Figure 1.
Crystals of the ancSR2–progesterone–mifepristone complex and in vitro activation data.
A. The ancSR2–progesterone–mifepristone ternary complex crystals measured approximately 50 x 20 x 20 microns. B. In luciferase reporter assays, ancSR2 is strongly activated by mifepristone (EC50 = 56 ± 1.2 nM) as well as progesterone (EC50 = 78 ± 1.7 pM).
Figure 2.
Overall structure of the ancSR2–progesterone–mifepristone complex.
Overall structure of the ancSR2 LBD with bound progesterone and mifepristone shown as green and magenta, respectively with oxygens, colored red. Helices are blue, β-sheets are yellow, loops are white. Figures were generated in PyMol (Schödenger, LLC).
Figure 3.
Fo-Fc electron density (green) contoured to 2 σ showing evidence for bound ligand. Omit maps were generated by removal of the ligand from the structure and running 3 cycles of gradient energy minimization and B-factor optimization in PHENIX (version dev-1423) to minimize model bias. A. Electron density within the LBP corresponding to the volume of progesterone. B. Electron density at the base of the receptor corresponding to the volume of mifepristone (site-one). C. Electron density at the coactivator cleft corresponding to the volume of mifepristone (site-two).
Figure 4.
Mifepristone binding site interactions.
ancSR2 is shown in slate blue; mifepristone is shown in magenta (oxygens, red; nitrogens, blue). Residues within 4.2 Å of the ligand are shown. A. Site-one mifepristone interacts with both the monomer in the asymmetric unit as well as residues in a symmetry mate (forest green). B. Site-two mifepristone interacts with residues in the ancSR2 coactivator cleft.
Figure 5.
Mifepristone occupies the coactivator protein space.
A. ancSR2–progesterone–mifepristone (protein, slate blue; mifepristone, magenta) was overlaid with ancestral corticoid receptor–deoxycorticosterone–hSHP NRBox1 (PDB accession code: 2Q3Y) complex. Mifepristone occupies the same space as the hSHP NRBox1 peptide (green, leucine side chains shown as sticks). B. Sequence alignment of ancestral and extant 3-ketosteroid receptor coactivator binding clefts. Sequence alignments of the AncSR2, AncCR, Progesterone Receptor (PR), Androgen Receptor (AR), Glucocorticoid Receptor (GR), and Mineralocorticoid Receptor (MR) coactivator binding clefts. Hydrophobic interactions (green) and hydrogen bonds (red) are shown for the interaction between AncSR2–mifepristone site-two and AncCR–SHP. Conserved residues across the SR lineage are indicated by a black box.
Figure 6.
Global alignment of progesterone-bound steroid receptors.
A cartoon representation of the ancSR2–progesterone–mifepristone (slate blue), ancSR2–progesterone (orange, PDB accession code: 4FN9), and progesterone receptor–progesterone (purple, PDB accession code 1A28) complexes overlay with high overall structural similarity. B. Ligand adopts an identical position and conformation in two progesterone-bound receptor structures. The ancSR2–progesterone–mifepristone (ligand – slate blue, receptor – light blue) and progesterone receptor–progesterone (ligand – magenta, receptor – light pink, PDB accession code 1A28) structures show identical positioning and conformation of the ligand within the ligand binding pocket. Progesterone makes hydrogen bonds with Arg82 and Thr210 in both structures (dashed red lines).
Figure 7.
Mifepristone and 4-hydroxytamoxifen show similar binding modes to the steroid receptor coactivator binding cleft.
Alignment of the ancSR2–progesterone–mifepristone crystal structure and the ERβ–tamoxifen structure shows both mifepristone and 4-hydroxytamoxifen bound to the coactivator cleft. ancSR2 – slate blue, mifepristone – magenta, ERβ – light green, tamoxifen – orange.