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PubMed1996

Cooperativity and anti-cooperativity between ligand binding and the dimerization of ristocetin A: asymmetry of a homodimer complex and implications for signal transduction.

BACKGROUND: Recent work has indicated that dimerization is important in the mode of action of the vancomycin group of glycopeptide antibiotics. NMR studies have shown that one member of this group, ristocetin A, forms an asymmetric dimer with two physically different binding sites for cell wall peptides. Ligand binding by ristocetin A and dimerization are slightly anti-cooperative. In contrast, for the other glycopeptide antibiotics of the vancomycin group that have been examined so far, binding of cell wall peptides and dimerization are cooperative. RESULTS: Here we show that the two halves of the asymmetric homodimer formed by ristocetin A have different affinities for ligand binding. One of these sites is preferentially filled before the other, and binding to this site is cooperative with dimerization. Ligand binding to the other, less favored half of the dimer, is anti-cooperative with dimerization. CONCLUSIONS: In dimer complexes, anti-cooperativity of dimerization upon ligand binding can be a result of asymmetry, in which two binding sites have different affinities for ligands. Such a system, in which one binding site is filled preferentially, may be a mechanism by which the cooperativity between ligand binding and dimerization is fine tuned and may thus have relevance to the control of signal transduction in biological systems.

Binding Sites↗
PubMed2001

Regulation of the ligand binding activity of the human very low density lipoprotein receptor by protein kinase C-dependent phosphorylation.

The very low density lipoprotein receptor (VLDL-R) binds and internalizes several ligands, including very low density lipoprotein (VLDL), urokinase-type plasminogen activator:plasminogen activator inhibitor type 1 complexes, lipoprotein lipase, and the 39-kDa receptor-associated protein that copurifies with the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor. Although several agonists regulate VLDL-R mRNA and/or protein expression, post-transcriptional regulation of receptor activity has not been described. Here, we report that the ligand binding activity of the VLDL-R in THP-1 monocytic cells, endothelial cells, smooth muscle cells, and VLDL-R-transfected HEK 293 cells is diminished after treatment with phorbol 12-myristate 13-acetate. This response was blocked by inhibitors of protein kinase C (PK-C), including a specific inhibitor of the PK-C beta II isoform, and was associated with phosphorylation of serine residues in the cytoplasmic domain of the receptor. Culture of endothelial cells in the presence of high glucose concentrations, which stimulate diacylglycerol synthesis and PK-C beta II activation, also induced a PK-C-dependent loss of VLDL-R ligand binding activity. Taken together, these studies demonstrate that the ligand binding activity of the VLDL-R is regulated by PK-C-dependent phosphorylation and that hyperglycemia may diminish VLDL-R activity.

Cell Line↗
PubMed2004

The family 6 carbohydrate binding module CmCBM6-2 contains two ligand-binding sites with distinct specificities.

The microbial degradation of the plant cell wall is an important biological process, representing a major component of the carbon cycle. Enzymes that mediate the hydrolysis of this composite structure are modular proteins that contain non-catalytic carbohydrate binding modules (CBMs) that enhance catalytic activity. CBMs are grouped into sequence-based families, and in a previous study we showed that a family 6 CBM (CBM6) that interacts with xylan contains two potential ligand binding clefts, designated cleft A and cleft B. Mutagenesis and NMR studies showed that only cleft A in this protein binds to xylan. Family 6 CBMs bind to a range of polysaccharides, and it was proposed that the variation in ligand specificity observed in these proteins reflects the specific cleft that interacts with the target carbohydrate. Here the biochemical properties of the C-terminal cellulose binding CBM6 (CmCBM6-2) from Cellvibrio mixtus endoglucanase 5A were investigated. The CBM binds to the beta1,4-beta1,3-mixed linked glucans lichenan and barley beta-glucan, cello-oligosaccharides, insoluble forms of cellulose, the beta1,3-glucan laminarin, and xylooligosaccharides. Mutagenesis studies, informed by the crystal structure of the protein (presented in the accompanying paper, Pires, V. M. R., Henshaw, J. L., Prates, J. A. M., Bolam, D., Ferreira, L. M. A. Fontes, C. M. G. A., Henrissat, B., Planas, A., Gilbert, H. J., Czjzek, M. (2004) J. Biol. Chem. 279, 21560-21568), show that both cleft A and B can accommodate cello-oligosaccharides and laminarin displays a preference for cleft A, whereas xylooligosaccharides exhibit absolute specificity for this site, and the beta1,4,-beta1,3-mixed linked glucans interact only with cleft B. The binding of CmCBM6-2 to insoluble cellulose involves synergistic interactions between cleft A and cleft B. These data show that CmCBM6-2 contains two binding sites that display differences in ligand specificity, supporting the view that distinct binding clefts with different specificities can contribute to the variation in ligand recognition displayed by family 6 CBMs. This is in sharp contrast to other CBM families, where variation in ligand binding is a result of changes in the topology of a single carbohydrate-binding site.

