Localization Of Binding Sites Research Articles

InCa-SiteFinder: A method for structure-based prediction of inositol and carbohydrate binding sites on proteins

Carbohydrate binding sites are considered important for cellular recognition and adhesion and are important targets for drug design. In this paper we present a new method called InCa-SiteFinder for predicting non-covalent inositol and carbohydrate binding sites on the surface of protein structures. It uses the van der Waals energy of a protein–probe interaction and amino acid propensities to locate and predict carbohydrate binding sites. The protein surface is searched for continuous volume envelopes that correspond to a favorable protein–probe interaction. These volumes are subsequently analyzed to demarcate regions of high cumulative propensity for binding a carbohydrate moiety based on calculated amino acid propensity scores. InCa-SiteFinder 1 1 Access to InCa-SiteFinder is freely available at: http://www.modelling.leeds.ac.uk/InCaSiteFinder/. was tested on an independent test set of 80 protein–ligand complexes. It efficiently identifies carbohydrate binding sites with high specificity and sensitivity. It was also tested on a second test set of 80 protein–ligand complexes containing 40 known carbohydrate binders (having 40 carbohydrate binding sites) and 40 known drug-like compound binders (having 58 known drug-like compound binding sites) for the prediction of the location of the carbohydrate binding sites and to distinguish these from the drug-like compound binding sites. At 73% sensitivity the method showed 98% specificity. Almost all of the carbohydrate and drug-like compound binding sites were correctly identified with an overall error rate of 12%.

Crystal Structure of the LG1-3 Region of the Laminin α2 Chain

Laminins are large heterotrimeric glycoproteins with many essential functions in basement membrane assembly and function. Cell adhesion to laminins is mediated by a tandem of five laminin G-like (LG) domains at the C terminus of the α chain. Integrin binding requires an intact LG1-3 region, as well as contributions from the coiled coil formed by the α, β, and γ chains. We have determined the crystal structure at 2.8-Å resolution of the LG1-3 region of the laminin α2 chain (α2LG1-3). The three LG domains adopt typical β-sandwich folds, with canonical calcium binding sites in LG1 and LG2. LG2 and LG3 interact through a substantial interface, but LG1 is completely dissociated from the LG2-3 pair. We suggest that the missing γ chain tail may be required to stabilize the interaction between LG1 and LG2-3 in the biologically active conformation. A global analysis of N-linked glycosylation sites shows that the β-sandwich faces of LG1 are free of carbohydrate modifications in all five laminin α chains, suggesting that these surfaces may harbor the integrin binding site. The α2LG1-3 structure provides the first atomic view of the integrin binding region of laminins.

Genome-Wide Identification of Direct Target Genes Implicates Estrogen-Related Receptor α as a Determinant of Breast Cancer Heterogeneity

Estrogen-related receptor alpha (ERRalpha) is an orphan nuclear receptor, the expression of which correlates with negative prognosis in breast cancer. ERRalpha shares functional features with the estrogen receptor alpha (ERalpha) and its activity is modulated by the ERBB2 signaling pathway. Using genome-wide binding sites location analyses in ERalpha-positive and ERalpha-negative breast cancer cell lines, we show that ERRalpha and ERalpha display strict binding site specificity and maintain independent mechanisms of transcriptional activation. Nonetheless, ERRalpha and ERalpha coregulate a small subset of common target genes via binding either to a dual-specificity binding site or to distinct cognate binding sites located within the extended promoter region of the gene. Although ERRalpha signaling in breast cancer cells is mostly independent of ERalpha, the small fraction of common ERRalpha/ERalpha targets comprises genes with high relevance to breast tumor biology, including genes located within the ERBB2 amplicon and GATA3. Finally, unsupervised hierarchical clustering based on the expression profiling of ERRalpha direct target genes in human breast tumors revealed four main clusters that recapitulate established tumor subtypes. Taken together, the identification and functional characterization of the ERRalpha transcriptional network implicate ERRalpha signaling as a determinant of breast cancer heterogeneity.

