EfeO-cupredoxins: major new members of the cupredoxin superfamily with roles in bacterial iron transport

The EfeUOB system of Escherichia coli is a tripartite, low pH, ferrous iron transporter. It resembles the high-affinity iron transporter (Ftr1p-Fet3p) of yeast in that EfeU is homologous to Ftr1p, an integral-membrane iron-permease. However, EfeUOB lacks an equivalent of the Fet3p component—the multicopper oxidase with three cupredoxin-like domains. EfeO and EfeB are periplasmic but their precise roles are unclear. EfeO consists primarily of a C-terminal peptidase-M75 domain with a conserved ‘HxxE’ motif potentially involved in metal binding. The smaller N-terminal domain (EfeO-N) is predicted to be cupredoxin (Cup) like, suggesting a previously unrecognised similarity between EfeO and Fet3p. Our structural modelling of the E. coli EfeO Cup domain identifies two potential metal-binding sites. Site I is predicted to bind Cu2+ using three conserved residues (C41 and 103, and E66) and M101. Of these, only one (C103) is conserved in classical cupredoxins where it also acts as a Cu ligand. Site II most probably binds Fe3+ and consists of four well conserved surface Glu residues. Phylogenetic analysis indicates that the EfeO-Cup domains form a novel Cup family, designated the ‘EfeO-Cup’ family. Structural modelling of two other representative EfeO-Cup domains indicates that different subfamilies employ distinct ligand sets at their proposed metal-binding sites. The ~100 efeO homologues in the bacterial sequence databases are all associated with various iron-transport related genes indicating a common role for EfeO-Cup proteins in iron transport, supporting a new copper-iron connection in biology.

Evidence of a possible role for Lys-gamma 3-MSH in the regulation of adipocyte function

Lys-gamma 3-MSH is a melanocortin peptide derived from the C-terminal of the 16 kDa fragment of POMC. The physiological role of Lys-gamma 3-MSH is unclear, although it has previously been shown that, although not directly steroidogenic, it can act to potentiate the steroidogenic response of adrenal cortical cells to ACTH. This synergistic effect appears to be correlated with an ability to increase the activity of hormone sensitive lipase (HSL) and therefore the rate of cholesterol ester hydrolysis. Ligand binding studies have suggested that high-affinity binding sites for Lys-gamma 3-MSH exist in the adrenal gland and a number of other rat tissues that express HSL, including adipose, skeletal muscle and testes. To investigate the hypothesis that Lys-gamma 3-MSH may play a wider role in cholesterol and lipid metabolism, we tested the effect of Lys-gamma 3-MSH on lipolysis, an HSL-mediated process, in 3T3-L1 adipocytes. In comparison with other melanocortin peptides, Lys-gamma 3-MSH was found to be a potent stimulator of lipolysis. It was also able to phosphorylate HSL at key serine residues and stimulate the hyper-phosphorylation of perilipin A. The receptor through which the lipolytic actions of Lys-gamma 3-MSH are being mediated is not clear. Attempts to characterise this receptor suggest that either the pharmacology of the melanocortin receptor 5 in 3T3-L1 adipocytes is different from that described when expressed in heterologous systems or the possibility that a further, as yet uncharacterised, receptor exists.

DNA interaction and phosphotransfer of the C-4-dicarboxylate-responsive DcuS-DcuR two-component regulatory system from Escherichia coli

The DcuS-DcuR system of Escherichia coli is a two-component sensor-regulator that controls gene expression in response to external C-4-dicarboxylates and citrate. The DcuS protein is particularly interesting since it contains two PAS domains, namely a periplasmic C-4-dicarboxylate-sensing PAS domain (PASp) and a cytosolic PAS domain (PASc) of uncertain function. For a study of the role of the PASc domain, three different fragments of DcuS were overproduced and examined: they were PASc-kinase, PASc, and kinase. The two kinase-domain-containing fragments were autophosphorylated by [gamma-P-32]ATP. The rate was not affected by fumarate or succinate, supporting the role of the PASp domain in C-4-dicarboxylate sensing. Both of the phosphorylated DcuS constructs were able to rapidly pass their phosphoryl groups to DcuR, and after phosphorylation, DcuR dephosphorylated rapidly. No prosthetic group or significant quantity of metal was found associated with either of the PASc-containing proteins. The DNA-binding specificity of DcuR was studied by use of the pure protein. It was found to be converted from a monomer to a dimer upon acetylphosphate treatment, and native polyacrylamide gel electrophoresis suggested that it can oligomerize. DcuR specifically bound to the promoters of the three known DcuSR-regulated genes (dctA, dcuB, and frdA), with apparent K(D)s of 6 to 32 muM for untreated DcuR and less than or equal to1 to 2 muM for the acetylphosphate-treated form. The binding sites were located by DNase I footprinting, allowing a putative DcuR-binding motif [tandemly repeated (T/A)(A/T)(T/C)(A/T)AA sequences] to be identified. The DcuR-binding sites of the dcuB, dctA, and frdA genes were located 27, 94, and 86 bp, respectively, upstream of the corresponding +1 sites, and a new promoter was identified for dcuB that responds to DcuR.

