though they catalyze the same basic chemical reaction. Structural comparison of the active sites of mammalian and E. coli PNPs would provide an improved basis for the design of potential prodrugs that are specific for E. coli PNP. RESULTS: The crystal structure of E. coli PNP at 2.0 A resolution shows that the overall subunit topology and active-site location within the subunit are similar to those of the subunits from human PNP and E. coli uridine phosphorylase. Nevertheless, even though the overall geometry of the E. coli PNP active site is similar to human PNP, the active-site residues and subunit interactions are strikingly different. In E. coli PNP, the purine- and ribose-binding sites are generally hydrophobic, although a histidine residue from an adjacent subunit probably forms a hydrogen bond with a hydroxyl group of the sugar. The phosphate-binding site probably consists of two main-chain nitrogen atoms and three arginine residues. In addition, the active site in hexameric PNP is much more accessible than in trimeric PNP. CONCLUSIONS: The structures of human and E. coli PNP define two possible classes of nucleoside phosphorylase, and help to explain the differences in specificity and efficiency between trimeric and hexameric PNPs. This structural data may be useful in designing prodrugs that can be activated by E. coli PNP but not the human enzyme.