Molecular determinants of local anesthetic action of beta-blocking drugs: Implications for therapeutic management of long QT syndrome variant 3

The congenital long QT syndrome (LQTS) is a heritable arrhythmia in which mutations in genes coding for ion channels or ion channel associated proteins delay ventricular repolarization and place mutation carriers at risk for serious or fatal arrhythmias. Triggers and therapeutic management of LQTS arrhythmias have been shown to differ in a manner that depends strikingly on the gene that is mutated. Additionally, beta-blockers, effective in the management of LQT-1, have been thought to be potentially proarrhythmic in the treatment of LQT-3 because of concomitant slowing of heart rate that accompanies decreased adrenergic activity. Here we report that the beta-blocker propranolol interacts with wild type (WT) and LQT-3 mutant Na + channels in a manner that resembles the actions of local anesthetic drugs. We demonstrate that propranolol blocks Na + channels in a use-dependent manner; that propranolol efficacy is dependent on the inactivated state of the channel; that propranolol blocks late non-inactivating current more effectively than peak sodium current; and that mutation of the local anesthetic binding site greatly reduces the efficacy of propranolol block of peak and late Na + channel current. Furthermore our results indicate that this activity, like that of local anesthetic drugs, differs both with drug structure and the biophysical changes in Na + channel function caused by specific LQT-3 mutations.

Number and Locations of Agonist Binding Sites Required to Activate Homomeric Cys-Loop Receptors

Homo-pentameric Cys-loop receptors contain five identical agonist binding sites, each formed at a subunit interface. To determine the number and locations of binding sites required to generate a stable active state, we constructed a receptor subunit with a mutation that disables the agonist binding site and a reporter mutation that alters unitary conductance and coexpressed mutant and nonmutant subunits. Although receptors with a range of different subunit compositions are produced, patch-clamp recordings reveal that the amplitude of each single-channel opening event reports the number and, for certain subunit combinations, the locations of subunits with intact binding sites. We find that receptors with three binding sites at nonconsecutive subunit interfaces exhibit maximal mean channel open time, receptors with binding sites at three consecutive or two nonconsecutive interfaces exhibit intermediate open time, and receptors with binding sites at two consecutive or one interface exhibit brief open time. Macroscopic recordings after rapid application of agonist reveal that channel activation slows and the extent of desensitization decreases as the number of binding sites per receptor decreases. The overall results provide a framework for defining mechanisms of activation and drug modulation for homo-pentameric Cys-loop receptors.

Assay dependent activity of the sodium channel gating modifier protoxin‐I: implications for sodium channel drug discovery

Sodium channels (NaV1.7) are extremely attractive analgesic drug targets. The Holy Grail in NaV1.7 drug discovery (in‐vitro) is to discover selective NaV1.7 antagonists. Most compounds, if not all bind to a highly conserved, "local anesthetic binding site". Gating modifiers may exert selectivity due to interaction at less conserved allosteric sites. Therefore, we were interested to determine if several of the most commonly used sodium channel screening assays can identify such allosteric hits. Using protoxin‐I (ProTx‐I) we show the ability to detect gating modifiers is assay dependent. Using a voltage‐sensitive dye in FLIPR, ProTX‐I was unable to inhibit veratridine activation of NaV1.7 at concentrations up to 1 μM. Widely used pore blockers (amitriptyline, tetracaine) were effective in this assay format with a pIC50 of 6. Likewise, ProTX‐I was inactive in a radioligand‐binding assay using [3H]‐batrachotoxin as a tracer. In a VIPR FRET assay, ProTX‐1 caused only a small 'delay' in veratridine activation. In contrast , protoxin‐I was a potent blocker (pIC50 of 6.4) of the channel in two automated patch‐clamp assays (IonWorks Quattro and PatchXpress). These findings suggest that gating modulators may be missed in commonly used assays such as FLIPR that use an end‐point read. Carefully optimized VIPR assays or HTEP assays may be the preferred platform to identify sub‐type selective sodium channel blockers.

