PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “ligand binding”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed2003

Full-length estrogen receptor alpha and its ligand-binding domain adopt different conformations upon binding ligand.

The binding of ligand to a nuclear receptor causes conformational changes that can result in coactivator or corepressor recruitment and subsequent regulation of transcription. Several peptides have previously been identified that bind to the liganded estrogen receptor (ER). One interacting peptide, pepalphaII, was used in the present studies to assess the ability of ligands to induce spatial changes within both the full-length human estrogen receptor alpha (ER-alpha) and a truncated receptor containing the ligand-binding domain (LBD). pepalphaII interacted weakly with the full-length estrogen receptor alpha in the presence of both agonists and antagonists. In contrast, the interaction of pepalphaII with the truncated receptor containing the ligand-binding domain was strongly induced by antagonists and only weakly induced by agonists. Thus, the same ligand can induce different spatial configurations of the full-length and ligand-binding domain of estrogen receptor alpha as measured by pepalphaII affinity. Crystal structures of nuclear hormone receptors solved to date have used ligand-binding domains and therefore may not accurately predict surface interaction domains present in the liganded full-length receptor. Furthermore, the ability of a ligand to induce a strong interaction of pepalphaII with the estrogen receptor alpha ligand-binding domain predicts that the ligand will have greater antagonist activity on the full-length receptor.

Animals↗
PubMed1976

Determination of ligand binding: partial and full saturation of aspartate transcarbamylase. Applicability of a filter assay to weakly binding ligands.

Carbamyl phosphate and succinate each bind to six sites in the hexameric aspartate transcarbamylase from Escherichia coli when both ligands are present in saturating concentrations. Their respective dissociation constants are 2.4 and 1400 muM. Positive homotropic interaction, shown earlier for the association of succinate with the enzyme in the presence of carbamyl phosphate (Changeux, J.-P., Gerhart, J.C., and Schachamn, H.K. (1968) Biochemistry 7, 513-538), is also found for carbamyl phosphate binding in the presence of succinate. Apparent half-of-the-sites saturation, previously described for carbamyl phosphate binding in the absence of succinate (Rosenbusch, J.P., and Griffin, J.H. (1973) J. Biol, Chem. 248, 5063-5066), also occurs when succinate binds to the enzyme in the absence of carbamyl phosphate. A second class of three low affinity sites for carbamyl phosphate could be detected in the enzyme when succinate was absent. These results indicate that aspartate transcarbamylase exists in an asymmetric state under several defined conditions. The results reported were obtained with a highly sensitive filter bonding assay, modified to allow the study of the protein-ligand interactions with dissociation constants in the millimolar range. The assay is described in detail. Its validity is demonstrated by the good correlation of the results obtained with those observed with independent methods.

Binding Sites↗
PubMed2001

Competition of tamoxifen with thyroxine for TBG binding: ligand binding assay and computational data.

OBJECTIVE: Tamoxifen, a nonesteroidal antiesterogen, is widely used in the treatment of breast cancer. Recently, the effect of tamoxifen on thyroid function has caused considerable concern, yet the results of different studies are controversial and the precise mechanism of such influence is obscure. In view of the fact that some drugs such as furosemide, diclofenac and mefenamic acid, based on the structural similarities to thyroxine could compete for binding to thyroxine binding globulin (TBG) and appears that there are some structural similarities between tamoxifen and thyroxine, one can hypothesize that tamoxifen is also able to compete for TBG binding and thereby affecting thyroid function tests. DESIGN AND METHODS: In this study, we designed an in vitro binding assay as well as computational methods using MOPAC 7 package for evaluation of competitive potency of tamoxifen for TBG binding in comparison with well-known TBG competitors (including furosemide, mefenamic acid and diclofenac). RESULTS: The result of competition assay and Scatchard analysis revealed that tamoxifen does not bind to TBG at the T4 binding site, thus it is not a thyroxine competitor. Computational results also indicated that structural characteristics of tamoxifen are significantly different from those of T4 and its well-known competitors. CONCLUSION: In conclusion, the probability of competition between tamoxifen and T4 is ruled out by these results.

Binding, Competitive↗
PubMed2000

Side-chain flexibility in proteins upon ligand binding.