Amino Acid Substitution↗
PubMed1988

Mutational analysis of the ligand binding domain of the low density lipoprotein receptor.

The ligand binding domain of the low density lipoprotein (LDL) receptor contains seven imperfect repeats of a 40-amino acid cysteine-rich sequence. Each repeat contains clustered negative charges that have been postulated as ligand-binding sites. The adjacent region of the protein, the growth factor homology region, contains three cysteine-rich repeats (A-C) whose sequence differs from those in the ligand binding domain. To dissect the contribution of these different cysteine-rich repeats to ligand binding, we used oligonucleotide-directed mutagenesis to alter expressible cDNAs for the human LDL receptor which were then introduced into monkey COS cells by transfection. We measured the ability of the mutant receptors to bind LDL, which contains a single protein ligand for the receptor (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains apoB-100 plus multiple copies of another ligand (apoE). The results show that repeat 1 is not required for binding of either ligand. Repeats 2 plus 3 and repeats 6 plus 7 are required for maximal binding of LDL, but not beta-VLDL. Repeat 5 is required for binding of both ligands. Repeat A in the growth factor homology region is required for binding of LDL, but not beta-VLDL. Repeat B is not required for ligand binding. These results support a model for the LDL receptor in which various repeats play additive roles in ligand binding, each repeat making a separate contribution to the binding event.

Amino Acid Sequence↗
PubMed1985

The relationship between zeros and factors of binding polynomials and cooperativity in protein-ligand binding.

Cooperativity in the protein-ligand binding process is discussed in terms of the zeros of the binding polynomial and the corresponding possible factorizations of the binding polynomial into polynomials having non-negative coefficients. Particular attention is paid to the case in which the real parts of all zeros are negative (Hurwitz polynomial) and the case in which the binding polynomial admits no positive factorization (positive irreducible polynomial). Such factorizations are then interpreted as site linkage patterns and related to cooperativity. The possible combinations of zeros of the binding polynomials for the MWC and KNF tetrahedral, square and linear models are determined and the corresponding factorization and linkage patterns analyzed. An application and interpretation are then made for data obtained from Trout I hemoglobin.

Animals↗
PubMed2005

Protein flexibility and ligand rigidity: a thermodynamic and kinetic study of ITAM-based ligand binding to Syk tandem SH2.

