Identification of the hydrophobic ligand binding pocket of the S1P1 receptor

Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P1 GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head group-interacting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [32P]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P1 to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P1.     

An activation switch in the ligand binding pocket of the C5a receptor

Although agonists are thought to occupy binding pockets within the seven-helix core of serpentine receptors, the topography of these binding pockets and the conformational changes responsible for receptor activation are poorly understood. To identify the ligand binding pocket in the receptor for complement factor 5a (C5aR), we assessed binding affinities of hexapeptide ligands, each mutated at a single position, for seven mutant C5aRs, each mutated at a single position in the putative ligand binding site. In ChaW (an antagonist) and W5Cha (an agonist), the side chains at position 5 are tryptophan and cyclohexylalanine, respectively. Comparisons of binding affinities indicated that the hexapeptide residue at this position interacts with two C5aR residues, Ile-116 (helix III) and Val-286 (helix VII); in a C5aR model these two side chains point toward one another. Both the I116A and the V286A mutations markedly increased binding affinity of W5Cha but not that of ChaW. Moreover, ChaW, the antagonist hexapeptide, acted as a full agonist on the I116A mutant. These results argue that C5aR residues Ile-116 and Val-286 interact with the side chain at position 5 of the hexapeptide ligand to form an activation switch. Based on this and previous work, we present a docking model for the hexapeptide within the C5aR binding pocket. We propose that agonists induce a small change in the relative orientations of helices III and VII and that these helices work together to allow movement of helix VI away from the receptor core, thereby triggering G protein activation.     

Alanine scanning mutational analysis of the ligand binding pocket of the human Vitamin D receptor

We achieved exhaustive alanine scanning mutational analysis of the amino acid residues lining the ligand binding pocket of the Vitamin D receptor to investigate the mechanism of the ligand recognition by the receptor. This is the first exhaustive analysis in the nuclear receptor superfamily. Our results demonstrated the role and importance of all the residues lining the ligand binding pocket. In addition, this analysis was found to indicate ligand-specific ligand-protein interactions, which have key importance in determining the transactivation potency of the individual ligands. Thus, the analysis using 1beta-methyl-1alpha,25-dihydroxyvitamin D(3) revealed the specific van der Waals interactions of 1beta-methyl group with the receptor.     

Mapping the ligand binding pocket in the cellular retinaldehyde binding protein

Retinoid interactions determine the function of the cellular retinaldehyde binding protein (CRALBP) in the rod visual cycle where it serves as an 11-cis-retinol acceptor for the enzymatic isomerization of all-trans- to 11-cis-retinol and as a substrate carrier for 11-cis-retinol dehydrogenase (RDH5). Based on preliminary NMR studies suggesting retinoid interactions with Met and Trp residues, human recombinant CRALBP (rCRALBP) with altered Met or Trp were produced and analyzed for ligand interactions. The primary structures of the purified proteins were verified for mutants M208A, M222A, M225A, W165F, and W244F, then retinoid binding properties and substrate carrier functions were evaluated. All the mutant proteins bound 11-cis- and 9-cis-retinal and therefore were not grossly misfolded. Altered UV-visible spectra and lower retinoid binding affinities were observed for the mutants, supporting modified ligand interactions. Altered kinetic parameters were observed for RDH5 oxidation of 11-cis-retinol bound to rCRALBP mutants M222A, M225A, and W244F, supporting impaired substrate carrier function. Heteronuclear single quantum correlation NMR analyses confirmed localized structural changes upon photoisomerization of rCRALBP-bound 11-cis-retinal and demonstrated ligand-dependent conformational changes for residues Met-208, Met-222, Trp-165, and Trp-244. Furthermore, residues Met-208, Met-222, Met-225, and Trp-244 are within a region exhibiting high homology to the ligand binding cavity of phosphatidylinositol transfer protein. Overall the data implicate Trp-165, Met-208, Met-222, Met-225, and Trp-244 as components of the CRALBP ligand binding cavity.     

