Structure Of Ligand-binding Domain Research Articles

Crystal Structure of Human Lectin-like, Oxidized Low-Density Lipoprotein Receptor 1 Ligand Binding Domain and Its Ligand Recognition Mode to OxLDL

Lectin-like, oxidized low-density lipoprotein (LDL) receptor 1, LOX-1, is the major receptor for oxidized LDL (OxLDL) in endothelial cells. We have determined the crystal structure of the ligand binding domain of LOX-1, with a short stalk region connecting the domain to the membrane-spanning region, as a homodimer linked by an interchain disulfide bond. In vivo assays with LOX-1 mutants revealed that the "basic spine," consisting of linearly aligned arginine residues spanning over the dimer surface, is responsible for ligand binding. Single amino acid substitution in the dimer interface caused a severe reduction in LOX-1 binding activity, suggesting that the correct dimer arrangement is crucial for binding to OxLDL. Based on the LDL model structure, possible binding modes of LOX-1 to OxLDL are proposed.

The X-ray Structure of a Hemipteran Ecdysone Receptor Ligand-binding Domain: COMPARISON WITH A LEPIDOPTERAN ECDYSONE RECEPTOR LIGAND-BINDING DOMAIN AND IMPLICATIONS FOR INSECTICIDE DESIGN

The ecdysone receptor is a hormone-dependent transcription factor that plays a central role in regulating the expression of vast networks of genes during development and reproduction in the phylum Arthropoda. The functional receptor is a heterodimer of the two nuclear receptor proteins ecdysone receptor (EcR) and ultraspiracle protein. The receptor is the target of the environmentally friendly bisacylhydrazine insecticides, which are effective against Lepidoptera but not against Hemiptera or several other insect orders. Here we present evidence indicating that much of the selectivity of the bisacylhydrazine insecticides can be studied at the level of their binding to purified ecdysone receptor ligand-binding domain (LBD) heterodimers. We report the crystal structure of the ecdysone receptor LBD heterodimer of the hemipteran Bemisia tabaci (Bt, sweet potato whitefly) in complex with the ecdysone analogue ponasterone A. Although comparison with the corresponding known LBD structure from the lepidopteran Heliothis virescens (Hv) ecdysone receptor revealed the overall mode of ponasterone A binding to be very similar in the two cases, we observed that the BtEcR ecdysteroid-binding pocket is structured differently to that of HvEcR in those parts that are not in contact with ponasterone A. We suggest that these differences in the ligand-binding pocket may provide a molecular basis for the taxonomic order selectivity of bisacylhydrazine insecticides.

Structural Basis for an Unexpected Mode of SERM-Mediated ER Antagonism

Tamoxifen is effective for the prevention and treatment of estrogen-dependent breast cancers, but is associated with an increased incidence of endometrial tumors. We report the crystal structure of the estrogen receptor alpha (ERalpha) ligand binding domain (LBD) bound to the structurally similar compound GW5638, which has therapeutic potential and does not stimulate the uterus. Like tamoxifen, GW5638 relocates the carboxy-terminal helix (H12) to the known coactivator-docking site in the ERalpha LBD. However, GW5638 repositions residues in H12 through specific contacts with the N terminus of this helix. In contrast to tamoxifen, the resulting increase in exposed hydrophobic surface of ERalpha LBD correlates with a significant destabilization of ERalpha in MCF-7 cells. Thus, the GW5638-ERalpha LBD structure reveals an unexpected mode of SERM-mediated ER antagonism, in which the stability of ERalpha is decreased through an altered position of H12. This dual mechanism of antagonism may explain why GW5638 can inhibit tamoxifen-resistant breast tumors.

The Nuclear Receptor Ligand-Binding Domain: A Family-Based Structure Analysis

Nuclear receptors (NRs) are ligand-dependent transcription factors that play a central role in various physiological processes. The pharmaceutical industry has great interest in this gene-family for the discovery of novel or improved drugs for treatment of, for example, cancer, infertility, or diabetes. The usage of three-dimensional coordinates of protein structures to analyse and predict interactions with ligands is an important aspect of this process. All NR ligand-binding domains have a similar fold, which allows for comparison of the structures of their three main functional sites: the ligand-binding pocket, the cofactor-binding groove, and the dimerization interface. We performed an analysis of nearly one hundred NR ligand-binding domain structures, and identified the functionally important residues. The combined knowledge about the shape of the binding sites and the residues involved in the binding is important for drug design in two ways. First, knowledge about the location of residues that interact with a ligand in all crystal structures or in certain subfamilies assists in the design and docking of drugs. Second, similarities and differences in the residue types of the most frequent ligand- and cofactor-binding residues provide insight about potential cross-reactivity of ligands or cofactors.

