Mechanism for a transcriptional activator that works at the isomerization step

Transcriptional activators in prokaryotes have been shown to stimulate different steps in the initiation process including the initial binding of RNA polymerase (RNAP) to the promoter and a postbinding step known as the isomerization step. Evidence suggests that activators that affect initial binding can work by a cooperative binding mechanism by making energetically favorable contacts with RNAP, but the mechanism by which activators affect the isomerization step is unclear. A well-studied example of an activator that normally exerts its effect exclusively on the isomerization step is the bacteriophage λ cI protein (λcI), which has been shown genetically to interact with the C-terminal region of the σ70 subunit of RNAP. We show here that the interaction between λcI and σ can stimulate transcription even when the relevant portion of σ is transplanted to another subunit of RNAP. This activation depends on the ability of λcI to stabilize the binding of the transplanted σ moiety to an ectopic −35 element. Based on these and previous findings, we discuss a simple model that explains how an activator's ability to stabilize the binding of an RNAP subdomain to the DNA can account for its effect on either the initial binding of RNAP to a promoter or the isomerization step.

Structural evidence for a programmed general base in the active site of a catalytic antibody

The crystal structure of the complex of a catalytic antibody with its cationic hapten at 1.9-Å resolution demonstrates that the hapten amidinium group is stabilized through an ionic pair interaction with the carboxylate of a combining-site residue. The location of this carboxylate allows it to act as a general base in an allylic rearrangement. When compared with structures of other antibody complexes in which the positive moiety of the hapten is stabilized mostly by cation–π interactions, this structure shows that the amidinium moiety is a useful candidate to elicit a carboxylate in an antibody combining site at a predetermined location with respect to the hapten. More generally, this structure highlights the advantage of a bidentate hapten for the programmed positioning of a chemically reactive residue in an antibody through charge complementarity to the hapten.

The domain structure of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides

Protoporphyrinogen oxidase (EC 1–3-3–4), the 60-kDa membrane-bound flavoenzyme that catalyzes the final reaction of the common branch of the heme and chlorophyll biosynthesis pathways in plants, is the molecular target of diphenyl ether-type herbicides. It is highly resistant to proteases (trypsin, endoproteinase Glu-C, or carboxypeptidases A, B, and Y), because the protein is folded into an extremely compact form. Trypsin maps of the native purified and membrane-bound yeast protoporphyrinogen oxidase show that this basic enzyme (pI > 8.5) was cleaved at a single site under nondenaturing conditions, generating two peptides with relative molecular masses of 30,000 and 35,000. The endoproteinase Glu-C also cleaved the protein into two peptides with similar masses, and there was no additional cleavage site under mild denaturing conditions. N-terminal peptide sequence analysis of the proteolytic (trypsin and endoproteinase Glu-C) peptides showed that both cleavage sites were located in putative connecting loop between the N-terminal domain (25 kDa) with the βαβ ADP-binding fold and the C-terminal domain (35 kDa), which possibly is involved in the binding of the isoalloxazine moiety of the FAD cofactor. The peptides remained strongly associated and fully active with the Km for protoporphyrinogen and the Ki for various inhibitors, diphenyl-ethers, or diphenyleneiodonium derivatives, identical to those measured for the native enzyme. However, the enzyme activity of the peptides was much more susceptible to thermal denaturation than that of the native protein. Only the C-terminal domain of protoporphyrinogen oxidase was labeled specifically in active site-directed photoaffinity-labeling experiments. Trypsin may have caused intramolecular transfer of the labeled group to reactive components of the N-terminal domain, resulting in nonspecific labeling. We suggest that the active site of protoporphyrinogen oxidase is in the C-terminal domain of the protein, at the interface between the C- and N-terminal domains.

Identification of the gene (lgtG) encoding the lipooligosaccharide β chain synthesizing glucosyl transferase from Neisseria gonorrhoeae

The lipooligosaccharide from Neisseria gonorrhoeae (GC), consists of lipid A, an oligosaccharide core and three branches, α, β, and γ. We report the cloning of the gene (lgtG, lipooligosaccharide glycosyl transferase G) encoding the glucosyl transferase of GC that initiates the β chain which consists of a lactosyl moiety. This gene contains a homopolymeric tract of cytidine [poly(C)] and we demonstrate that changes in the number of Cs in poly(C) account for the variation of β chain expression in different GC strains. Biochemical analyses and mass spectrometry clearly attribute the reactivity of mAb 2C7 to the presence of the lactosyl β chain. In addition, we demonstrate that in the absence of the lactosyl group, a phosphoethanolamine is added to generate a new antigenic epitope as evidenced by the gain of reactivity to mAb 2-L1–8. These results show that, like the α chain, the β chain of lipooligosaccharide is subject to antigenic variation.

