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PubMed1992

Importance of thiol groups in ligand binding to D2 dopamine receptors from brain and anterior pituitary gland.

The effects of the thiol group reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) on D2 dopamine receptors have been examined in three brain regions (caudate nucleus, putamen and olfactory tubercle), and in the anterior and neurointermediate lobes of the pituitary gland. Whereas the receptors in brain were insensitive to DTNB, a dose-dependent inhibition of [3H]spiperone binding to D2 receptors in both lobes of the pituitary gland was observed. The effects in the pituitary could be reversed by subsequent treatment with dithiothreitol and could be prevented by prior occupancy of the receptor binding site with a ligand. The effect of DTNB was to decrease the number of ligand-binding sites without altering the affinity of those remaining. The results show that modification of a thiol group of the D2 dopamine receptor in the two lobes of the pituitary gland tested here significantly affects ligand binding. There is therefore a difference between the D2 dopamine receptor populations in brain and pituitary in their sensitivity to modification by DTNB, and this may imply the existence of different receptor isoforms in the two issues.

Animals↗
PubMed1991

A 25-kDa stretch of the extracellular domain of the human interferon gamma receptor is required for full ligand binding capacity.

We investigated which is the shortest fragment of the interferon gamma receptor with ligand binding capacity. A recombinant soluble interferon gamma receptor produced in Escherichia coli was subjected to controlled digestion with several proteolytic enzymes. The fragments generated were assayed by four approaches for interferon gamma binding. A 25-kDa polypeptide comprising residues 6-227 of the mature protein was produced by sequential digestion with trypsin and proteinase K. It was identified as the shortest receptor domain with full interferon gamma binding capacity as judged by ligand blots. The proteolytic fragments were further tested for ligand binding by interferon gamma affinity chromatography. A 15-kDa polypeptide comprising amino acids 94-227 produced by digestion with endoproteinase Glu-C was found to bind with low affinity to immobilized interferon gamma. This fragment, which does not show ligand binding capacity on protein blots, was immunoprecipitated as a complex with interferon gamma by anti-interferon gamma antibodies. It also competed for the binding of radiolabeled interferon gamma to the cell surface receptor when it was assayed as a mixture of the proteolytic products, but not after separation from the cleaved rest of the molecule. The 15-kDa polypeptide probably carries the ligand-binding domain or part of it, but it lacks sequences essential for full interferon gamma binding capacity. The stretch between amino acids 6 and 21 which does not include any disulfide bonds seems to be essential for the receptor to show full activity. The digestion with endoproteinase Glu-C revealed that cysteine residues 60 and 68 of the interferon gamma receptor form a disulfide bond.

Amino Acid Sequence↗
PubMed2000

Identification of discrete domains within gonococcal transferrin-binding protein A that are necessary for ligand binding and iron uptake functions.

The availability of free iron in vivo is strictly limited, in part by the iron-binding protein transferrin. The pathogenic Neisseria spp. can sequester iron from this protein, dependent upon two iron-repressible, transferrin-binding proteins (TbpA and TbpB). TbpA is a TonB-dependent, integral, outer membrane protein that may form a beta-barrel exposing multiple surface loops, some of which are likely to contain ligand-binding motifs. In this study we propose a topological model of gonococcal TbpA and then test some of the hypotheses set forth by the model by individually deleting three putative loops (designated loops 4, 5, and 8). Each mutant TbpA could be expressed without toxicity and was surface exposed as assessed by immunoblotting, transferrin binding, and protease accessibility. Deletion of loop 4 or loop 5 abolished transferrin binding to whole cells in solid- and liquid-phase assays, while deletion of loop 8 decreased the affinity of the receptor for transferrin without affecting the copy number. Strains expressing any of the three mutated TbpAs were incapable of growth on transferrin as a sole iron source. These data implicate putative loops 4 and 5 as critical determinants for receptor function and transferrin-iron uptake by gonococcal TbpA. The phenotype of the DeltaL8TbpA mutant suggests that high-affinity ligand interaction is required for transferrin-iron internalization.

Amino Acid Sequence↗
PubMed1999

Conformational changes in tertiary structure near the ligand binding site of an integrin I domain.

