Low-resolution structures of thyroid hormone receptor dimers and tetramers in solution

High-resolution X-ray structures of thyroid hormone (TH) receptor (TR) DNA and ligand binding domains (DBD and LBD) have yielded significant insights into TR action. Nevertheless, the TR DBD and LBD act in concert to mediate TH effects upon gene expression, and TRs form multiple oligomers; however, structures of full-length TRs or DBD-LBD constructs that would clarify these influences are not available. Here, we report low-resolution X-ray structures of the TRbeta DBD-LBD construct in solution which define the shape of dimers and tetramers and likely positions of the DBDs and LBDs. The holo TRbeta DBD-LBD construct forms a homodimer with LBD-DBD pairs in close contact and DBDs protruding from the base in the same direction. The DBDs are connected to the LBDs by crossed extended D domains. The apo hTRbeta DBD-LBD construct forms tetramers that resemble bulged cylinders with pairs of LBD dimers in a head-to-head arrangement with DBD pairs packed tightly against the LBD core. Overall, there are similarities with our previous low-resolution structures of retinoid X receptors, but TRs exhibit two unique features. First, TR DBDs are closely juxtaposed in the dimer and tetramer forms. Second, TR DBDs are closely packed against LBDs in the tetramer, but not the dimer. These findings suggest that TRs may be able to engage in hitherto unknown interdomain interactions and that the D domain must rearrange in different oligomeric forms. Finally, the data corroborate our suggestion that apo TRs form tetramers in solution which dissociate into dimers upon hormone binding.     

MDA5 cooperatively forms dimers and ATP-sensitive filaments upon binding double-stranded RNA

Melanoma differentiation-associated gene-5 (MDA5) detects viral double-stranded RNA in the cytoplasm. RNA binding induces MDA5 to activate the signalling adaptor MAVS through interactions between the caspase recruitment domains (CARDs) of the two proteins. The molecular mechanism of MDA5 signalling is not well understood. Here, we show that MDA5 cooperatively binds short RNA ligands as a dimer with a 16-18-basepair footprint. A crystal structure of the MDA5 helicase-insert domain demonstrates an evolutionary relationship with the archaeal Hef helicases. In X-ray solution structures, the CARDs in unliganded MDA5 are flexible, and RNA binds on one side of an asymmetric MDA5 dimer, bridging the two subunits. On longer RNA, full-length and CARD-deleted MDA5 constructs assemble into ATP-sensitive filaments. We propose a signalling model in which the CARDs on MDA5-RNA filaments nucleate the assembly of MAVS filaments with the same polymeric geometry.     

Human adenosine deaminase as an allosteric modulator of human A(1) adenosine receptor: abolishment of negative cooperativity for [H](R)-pia binding to the caudate nucleus

It has been shown that adenosine deaminase (ADA; EC 3.5.4.4) behaves as an ecto-enzyme anchored to membrane proteins, among them A(1) adenosine receptors (A(1)Rs). Bovine ADA interacts with A(1)Rs from many species and regulates agonists binding to receptors in an activity-independent form. However, it was not known whether human ADA exerted any effect on the agonist binding to human A(1)Rs, because of both technical difficulties in obtaining pure human ADA and tissues containing human A(1)Rs. In this study, human ADA was purified to homogeneity. Taking in consideration that A(1)Rs form homodimers and taking advantage of a new procedure to fit binding data to receptors dimers, which allows to calculate ligand dissociation constants and the degree of cooperativity between the two subunits in the dimer, here it is demonstrated that human ADA markedly enhances the agonist and antagonist affinity and abolishes the negative cooperativity on agonist binding to human striatal A(1)Rs. ADA also increases the ability of the agonist to decrease the forskolin-induced cAMP levels. The results show that human ADA, apart from reducing the adenosine concentration and thus preventing A(1)R desensitization, binds to A(1)R behaving as an allosteric effector that markedly enhances agonist affinity and increases receptor functionality. The physiological role of the interaction is to make receptors more sensitive to adenosine. This powerful regulation has important implications for the physiology and pharmacology of neuronal A(1)Rs.     