Conformational adaptation of nuclear receptor ligand binding domains to agonists: Potential for novel approaches to ligand design

Ligands occupy the core of nuclear receptor (NR) ligand binding domains (LBDs) and modulate NR function. X-ray structures of NR LBDs reveal most NR agonists fill the enclosed pocket and promote packing of C-terminal helix 12 (H12), whereas the pockets of unliganded NR LBDs differ. Here, we review evidence that NR pockets rearrange to accommodate different agonists. Some thyroid hormone receptor (TR) ligands with 5′ extensions designed to perturb H12 act as antagonists, but many are agonists. One mode of adaptation is seen in a TR/thyroxine complex; the pocket expands to accommodate a 5′ iodine extension. Crystals of other NR LBDs reveal that the pocket can expand or contract and some agonists do not fill the pocket. A TRβ structure in complex with an isoform selective drug (GC-24) reveals another mode of adaptation; the LBD hydrophobic interior opens to accommodate a bulky 3′ benzyl extension. We suggest that placement of extensions on NR agonists will highlight unexpected areas of flexibility within LBDs that could accommodate extensions; thereby enhancing the selectivity of agonist binding to particular NRs. Finally, agonists that induce similar LBD structures differ in their activities and we discuss strategies to reveal subtle structural differences responsible for these effects.

Evidence for Ligand-independent Transcriptional Activation of the Human Estrogen-related Receptor α (ERRα): CRYSTAL STRUCTURE OF ERRα LIGAND BINDING DOMAIN IN COMPLEX WITH PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR COACTIVATOR-1α

The crystal structure of the ligand binding domain (LBD) of the estrogen-related receptor alpha (ERRalpha, NR3B1) complexed with a coactivator peptide from peroxisome proliferator-activated receptor coactivator-1alpha (PGC-1alpha) reveals a transcriptionally active conformation in the absence of a ligand. This is the first x-ray structure of ERRalpha LBD, solved to a resolution of 2.5 A, and the first structure of a PGC-1alpha complex. The putative ligand binding pocket (LBP) of ERRalpha is almost completely occupied by side chains, in particular with the bulky side chain of Phe328 (corresponding to Ala272 in ERRgamma and Ala350 in estrogen receptor alpha). Therefore, a ligand of a size equivalent to more than approximately 4 carbon atoms could only bind in the LBP, if ERRalpha would undergo a major conformational change (in particular the ligand would displace H12 from its agonist position). The x-ray structure thus provides strong evidence for ligand-independent transcriptional activation by ERRalpha. The interactions of PGC-1alpha with ERRalpha also reveal for the first time the atomic details of how a coactivator peptide containing an inverted LXXLL motif (namely a LLXYL motif) binds to a LBD. In addition, we show that a PGC-1alpha peptide containing this nuclear box motif from the L3 site binds ERRalpha LBD with a higher affinity than a peptide containing a steroid receptor coactivator-1 motif and that the affinity is further enhanced when all three leucine-rich regions of PGC-1alpha are present.

Structure and gating of the glutamate receptor ion channel

Ionotropic glutamate receptors are ligand-gated ion channels that mediate rapid information transfer at most excitatory synapses in the brain. Crystal structures are now available for the ligand-binding domain, but the structure of the ion channel itself remains unknown. The core of the ion channel shares structural features with an inverted K+ channel. In detail, however, differences are emerging. Most notable in the glutamate receptor ion channel are distinct structural and functional properties of a major pore-lining domain, the requirement of an additional transmembrane segment (M4), and twofold rather than fourfold symmetry. Together with ligand-binding domain structures and kinetic experiments, these findings have started to define the basic principles of channel gating in glutamate receptors.