Location of the Antidepressant Binding Site in the Serotonin Transporter

The serotonin transporter (SERT) regulates extracellular levels of serotonin (5-hydroxytryptamine, 5HT) in the brain by transporting 5HT into neurons and glial cells. The human SERT (hSERT) is the primary target for drugs used in the treatment of emotional disorders, including depression. hSERT belongs to the solute carrier 6 family that includes a bacterial leucine transporter (LeuT), for which a high resolution crystal structure has become available. LeuT has proved to be an excellent model for human transporters and has advanced the understanding of solute carrier 6 transporter structure-function relationships. However, the precise structural mechanism by which antidepressants inhibit hSERT and the location of their binding pockets are still elusive. We have identified a residue (Ser-438) located within the 5HT-binding pocket in hSERT to be a critical determinant for the potency of several antidepressants, including the selective serotonin reuptake inhibitor citalopram and the tricyclic antidepressants imipramine, clomipramine, and amitriptyline. A conservative mutation of Ser-438 to threonine (S438T) selectively increased the K(i) values for these antidepressants up to 175-fold. The effects of introducing a protein methyl group into the 5HT-binding pocket by S438T were absent or reduced for analogs of these antidepressants lacking a single methyl group. This suggests that these antidepressants interact directly with Ser-438 during binding to hSERT, implying an overlapping localization of substrate- and inhibitor-binding sites in hSERT suggesting that antidepressants function by a mechanism that involves direct occlusion of the 5HT-binding site.

Lighting Up Individual DNA Binding Proteins with Quantum Dots

The ability to determine the precise loci and occupancy of DNA-binding proteins is instrumental to our understanding of cellular processes like gene expression and regulation. We propose a single-molecule approach for the direct visualization of proteins bound to their template DNA. Fluorescent quantum dots (QD) are used to label proteins bound to DNA, allowing multicolor, nanometer-resolution localization. Protein-DNA complexes are linearly extended and imaged to determine the precise location of the protein binding sites. The method is demonstrated by detecting individual QD-labeled T7-RNA polymerases on the T7 bacteriophage genome. This work demonstrates the potential of this approach to precisely read protein binding position or, alternatively, "write" such information on extended DNA with QDs via sequence-specific molecular recognition.

Studies of imipramine binding to human serum albumin by high-performance affinity chromatography

Binding by the drug imipramine to the protein human serum albumin (HSA) was studied by using high-performance affinity chromatography. The association equilibrium constants and number of binding sites for imipramine with HSA were first estimated by utilizing frontal analysis. Imipramine was found to have one major binding site on HSA with an association equilibrium constant of 1.6 × 10 5 M −1 at pH 7.4 and 37 °C, as well as a second group of weaker and non-specific binding regions (8–9 mol/mol HSA) with an average association equilibrium constant of 1.5 × 10 3 M −1. Competition studies based on zonal elution were performed to identify the location of the major binding site for imipramine on HSA. Imipramine was found to have direct competition with L-tryptophan, which indicated that imipramine was interacting with Sudlow site II, or the indole-benzodiazepine site of HSA. No competition or allosteric effects were noted between imipramine and warfarin, a probe for Sudlow site I or the warfarin-azapropazone site of HSA. The association equilibrium constant found for imipramine at its site of competition with L-tryptophan also agreed with the value that was obtained for the major binding site of imipramine in the frontal analysis studies. These results confirmed that Sudlow site II was the location of the major binding site for imipramine on HSA. These results gave good agreement with previous observations made in the literature and should provide a more detailed description of how imipramine is transported in blood and of how it may interact with other drugs in the body.

Molecular Determinants of Coupling Between the Domain III Voltage-sensor and Local Anesthetic Binding Site in the Skeletal Sodium Channel

