The structure of an unconventional HD-GYP protein from Bdellovibrio reveals the roles of conserved residues in this class of cyclic-di-GMP phosphodiesterases

UNLABELLED: Cyclic-di-GMP is a near-ubiquitous bacterial second messenger that is important in localized signal transmission during the control of various processes, including virulence and switching between planktonic and biofilm-based lifestyles. Cyclic-di-GMP is synthesized by GGDEF diguanylate cyclases and hydrolyzed by EAL or HD-GYP phosphodiesterases, with each functional domain often appended to distinct sensory modules. HD-GYP domain proteins have resisted structural analysis, but here we present the first structural representative of this family (1.28 Å), obtained using the unusual Bd1817 HD-GYP protein from the predatory bacterium Bdellovibrio bacteriovorus. Bd1817 lacks the active-site tyrosine present in most HD-GYP family members yet remains an excellent model of their features, sharing 48% sequence similarity with the archetype RpfG. The protein structure is highly modular and thus provides a basis for delineating domain boundaries in other stimulus-dependent homologues. Conserved residues in the HD-GYP family cluster around a binuclear metal center, which is observed complexed to a molecule of phosphate, providing information on the mode of hydroxide ion attack on substrate. The fold and active site of the HD-GYP domain are different from those of EAL proteins, and restricted access to the active-site cleft is indicative of a different mode of activity regulation. The region encompassing the GYP motif has a novel conformation and is surface exposed and available for complexation with binding partners, including GGDEF proteins.

IMPORTANCE: It is becoming apparent that many bacteria use the signaling molecule cyclic-di-GMP to regulate a variety of processes, most notably, transitions between motility and sessility. Importantly, this regulation is central to several traits implicated in chronic disease (adhesion, biofilm formation, and virulence gene expression). The mechanisms of cyclic-di-GMP synthesis via GGDEF enzymes and hydrolysis via EAL enzymes have been suggested by the analysis of several crystal structures, but no information has been available to date for the unrelated HD-GYP class of hydrolases. Here we present the multidomain structure of an unusual member of the HD-GYP family from the predatory bacterium Bdellovibrio bacteriovorus and detail the features that distinguish it from the wider structural family of general HD fold hydrolases. The structure reveals how a binuclear iron center is formed from several conserved residues and provides a basis for understanding HD-GYP family sequence requirements for c-di-GMP hydrolysis.

Molecular diagnosis for heterogeneous genetic diseases with high-throughput DNA sequencing applied to retinitis pigmentosa

BACKGROUND:
The genetic heterogeneity of many Mendelian disorders, such as retinitis pigmentosa which results from mutations in over 40 genes, is a major obstacle to obtaining a molecular diagnosis in clinical practice. Targeted high-throughput DNA sequencing offers a potential solution and was used to develop a molecular diagnostic screen for patients with retinitis pigmentosa.
METHODS:
A custom sequence capture array was designed to target the coding regions of all known retinitis pigmentosa genes and used to enrich these sequences from DNA samples of five patients. Enriched DNA was subjected to high-throughput sequencing singly or in pools, and sequence variants were identified by alignment of up to 10 million reads per sample to the normal reference sequence. Potential pathogenicity was assessed by functional predictions and frequency in controls.
RESULTS AND CONCLUSIONS:
Known homozygous PDE6B and compound heterozygous CRB1 mutations were detected in two patients. A novel homozygous missense mutation (c.2957A?T; p.N986I) in the cyclic nucleotide gated channel ß1 (CNGB1) gene predicted to have a deleterious effect and absent in 720 control chromosomes was detected in one case in which conventional genetic screening had failed to detect mutations. The detection of known and novel retinitis pigmentosa mutations in this study establishes high-throughput DNA sequencing with DNA pooling as an effective diagnostic tool for heterogeneous genetic diseases.

Effects of the novel (Pro(3))GIP antagonist and exendin(9-39)amide on GIP- and GLP-1-induced cyclic AMP generation, insulin secretion and postprandial insulin release in obese diabetic (ob/ob) mice: evidence that GIP is the major physiological incretin

AIMS/HYPOTHESIS: This study examined the biological effects of the GIP receptor antagonist, (Pro3)GIP and the GLP-1 receptor antagonist, exendin(9-39)amide.

METHODS: Cyclic AMP production was assessed in Chinese hamster lung fibroblasts transfected with human GIP or GLP-1 receptors, respectively. In vitro insulin release studies were assessed in BRIN-BD11 cells while in vivo insulinotropic and glycaemic responses were measured in obese diabetic ( ob/ ob) mice.

RESULTS: In GIP receptor-transfected fibroblasts, (Pro(3))GIP or exendin(9-39)amide inhibited GIP-stimulated cyclic AMP production with maximal inhibition of 70.0+/-3.5% and 73.5+/-3.2% at 10(-6) mol/l, respectively. In GLP-1 receptor-transfected fibroblasts, exendin(9-39)amide inhibited GLP-1-stimulated cyclic AMP production with maximal inhibition of 60+/-0.7% at 10(-6) mol/l, whereas (Pro(3))GIP had no effect. (Pro(3))GIP specifically inhibited GIP-stimulated insulin release (86%; p<0.001) from clonal BRIN-BD11 cells, but had no effect on GLP-1-stimulated insulin release. In contrast, exendin(9-39)amide inhibited both GIP and GLP-1-stimulated insulin release (57% and 44%, respectively; p<0.001). Administration of (Pro(3))GIP, exendin(9-39)amide or a combination of both peptides (25 nmol/kg body weight, i.p.) to fasted (ob/ob) mice decreased the plasma insulin responses by 42%, 54% and 49%, respectively (p<0.01 to p<0.001). The hyperinsulinaemia of non-fasted (ob/ob) mice was decreased by 19%, 27% and 18% (p<0.05 to p<0.01) by injection of (Pro3)GIP, exendin(9-39)amide or combined peptides but accompanying changes of plasma glucose were small.

