Molecular dynamics simulations reveal multiple pathways of ligand dissociation from thyroid hormone receptors

Nuclear receptor (NR) ligands occupy a pocket that lies within the core of the NR ligand-binding domain (LBD), and most NR LBDs lack obvious entry/exit routes upon the protein surface. Thus, significant NR conformational rearrangements must accompany ligand binding and release. The precise nature of these processes, however, remains poorly understood. Here, we utilize locally enhanced sampling (LES) molecular dynamics computer simulations to predict molecular motions of x-ray structures of thyroid hormone receptor (TR) LBDs and determine events that permit ligand escape. We find that the natural ligand 3,5,3'-triiodo-L-thyronine (T(3)) dissociates from the TRalpha1 LBD along three competing pathways generated through i), opening of helix (H) 12; ii), separation of H8 and H11 and the Omega-loop between H2 and H3; and iii), opening of H2 and H3, and the intervening beta-strand. Similar pathways are involved in dissociation of T(3) and the TRbeta-selective ligand GC24 from TRbeta; the TR agonist IH5 from the alpha- and beta-TR forms; and Triac from two natural human TRbeta mutants, A317T and A234T, but are detected with different frequencies in simulations performed with the different structures. Path I was previously suggested to represent a major pathway for NR ligand dissociation. We propose here that Paths II and III are also likely ligand escape routes for TRs and other NRs. We also propose that different escape paths are preferred in different situations, implying that it will be possible to design NR ligands that only associate stably with their cognate receptors in specific cellular contexts.     

Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster

Mutants created by site-directed mutagenesis were used to elucidate the function of amino acids involved in ligand binding to ecdysteroid receptor (EcR) and heterodimer formation with ultraspiracle (USP). The results demonstrate the importance of the C-terminal part of the D-domain and helix 12 of EcR for hormone binding. Some amino acids are involved either in ligand binding to EcR (E476, M504, D572, I617, N626) or ligand-dependent heterodimerization as determined by gel mobility shift assays (A612, L615, T619), while others are involved in both functions (K497, E648). Some amino acids are suboptimal for ligand binding (L615, T619), but mediate ligand-dependent dimerization. We conclude that the enhanced regulatory potential by ligand-dependent modulation of dimerization in the wild type is achieved at the expense of optimal ligand binding. Mutation of amino acids (K497, E648) involved in the salt bridge between helix 4 and 12 impair ligand binding to EcR more severely than hormone binding to the heterodimer, indicating that to some extent heterodimerization compensates for the deleterious effect of certain mutations. Different effects of the same point mutations on ligand binding to EcR and EcR/USP (R511, A612, L615, I617, T619, N626) indicate that the ligand-binding pocket is modified by heterodimerization.     

Dynamic of ligand binding to Drosophila melanogaster ecdysteroid receptor

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

Transformation of the 8-9 S molybdate-stabilized estrogen receptor from low-affinity to high-affinity state without dissociation into subunits

The rate of dissociation of labeled estradiol from [3H] estradiol-8-9 S receptor complexes ([3H]E2-8-9 S ER) molybdate-stabilized was determined in the presence of either an excess of unlabeled hormone ("chase") or of charcoal/dextran suspension ("stripping"). Biphasic dissociation of the hormone was observed in both cases, but the fraction of the fast-dissociating component was dramatically reduced (5% instead of 60%) when stripping was used. As the dissociation patterns were independent of the degree of saturation of the receptor, the results do not favor the possibility of cooperative effects between binding sites in the 8-9 S ER. After pretreatment of cytosol by charcoal at 28 degrees C for 15 min, the dissociation studied by chase displayed only the slowly dissociating component (t1/2 approximately 65 min). This effect was dependent on temperature and influenced by the ligand bound to 8-9 S ER, being pronounced with estradiol (E2) and absent with [3H]4-hydroxytamoxifen. The slow-dissociating component obtained after charcoal treatment was reconverted to fast-dissociating state by adding dithiothreitol or by incubation with cytosol at 20 degrees C. The charcoal treatment did not change the sedimentation coefficient (approximately 9 S) and the Stokes radius (approximately 7 nm) of the [3H]E2-8-9 S ER, and the slow-dissociating form obtained did not bind to DNA-cellulose either in the presence or absence of molybdate ions. Thus there are likely small but functionally significant changes of structure in the 8-9 S ER which remain in a non-DNA-binding form, whereas the rate of estradiol dissociation is modified.     