Molecular dynamics simulations of apo and holo forms of fatty acid binding protein 5 and cellular retinoic acid binding protein II reveal highly mobile protein,retinoic acid ligand,and water molecules

Structural and dynamic properties from a series of 300 ns molecular dynamics, MD, simulations of two intracellular lipid binding proteins, iLBPs, (Fatty Acid Binding Protein 5, FABP5, and Cellular Retinoic Acid Binding Protein II, CRABP-II) in both the apo form and when bound with retinoic acid reveal a high degree of protein and ligand flexibility. The ratio of FABP5 to CRABP-II in a cell may determine whether it undergoes natural apoptosis or unrestricted cell growth in the presence of retinoic acid. As a result, FABP5 is a promising target for cancer therapy. The MD simulations presented here reveal distinct differences in the two proteins and provide insight into the binding mechanism. CRABP-II is a much larger, more flexible protein that closes upon ligand binding, where FABP5 transitions to an open state in the holo form. The traditional understanding obtained from crystal structures of the gap between two β-sheets of the β-barrel common to iLBPs and the α-helix cap that forms the portal to the binding pocket is insufficient for describing protein conformation (open vs. closed) or ligand entry and exit. When the high degree of mobility between multiple conformations of both the ligand and protein are examined via MD simulation, a new mode of ligand motion that improves understanding of binding dynamics is revealed.     

Effects of hyperosmolarity on ligand processing and receptor recycling in the hepatic galactosyl receptor system

Binding, endocytosis, and degradation of asialo-orosomucoid (ASOR) mediated by the galactosyl (Gal) receptor were examined in isolated rat hepatocytes in complete media supplemented with an osmolite. The specific binding of 125I-ASOR to cells at 4 degrees C was unaffected by up to 0.4 M sucrose or NaCl. Unlike sucrose or NaCl, mannitol stimulated 125I-ASOR binding at low concentrations but inhibited binding at higher concentrations. Continuous internalization at 37 degrees C, which requires receptor recycling, was completely blocked at 0.2 M sucrose or 0.15 M NaCl, corresponding in each case to a total osmolality of about 550 mmol/kg. This effect was reversed and endocytic function was restored by washing the cells, indicating that cell viability was unaffected. The rate of degradation of internalized 125I-ASOR was also inhibited by increasing sucrose concentrations. This inhibition is due to a block in the delivery of ligand to lysosomes and not an effect on degradation per se. In the presence of 0.2 M sucrose, the rate and extent of endocytosis of surface-bound 125I-ASOR were, respectively, 33.0 +/- 8.1% and 69.4 +/- 10.5% (n = 8) of the control without sucrose. Under these conditions, the dissociation of internalized receptor-ASOR complexes was completely inhibited. When sucrose was added, the effect on the endocytosis of surface-bound 125I-ASOR was virtually immediate. Previous studies showed that about 40% of the surface-bound 125I-ASOR which is internalized can return to the cell surface still bound to receptor (Weigel and Oka: J Biol Chem 259:1150, 1984). If 0.2 M sucrose was added after endocytosis occurred, 125I-ASOR still returned to the cell surface, although the rate and extent of return were inhibited by more than 50%. Interestingly, hyperosmolarity is the only treatment we have found which can reversibly inhibit, although only partially, the endocytosis of surface-bound 125I-ASOR.     

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.     

