A long-acting GH receptor antagonist through fusion to GH binding protein.

Unlabelled: Acromegaly is a human disease of growth hormone (GH) excess with considerable morbidity and increased mortality. Somatostatin analogues are first line medical treatment but the disease remains uncontrolled in up to 40% of patients. GH receptor (GHR) antagonist therapy is more effective but requires frequent high-dose injections.

Direct observation of DNA threading in flap endonuclease complexes.

Maintenance of genome integrity requires that branched nucleic acid molecules be accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki-fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates and products, at resolutions of 1.

COGNAC: a web server for searching and annotating hydrogen-bonded base interactions in RNA three-dimensional structures.

Hydrogen bonds are crucial factors that stabilize a complex ribonucleic acid (RNA) molecule's three-dimensional (3D) structure. Minute conformational changes can result in variations in the hydrogen bond interactions in a particular structure. Furthermore, networks of hydrogen bonds, especially those found in tight clusters, may be important elements in structure stabilization or function and can therefore be regarded as potential tertiary motifs.

Cyclic-AMP and bacterial cyclic-AMP receptor proteins revisited: adaptation for different ecological niches.

Escherichia coli cyclic-AMP receptor protein (CRP) represents one of the paradigms of bacterial gene regulation. Yet despite decades of intensive study, new information continues to emerge that prompts reassessment of this classic regulatory system. Moreover, in recent years CRPs from several other bacterial species have been characterized, allowing the general applicability of the CRP paradigm to be tested.

Structure of the NheA component of the Nhe toxin from Bacillus cereus: implications for function.

The structure of NheA, a component of the Bacillus cereus Nhe tripartite toxin, has been solved at 2.05 Å resolution using selenomethionine multiple-wavelength anomalous dispersion (MAD). The structure shows it to have a fold that is similar to the Bacillus cereus Hbl-B and E.

The structure of Escherichia coli ExoIX--implications for DNA binding and catalysis in flap endonucleases.

Escherichia coli Exonuclease IX (ExoIX), encoded by the xni gene, was the first identified member of a novel subfamily of ubiquitous flap endonucleases (FENs), which possess only one of the two catalytic metal-binding sites characteristic of other FENs. We have solved the first structure of one of these enzymes, that of ExoIX itself, at high resolution in DNA-bound and DNA-free forms. In the enzyme-DNA cocrystal, the single catalytic site binds two magnesium ions.

IMAAAGINE: a webserver for searching hypothetical 3D amino acid side chain arrangements in the Protein Data Bank.

We describe a server that allows the interrogation of the Protein Data Bank for hypothetical 3D side chain patterns that are not limited to known patterns from existing 3D structures. A minimal side chain description allows a variety of side chain orientations to exist within the pattern, and generic side chain types such as acid, base and hydroxyl-containing can be additionally deployed in the search query. Moreover, only a subset of distances between the side chains need be specified.

Structure-function dissection of the frizzled receptor in Drosophila melanogaster suggests different mechanisms of action in planar polarity and canonical Wnt signaling.

Members of the Frizzled family of sevenpass transmembrane receptors signal via the canonical Wnt pathway and also via noncanonical pathways of which the best characterized is the planar polarity pathway. Activation of both canonical and planar polarity signaling requires interaction between Frizzled receptors and cytoplasmic proteins of the Dishevelled family; however, there has been some dispute regarding whether the Frizzled-Dishevelled interactions are the same in both cases. Studies looking at mutated forms of Dishevelled suggested that stable recruitment of Dishevelled to membranes by Frizzled was required only for planar polarity activity, implying that qualitatively different Frizzled-Dishevelled interactions underlie canonical signaling.

Expression of a glycosylphosphatidylinositol-anchored ligand, growth hormone, blocks receptor signalling.

We have investigated the interaction between GH (growth hormone) and GHR (GH receptor). We previously demonstrated that a truncated GHR that possesses a transmembrane domain but no cytoplasmic domain blocks receptor signalling. Based on this observation we investigated the impact of tethering the receptor's extracellular domain to the cell surface using a native lipid GPI (glycosylphosphatidylinositol) anchor.

