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At least 37 records · Page 2Linked to original sources
PubMed1991

Nature of protamine-DNA complexes. A special type of ligand binding co-operativity.

The mode of protamine binding to DNA double helices has been analyzed for the example of clupein Z from herring and DNA samples from bacteriophages lambda and PM2 by measurements of light-scattering intensities, ultracentrifugation and kinetics. The light-scattering intensity of DNA increases co-operatively at a threshold clupein concentration suggesting co-operative binding of clupein to double helices. These data are first analyzed in terms of a model with a transition at a threshold degree of binding. The parameters resulting from this analysis appear to be reasonable, but are shown to be in contrast with data on the absolute degree of clupein binding to DNA obtained by centrifugation experiments. An analysis of the kinetics associated with clupein binding to DNA by measurements of the time-dependence of light-scattering intensities in the time range of seconds demonstrates directly that clupein-induced intermolecular interactions of DNA molecules are essential. The rate constants of DNA association increase co-operatively at threshold clupein concentrations, which correspond to those observed in the equilibrium titrations. Above the threshold, the rate constants arrive at a level that is almost constant, but shows some decrease with increasing clupein concentrations. These results are described by a model with a monomer and a dimer state of DNA, which bind ligands with different affinities according to an excluded-site binding scheme. When the ligand binding constant is larger for the dimer than for the monomer state, as should be expected, binding of ligands drives the DNA from the monomer to the dimer state, even if the dimerization equilibrium in the absence of ligands is far in favor of the monomer. The transition from the monomer to the dimer state proves to be strongly co-operative. When the ligand concentration is increased to higher values, the dimers may be converted back to monomers due to an increased extent of ligand binding to the monomer state. The model is consistent with the available experimental data. The analysis of the data by the model indicates the existence of a reaction unit much below the DNA chain length, corresponding to about 80 nucleotide residues. The present model describes ligand driven intermolecular association; an analogous model is applicable to ligand driven intramolecular association. In summary, the co-operativity of clupein binding to DNA double helices is not due to nearest neighbor interactions, but results from thermodynamic coupling of clupein binding with clupein-induced DNA association.

Bacteriophages↗
PubMed2004

Hydrogen exchange and ligand binding: ligand-dependent and ligand-independent protection in the Src SH3 domain.

Amide hydrogen-deuterium exchange has proven to be a powerful tool for detecting and characterizing high-energy conformations in protein ensembles. Since interactions with ligands can modulate these high-energy conformations, hydrogen exchange appears to be an ideal experimental probe of the physical mechanisms underlying processes like allosteric regulation. The chemical mechanism of hydrogen exchange, however, can complicate such studies. Here, we examine hydrogen exchange rates in a simple model system, the c-Src SH3 domain interacting with a short peptide ligand. Addition of ligand slows the rates of hydrogen exchange at nearly every amide for which we can obtain data. Careful analysis, however, reveals that this slowing is due primarily to a reduction in the population of free protein in the system, and not to any specific property of the complex. We present a method to separate the contributions of free and bound protein to the exchange kinetics that has allowed us to identify the subset of amides where exchange arises directly from the complex. These results demonstrate that the slowing of hydrogen exchange induced by ligand interactions should be interpreted with caution, and more extensive experiments are required to correlate changes in hydrogen exchange with changes in structure or internal dynamics.

Animals↗
PubMed1997

Overexpression, purification, and use of a soluble human interleukin-4 receptor alpha-chain/Ig gamma 1 fusion protein for ligand binding studies. Characterization of ligand binding to soluble IL-4 receptor alpha-chain by surface plasmon resonance measurements and by microtiter-plate-based ELISA with biotinylated IL-4.

The pleiotropic cytokine IL-4 transmits cellular signals mainly via the IL-4 receptor complex, with the alpha-chain as the high affinity binding subunit. Here we describe the overexpression of a soluble IL-4R alpha-chain (sIL-4R) as a fusion to immunoglobulin gamma 1 heavy chain, consisting of the H-CH2-CH3 domains, in baby hamster kidney cells. The dimeric fusion protein named sIL-4R:E gamma 1 was purified from culture supernatant by protein-A affinity chromatography, yielding up to 10 mg/l homogenous protein which was highly stable. The antibody-like features of the sIL-4R:E gamma 1 fusion protein allowed immobilization on a biosensor matrix for surface plasmon resonance measurements by direct amine coupling as well as immobilization on microtiter plates coated with protein A for displacement binding. Kinetic parameters (kon and koff) for binding of IL-4 or the antagonistic mutant IL-4(Y124D) to the sIL-4R:E gamma 1 fusion protein on the chip as determined with the BIAcore instrument showed a high affinity binding with KD = 239 +/- 35 pM and KD=148 +/- 33 pM, respectively. The extremely high kon rate and the relatively slow koff rate for both ligands highlighted the limits of the BIAcore technology. The binding affinity as calculated in displacement binding studies with biotinylated IL-4 was similar for IL-4 and IL-4(Y124D) (IC50=1.1nM), thus offering a simple alternative for initial characterization of IL-4 mutants.