Sodium channels are a major target for many toxins and drugs including local anesthetics (LA). Gating current (Sheets and Hanck, J. Gen. Physiol.; 121(2), 2003) and fluorescence measurements (Muroi and Chanda, Biophysical Society Meeting, 2008) show that LA binding to the pore mainly stabilizes the voltage-sensor of domains III of sodium channel in an activated conformation. The midpoints of the fluorescence-voltage (F-V) curves of probes attached to the voltage-sensor of domain III are left shifted by as much as 50 mV upon LA binding. How does the binding of LA to the pore affect the movement of the S4-domain III voltage-sensor? To identify the molecular determinants of interaction between the voltage-sensor and LA binding site, we systematically substituted tryptophan residues in the S4-S5 linker and S6 of domain III of skeletal Na+ channel and examined the effect of these substitutions on the movement of DIII-voltage sensor in the absence and presence of LA by voltage-clamp fluorometry technique. A number of mutations in the S4-S5 linker and S6 either showed a significantly diminished or no shift in the F-V curve upon LA binding, suggesting that those residues are involved in coupling the voltage sensor and LA binding site of the sodium channel.Support: National Institutes of Health, AHA and Shaw Scientific Award.

Electrical Fingerprinting Reveals Agonist Binding Sites Required for Activation of Homo-pentameric Cys-loop Receptors

Ancestral neurotransmitter Cys-loop receptors were homo-pentamers harboring five identical agonist binding sites but most present day receptors are hetero-pentamers with only two binding sites. To understand why Cys-loop receptors evolved to utilize fewer than five binding sites, we disabled different numbers of sites and developed a method to monitor lifetimes of individual active receptors and the corresponding number of functional binding sites. We find that maximal open-channel lifetime is achieved when the neurotransmitter occupies three non-consecutive binding sites. Occupancy of one site allows receptor activation, although the open state is unstable; occupancy of two non-consecutive sites produces a much longer-lived open state appropriate for efficient activation. However, occupancy of a third site further increases channel lifetime, thus providing optimal stabilization of the active state. Maximal activation of homomeric receptors by agonist occupancy of less than the five potential sites enhances the rate of channel opening and increases agonist sensitivity. The results reveal that allosteric requirements dictated the number and location of the agonist binding sites, and provide an indispensable framework for further progress in drug design.

Metal binding to alpha-synuclein peptides and its contribution to toxicity

Recent studies have suggested that alpha-synuclein (AS) is a metal binding protein. Metals also induce protein aggregation. In order to clarify controversy over the location of the metal binding sites six peptide fragments spanning the full length of the protein were analysed to identify metal binding domains. Our results indicated that both the C-terminus of the protein and a region around histidine 50 play a role in copper binding. We suggest that the true binding domain is a nonlinear site composed of both areas acting together to bind copper. The toxicity of these peptides to SH-SY5Y cells was also studied. There was a copper-independent component associated with the NAC domain of the protein and a copper-dependent component associated with the C-terminus of the protein and potentiated by involvement of the N-terminus. We hypothesise that copper binding can cause conversion of AS to a neurotoxic form via inter-protein interactions.

The Primed Ebolavirus Glycoprotein (19-Kilodalton GP 1,2 ): Sequence and Residues Critical for Host Cell Binding

Entry of ebolavirus (EBOV) into cells is mediated by its glycoprotein (GP(1,2)), a class I fusion protein whose structure was recently determined (J. E. Lee et al., Nature 454:177-182, 2008). Here we confirmed two major predictions of the structural analysis, namely, the residues in GP(1) and GP(2) that remain after GP(1,2) is proteolytically primed by endosomal cathepsins for fusion and residues in GP(1) that are critical for binding to host cells. Mass spectroscopic analysis indicated that primed GP(1,2) contains residues 33 to 190 of GP(1) and all residues of GP(2). The location of the receptor binding site was determined by a two-pronged approach. We identified a small receptor binding region (RBR), residues 90 to 149 of GP(1), by comparing the cell binding abilities of four RBR proteins produced in high yield. We characterized the binding properties of the optimal RBR (containing GP(1) residues 57 to 149) and then conducted a mutational analysis to identify critical binding residues. Substitutions at four lysines (K95, K114, K115, and K140) decreased binding and the ability of RBR proteins to inhibit GP(1,2)-mediated infection. K114, K115, and K140 lie in a small region modeled to be located on the top surface of the chalice following proteolytic priming; K95 lies deeper in the chalice bowl. Combined with those of Lee et al., our findings provide structural insight into how GP(1,2) is primed for fusion and define the core of the EBOV RBR (residues 90 to 149 of GP(1)) as a highly conserved region containing a two-stranded beta-sheet, the two intra-GP(1) disulfide bonds, and four critical Lys residues.