Control of estrogen receptor ligand binding by Hsp90

The molecular chaperone Hsp90 interacts with unliganded steroid hormone receptors and regulates their activity. We have analyzed the function of yeast and mammalian Hsp90 in regulating the ability of the human estrogen receptor (ER) to bind ligands in vivo and in vitro. Using the yeast system, we show that the ER expressed in several different hsp82 mutant strains binds reduced amounts of the synthetic estrogen diethylstilbestrol compared to the wild type. This defect in hormone binding occurs without any significant change in the steady state levels of ER protein. To analyze the role of mammalian Hsp90, we synthesized the human ER in rabbit reticulocyte lysates containing geldanamycin, an Hsp90 inhibitor. At low concentrations of geldanamycin we observed reduced levels of hormone binding by the ER. At higher concentrations, we found reduced synthesis of the receptor. These data indicate that Hsp90 functions to maintain the ER in a high affinity hormone-binding conformation.     

A high-throughput fluorescent polarization assay for nuclear receptor binding utilizing crude receptor extract.

A homogenous high-throughput assay has been developed to measure the binding between nuclear receptors and test compounds. This assay applies a fluorescence polarization (FP) detection method using human glucocorticoid receptor (GR) as a model system. Crude receptor extract, which requires no additional purification, is used in the assay. The binding conditions (i.e., DMSO tolerance, temperature, stability, and variability) have been investigated and validated. At the optimized conditions, a signal-to-background ratio of 2:1 and a Z'-factor of 0.7 was achieved in a 384-well format. Several known strong and weak GR ligands have been evaluated in this system. Possible interference of fluorescent compounds and methods to identify false positives are also discussed. This FP-based assay system can potentially be used for many soluble nuclear receptors in high-throughput binding assays.     

4Ca2+.troponin C forms dimers in solution at neutral pH that dissociate upon binding various peptides: small-angle X-ray scattering studies of peptide-induced structural changes.

Small-angle X-ray scattering data have been measured for rabbit skeletal muscle troponin C and its complexes with the venom peptides melittin and mastoparan as well as synthetic peptides based on regions of the troponin I sequence implicated in troponin C binding. At the neutral pH used in this study (pH 6.8), troponin C shows a tendency to form dimers in the presence of 4 mol equiv of Ca2+, but is monomeric in solution when 2 or less mol equiv of Ca2+ is present. The 4Ca2+.troponin C dimers dissociate upon binding melittin, mastoparan, and peptides based on residues 96-115, 1-30, and 1-40 in the troponin I sequence. This result suggests that the peptide-binding sites overlap with the regions of contact between troponin C molecules forming a dimer. Like the structurally homologous calcium-binding protein calmodulin, troponin C shows conformational flexibility upon binding different peptides. Upon binding melittin, troponin C contracts in a similar manner to calmodulin when it binds peptides known to form amphiphilic helices (e.g., melittin, mastoparan, or MLCK-I). In contrast, mastoparan binding to troponin C does not result in a contracted structure. The scattering data indicate troponin C also remains in an extended structure upon binding the inhibitory peptides having the same sequence as residues 96-115 in troponin I.     

Order changes within receptor systems upon ligand binding: receptor tightening/oligomerisation and the interpretation of binding parameters

Recent hydrogen-deuterium exchange experiments have highlighted tightening and loosening of protein structures upon ligand binding, with changes in bonding (DeltaH) and order (DeltaS) which contribute to the overall thermodynamics of ligand binding. Tightening and loosening show that ligand binding respectively stabilises or destabilises the internal structure of the protein, i.e. it shows positive or negative cooperativity between ligand binding and the receptor structure. In the case of membrane-bound receptors, such as G protein-coupled receptors (GPCRs) and ligand gated ion channel receptors (LGICRs), most binding studies have focussed on association/dissociation constants. Where these have been broken down into enthalpic and entropic contributions, the phenomenon of "thermodynamic discrimination" between antagonists and agonists has often been noted; e.g. for a receptor where agonist binding is predominantly enthalpy driven, antagonist binding is predominantly entropy driven and vice versa. These data have not previously been considered in terms of the tightening, or loosening, of receptor structures that respectively occurs upon positively, or negatively, cooperative binding of ligand. Nor have they been considered in light of the homo- and hetero-oligomerisation of GPCRs and the possibility of ligand-induced changes in oligomerisation. Here, we argue that analysis of the DeltaH and DeltaS of ligand binding may give useful information on ligand-induced changes in membrane-bound receptor oligomers, relevant to the differing effects of agonists and antagonists.     

