Improved display of synthetic IgG-binding domains on the baculovirus surface

Improved display of foreign protein moieties in combination with beneficial alteration of the viral surface properties should be of value for targeted and enhanced gene delivery. Here, we describe a vector based on Autographa californica multiple nucleopolyhedrovirus (AcMNPV) displaying synthetic IgG-bincling domains (ZZ) of protein A fused to the transmembrane anchor of vesicular stomatitis virus (VSV) G protein. This display vector was equipped with a GFP/EGFP expression cassette enabling fluorescent detection in both insect and mammalian cells. The virus construct displayed the biologically active fusion protein efficiently and showed increased binding capacity to IgG. As the display is carried out using a membrane anchor of foreign origin, gp64 is left intact for virus entry, which may increase gene expression in the transduced mammalian cells. In addition, the viral vector can be targeted to any desired cell type via binding of ZZ domains when an appropriate IgG antibody is available.

Interactions of decay-accelerating factor (DAF) with haemagglutinating human enteroviruses: utilizing variation in primate DAF to map virus binding sites

A cellular receptor for the haemagglutinating enteroviruses (HEV), and the protein that mediates haemagglutination, is the membrane complement regulatory protein decay accelerating factor (DAF; CD55). Although primate DAF is highly conserved, significant differences exist to enable cell lines derived from primates to be utilized for the characterization of the DAF binding phenotype of human enteroviruses. Thus, several distinct DAF-binding phenotypes of a selection of HEVs (viz. coxsackievirus A21 and echoviruses 6, 7, 11-13, 29) were identified from binding and infection assays using a panel of primate cells derived from human, orang-utan, African Green monkey and baboon tissues. These studies complement our recent determination of the crystal structure of SCR(34) of human DAF [Williams, P., Chaudhry, Y., Goodfellow, I. G., Billington, J., Powell, R., Spiller, O. B., Evans, D. J. & Lea, S. (2003). J Biol Chem 278, 10691-10696] and have enabled us to better map the regions of DAF with which enteroviruses interact and, in certain cases, predict specific virus-receptor contacts.

Protein-disulfide isomerase-mediated reduction of two disulfide bonds of HIV envelope glycoprotein 120 occurs post-CXCR4 binding and is required for fusion

The human immunodeficiency virus (HIV) envelope (Env) glycoprotein (gp) 120 is a highly disulfide-bonded molecule that attaches HIV to the lymphocyte surface receptors CD4 and CXCR4. Conformation changes within gp120 result from binding and trigger HIV/cell fusion. Inhibition of lymphocyte surface-associated protein-disulfide isomerase (PDI) blocks HIV/cell fusion, suggesting that redox changes within Env are required. Using a sensitive assay based on a thiol reagent, we show that (i) the thiol content of gp120, either secreted by mammalian cells or bound to a lymphocyte surface enabling CD4 but not CXCR4 binding, was 0.5-1 pmol SH/pmol gp120 (SH/gp120), whereas that of gp120 after its interaction with a surface enabling both CD4 and CXCR4 binding was raised to 4 SH/gp120; (ii) PDI inhibitors prevented this change; and (iii) gp120 displaying 2 SH/gp120 exhibited CD4 but not CXCR4 binding capacity. In addition, PDI inhibition did not impair gp120 binding to receptors. We conclude that on average two of the nine disulfides of gp120 are reduced during interaction with the lymphocyte surface after CXCR4 binding prior to fusion and that cell surface PDI catalyzes this process. Disulfide bond restructuring within Env may constitute the molecular basis of the post-receptor binding conformational changes that induce fusion competence.