<em>In silico</em> modeling of toluene binding site in the pore of voltage-gate sodium channel

In silico modeling of toluene binding site in the pore of voltage-gate sodium channel Thomas RF Scior1, Evelyn Martínez-Morales2, Silvia L Cruz3, Eduardo M Salinas-Stefanon21Departamento de Farmacia; 2Instituto de Fisiología, Benemérita Universidad Autónoma de Puebla, Ciudad Universitaria, Puebla, Pue, México; 3Departamento de Farmacobiología, CINVESTAV-IPN, Calz. Col. Granjas Coapa, MéxicoAbstract: Toluene is a commonly used organic solvent in commercial products and is sometimes abused as an inhalative hallucinogen, causing arrythmogenic toxicity. At a molecular level we investigated whether a hypothetical interaction model could be devised for the reported myo- and cardiotoxic effects of toluene. Three lines of computed evidence support our hypothesis on the interaction mechanism: (i) Toluene binds at the local anesthetic binding site (LABS), on the wild type (WT) but not on its F1579A mutation, confirming our experimental findings that it inhibits only the WT of skeletal muscle or cardiac isoforms (Nav 1.4 or 1.5). (ii) Typically for small alkylaryl moiety, multiple binding modes were detected during docking. Toluene is trapped in the tryptophane-rich area at the extracellular vestibule by hydrophobic interaction, mainly π–π stacking, or bound to the LABS with equal binding strength and number of solved poses, mostly by edge-to-face contacts. (iii) The computed loss of toluene binding at the LABS on the mutant model parallels clearly the observed loss of toluene effects on Nav 1.4. Moreover, we inspected the complete primary sequences with the omitted loops in the 3D models to identify the possible interacting amino acids among the 16% nonidentical ones, and thus confirmed the observed toxicity effects.Keywords: Toluene, Na+ channel, Nav1.4 isoform, Nav1.5 isoform, in-silico simulation, cardiotoxicity

A comparative study of sodium channel inhibitor drugs: Distinct types of pH-dependence

Event Abstract Back to Event A comparative study of sodium channel inhibitor drugs: Distinct types of pH-dependence Árpád Mike1*, P. Lukacs1, Nóra Lenkey1, R. Karoly1 and Szilveszter E. Vizi1 1 Institute of Experimental Medicine, Hungary Clinically used sodium channel inhibitors (SCIs) are generally considered to cause use-dependent inhibition by binding to the (intracellularly accessible) local anesthetic binding site, and obstructing sodium ion conduction. However, inhibition of conduction is possible by mechanisms other than classic channel block, and evidence for alternative binding sites is accumulating. For most SCIs neither the mechanism of inhibition, nor the location of the binding site is clear thus far. Most SCI drugs contain an amine moiety, in which the positive charge is pH-dependent. Thus by manipulating the pH, the charge and hydrophobicity of drug molecules can be controlled, and information can be obtained regarding the chemical nature of the binding site and/or the access pathway to the binding site. In this study we investigated the pH-dependence of inhibition using 32 different SCI drugs, using rNav expressing HEK 293 cells, and the QPatch HT system (Sophion Bioscience, Denmark). Drugs included local anesthetics, anticonvulsants, antiarrhythmics, antidepressants, antipsychotics, antispastic agents, neuroprotectants, and drugs of other indications. Onset of inhibition as well as recovery from inhibition was studied at three different pH values: 6.0, 7.3, and 8.6. Most drugs showed a definite pH-dependence, being much more efficient at pH = 8.6. However, the pH-dependence of inhibition did not correlate with the pH-dependence of partition coefficient, suggesting that chemical interactions which are more complex than membrane permeation are involved. Clusters formed on the basis of pH dependence of inhibition, and of the pH dependent recovery rate from inhibition, were found to considerably overlap with therapeutic categories. Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Developmental neurobiology and subcortical functions Citation: Mike Á, Lukacs P, Lenkey N, Karoly R and Vizi SE (2009). A comparative study of sodium channel inhibitor drugs: Distinct types of pH-dependence. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.066 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 02 Mar 2009; Published Online: 02 Mar 2009. * Correspondence: Árpád Mike, Institute of Experimental Medicine, Budapest, Hungary, arpadmike1@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Árpád Mike P. Lukacs Nóra Lenkey R. Karoly Szilveszter E Vizi Google Árpád Mike P. Lukacs Nóra Lenkey R. Karoly Szilveszter E Vizi Google Scholar Árpád Mike P. Lukacs Nóra Lenkey R. Karoly Szilveszter E Vizi PubMed Árpád Mike P. Lukacs Nóra Lenkey R. Karoly Szilveszter E Vizi Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Differential Interactions of Thrombospondin-1, -2, and -4 with CD47 and Effects on cGMP Signaling and Ischemic Injury Responses