Animals↗
PubMed2002

Investigation of the PDZ domain ligand binding site using chemically modified peptides.

Several chemically modified analogues to a tightly binding ligand for the second PDZ domain of MAGI-3 were synthesized and evaluated for their ability to compete with native peptide ligands. N-methyl scanning of the ligand backbone amides revealed the energetically important hydrogen bonds between the ligand backbone and the PDZ domain. Analogues to the ligand's conserved threonine/serine(-2) residue, involved in a side chain to side chain hydrogen bond with a conserved histidine in the PDZ domain, revealed that the interaction is highly sensitive to the steric structure around the hydroxyl group of this residue. Analogues of the ligand carboxy terminus revealed that the full hydrogen bond network of the GLGF loop is important in ligand binding.

Amino Acid Sequence↗
PubMed2000

Structure-function analysis of the Rev-erbA and RVR ligand-binding domains reveals a large hydrophobic surface that mediates corepressor binding and a ligand cavity occupied by side chains.

Rev-erbA/RVR are closely related orphan nuclear receptors (NRs) functioning as dominant transcriptional silencers through an association with the nuclear receptor corepressor N-CoR. In contrast with ligand-regulated NRs, Rev-erbA/RVR lack the ligand-binding domain (LBD) C-terminal activation helix, H12. In the case of retinoid acid receptor and thyroid hormone receptor, ligand binding is thought to reposition H12, causing corepressor dissociation and coactivator recruitment, thus leading to transcriptional activation. Here we present homology models of the Rev-erbA/RVR LBDs, which show that the putative ligand cavity is occupied by side chains, suggesting the absence of endogenous ligands. Modeling also revealed a very hydrophobic surface due to the absence of H12, exposing residues from H3, loop 3-4, H4, and H11. Mutation of specific residues from this surface severely impaired the in vitro and in vivo interaction of the Rev-erbA/RVR LBD with the receptor-interacting domain of the corepressors N-CoR or its splice variant RIP13delta1, reinforcing the view of the physical association of N-CoR with a LBD surface encompassing H3-H4 and H11. Furthermore, mutations in the LBD surface significantly reduced the ability of Rev-erbA and RVR to function as repressors of transcription. Interestingly, a hydrophobic surface comprised of H3-H4 and H12 in liganded NRs mediates the interaction with coactivators. Hence, it appears that corepressors and coactivators bind to overlapping surfaces of NR LBDs, the conformational change associated with H12 upon ligand binding resulting in a switch from a corepressor- to a coactivator-binding surface.

Amino Acid Sequence↗
PubMed1989

A monoclonal antibody against the ligand binding site of the receptor for mouse interferon-gamma.

A rat monoclonal IgG2a antibody, GR-20, has been produced against the receptor for mouse interferon-gamma (MuIFN-gamma). Comparison of competitive binding studies performed with either 125I-labeled GR-20 or recombinant (r) MuIFN-gamma, as well as a variety of other studies, suggested that the epitope recognized by the monoclonal antibody (mAb) is in the domain of the receptor that binds ligand. The binding of GR-20 to cells of the monomyelocytic line WEHI-3 was of high affinity (1-2 nM). Approximately 20,000 binding sites were found per cell, a value that is in close agreement with the number of MuIFN-gamma receptors quantified on cells of the same type by ligand binding studies. The mAb also bound to a variety of other mouse cells, suggesting that the same epitope is shared by receptors for MuIFN-gamma, regardless of cell type. The epitope was not detected on two human cell types that were tested, while cells of a rat cell line shown to be minimally responsive to rMuIFN-gamma gave equivocal binding results when they were interacted with GR-20. Binding of the mAb to the receptor did not mimic the effects of ligand. In fact, the opposite was true: binding blocked the induction of three biological effects of MuIFN-gamma, including priming of macrophages for tumor cell killing, upregulation of the expression of class II major histocompatibility antigens (Ia) on the same cell type, and induction of antiviral activity in L cells. Following intravenous injection, initial removal of GR-20 was precipitous, followed after 1 h by a phase which was more gradual, resulting in 5-10% of biologically active mAb remaining in the circulation after 24 h. Such retention should make this mAb useful in a variety of studies in vivo.