Structure of the snake-venom toxin convulxin

Snake venoms contain a number of proteins that interact with components of the haemostatic system that promote or inhibit events leading to blood- clot formation. The snake- venom protein convulxin ( Cvx) binds glycoprotein ( GP) VI, the platelet receptor for collagen, and triggers signal transduction. Here, the 2.7 Angstrom resolution crystal structure of Cvx is presented. In common with other members of this snake-venom protein family, Cvx is an alphabeta- heterodimer and conforms to the C- type lectin- fold topology. Comparison with other family members allows a set of Cvx residues that form a concave surface to be putatively implicated in GPVI binding. Unlike other family members, with the exception of flavocetin- A ( FL- A), Cvx forms an (alphabeta)(4) tetramer. This oligomeric structure is consistent with Cvx clustering GPVI molecules on the surface of platelets and as a result promoting signal transduction activity. The Cvx structure and the location of the putative binding sites suggest a model for this multimeric signalling assembly.

A comparative study on the relationship between acetyl cholinesterase activity and acute toxicity in Daphnia magna exposed to anticholinesterase insecticides

Acetylcholinesterase (AChE) activity was measured in Daphnia magna that had been exposed to four organophosphates (OPs; parathion, chlorpyrifos, malathion, and acephate) and one carbamate (propoxur) for 48 h. These results were related to acute toxicity (median effective concentration [EC50] for immobility). For the four OPs, the EC50s were 7.03 pM, 3.17 pM, 10.56 pM, and 309.82 muM, respectively. The EC50 for propoxur was 449.90 pM. Reduction in AChE activity was directly related to an increase in immobility in all chemicals tested. However, the ratio between the EC50 and the AChE median inhibiting concentration ranged from 0.31 to 0.90. A 50% reduction in AChE activity generally was associated with detrimental effects on mobility. However, for acephate, high levels of AChE inhibition (70%) were observed in very low concentrations and were not associated with immobility. In addition, increasing the concentration of acephate further had a slight negative effect oil AChE activity but a Strong detrimental effect on mobility. Binding sites other than AChE possibly are involved in acephate toxicity to D. magna. Our findings demonstrate different associations between AChE inhibition and toxicity when different chemicals are compared. Therefore, the value of using AChE activity as a biomarker in D. magna will be dependent on the chemical tested.

Pharmacological analysis of a dopamine D-2Short: G(alpha o) fusion protein expressed in Sf9 cells

A dopamine D-2Short receptor:G(alphao) fusion protein was expressed in Sf9 cells using the baculovirus expression system. [H-3]Spiperone bound to D-2Short:G(alphao) with a pK(d) approximate to 10. Dopamine stimulated the binding of [S-35]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) to D-2Short:G(alphao) expressed with Gbeta(1)gamma(2) (E-max > 460%; pEC(50) 5.43 +/- 0.06). Most of the putative D-2 antagonists behaved as inverse agonists (suppressing basal [S-35]GTPgammaS binding) at D-2Short:G(alphao)/Gbeta(1)gamma(2) although (-)-suipiride and ziprasidone were neutral antagonists. Competition of [H-3]spiperone binding by dopamine and 10,11-dihydroxy-N-n-propylnorapo-morphine revealed two, binding sites of different affinities, even in the presence of GTP (100 muM). The D-2Short:G(alphao) fusion protein is therefore a good model for characterising D-2 receptors. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Stereochemically non-rigid helical mercury(II) complexes

Reactions of the 1: 2 condensate (L) of benzil dihydrazone and 2-acetylpyridine with Hg(ClO4)(2) center dot xH(2)O and HgI2 yield yellow [HgL2](ClO4)(2) (1) and HgLI2 (2), respectively. Homoleptic 1 is a 8-coordinate double helical complex with a Hg(II)N-8 core crystallising in the space group Pbca with cell dimensions: a = 16.2250(3), b = 20.9563(7), c = 31.9886(11) angstrom. Complex 2 is a 4-coordinate single helical complex having a Hg(II)N2I2 core crystallising in the space group P2(1)/n with cell dimensions a = 9.8011(3), b = 17.6736(6), c = 16.7123(6) angstrom and b = 95.760(3). In complex 1, the N-donor ligand L uses all of its binding sites to act as tetradentate. On the other hand, it acts as a bidentate N-donor ligand in 2 giving rise to a dangling part. From variable temperature H-1 NMR studies both the complexes are found to be stereochemically non-rigid in solution. In the case of 2, the solution process involves wrapping up of the dangling part of L around the metal. (C) 2008 Elsevier B.V. All rights reserved.