Insights into the effects on metal binding of the systematic substitution of five key glutamate ligands in the ferritin of Escherichia coli

Ferritins are nearly ubiquitous iron storage proteins playing a fundamental role in iron metabolism. They are composed of 24 subunits forming a spherical protein shell encompassing a central iron storage cavity. The iron storage mechanism involves the initial binding and subsequent O-2-dependent oxidation of two Fe2+ ions located at sites A and B within the highly conserved dinuclear "ferroxidase center" in individual subunits. Unlike animal ferritins and the heme-containing bacterioferritins, the Escherichia coli ferritin possesses an additional iron-binding site (site C) located on the inner surface of the protein shell close to the ferroxidase center. We report the structures of five E. coli ferritin variants and their Fe3+ and Zn2+ (a redox-stable alternative for Fe2+) derivatives. Single carboxyl ligand replacements in sites A, B, and C gave unique effects on metal binding, which explain the observed changes in Fe2+ oxidation rates. Binding of Fe2+ at both A and B sites is clearly essential for rapid Fe2+ oxidation, and the linking of Fe-B(2+) to Fe-C(2+) enables the oxidation of three Fe2+ ions. The transient binding of Fe2+ at one of three newly observed Zn2+ sites may allow the oxidation of four Fe2+ by one dioxygen molecule.

Mapping CD55 function: the structure of two pathogen-binding domains at 1.7 A

Decay-accelerating factor (CD55), a regulator of the alternative and classical pathways of complement activation, is expressed on all serum-exposed cells. It is used by pathogens, including many enteroviruses and uropathogenic Escherichia coli, as a receptor prior to infection. We describe the x-ray structure of a pathogen-binding fragment of human CD55 at 1.7 A resolution containing two of the three domains required for regulation of human complement. We have used mutagenesis to map biological functions onto the molecule; decay-accelerating activity maps to a single face of the molecule, whereas bacterial and viral pathogens recognize a variety of different sites on CD55.

1-(2 '-pyridylazo)-2-naphtholate complexes of ruthenium: synthesis, characterization, and DNA binding properties

Reaction of 1-(2'-pyridylazo)-2 -naphthol (Hpan) with [Ru(dmso)(4)Cl-2] (dmso=dimethylsulfoxide), [Ru(trpy)Cl-3] (trpy=2,2',2 ''-terpyridine), [Ru(bpy)Cl-3] (bpy=2,2'-bipyridine) and [Ru(PPh3)(3)Cl-2] in refluxing ethanol in the presence of a base (NEt3) affords, respectively, the [Ru(pan)(2)], [Ru(trpy)(pan)](+) (isolated as perchlorate salt), [Ru(bpy)(pan)Cl] and [Ru(PPh3)(2)(pan)Cl] complexes. Structures of these four complexes have been determined by X-ray crystallography. in each of these complexes, the pan ligand is coordinated to the metal center as a monoanionic tridentate N,N,O-donor. Reaction of the [Ru(bpy)(pan)Cl] complex with pyridine (py) and 4-picoline (pic) in the presence of silver ion has yielded the [Ru(bpy)(pan)(py)](+) and [Ru(bpy)(pan)(pic)](+) complexes (isolated as perchlorate salts), respectively. All the complexes are diamagnetic (low-spin d(6), S = 0) and show characteristic H-1 NMR signals and intense MLCT transitions in the visible region. Cyclic voltammetry on all the complexes shows a Ru(II)-Ru(III) oxidation on the positive side of SCE. Except in the [Ru(pan)(2)] complex, a second oxidative response has been observed in the other five complexes. Reductions of the coordinated ligands have also been observed on the negative side of SCE. The [Ru(trpy)(pan)]ClO4, [Ru(bpy)(pan)(py)]ClO4 and [Ru(bpy) (pan)(pic)]ClO4 complexes have been observed to bind to DNA, but they have not been able to cleave super-coiled DNA on UV irradiation. (c) 2008 Elsevier Ltd. All rights reserved.

Pyrrole-based metallo-macrocycles and cryptands

The synthesis of dithiocarbamate ligands based on a pyrrole framework is reported. These ligands self-assemble with zinc(II), nickel(II) and copper(II) to afford neutral, dinuclear metallomacrocycles and trinuclear metallocryptands. The assembled metallo compounds have been characterised by a range of techniques, including H-1 NMR, UV-vis spectroscopy, elemental analysis, mass spectrometry and X-ray crystallography. Some preliminary anion binding studies have also been conducted, using electronic spectroscopy and electrochemistry. The nickel macrocycles showed some affinity for acetate, whereas the copper cryptand showed affinity for benzoate anions. The copper cryptand also exhibited a significant electrochemical response to a range of anions.