Crystal Structure of the Human RORα Ligand Binding Domain in Complex with Cholesterol Sulfate at 2.2 Å

The retinoic acid-related orphan receptor alpha (RORalpha) is an orphan member of the subfamily 1 of nuclear hormone receptors. Our recent structural and functional studies have led to the hypothesis that cholesterol or a cholesterol derivative is the natural ligand of RORalpha. We have now solved the x-ray crystal structure of the ligand binding domain of RORalpha in complex with cholesterol-3-O-sulfate following a ligand exchange experiment. In contrast to the 3-hydroxyl of cholesterol, the 3-O-sulfate group makes additional direct hydrogen bonds with three residues of the RORalpha ligand binding domain, namely NH-Gln(289), NH-Tyr(290), and NH1-Arg(370). When compared with the complex with cholesterol, seven well ordered water molecules have been displaced, and the ligand is slightly shifted toward the hydrophilic part of the ligand binding pocket, which is ideally suited for interactions with a sulfate group. These additional ligand-protein interactions result in an increased affinity of cholesterol sulfate when compared with cholesterol, as shown by mass spectrometry analysis done under native conditions and differential scanning calorimetry. Moreover, mutational studies show that the higher binding affinity of cholesterol sulfate translates into an increased transcriptional activity of RORalpha. Our findings suggest that cholesterol sulfate could play a crucial role in the regulation of RORalpha in vivo.

Peroxisome proliferator-activated receptors target family landscape: a chemometrical approach to ligand selectivity based on protein binding site analysis.

The Peroxisome Proliferator-Activated Receptors (PPARs) are nuclear receptors which over the last couple of years have been the focus of considerable research efforts aiming to identify compounds with well-defined selectivity profiles for the treatment of metabolic diseases. The ligand binding domains (LBD) of the three known PPAR subtypes exhibit between 60 and 70% sequence identity. To gain insight into the structural determinants of selectivity for the PPAR subtypes, a set of 13 crystal structures of PPAR LBD were classified, using the GRID/CPCA approach. As a result, nearly all of the crystal structures of each different PPAR subtype were found clustered in different regions of the CPCA score plots, and hydrophobic as well as steric interactions were identified as the major determinants of PPAR subtypes selectivity. Furthermore, interpretation of the GRID/CPCA model in structural terms led to the identification of LBD regions which could be targeted to improve the selectivity for a given PPAR subtype. Our findings are consistent with published structure-activity relationships for PPAR ligands as well as with site-directed mutagenesis results.

X-ray structure of the orphan nuclear receptor RORbeta ligand-binding domain in the active conformation.

The retinoic acid-related orphan receptor beta (RORbeta) exhibits a highly restricted neuronal-specific expression pattern in brain, retina and pineal gland. So far, neither a natural RORbeta target gene nor a functional ligand have been identified, and the physiological role of the receptor is not well understood. We present the crystal structure of the ligand-binding domain (LBD) of RORbeta containing a bound stearate ligand and complexed with a coactivator peptide. In the crystal, the monomeric LBD adopts the canonical agonist-bound form. The fatty acid ligand-coactivator peptide combined action stabilizes the transcriptionally active conformation. The large ligand-binding pocket is strictly hydrophobic on the AF-2 side and more polar on the beta-sheet side where the carboxylate group of the ligand binds. Site-directed mutagenesis experiments validate the significance of the present structure. Homology modeling of the other isotypes will help to design isotype-selective agonists and antagonists that can be used to characterize the physiological functions of RORs. In addition, our crystallization strategy can be extended to other orphan nuclear receptors, providing a powerful tool to delineate their functions.

Structure of the PPARα and -γ Ligand Binding Domain in Complex with AZ 242; Ligand Selectivity and Agonist Activation in the PPAR Family

Background: The peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors belonging to the nuclear receptor family. The roles of PPARα in fatty acid oxidation and PPARγ in adipocyte differentiation and lipid storage have been characterized extensively. PPARs are activated by fatty acids and eicosanoids and are also targets for antidyslipidemic drugs, but the molecular interactions governing ligand selectivity for specific subtypes are unclear due to the lack of a PPARα ligand binding domain structure. Results: We have solved the crystal structure of the PPARα ligand binding domain (LBD) in complex with the combined PPARα and -γ agonist AZ 242, a novel dihydro cinnamate derivative that is structurally different from thiazolidinediones. In addition, we present the crystal structure of the PPARγ_LBD/AZ 242 complex and provide a rationale for ligand selectivity toward the PPARα and -γ subtypes. Heteronuclear NMR data on PPARα in both the apo form and in complex with AZ 242 shows an overall stabilization of the LBD upon agonist binding. A comparison of the novel PPARα/AZ 242 complex with the PPARγ/AZ 242 complex and previously solved PPARγ structures reveals a conserved hydrogen bonding network between agonists and the AF2 helix. Conclusions: The complex of PPARα and PPARγ with the dual specificity agonist AZ 242 highlights the conserved interactions required for receptor activation. Together with the NMR data, this suggests a general model for ligand activation in the PPAR family. A comparison of the ligand binding sites reveals a molecular explanation for subtype selectivity and provides a basis for rational drug design.

