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PubMed2006

Stepped changes of monovalent ligand-binding force during ligand-induced clustering of integrin alphaIIB beta3.

Recent evidence demonstrated that conformational changes of the integrin during receptor activation affected its binding to extracellular matrix; however, experimental assessment of ligand-receptor binding following the initial molecular interaction has rarely been carried out at a single-molecule resolution. In the present study, laser tweezers were used to measure the binding force exerted by a live Chinese hamster ovary cell that expressed integrin alphaIIb beta3 (CHO alphaIIb beta3), to the bead carrier coated with the snake venom rhodostomin that served as an activated ligand for integrin alphaIIb beta3. A progressive increase of total binding force over time was noticed when the bead interacted with the CHO alphaIIb beta3 cell; such an increase was due mainly to the recruitment of more integrin molecules to the bead-cell interface. When the binding strength exerted by a single ligand-receptor pair was derived from the "polyvalent" measurements, surprisingly, a stepped decrease of the "monovalent binding force" was noted (from 4.15 to 2.54 piconewtons (pN)); such decrease appeared to occur during the ligand-induced integrin clustering process. On the other hand, the mutant rhodostomin defective in clustering integrins exhibited only one (1.81 pN) unit binding strength.

Animals↗
PubMedDate not supplied

Alternative splicing within the ligand binding domain of the human constitutive androstane receptor.

The human constitutive androstane receptor (hCAR; NR1I3) is a member of the nuclear receptor superfamily. The activity of hCAR is regulated by a variety of xenobiotics including clotrimazole and acetaminophen metabolites. hCAR, in turn, regulates a number of genes responsible for xenobiotic metabolism and transport including several cytochrome P450s (CYP 2B5, 2C9, and 3A4) and the multidrug resistance-associated protein 2 (MRP2, ABCC2). Thus, hCAR is believed to be a mediator of drug-drug interactions. We identified two novel hCAR splice variants: hCAR2 encodes a receptor in which alternative splice acceptor sites are utilized resulting in a 4 amino acid insert between exons 6 and 7, and a 5 amino acid insert between 7 and 8, and hCAR3 encodes a receptor with exon 7 completely deleted resulting in a 39 amino acid deletion. Both hCAR2 and hCAR3 mRNAs are expressed in a pattern similar to the initially described MB67 (hCAR1) with some key distinctions. Although the levels of expression vary depending on the tissue examined, hCAR2 and hCAR3 contribute 6-8% of total hCAR mRNA in liver. Analysis of the activity of these variants indicates that both hCAR2 and hCAR3 lose the ability to heterodimerize with RXR and lack transactivation activity in cotransfection experiments where either full-length receptor or GAL4 DNA-binding domain/CAR ligand binding domain chimeras were utilized. Although the role of hCAR2 and hCAR3 is currently unclear, these additional splice variants may provide for increased diversity in terms of responsiveness to xenobiotics.

Alternative Splicing↗
PubMed2001

Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors.

Pentameric ligand gated ion-channels, or Cys-loop receptors, mediate rapid chemical transmission of signals. This superfamily of allosteric transmembrane proteins includes the nicotinic acetylcholine (nAChR), serotonin 5-HT3, gamma-aminobutyric-acid (GABAA and GABAC) and glycine receptors. Biochemical and electrophysiological information on the prototypic nAChRs is abundant but structural data at atomic resolution have been missing. Here we present the crystal structure of molluscan acetylcholine-binding protein (AChBP), a structural and functional homologue of the amino-terminal ligand-binding domain of an nAChR alpha-subunit. In the AChBP homopentamer, the protomers have an immunoglobulin-like topology. Ligand-binding sites are located at each of five subunit interfaces and contain residues contributed by biochemically determined 'loops' A to F. The subunit interfaces are highly variable within the ion-channel family, whereas the conserved residues stabilize the protomer fold. This AChBP structure is relevant for the development of drugs against, for example, Alzheimer's disease and nicotine addiction.

Acetylcholine↗
PubMed2006

Kinetics of ligand binding to nucleic acids.

Ligand binding to nucleic acids (NA) is considered as a stationary Markov process. It is shown that the probabilistic description of ligand-NA binding allows one to describe not only the kinetics of the change of number of bound ligands at arbitrary fillings but also to calculate stationary values of the number of bound ligands and its dispersion. The general analysis of absorption isotherms and kinetics of ligand binding to NA make it possible to determine of rate constants of ligand-NA complex formation and dissociation.

