1. Volume 9, Issue 2, Pages 303-313 (February 2002)
Structural and Functional Evidence for Ligand-Independent Transcriptional Activation by the Estrogen-Related Receptor 3 
Holger Greschik, Jean-Marie Wurtz, Sarah Sanglier, William Bourguet, Alain van Dorsselaer, Dino Moras, Jean-Paul Renaud 
Molecular Cell 
Volume 9, Issue 2, Pages (February 2002)
DOI: /S (02)

2. Figure 1 Schematic Representation of the ERR3 LBD Crystal Structure
(A) The ERR3 LBD forms a homodimer with a dimer interface similar to that of ERα. The ERR3 LBD adopts a transcriptionally active conformation with a SRC-1 coactivator peptide (NR box 2) bound in an α-helical conformation (depicted in green). The peptide interacts with the ERR3 LBD as previously observed for other LBD-coactivator peptide complexes.
(B) Alignment of the LBD sequences of human ERR1, 2, and 3, and human ERα. Amino acid residues conserved between all four receptors are marked in yellow, and residues conserved in all ERR family members are marked in blue. Secondary structure elements of the LBDs of hERR3 and hERα are shown above and below the amino acid sequence alignment, respectively. Amino acid numbering is given for hERR3 and hERα. To obtain the residue numbering of hERR1 and hERR2, add the following offset to the hERR3 residue numbering: +56 and –25, respectively. (Note the insertion in ERR1 between H9 and H10.) Green and blue dots mark the residues that line the ligand binding cavities of hERR3 and hERα, respectively. SwissProt accession numbers are O75454 (hERR3), P11474 (hERR1), O95718 (hERR2), and P03372 (hERα).
Molecular Cell 2002 9, DOI: ( /S (02) )

3. Figure 5
ESI-MS Analysis of Estradiol Binding to Wild-Type and Mutant ERR3 LBDs
About 3.7 nmol of WT or mutant ERR3 LBD was incubated for 15 min on ice with a 1.7 molar excess of E2. ESI-MS analysis was performed as described in Experimental Procedures. The spectrum of the mutant L345I-F435L was aligned with those of the WT LBD and the mutant L345I to compensate for the difference in mass. The relative abundance of unliganded or E2-bound species is indicated above the spectra.
Molecular Cell 2002 9, DOI: ( /S (02) )

4. Figure 2
Schematic Representation of the Ligand binding Pocket of the ERR3 LBD
(A) In the crystal structure, the positions of all shown side chains are well defined, with the exception of that of E275, which exhibits high temperature factors. All atoms are colored according to the following code: carbon, gray; oxygen, red; nitrogen, blue; sulfur, yellow.
(B) View as in (A) with E2 docked into the empty ligand binding cavity of ERR3. The position of E2 results from the superposition of the ERR3 LBD and the ERα LBD/E2 complex. Steric interference with the D-ring of E2 is mainly due to the presence of F435 (L525 in hERα) and L345 (I424 in hERα) in ERR3.
(C) Schematic comparison of amino acid residues that form the ligand binding pocket in hERR3 with the corresponding residues of hERR2, hERR1, and hERα. Residues that are conserved among all four receptors are depicted in yellow, and those conserved among the ERR isotypes are colored in blue. Amino acids of hERα that according to our modeling studies allow the binding of E2 to ERs but not to ERRs are highlighted in red. Important isotype-specific amino acid differences are colored in green.
Molecular Cell 2002 9, DOI: ( /S (02) )

5. Figure 3
Docking of E2, DES, and 4-OHT into the Ligand Binding Pocket of ERR3 and ERR1
(A) E2 is positioned within the enlarged ERR3 ligand binding pocket (transparent blue color) obtained upon moving the side chain of F435 in H11 from its position observed in the crystal structure (marked in green) to the position which induces an antagonist LBD conformation (marked in red). The “activation helix” (H12) is still depicted in the agonist position observed in the crystal structure but would have to move into an antagonist position to avoid a steric clash with the new rotamer of F435. Despite the conformational change of F435, E2 would still not significantly bind to ERR3 due to steric interference with L345 in H7.
(B and C) Docking of DES and 4-OHT, respectively, into the enlarged ligand binding pocket of ERR3. The antagonist action of DES mainly results from the conformational changes of F435 and subsequently H12 induced upon ligand binding. In comparison, the side chain containing the C-ring of 4-OHT also contributes to the antagonist action of this ligand due to steric interference with H12.
(D) Comparison of the ligand binding cavities of ERR3 and ERR1. F435 in H11 (colored green) is conserved between ERR1 and ERR3 and interferes with the positioning of DES (colored cyan). All other residues of ERR3 are shown in gray. Residues of ERR1 that account for isotype-specific differences in the ligand binding pocket are shown in magenta. Most importantly, A272 is replaced by phenylalanine, which in the given conformation fills the cavity. Therefore, multiple conformational changes are required to accommodate ligands such as DES in ERR1.
Molecular Cell 2002 9, DOI: ( /S (02) )

6. Figure 4
In Vitro Interaction Between Wild-Type or Mutant LBDs of ERR3 and the RID of SRC-1
Two micrograms (about 70 pmol) of partially purified His-tagged ERR3 LBD was preincubated for 10 min on ice with 4 μg (about 100 pmol) of GST-tagged SRC-1 RID or GST in the absence (A) or in the presence ([B] and [C]) of 10−4 M 4-OHT or 10−4 M DES. Complexes were separated on native polyacrylamide gradient gels.
Molecular Cell 2002 9, DOI: ( /S (02) )

7. Figure 6
Transcriptional Activities of Wild-Type and Mutant ERR3 Fusion Proteins in Transient Transfection Assays
(A) COS-1 cells were transfected in 24-well plates with 250 ng per well of Gal4(5×)-TATA-LUC reporter and 50 ng of WT or mutant pCMX-Gal4-ERR3 expression plasmid. The empty plasmid pCMX-Gal4 served as control. Experiments were performed in the absence or presence of 10−6 M 4-OHT or 10−5 M DES.
(B) Experiments were performed as described in (A) using 250 ng of Gal4(3×)-TK-LUC reporter plasmid and 50 ng of WT or mutant Gal4-ERR3 expression plasmid.
(C) Experiments were performed as described in (A) in the absence or presence of 10−5 M E2. Panels (A)–(C) represent an average of three independent experiments.
Molecular Cell 2002 9, DOI: ( /S (02) )