The Syk tandem Src homology 2 domain (Syk tSH2) constitutes a flexible protein module involved in the regulation of Syk kinase activity. The Syk tSH2 domain is assumed to function by adapting the distance between its two SH2 domains upon bivalent binding to diphosphotyrosine ligands. A thermodynamic and kinetic analysis of ligand binding was performed by using surface plasmon resonance (SPR). Furthermore, the effect of binding on the Syk tSH2 structural dynamics was probed by hydrogen/deuterium exchange and electrospray mass spectrometry (ESI-MS). Two ligands were studied: 1, a flexible peptide derived from the tSH2 recognition ITAM sequence at the gamma chain of the FcepsilonRI-receptor, and 2, a ligand in which the amino acids between the two SH2 binding motifs in ligand 1 have been replaced by a rigid linker of comparable length. Both ligands display comparable affinity for Syk tSH2 at 25 degrees C, yet a major difference in thermodynamics is observed. Upon binding of the rigid ligand, 2, the expected entropy advantage is not realized. On the contrary, 2 binds with a considerably higher entropy price of approximately 9 kcal mol-1, which is attributed to a further decrease in protein flexibility upon binding to this rigid ligand. The significant reduction in deuterium incorporation in the Syk tSH2 protein upon binding of either 1 or 2, as monitored by ESI-MS, indicates a major reduction in protein dynamics upon binding. The results are consistent with a two-step binding model: after an initial binding step, a rapid structural change of the protein occurs, followed by a second binding step. Such a bivalent binding model allows high affinity and fast dissociation kinetics, which are very important in transient signal-transduction processes.

Intracellular Signaling Peptides and Proteins↗
PubMed2004

Dynamic of ligand binding to Drosophila melanogaster ecdysteroid receptor.

Ligand binding to ecdysone receptor (EcR) is an autonomous function of the ligand binding domain (LBD) and is not modified by other receptor domains or tags fused to the LBD. Association and dissociation velocity of hormone to EcR was studied in the absence and presence of its main dimerization partner Ultraspiracle (USP). Mutational analysis of the EcR(LBD) revealed that ligand entry and exit is affected differently by the same point mutation, indicating that different pathways are used for association and dissociation of the ligand. Heterodimerization with wild type USP(LBD) increases ligand association to EcR(LBD) about fivefold and reduces dissociation 18-fold. Opposite effects of the same mutation (N626K) on dissociation velocity of ligand in EcR and EcR/USP indicate that not only hormone binding itself, but also the kinetic behaviour of ligand binding is modified by the dimerization partner. A general effect of the point mutations on the 3D architecture seems unlikely due to the highly selective effects on the kinetics of hormone binding.

Animals↗
PubMed1998

Different classes of coactivators recognize distinct but overlapping binding sites on the estrogen receptor ligand binding domain.

We have analyzed interaction of coactivators with the wild-type estrogen receptor alpha (ER), HEG0, and a mutant, L536P-HEG0, which is constitutively active in several transiently transfected cells and a HeLa line that stably propagates an estrogen-sensitive reporter gene. Different classes of coactivators do not recognize the ER ligand binding domain (LBD) in the same manner. Steroid receptor coactivator-1 (SRC-1), amplified in breast cancer-1 (AIB-1), transcriptional intermediary factor-1 (TIF-1), transcriptional intermediary factor-2 (TIF-2), and receptor interacting protein 140 (RIP140) interacted with HEG0 and L536P-HEG0 in the presence of estradiol, but generally not in the presence of anti-estrogens. However, ICI164,384 stimulated some interaction of RIP140 with LBDs. SRC-1, AIB-1, and RIP140 interacted constitutively with the L536P ER, whereas TIF-1 and TIF-2 interacted only weakly in the absence of hormone. Reciprocal competition for binding to the ER LBD was observed between different classes of coactivators. Moreover, coexpression of RIP140 blocked enhanced transactivation by HEG0 observed in the presence of TIF-2, suggesting that RIP140 may play a negative role in ER signaling. We conclude that constitutive activity of L536P-HEG0 is manifested to similar degrees in different cell types and likely arises from constitutive coactivator binding; different classes of coactivators recognize distinct but overlapping binding sites on the ER LBD. Finally, the observation that L536P-HEG0 interacted constitutively with AIB-1, a coactivator that has been implicated in ER signaling in breast and ovarian cancer, suggests that similar mutations in the ER may contribute to hormone-independent proliferation of breast and ovarian cells.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Imidazole binding to Rhodobacter capsulatus cytochrome c2. Effect of site-directed mutants on ligand binding.