Plasticity of the ligand binding pocket in the bitter taste receptor T2R7

Bitter taste receptors (T2Rs) are a group of 25 G protein-coupled receptors (GPCRs) in humans. The cognate agonists and the mechanism of ligand binding to the majority of the T2Rs remain unknown. Here we report the first structure-function analysis of T2R7 and study the ability of this receptor to bind to different agonists by site-directed mutagenesis. Screening of ligands for T2R7 in calcium based assays lead to the identification of novel compounds that activate this receptor. Quinine, diphenidol, dextromethorphan and diphenhydramine showed substantial activation of T2R7. Interestingly, these bitter compounds showed different pharmacological characteristics. To investigate the structural features in T2R7 that might contribute to the observed differences in agonist specificities, molecular model guided ligand docking and site-directed mutagenesis was pursued. Amino acids D65, D86, W89, N167, T169, W170, S181, T255 and E271 in the ligand-binding pocket were replaced and the mutants characterized pharmacologically. Our results suggest D86, S181 and W170 present on the extracellular side of transmembrane 3 (TM3), TM5 and in extracellular loop 2 (ECL2) are essential for agonist binding in T2R7. Mutations of these amino acids lead to loss-of-function. We also identified gain-of-function residues that are agonist specific. These results suggest that agonists bind at an extracellular site rather than deep within the TM core involving residues present in both ECL2 and TM helices in T2R7. Similar to majority of the Class A GPCRs, ECL2 in T2R7 plays a significant role in agonist binding and activation.     

Reduction of glucocorticoid receptor ligand binding by the 11-beta hydroxysteroid dehydrogenase type 2 inhibitor, Thiram

Endogenous and synthetic glucocorticoids (GCs), such as cortisol and dexamethasone (Dex), modulate airway inflammation, regulate the production of surfactant by lung epithelial cells, and influence fetal lung maturation. The 11-beta hydroxysteroid dehydrogenase type 2 (HSD2) enzyme catalyzes the oxidation of bioactive cortisol and Dex to their 11-keto metabolites. Thiram (tetramethylthiuram disulfide) specifically inhibits HSD2 activity by oxidizing cysteine residues located in the cofactor binding domain of the enzyme. During studies performed to define a potential role for HSD2 in modulating GC action in human lung epithelial cells, we observed that exposure of intact human lung epithelial cells (NCI-H441) to 50 microM Thiram significantly attenuated the down-stream effects of Dex (100 nM) on the expression of two GC-sensitive genes, pulmonary surfactant proteins A and B. This observation appeared to be inconsistent with simple inhibition of HSD2 activity. Although Thiram inhibited HSD2 oxidase activity in a dose-dependent manner without affecting HSD2 protein expression, Thiram also reduced specific binding of [3H]-Dex to the glucocorticoid receptor (GR). Pre-treatment of cells with 1 mM dithiothreitol (DTT), a thiol-reducing agent, completely blocked the inhibitory effect of Thiram on ligand binding. These results are suggestive that Thiram may alter the ligand-binding domain of the GR by oxidizing critical thiol-containing amino acid residues. Taken collectively, these data demonstrate that attenuated down-stream GC signaling, via decreased binding of ligand to the GR, is a novel cellular effect of Thiram exposure in human lung epithelial cells.     

The association between a negatively charged ligand and the electronegative binding pocket of its receptor.