Animals↗
PubMed2002

CADASIL Notch3 mutant proteins localize to the cell surface and bind ligand.

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a vascular dementia arising from abnormal arteriolar vascular smooth muscle cells. CADASIL results from mutations in Notch3 that alter the number of cysteine residues in the extracellular epidermal growth factor-like repeats, important for ligand binding. It is not known whether CADASIL mutations lead to loss or gain of Notch3 receptor function. To examine the functional consequences of CADASIL mutations, we engineered 4 CADASIL-like mutations into rat Notch3 and have shown that the presence of an unpaired cysteine does not impair cell-surface expression or ligand binding.

Amino Acid Motifs↗
PubMed1999

Identification of amino acid residues that determine pH dependence of ligand binding to the asialoglycoprotein receptor during endocytosis.

The rat hepatic asialoglycoprotein receptor mediates clearance of galactose- and N-acetylgalactosamine-terminated glycoproteins by endocytosis, binding ligands through a C-type, Ca(2+)-dependent carbohydrate-recognition domain (CRD) at extracellular pH and releasing them at lower pH in endosomes. At physiological Ca(2+) concentrations, the midpoint for ligand release from the CRD of the major subunit of the receptor is pH 7.1. In contrast, the midpoint is pH 5.0 for a galactose-binding derivative of the homologous C-type CRD of serum mannose-binding protein, which would thus not efficiently release ligand at an endosomal pH of 5.4. Site-directed mutagenesis of the CRD from the major subunit of the asialoglycoprotein receptor has been used to identify residues that are essential for efficient release of ligand at endosomal pH. The effects of changes to residues His(256), Asp(266), and Arg(270) singly and in combination indicate that these residues reduce the affinity of the CRD for Ca(2+), so that ligands are released at physiological Ca(2+) concentrations. The proximity of these three residues to the ligand-binding site at Ca(2+) site 2 of the domain suggests that they form a pH-sensitive switch for Ca(2+) and ligand binding. Introduction of histidine and aspartic acid residues into the mannose-binding protein CRD at positions equivalent to His(256) and Asp(266) raises the pH for half-maximal binding of ligand to 6.1. The results, as well as sequence comparisons with other C-type CRDs, confirm the importance of these residues in conferring appropriate pH dependence in this family of domains.

Amino Acid Sequence↗
PubMed1998

Mutagenesis study on human galanin receptor GalR1 reveals domains involved in ligand binding.

Many receptor mutants were generated and several NH2-terminally modified galanin analogs synthesized to define the regions of hGalR1 involved in galanin binding. Ligand binding properties and functionality of mutant receptors were evaluated. The His264Ala and Phe282Ala receptor mutants, although deficient in binding in the concentration range of galanin used, remained functional albeit at least 20-fold less efficient than the wild-type receptor in the inhibition of stimulated cAMP production. Hence, His264 and Phe282 of hGalR1 are directly involved in galanin binding. NH2-terminal carboxylic acid analogs of galanin (1-16) have a very low affinity for the wild-type receptor, but substantially increased affinity for the Glu271Lys-hGalR1, suggesting that the NH2-terminus of galanin binds to the receptor near the transmembrane (TM) VI. Based on these findings and computer-aided molecular modeling, we propose a binding site model for the hGalR1 receptor (possibly also for other galanin receptor subtypes): galanin binds with its NH2-terminus to the pocket between TM III and TM VI, Trp2 of galanin interacts with His264 of the receptor, and Tyr9 is involved in an aromatic-aromatic type of interaction with Phe282 of ECIII of GalR1.

Amino Acid Sequence↗
PubMed1998

Polyamine inhibition of estrogen receptor (ER) DNA-binding and ligand-binding functions.

Polyamines are known to inhibit sequence specific DNA-binding activity of several zinc-finger transcription factors, including estrogen receptor (ER) binding to its cognate estrogen response element (ERE). The mechanism accounting for this disruption of protein-DNA interaction is unknown, although polyamine induction of DNA conformational changes has been suggested. To determine if polyamines can directly impair ER action, we compared the effects of putrescine (Putr), spermidine (Spd), and spermine (Spm) on ER DNA-binding (ER-ERE complex formation), ER ligand-binding (estradiol), ER structure (circular dichroism and sucrose gradient sedimentation), and the capacity of ER to transactivate an ERE-tk-CAT reporter in transient transfection assays. Polyamine concentrations causing 50% inhibition of ER-ERE formation (IC50 values) were found to be 1 mM for Putr, 4 mM for Spd, and 3 mM for Spm. This loss of ER DNA-binding was associated with a direct and irreversible effect on the ER DNA-binding domain (ER-DBD). Additionally, polyamines were observed to inhibit ER ligand-binding with IC50 values of 10 mM for Putr, 2 mM for Spd, and < 0.1 mM for Spm; and this correlated with a measureable change in higher-order ER structure (5S to 3.5S sedimentation) and inhibition of intracellular ER transactivation. These findings suggest that in ER-positive human breast tumors with increased polyamine (especially Spm) content, ER structure and function may be directly altered by tight-ion polyamine complexing that results in loss of ER-mediated gene regulation.