Dinuclear copper and zinc complexes of a hexaazamacrocycle containing p-xylyl spacers and bridging anions: theoretical and spectroscopic studies

The hexaazamacrocycle 7,22-dimethyl-3,7,11,18,22,26-hexaazatricyclo[26.2.2.2(13,16)] tetratriaconta-1(30), 13,15,28,31,33- hexaene (Me-2[30] pbz(2)N(6)) was synthesized and characterised by single crystal X-ray diffraction. The macrocycle adopts a conformation with the two aromatic rings almost parallel at a distance of ca. 4.24 Angstrom, but displaced relative to each other by ca. 1.51 Angstrom. The protonation constants of this compound and the stability constants of its complexes with Cu2+ and Zn2+, were determined in water - methanol (9 : 1 v/v) at 25 degreesC with ionic strength 0.10 mol dm(-3) in KCl. The potentiometric and spectroscopic studies (NMR of zinc, cadmium and lead complexes, and EPR of the copper complexes) indicate the formation of only dinuclear complexes. The association constants of the dinuclear copper complex with anions ( thiocyanate, terephthalate and glyphosate) and neutral molecules (1,4-benzenedimethanol, p-xylylenediamine and terephthalic acid) were determined at 20 degreesC in methanol. The structural preferences of this ligand and of its dinuclear copper(II) complex with a variety of bridging ligands were evaluated theoretically by molecular mechanics calculations (MM) and molecular dynamics (MD) using quenching techniques.

Binding studies of a protonated dioxatetraazamacrocycle with carboxylate substrates

The tetraprotonated form of the dioxatetraazamacrocycle, 6,19-dioxa-3,9,16,22-tetraaza[22.2.2.2(11,14)]-triaconta-1(26),11,13,24, 27,29-hexaene, (H4L1)(4+), was used as the receptor for binding studies with carboxylate anionic substrates of different shapes, sizes, and charges [succinate (suc(2-)), cyclo- hexanetricarboxylate (cta(3-)), phthalate (ph(2-)), isophthalate (iph(2-)), terephthalate (tph(2-)), and benezenetricarboxylate (btc(3-))]. Association constants were determined by potentiometry in aqueous solution at 298.2 K and 0.10 M KCl and by H-1 NMR titration in D2O. The strongest association was found for the btc3- anion at 5-7 pH region. From both techniques it was possible to establish the binding preference trend of the receptor for the different substrates, and the H-1 NMR spectroscopy gave important suggestions about the type of interactions between partners and the location of the substrates in the supramolecular entities formed. The effective binding constants at pH 6 follow the order: btc(3-)>iph(2-)>cta(3-) =ph(2-)>tph(2-)>suc(2-). All the studies suggest that the anionic substrates bind to the receptor via N-H center dot center dot center dot O = C hydrogen bonds and electrostatic interactions, and the aromatic substrates can also establish pi-pi stacking interactions. The crystal structures of (H4L1)(4+) and its supramolecular assemblies with ph(2-) and tph(2-) were determined by X-ray diffraction. The last two structures showed that the association process in solid state occurs via multiple N-H center dot center dot center dot O = C hydrogen bonds with the anionic substrate located outside the macrocyclic cavity of the receptor. Molecular dynamics simulations carried out for the association of (H4L1)(4+) with tph(2-) and btC(3-) in water solution established at atomic level the existence of all interactions suggested by the experimental studies, which act cooperatively in the binding process. Furthermore, the binding free energies were estimated and the values are in agreement with the experimental ones, indicating that the binding of these two anionic substrates occurs into the receptor cavity. However, the tph(2-) has also propensity to leave the macrocyclic cavity and its molecular recognition can also happen at the top of the receptor. (C) 2008 Elsevier Ltd. All rights reserved.