Many examples exist of charged amino acids that play a role in attracting or holding a charged ligand toward or inside an oppositely charged binding pocket of the protein. For example, the enzymes superoxide dismutase, triose-phosphate isomerase, and acetylcholinesterase can steer ligands toward their oppositely charged binding pockets or gorges. Interestingly, in our Brownian dynamics simulations of a phosphate-binding protein, we discovered that negatively charged phosphate (HPO(2-)(4)) could make its way into the negatively charged binding pocket. In fact, the phosphate-binding protein exhibits counterintuitive kinetics of association. That is, one would expect that the rate of association would increase on increases to the ionic strength since the interaction between the ligand, with a charge of -2, and the electronegative binding pocket would be repulsive and greater screening should reduce this repulsion and increase the rate of association. However, the opposite is seen-i.e., the rate of association decreases on increases in the ionic strength. We used Brownian dynamics techniques to compute the diffusion limited association rate constants between the negatively charged phosphate ligand and several open forms of PBP (wild-type and several mutants based on an x-ray structure of open-form PBP, mutant T141D). With the appropriate choices of reaction criteria and molecular parameters, the ligand was able to diffuse into the binding pocket. A number of residues influence binding of the ligand within the pocket via hydrogen bonds or salt bridges. Arg135 partially neutralizes the charges on the HPO(2-)(4) ligand in the binding pocket, allowing it to enter. It is also found that the positive electrostatic patches above and below the binding entrance of PBP contribute the major attractive forces that direct the ligand toward the surface of the protein near the binding site.     

Evidence for preorganization of the glmS ribozyme ligand binding pocket

We have examined the tertiary structure of the ligand-activated glmS ribozyme by a combination of methods with the aim of evaluating the magnitude of RNA conformational change induced by binding of the cofactor, glucosamine 6-phosphate (GlcN6P). Hydroxyl radical footprinting of a trans-acting ribozyme complex identifies several sites of solvent protection upon incubation of the RNA in Mg(2+)-containing solutions, providing initial evidence of the tertiary fold of the ribozyme. Under these folding conditions and at GlcN6P concentrations that saturate the ligand-induced cleavage reaction, we do not observe changes to this pattern. Cross-linking with short-wave UV light of the complex yielded similar overall results. In addition, ribozyme-substrate complexes cross-linked in the absence of GlcN6P could be gel purified and then activated in the presence of ligand. One of these active cross-linked species links the base immediately 3' of the cleavage site to a highly conserved region of the ribozyme core and could be catalytically activated by ligand. Combined with recent studies that argue that GlcN6P acts as a coenzyme in the reaction, our data point to a riboswitch mechanism in which ligand binds to a prefolded active site pocket and assists in catalysis via a direct participation in the reaction chemistry, the local influence on the geometry of the active site constituents, or a combination of both mechanisms. This mode of action is different from that observed for other riboswitches characterized to date, which act by inducing secondary and tertiary structure changes.     

Influence of molybdate, ionic strength and pH on ligand binding to the glucocorticoid receptor

The addition of molybdate to rabbit liver cytosol increased significantly the affinity of the glucocorticoid receptor for [3H] dexamethasone without influencing the concentration of binding sites. This effect was concentration dependent. Analysis of the binding data by curve-fitting and Scatchard plot revealed the occurrence of a complex binding process in the presence of molybdate. The pH-dependence curve of the binding was shifted towards alkaline values by the oxyanion. Taken together, these data suggest that molybdate exerts its effects via an interaction with the receptor molecule.     

Fluorescence analysis of the size of a binding pocket of a peptide receptor at natural abundance

We have studied the topography of interaction of a family of fluorescent formyl peptides containing four (CHO-Met-Leu-Phe-Lys-fluorescein), five (CHO-Met-Leu-Phe-Phe-Lys- fluorescein), and six (CHO-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein and CHO-Met-Leu-Phe-Phe-Phe-Lys- fluorescein) amino acids with their receptor using spectroscopic methods adapted to small sample volumes. Only the fluorescent peptides containing four and five amino acids were quenched upon binding to the receptor, indicating physical contact of the chromophore with the receptor. In contrast, only the hexapeptides were accessible to antibodies to fluorescein. Taken together, these results suggest that the carboxy terminus of the tetrapeptide or the pentapeptide is protected in the receptor binding pocket while the fluorescein on the carboxy terminus of either hexapeptide is exposed and recognized by the antibody to fluorescein. These results indicate that the binding pocket accommodates at least five but no more than six amino acids.     