Identification of a second aryl phosphate-binding site in protein-tyrosine phosphatase 1B: A paradigm for inhibitor design

The structure of the catalytically inactive mutant (C215S) of the human protein-tyrosine phosphatase 1B (PTP1B) has been solved to high resolution in two complexes. In the first, crystals were grown in the presence of bis-(para-phosphophenyl) methane (BPPM), a synthetic high-affinity low-molecular weight nonpeptidic substrate (Km = 16 μM), and the structure was refined to an R-factor of 18.2% at 1.9 Å resolution. In the second, crystals were grown in a saturating concentration of phosphotyrosine (pTyr), and the structure was refined to an R-factor of 18.1% at 1.85 Å. Difference Fourier maps showed that BPPM binds PTP1B in two mutually exclusive modes, one in which it occupies the canonical pTyr-binding site (the active site), and another in which a phosphophenyl moiety interacts with a set of residues not previously observed to bind aryl phosphates. The identification of a second pTyr molecule at the same site in the PTP1B/C215S–pTyr complex confirms that these residues constitute a low-affinity noncatalytic aryl phosphate-binding site. Identification of a second aryl phosphate binding site adjacent to the active site provides a paradigm for the design of tight-binding, highly specific PTP1B inhibitors that can span both the active site and the adjacent noncatalytic site. This design can be achieved by tethering together two small ligands that are individually targeted to the active site and the proximal noncatalytic site.

Molecular determinants of tissue selectivity in estrogen receptor modulators

Interaction of the estrogen receptor/ligand complex with a DNA estrogen response element is known to regulate gene transcription. In turn, specific conformations of the receptor-ligand complex have been postulated to influence unique subsets of estrogen-responsive genes resulting in differential modulation and, ultimately, tissue-selective outcomes. The estrogen receptor ligands raloxifene and tamoxifen have demonstrated such tissue-specific estrogen agonist/antagonist effects. Both agents antagonize the effects of estrogen on mammary tissue while mimicking the actions of estrogen on bone. However, tamoxifen induces significant stimulation of uterine tissue whereas raloxifene does not. We postulate that structural differences between raloxifene and tamoxifen may influence the conformations of their respective receptor/ligand complexes, thereby affecting which estrogen-responsive genes are modulated in various tissues. These structural differences are 4-fold: (A) the presence of phenolic hydroxyls, (B) different substituents on the basic amine, (C) incorporation of the stilbene moiety into a cyclic benzothiophene framework, and (D) the imposition of a carbonyl “hinge” between the basic amine-containing side chain and the olefin. A series of raloxifene analogs that separately exemplify each of these differences have been prepared and evaluated in a series of in vitro and in vivo assays. This strategy has resulted in the development of a pharmacophore model that attributes the differences in effects on the uterus between raloxifene and tamoxifen to a low-energy conformational preference imparting an orthogonal orientation of the basic side chain with respect to the stilbene plane. This three-dimensional array is dictated by a single carbon atom in the hinge region of raloxifene. These data indicate that differences in tissue selective actions among benzothiophene and triarylethylene estrogen receptor modulators can be ascribed to discrete ligand conformations.

Characterization and mutational analysis of yeast Dbp8p, a putative RNA helicase involved in ribosome biogenesis

RNA helicases of the DEAD box family are involved in almost all cellular processes involving RNA molecules. Here we describe functional characterization of the yeast RNA helicase Dbp8p (YHR169w). Our results show that Dbp8p is an essential nucleolar protein required for biogenesis of the small ribosomal subunit. In vivo depletion of Dbp8p resulted in a ribosomal subunit imbalance due to a deficit in 40S ribosomal subunits. Subsequent analyses of pre-rRNA processing by pulse–chase labeling, northern hybridization and primer extension revealed that the early steps of cleavage of the 35S precursor at sites A1 and A2 are inhibited and delayed at site A0. Synthesis of 18S rRNA, the RNA moiety of the 40S subunit, is thereby blocked in the absence of Dbp8p. The involvement of Dbp8p as a bona fide RNA helicase in ribosome biogenesis is strongly supported by the loss of Dbp8p in vivo function obtained by site-directed mutagenesis of some conserved motifs carrying the enzymatic properties of the protein family.