Ligand binding may involve a wide range of structural changes in the receptor protein, from hinge movement of entire domains to small side-chain rearrangements in the binding pocket residues. The analysis of side chain flexibility gives insights valuable to improve docking algorithms and can provide an index of amino-acid side-chain flexibility potentially useful in molecular biology and protein engineering studies. In this study we analyzed side-chain rearrangements upon ligand binding. We constructed two non-redundant databases (980 and 353 entries) of "paired" protein structures in complexed (holo-protein) and uncomplexed (apo-protein) forms from the PDB macromolecular structural database. The number and identity of binding pocket residues that undergo side-chain conformational changes were determined. We show that, in general, only a small number of residues in the pocket undergo such changes (e.g., approximately 85% of cases show changes in three residues or less). The flexibility scale has the following order: Lys > Arg, Gln, Met > Glu, Ile, Leu > Asn, Thr, Val, Tyr, Ser, His, Asp > Cys, Trp, Phe; thus, Lys side chains in binding pockets flex 25 times more often then do the Phe side chains. Normalizing for the number of flexible dihedral bonds in each amino acid attenuates the scale somewhat, however, the clear trend of large, polar amino acids being more flexible in the pocket than aromatic ones remains. We found no correlation between backbone movement of a residue upon ligand binding and the flexibility of its side chain. These results are relevant to 1. Reduction of search space in docking algorithms by inclusion of side-chain flexibility for a limited number of binding pocket residues; and 2. Utilization of the amino acid flexibility scale in protein engineering studies to alter the flexibility of binding pockets.

Amino Acids↗
PubMed2006

Molecular mechanics methods for predicting protein-ligand binding.

Ligand binding affinity prediction is one of the most important applications of computational chemistry. However, accurately ranking compounds with respect to their estimated binding affinities to a biomolecular target remains highly challenging. We provide an overview of recent work using molecular mechanics energy functions to address this challenge. We briefly review methods that use molecular dynamics and Monte Carlo simulations to predict absolute and relative ligand binding free energies, as well as our own work in which we have developed a physics-based scoring method that can be applied to hundreds of thousands of compounds by invoking a number of simplifying approximations. In our previous studies, we have demonstrated that our scoring method is a promising approach for improving the discrimination between ligands that are known to bind and those that are presumed not to, in virtual screening of large compound databases. In new results presented here, we explore several improvements to our computational method including modifying the dielectric constant used for the protein and ligand interiors, and empirically scaling energy terms to compensate for deficiencies in the energy model. Future directions for further improving our physics-based scoring method are also discussed.

Algorithms↗
PubMed2004

Following ligand binding and ligand reactions in proteins via Raman crystallography.

Raman crystallography permits the monitoring of chemical events in single-protein crystals in real time. Using a Raman microscope, it is possible to obtain protein Raman spectroscopic data of unprecedented quality and stability. The latter features allow us to obtain the Raman spectrum for small molecules soaking into crystals under normal (nonresonance) Raman conditions. Thus, via an approach utilizing Raman difference spectroscopy, we can quantitate the amount of ligand in the crystal, determine the chemistry of inhibitor-protein interactions, and follow chemical reactions in the active site on the time scale of minutes. While providing unique chemical insights, these data also provide an invaluable guide for determining the conditions for flash-freezing crystals for X-ray crystallographic analysis. In addition, the Raman difference spectra often contain contributions from protein modes due to protein conformational changes occurring upon ligand binding. These features allow us to probe events ranging from small cooperative conformational changes to massive and unexpected secondary structure changes in the crystal. An experimental advantage of Raman crystallography is that the data can be collected from crystals in situ, in sitting or hanging drops, under the conditions used to grow the crystals.

Ligands↗
PubMed1997

Interaction of minor groove binding ligands with long AT tracts.

We have used quantitative DNase I footprinting to examine the ability of distamycin and Hoechst 33258 to discriminate between different arrangements of AT residues, using synthetic DNA fragments containing multiple blocks of (A/T)6or (A/T)10in identical sequence environments. Previous studies have shown that these ligands bind less well to (A/T)4sites containing TpA steps. We find that in (A/T)6tracts distamycin shows little discrimination between the various sites, binding approximately 2-fold stronger to TAATTA than (TA)3, T3A3and GAATTC. In contrast, Hoechst 33258 binds approximately 20-fold more tightly to GAATTC and TAATTA than T3A3and (TA)3. Hydroxyl radical footprinting reveals that both ligands bind in similar locations at the centre of each AT tract. At (A/T)10sites distamycin binds with similar affinity to T5A5, (TA)5and AATT, though bands in the centre of (TA)5are protected at approximately 50-fold lower concentration than those towards the edges. Hoechst 33258 shows a similar pattern of preference, with strong binding to AATT, T5A5and the centre of (TA)5. Hydroxyl radical footprinting reveals that at low concentrations both ligands bind at the centre of (TA)5and A5T5, while at higher concentrations ligand molecules bind to each end of the (A/T)10tracts. At T5A5two ligand molecules bind at either end of the site, even at the lowest ligand concentration, consistent with the suggestion that these compounds avoid the TpA step. Similar DNase I footprinting experiments with a DNA fragment containing T n (n = 3-6) tracts reveals that both ligands bind in the order T3< T4 << T5 = T6.