For efficient ligand binding, integrins must be activated. Specifically, a conformational change has been proposed in a ligand binding domain present within some integrins, the inserted (I) domain [Lee, J., Bankston, L., Arnaout, M. & Liddington, R. C. (1995) Structure (London) 3, 1333-1340]. This proposal remains controversial, however, despite extensive crystal structure studies on the I domain [Lee, J., Bankston, L., Arnaout, M. & Liddington, R. C. (1995) Structure (London) 3, 1333-1340; Liddington, R. & Bankston, L. (1998) Structure (London) 6, 937-938; Qu, A. & Leahy, D. J. (1996) Structure (London) 4, 931-942; and Baldwin, E. T., Sarver, R. W., Bryant, G. L., Jr., Curry, K. A., Fairbanks, M. B., Finzel, B. C. , Garlick, R. L., Heinrikson, R. L., Horton, N. C. & Kelly, L. L. (1998) Structure (London) 6, 923-935]. By defining the residues present in the epitope of a mAb against the human Mac-1 integrin (alphaMbeta2, CD11b/CD18) that binds only the active receptor, we provide biochemical evidence that the I domain itself undergoes a conformational change with activation. This mAb, CBRM1/5, binds the I domain very close to the ligand binding site in a region that is widely exposed regardless of activation as judged by reactivity with other antibodies. The conformation of the epitope differs in two crystal forms of the I domain, previously suggested to represent active and inactive receptor. Our data suggests that conformational differences in the I domain are physiologically relevant and not merely a consequence of different crystal lattice interactions. We also demonstrate that the transition between the two conformational states depends on species-specific residues at the bottom of the I domain, which are proposed to be in an interface with another integrin domain, and that this transition correlates with functional activity.

Amino Acid Sequence↗
PubMed2001

Expression pattern of the type 1 sigma receptor in the brain and identity of critical anionic amino acid residues in the ligand-binding domain of the receptor.

The type 1 sigma receptor (sigmaR1) has been shown to participate in a variety of functions in the central nervous system. To identify the specific regions of the brain that are involved in sigmaR1 function, we analyzed the expression pattern of the receptor mRNA in the mouse brain by in situ hybridization. SigmaR1 mRNA was detectable primarily in the cerebral cortex, hippocampus, and Purkinje cells of cerebellum. To identify the critical anionic amino acid residues in the ligand-binding domain of sigmaR1, we employed two different approaches: chemical modification of anionic amino acid residues and site-directed mutagenesis. Chemical modification of anionic amino acids in sigmaR1 with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide reduced the ligand-binding activity markedly. Since it is known that a splice variant of this receptor which lacks exon 3 does not have the ability to bind sigma ligands, the ligand-binding domain with its critical anionic amino acid residues is likely to be present in or around the region coded by exon 3. Therefore, each of the anionic amino acids in this region was mutated individually and the influence of each mutation on ligand binding was assessed. These studies have identified two anionic amino acids, D126 and E172, that are obligatory for ligand binding. Even though the ligand-binding function was abolished by these two mutations, the expression of these mutants was normal at the protein level. These results show that sigmaR1 is expressed at high levels in specific areas of the brain that are involved in memory, emotion and motor functions. The results also provide important information on the chemical nature of the ligand-binding site of sigmaR1 that may be of use in the design of sigmaR1-specific ligands with potential for modulation of sigmaR1-related brain functions.

Amino Acid Motifs↗
PubMed1999

Calorimetric studies on the stability of the ribosome-inactivating protein abrin II: effects of pH and ligand binding.