Although ligand binding in c-type cytochromes is not directly related to their physiological function, it has the potential to provide valuable information on protein stability and dynamics, particularly in the region of the methionine sixth heme ligand and the nearby peptide chain that has been implicated in electron transfer. Thus, we have measured the equilibrium and kinetics of binding of imidazole to eight mutants of Rhodobacter capsulatus cytochrome c2 that differ in overall protein stability. We found that imidazole binding affinity varies 70-fold, but does not correlate with overall protein stability. Instead, each mutant exerts an effect at the local level, with the largest change due to mutant G95E (glycine substituted by glutamate), which shows 30-fold stronger binding as compared with the wild-type protein. The kinetics of imidazole binding are monophasic and reach saturation at high ligand concentrations for all the mutants and wild-type protein, which is attributed to a rate-limiting conformational change leading to breakage of the iron-methionine bond and providing a binding site for imidazole. The mutants show as much as an 18-fold variation in the first-order rate constant for the conformational change, with the largest effect found with mutant G95E. The kinetics also show a lack of correlation with overall protein stability, but are consistent with localized effects on the dynamics of hinge region 88-102 of the protein, which changes conformation to permit ligand binding. These results are consistent with R. capsulatus cytochrome c2 stabilizing the complex through hydrogen bonding to the imidazole. The larger effects of mutant G95E on equilibrium and kinetics are likely to be due to its location within the hinge region adjacent to heme ligand methionine 96, which is displaced by imidazole.

Bacterial Proteins↗
PubMed2005

Structure of a mannan-specific family 35 carbohydrate-binding module: evidence for significant conformational changes upon ligand binding.

Enzymes that digest plant cell wall polysaccharides generally contain non-catalytic, carbohydrate-binding modules (CBMs) that function by attaching the enzyme to the substrate, potentiating catalytic activity. Here, we present the first structure of a family 35 CBM, derived from the Cellvibrio japonicus beta-1,4-mannanase Man5C. The NMR structure has been determined for both the free protein and the protein bound to mannopentaose. The data show that the protein displays a typical beta-jelly-roll fold. Ligand binding is not located on the concave surface of the protein, as occurs in many CBMs that display the jelly-roll fold, but is formed by the loops that link the two beta-sheets of the protein, similar to family 6 CBMs. In contrast to the majority of CBMs, which are generally rigid proteins, CBM35 undergoes significant conformational change upon ligand binding. The curvature of the binding site and the narrow binding cleft are likely to be the main determinants of binding specificity. The predicted solvent exposure of O6 at several subsites provides an explanation for the observed accommodation of decorated mannans. Two of the key aromatic residues in Man5C-CBM35 that interact with mannopentaose are conserved in mannanase-derived CBM35s, which will guide specificity predictions based on the primary sequence of proteins in this CBM family.

Amino Acid Sequence↗
PubMed2004

The important role for betaVLDLs binding at the fourth cysteine of first ligand-binding domain in the low-density lipoprotein receptor.

The low-density lipoprotein (LDL) receptor (LDLR) is a crucial role for binding and uptaking apolipoprotein (apo) B-containing lipoproteins, such as very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and LDL. The defect function of the LDLR causes familial hypercholesterolemia (FH), the phenotype of which is elevated plasma cholesterol and premature coronary heart disease (CHD). In the present study, we characterize the role of the cysteine residue of the ligand-binding domain of the LDLR. The mutant LDLR protein of cysteine for serine at codon 25 (25S-LDLR) was expressed in Chinese hamster ovary (CHO) cell line, ldl-A7. By Western blot analysis, the 25S-LDLR was detected with monoclonal antibody IgG-12D10, which reacts with the linker site of the LDLR but not with IgG-C7, which reacts with the NH2 terminus of the receptor. The 25S-LDLR bound LDL similarly to the wild-type LDLR, but the rate of uptake of LDL by the mutant receptor was only about half of that by the wild-type receptor. In contrast, the 25S-LDLR bound and internalized beta VLDL more avidly than LDL. These results suggest that the fourth cysteine residue of the first ligand-binding domain of the LDLR might be important for the internalization of atherogenic lipoproteins by vascular cells despite reduced LDL uptake, leading to atherosclerosis and premature cardiovascular disease.