Ligand unbinding pathways from the vitamin D receptor studied by molecular dynamics simulations

Molecular dynamics simulation techniques have been used to study the unbinding pathways of 1α,25-dihydroxyvitamin D₃ from the ligand-binding pocket of the vitamin D receptor (VDR). The pathways observed in a large number of relatively short (<200 ps) random acceleration molecular dynamics (RAMD) trajectories were found to be in fair agreement, both in terms of pathway locations and deduced relative preferences, compared to targeted molecular dynamics (TMD) and streered molecular dynamics simulations (SMD). However, the high-velocity ligand expulsions of RAMD tend to favor straight expulsion trajectories and the observed relative frequencies of different pathways were biased towards the probability of entering a particular exit channel. Simulations indicated that for VDR the unbinding pathway between the H1–H2 loop and the β-sheet between H5 and H6 is more favorable than the pathway located between the H1–H2 loop and H3. The latter pathway has been suggested to be the most likely unbinding path for thyroid hormone receptors (TRs) and a likely path for retinoic acid receptor. Ligand entry/exit through these two pathways would not require displacement of H12 from its agonistic position. Differences in the packing of the H1, H2, H3 and β-sheet region explain the changed relative preference of the two unbinding pathways in VDR and TRs. Based on the crystal structures of the ligand binding domains of class 2 nuclear receptors, whose members are VDR and TRs, this receptor class can be divided in two groups according to the packing of the H1, H2, H3 and β-sheet region. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Activation of the estrogen receptor from N-nitrosomethylurea-induced rat mammary tumors

The activation of the estrogen receptor (ER) from N-nitrosomethylurea (NMU)-induced rat mammary tumors was studied in vitro. The activation of the receptor induced by heating of the cytosol containing occupied ER was measured by a 3-4-fold increase of receptor binding to nuclei in comparison with the nuclear binding of the nonactivated ER. The activation of the ER was further shown by alteration of the elution profile from DEAE-cellulose. A shift of the receptor peak from 234 mM (Peak II, nonactivated ER) to 70 mM (Peak I, activated ER) phosphate buffer could be obtained. The overall recoveries of activated ER following chromatography on DEAE-cellulose were significantly lower than the recoveries of the nonactivated ER, 71 and 85%, respectively. Binding of the activated ER to nuclei and chromatography of the supernatant which is not able to bind to nuclei on DEAE-cellulose resulted in a decrease of Peak I and in an increase of the overall recovery. These findings suggest that the nuclear bound ER consists of two parts. One is represented partially by Peak I of the elution profile and the other one by that part of the receptor which can not be eluted from the column under the conditions used. Furthermore, the dissociation of tritiated estradiol (E3H) from the nonactivated ER followed a two component exponential function whereas after activation a monophasic dissociation curve could be observed. The mean half times for the dissociation of E3H from the activated and nonactivated ER were 101 and 7.2 min, respectively. Finally, the nonactivated molybdate stabilized ER sedimented in 5-20% sucrose density gradients as two peaks, one at 9.5 S and the other at 4 S. After activation of the ER only the smaller 4 S peak was evident. Molybdate inhibited the activation of the ER measured by nuclear binding assays, sucrose density gradient analysis, dissociation kinetics or ion exchange chromatography but not completely in every case.     

In vivo characterization of estrogen receptor modulators with reduced genomic versus nongenomic activity in vitro

Estrogen receptor (ER) ligands that are able to prevent postmenopausal bone loss, but have reduced activity in the uterus and the mammary gland might be of great value for hormone therapy. It is well established that the classical ER can activate genomic as well as nongenomic signal transduction pathways. In this study, we analyse the in vivo behaviour of ER ligands that stimulate nongenomic ER effects to the same extent as estradiol, but show clearly reduced activation of genomic ER effects in vitro. Using different readout parameters such as morphological changes, cellular proliferation, and target gene induction, we are able to demonstrate that ER ligands with reduced genomic activity in vitro show a better dissociation of bone versus uterine and mammary gland effects than estradiol that stimulates genomic and nongenomic effects to the same extent. We conclude that pathway-selective ER ligands may represent an interesting option for hormone therapy.     

Functional studies on the ligand-binding domain of Ultraspiracle from Drosophila melanogaster

The functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and characterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the heterodimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acid-binding residue within the ligand-binding pocket, 1323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligand-binding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein.     

A simple method for calculating the dissociation constant of a receptor (or enzyme).unlabeled ligand complex from radioligand displacement measurements

A general procedure is described for determining the dissociation constant of a receptor (or enzyme).unlabeled ligand complex (EI) by analyzing the I-dependent displacement of bound radioligand (A*) from EA*. The procedure (which involves measuring free A* in the presence of I) requires a knowledge of the total concentrations of receptor ([E]t), unlabeled ligand ([I]t) and radioligand ([A*]t), and the dissociation constant of the EA* complex. The unknown Kd is obtained from five simple, sequential calculations which are valid for either high or low affinity competitive unlabeled ligands and are independent of total receptor concentration or initial degree of saturation with A*. The procedure also provides the information needed to construct a distribution curve of all enzyme and ligand species (E, EA*, EI, A*, I) as [I]t is varied.