Ligand-binding Unit Research Articles

Comparative molecular dynamics simulation analysis of G20 and C92 mutations in c-di-GMP I riboswitch and the wild type with docked c-di-GMP ligand.

Riboswitch, a bacterial regulatory RNA consists of an aptamer (specific ligand binding unit) and an expression platform (gene expression modulation unit), which act as a potential drug target as it regulates critical genes. Therefore, it is of interest to glean information on the binding of c-di-GMP ligand to mutated conserved G20 and C92 residues of cyclic diguanosine monophosphate I (c-di-GMP I) riboswitch using molecular dynamics simulation. The result shows that the binding energy of wild/native type riboswitch-ligand complex (3IRW) is lower than the mutant complexes suggesting that the binding affinity for c-di-GMP ligand decreases in case of mutant riboswitches. The hydrogen bonding interactions analysis also showed a high number of hydrogen bonds formation in the wild type riboswitch-ligand complex as compared to the mutant complexes illustrating stronger interaction of ligand to wild type riboswitch than the mutants. The simulation result shows that the mutations affected riboswitch-ligand interactions. The residues G14, G21, C46, A47, and U92 were identified as the key residues which contributed effectively to the binding of c-di-GMP I riboswitch with the natural ligand.

MERIT40-dependent recruitment of tankyrase to damaged DNA and its implication for cell sensitivity to DNA-damaging anticancer drugs.

Tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, regulates various intracellular responses, such as telomere maintenance, Wnt/β-catenin signaling and cell cycle progression through its interactions with multiple target proteins. Tankyrase contains a long stretch of 24 ankyrin repeats that are further divided into five subdomains, called ANK repeat clusters (ARCs). Each ARC works as an independent ligand-binding unit, which implicates tankyrase as a platform for multiple protein-protein interactions. Furthermore, tankyrase distributes to various intracellular loci, suggesting potential distinct but yet unidentified physiological functions. To explore the novel functions of tankyrase, we performed liquid chromatography-mass spectrometry analysis and identified the BRE-BRCC36-MERIT40 complex, a regulator of homologous recombination, as tankyrase-binding proteins. Among the complex components, MERIT40 was directly associated with tankyrase via a tankyrase-binding consensus motif, as previously reported. In X-ray-irradiated non-small cell lung cancer cells, tankyrase localized to DNA double-stranded break sites in a MERIT40-dependent manner. MERIT40 knockdown increased the cell sensitivity to X-ray, whereas the wild-type, but not the tankyrase-unbound mutant, MERIT40 rescued the phenotype of the knockdown cells. Tankyrase inhibitors, such as G007-LK and XAV939, increased the cellular sensitivity to X-ray irradiation and anticancer drugs that induce DNA double-stranded breaks. These observations suggest that tankyrase plays a role in the DNA damage repair response and implicates a potential therapeutic utility of tankyrase inhibitors in combination treatments with DNA-damaging anticancer drugs.

Reversible ion transportation switch by a ligand-gated synthetic supramolecular ion channel.

Inspired by the regulation of cellular activities found in the ion channel proteins, here we developed membrane-embedded synthetic chiral receptors 1 and 2 with different terminal structures, where receptor 1 has hydrophobic triisopropylsilyl (TIPS) groups and receptor 2 has hydrophilic hydroxy groups. The receptors have ligand-binding units that interact with cationic amphiphiles such as 2-phenethylamine (PA). Conductance study revealed that the receptors hardly show ion transportation at the ligand-free state. After ligand binding involving a conformational change, receptor 1 bearing TIPS termini displays a significant current enhancement due to ion transportation. The current substantially diminishes upon addition of β-cyclodextrin (βCD) that scavenges the ligand from the receptor. Importantly, the receptor again turns into the conductive state by the second addition of PA, and the activation/deactivation of the ion transportation can be repeated. In contrast, receptor 2 bearing the hydroxy terminal groups hardly exhibits ion transportation, suggesting the importance of terminal TIPS groups of 1 that likely anchor the receptor in the membrane.

