Intracellular receptors
ContentsSignal recognition particle receptorGTP binding site. Water molecules are shown as red spheres. Receptor for activated C kinase 1Nuclear receptorsThe thyroid hormone binds in the ligand niche making several interactions with TR The all-trans retinoid acid binds to the hydrophobic pocket of RARγ.
RARα-RXRα interaction (PDB entry 1dkf). The Ligand binding domain for each piece of the dimer has a nearly identical structure of an Tα-helical sandwich. These α-helices form 12 domains per protein (H1-12), with an additional 2 β-sheets. The α-helical sandwich bind All-Trans Retinoic Acid (ATRA), the isomer of RA used by the body. Both monomers contain two regions of activity, the dimerization interface and the ligand binding pocket . When RARα/RXRα proteins form a heterodimer, the overall structure of the larger dimer is comparable to that of an RXRα homodimer, likely due to the many similarities these 2 molecules share. RARα and RXRα rely on residues from the H7-10, L8-9, and L9-10 domains of both molecules to form the heterodimer interface. The sequence identity between the 2 molecules on the dimer interface is 0.33. The residues of RAR-α that are interacting in the heterodimer: Hydrophobic residues: L356, F374, P375, L378, M379, I381 and A389 (yellow); Negatively charged residues: D338, D349, E353, E357, D383, and E393 (red); Positively charged residues: K360, R364, H372, K376, K380, and R385 (blue); Hydrophilic residues: Q315, Q352, T382, and S386 (green). The residues of RXR-α that are interacting in the heterodimer are as follows: Hydrophobic residues: Y402, P417, F420, A421, L424, L425, L427, P428, A429, and L435 (yellow); Negatively charged residues: E357, D384, E395, E399, E406, and E439 (red); Positively charged residues: R353, K361, R398, K410, K422, R426, R431, and K436 (blue); Hydrophilic residues: S432 (green). Upon binding of the ligand ATRA in the cytoplasm, RARα and RXRα form a heterodimer and alter the C-terminals on domain H12 of both subunits in a manner that allows them to change the conformation of their DNA binding domains. The 2 proteins have 29% identity in their LBD. For the ligand used in RARα crystallization, BMS614, 21 primarily hydrophobic residues form the RARα ligand binding pocket. BMS614 is not the natural ligand for this molecule, but acts as stable agonist for crystallization. The largest difference between BMS614 and ATRA upon binding to the pocket are at Ile412, where BMS614 pushes much closer to the amino acid than ATRA does. Residues that form the binding pocket are found on H1, H3, H5, H11, L6-7, and L11-12 on RARα. The major differences between RARα, RARβ and RARγ are present in this area: Residue 270: α:Ile β:Ile γ:Met; Residue 232: α:Ser β:Ala γ:Ala; Residue 395: α:Val β:Val γ:Ala The RXRα binding pocket is comprised of 16 primarily hydrophobic residues, found on the H3, H5, H7, H11, and L11-12 domains. The ligand used in the crystal, Oleic Acid, is similar to RA, and RA is capable of binding to the RXRα pocket. Crystal structure of RXRα-DNA complex (1by4). When RXRα homodimers assemble on DNA, they form a 4 poplypeptide complex assembled via head to tail interactions along DR-1 repeated sequences. The α-helical structures of the polypeptides sit in the major grooves of the DNA chain, allowing for interaction with specific bases, giving a sequence specificity for the protein. The 2 Zn containing domains do not alter their configuration upon DNA binding, but are used to guide the DNA into the correct position. Upon binding to DNA, the C-terminal end of the protein, referred to as the "T-box" alters its conformation from α-helical to an extended conformation. This extended conformation allows Glu74 to move away from the DNA binding pocket and moves it so it interacts with the Zn(II) domain of the next polypeptide. RXRα homodimers preferentially assemble on DR-1 repeat sequences. DR-1 sequences are composed of an AGGTCA tandem repeat, with a single nucleotide spacer in between the repeats. Only 4 residues Lys22, Lys26, Glu19 and Arg27 interact with the DNA bases directly. 7 residues interact with the phosphate backbone of the DNA molecule, making sure it is in position for base recognition. RXRα homodimers should to assemble on DR-2 tandem repeats, sequences with the same organization as DR-1, but with 2 nucleotides as a spacer. The DNA interaction is similar with DR-2 repeats, just spaced further apart. Peroxisome proliferator-activated receptor γ ((PPAR)γ) is a protein in the nuclear receptors subfamily. It is 1 of 3 isotypes (-α, -β/ δ, and -γ) of PPAR receptors and has 2 protein isoforms governed by splice variations, which result in differences in the length of the N-terminal region (PPARγ1 and PPARγ2). PPARγ is involved in transcriptional regulation of glucose and lipid homeostasis, and helps regulate adipocyte differentiation. It has a large binding pocket, which allows it to interact with a wide array of ligands. Ligand binding triggers a conformational change of