Kinetics↗
PubMed1990

Odorant-binding protein. Characterization of ligand binding.

We have characterized the odorant binding properties of purified bovine odorant-binding protein (OBP) using as a ligand [3H]3,7-dimethyloctan-1-ol ([3H]DMO). A broad variety of odorants, including terpenes, aldehydes, esters, and musks, bind to OBP with affinities of 0.2 to 100 microM. Odorant affinities for OBP correlate most closely with their stimulation of an odorant-sensitive adenylyl cyclase as well as hydrophobicity. We also measured the kinetics of binding for the ligands, [3H]DMO and 2-isobutyl-3-[3H]methoxypyrazine. Dissociation of both is markedly accelerated in the presence of excess unlabeled ligand. Competition curves of displacers for [3H]DMO binding are shallow, and saturation binding isotherms for 3H-odorants are curvilinear. These kinetic and equilibrium binding properties suggest that OBP interactions with odorant ligands are negatively cooperative.

Animals↗
PubMed1992

The mechanism of ligand binding to the periplasmic C4-dicarboxylate binding protein (DctP) from Rhodobacter capsulatus.

The kinetics of ligand binding to the periplasmic C4-dicarboxylate binding protein (DctP) from Rhodobacter capsulatus were investigated by exploiting the changes in the intrinsic fluorescence of the protein upon binding ligands. Steady state measurements have shown that L-malate, succinate, and fumarate are all bound with sub-micromolar Kd values, whereas D-malate is bound 2 orders of magnitude more weakly. Stopped-flow studies have revealed that the binding process involves at least three steps. In the absence of ligand, the protein is in equilibrium between an essentially nonbinding form, BP1, and the binding form, BP2. Ligands bind to the BP2 form, shifting the equilibrium toward the BP2-L conformation, and also inducing a further isomerization of the protein, to the BP3-L form. The kinetic properties of the four different conformational states of the DctP protein identified in this study would be consistent with their identification as the closed-conformation, the open-conformation, an open-liganded conformation, and a closed-liganded conformation. The latter three states have been identified by x-ray crystallographic studies of binding proteins, but no kinetic or structural data have been presented previously to support the possibility of a closed but unliganded conformation.

Bacterial Proteins↗
PubMed2006

Retinol modulates site-specific mobility of apo-cellular retinol-binding protein to promote ligand binding.

A fundamental question in protein science is how the inherent dynamics of a protein influence its function. If this function involves interactions with a ligand, the protein-ligand encounter has the potential to modulate the protein dynamics. This study reveals how site-specific mobility can be modulated by the ligand to facilitate high affinity binding. We have investigated the mechanism of retinol uptake by the cellular retinol-binding protein type I (CRBP) using line shape analysis of NMR signals. The highly similar structures of apo- and holo-CRBP exhibit closed conformations that seemingly offer no access to ligand, yet the protein binds retinol rapidly and with high affinity. NMR line shape analysis reveals how protein dynamics resolve this apparent paradox. An initial nonspecific encounter with the ligand induces the formation of long-lived conformers in the portal region of CRBP suggesting a mechanism how retinol accesses the cavity.

Binding Sites↗
PubMed1995

Distinction between binding of [3H]triamcinolone acetonide to a ligand binding domain on the glucocorticoid receptor complex in cytosol fractions of brain and liver from the rat with intact adrenals.

Binding of [3H]triamcinolone acetonide (TA) was examined to evaluate possible differences in biochemical and pharmacological profiles of a ligand binding domain on the naive glucocorticoid (GC) receptor complex between cytosol fractions of brain and liver obtained from the rat with intact adrenals. Unlabeled TA was 3-times less potent in displacing [3H]TA binding at equilibrium in the presence of Na2MoO4 at 2 degrees C in brain than in liver, while dexamethasone was more than 15-times more potent in displacing brain binding than liver binding. Both progesterone and aldosterone caused more than 20-times more potent displacement of [3H]TA binding in brain than in liver. However, zinc ions were effective in similarly inhibiting binding at equilibrium via lowering the affinity without affecting the density. In contrast to these experiments done at 2 degrees C, the addition of Na2MoO4 potentiated binding in brain and liver in a transient manner at 30 degrees C. Prolongation of incubation period from 30 min to 5 h induced a rightward shift of concentration-response curves of Na2MoO4 for [3H]TA binding at 30 degrees C in brain and liver in an irreversible fashion. The potentiation by Na2MoO4 was prevented by the addition of leupeptin but not 5 other protease inhibitors in brain cytosol fractions. Ion exchange chromatography revealed that elution profiles were entirely different from each other for [3H]TA binding between brain and liver cytosol fractions. These results suggest that GC receptors may respond to a variety of signals mediated by different steroid hormones through broader pharmacological spectrum of the ligand binding domain in brain than in liver.

