Intracellular receptors: Difference between revisions

<StructureSection load='' size='300' side='right' caption='Human androgen receptor ligand-binding domain complex with modulator (PDB code [[3b5r]])' scene='54/543362/Cv/1'>

The Ligand binding domain for each piece of the dimer has a nearly identical structure of an <scene name='RA_Mediated_T-reg_Differentiaition/Alpha-helical_domains/2'>Tα-helical sandwich</scene>. 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 <scene name='RA_Mediated_T-reg_Differentiaition/Dimerization_interface/3'>dimerization interface</scene> and the <scene name='RA_Mediated_T-reg_Differentiaition/Ligand_binding_pockets/1'>ligand binding pocket </scene>.

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 <scene name='RA_Mediated_T-reg_Differentiaition/Dimerization_interface/1'>heterodimer interface</scene>. The sequence identity between the 2 molecules on the dimer interface is 0.33.  

The residues of <scene name='RA_Mediated_T-reg_Differentiaition/Rar_dimer_interface/2'> RAR-</scene>α that are interacting in the heterodimer:

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 <scene name='RA_Mediated_T-reg_Differentiaition/Ligand_binding_pockets/1'>LBD</scene>. For the ligand used in RARα crystallization, BMS614, 21 primarily hydrophobic residues form the <scene name='RA_Mediated_T-reg_Differentiaition/Rar-ligand_binding_pocket/1'>RARα ligand binding pocket</scene>. 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 <scene name='RA_Mediated_T-reg_Differentiaition/Rar-ligand_binding_pocket2/1'>major differences</scene> 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 <scene name='RA_Mediated_T-reg_Differentiaition/Rxr-ligand_binding_pocket/1'>RXRα binding pocket</scene> 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.

<scene name='51/519788/Cv/2'>Crystal structure of RXRα-DNA complex</scene> ([[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  

<scene name='RA_Mediated_T-reg_Differentiaition/Rxr_dbd_alpha_helices/1'>α-helical</scene> 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 <scene name='RA_Mediated_T-reg_Differentiaition/Rxr_dbd_zn_domains/2'>Zn containing domains</scene> 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 <scene name='RA_Mediated_T-reg_Differentiaition/Rxr_dbd_t-box/1'>"T-box"</scene> 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 <scene name='RA_Mediated_T-reg_Differentiaition/Rxr-dna_base_interact/1'>4 residues</scene> Lys22, Lys26, Glu19 and Arg27 interact with the DNA bases directly. <scene name='RA_Mediated_T-reg_Differentiaition/Rxr-dna_backbone_interact/1'>7 residues</scene> 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 (<scene name='PPAR-gamma/Ppar_gamma/3'>(PPAR)</scene>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 <scene name='PPAR-gamma/Binding_pocket/1' target='1'>large binding pocket</scene>, which allows it to interact with a wide array of ligands. <scene name='PPAR-gamma/Interacting_residues/3'>Ligand binding</scene> triggers a conformational change of PPARγ, in the activation function-2 <scene name='PPAR-gamma/Af-2_domain/2'>(AF-2) domain</scene>, which aids in the recruitment of co-regulatory factors to regulate gene transcription. PPARγ can form a <scene name='PPAR-gamma/Ppar_rxr/3'>heterodimer</scene> 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 <scene name='PPAR-gamma/Lbd/2' target='2'>ligand binding domain</scene> 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 <scene name='PPAR-gamma/Coactivator_bound/2'>coactivator site</scene> 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.

<scene name='56/562378/Vit_d_receptor_3m7r/3'>Vitamin D receptor (VDR)</scene> 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 <scene name='56/562378/Vit_d_receptor_ligand/1'>vitamin D hormone</scene> (green) binds to receptors in its target cells, controlling the synthesis of many different proteins involved in Ca transport and utilization.

<scene name='51/517370/Cv/2'>Vitamin D hormone binding site</scene>.  

 

<scene name='51/517370/Cv/3'>Vitamin D hormone is located in deep pocket</scene>. VDR contains 2 domains: a <scene name='56/562378/Lbd/1'>ligand binding domain (LBD)</scene>, that binds to the hormone (grey) and <scene name='56/562378/Dbd/2'>DNA-binding domain (DBD)</scene> 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 <scene name='56/562378/Serine_final/1'>serine</scene> is mutated it is replaced with a <scene name='56/562378/Glycine_final/1'>glycine</scene> 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. <scene name='56/562378/Serine_final/1'>Serine</scene> is replaced with <scene name='56/562378/Asparticacid_final/1'>aspartic acid</scene> 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 <scene name='56/562378/3a3z/1'>the synthetic analogue (20S,23S)-epoxymethano-1α,25-dihydroxyvitamin D(3) (2a)</scene> 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.

 

 

<scene name='71/714947/Er_bound_to_dna/4'>ER bound to DNA</scene>. 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.

 

<scene name='71/714947/Agonist_ferutinine_bound_er/5'>Agonist ferutinine bound ER</scene>. 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. <scene name='71/714947/Er_ligand_binding_domain_estra/1'>ER ligand binding domain bound to estradiol</scene>.

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. <scene name='71/714947/Agonist_estradiol_bound_er/2'>Agonist estradiol bound er</scene> 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. <scene name='71/714947/Partial_agonist_genistein_er/3'>Partial Agonist genistein bound ER</scene> 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. <scene name='71/714947/Antagonist_tamoxifen_bound_er/5'>Antagonist tamoxifen bound ER</scene> The most drastic difference is noticeable between agonist and antagonist ligands. Compare the agonist scene to the <scene name='71/714947/Agonist_estradiol_bound_er/2'>Agonist estradiol bound er</scene>. 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.