Breast↗
PubMed1986

A simple and rapid orientation procedure for the estimation of affinities (1/Kd) and maximum binding capacities (Bmax) of ligand binding to two receptor subpopulations.

This paper describes a simple and rapid procedure for the estimation of specific parameters (dissociation constants, Kd and maximum binding capacities, Bmax) of ligand binding to two receptor subpopulations. This procedure provides, in a few minutes, the investigator, performing the actual binding studies, the necessary information about receptor heterogeneity, enabling the investigator to plan further experiments. The procedure is based on the graphical comparison of experimental binding data (ligand binding to one or two receptor subpopulations) with the theoretical values of ligand binding to one receptor population at four levels). The values of Kd and Bmax for high- and low-affinity receptors are derived from 4 horizontal deviations of experimental data from a theoretical data plot at these levels by their comparison with tabulated deviations. The correctness of the estimated parameters can be confirmed by the comparison of experimental data with those simulated on the basis of applying the values of Kd and Bmax found in the formula for ligand binding to two receptor subpopulations. The practical applicability of this procedure was demonstrated both on simulated and experimental data, and confirmed by the well known computer programs for evaluating receptor heterogeneity, namely "LIGAND" and "Affinity spectra".

Kinetics↗
PubMed1998

Azoospermia associated with a mutation in the ligand-binding domain of an androgen receptor displaying normal ligand binding, but defective trans-activation.

Although male infertility affects a significant proportion of couples trying to conceive, the cause of defective spermatogenesis is not known in a large number of cases. Ligand binding studies indicate that a number of these subjects may have defects of the androgen receptor (AR). Genetic screening in subjects with defective spermatogenesis and in 110 fertile controls identified an azoospermic (no sperm in any ejaculates) patient with an amino acid substitution (Gln-->Glu) in residue 798 of the AR gene. This germline mutation was pathogenic because it was not observed in fertile controls, was associated with features of minimal androgen insensitivity in our patient, has been related to more severe grades of androgen insensitivity, and caused a subtle, but significant, decrease in receptor trans-activation function in vitro that is consistent with the phenotype. Despite being located in the middle of the ligand-binding domain of the receptor, the Q798E mutation did not cause any ligand binding defect, indicating that this highly conserved residue has a trans-activation function but does not directly form part of the ligand binding pocket of the receptor. The trans-activation defect of the mutant receptor can be rectified in vitro with the androgenic drug, fluoxymesterone, but not with mesterolone or nortestosterone. Further studies are required to determine the therapeutic relevance of this finding.

Adult↗
PubMed1997

Kinetic pathway for the slow to fast transition of thrombin. Evidence of linked ligand binding at structurally distinct domains.

The kinetic pathway for the Na+-induced slow --> fast transition of thrombin was characterized. The slow form was shown to consist of two conformers in a 3:1 ratio (ES2:ES1) at 5 degrees C, pH 7.4, Gamma/2 0.3. ES2 binds Na+ 3 orders of magnitude faster than does ES1. The small molecule active site-directed inhibitor L-371,912, and the exosite I binding ligand hirugen, like Na+, bind selectively to ES2 and induce the slow --> fast conversion of thrombin. The slow --> fast transition is limited by the rate of conversion of ES1 to ES2 (k approximately 28 s-1 at 5 degrees C). Replacement of Arg-221a or Lys-224 at the Na+ binding site with Ala appears to selectively alter the slow form and reduce the apparent affinity of the mutants for Na+ and L-371,912. This replacement, however, has little effect on the affinity for the inhibitor in the presence of saturating concentrations of Na+. The kinetically linked ligand binding at the Na+ binding site, exosite I, and the active site of thrombin characterized in the present study indicates the basis for the plasticity of this important enzyme, and suggests the possibility that the substrate specificity and, therefore, the procoagulant and anticoagulant activities of thrombin may be subject to allosteric regulation by as yet unidentified physiologically important effectors.

Allosteric Regulation↗
PubMed1991

Epidermal growth factor (EGF) induces oligomerization of soluble, extracellular, ligand-binding domain of EGF receptor. A low resolution projection structure of the ligand-binding domain.