Dihydrogen in cation-substituted zeolites X - an inelastic neutron scattering study

An inelastic neutron scattering (INS) study of the rotational - vibrational spectrum of dihydrogen sorbed by zeolite X having substituted sodium, calcium and zinc cations is reported. The rotational - vibrational spectrum of H-2 was observed at low energy transfer ( below ca. 25 meV, 202 cm(-1)); the vibration was that of the H-2 molecule against the binding site (H-2 - X, not H - H). The vibration frequency was proportional to the polarising power of the cation (Na+ < Ca2+ < Zn2+). Polarisation of the H-2 molecule dominated the interaction of H-2 with this binding site. The total scattering intensity was proportional to the dihydrogen dose. However the vibrational intensities became constant at ca. 0.3 wt% showing that the H-2 binding sites had saturated. Additional dihydrogen appeared as unbound or weakly bound dihydrogen exhibiting recoil.

Enantioselective detection of L-serine

An acoustic wave sensor coated with an artificial biomimetic recognition element has been developed to selectively detect the amino acid L-serine. A highly specific non-covalently imprinted polymer was cast on one electrode of a quartz crystal microbalance (QCM) as a thin permeable film. Selective rebinding of the L-serine was observed as a frequency shift in the QCM with a detection limit of 2 ppb and for concentrations up to 0.4 ppm. The sensor binding is shown to be capable of discrimination between L- and D-stereoisomers of serine as a result of the enantioselectivity of the imprinted binding sites. (C) 2002 Elsevier Science B.V. All rights reserved.

The interactions of flavonoids within neuronal signalling pathways

Emerging evidence suggests that dietary phytochemicals, in particular flavonoids, may exert beneficial effects in the central nervous system by protecting neurons against stress-induced injury, by suppressing neuroinflammation and by promoting neurocognitive performance, through changes in synaptic plasticity. It is likely that flavonoids exert such effects in neurons, through selective actions on different components within a number of protein kinase and lipid kinase signalling cascades, such as phosphatidylinositol-3 kinase (PI3K)/Akt, protein kinase C and mitogen-activated protein kinase. This review details the potential inhibitory or stimulatory actions of flavonoids within these pathways, and describes how such interactions are likely to affect cellular function through changes in the activation state of target molecules and/or by modulating gene expression. Although, precise sites of action are presently unknown, their abilities to: (1) bind to ATP binding sites on enzymes and receptors; (2) modulate the activity of kinases directly; (3) affect the function of important phosphatases; (4) preserve neuronal Ca2+ homeostasis; and (5) modulate signalling cascades lying downstream of kinases, are explored. Future research directions are outlined in relation to their precise site(s) of action within the signalling pathways and the sequence of events that allow them to regulate neuronal function in the central nervous system.

Probing protein-tannin interactions by isothermal titration microcalorimetry

Isothermal titration microcalorimetry (ITC) has been applied to investigate protein-tannin interactions. Two hydrolyzable tannins were studied, namely myrabolan and tara tannins, for their interaction with bovine serum albumin (BSA), a model globular protein, and gelatin, a model proline-rich random coil protein. Calorimetry data indicate that protein-tannin interaction mechanisms are dependent upon the nature of the protein involved. Tannins apparently interact nonspecifically with the globular BSA, leading to binding saturation at estimated tannin/BSA molar ratios of 48:1 for tara- and 178:1 for myrabolan tannins. Tannins bind to the random coil protein gelatin by a two-stage mechanism. The energetics of the first stage show evidence for cooperative binding of tannins to the protein, while the second stage indicates gradual saturation of binding sites as observed for interaction with BSA. The structure and flexibility of the tannins themselves alters the stoichiometry of the interaction, but does not appear to have any significant affect on the overall binding mechanism observed. This study demonstrates the potential of ITC for providing an insight into the nature of protein-tannin interactions.

