Crystal Structure of the Glucocorticoid Receptor Ligand Binding Domain Reveals a Novel Mode of Receptor Dimerization and Coactivator Recognition

Edwin Ramirez '20 and Nicole Steady '21


Contents:


I. Introduction

Nuclear receptors are a group of proteins found in cells that regulate expression of genes that affect the development and homeostasis of the cell through sensing different types of steroids, hormones and other types of molecules. The is a type of nuclear receptor found in humans that can regulate: bone turnover, cell differentiation, lung maturation, inflammation, etc.[1]. As found in Vareen et al. (1998), the glucocorticoid receptor can regulate gene expression via: glucocorticoid/GR complexbinding directly to specific DNA sequences and regulating or activating certain genes, affecting the mRNA stability, and through interactions with transcriptional factors. This receptor is very often targeted by common ligands such as dexamethasone and other glucocorticoid analogs which can treat can treat medical conditions, such as the ones listed before but the protein is most commonly targeted to treat inflammatory diseases such as asthma.


II. Mechanism

There is a myriad of GR isoforms, but the two most common isoforms of the GR are the alpha and the beta GR, which can be found in the lining of the bronchioli of the lung. The GR is composed of three main domains, the N-terminal activation function 1-domain (AF-1), the central DNA binding domain (DBD), and the C-terminal ligand binding domain (LBD). The DBD bind to the DNA at the Glucocorticoid Responsive Elements, which is a promoter region on the DNA. The dimer can bind positively with a palindromic consensus sequence pf GGTACAnnnTGTTCT to cause transcriptional activation, or negatively where the binding sequence can vary to cause repression of a gene. Glucocorticoids can also regulate gene expression through an enhanced mechanism that allows for ribonucleases to degrade specific mRNA that contains AU sequences in the untranslated 3’ region. The final way in which glucocorticoids regulate gene expression is interactions between the GR/glucocorticoid complex and transcriptional factors such as AP-1 and NF-kB which are used to mediate the induction of inflammatory genes. These protein-protein interactions result in downregulation of receptors for these transcriptional factors.

Velden et al. (1998)


III. C-Terminal Ligand Binding Domain

The composition of the GR LBDmonomers forms a that is stabilized by a series of , in an antiparallel conformation, formed by residues 547-551 of helices 1 and 3 from each LBD. The structure of the LBD involves 11 alpha helices and 4 beta strands that make up a three-layer helical domain. Helices 1 and 3 for the top layer, while helices 7 and 10 construct the bottom layer, and, lastly, helices 4,5,8, and 9 form the middle layer but are found closer to the top layer than the bottom layer.[1] The space that is left open in the bottom half of the helical sandwich contains the hydrophobic pocket to which a ligand, , can bind to. The contains reciprocal interactions between residues P625 and I628 of beta strands 3 and 4 of the LBD. The LBD also contains a C-terminal which activates the LBD by creating a conformational change by packing together the helices 3,4 and 10. The AF-2 helix is stabilized by an that forms a with the beta strand of helices 8 and 9.


IV. Dexamethasone and TIF2 Recognition

Most coactivators such as , bind to a nuclear receptor through three LXXLL motifs, but most NR have had only two out of the three motifs crystallized in complex. Bledsoe et al discovered the third motif of the GR and TIF2 complex to be , which also forms a two-turn alpha helix that orients the hydrophobic leucine side chains into a groove. The N- and C-terminal ends of the coactivator helix are stabilized by the E755 residue of the AF-2 helix and the K57 residue of helix 3 of the GR LBD, which serves as a . Bledsoe et al all support the presence of a between residues D590 and R585 of the GR and +2 and the +6 sites of the LXXLL motif. When in the hydrophobic pocket, the ligand has both hydrophobic and hydrophilic interactions with helices 3,4,5,6,7,10, AF-2, and residues from beta strands 1 and 2. Dexamethasone has its A ring facing towards Beta strands 1 and 2, and its D D ring facing towards the AF-2 helix . With this orientation comes an extensive number of and with almost every atom in dexamethasone which ultimately helps to stabilize the ligand in this binding center. Lastly, the ligand makes with residue L753 of the AF-2 helix, and the residues I747 and F749 of the loop that proceeds the AF-2 helix in order to keep the AF-2 in the active binding site conformation.

Bledsoe et al. (2002)


V. N-terminal Activation Function-1 domain and DNA Binding Domain

The N-terminal can be as small as 24 amino acid residues or as long as 600 amino acid residues and it is understood that this component of the GR is significant for transcriptional activation. AF-1 domain activates transcription by binding to sites on DNA that are similar to those used for the DBD, GREs. The DBD has two zinc ions that each contain different interactions with the DBD residues. Both contain 4 cytosine interactions with the zinc ions, which creates “zinc fingers”, but one is followed by the H1 helix, the DNA reading helix, which contains an arginine residue that is used to bind to specific bases of the major groove of the DNA, whereas the other “zinc finger” is followed by the H2 helix which makes contact to the minor groove of the DNA and contains peptide loop, “D loop”, that is used to stabilize the dimer of the DBD. Both zinc fingers are connected by “lever arm” loop.

Burgess et al. (2017)



VI. References

Álvarez, L. D., Presman, D. M., & Pecci, A. (2017). Molecular dynamics simulations of the glucocorticoid receptor DNA-binding domain suggest a role of the lever-arm mobility in transcriptional output. PLoS ONE, 12(12), 1–18

Bledsoe, Randy K., Montana,Valerie G., Stanley,Thomas B., Delves,Chris J, Apolito, Christopher J., McKee, David D., Consler, Thomas G., Parks, Derek J. Stewart, Eugene L., Willson, Timothy M.,Lambert, Millard H., Moore, John T. 2002. Crystal Structure of the Glucocorticoid Receptor Ligand Binding Domain Reveals a Novel Mode of Receptor Dimerization and Coactivator Recognition. Cell Press, Vol. 110: 93–105.

Burgess, A., Shah, K., Hough, O., & Hynynen, K. (2016). HHS Public Access, 15(5), 477–491. https://doi.org/10.1586/14737175.2015.1028369.Focused

Velden, V. H. J. van der. 1998. Glucocorticoids: mechanisms of action and anti-inflammatory potential in asthma. Mediators of Inflammation, Vol.7: 229–237.

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