PPARγ, in the activation function-2 (AF-2) domain, which aids in the recruitment of co-regulatory factors to regulate gene transcription. PPARγ can form a heterodimer with retinoic X receptor α (RXRα), a process necessary for most PPARγ-DNA interactions. PPARγ is a molecular target for antidiabetic drugs such as thiazolidinediones (TZDs), which makes the protein a target for Type II Diabetes (T2D) drug research. PPARγ is composed of the ligand-independent activation domain (AF-1 region and A/B-domain), a DNA-binding domain (DBD) (C-domain), a hinge region (D-domain), and a ligand-dependent ligand-binding domain (LBD) (E/F-domain and AF-2 region). The 2 PPARγ isoforms, PPARγ1 and PPARγ2, differ by only 30 amino acids at the N-terminal end. These added amino acids on PPARγ2 result in increased potency and adipose-selectivity, which makes this protein a key player of adipocyte differentiation. The ligand binding domain is composed of 13 α-helices and 4 short β-strands. It has a T-shaped binding pocket with a volume of ~1440 Å3, which is larger than that of most nuclear receptors, allowing for interactions with a variety of ligands. The PPARγ LBD is folded into a helical sandwich to provide a binding site for ligands. It is located at the C-terminal end of PPARγ and is composed of ~ 250 amino acids. Activation by full agonists occurs through hydrogen bond interactions between the S289, H323, Y473, and H449 residues of the PPARγ-LBD and polar functional groups on the ligand which are typically carbonyl or carboxyl oxygen atoms. Agonist binding results in a conformational change of the LBD AF-2 region, which is necessary for coactivator recruitment. This change can either be dramatic or subtle, which leads to stabilization of a charge clamp between helices H3 and H12 to aid in associations with the LXXLL (L, leucine; X, any amino acid) motif of the coactivator. Ligand binding of PPARγ is regulated by communication between the N-terminal A/B domain, which is adjacent to the DBD, and the carboxyl-terminal LBD. The coactivator site of PPARγ is a groove created by hydrophobic residues of the H3, H3’, H4, and H12 helices. Stabilization of the AF-2 domain is important for coactivator interactions, and is achieved through ligand binding. Upon agonist binding, coactivators and other chromatin-remodeling cofactors, like histone deacetylases, are recruited and transcription is activated. Coactivators can be regulated at the transcriptional and post-transcriptional levels, as well as by protein-kinase cascades. PPARγ can actively silence genes it is bound to by recruiting a corepressor in the absence of a ligand. Once this occurs, an antagonist binds to stabilize the AF-2 region, preventing interactions with coactivators and activation of transcription. Corepressor binding creates a 3-turn α-helix corepressor motif important for preventing the AF-2 domain from assuming an active conformation. Common coactivators of PPARγ include CBP/p300, the SRC family, and TRAP220. Common corepressors include SMART, NCoR, and RIP140.
Human liver X receptor β ligand binding domain complex with retinoic X receptor β and modulator. LXR modulator binds to the LXR in the LXR/retinoic X receptor β heterodimer in a hydrophobic pocket. Structure of human FXR ligand-binding domain (deeppink) complex with non-steroidal agonist, nuclear receptor coactivator 1 peptide (cyan) and sulfate ions (3ruu). Vitamin D receptor (VDR) is a transcription factor. Upon binding to vitamin D, VDR forms a heterodimer with retinoid-X receptor and binds to hormone response receptors on DNA causing gene expression. The vitamin D hormone (green) binds to receptors in its target cells, controlling the synthesis of many different proteins involved in Ca transport and utilization. Vitamin D hormone binding site. Vitamin D hormone is located in deep pocket. VDR contains 2 domains: a ligand binding domain (LBD), that binds to the hormone (grey) and DNA-binding domain (DBD) that binds to DNA (green and blue are 2 same VDR structures). It pairs up with a similar protein, 9-cis retinoic acid receptor (RXR), and together they bind to the DNA, activating synthesis in some cases and repressing it in others. When serine is mutated it is replaced with a glycine which results in an inhibition of transcriptional activation. When transcription is inhibited it results in p53 accumulation, which activates and promotes p53 translocation into mitochondria leading to apoptosis. Serine is replaced with aspartic acid when mutated creating a negative charge. The negative charge at the residue inhibits DNA binding which cause a downregulation of VDR activity. VDR needs DNA binding in order for it to be activated which is only possible with a serine residue. The vitamin D nuclear receptor is a ligand-dependent transcription factor that controls multiple biological responses such as cell proliferation, immune responses, and bone mineralization. Numerous 1 α,25(OH)(2)D(3) analogues, which exhibit low calcemic side effects and/or