A comparative study on the relationship between acetylcholinesterase activity and acute toxicity in Daphnia magna exposed to anticholinesterase insecticides

Acetylcholinesterase (AChE) activity was measured in Daphnia magna that had been exposed to four organophosphates (OPs; parathion, chlorpyrifos, malathion, and acephate) and one carbamate (propoxur) for 48 h. These results were related to acute toxicity (median effective concentration [EC50] for immobility). For the four OPs, the EC50s were 7.03 pM, 3.17 pM, 10.56 pM, and 309.82 microM, respectively. The EC50 for propoxur was 449.90 pM. Reduction in AChE activity was directly related to an increase in immobility in all chemicals tested. However, the ratio between the EC50 and the AChE median inhibiting concentration ranged from 0.31 to 0.90. A 50% reduction in AChE activity generally was associated with detrimental effects on mobility. However, for acephate, high levels of AChE inhibition (70%) were observed in very low concentrations and were not associated with immobility. In addition, increasing the concentration of acephate further had a slight negative effect on AChE activity but a strong detrimental effect on mobility. Binding sites other than AChE possibly are involved in acephate toxicity to D. magna. Our findings demonstrate different associations between AChE inhibition and toxicity when different chemicals are compared. Therefore, the value of using AChE activity as a biomarker in D. magna will be dependent on the chemical tested.

Trapping of palindromic ligands within native transthyretin prevents amyloid formation

Transthyretin (TTR) amyloidosis is a fatal disease for which new therapeutic approaches are urgently needed. We have designed two palindromic ligands, 2,2’-(4,4’-(heptane 1,7-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (mds84) and 2,2’-(4,4’-(undecane-1,11-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (4ajm15), that are rapidly bound by native wild-type TTR in whole serum and even more avidly by amyloidogenic TTR variants. One to one stoichiometry, demonstrable in solution and by MS, was confirmed by X-ray crystallographic analysis showing simultaneous occupation of both T4 binding sites in each tetrameric TTR molecule by the pair of ligand head groups. Ligand binding by native TTR was irreversible under physiological conditions, and it stabilized the tetrameric assembly and inhibited amyloidogenic aggregation more potently than other known ligands. These superstabilizers are orally bioavailable and exhibit low inhibitory activity against cyclooxygenase (COX). They offer a promising platform for development of drugs to treat and prevent TTR amyloidosis.

Twists and turns: a tale of two shikimate-pathway enzymes

We are studying two enzymes from the shikimate pathway, dehydroquinate synthase (DHQS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Both enzymes have been the subject of numerous studies to elucidate their reaction mechanisms. Crystal structures of DHQS and EPSPS in the presence and absence of substrates, cofactors and/or inhibitors are now available. These structures reveal movements of domains, rearrangements of loops and changes in side-chain positions necessary for the formation of a catalytically competent active site. The potential for using complementary small-angle X-ray scattering (SAXS) studies to confirm the presence of these structural differences in solution has also been explored. Comparative analysis of crystal structures, in the presence and absence of ligands, has revealed structural features critical for substrate-binding and catalysis. We have also analysed these structures by generating GRID energy maps to detect favourable binding sites. The combination of X-ray crystallography, SAXS and computational techniques provides an enhanced analysis of structural features important for the function of these complex enzymes.

Dimetallic complexes of macrocycles with two rigid dibenzofuran units as receptors for detection of anionic substrates

The hexaazamacrocycles [28](DBF)2N6 {cyclo[bis(4,6-dimethyldibenzo[b,d]furaniminoethyleneiminoethylene]} and [32](DBF)2N6 {cyclo[bis(4,6-dimethyldibenzo[b,d]furaniminopropyleneiminopropylene]} form stable dinuclear copper(II) complexes suitable to behave as receptors for several anionic substrates. These two receptors were used to study the binding interactions with several substrates, such as imidazole (Him) and some carboxylates [benzoate (bz−), oxalate (ox2−), malonate (mal2−), phthalate (ph2−), isophthalate (iph2−), and terephthalate (tph2−)] by spectrophotometric titrations and EPR spectroscopy in MeOH (or H2O):DMSO (1:1 v/v) solution. The largest association constant was found for ox2− with Cu2[32](DBF)2N64+, whereas for the aromatic dicarboxylate anions the binding constants follow the trend ph2− > iph2− > tph2−, i.e. decrease with the increase of the distance of the two binding sites of the substrate. On the other hand, the large blue shift of 68 nm observed by addition of Him to Cu2[32](DBF)2N64+ points out for the formation of the bridged CuimCu cascade complex, indicating this receptor as a potential sensor for the detection and determination of imidazole in solution. The X-band EPR spectra of the Cu2[28](DBF)2N64+ and Cu2[32](DBF)2N6]4+ complexes and the cascade complexes with the substrates, performed in H2O:DMSO (1:1 v/v) at 5 to 15 K, showed that the CuCu distance is slightly larger than the one found in crystal state and that this distance increases when the substrate is accommodated between the two copper centres. The crystal structure of [Cu2[28](DBF)2N6(ph)2]·CH3OH was determined by X-ray diffraction and revealed the two copper centres bridged by two ph2− anions at a Cu···Cu distance of 5.419(1) Å. Each copper centre is surrounded by three carboxylate oxygen atoms from two phthalate anions and three contiguous nitrogen atoms of the macrocycle in a pseudo octahedral coordination environment.