Adrenal Glands↗
PubMed2006

Ligand binding by TPR domains.

Tetratricopeptide repeat (TPR) domains bind specific peptide ligands and are thought to mediate protein-protein interactions in a variety of biological systems. Here we compare peptide ligand-binding by several different TPR domains. We present specific examples that demonstrate that TPR domains typically undergo little or no structural rearrangement upon ligand binding. Our data suggest that, contrary to a recent proposal, coupled folding and binding is not the common mechanism of ligand recognition by TPR domains.

Binding Sites↗
PubMed1999

Temperature dependence of estrogen binding: importance of a subzone in the ligand binding domain of a novel piscine estrogen receptor.

The full length estrogen receptor from Oreochromis aureus (OaER) was cloned and expressed in vitro and in vivo as a functional transcription factor. Amino acid residues involved in the thermal stability of the receptor are located at/near subzones beta1 and beta3, which are highly conserved in other non-piscine species but not in OaER. Hormone binding studies, however, indicate that OaER is thermally stable but exhibited a approximately 3-fold reduced affinity for estrogen at elevated temperatures. Transfection of OaER into various cell lines cultured at different temperatures displayed a significant estrogen dose-response shift compared with that of chicken ER (cER). At 37 degrees C, OaER requires approximately 80-fold more estrogen to achieve half-maximal stimulation of CAT. Lowering of the incubation temperature from 37 degrees C to 25 degrees C or 20 degrees C resulted in a 4-fold increase in its affinity for estrogen. The thermally deficient transactivation of OaER at temperatures above 25 degrees C was fully prevented by high levels of estrogen. Thus, compared to cER, the OaER exhibits reduced affinity for estrogen at elevated temperature as reflected in its deficient transactivation capability. Amino acid replacements of OaER beta3 subzones with corresponding amino acids from cER could partially rescue this temperature sensitivity. The three-dimensional structure of the OaER ligand binding domain (LBD) was modelled based on conformational similarity and sequence homology with human RXRalpha apo, RARgamma holo and ERalpha LBDs. Unliganded and 17beta-estradiol-liganded OaER LBD retained the overall folding pattern of the nuclear receptor LBDs. The residues at/near the subzone beta3 of the LBD constitute the central core of OaER structure. Thus, amino acid alteration at this region potentially alters the structure and consequently its temperature-dependent ligand binding properties.

Amino Acid Sequence↗
PubMed2000

Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core.

Crystal structures of the GluR2 ligand binding core (S1S2) have been determined in the apo state and in the presence of the antagonist DNQX, the partial agonist kainate, and the full agonists AMPA and glutamate. The domains of the S1S2 ligand binding core are expanded in the apo state and contract upon ligand binding with the extent of domain separation decreasing in the order of apo > DNQX > kainate > glutamate approximately equal to AMPA. These results suggest that agonist-induced domain closure gates the transmembrane channel and the extent of receptor activation depends upon the degree of domain closure. AMPA and glutamate also promote a 180 degrees flip of a trans peptide bond in the ligand binding site. The crystal packing of the ligand binding cores suggests modes for subunit-subunit contact in the intact receptor and mechanisms by which allosteric effectors modulate receptor activity.

Binding Sites↗
PubMed1999

Structural independence of ligand-binding modules five and six of the LDL receptor.