Animals↗
PubMed2001

Definition of a dioxin receptor mutant that is a constitutive activator of transcription: delineation of overlapping repression and ligand binding functions within the PAS domain.

The intracellular dioxin (aryl hydrocarbon) receptor is a ligand-activated transcription factor that mediates the adaptive and toxic responses to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and structurally related congeners. Whereas the ligand-free receptor is characterized by its association with the molecular chaperone hsp90, exposure to ligand initiates a multistep activation process involving nuclear translocation, dissociation from the hsp90 complex, and dimerization with its partner protein Arnt. In this study, we have characterized a dioxin receptor deletion mutant lacking the minimal ligand-binding domain of the receptor. This mutant did not bind ligand and localized constitutively to the nucleus. However, this protein was functionally inert since it failed to dimerize with Arnt and to bind DNA. In contrast, a dioxin receptor deletion mutant lacking the minimal PAS B motif but maintaining the N-terminal half of the ligand-binding domain showed constitutive dimerization with Arnt, bound DNA, and activated transcription in a ligand-independent manner. Interestingly, this mutant showed a more potent functional activity than the dioxin-activated wild-type receptor in several different cell lines. In conclusion, the constitutively active dioxin receptor may provide an important mechanistic tool to investigate receptor-mediated regulatory pathways in closer detail.

Animals↗
PubMed1993

Mutational analysis of the human KDEL receptor: distinct structural requirements for Golgi retention, ligand binding and retrograde transport.

The KDEL receptor is a seven-transmembrane-domain protein that is responsible for the retrieval of endoplasmic reticulum (ER) proteins from the Golgi complex. It is a temporary resident of the Golgi apparatus: upon binding a KDEL-containing ligand, it moves to the ER, where the ligand is released. We have expressed mutant forms of the human receptor in COS cells and examined their intracellular locations and ligand-binding capacities. We show that ligand binding is dependent on charged residues within the transmembrane domains. Surprisingly, retrograde transport of occupied receptor is unaffected by most mutations in the cytoplasmic loops, but is critically dependent upon an aspartic acid residue in the seventh transmembrane domain. Retention in the Golgi apparatus requires neither ligand binding nor this aspartate residue, and thus is independent of receptor recycling. We suggest that movement of the receptor is controlled by conformational changes and intermolecular interactions within the membrane bilayer.

Amino Acid Sequence↗
PubMed1992

Studies on the structure of the ligand-binding site of the brain D1 dopamine receptor.

A series of group-specific modifying reagents were tested for their effects on [3H]SCH23390 binding to brain D1 dopamine receptors in order to identify amino acid residues at the ligand binding site of the D1 dopamine receptor that are critical for ligand binding. The dependence of ligand binding on the pH of the incubation medium was also examined. The histidine-selective reagent, diethylpyrocarbonate did affect ligand binding but this is probably not due to an effect at the ligand binding site. Experiments with N-acetylimidazole and ethylacetimidate indicated that modification of tyrosine and amino residues did not exert major influences at the ligand binding site. The use of the thiol dithiothreitol indicated that breakage of a disulphide bond altered ligand binding, probably by affecting the receptor conformation, and the use of the sulphydryl reagent 5,5'-dithio-bis-nitrobenzoic acid showed that modification of a sulphydryl group on the receptor inhibited ligand binding. The carboxyl reagent N,N'-dicyclohexyl carbodiimide (DCCD) potently inhibited ligand binding and the effect could be prevented by occupancy of the receptor site by an agonist or antagonist so that there is an important carboxyl group at the receptor binding site. The total number of D1 receptors was reduced after the modification by DCCD and 70% of the residual receptors showed a reduced affinity for binding [3H]SCH23390, the remainder having the same affinity as untreated receptors. [3H]SCH23390 binding is also reduced by a decrease of pH and this effect seems to depend on the protonation of a group of pKa 6.9. Saturation analysis of [3H]SCH23390 binding performed at pH 7.5 shows a single class of high affinity sites whereas at pH 6.0, two classes of sites with higher and lower affinities are seen. These studies suggested a model whereby [3H]SCH23390 binding is to two receptor isoforms with different pH dependencies for [3H]SCH23390 binding.