CONCLUSIONS/INTERPRETATION: These data show that (Pro(3))GIP is a specific GIP receptor antagonist. Furthermore, feeding studies in one commonly used animal model of obesity and diabetes, (ob/ob) mice, suggest that GIP is the major physiological component of the enteroinsular axis, contributing approximately 80% to incretin-induced insulin release.

2,4-dinitrophenol as an activating reagent in a facile preparation of cyclic phosphate trimesters

Abstract 2,4-Dinitrophenol was employed with benzyloxy-bis-(diisopropylamino)phosphine to synthesise the cyclic phosphate derivatives of a series of alkane diols (HO–(CH2)n–OH, n=2–6) in good isolated yields. Tetrazole and DNP were compared by 31P NMR spectroscopy for their ability to catalyse the cyclisation at the P(III) stage. Investigation of the phosphate triester stability under various oxidation and chromatographic conditions resulted in the optimisation of the isolation procedures of the chemically unstable cyclic compounds. Conditions for debenzylation were developed to yield the corresponding cyclic phosphodiesters quantitatively. The methodology was further applied to the preparation and isolation of the cyclic phosphate derivative of a carbohydrate.

Equilibrium polymerization of cyclic carbonate oligomers. III. Chain branching and the gel transition

Ring-opening polymerization of cyclic polycarbonate oligomers, where monofunctional active sites act on difunctional monomers to produce an equilibrium distribution of rings and chains, leads to a "living polymer." Monte Carlo simulations [two-dimensional (2D) and three-dimensional (3D)] of the effects of single [J. Chem. Phys. 115, 3895 (2001)] and multiple active sites [J. Chem. Phys. 116, 7724 (2002)] are extended here to trifunctional active sites that lead to branching. Low concentrations of trifunctional particles c(3) reduce the degree of polymerization significantly in 2D, and higher concentrations (up to 32%) lead to further large changes in the phase diagram. Gel formation is observed at high total density and sizable c(3) as a continuous transition similar to percolation. Polymer and gel are much more stable in 3D than in 2D, and both the total density and the value of c(3) required to produce high molecular weight aggregates are reduced significantly. The degree of polymerization in high-density 3D systems is increased by the addition of trifunctional monomers and reduced slightly at low densities and low c(3). The presence of branching makes equilibrium states more sensitive (in 2D and 3D) to changes in temperature T. The stabilities of polymer and gel are enhanced by increasing T, and-for sufficiently high values of c(3)-there is a reversible polymer-gel transformation at a density-dependent floor temperature. (C) 2002 American Institute of Physics.

Equilibrium polymerization of cyclic carbonate oligomers. II. Role of multiple active sites

Ring opening polymerization of bisphenol A polycarbonate is studied by Monte Carlo simulations of a model comprising a fixed number of Lennard-Jones particles and harmonic bonds [J. Chem. Phys. 115, 3895 (2001)]. Bond interchanges produced by a low concentration (0.10%less than or equal toc(a)less than or equal to0.36%) of chemically active particles lead to equilibrium polymerization. There is a continuous transition in both 2D and 3D from unpolymerized cyclic oligomers at low density to a system of linear chains at high density, and the polymeric phase is much more stable in three dimensions than in two. The steepness of the polymerization transition increases rapidly as c(a) decreases, suggesting that it is discontinuous in the limit c(a)-->0. The transition is entropy driven, since the average potential energy increases systematically upon polymerization, and there is a steady decline in the degree of polymerization as the temperature is lowered. The mass distribution functions for open chains and for rings are unimodal, with exponentially decaying tails that can be fitted by Zimm-Schulz functions and simpler exponential forms. (C) 2002 American Institute of Physics.

Equilibrium polymerization of cyclic carbonate oligomers

A model of the polymerization of ring oligomers of bisphenol A polycarbonate (BPA-PC) is used to investigate the influence of dimensionality (2D or 3D), density and temperature on the size distribution of the polymer chains. The polymerization step is catalyzed by a single active particle, conserves the number and type of the chemical bonds, and occurs without a significant gain in either potential energy or configurational entropy. Monte Carlo and molecular dynamics simulations show that polymerization of cyclic oligomers occurs readily at high density and is driven by the entropy associated with the distribution of interparticle bonds. Polymerization competes at lower densities with long range diffusion, which favors small molecular species, and is prevented if the system is sufficiently dilute. Polymerization occurs in 2D via a weakly first order transition as a function of density and is characterized by low hysteresis and large fluctuations in the size of polymer chains. Polymerization occurs more readily in 3D than in 2D, and is favored by increasing temperature, as expected for an entropy-driven process. (C) 2001 American Institute of Physics.