The effects of pH and ligand binding on the stability of abrin II, a heterodimeric ribosome-inactivating protein, and its subunits have been studied using high-sensitivity differential scanning calorimetry. At pH7.2, the calorimetric scan consists of two transitions, which correspond to the B-subunit [transition temperature (Tm) 319.2K] and the A-subunit (Tm 324.6K) of abrin II, as also confirmed by studies on the isolated A-subunit. The calorimetric enthalpy of the isolated A-subunit of abrin II is similar to that of the higher-temperature transition. However, its Tm is 2.4K lower than that of the higher-temperature peak of intact abrin II. This indicates that there is some interaction between the two subunits. Abrin II displays increased stability as the pH is decreased to 4.5. Lactose increases the Tm values as well as the enthalpies of both transitions. This effect is more pronounced at pH7.2 than at pH4.5. This suggests that ligand binding stabilizes the native conformation of abrin II. Analysis of the B-subunit transition temperature as a function of lactose concentration suggests that two lactose molecules bind to one molecule of abrin II at pH7.2. The presence of two binding sites for lactose on the abrin II molecule is also indicated by isothermal titration calorimetry. Plotting DeltaHm (the molar transition enthalpy at Tm) against Tm yielded values for DeltaCp (change in excess heat capacity) of 27+/-2 kJ.mol-1.K-1 for the B-subunit and 20+/-1 kJ.mol-1.K-1 for the A-subunit. These values have been used to calculate the thermal stability of abrin II and to surmise the mechanism of its transmembrane translocation.

Abrin↗
PubMed1990

Calcium channel ligand binding to intact, concanavalin A and cyclic AMP-treated cells of the immune system.

To examine whether the cells of immune system express calcium channel-forming proteins, we studied the binding of calcium channel ligands, known to detect certain types of the above channels in excitable tissues, to murine splenic and human peripheral blood mononuclear cells. Specific (i.e., displaceable by excess cold ligand) binding of the 3H-labelled dihydropyridine drugs PN200-110 and nitrendipine, was not detected in these cells. Specific binding of a phenylalkylamine drug, [3H]verapamil, was detected, but cannot be attributed to the existence of certain specialized receptors, since multiple [3H]verapamil binding sites (about 10(6) per cell) appeared to be occupied. [3H]Verapamil binding to murine splenic mononuclear cells was inhibited following exposure to either the polyclonal T-cell activator, concanavalin A, or a cell-permeable analogue of the second messenger, cyclic AMP, suggesting that processes of lymphocyte activation and/or intracellular signalling may down-modulate at least some of calcium channel ligand binding sites.

Animals↗
PubMed2001

A model for ligand binding to hexacoordinate hemoglobins.

Hexacoordinate hemoglobins are heme proteins capable of reversible intramolecular coordination of the ligand binding site by an amino acid side chain from within the heme pocket. Examples of these proteins are found in many living organisms ranging from prokaryotes to humans. The nonsymbiotic hemoglobins (nsHbs) are a class of hexacoordinate heme proteins present in all plants. The nsHb from rice (rHb1) has been used as a model system to develop methods for determining rate constants characterizing binding and dissociation of the His residue responsible for hexacoordination. Measurement of these reactions exploits laser flash photolysis to initiate the reaction from the unligated, pentacoordinate form of the heme protein. A model for ligand binding is presented that incorporates the reaction following rapid mixing with the reaction starting from the pentacoordinate hemoglobin (Hb). This model is based on results indicating that ligand binding to hexacoordinate Hbs is not a simple combination of competing first order (hexacoordination) and second order (exogenous ligand binding) reactions. Ligand binding following rapid mixing is a multiphasic reaction displaying time courses ranging from milliseconds to minutes. The new model incorporates a "closed", slow reacting form of the protein that is not at rapid equilibrium with the reactive conformation. It is also demonstrated that formation of the closed protein species is not dependent on hexacoordination.

Animals↗
PubMed2006

Ion dependence of ligand binding to metabotropic glutamate receptors.

The ionic requirements for ligand binding to metabotropic glutamate receptors were carried out on secreted truncated receptors containing only the extracellular ligand binding domains of the receptors. The influence of ions on agonist binding was examined in mGluR1, mGluR3, and mGluR4 representing Group I, II, and III metabotropic glutamate receptors, respectively. [(3)H]Quisqualic acid binding to mGluR1 required the presence of calcium (or magnesium) ions but not sodium or chloride ions while [(3)H]DCG-IV binding to mGluR3 was dependent upon both cations and anions. [(3)H]L-AP4 binding to mGluR4 required chloride ions but not monovalent or divalent cations. The EC(50) for chloride facilitation of L-AP4 binding to mGluR4 was 63mM; this value is approximately one-half of the normal resting extracellular chloride concentration. These results demonstrate that metabotropic glutamate receptor subtypes require different complements of ions for ligand binding and suggest that natural physiological fluctuations in synaptic ion concentrations may regulate receptor binding and activation.