The Human C1q Globular Domain: Structure and Recognition of Non-Immune Self Ligands

C1q, the ligand-binding unit of the C1 complex of complement, is a pattern recognition molecule with the unique ability to sense an amazing variety of targets, including a number of altered structures from self, such as apoptotic cells. The three-dimensional structure of its C-terminal globular domain, responsible for its recognition function, has been solved by X-ray crystallography, revealing a tightly packed heterotrimeric assembly with marked differences in the surface patterns of the subunits, and yielding insights into its versatile binding properties. In conjunction with other approaches, this same technique has been used recently to decipher the mechanisms that allow this domain to interact with various non-immune self ligands, including molecules known to provide eat-me signals on apoptotic cells, such as phosphatidylserine and DNA. These investigations provide evidence for a common binding area for these ligands located in subunit C of the C1q globular domain, and suggest that ligand recognition through this area down-regulates C1 activation, hence contributing to the control of the inflammatory reaction. The purpose of this article is to give an overview of these advances which represent a first step toward understanding the recognition mechanisms of C1q and their biological implications.

Pyrrolidine Dithiocarbamate Inhibits Interleukin-6 Signaling through Impaired STAT3 Activation and Association with Transcriptional Coactivators in Hepatocytes

Pyrrolidine Dithiocarbamate Inhibits Interleukin-6 Signaling through Impaired STAT3 Activation and Association with Transcriptional Coactivators in Hepatocytes

Mixed‐Valence, Mixed‐Spin‐State, and Heterometallic [2×2] Grid‐type Arrays Based on Heteroditopic Hydrazone Ligands: Synthesis and Electrochemical Features

An extended family of heterometallic [(M1)2(M2)2(L-)4](n+) [2x2] grid-type arrays 1-9 has been prepared. The three-tiered synthetic route encompasses regioselective, redox and enantioselective features and is based on the stepwise construction of heteroditopic hydrazone ligands A-C. These ligands contain ionisable NH and nonionisable NMe hydrazone units, which allows the metal redox properties to be controlled according to the charge on the ligand binding pocket. The 2-pyrimidine (R) and 6-pyridine (R') substituents have a significant effect on complex geometry and influence both the electrochemical and magnetic behaviour of the system. 1H NMR spectroscopic studies show that the Fe(II) ions in the grid can be low spin, high spin or spin crossover depending on the steric effect of substituents R and R'. This steric effect has been manipulated to construct an unusual array possessing two low-spin and two spin-crossover Fe(II) centres (grid 8). Electrochemical studies were performed for the grid-type arrays 1-9 and their respective mononuclear precursor complexes 10-13. The grids function as electron reservoirs and display up to eight monoelectronic, reversible reduction steps. These processes generally occur in pairs and are assigned to ligand-based reductions and to the Co(III)/Co(II) redox couple. Individual metal ions in the heterometallic grid motif can be selectively addressed electrochemically (e.g., either the Co(III) or Fe(II) ions can be targeted in grids 2 and 5). The Fe(II) oxidation potential is governed by the charge on the ligand binding unit, rather than the spin state, thus permitting facile electrochemical discrimination between the two types of Fe(II) centre in 7 or in 8. Such multistable heterometallic [2x2] gridlike arrays are of great interest for future supramolecular devices incorporating multilevel redox activity.

LRP5 and Wnt signaling: a union made for bone.

THE IDENTIFICATION OF mutations in the human lowdensity lipoprotein (LDL) receptor–related protein 5 (LRP5) gene that give rise to conditions of low bone mass and increased bone mass has brought what just a few years ago were two seemingly unrelated fields crashing together, namely bone biology and Wnt signaling. It is now clear that the Wnt signaling pathway is another key pathway involved in the regulation of bone mass. This is not to diminish the importance of any other pathway that is known to play a role; rather, this discovery only highlights how little we probably really know about bone mass regulation. Certainly, a big challenge we now face will be to integrate the Wnt signaling pathway with all of its complexities and subtleties into the already complex nature of the other pathways that have been studied, with the ultimate goal of developing a composite understanding of their collective roles and interactions. In this article, we will summarize the main points of our current understanding of LRP5, Wnt’s and Wnt signaling, and the role they may play in bone. This is by no means comprehensive, and there are more unanswered questions than answers.

Megalin and cubilin, role in proximal tubule function and during development.

Epithelia lining the proximal convoluted epithelium and the visceral yolk sac in rodents are characterized by their ability to internalize and degrade many proteins facing their apical pole. It has become apparent over the last decade that this property is mediated through a very high endocytic activity and the expression of at least two distinct multiligand receptors, cubilin and megalin, characterized by the accumulation of ligand-binding units, LDL receptor class A repeats and CUB domains, respectively. We review here evidence that both receptors are involved in proximal tubule function and embryonic development.