antitumoral properties, have been synthesized. It was shown that the synthetic analogue (20S,23S)-epoxymethano-1α,25-dihydroxyvitamin D(3) (2a) acts as a 1α,25(OH)(2)D(3) superagonist and exhibits both antiproliferative and prodifferentiating properties in vitro. Using this information and on the basis of the crystal structures of human VDR ligand binding domain (hVDR LBD) bound to 1α,25(OH)(2)D(3), 2α-methyl-1α,25(OH)(2)D(3), or 2a, a novel analogue, 2α-methyl-(20S,23S)-epoxymethano-1α,25-dihydroxyvitamin D(3) (4a) was designed, in order to increase its transactivation potency. PXR ligand-binding pocket is flexible and allows binding to structurally and chemically distinct ligands among them the macrolide antibiotic rifampicin which is used in testing tuberculosis. 9-cis retinoid acid binds to RXRα LBD in a hydrophobic pocket. Hydrophobic, Polar. 9-cis retinoid acid is in a hydrophobic pocket. The ligand-binding residues are conserved in the 3 classes of RXR. Click here to see the difference between conformations of estrogen receptor α complexed with raloxifene and a corepressor peptide (morph was taken from Gallery of Morphs of the Yale Morph Server). Structure of estradiol metal chelate and estrogen receptor complex: The basis for designing a new class of SERMs[1]. Selective estrogen receptor modulators, such as estradiol 17-derived metal complexes, have been synthesized as targeted probes for the diagnosis and treatment of breast cancer. The detailed 3D structure of estrogen receptor α ligand-binding domain (ER-LBD) bound with a novel estradiol-derived metal complex, estradiol-pyridinium tetra acetate europium (III) (EPTA-Eu) at 2.6Å resolution was reported (2yat). The residues Glu353, Arg394 and His524 and the conserved water molecule (W1006) form hydrogen bonds with EPTA-Eu. The hydrogen bonds are shown as white dashed lines. Superposition of this structure with the structure of native ligand 17β-estradiol (E2) in the complex of E2/ERα-LBD complex (1ere) reveals that the E2 core of EPTA-Eu overlaps closely with that of E2 itself. The hydrogen bonds network made by additional estrogen receptor residues (e.g. Glu419 of H7 and Glu339 of H3, this depends on subunit), may work together with the E2 17β hydroxyl-His524 hydrogen bond and tighten the neck of the LBP upon binding of the endogenous ligand E2. 4-Hydroxytamoxifen (OHT) is an other selective estrogen receptor modulator. Superposition of EPTA-Eu/ERα-LBD complex on OHT/ERα-LBD complex (3ert) shows that there is similar network of hydrogen bonds in both complexes, except for His524 which does not form hydrogen bond with OHT in the OHT/ERα-LBD complex. Superposition of structures of all these three complexes: E2/ERα-LBD (1ere), OHT/ERα-LBD (3ert) and EPTA-Eu/ERα-LBD shows that they overlap well in the majority portions of the domain, but differ significantly in the region of the 'omega loop'. They display different synergistic reciprocating movements, depending on the specific nature of the ligand bound. The structure of estrogen receptor complexed with EPTA-Eu provides important information pertinent to the design of novel functional ER targeted probes for clinical applications. ER is a modular protein composed of a ligand binding domain, a DNA binding domain and a transactivation domain. ER is a DNA-binding transcription factor. ER bound to DNA. The DNA binding domain can be clearly observed in this scene; the highlighted yellow helix in close proximity to the DNA is part of the DNA binding domain. The blue beta sheet close to the yellow DNA binding alpha helix is also part of the DNA binding domain. The transactivation domain forms an alpha helix which is colored in purple. The transactivation domain activates RNA polymerase when the receptor binds to DNA. The ligand binding domain may be observed here with the following scene. Agonist ferutinine bound ER. The ligand ferutinine (highlighted in pink) is bound by the ligand binding domain, composed of the blue colored alpha helices immediately surrounding the purple ligand. Another view of the ligand binding domain is shown here, with estradiol bound. ER ligand binding domain bound to estradiol. ER is functional as a ligand-dependent transcription factor. ER responds to both agonist and antagonist ligands and can associate with the nuclear matrix. Differences in the structure of the receptor are observed depending on what ligand ER has bound (if any). Through comparisons of ER bound to agonist and antagonist ligands, some structural components may be highlighted. Agonist estradiol bound er The specific conformation of this tight loop of alpha helices and beta sheets around the ligand shows a complex capable of activating ER's transcription loci. This complex allows for the activation signal that will stimulate normal growth. Normal growth is stimulated when an agonist bound ER binds DNA. This occurs with the assistance of chaperon proteins. These chaperons are capable of recognizing estrogen receptor ligand complexes. When ER has bound a