High activity,soluble, bacterially expressed human vitamin D receptor and its ligand binding domain

The effects of 1α,25(OH)₂vitamin D₃ on cell growth and differentiation are primarily mediated by the nuclear vitamin D receptor (VDR). In order to study aspects of receptor function and ultimately the structural basis of the VDR-ligand interaction, it is necessary to produce large quantities of purified VDR. To achieve this, we have expressed the human VDR and its ligand binding domain in E. coli as fusion proteins with the maltose binding protein using the expression vector pMal-c2. In this system high level expression of both fusion proteins in a soluble form was achieved, whereas previous attempts to express the VDR in E. coli have resulted in an insoluble product. After affinity purification on amylose resin, the fusion proteins were isolated with yields of 10–20 mg/l of culture. Both forms of the recombinant receptor bound 1α,25(OH)₂vitamin D₃ with high affinity; estimated K_d values from Scatchard analysis for the purified full-length receptor and the ligand binding domain were 0.16 ± 0.07 nM and 0.04 ± 0.02 nM, respectively. The nonhypercalcemic analogs of vitamin D, MC903 and Δ22-1,25S,26(OH)₃vitamin D₃, bound the recombinant fusion proteins with a similar affinity to the native ligand, 1α,25(OH)₂vitamin D₃. In addition, the full-length VDR fusion protein was shown by gel shift analysis to bind weakly to the human osteocalcin gene vitamin D response element, an interaction greatly facilitated by addition of RXRα. These results show that the bacterial expression system detailed here is readily able to produce soluble and functional VDR and its ligand binding domain in high yield. These proteins are easily purified and should be suitable for further structural and functional analysis. © 1996 Wiley-Liss, Inc.     

Transmembrane domain of EphA1 receptor forms dimers in membrane-like environment

Eph receptor tyrosine kinases (RTKs) are activated by a ligand-mediated dimerization in the plasma membrane and subjected to clusterization at a high local density of receptors and their membrane-anchored ligands. Interactions between transmembrane domains (TMDs) were recognized to assist to the ligand-binding extracellular domains in the dimerization of some RTKs, whereas a functional role of Eph-receptor TMDs remains unknown. We have studied a propensity of EphA1-receptor TMDs (TMA1) to self-association in membrane-mimetic environment. Dimerization of TMA1 in SDS environment was revealed by SDS-PAGE and confirmed by FRET analysis of the fluorescently labeled peptide (K_d = 7.2 ± 0.4 μM at 1.5 mM SDS). TMA1 dimerization was also found in 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes (ΔG = −15.4 ± 0.5 kJ/mol). Stability of TMA1 dimers is comparable to the reported earlier stability of TMD dimers of fibroblast growth factor receptor 3 and tenfold weaker than the stability of TMD dimers of glycophorin A possessing high propensity to dimerization. Our results suggest that EphA1-receptor TMD contribute to the dimerization-mediated receptor activation. An assumed role of the TMD interactions is the efficient signal transduction due to TMD-driving mutual orientation of kinase domains in dimers, while a relatively low force of the TMD interactions does not prevent a ligand-controlled regulation of the receptor dimerization.     