Association of non-receptor protein tyrosine kinases with the Fc gamma RI/gamma-chain complex in monocytic cells.

To characterize the functional macromolecular complex of the high affinity receptor for IgG (Fc gamma RI), we have undertaken the identification of the molecules associated with the ligand binding unit and its associated subunit, gamma-chain, in a monocyte cell line, U937. Comparison of the pattern of tyrosine-phosphorylated proteins that coprecipitate with anti-Fc gamma RI or anti-gamma-chain Abs after Fc gamma RI clustering suggests that, like other receptor systems, the different units of the receptor associate or recruit different elements of the signaling pathway. Syk associates preferentially with the gamma-chain subunit and not with Fc gamma RI itself. This association is dependent on receptor clustering and correlates with gamma-chain phosphorylation. The Src kinase Lyn is also part of the receptor complex. Lyn associates with both units of the receptor, and this association is independent of receptor engagement; however, the level of tyrosine phosphorylation of the kinase increases after Fc gamma RI clustering. Association of a 35-kDa phosphoprotein with the binding unit was also observed after receptor clustering. We further found that after Fc gamma RI clustering, tyrosine-phosphorylated Syk associates with Lyn. These data suggest that several levels of interaction occur between these molecules or that different pools of tyrosine kinases become activated after Fc gamma RI clustering.

A SOLUBLE INTERLEUKIN 6 RECEPTOR ISOLATED FROM CONDITIONED MEDIUM OF HUMAN BREAST CANCER CELLS IS ENCODED BY A DIFFERENTIALLY SPLICED mRNA

The human interleukin 6 receptor (IL-6R) is expressed on cells as a transmembrane protein of 80 kDa (gp80, ligand binding unit), or as a smaller soluble counterpart (sIL-6R, 55 kDa). Recombinant or natural sIL-6Rs bind IL-6 and stimulate biological activity by association with the signal transducing subunit gp130 at the cell surface. The origin of sIL-6Rs is not clear. Haematopoietic cells express, in addition to the gp80 mRNA, an IL-6R mRNA where the transmembrane domain is spliced out, predicting a shorter protein with a modified basic sequence at the C-terminus. We show that the spliced mRNA is expressed in human T47D breast carcinoma cells and soluble IL-6R protein is indeed secreted by these cells. An antibody against the C-terminus of the spliced protein detects a 55–65 kDa glycosylated species in sIL-6R purified from T47D supernatant by classical and immunoaffinity chromatography. The spliced T47D IL-6R, glycosylated or after removal of O- and N-linked polysaccharides, has the same size as a recombinant spliced IL-6R from CHO cells. The recombinant spliced IL-6R acts on cells as an IL-6 agonist to stimulate transcription from IL-6 inducible enhancers.

Glycosyl-phosphatidylinositol-anchored or integral membrane forms of CD14 mediate identical cellular responses to endotoxin.

Endotoxin stimulates leukocytes to release cytokines that initiate septic shock in humans and animals. CD14, a glycosyl-phosphatidylinositol-anchored membrane glycoprotein, is an endotoxin receptor on leukocytes, and endotoxin binding to CD14 induces cytokine production. Here we show that glycosyl-phosphatidylinositol-anchored or integral membrane CD14 mediates identical cellular responses to endotoxin, including NF-kappa B activation and protein tyrosine phosphorylation. We also show that an anti-CD14 monoclonal antibody that does not block endotoxin binding to CD14 nonetheless inhibits cell activation by endotoxin. These findings suggest that binding of endotoxin to cell-surface CD14 is followed by subsequent interactions of the endotoxin-CD14 complex with additional membrane component(s) that enable transmembrane signaling. This function of CD14 may be prototypic for other members of the glycosyl-phosphatidylinositol-anchored family of proteins that do not play a primary role in signal transduction but rather are the principal ligand-binding units of membrane-bound receptor complexes.

Double antenna structure of chicken prolactin receptor deduced from the cDNA sequence

Chicken prolactin receptor (cPRLR) deciphere from the cDNA sequence showed a unique double antenna structure in its extracellular domain. The predicted cPRLR preprotein was composed of 831 amino acids and contained a signal peptide and a transmembrane region. The extracellular domain comprised 438 residues, and was divided into two tandemly repeated, highly homologous units, each of which corresponded to the extracellular domains of mammalian prolactin receptors. Both extracellular units of cPRLR possessed two structural features characteristic of the ligand binding units of cytokine/prolactin receptor family, namely two pairs of cysteine residues and a WSXWS motif. These findings strongly suggest that cPRLR contains two repeated ligand binding units, that is a double antenna structure. The cPRLR gene is expressed in a wide range of tissues of laying hen.