Binding Sites↗
PubMed1987

Disparity between expression of transferrin receptor ligand binding and non-ligand binding domains on human lymphocytes.

We compared transferrin receptor (TfR) expression on human peripheral blood lymphocytes (PBL) activated by phorbol myristate acetate (PMA) or L-phytohemagglutinin (LPHA) using two techniques: (1) 125I-iron-saturated transferrin (FeTf) binding, (2) reactivity with monoclonal anti-TfR antibodies--OKT9 and B3/25. These monoclonal antibodies do not block FeTf binding, and therefore bind to TfR domains separate from the ligand binding site. Unstimulated PBL bound fewer than 1,000 molecules of 125I-FeTf per cell, and less than 5% of cells expressed TfR antigens detected by OKT9 or B3/25. 125I-FeTf binding and antibody binding increased in parallel on LPHA-activated PBL. After exposure to LPHA for 72 hr, 125I-FeTf binding increased 100-fold to 10(5) molecules per cell and greater than 50% of cells expressed TfR antigens. By contrast, PMA activation of PBL markedly increased binding of OKT9 and B3/25 but not the binding of 125I-FeTf. Cell surface expression of TfR antigens seen by OKT9 and B3/25 did not differ between LPHA- and PMA-activated PBL. However, after 72 hr with PMA, 125I-FeTf binding increased only 6-fold and consistently remained at less than 10(4) molecules per cell. Therefore, PMA induced a disparity between expression of TfR ligand binding domains and immunological domains at the cell surface. Cell proliferation assessed by fluorescent DNA analysis was similar in cultures stimulated by LPHA or PMA. These data indicate that lymphoid cells may possess a mechanism for modulating TfR expression in which down-regulation of FeTf binding occurs without receptor internalization. Alternatively, it is possible that this observation may reflect a membrane perturbation effect of PMA.

Antibodies, Monoclonal↗
PubMed1997

The lysine cluster in the collagen-like domain of the scavenger receptor provides for its ligand binding and ligand specificity.

Scavenger receptors bind modified low-density lipoproteins (LDL) on a collagen-like domain which possesses a lysine cluster at the carboxy end. We previously constructed a receptor model peptide containing the lysine cluster. In the present study, we evaluated the ligand specificity of the receptor model peptide. It selectively bound modified-LDLs, and not LDL. The binding of acetylated-LDL (Ac-LDL) was inhibited by dextran sulfate, fucoidan, and sulfatides in a manner similar to that of the natural receptor. Both polyguanylic and polyinosinic acids inhibit the Ac-LDL binding whereas polycytidylic acid did not. These results indicate that the lysine cluster in the collagen-like domain has important roles in both ligand binding and ligand specificity.

Amino Acid Sequence↗
PubMed1997

Identification of a region in the integrin beta3 subunit that confers ligand binding specificity.

Many integrin adhesion receptors bind ligands containing the Arg-Gly-Asp (RGD) peptide motif. Most integrins exhibit considerable specificity for particular ligands and can distinguish among the many conformations of RGD. In this study we identify the domain of the integrin beta subunit involved in determining ligand binding specificity. Chimeras of beta3 and beta5, the most homologous integrin beta subunits, were expressed with alphav on the surface of human 293 cells. The ligand binding phenotype of each chimera was assessed using the ligands Fab-9 and fibrinogen, both of which have a binding preference for alphavbeta3. The results of the study show that when exons C and D of the beta3 subunit (residues 95-233) are substituted into beta5, the chimera gained the ability to bind Fab-9 with an affinity close to that of wild-type alphavbeta3. This chimera was able to mediate cell adhesion to fibrinogen. Furthermore, the swap of only a 39-residue segment of this larger domain, beta3 residues 164-202, into the backbone of beta5 enabled the chimeric integrin to bind soluble Fab-9. This small domain is highly divergent among the integrin beta subunits, suggesting that it may play a role in determining ligand selection by all integrins.

Amino Acid Sequence↗
PubMed1999

Substitution of a conserved amino acid residue alters the ligand binding properties of peroxisome proliferator activated receptors.