ligand chaperons facilitate the trans-location of the complex to the nucleus. Eventually the chaperon ligand ER complex will reach specific euchromatin, at which point the chaperons facilitate the ligand ER complex to changes conformation. This conformation will facilitate the estrogen receptor to bind the DNA major groove at specific palindromic sequences. Estradiol is a normal ligand for ER and allows for binding in the major groove of DNA. If the ligand is an antagonist the transcription factor function of estrogen receptor becomes hindered. Partial Agonist genistein bound ER The conformation of ER bound to the partial agonist genistein has a loop which is not as tight around the ligand as those found on ER with a complete agonist ligand. The ligands themselves take up different amounts of space and have varying interactions within ER. This slight difference effects the ability of the chaperon to be able to bind the receptor ligand complex to the major groove of DNA. There is a noticeable difference in the size of the pure agonist vs partial agonist scenes. Specifically, look at the width of the agonist compared to the partial agonist. Similar differences may be observed between ER which has bound the partial agonist and complete antagonist ligands. Antagonist tamoxifen bound ER The most drastic difference is noticeable between agonist and antagonist ligands. Compare the agonist scene to the Agonist estradiol bound er. Special attention should be given to the bottom right alpha helices and beta sheets that are pushed out more in the antagonist compared to the agonist bound ER. Tamoxifen (an ER antagonist) is a drug created to bind ER and inhibit the transcription factor activity of ER. Tamoxifen is larger than the normal hormone ER binds (estradiol); for this reason added with the conformation estrogen receptor takes on, the activation loop is pushed into an inactive conformation. The picture below shows structural differences between the (left/blue) antagonist tamoxifen bound ER and the (right/tan) agonist estradiol bound ER. This blocks ER from giving the signal to grow. Antagonists are generally larger and cause estrogen receptors to be too hindered sterically to be able to bind to the major groove of DNA, inhibiting the receptor. The antagonist bound estrogen receptor is noticeably larger than the agonist bound version. Further inhibition occurs when ER has bound an antagonist ligand. Antagonist bound ER is still brought to Euchromatin in the nucleus. The larger than agonist bound ER ligand chaperon complex is not capable of binding the major groove of DNA, but still occupies the space around specific palindromic sites which ER binds and modifies the transcription of local genes to these palindromic sequence areas. This blocks agonist bound ER from being able to reach these specific palindromic major groove target loci in DNA. Estrogen receptor β (ER-β) is 1 of the 2 isoforms of the estrogen receptor, a ligand-activated transcription factor which regulates the biological effects of the steroid hormone 17 β-estradiol, or estrogen, in both males and females. The complex is a hetero-tetrameric assembly consisting of 4 molecules and a ligand: 2 copies of estrogen receptor β, 2 copies of steroid receptor coactivator-1, and the ligand, Genistein. Once the ligand is bound, the complex recruits the steroid receptor coactivators, which recruit other proteins to form the transcriptional complex for initiation of transcription. This activates expression of reporter genes containing estrogen response elements. Genistein is a phytoestrogen with structural similarity to estrogen which competes for estrogen receptors. Although estrogen receptor β is widely expressed, it is not the primary estrogen receptor in most tissues. As a result, it has become a target for drug delivery, especially since it is 40x more selective for genistein than the α isoform. This enhanced selectivity may be caused by differences in residues 336 and 373 between the 2 isoforms, allowing ER-β to accommodate more polar substituents in its binding pocket. ER-β differs greatly from ER-α at the N-terminal domains, which can be seen located at opposite ends from the C termini in this rainbow representation. The protein is composed of 3 sections: a modulating N-terminal domain, a DNA-binding domain and a C-terminal ligand-binding domain.
Each ERβ contains several domains with specific functions: an N-terminal domain (NTD), a DNA-binding domain (DBD), a flexible hinge region and a C-terminal Ligand-binding domain (LBD). The complex overall is about 66% helical (10 helices; 160 residues) and 3% β-sheet (2 strands; 9 residues). The NTD is the 1st activation function (AF-1) domain that consists mostly of random coils and a small portion of helices (red) and sheets (green); it is a variable region. This lack of structure allows the region to recruit and bond many different interaction partners. This region also has the capacity to transactivate transcription without binding estrogen.
