Interaction Of Ligand-binding Domain Research Articles

In-silico Docking and Dynamics Simulation Analysis of Peroxisome Proliferator-Activated Receptor-Gamma and β-Carotene

Background:: Peroxisome proliferator-activated receptor-gamma (PPAR-γ) plays a crucial role in regulating lipid and glucose metabolism, cancer, and inflammation, making it an attractive target for drug development. Meanwhile, β-Carotene, known for its antioxidant, anticancer and antiinflammatory properties, holds promise for modulating PPAR-γ activity. Understanding their interaction is crucial. Objective:: This study aims to explore the therapeutic potential of β-carotene in modulating PPAR-γ activity by investigating their binding interactions. objective: To explore the potential therapeutic applications of β-carotene in modulating PPAR-γ activity, it is of great interest to comprehend the binding interactions between PPAR-γ and β-carotene. Methods:: Screening of bioactive compounds from PubChem was conducted using GlideXP to identify potential PPAR-γ (PDB: 2PRG) ligands. During this screening, both protein and bioactive compounds were prepared following established protocols. Subsequently, the compounds were docked into the ligand binding domain (LBD) of the protein using XP docking. Rosiglitazone was used as an internal control. β-Carotene emerged as a lead based on Lipinski’s rule, docking score, free energy, and LBD interactions. Molinspiration analysis assessed its drug likeness. Molecular dynamics (MD) simulations utilizing Desmond with OPLS 2005 force field were employed to examine the dynamics and stability of the PPAR-γ/β-carotene complex. Results:: β-carotene had strong hydrophobic interactions with specific residues within the ligandbinding domain of PPAR-γ. The calculated binding affinity (-9.07 kcal/mol) indicated a strong interaction between β-carotene and PPAR-γ, suggesting that β-carotene may modulate the activity of PPAR-γ. On a time scale of 100 ns, the MD simulations provided insights into the conformational changes, flexibility, and intermolecular interactions within the complex. Conclusion:: In silico docking and dynamics simulation analyses show that PPAR-γ and β-carotene can form a stable complex, suggesting potential implications for metabolic modulation.

Computer Simulation Studies of a Kainate (GluK1) Receptor with Two Glutamate Analogues

Unprecedented advancements have been achieved to understand the underlying mechanisms that sustain life. The modulation of these mechanisms, especially in disease conditions, could lead to the development of new medical applications. However, to attain this goal, we need to characterize life processes at the molecular level. Unfortunately, the majority of current experimental techniques used in life sciences lack this resolution. In this work, we have used molecular dynamics, a computational “microscopy”, to gain insight into the mechanism of interaction, at the atomic level, between two bicyclic glutamate analogues with the ligand–binding domain (LBD) of a kainate receptor 1 (GluK1). This protein receptor plays a crucial role in the development of various central nervous system (CNS) disorders such as Alzheimer’s disease, epilepsy and depression. Here we report the outcome of Molecular Dynamics (MD) simulations to calculate the affinity of binding of two ligands, the glutamate analogues LM-12b and CIP-AS, toward the LBD of GluK1 (GluK1–LBD) and to unravel, at the atomic level, the structural dynamics of such interactions. Our computational approach not only was capable of ranking correctly the binding affinity of analyzed ligands toward the protein receptor, but also to reveal, at atomic resolution, the dynamic nature of such ligand–LBD interaction. Our studies showed that the methyl group of LM-12b is crucial to stabilize structurally the LBD pocket. In contrast, the LBD–CIP-AS complex lacked this interaction, which may explain its weaker affinity. Revealing the structural and dynamics bases that underlie the mechanism of ligand–kainate receptor interaction may ultimately drive the identification of new modulators aimed at the treatment of CNS disorders.

Ligand binding domain interface: A tipping point for pharmacological agents binding with GluN1/2A subunit containing NMDA receptors