Effects of ligand binding on the association properties and conformation in solution of retinoic acid receptors RXR and RAR

In higher eukaryotes, vitamin A derived metabolites such as 9-cis and all-trans retinoic acid (RA), are involved in the regulation of several essential physiological processes. Their pleiotropic physiological effects are mediated through direct binding to cognate nuclear receptors RXRs and RARs that act as regulated transcription factors belonging to the superfamily of nuclear hormone receptors. Hormone binding to the structurally conserved ligand-binding domain (LBD) of these receptors triggers a conformational change that principally affects the conserved C-terminal transactivation helix H12 involved in transcriptional activation.We report an extensive biophysical solution study of RARα, RXRα LBDs and their corresponding RXRα/RARα LBD heterodimers combining analytical ultracentrifugation (AUC), small-angle X-ray and neutron scattering (SAXS and SANS) and ab initio three-dimensional shape reconstruction at low resolution. We show that the crystal structures of RXRs and RARs LBDs correlate well with the average conformations observed in solution. Furthermore we demonstrate the effects of 9-cisRA and all-transRA binding on the association properties and conformations of RXRα and RARα LBDs in solution.The present study shows that in solution RARα LBD behaves as a monomer in both unliganded and liganded forms. It confirms the existence in solution of a ligand-induced conformational change towards a more compact form of the LBD. It also confirms the stability of the predicted RXRα/RARα LBD heterodimers in solution. SAS measurements performed on three different types of RXRα/RARα LBD heterodimers (apo/apo, apo/holo and holo/holo) with respect to their ligand-binding site occupancy show the existence of three conformational states depending on the progressive binding of RA stereoisomers on RARα and RXRα LBD subunits in the heterodimeric context. These results suggest that the subunits are structurally independent within the heterodimers.Our study also underlines the particular behaviour of RXRα LBD. In solution unliganded RXRα LBD is observed as two species that are unambiguously identified as homotetramers and homodimers. Molecular modelling combined with SAS data analysis allows us to propose a structural model for this autorepressed apo-tetramer. In contrast to the monomeric state observed in the crystal structure, our data show that in solution active holo-RXRα LBD bound to 9-cisRA is a homodimer regardless of the protein concentration. This study demonstrates the crucial role of ligands in the regulation of homodimeric versus heterodimeric association state of RXR in the NR signalling pathways.

Crystal Structure of a Mutant hERα Ligand-binding Domain Reveals Key Structural Features for the Mechanism of Partial Agonism

The crystal structure of a triple cysteine to serine mutant ERalpha ligand-binding domain (LBD), complexed with estradiol, shows that despite the presence of a tightly bound agonist ligand, the protein exhibits an antagonist-like conformation, similar to that observed in raloxifen and 4-hydroxytamoxifen-bound structures. This mutated receptor binds estradiol with wild type affinity and displays transcriptional activity upon estradiol stimulation, but with limited potency (about 50%). This partial activity is efficiently repressed in antagonist competition assays. The comparison with available LBD structures reveals key features governing the positioning of helix H12 and highlights the importance of cysteine residues in promoting an active conformation. Furthermore the present study reveals a hydrogen bond network connecting ligand binding to protein trans conformation. These observations support a dynamic view of H12 positioning, where the control of the equilibrium between two stable locations determines the partial agonist character of a given ligand.

Dynamic Stabilization of Nuclear Receptor Ligand Binding Domains by Hormone or Corepressor Binding

We have developed a novel assembly assay to examine structural changes in the ligand binding domain (LBD) of the thyroid hormone receptor (TR). Fragments including the first helix of the TR LBD interact only weakly with the remainder of the LBD in the absence of hormone, but this interaction is strongly enhanced by the addition of either hormone or the corepressor NCoR. Since neither the ligand nor the corepressor shows direct interaction with this helix, we propose that both exert their effects by stabilizing the overall structure of the LBD. Current models of activation of nuclear hormone receptors focus on a ligand-induced allosteric shift in the position of the C-terminal helix 12 that generates the coactivator binding site. Our results suggest that ligand binding also has more global effects that dynamically alter the structure of the receptor LBD.