Specific EGF repeats of Notch mediate interactions with Delta and serrate: Implications for notch as a multifunctional receptor

The neurogenic loci Notch and Delta, which both encode EGF-homologous transmembrane proteins, appear to function together in mediating cell-cell communication and have been shown to interact at the cell surface in vitro. To examine the role of the EGF repeats in this interaction, we performed an extensive deletion mutagenesis of the extracellular domain of Notch. We find that of the 36 EGF repeats of Notch, only two, 11 and 12, are both necessary and sufficient to mediate interactions with Delta. Furthermore, this Delta binding ability is conserved in the corresponding two repeats from the Xenopus Notch homolog. We report a novel molecular interaction between Notch and Serrate, another EGF-homologous transmembrane protein containing a region of striking similarity to Delta, and show that the same two EGF repeats of Notch also constitute a Serrate binding domain. These results suggest that Notch may act as a multifunctional receptor whose 36 EGF repeats form a tandem array of discrete ligand-binding units, each of which may potentially interact with several different proteins during development.

Hepatic Ah Receptor from the Wistar Rat: Role of Solvation in Receptor Structure and Inactivation

Repeated freezing and thawing, the addition of salts, and elevated temperatures all promote the inactivation of the rat hepatic Ah receptor. The reduced availability of bulk water to solvate the protein is proposed to be the factor linking all these routes for inactivation. Prospective protocols for purification of unliganded Ah receptor should therefore minimize the number of freeze/thaw cycles; long-term freezing of cytosolic samples at -20 degrees C is inadequate to maintain long-term viability of the unliganded receptor. The stability of rat hepatic receptor is greatly increased upon binding the ligand, and the extent of ligand-induced stabilization is much greater than what is observed with steroid hormone receptors. Concentrations of NaCl and K2HPO4 up to 0.5 M inactivate the unbound Ah receptor irreversibly, with the loss of approximately 50% of the specific binding. At 2.0 M NaCl, a further reversible reduction in ligand binding activity is observed. The results at lower salt concentrations are interpreted in terms of the irreversible dissociation of a single binding unit from the trimeric cytosolic Ah receptor (which consists of two ligand-binding units and a 90-kDa heat shock protein), with the release of bound ligand from that subunit.

Oestrogen-receptors: an overview of recent advances in their structure and function

SynopsisRecent advances in our understanding of the receptor components involved in the initial events of oestradiol action are reviewed with an emphasis on aspects of clinical relevance. Two main features of the models have evolved: (1) the domain structure of the ligand-binding unit, with distinct parts of the molecule being concerned with oestrogen-binding and interaction with DNA, and other parts of the molecule having important regulatory functions; (2) the regulatory regions (oestrogen-response elements) of oestrogen-sensitive genes which specifically interact with the receptor protein.The homologies between different proteins capable of binding ligands as diverse as oestradiol, retinoic acid and thyroid hormone point to possible interactions between such ligands. Oestrogen-response elements can recognise oestradiol receptor proteins complexed with either agonists or antagonists. Interactions with DNA may play an important rôle in determining gene expression additional to those seen at the receptor level.

Purification of the D-2 dopamine receptor from bovine striatum

The D-2 dopamine receptor has been purified 21500 fold from bovine striatal membranes. Solubilized receptor preparation was partially purified by affinity chromatography on a haloperidol adsorbent followed by gel filtration on a Sephacryl S-300 column. The fractions eluted from this column which contained the ligand binding activity were further chromatographed on wheat germ agglutinin conjugated to Sepharose. The resulting receptor preparation displays a major polypeptide band of an apparent molecular weight of 92 kDa, and exhibits a specific binding activity of 2490 pmol spiperone per mg protein. This purified receptor preparation can reabsorb specifically to the haloperidol affinity column indicating that the 92 kDa polypeptide represents the ligand binding unit of the D-2 dopamine receptor.