Animals↗
PubMedDate not supplied

New insights into receptor ligand binding domains from a novel assembly assay.

Previous studies have demonstrated that hormone binding stabilizes the ligand binding domain (LBD) of the nuclear hormone receptors against proteolysis. We have confirmed and extended this observation using a newly developed assembly assay. In this assay, the LBD is divided into two parts, of which one includes the first helix of this domain and the other corresponds to the remainder of the LBD. Several independent criteria demonstrate that these two fragments can assemble into a functional LBD in the presence of a ligand, but not in its absence, and that this is a reflection of the stabilizing effect of ligand. We have also used this assay to demonstrate that binding of the nuclear receptor corepressor NCoR can directly stabilize the LBD. Overall, these results highlight the dynamic nature of the LBD and suggest that current models for activation based solely on allosteric effects on the C-terminal helix may be too limited.

Hydrolysis↗
PubMed2004

Coupling of protein relaxation to ligand binding and migration in myoglobin.

Protein relaxation, ligand binding, and ligand migration into a hydrophobic cavity in myoglobin are unified by a bounded diffusion model which produces an accurate fit to complex ligand rebinding data over eight decades in time and a 160 K temperature range, in qualitative agreement with time-resolved x-ray crystallography. Protein relaxation operates in a cyclic manner to move the ligand away from the binding site.

Animals↗
PubMed2002

Mutations at positions 547-553 of rat glucocorticoid receptors reveal that hsp90 binding requires the presence, but not defined composition, of a seven-amino acid sequence at the amino terminus of the ligand binding domain.

Glucocorticoid receptors (GRs) must heterocomplex with hsp90 to have an open steroid binding cleft that can be accessed by steroid. We reported that a seven-amino acid sequence (547-553 of rat GR) overlapping the amino-terminal end of the ligand binding domain is required for hsp90 binding to GR. We have now conducted saturation mutagenesis of this sequence, which appears to be part of the surface where the ligand binding cleft merges with the surface of the ligand binding domain. No single point mutation causes significant changes in any of a variety of biochemical and biological properties in addition to hsp90 binding. A triple mutation (P548A/T549A/V551A) increases by >100-fold the steroid concentration required for half-maximal induction without affecting the level of maximal induction or coactivator response. Interestingly, this triple mutant displays reduced binding of steroid and hsp90 in whole cells, but it possesses wild type affinity for steroid and normal hsp90 binding capacity under cell-free conditions. This phenotype of a dramatic shift in the dose response for transactivation would be expected from an increase in the rate of disassembly of the triple mutant GR.hsp90 heterocomplex in the cell. Mutation of the entire seven-amino acid region to CAAAAAC maintains the presence of a critical alpha-helical structure and heterocomplex formation with hsp90 but eliminates steroid binding and transcriptional activation, thus disconnecting hsp90 binding from opening of the ligand binding cleft and steroid binding.

Animals↗
PubMed1994

Identification of a ligand-binding site of the human endothelin-A receptor and specific regions required for ligand selectivity.

To investigate the ligand-binding site of the human endothelin-A-receptor subtype (ETA), we have produced various chimeric and mutated receptors in chinese hamster ovary cells. The substitution of Lys140 with Ile located in the C-terminus of the second transmembrane region caused a 13-fold reduction in affinity for endothelin-1 (ET-1) and 3.6-fold lower Bmax than those values for the original receptor. Correspondingly, the mutated ETA receptor with the Lys140-->Ile substitution failed to induce an increase in the intracellular calcium concentration in the presence of 1 nM ET-1. Thus, the Lys140 in the ETA receptor is important in ligand binding. ETA and ETB receptors possess the ET isopeptides selective and non-selective binding activities, respectively. Displacement experiments and the binding of 125I-ET-3 to various chimera receptors demonstrated that both the third and fourth extracellular regions, including the flanking transmembrane regions, are responsible for the ligand-binding selectivity of the ETA receptor.