Crystallization and preliminary crystallographic analysis of the NheA component of the Nhe toxin from Bacillus cereus.

The nonhaemolytic enterotoxin (Nhe) of Bacillus cereus plays a key role in cases of B. cereus food poisoning. The toxin is comprised of three different proteins: NheA, NheB and NheC.

NASSAM: a server to search for and annotate tertiary interactions and motifs in three-dimensional structures of complex RNA molecules.

Similarities in the 3D patterns of RNA base interactions or arrangements can provide insights into their functions and roles in stabilization of the RNA 3D structure. Nucleic Acids Search for Substructures and Motifs (NASSAM) is a graph theoretical program that can search for 3D patterns of base arrangements by representing the bases as pseudo-atoms. The geometric relationship of the pseudo-atoms to each other as a pattern can be represented as a labeled graph where the pseudo-atoms are the graph's nodes while the edges are the inter-pseudo-atomic distances.

SPRITE and ASSAM: web servers for side chain 3D-motif searching in protein structures.

Similarities in the 3D patterns of amino acid side chains can provide insights into their function despite the absence of any detectable sequence or fold similarities. Search for protein sites (SPRITE) and amino acid pattern search for substructures and motifs (ASSAM) are graph theoretical programs that can search for 3D amino side chain matches in protein structures, by representing the amino acid side chains as pseudo-atoms. The geometric relationship of the pseudo-atoms to each other as a pattern can be represented as a labeled graph where the pseudo-atoms are the graph's nodes while the edges are the inter-pseudo-atomic distances.

Structure of the human obesity receptor leptin-binding domain reveals the mechanism of leptin antagonism by a monoclonal antibody.

Leptin regulates energy homeostasis, fertility, and the immune system, making it an important drug target. However, due to a complete lack of structural data for the obesity receptor (ObR), leptin's mechanism of receptor activation remains poorly understood. We have crystallized the Fab fragment of a leptin-blocking monoclonal antibody (9F8), both in its uncomplexed state and bound to the leptin-binding domain (LBD) of human ObR.

Novel base triples in RNA structures revealed by graph theoretical searching methods.

Background: Highly hydrogen bonded base interactions play a major part in stabilizing the tertiary structures of complex RNA molecules, such as transfer-RNAs, ribozymes and ribosomal RNAs.

Results: We describe the graph theoretical identification and searching of highly hydrogen bonded base triples, where each base is involved in at least two hydrogen bonds with the other bases. Our approach correlates theoretically predicted base triples with literature-based compilations and other actual occurrences in crystal structures.

A Burkholderia pseudomallei toxin inhibits helicase activity of translation factor eIF4A.

The structure of BPSL1549, a protein of unknown function from Burkholderia pseudomallei, reveals a similarity to Escherichia coli cytotoxic necrotizing factor 1. We found that BPSL1549 acted as a potent cytotoxin against eukaryotic cells and was lethal when administered to mice. Expression levels of bpsl1549 correlate with conditions expected to promote or suppress pathogenicity.

Assessing the role of Asp 194 in the transmembrane domains of the α-chain of the high-affinity receptor complex for immunoglobulin E in signal transduction.

The high-affinity receptor complex for IgE plays a pivotal role in allergic responses since cross-linking of the high-affinity IgE receptor (FcɛRI) on target cells initiates a signaling cascade facilitating release of inflammatory mediators causing allergic responses. The transmembrane regions of the ligand binding domains of the high-affinity IgE and low-affinity IgG receptors share an invariant motif (LFAVDTGL) containing a polar aspartate within a predominantly non-polar setting. The functional importance of this aspartate residue (D194) in FcɛRI-mediated receptor signaling was assessed by site-directed mutagenesis.

Hemolysin E (HlyE, ClyA, SheA) and related toxins.