A nicked duplex decamer DNA with a PEG6 tether

A dumbbell double-stranded DNA decamer tethered with a hexaethylene glycol linker moiety (DDSDPEG), with a nick in the centre of one strand, has been synthesised. The standard NMR methods, E.COSY, TOCSY, NOESY and HMQC, were used to measure 1H, 31P and T1 spectral parameters. Molecular modelling using rMD-simulated annealing was used to compute the structure. Scalar couplings and dipolar contacts show that the molecule adopts a right-handed B-DNA helix in 38 mM phosphate buffer at pH 7. Its high melting temperature confirms the good base stacking and stability of the duplex. This is partly attributed to the presence of the PEG6 linker at both ends of the duplex that restricts the dynamics of the stem pentamers and thus stabilises the oligonucleotide. The inspection of the global parameters shows that the linker does not distort the B-DNA geometry. The computed structure suggests that the presence of the nick is not disturbing the overall tertiary structure, base pair geometry or duplex base pairing to a substantial extent. The nick has, however, a noticeable impact on the local geometry at the nick site, indicated clearly by NMR analysis and reflected in the conformational parameters of the computed structure. The 1H spectra also show much sharper resonances in the presence of K+ indicating that conformational heterogeneity of DDSDPEG is reduced in the presence of potassium as compared to sodium or caesium ions. At the same time the 1H resonances have longer T1 times. This parameter is suggested as a sensitive gauge of stabilisation.

Toward optimized carbohydrate-based anticancer vaccines: Epitope clustering, carrier structure, and adjuvant all influence antibody responses to Lewisy conjugates in mice

The feasibility of using carbohydrate-based vaccines for the immunotherapy of cancer is being actively explored at the present time. Although a number of clinical trials have already been conducted with glycoconjugate vaccines, the optimal design and composition of the vaccines has yet to be determined. Among the candidate antigens being examined is Lewisy (Ley), a blood group-related antigen that is overexpressed on the majority of human carcinomas. Using Ley as a model for specificity, we have examined the role of epitope clustering, carrier structure, and adjuvant on the immunogenicity of Ley conjugates in mice. A glycolipopeptide containing a cluster of three contiguous Ley-serine epitopes and the Pam3Cys immunostimulating moiety was found to be superior to a similar construct containing only one Ley-serine epitope in eliciting antitumor cell antibodies. Because only IgM antibodies were produced by this vaccine, the effect on immunogenicity of coupling the glycopeptide to keyhole limpet hemocyanin was examined; although both IgM and IgG antibodies were formed, the antibodies reacted only with the immunizing structure. Reexamination of the clustered Ley-serine Pam3Cys conjugate with the adjuvant QS-21 resulted in the identification of both IgG and IgM antibodies reacting with tumor cells, thus demonstrating the feasibility of an entirely synthetic carbohydrate-based anticancer vaccine in an animal model.

Phosphatidylcholine Synthesis Influences the Diacylglycerol Homeostasis Required for Sec14p-dependent Golgi Function and Cell Growth

Phosphatidylcholine and phosphatidylethanolamine are the most abundant phospholipids in eukaryotic cells and thus have major roles in the formation and maintenance of vesicular membranes. In yeast, diacylglycerol accepts a phosphocholine moiety through a CPT1-derived cholinephosphotransferase activity to directly synthesize phosphatidylcholine. EPT1-derived activity can transfer either phosphocholine or phosphoethanolamine to diacylglcyerol in vitro, but is currently believed to primarily synthesize phosphatidylethanolamine in vivo. In this study we report that CPT1- and EPT1-derived cholinephosphotransferase activities can significantly overlap in vivo such that EPT1 can contribute to 60% of net phosphatidylcholine synthesis via the Kennedy pathway. Alterations in the level of diacylglycerol consumption through alterations in phosphatidylcholine synthesis directly correlated with the level of SEC14-dependent invertase secretion and affected cell viability. Administration of synthetic di8:0 diacylglycerol resulted in a partial rescue of cells from SEC14-mediated cell death. The addition of di8:0 diacylglycerol increased di8:0 diacylglycerol levels 20–40-fold over endogenous long-chain diacylglycerol levels. Di8:0 diacylglcyerol did not alter endogenous phospholipid metabolic pathways, nor was it converted to di8:0 phosphatidic acid.

In Vitro Biosynthesis of Phosphorylated Starch in Intact Potato Amyloplasts1

Intact amyloplasts from potato (Solanum tuberosum L.) were used to study starch biosynthesis and phosphorylation. Assessed by the degree of intactness and by the level of cytosolic and vacuolar contamination, the best preparations were selected by searching for amyloplasts containing small starch grains. The isolated, small amyloplasts were 80% intact and were free from cytosolic and vacuolar contamination. Biosynthetic studies of the amyloplasts showed that [1-14C]glucose-6-phosphate (Glc-6-P) was an efficient precursor for starch synthesis in a manner highly dependent on amyloplast integrity. Starch biosynthesis from [1-14C]Glc-1-P in small, intact amyloplasts was 5-fold lower and largely independent of amyloplast intactness. When [33P]Glc-6-P was administered to the amyloplasts, radiophosphorylated starch was produced. Isoamylase treatment of the starch followed by high-performance anion-exchange chromatography with pulsed amperometric detection revealed the separated phosphorylated α-glucans. Acid hydrolysis of the phosphorylated α-glucans and high-performance anion-exchange chromatography analyses showed that the incorporated phosphate was preferentially positioned at C-6 of the Glc moiety. The incorporation of radiolabel from Glc-1-P into starch in preparations of amyloplasts containing large grains was independent of intactness and most likely catalyzed by starch phosphorylase bound to naked starch grains.

Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: Use of a photoactivatable reagent

Interaction of light-activated rhodopsin with transducin (T) is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide. The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfide-exchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at λ >495 nm. Subsequent irradiation of the complex at λ310 nm generates covalent crosslinks between the two proteins. Crosslinking was demonstrated between T and a number of single cysteine rhodopsin mutants. However, sites of crosslinks were investigated in detail only between T and the rhodopsin mutant S240C (cytoplasmic loop V-VI). Crosslinking occurred predominantly with Tα. For identification of the sites of crosslinks in Tα, the strategy used involved: (i) derivatization of all of the free cysteines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction of the disulfide bond linking the two proteins and isolation of all of the Tα species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the N-maleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting Tα derivatives and isolation of Tα peptides carrying maleimido-butyryl-biocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in Tα containing the amino acid sequences 310–313 and 342–345.

Mapping of contact sites in complex formation between light-activated rhodopsin and transducin by covalent crosslinking: Use of a chemically preactivated reagent

Contact sites in interaction between light-activated rhodopsin and transducin (T) have been investigated by using a chemically preactivated crosslinking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate. The 3 propionyl-N-succinimidyl group in the reagent was attached by a disulfide exchange reaction to rhodopsin mutants containing single reactive cysteine groups in the cytoplasmic loops. Complex formation between the derivatized rhodopsin mutants and T was carried out by illumination at λ > 495 nm. Subsequent increase in pH (from 6 to 7.5 or higher) of the complex resulted in crosslinking of rhodopsin to the Tα subunit. Crosslinking to Tα was demonstrated for the rhodopsin mutants K141C, S240C, and K248C, and the crosslinked sites in Tα were identified for the rhodopsin mutant S240C. The peptides carrying the crosslinking moiety were isolated from the trypsin-digested peptide mixture, and their identification was carried out by matrix-assisted laser desorption ionization–time of flight mass spectrometry. The main site of crosslinking is within the peptide sequence, Leu-19–Arg-28 at the N-terminal region of Tα. The total results show that both the N and the C termini of Tα are in close vicinity to the third cytoplasmic loop of rhodopsin in the complex between rhodopsin and T.

Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1

Nox1, a homologue of gp91phox, the catalytic moiety of the superoxide (O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document})-generating NADPH oxidase of phagocytes, causes increased O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document} generation, increased mitotic rate, cell transformation, and tumorigenicity when expressed in NIH 3T3 fibroblasts. This study explores the role of reactive oxygen species (ROS) in regulating cell growth and transformation by Nox1. H2O2 concentration increased ≈10-fold in Nox1-expressing cells, compared with <2-fold increase in O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document}. When human catalase was expressed in Nox1-expressing cells, H2O2 concentration decreased, and the cells reverted to a normal appearance, the growth rate normalized, and cells no longer produced tumors in athymic mice. A large number of genes, including many related to cell cycle, growth, and cancer (but unrelated to oxidative stress), were expressed in Nox1-expressing cells, and more than 60% of these returned to normal levels on coexpression of catalase. Thus, H2O2 in low concentrations functions as an intracellular signal that triggers a genetic program related to cell growth.

A type IB topoisomerase with DNA repair activities

Previously we have characterized type IB DNA topoisomerase V (topo V) in the hyperthermophile Methanopyrus kandleri. The enzyme has a powerful topoisomerase activity and is abundant in M. kandleri. Here we report two characterizations of topo V. First, we found that its N-terminal domain has sequence homology with both eukaryotic type IB topoisomerases and the integrase family of tyrosine recombinases. The C-terminal part of the sequence includes 12 repeats, each repeat consisting of two similar but distinct helix-hairpin-helix motifs; the same arrangement is seen in recombination protein RuvA and mammalian DNA polymerase β. Second, on the basis of sequence homology between topo V and polymerase β, we predict and demonstrate that topo V possesses apurinic/apyrimidinic (AP) site-processing activities that are important in base excision DNA repair: (i) it incises the phosphodiester backbone at the AP site, and (ii) at the AP endonuclease cleaved AP site, it removes the 5′ 2-deoxyribose 5-phosphate moiety so that a single-nucleotide gap with a 3′-hydroxyl and 5′-phosphate can be filled by a DNA polymerase. Topo V is thus the prototype for a new subfamily of type IB topoisomerases and is the first example of a topoisomerase with associated DNA repair activities.