Conformational change in the human glucocorticoid receptor induced by ligand binding is altered by mutation of isoleucine 747 by a threonine

Limited proteolysis experiments were performed to study conformation changes induced by ligand binding on in vitro produced wild-type and I747T mutant glucocorticoid receptors. Dexamethasone-induced conformational changes were characterized by two resistant proteolysis fragments of 30 and 27 kDa. Although dexamethasone binding affinity was only slightly altered by the I747T substitution (Roux, S., Térouanne, B., Balaguer, P., Loffreda-Jausons, N., Pons, M., Chambon, P., Gronemeyer, H., and Nicolas, J.-C. (1996) Mol. Endocrinol. 10, 1214-1226), higher dexamethasone concentrations were required to obtain the same proteolysis pattern. This difference was less marked when proteolysis experiments were conducted at 0 degrees C, indicating that a step of the conformational change after ligand binding was affected by the mutation. In contrast, RU486 binding to the wild-type receptor induced a different conformational change that was not affected by the mutation. Analysis of proteolysis fragments obtained in the presence of dexamethasone or RU486 indicated that the RU486-induced conformational change affected the C-terminal part of the ligand binding domain differently. These data suggest that the ligand-induced conformational change occurs via a multistep process. In the first step, characterized by compaction of the ligand binding domain, the mutation has no effect. The second step, which stabilizes the activated conformation and does not occur at 4 degrees C, seems to be a key element in the activation process that can be altered by the mutation. This step could involve modification of the helix H12 position, explaining why the conformation induced by RU486 is not affected by the mutation.     

Evaluation of the role of ligand and thermal activation of specific DNA binding by in vitro synthesized human glucocorticoid receptor

We have used a DNA-binding/immunoprecipitation assay to analyze the capacity of human glucocorticoid receptor (hGR), generated in rabbit reticulocyte lysates, to bind DNA. In vitro translated hGR was indistinguishable from native hGR, as determined by migration on sodium dodecyl sulfate-polyacrylamide gels, sedimentation on sucrose density gradients, and reactivity with antipeptide antibodies generated against hGR. In addition, cell-free synthesized hGR was capable of specific binding to glucocorticoid response element (GRE)-containing DNA fragments. Using this assay system, we have evaluated the contributions of ligand binding and heat activation to DNA binding by these glucocorticoid receptors. In vitro translated hGR was capable of selective DNA binding even in the absence of glucocorticoid. Treatment with dexamethasone or the antiglucocorticoid RU486 had no additional effect on the DNA-binding capacity when receptor preparations were maintained at 0 C (no activation). In contrast, addition of either ligand or antagonist in combination with a heat activation step promoted DNA binding by approximately 3-fold over that of heat-activated unliganded receptors. Agonist (dexamethasone) was slightly more effective in supporting specific DNA binding than antagonist (RU486). DNA binding by in vitro synthesized GR was blocked by the addition of sodium molybdate to the receptor preparations before steroid addition and thermal activation. Addition of KCl resulted in less DNA binding either due to blockage of DNA-receptor complex formation or disruption of the complexes. The specificity of DNA binding by cell-free synthesized hGR was analyzed further by examining the abilities of various DNAs to compete for binding to a naturally occurring GRE found in the mouse mammary tumor virus-long terminal repeat. Oligonucleotides containing the consensus GRE were the most efficient competitors, and fragments containing regulatory sequences from glucocorticoid-repressible genes were somewhat competitive, whereas single stranded oligonucleotides were unable to compete for mouse mammary tumor virus-long terminal repeat DNA binding, except when competitor was present at extremely high concentrations. Together these studies indicate that hGR synthesized in rabbit reticulocyte lysates displays many of the same properties, including GRE-specific DNA binding, observed for glucocorticoid receptor present in cytosolic extracts of mammalian cells and tissues. Similarities between the effects of dexamethasone and RU486 suggest that the antiglucocorticoid properties of RU486 do not occur at the level of specific DNA binding.     