Certain strains of Escherichia coli, Salmonella enterica and Shigella flexneri produce a pore-forming toxin hemolysin E (HlyE), also known as cytolysin A (ClyA) and silent hemolysin, locus A (SheA). HlyE lyses erythrocytes and mammalian cells, forming transmembrane pores with a minimum internal diameter of-25 A. We review the current knowledge of HlyE structure and function in its solution and pore forms, models for membrane insertion, its potential use in biotechnology applications and its relationship to a wider superfamily of toxins.

Immunogenicity, toxicology, pharmacokinetics and pharmacodynamics of growth hormone ligand-receptor fusions.

A fundamental concern for all new biological therapeutics is the possibility of inducing an immune response. We have recently demonstrated that an LR-fusion (ligand-receptor fusion) of growth hormone generates a potent long-acting agonist; however, the immunogenicity and toxicity of these molecules have not been tested. To address these issues, we have designed molecules with low potential as immunogens and undertaken immunogenicity and toxicology studies in Macaca fascicularis and pharmacokinetic and pharmacodynamic studies in rats.

Mycobacterium tuberculosis cAMP receptor protein (Rv3676) differs from the Escherichia coli paradigm in its cAMP binding and DNA binding properties and transcription activation properties.

The pathogen Mycobacterium tuberculosis produces a burst of cAMP upon infection of macrophages. Bacterial cyclic AMP receptor proteins (CRP) are transcription factors that respond to cAMP by binding at target promoters when cAMP concentrations increase. Rv3676 (CRP(Mt)) is a CRP family protein that regulates expression of genes (rpfA and whiB1) that are potentially involved in M.

The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion.

Fumarate and nitrate reduction regulatory (FNR) proteins are bacterial transcription factors that coordinate the switch between aerobic and anaerobic metabolism. In the absence of O(2), FNR binds a [4Fe-4S](2+) cluster (ligated by Cys-20, 23, 29, 122) promoting the formation of a transcriptionally active dimer. In the presence of O(2), FNR is converted into a monomeric, non-DNA-binding form containing a [2Fe-2S](2+) cluster.

The formation and structure of Escherichia coli K-12 haemolysin E pores.

Some enteric bacteria synthesize a pore-forming toxin, HlyE, which is cytolytic and cytotoxic to host cells. Measurement of HlyE binding to erythrocyte ghosts and the kinetics of HlyE-mediated erythrocyte lysis suggests that interaction with target membranes is not the rate-limiting step in the formation of HlyE pores, but that there is a temperature-dependent lag phase before a functional pore is formed. Circular dichroism and fluorescence energy transfer analyses show that HlyE protomers retain an alpha-helical structure when oligomerized to form a pore consisting of parallel HlyE protomers.

A ligand-receptor fusion of growth hormone forms a dimer and is a potent long-acting agonist.

Cytokine hormones have a short plasma half-life and require frequent administration. For example, growth hormone replacement involves daily injections. In common with other cytokines, the extracellular domain of the growth hormone receptor circulates as a binding protein, which naturally prolongs the biological half-life of growth hormone.

Structure of the hemolysin E (HlyE, ClyA, and SheA) channel in its membrane-bound form.

Hemolysin E (HlyE, ClyA, SheA) is a pore-forming protein toxin isolated from Escherichia coli. The three-dimensional structure of its water-soluble form is known, but that of the membrane-bound HlyE complex is not. We have used electron microscopy and image processing to show that the pores are predominantly octameric.

The crystal structures of Lactococcus lactis MG1363 Dps proteins reveal the presence of an N-terminal helix that is required for DNA binding.

Dps proteins play a major role in the protection of bacterial DNA from damage by reactive oxygen species. Previous studies have implicated the extended lysine-containing N-terminal regions of Dps subunits in DNA binding, but this part of the structure has not previously been observed crystallographically. Here the structures of two Dps proteins (DpsA and DpsB) from Lactococcus lactis MG1363 reveal for the first time the presence of an N-terminal alpha helix that extends from the core of the Dps subunit.