N-methyl D-aspartate (NMDA) receptors play a crucial role in normal brain function, pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDA receptor contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that evolve from six different genes including four GluN2 (A-D) and two GluN3 (A-B) subunits. Since NMDA receptors confer varied physiological properties and spatiotemporal distributions in the brain, pharmacological agents that target NMDA receptors with specific GluN2 subunits have significant potential for therapeutic applications. In the present work, by using electrophysiology techniques, we have studied the role of ligand binding domain (LBD) interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, channel blockers and an allosteric modulator. Remarkably, point mutations at the distal end (site-II&III) of GluN1 LBD interface increased memantine potency up to sevenfold when co-expressed with wild type GluN2A receptors but exhibit no effect on Mg2+ activity. Conversely, mutations at the proximal end (site-I) of the LBD interface did not affect the memantine but altered Zn2+ and Mg2+ potency towards opposite directions. These results indicate that GluN1/2A LBD interface interactions play a key role in determining channel function. Further, subtle changes in LBD interaction can be readily translated to the downstream extracellular vestibule of channel pore to adopt a conformation that may affect memantine, Zn2+ and Mg2+ binding. Further studies on NMDA receptor LBD to transmembrane domain signal propagation mechanisms will help develop GluN2 subunit selective biomolecules that can be used for the treatment of neurological and psychiatric disorders.

Multidomain architecture of estrogen receptor reveals interfacial cross-talk between its DNA-binding and ligand-binding domains

Human estrogen receptor alpha (hERα) is a hormone-responsive nuclear receptor (NR) involved in cell growth and survival that contains both a DNA-binding domain (DBD) and a ligand-binding domain (LBD). Functionally relevant inter-domain interactions between the DBD and LBD have been observed in several other NRs, but for hERα, the detailed structural architecture of the complex is unknown. By utilizing integrated complementary techniques of small-angle X-ray scattering, hydroxyl radical protein footprinting and computational modeling, here we report an asymmetric L-shaped “boot” structure of the multidomain hERα and identify the specific sites on each domain at the domain interface involved in DBD–LBD interactions. We demonstrate the functional role of the proposed DBD–LBD domain interface through site-specific mutagenesis altering the hERα interfacial structure and allosteric signaling. The L-shaped structure of hERα is a distinctive DBD–LBD organization of NR complexes and more importantly, reveals a signaling mechanism mediated by inter-domain crosstalk that regulates this receptor’s allosteric function.

Positive Modulatory Interactions of NMDA Receptor GluN1/2B Ligand Binding Domains Attenuate Antagonists Activity.

N-methyl D-aspartate receptors (NMDAR) play crucial role in normal brain function and pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDAR contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that include six different gene products; four GluN2 (A–D) and two GluN3 (A–B) subunits. The heterogeneity of subunit combination facilities the distinct function of NMDARs. All GluN subunits contain an extracellular N-terminal Domain (NTD) and ligand binding domain (LBD), transmembrane domain (TMD) and an intracellular C-terminal domain (CTD). Interaction between the GluN1 and co-assembling GluN2/3 subunits through the LBD has been proven crucial for defining receptor deactivation mechanisms that are unique for each combination of NMDAR. Modulating the LBD interactions has great therapeutic potential. In the present work, by amino acid point mutations and electrophysiology techniques, we have studied the role of LBD interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, and allosteric modulators. The results reveal that agonists (glycine and glutamate) potency was altered based on mutant amino acid sidechain chemistry and/or mutation site. Most antagonists inhibited mutant receptors with higher potency; interestingly, clinically used NMDAR channel blocker memantine was about three-fold more potent on mutated receptors (N521A, N521D, and K531A) than wild type receptors. These results provide novel insights on the clinical pharmacology of memantine, which is used for the treatment of mild to moderate Alzheimer's disease. In addition, these findings demonstrate the central role of LBD interactions that can be exploited to develop novel NMDAR based therapeutics.

Challenges in enrolling to metastatic castration-resistant prostate cancer (mCRPC) studies that require androgen receptor splice variant (AR-V) positivity.