Functional analysis of R651 mutations in the putative helix 6 of rat glucocorticoid receptors

Trypsin digestion of steroid-free, but not steroid-bound, rat glucocorticoid receptor (GR) has recently been reported to occur at arginine-651 (R651). This residue is close to the affinity labeled Cys-656 and thus could be a sensitive probe of steroid binding. This hypothesis is supported by the current model of the GR ligand binding domain (LBD), which is based on the X-ray structures of several related receptor LBDs and places R651 in the middle of the putative α-helix 6 (649-EQRMS-653 of rat GR), close to the bound steroid. To test this model, R651, which could be involved in hydrophilic and/or hydrogen bonding, was mutated to alanine (A), which favors α-helices, the helix breakers proline (P) and glycine (G), or tryptophan (W). All receptors were expressed at about the same level, as determined by Western blots, but the cell-free binding activity of R651P was reduced twofold. The cell-free binding affinities were all within a factor of 10 of wild type receptors. Whole cell biological activity with transiently transfected receptors was determined with a variety of GR agonists (dexamethasone and deacylcortivazol) or antagonists (dexamethasone mesylate, RU486, and progesterone). Reporters containing both simple (GRE) and complex (MMTV) enhancers were used to test for alterations in GR interactions with enhancer/promoter complexes. Surprisingly, no correlation was observed between biological activity and ability to preserve α-helical structures for each point mutation. Finally, similar trypsin digestion patterns indicated no major differences in the tertiary structure of the mutant receptors. Collectively, these results argue that the polar/ionizable residue R651 is not required for GR activity and is not part of an α-helix in the steroid-free or bound GR. The effect of these mutations on GR structure and activity may result from a cascade of initially smaller perturbations. These LBD alterations were the most varied for interactions with deacylcortivazol and RU 486, which have recently been predicted to be sub-optimal binders due to their large size. However, further analyses of ligand size versus affinity suggest that there is no narrowly defined optimal size for ligand binding, although larger ligands may be more sensitive to modifications of LBD structure. Finally, the changes in GR activity with the various mutations seem to result from altered LBD interactions with common, as opposed to enhancer specific, transcription factors.

Molecular and structural biology of thyroid hormone receptors.

1. Thyroid hormone receptors (TR) are expressed from two separate genes (alpha and beta) and belong to the nuclear receptor superfamily, which also contains receptors for steroids, vitamins and prostaglandins. 2. Unliganded TR are bound to DNA thyroid hormone response elements (TRE) predominantly as homodimers, or as heterodimers with retinoid X-receptors (RXR), and are associated with a complex of proteins containing corepressor proteins. Ligand binding promotes corepressor dissociation and binding of a coactivator. 3. Recent studies from our group have focused on the acquisition and use of X-ray crystallographic structures of ligand-binding domains (LBD) of both the rat (r) TR alpha and the human (h) TR beta bound to several different ligands. We have also developed ligands that bind selectively to the TR beta, which may provide ways to explore the differential functions of TR alpha compared with TR beta isoforms. 4. The LBD is comprised mostly of alpha-helices. The ligand is completely buried in the receptor and forms part of its hydrophobic core. Kinetic studies suggest that the limiting step in formation of high-affinity ligand-receptor complexes is the rate of folding of the receptor around the ligand. Ligands can be fitted tightly in the ligand-binding pocket and small differences in this fitting may explain many structure-activity relationships. Interestingly, analysis of the structures of antagonists suggests that they have chemical groups, 'extensions', that could impair receptor folding around them and, thus, prevent the agonist-induced conformation changes in the receptor. 5. The TR structures allowed us to see that the mutations that occur in the syndrome of generalized resistance to thyroid hormone are located in the vicinity of the ligand-binding pocket. 6. X-ray structure of the TR has also been used to guide construction of mutations in the TR surface that block binding of various proteins important for receptor function. Studies with these TR mutants reveal that the interfaces for homo- and heterodimerization map to similar residues in helix 10 and 11 and also allow the definition of the surface for binding of coactivators, which appears to be general for nuclear receptors. Formation of this surface, which involves packing of helix 12 of the TR into a scaffold formed by helices 3 and 5, appears to be the major change in the receptor structure induced by hormone occupancy.

Atomic structure of progesterone complexed with its receptor.