Amino Acid Sequence↗
PubMed1998

Regulation of the extracellular ligand binding activity of integrins.

Integrins are a large heterodimeric family of cell surface adhesion receptors that bind extracellular matrix and cell surface ligands. The extracellular ligand binding activity of integrins is a dynamic and highly regulated event involving the induction of conformational changes within the integrin structure. The adhesive properties of integrins can be controlled by altering the activation state of the integrin, either through conformational change or receptor clustering, using mechanisms that are regulated by intracellular proteins. In this review, we will discuss what is currently known about integrin structure and the ligand binding sites present within the receptor. In addition, the mechanisms by which the ligand binding event is regulated through conformational change will be addressed, and the potential role of intracellular cytoplasmic proteins will be discussed.

Animals↗
PubMed1995

Rapid substrate translocation by the multisubunit, erythroid glucose transporter requires subunit associations but not cooperative ligand binding.

The human erythroid glucose transporter is a GLUT1 homotetramer whose structure and function are stabilized by noncovalent, cooperative subunit interactions. The present study demonstrates that exofacial tryptic digestion of GLUT1 abolishes cooperative interactions between substrate binding sites on adjacent subunits under circumstances where subunit associations and high catalytic turnover are maintained. Extracellular trypsin produces rapid, quantitative cleavage of the human red cell-resident sugar transport protein, GLUT1. One major carboxyl-terminal peptide of M(r)(app) 25,000 is detected by immunoblot analysis. Endofacial tryptic digestion of GLUT1 results in the complete loss of GLUT1 carboxyl-terminal structure. GLUT1-mediated erythrocyte sugar uptake, transport inhibition by cytochalasin B, and GLUT1 oligomeric structure are unaffected by exofacial GLUT1 proteolysis. In contrast, the cytochalasin B binding capacity of GLUT1 and the Kd(app) for cytochalasin B binding to the transporter are doubled following exofacial tryptic digestion of GLUT1. Photoaffinity labeling experiments show that increased cytochalasin B binding results from increased ligand binding to the 25 kDa carboxyl-terminal GLUT1 peptide. Proteolysis abolishes allosteric interactions between sugar import (maltose binding) and sugar export (cytochalasin B binding) sites that normally exist on adjacent subunits within the transporter complex, but interact with negative cooperativity. Following exofacial proteolysis, these sites become mutually exclusive. Dithiothreitol disrupts GLUT1 quaternary structure, inhibits 3-O-methylglucose transport, and abolishes cooperative interactions between sugar import and export sites in control cells. Studies with reconstituted purified GLUT1 confirm that the action of trypsin on cytochalasin B binding is direct, show that proteolysis increases the apparent affinity of the sugar efflux site for transported sugars, and suggest that the membrane bilayer stabilizes GLUT1 noncovalent structure and catalytic function following GLUT1 proteolysis. Collectively, these findings demonstrate that GLUT1 does not require an intact polypeptide backbone for catalytic function. They show that the multisite sugar transporter mechanism is converted to a simple ping-pong carrier mechanism following exofacial GLUT1 proteolysis. They reveal that subunit cooperativity can be lost under circumstances where cohesive structural interactions between transporter subunits are maintained. They also refute the hypothesis [Hebert, D. N., & Carruthers, A. (1992) J. Biol. Chem. 267, 23829-23838] that rapid substrate translocation by the multisubunit erythroid glucose transporter requires cooperative interactions between subunit ligand binding sites.

3-O-Methylglucose↗
PubMed1974

A probabilistic approach to cooperativity of ligand binding by a polyvalent molecule.

Cooperativity of ligand binding is defined operationally in terms of the Hill parameter n, whose values uniquely define the shape of the binding curve. When n is everywhere equal to 1 the system behaves as if the sites were all the same and independent. If at any point n is unequal to 1 the system is either cooperative (n > 1) or anticooperative (n < 1) at that point. This means that the binding of ligand at any site is influenced by the presence or absence of ligand at other sites. In the present paper this influence is formulated in terms of probabilities of the various binding events and of conditional probabilities which relate them. The result is a network of linked probabilities which determine the cooperativity or anticooperativity of the system. This probabilistic formulation supplements the more usual one in terms of free energies and places the subject in a broader perspective.