264 Background: AR-Vs are truncated androgen receptor (AR) isoforms that lack the ligand-binding domain (LBD) and are associated with resistance to drugs targeting the ligand-AR LBD interaction (e.g. abiraterone [Abi], enzalutamide [Enza]). Preclinical studies have shown that niclosamide, an anthelminticsdrug, is able to overcome AR-V mediated drug resistance. Methods: We are conducting a Phase I dose-escalation study testing high-dose niclosamide in combination with the FDA-approved dose of Enza. Niclosamide is given three-times-daily by mouth at one of the following dose-levels: 500, 1000 or 1500 mg. All patients are required to have mCRPC, have received prior Abi, and have detectable AR-V transcripts in circulating tumor cells (CTC) as determined using the AdnaTest ProstateCancerSelect/ProstateCancerDetect assay. Patients are permitted to start Enza prior to the addition of niclosamide. The primary objective is to assess safety. Niclosamide’s pharmacokinetic profile will also be determined. Results: From December 2015 to October 2016 we screened 14 patients. All patients previously progressed on Enza, with 13/14 patients demonstrating a rising PSA on Enza at screening. Two had previously documented AR-Vs as determined from prior transcript profiling studies; however, only one of these patients was AR-V positive (AR-V5-6) at the time of study enrollment. Of the 12 patients without prior evidence of AR-V, none were found to be AR-V+. All patients (14/14) were positive for actin and 10/14 were positive for full-length AR using the AdnaTest. To date, one patient has completed treatment with high-dose niclosamide. No study drug-related adverse events have been observed. Conclusions: Rates of AR-V positivity have been lower than expected in our cohort of patients that have progressed on Abi and Enza – challenging study accrual. Multicenter studies are needed to confirm rates of AR-V positivity in mCRPC populations. Furthermore, in order to ensure enrollment expectations are met, novel biomarkers (e.g. AR-V status as determined using AdnaTest) should undergo pre-study confirmatory testing within institutions prior to use as enrollment criteria. Clinical trial information: NCT02532114.

Tetrabromobisphenol-A disrupts thyroid hormone receptor alpha function in vitro: Use of fluorescence polarization to assay corepressor and coactivator peptide binding

Thyroid hormone receptors (TRs) recruit corepressor or coactivator factors to the promoters of target genes to regulate their transcription. Corepressors such as nuclear hormone receptor corepressor (NCoR) are recruited by unliganded TRs, whereas coactivators such as steroid receptor coactivator-2 (SRC2) are recruited when triiodothyronine (T3) is bound to TRs. These coregulator proteins interact with the ligand binding domain (LBD) of TRs via short, conserved peptide sequences that can be used to probe the conformational changes induced in TR LBD by TR ligands. Recombinant LBD of the human TRα1 isoform (hTRα1 LBD) was produced as a fusion with glutathione S-transferase, and used to develop assays based on fluorescence polarization to quantify the binding of either NCoR- or SRC2-derived fluorescent peptides to the hTRα1 LBD. The optimum concentrations of recombinant hTRα1 LBD, and of peptide probes were adjusted in order to produce the greatest possible T3-dependent signal variations in fluorescence polarization. Under these conditions, T3 induced a dose-dependent decrease in NCoR peptide binding, and a reciprocal dose-dependent increase in SRC2 peptide binding, in both cases at similar 50%-effective doses. The TR agonists triiodothyroacetic acid and thyroxine were also effective in preventing NCoR peptide binding and increasing SRC2 peptide binding, whereas reverse-triiodothyronine was less efficient and the biologically inactive thyronine had no effect on either process. These experiments validate cell-free assays based on the use of binding of corepressor or coactivator peptide probes, as measured by fluorescence polarization, for investigating the conformational changes of TRα1 LBD induced by potentially TR-interfering compounds. Both these methods were used to elucidate the mechanism of the disrupting effects of tetrabromobisphenol-A (TBBPA) on the hTRα1 LBD conformation related to the transcriptional activity of the receptor. TBBPA is a flame retardant that is released into the environment, and is a suspected disrupter of thyroid homeostasis. The present results indicate that TBBPA did indeed interfere with the ability of the hTRα1 LBD to bind both NCoR and SRC2. TBBPA behaved similarly to T3 in promoting the release of NCoR from LBD, whereas it failed to promote LBD interactions with SRC2. However, it did reduce the T3-induced interactions between LBD and the coactivator peptide. This study therefore suggests that TBBPA in the micromolar range can affect the regulation of transcription by both the apo- and the holo-TRα1, with potential disruption of the expression of genes that are either up- or down-regulated by T3.

Ligand-Dependent Assembly of Pregnane X Receptor, Constitutive Androstane Receptor and Liver X Receptor Is Applicable to Identify Ligands

Pregnane X receptor (PXR), constitutive androstane receptor (CAR) and liver X receptor (LXR) are intracellular sensors for foreign chemicals and/or endogenous compounds. Docking of a ligand into the ligand-binding domain (LBD) of a nuclear receptor induces conformational changes and switches the nuclear receptor into an active conformation. In this study, we examined whether assembly assays to exploit the ligand-dependent interaction of N- and C-terminal regions of the LBD could be used for detection of ligands for PXR, CAR and LXR. Rifampicin, CITCO and T1317 significantly enhanced interactions for human PXR, human CAR and human LXR, respectively. The effects of ligands on the interaction of LBDs in PXR and CAR reflected the species differences in ligand response of PXR and CAR. In conclusion, it appears that the present assay, which exploits the interaction between N- and C-terminal regions of LBDs, is applicable to identify ligands.