The physiological effects of progestins are mediated by the progesterone receptor, a member of the steroid/nuclear receptor superfamily. As progesterone is required for maintenance of pregnancy, its receptor has been a target for pharmaceuticals. Here we report the 1.8 A crystal structure of a progesterone-bound ligand-binding domain of the human progesterone receptor. The nature of this structure explains the receptor's selective affinity for progestins and establishes a common mode of recognition of 3-oxy steroids by the cognate receptors. Although the overall fold of the progesterone receptor is similar to that found in related receptors, the progesterone receptor has a quite different mode of dimerization. A hormone-induced stabilization of the carboxy-terminal secondary structure of the ligand-binding domain of the progesterone receptor accounts for the stereochemistry of this distinctive dimer, explains the receptor's characteristic pattern of ligand-dependent protease resistance and its loss of repression, and indicates how the anti-progestin RU486 might work in birth control. The structure also indicates that the analogous 3-keto-steroid receptors may have a similar mechanism of action.

Structural determinants of cortisol resistance in the guinea pig glucocorticoid receptor.

The guinea pig exhibits resistance to glucocorticoids in vivo which results from the guinea pig glucocorticoid receptor (GR) having a lower affinity for cortisol than the human GR. Cloning of the guinea pig GR has revealed that the amino acid sequence of the ligand-binding domain (LBD) differs from the human GR at 24 residues. The present study confirms that the decreased sensitivity and binding affinity of the guinea pig GR are conferred in vitro by the LBD. Further, the substitutions in the LBD do not confer altered relative steroid sensitivity or selectivity compared with the human GR. The altered sensitivity and binding of dexamethasone are confined to the first third of the LBD, which contains 5 nonconservative substitutions in a region that is otherwise highly conserved across several species of GR. These residues, either alone or in combination, were targeted for site-directed mutagenesis in both the human and guinea pig LBD. Trans-activation studies with these mutant GR failed to exclusively implicate or exclude any of the residues in the observed resistance. Rather, the changes, with 1 exception, caused a decrease in sensitivity, suggesting that critical intramolecular interactions involving at least 4 of these residues determine the correct conformation of this region. Recent molecular modeling of the GR LBD structure suggests that although the above region is not part of the core ligand-binding pocket, it is required to maintain the conformation of the binding pocket.

Partial androgen insensitivity and correlations with the predicted three dimensional structure of the androgen receptor ligand-binding domain

Genetic defects of the human androgen receptor (AR) can cause a wide spectrum of androgen insensitivity syndromes (AIS) ranging from phenotypic females in those with complete AIS; ambiguous genitalia in partial AIS; to male infertility in minimal AIS. The majority of these defects are due to point mutations resulting in amino acid substitutions. It is however unclear why certain mutations result in partial AIS, whereas others in the same exon cause the complete syndrome. We present a case of partial AIS due to a point mutation affecting codon 758 of the AR ligand-binding domain (LBD) that changed the sense of the codon from asparagine to threonine (N758T). The mutant receptor displayed normal binding affinity to DHT but abnormal dissociation kinetics in both patient’s fibroblasts and transfected COS-7 cells. The mutant AR was thermolabile, and resulted in ≈50% reduction in receptor transactivation capacity when examined with a reporter gene incorporating an androgen-response-element. Although the 3-D structure of AR LBD is not known, the homologous region in a member of the steroid receptor superfamily, retinoid-X receptor (RXR- α), has been crystalized, allowing comparison of aligned amino-acid sequences of RXR- α and AR. The mutation, N758T, lies in a predicted linker region between the fifth α-helix (H5) and the first β-strand (S1). Generally, mutations leading to partial AIS tend to cluster in the predicted linker regions located between the structural helices of the AR LBD. Most strikingly, the predicted linker regions contain over 70% of the mutant ARs associated with prostate cancer in the LBD. The occurrence of mutations associated with both partial AIS and prostate cancer in the same predicted linker regions, suggest that this clustering is not coincidental and that the predicted linker regions are likely to have important, but subtle, roles in defining androgen binding and ligand specificity.

Different positioning of the ligand-binding domain helix 12 and the F domain of the estrogen receptor accounts for functional differences between agonists and antagonists.

The estrogen receptor is capable of binding a diverse set of ligands that are broadly categorized as agonists or antagonists, depending on their abilities to induce or interfere with transcriptional responsiveness. We show, using a fusion protein assay for ligand-binding which does not rely on transcriptional responsiveness, that agonists and antagonists differently position the C-terminus of the ligand-binding domain (helix 12) and the F domain. Upon antagonist binding, the F domain interferes with the fusion protein activity. Mutational disruption of helix 12 alters the position of the F domain, imposing interference after agonist or antagonist binding. Genetically selected inversion mutations where only agonists, but not antagonists, induce interference are similarly reliant on helix 12 and F domain positioning. Our results demonstrate that agonists and antagonists differently position helix 12 and implicate the F domain in mechanisms of antagonist action.