Binding Sites↗
PubMed1994

Spectroscopic, electrochemical, and ligand binding properties of the horse heart metmyoglobin His64-Tyr variant.

The distal histidine (E7) of horse heart myoglobin (Mb) has been replaced by tyrosine using site-specific mutagenesis. The resulting green Mb variant (His64-Tyr) was expressed in Escherichia coli JM101, isolated and purified to homogeneity. Spectrophotometric pH titrations of the variant exhibit a change in spectrum that occurs with a pKa of 4.7 (25 degrees C). The midpoint reduction potential of the variant is 20 mV (vs. SHE at pH 7, 25 degrees C). Cyanide and azide binding measurements indicate that the oxidized variant binds these anionic ligands with much greater affinity at pH 4.0 than at neutral pH. Extended X-ray absorption fine structure (EXAFS) spectroscopy establishes that the variant is six coordinate at pH 7.0 and pH 4.2. Higher shell contributions to the iron EXAFS observed at pH 7.0 are attributed to tyrosine. These contributions are absent at pH 4.2. Thus, the sixth heme iron ligand of the oxidized variant Mb at pH 7.0 is attributed to oxygen from the hydroxyl group of tyrosine and the sixth ligand present at pH 4.2 is attributed to the oxygen atom of a coordinated water. The EXAFS spectra, electronic absorption spectra, and ligand binding properties of the His64-Tyr Mb variant are consistent with the binding of Tyr-64 as the sixth heme iron ligand between pH 5 and 12 and with the replacement of Tyr-64 by a water molecule at low pH with a pKa of 4.7.

Animals↗
PubMed1995

Aspartate mutation distinguishes ETA but not ETB receptor subtype-selective ligand binding while abolishing phospholipase C activation in both receptors.

The endothelin receptors, ETA and ETB, are G protein-coupled receptors (GPCR) that show distinctively different binding profiles for the endothelin peptides and other ligands. We recently reported that Tyr129 in the second transmembrane region (TM2) of the ETA receptor was critical for subtype-specific ligand binding [Krystek, S.R. et al. (1994) J. Biol. Chem. 269, 12383-12386]. Receptor models indicated that aspartic acids located one helical turn above (Asp133) and below (Asp126) Tyr129 in ETA had their side chains directed toward the putative binding cavity. Similarly in ETB, Asp147 and Asp154 are located one turn below and above His150, the residue that corresponds to Tyr129. Asp126 in ETA and Asp147 in ETB correspond to the highly conserved aspartate present in TM2 of many GPCR that has frequently been shown to be crucial for agonist efficacy. Mutagenesis of Asp126 of the human ETA receptor to alanine resulted in an unaltered affinity for ET-1, a 160-fold increase in ET-3 affinity and a decrease in affinity for the ETA selective naphthalenesulfonamide, BMS-182874. ET-1 activation of phospholipase C was abolished. In addition, despite the gain in binding affinity, ET-3 failed to activate phospholipase C, suggesting that Asp126 is required for signal transduction. Mutagenesis of Asp133 to alanine indicated that it was critical only for the binding of BMS-182874. In the ETB receptor, mutation of His150 to alanine or tyrosine indicated that it plays a minor role in ETB subtype-selective ligand binding; mutation of the aspartates in TM2 of ETB did not alter ligand binding. As in the Asp126 Ala ETA variant, ET-1 and ET-3 failed to increase intracellular levels of inositol phosphates in the Asp147Ala ETB mutant. Taken together, these data support the hypothesis that Asp126 and Asp133 flanking Tyr129 in TM2 of the ETA receptor play a role in defining ETA subtype-selective ligand binding but Asp147 and Asp154 that flank the His150 in TM2 of the ETB receptor do not. Furthermore, these data indicate that Asp126 in ETA and Asp147 in ETB are important for transmembrane signaling via phospholipase C.

Alanine↗