Ca 2+-dependent proteolysis of the Ah receptor

We previously reported ( J. Biol. Chem. (1986) 261, 6352–6465) that the photoaffinity ligand for the Ah receptor, [ 125I]-2-azido-3-iodo-7,8-dibromodibenzo- p-dioxin, upon incubation with the liver cytosol fraction from C57BL/6 mice, labeled in a 1:1 ratio two peptides that had apparent molecular masses of 95 and 70 kDa and similar proteolytic fragmentation patterns. In the cytosolic fraction of Hepa 1 cells, a cloned murine hepatoma cell line, the product of photoaffinity labeling is almost exclusively a 95-kDa peptide which is rapidly hydrolyzed by a Ca 2+-dependent proteinase to a 70-kDa peptide as well as other fragments. Thus, the ligand binding unit of the Ah receptor in C57BL/6 mouse liver and Hepa 1 cell is a 95-kDa peptide, and the 70-kDa fragment is a proteolytic artifact. The Ca 2+-dependent proteinase which hydrolyzes the 95-kDa peptide has the properties of calpain II: (i) an absolute requirement for Ca 2+, with maximal activity at 0.5 to 1.0 m m Ca 2+; (ii) a pH optimum of 7.5 to 8.0; (iii) inhibition by EDTA, iodoacetamide, leupeptin and l- trans-epoxysuccinylleucylamido(4-guanidino)butane, but not by soybean trypsin inhibitor, aprotinin, or phenylmethanesufonyl fluoride. Upon Chromatographic separation of the liver cytosol of C57BL/6 mice on DEAE-Sephacel, Ca 2+-dependent proteinase activity (using casein or the labeled 95-kDa peptide as substrates) elutes with 0.25 m NaCl, and a specific proteinase inhibitor elutes with 0.15 m NaCl. Ca 2+-dependent proteinase activity that hydrolyzes the 95-kDa peptide is found in the liver cytosols of several mammalian species.

Structure and function of steroid receptors.

Hormone Biochemistry Department, Imperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, London wc2a 3px received 19 February 1987 Introduction Steroids combine with a cytosolic 8S, oligomeric receptor protein which, after activation, binds to nuclear chromatin and elicits a biological response. This statement in one form or another can be found in numerous articles and text books. However, the generation of antibodies against the ligand binding unit of several steroid receptors, together with the resultant identification of receptor mRNAs, cDNAs and genes has led to the questioning of some and the validation of other parts of that dogma. In this review, it will not be possible to survey each class of receptors separately and a more general approach will be adopted, although where differences exist they will be identified. An exhaustive assessment of the literature will not be attempted; references will be selected to illustrate the point under discussion.

Evidence for a diprotomeric structure of Na,K-ATPase: Accurate determination of protein concentration and quantitative end-group analysis

Three methods were used to assess protein concentration in membrane-bound Na,K-ATPase preparations: standard Lowry assay, Kjeldahl nitrogen determination and amino acid analysis. While the first two methods showed excellent agreement, the third one always gave a lower value which varied drastically depending on the condition of sample treatment before amino acid analysis. This result reinforces the Lowry method in assessing the true concentration of Na,K-ATPase protein and suggests 250 kDa to be a true estimate of the molecular mass of the smallest ligand-binding unit of the enzyme. The cyanate method reveals two NH 2-terminal residues of the β-subunit (NH 2-Ala) and one such residue of the α-subunit (NH 2-Gly) per ligand-binding unit. From the data on equimolarity of the α- and β-subunits in Na,K-ATPase this suggests that the enzyme molecule is composed of two αβ-protomers, one possessing a modified (presumably an N-blocked) α-subunit.

Dopamine Agonists in the Treatment of Schizophrenia

Behavioral, neurochemical, ligand binding, and single cell unit recording studies suggest the existence of two distinct classes of dopamine (DA) receptors: autoreceptors, which are present on the membranes of dopamine neurons, and postsynaptic receptors located on the neurons with which dopamine neurons synapse. Various pharmacological studies have suggested an overactivity of dopaminergic neurons in schizophrenic patients, however, studies on the concentrations of dopamine metabolites in cerebrospinal fluid (CSF) and post-mortem brain tissue, and clinical studies with schizophrenic patients who are free of drugs do not support this interpretation. Attention has therefore turned to the studies on the central dopamine receptors in schizophrenia. The use of binding techniques with tritiated ligands to study dopamine receptor density in post-mortem brain tissue has produced a number of interesting results. At present, it is accepted that the density of dopamine receptors in post-mortem brain tissue from schizophrenic patients is significantly increased in the striatum and in the nucleus accumbens.