The low-density lipoprotein receptor (LDLR) is the primary mechanism for the uptake of plasma cholesterol into cells and serves as a prototype for a growing family of cell surface receptors. These receptors all utilize tandemly repeated LDL-A modules to bind their ligands. Each LDL-A module is about 40 residues long, has six conserved cysteine residues, and contains a conserved acidic region near the C-terminus which serves as a calcium-binding site. The structure of the interface presented for ligand binding by these modules, and the basis for their specificity and affinity in ligand binding, is not yet known. We have purified recombinant molecules corresponding to LDL-A modules five (LR5), six (LR6), and the module five-six pair (LR5-6) of the LDL receptor. Calcium is required to establish native disulfide bonds and to maintain the structural integrity of LR5, LR6, and the LR5-6 module pair. Folding studies of the I189D and D206Y mutations within LR5 indicate that each change leads to misfolding of the module, explaining the previous observation that each of these changes mimics the functional effect of deletion of the entire module [Russell, D. W., Brown, M. S., and Goldstein, J. L. (1989) J. Biol. Chem. 264, 21682-21688]. By fluorescence, the affinity of LR5 for calcium, which is crucial for folding and function of these modules, remains approximately 40 nM whether LR6 is attached. Comparison of proton and multidimensional heteronuclear NMR spectra of individual modules to those of the module pair indicates that most of the significant spectroscopic changes lie within the linker region between modules and that little structural interaction occurs between the cores of modules five and six in the 5-6 pair. These findings strongly support a model in which each module is essentially structurally independent of the other.

Amino Acid Sequence↗
PubMed2004

Three small integrin binding ligand N-linked glycoproteins (SIBLINGs) bind and activate specific matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are critical for development, wound healing, and for the progression of cancer. It is generally accepted that MMPs are secreted in a latent form (proMMP) and are activated only upon removal of their inhibitory propeptides. This report shows that three members of the SIBLING (Small, Integrin-Binding LIgand, N-linked Glycoprotein) family can specifically bind (Kd approximately equal nM) and activate three different MMPs. Binding of SIBLING to their corresponding proMMPs is associated with structural changes as indicated by quenching of intrinsic tryptophan fluorescence, increased susceptibility to plasmin cleavage, and decreased inhibition by specific natural and synthetic inhibitors. Activation includes both making the proMMPs enzymatically active and the reactivation of the TIMP (tissue inhibitors of MMP) inhibited MMPs. Bone sialoprotein specifically binds proMMP-2 and active MMP-2, while osteopontin binds proMMP-3 and active MMP-3, and dentin matrix protein-1 binds proMMP-9 and active MMP-9. Both pro and active MMP-SIBLING complexes are disrupted by the abundant serum protein, complement Factor H, thereby probably limiting SIBLING-mediated activation to regions immediately adjacent to sites of secretion in vivo. These data suggest that the SIBLING family offers an alternative method of controlling the activity of at least three MMPs.

Complement Factor H↗
PubMed2000

The pre-ligand binding assembly domain: a potential target of inhibition of tumour necrosis factor receptor function.

Signalling by the tumour necrosis factor receptors (TNFR) is thought to be mediated by the binding of the trimeric ligand TNF to three monomeric subunits of the receptor. This ligand induced trimerisation model of TNFR signalling is mainly supported by crystallographic data of the p60 TNFR-1 and TNFbeta complex in which the trimeric ligand interdigitates between the individual receptor chains and prevents the receptor subunits from interacting with each other. Recently, a domain NH(2)-terminal to the ligand binding domain in the extracellular region of p60 TNFR-1, p80 TNFR-2 and Fas was identified that mediates receptor self association before ligand binding. This pre-ligand binding assembly domain or PLAD is critical for assembly of functional receptor complexes on the cell surface and may provide a potential target in the design of future novel therapeutics against diseases mediated by members of the TNFR family of receptors.

Arthritis, Rheumatoid↗
PubMed2005

Observation of ligand binding to cytochrome P450 BM-3 by means of solid-state NMR spectroscopy.

A broad understanding of the binding modes of ligands and inhibitors to cytochrome P450 is vital for the development of new drugs. We investigated ligand binding in a site-specific fashion on cytochrome P450 BM-3 from Bacillus megaterium, a 119 kDa paramagnetic enzyme, using solid-state magic angle spinning nuclear magnetic resonance methods. Selective labeling and longitudinal relaxation effects were utilized to identify the peaks in a site-specific fashion and to provide evidence for binding. Well-resolved one-dimensional and two-dimensional NMR spectra of cytochrome P450 BM-3 reveal shifts upon binding of its substrate, N-palmitoylglycine. These data are consistent with the crystallographic result that a biochemically important amino acid residue, Phe87, moves upon ligation. This experimental scheme provides a tool for probing ligand binding for complex systems.