Benzazepines↗
PubMed2006

CXCR3 requires tyrosine sulfation for ligand binding and a second extracellular loop arginine residue for ligand-induced chemotaxis.

CXCR3 is a G-protein-coupled seven-transmembrane domain chemokine receptor that plays an important role in effector T-cell and NK cell trafficking. Three gamma interferon-inducible chemokines activate CXCR3: CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-TAC). Here, we identify extracellular domains of CXCR3 that are required for ligand binding and activation. We found that CXCR3 is sulfated on its N terminus and that sulfation is required for binding and activation by all three ligands. We also found that the proximal 16 amino acid residues of the N terminus are required for CXCL10 and CXCL11 binding and activation but not CXCL9 activation. In addition, we found that residue R216 in the second extracellular loop is required for CXCR3-mediated chemotaxis and calcium mobilization but is not required for ligand binding or ligand-induced CXCR3 internalization. Finally, charged residues in the extracellular loops contribute to the receptor-ligand interaction. These findings demonstrate that chemokine activation of CXCR3 involves both high-affinity ligand-binding interactions with negatively charged residues in the extracellular domains of CXCR3 and a lower-affinity receptor-activating interaction in the second extracellular loop. This lower-affinity interaction is necessary to induce chemotaxis but not ligand-induced CXCR3 internalization, further suggesting that different domains of CXCR3 mediate distinct functions.

Amino Acid Sequence↗
PubMed1999

Evolutionary constraint networks in ligand-binding domains: an information-theoretic approach.

Ligand-binding sites in homologous protein domains can diverge greatly during evolution. This poses a particularly interesting problem in those cases where the ligand-binding site is situated in, or close to, the domain core, or where ligand-docking induces dramatic conformational changes. These features are present in many receptors and enzymes; the hormone-binding domain of the nuclear receptors for steroids and retinoids, for example, exhibits both characteristics. It is therefore of great interest to determine how binding sites for diverse ligands evolve in core regions of structurally dynamic domains. Are evolutionary changes locally restricted to the ligand-binding site, or are they distributed throughout the domain? We describe here an information-theoretic approach for the study of covariation between ligand-contacting residues and compensatory mutations that preserve the structural integrity and the conformational dynamics of ligand-binding domains. We apply this method to the analysis of the nuclear receptor ligand-binding domain and show that the ligand-contacting residues in the hormone-binding pocket are evolutionarily linked to an extensive network of covarying positions.

Amino Acid Sequence↗
PubMed2001

Amino acid residues in the alpha IIb subunit that are critical for ligand binding to integrin alpha IIbbeta 3 are clustered in the beta-propeller model.

Several distinct regions of the integrin alpha(IIb) subunit have been implicated in ligand binding. To localize the ligand binding sites in alpha(IIb), we swapped all 27 predicted loops with the corresponding sequences of alpha(4) or alpha(5). 19 of the 27 swapping mutations had no effect on binding to both fibrinogen and ligand-mimetic antibodies (e.g. LJ-CP3), suggesting that these regions do not contain major ligand binding sites. In contrast, swapping the remaining 8 predicted loops completely blocked ligand binding. Ala scanning mutagenesis of these critical predicted loops identified more than 30 discontinuous residues in repeats 2-4 and at the boundary between repeats 4 and 5 as critical for ligand binding. Interestingly, these residues are clustered in the predicted beta-propeller model, consistent with this model. Most of the critical residues are located at the edge of the upper face of the propeller, and several critical residues are located on the side of the propeller domain. None of the predicted loops in repeats 1, 6, and 7, and none of the four putative Ca(2+)-binding predicted loops on the lower surface of the beta-propeller were important for ligand binding. The results map an important ligand binding interface at the edge of the top and on the side of the beta-propeller toroid, centering on repeat 3.

Amino Acid Sequence↗