Ligand-induced oligomerization is a universal phenomenon among growth factor receptors. Although the mechanism involved is yet to be defined, much evidence indicates that receptor oligomerization plays a crucial role in receptor activation and signal transduction. Here we show that epidermal growth factor (EGF) is able to stimulate the oligomerization of a recombinant, soluble, extracellular ligand-binding domain of EGF receptor. Covalent cross-linking experiments, analysis by sodium dodecyl sulfate-gel electrophoresis, size exclusion chromatography, and electron microscopy demonstrate that receptor dimers, trimers and larger multimers are formed in response to EGF. This establishes that receptor oligomerization is an intrinsic property of the extracellular ligand-binding domain of EGF receptor. Ligand-induced conformational change in the extracellular domain will stimulate receptor-receptor interactions. This may bring about the allosteric change involved in signal transduction from the extracellular domain across the plasma membrane, resulting in the activation of the cytoplasmic kinase domain. Electron microscopic images of individual extracellular ligand-binding domains appear as clusters of four similarly-sized stain-excluding areas arranged around a central, relatively less stain-excluded area. This suggests that the extracellular ligand-binding domain is structurally composed of four separate domains.

Base Sequence↗
PubMed1995

Ig domains 1 and 2 of murine CD22 constitute the ligand-binding domain and bind multiple sialylated ligands expressed on B and T cells.

Baby hamster kidney cells transfected with murine CD22 (mCD22) mediate adhesion to B- and T-lineage cells. To further characterize mCD22-mediated cell adhesion, we generated a panel of recombinant globulins (Rg) consisting of different extracellular Ig-like (Ig) domains of mCD22. FACS analysis using these mCD22.Rgs revealed that ligands for mCD22 are expressed on both B and T cell lines and also normal B and T cells. In B-lineage cells, the expression of mCD22 ligands began on sIgM- pre-B cells in bone marrow. The ligand-binding site of mCD22 for ligands was mapped to Ig domains 1 and 2: mCD22.Rgs containing Ig domains 1 and 2 bound target cells and immunoprecipitated sets of glycoproteins similar to Rgs containing Ig domains 1 to 3 or all 7 CD22 Ig domains, whereas Rgs containing Ig domains 2 to 3 or 3 to 7 did not bind either B or T cells. Furthermore, B cells apparently expressed higher levels of mCD22 ligands than that of T cells, suggesting a potential competition for CD22 binding between ligands expressed on the same B cell and those expressed on another B cell or T cells. Immunoprecipitation experiments using the mCD22.Rgs identified mCD22 itself and the B cell-specific isoform of mCD45RA (B220) as two of the mCD22 ligands expressed on B cells. Thus, mCD22 may potentially regulate B cell activation through interactions with itself or mCD45RA/B220.

Amino Acid Sequence↗
PubMed1998

A two-amino acid insertion in the Cys146- Cys167 loop of the alphaIIb subunit is associated with a variant of Glanzmann thrombasthenia. Critical role of Asp163 in ligand binding.

The ligand binding site(s) of the alpha subunit of integrin alphaIIb beta3 (GPIIb-IIIa), a prototypic non-I domain integrin, remains elusive. In this study, we have characterized a Japanese variant of Glanzmann thrombasthenia, KO, whose platelets express normal amounts of alphaIIb beta3. KO platelets failed to bind the activation-independent ligand-mimetic mAb OP-G2 and did not bind fibrinogen or the activation-dependent ligand-mimetic mAb PAC-1 following activation of alphaIIb beta3 under any condition examined. Sequence analysis of PCR fragments derived from KO platelet mRNA revealed a 6-bp insertion leading to a 2-amino-acid insertion (Arg-Thr) between residues 160 and 161 of the alphaIIb subunit. Introduction of the insertion into wild-type recombinant alphaIIb beta3 expressed in 293 cells led to the normal expression of alphaIIb beta3 having the defect in ligand binding function. The insertion is located within the small loop (Cys146-Cys167) in the third NH2-terminal repeat of the alphaIIb subunit. Alanine substitution of each of the oxygenated residues within the loop (Thr150, Ser152, Glu157, Asp159, Ser161, and Asp163) did not significantly affect expression of alphaIIbbeta3, and only Asp163AlaalphaIIb beta3 abolished the ligand binding function. In addition, Asp163AlaalphaIIb beta3 as well as KO mutant alphaIIb beta3 constitutively expressed the PMI-1 epitope. Our present data suggest that Asp163 of the alphaIIb subunit is one of the critical residues for ligand binding.

Adult↗