Bacillus megaterium↗
PubMed2005

Folding and binding integrity of variants of a prototype ligand-binding module from the LDL receptor possessing multiple alanine substitutions.

The LA repeats that comprise the ligand-binding domain of the LDL receptor are among the most common autonomously structured extracellular modules found in the nonredundant protein sequence database. Here, we investigate the information content of the amino acid sequence of a typical LA module by constructing sequences with alanine residues at nonconserved positions in the module. Starting with the sequence of the fifth ligand-binding repeat of the LDL receptor (LA5), we created generic LA modules with alanine substitutions of nonconserved residues in only the N-terminal lobe, only the C-terminal lobe, and throughout both lobes of the module. LA variants with alanine residues at as many as 18 of 37 positions fold to a preferred disulfide isomer in the presence of calcium. Indeed, the six cysteines, the C-terminal calcium coordinating residues, two hydrophobic residues involved in packing, two glycines, and five other residues that form side chain-intramodule hydrogen bonds are alone sufficient to specify the fold of an LA module when alanine residues are present at all other positions. The LA variants with multiple alanines in either the N- or C-terminal lobe were then exploited to identify residues of LA5 that contribute to the binding of apoE-containing ligands in LDL receptor-derived "minireceptors", implicating nonconserved residues of the N-terminal lobe of LA5 in recognition of apoE-DMPC. Our library of LA modules with multiple alanine substitutions should be generally useful for probing the roles of nonconserved side chains in ligand recognition by proteins of the LDL receptor family.

Alanine↗
PubMed1994

His145-Trp146 residues and the disulfide-linked loops in atrial natriuretic peptide receptor are critical for the ligand-binding activity.

To define the ligand-binding site of natriuretic peptide receptor (NPR), amino acid substitutions were carried out by site-directed mutagenesis at selected residues of bovine NPR-C. The mutant receptors were expressed in COS-1 cells and the effect of the mutations on the binding of ligand was analyzed by binding assay, affinity labeling, and immunoblotting. The replacement of His145-Trp146 (HW) by Leu145-Leu146 in the extracellular domain markedly reduced binding affinity, whereas mutations at charged amino acid residues in the vicinity of HW, such as Glu133 and Asp147, had little effect. Mutation of cysteine residues forming the Cys104-Cys132 and Cys209-Cys257 loops to Ser caused a reduction in the affinity of the receptor to bind ANP. These results suggest that HW residues and the two disulfide-linked loops in the extracellular domain contribute significantly to the ligand binding to NPR.

Amino Acid Sequence↗
PubMed2006

Vitamin D receptor agonists specifically modulate the volume of the ligand-binding pocket.

Existing crystal structure data has indicated that 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2) D(3)) and its analogues bind the ligand-binding pocket (LBP) of the human vitamin D receptor in a very similar fashion. Because docking of a ligand into the LBP is a more flexible process than crystallography can monitor, we analyzed 1alpha,25(OH)(2)D(3), its 20-epi derivative MC1288, the two side-chain analogues Gemini and Ro43-83582 (a hexafluoro-derivative) by molecular dynamics simulations in a complex with the vitamin D receptor ligand-binding domain and a co-activator peptide. Superimposition of the structures showed that the side chain of MC1288, the first side chain of the conformation II of Gemini, the second side chain of Ro43-83582 in conformation I and the first side chain of Ro43-83582 in conformation II take the same agonistic position as the side chain of 1alpha,25(OH)(2)D(3). Compared with the LBP of the natural hormone MC1288 reduced the volume by 17%, and Gemini expanded it by 19%. The shrinking of the LBP of MC1288 and its expansion to accommodate the second side chain of Gemini or Ro43-83582 is the combined result of minor movements of more than 30 residues and major movements of a few critical amino acids. The agonist-selective recognition of anchoring OH groups by the conformational flexible residues Ala-303, Leu-309, and His-397 was confirmed by in vitro assays. In summary, variations in the volume of agonists lead to adaptations in the volume of the LBP and alternative contacts of anchoring OH-groups.

Amino Acids↗