Novel FXXFF and FXXMF Motifs in Androgen Receptor Cofactors Mediate High Affinity and Specific Interactions with the Ligand-binding Domain

Upon hormone binding, a hydrophobic coactivator binding groove is induced in the androgen receptor (AR) ligand-binding domain (LBD). This groove serves as high affinity docking site for alpha-helical FXXLF motifs present in the AR N-terminal domain and in AR cofactors. Study of the amino acid requirements at position +4 of the AR FXXLF motif revealed that most amino acid substitutions strongly reduced or completely abrogated AR LBD interaction. Strong interactions were still observed following substitution of Leu+4 by Phe or Met residues. Leu+4 to Met or Phe substitutions in the FXXLF motifs of AR cofactors ARA54 and ARA70 were also compatible with strong AR LBD binding. Like the corresponding FXXLF motifs, interactions of FXXFF and FXXMF variants of AR and ARA54 motifs were AR specific, whereas variants of the less AR-selective ARA70 motif displayed increased AR specificity. A survey of currently known AR-binding proteins revealed the presence of an FXXFF motif in gelsolin and an FXXMF motif in PAK6. In vivo fluorescence resonance energy transfer and functional protein-protein interaction assays showed direct, efficient, and specific interactions of both motifs with AR LBD. Mutation of these motifs abrogated interaction of gelsolin and PAK6 proteins with AR. In conclusion, we have demonstrated strong interaction of FXXFF and FXXMF motifs to the AR coactivator binding groove, thereby mediating specific binding of a subgroup of cofactors to the AR LBD.

Androgen Receptor Ligand-Binding Domain Interaction and Nuclear Receptor Specificity of FXXLF and LXXLL Motifs as Determined by L/F Swapping

The androgen receptor (AR) ligand-binding domain (LBD) binds FXXLF motifs, present in the AR N-terminal domain and AR-specific cofactors, and some LXXLL motifs of nuclear receptor coactivators. We demonstrated that in the context of the AR FXXLF motif many different amino acid residues at positions +2 and +3 are compatible with strong AR LBD interaction, although a preference for E at +2 and K or R at +3 was found. Pairwise systematic analysis of F/L swaps at +1 and +5 in FXXLF and LXXLL motifs showed: 1) F to L substitutions in natural FXXLF motifs abolished AR LBD interaction; 2) binding of interacting LXXLL motifs was unchanged or increased upon L to F substitutions; 3) certain noninteracting LXXLL motifs became strongly AR-interacting FXXLF motifs; whereas 4) other nonbinders remained unaffected by L to F substitutions. All FXXLF motifs, but not the corresponding LXXLL motifs, displayed a strong preference for AR LBD. Progesterone receptor LBD interacted with some FXXLF motifs, albeit always less efficiently than corresponding LXXLL motifs. AR LBD interaction of most FXXLF and LXXLL peptides depended on classical charge clamp residue K720, whereas E897 was less important. Other charged residues lining the AR coactivator-binding groove, K717 and R726, modulated optimal peptide binding. Interestingly, these four charged residues affected binding of individual peptides independent of an F or L at +1 and +5 in swap experiments. In conclusion, F residues determine strong and selective peptide interactions with AR. Sequences flanking the core motif determine the specific mode of FXXLF and LXXLL interactions.

Distinct recognition modes of FXXLF and LXXLL motifs by the androgen receptor.

Among nuclear receptors, the androgen receptor (AR) is unique in that its ligand-binding domain (LBD) interacts with the FXXLF motif in the N-terminal domain, resembling coactivator LXXLL motifs. We compared AR- and estrogen receptor alpha-LBD interactions of the wild-type AR FXXLF motif and coactivator transcriptional intermediary factor 2 LXXLL motifs and variants of these motifs. Random mutagenesis revealed a key role for the F residues in FXXLF motifs in high-affinity and selective AR LBD interaction. The FXXLF motif in full-length AR and transcriptional intermediary factor 2 LXXLL motifs competed for an overlapping binding site. A computer model of the AR LBD/AR FXXLF complex showed that the bulky F residues are buried in a deep coactivator-binding groove. The corresponding groove in estrogen receptor alpha LBD is considerably shallower, explaining lack of binding of any of the FXXLF motifs tested. FXXLF and LXXLL motif interaction depended on different charged amino acid residues in the AR LBD present at opposite ends of the coactivator groove. In conclusion, our data demonstrate the importance of a deep hydrophobic groove and alternative usage of charged amino acids in specifying peptide binding to the AR LBD.