Evaluation of the binding ability of a novel dioxatetraazamacrocyclic receptor that contains two phenanthroline units: selective uptake of carboxylate anions

The novel dioxatetraaza macrocycle [26]phen(2)N(4)O(2), which incorporates two phenanthroline units, has been synthesized, and its acid-base behavior has been evaluated by potentiometric and H-1 NMR methods. Six protonation constants were determined, and the protonation sequence was established by NMR. The location of the fifth proton on the phen nitrogen was confirmed by X-ray determinations of the crystal structures of the receptor as bromide and chloride salts. The two compounds have the general molecular formula {(H-5[26]phen(2)N(4)O(2))X-n(H2O)(5-n)}X(n-1)(.)mH(2)O, where X = Cl, n = 3, and m = 6 or X = Br, n = 4, and m = 5.5. In the solid state, the (H-5[26]phen(2)N(4)O(2))(5+) cation adopts a "horseshoe" topology with sufficient room to encapsulate three or four halogen anions through the several N-(HX)-X-... hydrogen-bonding interactions. Two supermolecules {(H-5[26]phen(2)N(4)O(2))X-n(H2O)(5-n)}((5-n)+) form an interpenetrating dimeric species, which was also found by ESI mass spectrum. Binding studies of the protonated macrocycle with aliphatic (ox(2-), mal(2-), suc(2-), cit(3-), cta(3-)) and aromatic (bzc(-), naphc(-), anthc(-), pyrc(-), ph(2-), iph(2-), tph(2-), btc(3-)) anions were determined in water by potentiometric methods. These studies were complemented by H-1 NMR titrations in D2O of the receptor with selected anions. The H-i[26]phen(2)N(4)O(2)(i+) receptor can selectively uptake highly charged or extended aromatic carboxylate anions, such as btc(3-) and pyrc(-), in the pH ranges of 4.0-8.5 and < 4.0, respectively, from aqueous solution that contain the remaining anions as pollutants or contaminants. To obtain further insight into these structural and experimental findings, molecular dynamics (MD) simulations were carried out in water solution.

Carboxylate anions binding and sensing by a novel tetraazamacrocycle containing ferrocene as receptor

A tetraazamacrocycle containing ferrocene moieties has been synthesized and characterized. The tetraprotonated form of this compound was evaluated as a receptor (R) for anion recognition of several substrates (S), Cl-, PF6-, HSO4-, H2PO4- and carboxylates, such as p-nitrobenzoate (p-nbz(-)), phthalate (ph(2-)), isophthalate (iph(2-)) and dipicolinate (dipic(2-)). H-1 NMR titrations in CD3OD indicated that this receptor is not suitable for recognizing HSO4- and H2PO4-, but weakly binds p-nbz(-), and strongly interacts with ph(2-), dipic(2-), and iph(2-) anions forming 1 : 2 assembled species. The largest beta(2) binding constant was determined for ph(2-), followed by dipic(2-) and finally iph(2-). The effect of the anionic substrates on the electron-transfer process of the ferrocene units of R was evaluated using cyclic voltammetry (CV) and square wave voltammetry (SWV) in methanol solution and 0.1 mol dm(-3) (CH3)(4)NCl as the supporting electrolyte. Titrations of the receptor were undertaken by addition of anion solutions in their tetrabutylammonium or tetramethylammonium forms. The protonated ligand exhibits a reversible voltammogram, which shifts cathodically in the presence of the substrates. The data revealed kinetic constraints in the formation of the receptor/substrate entity for dipic(2-), ph(2-) and iph(2-) anions, but not for p-nbz(-). In spite of the slow kinetics of assembled species formation with the ph(2-) substrate, this anion provides the largest redox-response when the supramolecular entity is formed, followed by dipic(2-), iph(2-) and finally p-nbz(-) anions. This trend is in agreement with the H-1 NMR results and the values of the binding constants. Single crystal X-ray structures of the receptor with PF6-, ph(2-), iph(2-) and p-nbz(-) were carried out and showed that supermolecules with a RS2 stoichiometry are formed with the first three anions, but RS4 with p-nbz(-). In all cases the binding occurs outside the macrocyclic cavity via N-H center dot center dot center dot O=C hydrogen bonds for carboxylate anions and N - H center dot center dot center dot F hydrogen bonds for the PF6- anion, which is in agreement with the solution results. The macrocyclic framework adopts different conformations in order to interact with each substrate having Fe center dot center dot center dot Fe intramolecular distances ranging from 10.125(14) to 12.783(15) angstrom.