The DBD binds estrogen response elements (ERE) of target genes and recruits coactivator proteins responsible for the transcription of these genes. The ERE consist of a palindromic inverted repeat 5'GGTCAnnnTGACC-3' of target genes. The DBD is a highly conserved region. It is composed of 2 C4-type Zn fingers each containing 4 Cys residues coordinating to a Zn atom. The hinge region connects the DBD and LBD. LBD binds estrogen, coregulatory proteins, corepressors and coactivators. Genistein is not generated by the endocrine system that binds ERβ like estrogen; both ligands are completely buried within the hydrophobic core (Hydrophobic, Polar) of the ERβ complex. Binding at the LBD activates transcription mediated by the DBD. This domain is entirely helical; the LBD interacts with genistein through helices. The conformationally dynamic portion of this region gives rise to ERβ’s ligand-dependent transcriptional activation (AF-2) function. A key element of AF-2 is helix 12 (H12), which acts as a conformational switch; different receptor ligands influence the orientation of H12. Agonist ligands like genistein position H12 across the ligand-binding pocket of the LBD, which provides a coactivator docking surface. Geinstein binding allows the helices of AF-2 to form a shallow hydrophobic binding site for leucine-rich motifs of coactivators to bind. This conformation provides optimal interaction with coactivators and transcription is activated. Genistein's bicyclic form allows it to hydrogen bond on opposite sides with the hydroxyls of the histidine groups on the receptor. His475's binding to the receptor causes a conformational change and activates the receptor resulting in up-regulation for coactivators. Down-regulation will occur in the presence of corepressor as they bind to repressors and indirectly regulate gene expression. In order for the estrogen receptor β genistein to bind to a receptor and activate it there must be stabilization by a coactivator. The coactivator increases the gene expression and with this increase allows it to bind to an activator group consisting of a DNA binding domain. The estrogen receptor is found to be comprised of a dimer attached to a ligand and coactivator peptide which helps to stabilize the structure of each monomer. The conformational state of helix-12 can be modified by the binding of the coactivator. This scene depicts the hydrophobic and hydrophilic residues of the estrogen receptor. The hydrophobic regions are primarily on the inside of the protein surrounding genistein (red). Having the hydrophobic residues surrounding the binding pocket will stabilize the structure. The structure of this pocket is tertiary and do to the hydrophobic interactions inside the pocket and hydrophilic interactions on the outside help to stabilize this tertiary structure. The binding pocket is hydrophobic which means that an increase in lipophilicity would increase the affinity for ligands which in this case is genistein. The genistein structure has 3 hydroxyl groups, an ether and an ester. These 3 functional groups are polar and have many possibilities for hydrogen bonding. The His475 and Met336 residues in the binding pocket are capable of forming hydrogen bonds with genistein do to the many hydrogen bond forming functional groups. These residues are different from the residues found in ERα and so the selectivity of genistein is much greater for ERβ. Upon visualizing the estrogen receptor in an arrow representation, the structure can be classified as parallel or anti-parallel. Here is the zoomed primarily hydrophobic pocket. Binding of nuclear receptor corepressor 2 peptide and 4-hydroxytamoxifen to human estrogen-related receptor γ. The chemotherapeutic drugs bisphenol and tamoxifen are nestled between 4 alpha helices in the ERR active site. The MR ligand aldosterone binds in a fully enclosed pocket, contacting residues with six α-helices and a β-turn (Alpha Helices, Beta Strands , Loops , Turns). It forms hydrogen bonds with 4 MR residues[2]. Whole binding site. Water molecules are shown as red spheres. The structure of the complex of the ligand-binding domain of PR with RU486 - an abortion-inducing drug - shows the ligand bound at a similar location to other agonists or partial agonists with a flexible loop of PR being a possible entry route to the binding site and several PR-RU486 interactions AR structure is composed of 5 domains: A/B: N-terminal regulatory domain contains activation function 1 (residues 101-370), activation function 5 (residues 360-485) and dimerization surface (residues 1-36 and 370-494). C: DNA binding domain (DBD) D: Hinge region between DBD and LBD E: Ligand binding domain (LBD) containing an active site which binds intramolecularly the N-terminal FXXFL motif or coactivators with the same motif. F: C-terminal domain The ligand of LRH-1 binds deep in the binding pocket and is fully engulfed by it. Endoplasmic reticulum/Sarcoplasmic reticulum receptorsLigand-gated Calcium channelsInositol 1,4,5-Trisphosphate ReceptorRyanodine receptorSEE ALSO:
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