Thrombospondin-1 regulates nitric oxide (NO) signaling in vascular cells via CD47. Because CD47 binding motifs are conserved in the C-terminal signature domains of all five thrombospondins and indirect evidence has implied CD47 interactions with other family members, we compared activities of recombinant signature domains of thrombospondin-1, -2, and -4 to interact with CD47 and modulate cGMP signaling. Signature domains of thrombospondin-2 and -4 were less active than that of thrombospondin-1 for inhibiting binding of radiolabeled signature domain of thrombospondin-1 or SIRPalpha (signal-regulatory protein) to cells expressing CD47. Consistent with this binding selectivity, the signature domain of thrombospondin-1 was more potent than those of thrombospondin-2 or -4 for inhibiting NO-stimulated cGMP synthesis in vascular smooth muscle cells and downstream effects on cell adhesion. In contrast to thrombospondin-1- and CD47-null cells, primary vascular cells from thrombospondin-2-null mice lack enhanced basal and NO-stimulated cGMP signaling. Effects of endogenous thrombospondin-2 on NO/cGMP signaling could be detected only in thrombospondin-1-null cells. Furthermore, tissue survival of ischemic injury and acute recovery of blood flow in thrombospondin-2-nulls resembles that of wild type mice. Therefore, thrombospondin-1 is the dominant regulator of NO/cGMP signaling via CD47, and its limiting role in acute ischemic injury responses is not shared by thrombospondin-2.

Investigation of sodium channel inhibitor binding sites based on dissociation kinetics

Event Abstract Back to Event Investigation of sodium channel inhibitor binding sites based on dissociation kinetics Balázs Sas1*, Nóra Lenkey1, R. Karoly1, Szilveszter E. Vizi1 and A. Mike1 1 Institute of Experimental Medicine, Hungary Although most sodium channel inhibitor (SCI) drugs are considered to bind to the local anesthetic binding site (which is the only well characterized drug binding site of sodium channels), evidence for alternative binding sites is accumulating. Most importantly, SCI compounds are so heterogeneous chemically that it is very unlikely that a single binding site could suit all molecules. For the purpose of developing novel SCI drugs it would be essential to determine alternative binding sites, and to clarify inhibition mechanisms for different binding sites. We used an electrophysiology approach (whole cell patch clamp, using rNav1.2 channel expressing HEK cells) to investigate binding sites. We have observed that inhibition by specific SCIs differ in their recovery kinetics by several orders of magnitude. We tested if a specific slowly dissociating SCI can be displaced by applying high concentration of a rapidly dissociating SCI. Displacement could be detected by observing recovery kinetics. We found that flunarizine could not be displaced by lidocaine; therefore the binding site must be different. On the other hand we found interactions between crobenetine and fluoxetine, as well as between fluoxetine and lidocaine. Interactions may suggest either competition for the same binding sites, or an allosteric interaction between the two binding sites. Our results help to identify drug binding sites of sodium channels. Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Developmental neurobiology and subcortical functions Citation: Sas B, Lenkey N, Karoly R, Vizi SE and Mike A (2009). Investigation of sodium channel inhibitor binding sites based on dissociation kinetics. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.073 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 02 Mar 2009; Published Online: 02 Mar 2009. * Correspondence: Balázs Sas, Institute of Experimental Medicine, Budapest, Hungary, sas@koki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Balázs Sas Nóra Lenkey R. Karoly Szilveszter E Vizi A. Mike Google Balázs Sas Nóra Lenkey R. Karoly Szilveszter E Vizi A. Mike Google Scholar Balázs Sas Nóra Lenkey R. Karoly Szilveszter E Vizi A. Mike PubMed Balázs Sas Nóra Lenkey R. Karoly Szilveszter E Vizi A. Mike Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Specific binding sites for progesterone and 17beta-estradiol in cells of Triticum aestivum L.