Stabilization of C5a receptor–G-protein interactions through ligand binding

Binding of biotin-C5a to the C5a receptor in membrane fragments followed by detergent solubilization and purification with streptavidin-agarose affinity chromatography resulted in the isolation of a receptor complex with associated G-proteins. In contrast, when receptor was detergent-solubilized in the absence of C5a and purified by affinity chromatography with Affigel-C5a, G-proteins did not copurify. Since the results indicate that receptor ligation stabilized the receptor–G-protein interaction to allow purification of the complex, the findings emphasize the dynamic nature of the C5a receptor–effector interactions. When biotin-C5a–ligated receptor was purified from a mouse cell line overexpressing recombinant human receptor, both G_ialpha₂ and G_ialpha₃ subunits copurified, confirming that multiple transducing systems are linked to the C5a receptor. The method of stabilization of receptor-transducer complexes offers the opportunity to further elaborate the interactions of the C5a receptor with diverse transducing elements and second messenger systems. © 1994 Wiley-Liss, Inc.     

Different effects of lectins on the ligand binding of the NMDA receptors and sigma sites in rat brain hippocampus synaptic membranes

The effects of the lectins concanavalin A, WGA, ricin, abrin, and the mistletoe lectins from Viscum album MLI, MLII, and MLIII on the binding of ligands of the NMDA and sigma receptors in rat hippocampus synaptic plasma membranes were investigated. Binding of [³H]MK-801, [³H]glutamate, [³H]5,7-DCKA, and [³H]glycine to the membranes was decreased by 40-60% after addition of galactose-specific lectins (mistletoe lectins MLI, MLII, ricin, abrin) at concentrations of 0.01 mg/ml, but was not affected by the glucose- and mannose-specific lectin Con A, an acetylglucosamine-specific lectin WGA, or an acetylgalactosamine-specific lectin MLIII. The binding of [³H]SKF 10047 was decreased only in the presence of MLIII and did not change after addition of the other lectins. It is suggested that lectin-sensitive ligand binding sites of sigma- and NMDA receptors are located separately, and that the carbohydrate side chains of the sigma receptor do not participate in the modulation of the NMDA-receptor.     

Identification of amino acid residues responsible for the alpha5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABA(A) receptor

L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.     

Novel fluorescence based receptor binding assay method for receptors lacking ligand conjugates with preserved affinity: study on estrogen receptor alpha

In this study a novel general approach is presented that allows for a straightforward design of receptor binding assays. This principle of a receptor binding assay is applied to the estrogen receptor, which is important in the management of breast cancer and for the estimation of the estrogenic potency of chemicals in the environment. The inhibitory concentrations to reduce cell proliferation in 50% of controls for 17-beta-estradiol, 4-hydroxy tamoxifen, and tamoxifen are determined to be 61 nM, 33 nM, and 17 microM, respectively. The measurement time of the nanoparticle based immunoassay format is 3 s. The Z' factor, which is calculated to be 0.89, reflects the excellent assay performance.     

Invariant aspartic Acid in muscle nicotinic receptor contributes selectively to the kinetics of agonist binding

We examined functional contributions of interdomain contacts within the nicotinic receptor ligand binding site using single channel kinetic analyses, site-directed mutagenesis, and a homology model of the major extracellular region. At the principal face of the binding site, the invariant alphaD89 forms a highly conserved interdomain contact near alphaT148, alphaW149, and alphaT150. Patch-clamp recordings show that the mutation alphaD89N markedly slows acetylcholine (ACh) binding to receptors in the resting closed state, but does not affect rates of channel opening and closing. Neither alphaT148L, alphaT150A, nor mutations at both positions substantially affects the kinetics of receptor activation, showing that hydroxyl side chains at these positions are not hydrogen bond donors for the strong acceptor alphaD89. However substituting a negative charge at alphaT148, but not at alphaT150, counteracts the effect of alphaD89N, demonstrating that a negative charge in the region of interdomain contact confers rapid association of ACh. Interpreted within the structural framework of ACh binding protein and a homology model of the receptor ligand binding site, these results implicate main chain amide groups in the domain harboring alphaW149 as principal hydrogen bond donors for alphaD89. The specific effect of alphaD89N on ACh association suggests that interdomain hydrogen bonding positions alphaW149 for optimal interaction with ACh.