Mutation of glutamic acid 282 of PPARalpha to glycine has been shown to result in an increased EC(50) for a wide variety of PPAR activating compounds. This has suggested that mutant receptor has a reduced ability to bind ligand. In this study we show that this mutation reduces the affinity of mPPARalpha and hPPARgamma for the fluorescent fatty acid, cis-parinaric acid and that the mutant hPPARgamma protein has a reduced affinity for the radiolabelled compound, SB236636. These data confirm the role of this glutamic acid in ligand binding and support recent crystal structure observations regarding a proposed novel mode of ligand entry into the PPAR ligand binding cavities.

Amino Acid Sequence↗
PubMed1984

Identification of copper- and zinc-binding ligands in human and bovine milk.

The object of this study was to identify copper and zinc ligands detected during modified gel chromatography ( MGC ) of bovine and human milk ultrafiltrates. Isolation by anion-exchange chromatography and subsequent proton nuclear magnetic resonance (NMR) spectroscopy analysis demonstrated that the sole major low-molecular-weight ligand binding copper and zinc in bovine milk is citrate. Human milk apparently also contains citrate as a major metal-binding ligand but also contains amino acids, of which primarily glutamate was purified by anion-exchange chromatography. The amino acids bind copper well but are weak zinc-binding ligands. An artifactual MGC peak was seen in the milks, which was shown to be caused by the calcium present in the milk samples. No major differences in zinc-binding capacity were demonstrated between the low-molecular-weight fractions of the two milks. Although citrate may play a role in zinc uptake, it is apparently not the difference between the milks crucial to the acrodermatitis enteropathica individual. The difference in zinc availability between the milks may lie in some other aspect such as binding by proteins, which were noted to bind metal during MGC of nonultrafiltered milks.

Amino Acids↗
PubMed1993

Xenopus muscle acetylcholine receptor alpha subunits bind ligands with different affinities.

The acetylcholine receptor (AChR) from vertebrate skeletal muscle is a pentameric complex composed of two identical alpha (the ligand-binding subunit) and one each of beta, gamma, and delta subunits. All muscle-like alpha subunits appear to be encoded by a single gene except for Xenopus where two genes have been identified (X alpha 1a and X alpha 1b). By establishing stable cell lines expressing each Xenopus alpha subunit plus Torpedo beta gamma delta (XaT and XbT), we were able to investigate immunological and ligand-binding properties of these two Xenopus subunits. We determined that each subunit was capable of proper assembly with beta gamma delta subunits, each hybrid AChR cell line responded to bath-applied acetylcholine as determined by 22Na+ influx, but the two hybrid AChRs had distinct ligand-binding properties. The most striking difference between the two hybrids was observed with alpha-bungarotoxin (BuTx) binding. The KD for BuTx binding to XbT hybrids was virtually identical to that of Torpedo and most other muscle AChRs, approximately 10(-10)-10(-11) M. The KD for BuTx binding to XaT hybrids, however, was approximately 1000-fold weaker (6 x 10(-8) M). This weak binding was due primarily to a very rapid BuTx off-rate (4.4 x 10(-2) s-1 for XaT AChRs compared with approximately 1.6 x 10(-5) s-1 for all-Torpedo and XbT AChRs).

3T3 Cells↗
PubMed1997

Glycosylation of asparagine 24 of the natriuretic peptide receptor-B is crucial for the formation of a competent ligand binding domain.

UV cross-linking studies of the natriuretic peptide receptor-B (NPR-B) using radiolabeled C-type natriuretic peptide (CNP) indicate that only fully glycosylated receptors are capable of binding ligand. We therefore used site-directed mutagenesis to determine which potential glycosylation sites are occupied by carbohydrate, and the relevant mutants were characterized in order to understand the function of carbohydrate addition at those sites. Our results suggest that five of seven potential N-linked glycosylation sites are modified. In addition, mutation of asparagine 24 results in a loss of approximately 90% of receptor activity. This mutant is expressed at levels comparable to the wild-type receptor, and its activity is not significantly different from that of wild-type NPR-B in terms of EC50 for CNP. Ligand binding studies on this mutant further show that although there is no change in affinity for ligand, approximately 90% of receptor binding is lost. These data suggest that many of the mutant receptors are simply not properly folded. Our results indicate that glycosylation of asparagine 24 of NPR-B receptors may be critical for the formation of a competent ligand binding domain.

Amino Acid Sequence↗
PubMed1997

The glycosaminoglycan binding site governs ligand binding to the somatomedin B domain of vitronectin.