The presence and location of specific binding sites for progesterone and 17beta-estradiol in cells of wheat were estimated using radioligand binding assay. Membrane and cytosolic fractions of non-vernalized and vernalized plants were tested using tritium-labelled ligands. Specific binding of [(3)H]progesterone and [(3)H]17beta-estradiol occurs in wheat cells. The binding sites are located in membranes and in the cytosol. Specific binding of [(3)H]17beta-estradiol is higher in the membranes than in the cytosol. Specific binding of both ligands in the cytosolic fraction is higher in vernalized plants than in non-vernalized ones. The possibility of the occurrence of steroid binding proteins specific for progesterone and 17beta-estradiol, putative steroid receptors for these steroids in Triticum aestivum L., is discussed.

Predicting small ligand binding sites in proteins using backbone structure

Motivation: Specific non-covalent binding of metal ions and ligands, such as nucleotides and cofactors, is essential for the function of many proteins. Computational methods are useful for predicting the location of such binding sites when experimental information is lacking. Methods that use structural information, when available, are particularly promising since they can potentially identify non-contiguous binding motifs that cannot be found using only the amino acid sequence. Furthermore, a prediction method that can utilize low-resolution models is advantageous because high-resolution structures are available for only a relatively small fraction of proteins.Results: SitePredict is a machine learning-based method for predicting binding sites in protein structures for specific metal ions or small molecules. The method uses Random Forest classifiers trained on diverse residue-based site properties including spatial clustering of residue types and evolutionary conservation. SitePredict was tested by cross-validation on a set of known binding sites for six different metal ions and five different small molecules in a non-redundant set of protein–ligand complex structures. The prediction performance was good for all ligands considered, as reflected by AUC values of at least 0.8. Furthermore, a more realistic test on unbound structures showed only a slight decrease in the accuracy. The properties that contribute the most to the prediction accuracy of each ligand were also examined. Finally, examples of predicted binding sites in homology models and uncharacterized proteins are discussed.Availability: Binding site prediction results for all PDB protein structures and human protein homology models are available at http://sitepredict.org/.Contact: bordner.andrew@mayo.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Cooperative Synergy between NFAT and MyoD Regulates Myogenin Expression and Myogenesis

Calcineurin/NFAT signaling is involved in multiple aspects of skeletal muscle development and disease. The myogenic basic helix-loop-helix transcription factors, MyoD, myogenin, Myf5, and MRF4 specify the myogenic lineage. Here we show that calcineurin/NFAT (nuclear factor of activated T cells) signaling is required for primary myogenesis by transcriptional cooperation with the basic helix-loop-helix transcription factor MyoD. Calcineurin/NFAT signaling is involved in myogenin expression in differentiating myoblasts, where the myogenic regulatory factor MyoD synergistically cooperates with NFATc2/c3 at the myogenin promoter. Using gel shift and chromatin immunoprecipitation assays, we identified two conserved NFAT binding sites in the myogenin promoter that were occupied by NFATc3 upon skeletal muscle differentiation. The transcriptional integration between NFATc3 and MyoD is crucial for primary myogenesis in vivo, as myogenin expression is weak in myod:nfatc3 double null embryos, whereas myogenin expression is unaffected in embryos with null mutations for either factor alone. Thus, the combined findings provide a novel transcriptional paradigm for the first steps of myogenesis, where a calcineurin/NFATc3 pathway regulates myogenin induction in cooperation with MyoD during myogenesis.