Amino acids 3-13 and amino acids in and flanking the 23FxxLF27 motif modulate the interaction between the N-terminal and ligand-binding domain of the androgen receptor.

The N-terminal domain (NTD) and the ligand-binding domain (LBD) of the androgen receptor (AR) exhibit a ligand-dependent interaction (N/C interaction). Amino acids 3-36 in the NTD (AR3-36) play a dominant role in this interaction. Previously, it has been shown that a PhixxPhiPhi motif in AR3-36, 23FxxLF27, is essential for LBD interaction. We demonstrate in the current study that AR3-36 can be subdivided into two functionally distinct fragments: AR3-13 and AR16-36. AR3-13 does not directly interact with the AR LBD, but rather contributes to the transactivation function of the AR.NTD-AR.LBD complex. AR16-36, encompassing the 23FxxLF27 motif, is predicted to fold into a long amphipathic alpha-helix. A second PhixxPhiPhi candidate protein interaction motif within the helical structure, 30VREVI34, shows no affinity to the LBD. Within AR16-36, amino acid residues in and flanking the 23FxxLF27 motif are demonstrated to modulate N/C interaction. Substitution of Q24 and N25 by alanine residues enhances N/C interaction. Substitution of amino acids flanking the 23FxxLF27 motif by alanines are inhibitory to LBD interaction.

Human Androgen Receptor Mutation Disrupts Ternary Interactions between Ligand, Receptor Domains, and the Coactivator TIF2 (Transcription Intermediary Factor 2)

The androgen receptor (AR) is a ligand-dependent X-linked nuclear transcription factor regulating male sexual development and spermatogenesis. The receptor is activated when androgen binds to the C-terminal ligand-binding domain (LBD), triggering a cascade of molecular events, including interactions between the LBD and the N-terminal transactivation domain (TAD), and the recruitment of transcriptional coactivators. A nonconservative asparagine to lysine substitution in AR residue 727 was encountered in a phenotypically normal man with subfertility and depressed spermatogenesis. This N727K mutation, although located in the LBD, did not alter any ligand-binding characteristic of the AR in the patient's fibroblasts or when expressed in heterologous cells. Nonetheless, the mutant AR displayed only half of wild-type transactivation capacity when exposed to physiological or synthetic androgens. This transactivation defect was consistently present when examined with two different reporter systems in three cell lines, using three androgen-driven promoters (including the complex human prostate-specific antigen promoter), confirming the pathogenicity of the mutation. In mammalian two-hybrid assays, N727K disrupted LBD interactions with the AR TAD and with the coactivator, transcription intermediary factor 2 (TIF2). Strikingly, the transactivation defect of the mutant AR can be rectified in vitro with mesterolone, consistent with the ability of this androgen analog to restore sperm production in vivo. Mesterolone, but not the physiological androgen dihydrotestosterone, restored mutant LBD interactions with the TAD and with TIF2, when expressed as fusion proteins in the two-hybrid assay. Our data support an emerging paradigm with respect to AR mutations in the LBD and male infertility: pathogenicity is transmitted through reduced interdomain and coactivator interactions, and androgen analogs that are corrective in vitro may indicate hormonal therapy.

Both Coactivator LXXLL Motif-dependent and -independent Interactions Are Required for Peroxisome Proliferator-activated Receptor γ (PPARγ) Function

Nuclear receptor activation is dependent on recruitment of coactivators, including CREB-binding protein (CBP/p300) and steroid receptor coactivator-1 (SRC-1). A three-dimensional NMR approach was used to probe the coactivator binding interface in the peroxisome proliferator-activated receptor gamma (PPARgamma) ligand binding domain (LBD). In the presence of a CBP peptide, peaks corresponding to 20 residues in helices 3, 4, 5, and 12 of the LBD were attenuated. Alanine mutants revealed that K301A, V315A, Y320A, L468A, and E471A were required for binding of both CBP and SRC-1 and for cell-based transcription. Several additional amino acids in helix 4 of the PPARgammaLBD were defective with respect to CBP recruitment, but retained relatively normal SRC-1 recruitment. Thus these amino acid residues may be important determinants of specificity for nuclear receptor LBD interactions with discrete coactivator molecules.

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.