The ligand binding functions of vitronectin (Vn) are regulated by its conformational state/degree of multimerization. In the native plasma form of Vn, the C-terminal glycosaminoglycan (GAG) binding domain is believed to be cryptic. Here, evidence is provided that the addition of fucoidan or dextran sulfate to unfractionated plasma results in the formation of covalently and non-covalently stabilized Vn multimers. These multimers express conformationally sensitive antibody epitopes and ligand binding sites located in the N terminus of the Vn molecule. While heparin forms complexes with monomeric plasma Vn and induces conformational changes, a reduction in ionic strength is required for induction of multimerization. In addition, heparin serves as a template for the assembly of type 1 plasminogen activator inhibitor-induced disulfide-linked Vn multimers. These results support a new model for the structure of native Vn. The C-terminal GAG binding domain is predicted to be exposed in the native conformation, whereas the N terminus is cryptic. Ligand binding to the GAG binding site unfolds the N terminus, thereby exposing cryptic ligand binding sites.

Anticoagulants↗
PubMed2004

CADASIL-associated Notch3 mutations have differential effects both on ligand binding and ligand-induced Notch3 receptor signaling through RBP-Jk.

Mutations in the NOTCH3 gene are the cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary angiopathy leading to strokes and dementia. Pathogenic mutations remove or insert cysteine residues within epidermal growth factor (EGF) repeats in the extracellular domain of the Notch3 receptor (N3ECD). Vascular smooth muscle cells (VSMC) are the predominant site of Notch3 expression in adults. In CADASIL patients, VSMC degenerate and N3ECD is deposited within the vasculature. However, the mechanisms underlying VSMC degeneration and N3ECD accumulation are still unknown. In this study, we investigated the consequences of three pathogenic Notch3 mutations on the biological activity of the receptor by analyzing ligand (Delta-/Jagged-)-induced signaling via RBP-Jk. Two mutations (R133C and C183R) that are located outside the putative ligand binding domain (LBD) of the receptor were found to result in normal Jagged1-induced signaling in A7r5 VSMC, whereas the third mutation (C455R located within the putative LBD) showed strongly reduced signaling activity. Ligand binding assays with soluble Delta1 and Jagged1 revealed that C455R interferes with ligand binding through disruption of the LBD which, as we show here, is located in EGF repeats 10/11 of Notch3. All mutant receptors including Notch3C455R were targeted to the cell surface but showed an elevated ratio between the unprocessed full-length 280-kDa receptor and S1-cleaved receptor fragments. Taken together, these data indicate that CADASIL-associated Notch3 mutations differ with respect to their consequences both on ligand binding and ligand-induced signaling through RBP-Jk, whereas they have similar effects on receptor maturation. Moreover, the data suggest that ligand-induced receptor shedding may not be required for N3ECD deposition in CADASIL.

Adult↗
PubMed2003

Pausing of DNA polymerases on duplex DNA templates due to ligand binding in vitro.

Using the recently developed peptide nucleic acid (PNA)-assisted assay, which makes it possible to extend a primer on duplex DNA, we study the sequence-specific inhibition of the DNA polymerase movement along double-stranded DNA templates imposed by DNA-binding ligands. To this end, a plasmid vector has been prepared featuring the polylinker with two flanking priming sites to bi-directionally initiate the primer-extension reactions towards each other. Within this plasmid, we have cloned a set of random DNA sequences and analyzed the products of these reactions with several phage and bacterial DNA polymerases capable of strand-displacement synthesis. Two of them, ø29 and modified T7 (Sequenase 2.0) enzymes, were found to be most potent for primer extension in the presence of DNA-binding ligands. We used these enzymes for a detailed study of ligand-induced pausing effects with four ligands differing in modes of binding to the DNA double-helix. GC-specific intercalator actinomycin D and three minor groove-binders, chromomycin A(3) (GC-specific), distamycin A and netropsin (both AT-specific), have been chosen. In the presence of each ligand both selected DNA polymerases experienced multiple clear-cut pauses. Each ligand yielded its own characteristic pausing pattern for a particular DNA sequence. The majority of pausing sites could be located with a single-nucleotide resolution and corresponded to the preferred binding sites known from the literature for the ligands under study. Besides, DNA polymerases stalled exactly at the positions occupied by PNA oligomers that were employed to initiate the primer extension. These findings provide an important insight into the DNA polymerase performance. In addition, the high-resolution ligand-induced pausing patterns we obtained for the first time for DNA polymerase elongation on duplex DNA may become a valuable addition to the existing arsenal of methods used to monitor duplex DNA interactions with various DNA-binding ligands, including drugs.

Base Sequence↗