Statistical deconvolution of enthalpic energetic contributions to MHC-peptide binding affinity

Background: MHC Class I molecules present antigenic peptides to cytotoxic T cells, which forms an integral part of the adaptive immune response. Peptides are bound within a groove formed by the MHC heavy chain. Previous approaches to MHC Class I-peptide binding prediction have largely concentrated on the peptide anchor residues located at the P2 and C-terminus positions. Results: A large dataset comprising MHC-peptide structural complexes was created by remodelling pre-determined x-ray crystallographic structures. Static energetic analysis, following energy minimisation, was performed on the dataset in order to characterise interactions between bound peptides and the MHC Class I molecule, partitioning the interactions within the groove into van der Waals, electrostatic and total non-bonded energy contributions. Conclusion: The QSAR techniques of Genetic Function Approximation (GFA) and Genetic Partial Least Squares (G/PLS) algorithms were used to identify key interactions between the two molecules by comparing the calculated energy values with experimentally-determined BL50 data. Although the peptide termini binding interactions help ensure the stability of the MHC Class I-peptide complex, the central region of the peptide is also important in defining the specificity of the interaction. As thermodynamic studies indicate that peptide association and dissociation may be driven entropically, it may be necessary to incorporate entropic contributions into future calculations.

Zinc(II)-pyridylthioazophenol system: synthesis, characterization and crystal structure

Reactivities of pyridylthioazophenols (1) with zinc(II) salts have been studied and the complexes isolated in pure form and characterized. Pyridylthioazophenols react with zinc( II)acetate in MeOH/EtOH at room temperature to give a series of pyridylsulfinylazophenols (2)but no zinc( II) complex. The sulfoxides (2) have been characterized by IR and NMR. One of the pyridylsulfinylazophenols (2a) has been subjected to single-crystal X-ray analysis in order to confirm details of its structure. A series of dimeric zinc( II) complexes of tetradentate NSNO pyridylthioazophenolates has been isolated through reaction of zinc nitrate in MeOH followed by in situ reaction with azide ion, which acts as a mu-(1,1) bridge. All complexes have been characterized spectroscopically. The detailed structure of one of the dinuclear zinc( II) complexes has been established by a single-crystal X-ray structure determination. In complex 3a two octahedrally coordinated zinc( II) ions are bridged by two end-on azide ions. No reactions of pyridylthioazophenols with zinc chloride in refluxing EtOH have been observed.

A fluxional (CuN2O2)-N-I core: binding of a keto oxygen atom to Cu-I and Ag-I

[Cu(2-acetylpyridine)(2)]ClO4 (1), characterised here, has a novel Cu'N202 core in the solid state. Variable-temperature H-1 NMR studies show that the two chelate rings open up in solution at room temperature and the keto oxygen atoms dangle freely. As the temperature is lowered, the 0 atoms tend to bind to the metal atom. The corresponding silver(I) complex, [Ag(2-acetylpyridine)2]ClO4 (4), characterised by single-crystal X-ray crystallography, has an (AgN2)-N-I core in the solid state as well as in solution. Thus, while 1 is fluxional, 4 is not. In cyclic voltammetry, complex 1 displays a quasireversible Cu-II/I couple with a half-wave potential of 0.40 V vs. SCE. Complex I is easily oxidised by air and H2O2 in methanol to give rise to a dinuclear copper(II) complex where the ligand framework is not simple acetylpyridine. ((c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005).