Impact of ligand and protein desolvation on ligand binding to the S1 pocket of thrombin

In the present study, we investigate the impact of a tightly bound water molecule on ligand binding in the S1 pocket of thrombin. The S1 pocket contains a deeply buried deprotonated aspartate residue (Asp189) that is, due to its charged state, well hydrated in the uncomplexed state. We systematically studied the importance of this water molecule by evaluating a series of ligands that contains pyridine-type P1 side chains that could potentially alter the binding properties of this water molecule. All of the pyridine derivatives retain the original hydration state albeit sometimes with a slight perturbance. In order to prevent a direct H-bond formation with Asp189, and to create a permanent positive charge on the P1 side chain that is positioned adjacent to the Asp189 carboxylate anion, we methylated the pyridine nitrogen. This methylation resulted in displacement of water but was accompanied by a loss in binding affinity. Quantum chemical calculations of the ligand solvation free energy showed that the positively charged methylpyridinium derivatives suffer a large penalty of desolvation upon binding. Consequently, they have a substantially less favorable enthalpy of binding. In addition to the ligand desolvation penalty, the hydration shell around Asp189 has to be overcome, which is achieved in nearly all pyridinium derivatives. Only for the ortho derivative is a partial population of a water next to Asp189 found. Possibly, the gain of electrostatic interactions between the charged P1 side chain and Asp189 helps to compensate for the desolvation penalty. In all uncharged pyridine derivatives, the solvation shell remains next to Asp189, partly mediating interactions between ligand and protein. In the case of the para-pyridine derivative, a strongly disordered cluster of water sites is observed between ligand and Asp189.     

Modulation of DNA binding of glucocorticoid receptor by aurintricarboxylic acid

Effects of aurintricarboxylic acid (ATA) were examined on the DNA binding properties of rat liver glucocorticoid-receptor complex. The DNA-cellulose binding capacity of the glucocorticoid-receptor complex was completely abolished by a pretreatment of receptor preparation with 0.1-0.5 mM ATA at 4 degrees C. The half-maximal inhibition (i.d.50) in the DNA binding of [3H]triamcinolone acetonide-receptor complex [( 3H]TARc) was observed at 130- and 40 microM ATA depending upon whether the inhibitor was added prior to or following the receptor activation. The entire DNA-cellulose bound [3H]TARc could be extracted in a concentration-dependent manner by incubation with 2-100 microns ATA. The [3H]TARc remained intact under the above conditions, the receptor in both control and ATA-treated preparations sedimented in the same region in salt-containing 5-20% sucrose gradients. The action of ATA appeared to be on the receptor and not on DNA-cellulose. The DNA-binding capacity of ATA-treated receptor preparations could be recovered upon exhaustive dialysis. The treatment with ATA did not appear to change the ionic behavior of heat activated GRc; the receptor in both control and the ATA-treated preparations showed similar elution profiles. Therefore, ATA appears to alter the binding to and dissociation of glucocorticoid-receptor complex from DNA. The use of ATA should offer a good chemical probe for analysis of the DNA binding domain(s) of the glucocorticoid receptor.     

Alternate glucocorticoid receptor ligand binding structures influence outcomes in an in vivo tissue regeneration model

Since their characterization, glucocorticoids (GCs), the most commonly prescribed immunomodulatory drugs, have undergone numerous structural modifications designed to enhance their activity. In vivo assessment of these corticosteroid analogs is essential to understand the difference in molecular signaling of the ligands that share the corticosteroid backbone. Our research identified a novel function of GCs as modulators of tissue regeneration and demonstrated that GCs activate the glucocorticoid receptor (GR) to inhibit early stages of tissue regeneration in zebrafish (Danio rerio). We utilized this phenomenon to assess the effect of different GC analogs on tissue regeneration and identified that some GCs such as beclomethasone dipropionate (BDP) possess inhibitory properties, while others, such as dexamethasone and hydrocortisone have no effect on regeneration. We performed in silico molecular docking and dynamic studies and demonstrated that type and size of substitution at the C17 position of the cortisol backbone confer a unique stable conformation to GR on ligand binding that is critical for inhibitory activity. In the field of tissue regeneration, our study is one of the first Structure Activity Relationship (SAR) investigations performed in vertebrates demonstrating that the in vivo tissue regeneration model is a powerful tool to probe structure function relationships, to understand regenerative biology, and to assist in rational drug design.