1. Dynamic Stabilization of Nuclear Receptor Ligand Binding Domains by Hormone or Corepressor Binding 
Pavlos Pissios, Iphigenia Tzameli, Peter J. Kushner, David D. Moore 
Molecular Cell 
Volume 6, Issue 2, Pages (August 2000)
DOI: /S (00)

2. Figure 1
The Hinge of TR Interacts with the Remainder of the LBD in a Ligand-Dependent Manner
(A) Schematic representation of the different domains of TR and a detailed representation of the primary and secondary structure of the hinge region.
(B) Mammalian two-hybrid of Gal-TR hinge ( ) with progressive deletions of TR-LBD from the N terminus fused to VP16 activation domain. HepG2 cells were cotransfected with the above constructs along with G5E1B-Luc reporter containing five Gal4 sites in front of minimal E1B TATA box and treated with 10−6M of T3 or vehicle alone.
(C) In vitro interaction assay using a GST fusion of TR hinge (GST-TR ) with in vitro translated [35S]methionine-labeled TR-LBD lacking helix 1 (CMXFlag-TR ), top panel, or containing helix 1 (CMXFlag-TR ), bottom panel.
Molecular Cell 2000 6, DOI: ( /S (00) )

3. Figure 2
Full-Length TR Separated into Two Fragments Can Assemble in a Ligand-Dependent Manner In Vitro and In Vivo
(A) EMSA of full-length TR (1-461) and TR fragments (TR1-235 and TR ) on a DR4 oligonucleotide. In vitro translated full-length TR or the fragments were used in the presence or absence of 10−6 M T3. In vitro translated RXR was used in all lanes.
(B) Cotransfection of TR fragments (TR1-235 and FlagTR ) alone or in combination with 28T-TK-Luc reporter containing two IR0 elements in HepG2 cells in the absence or presence of T3.
Molecular Cell 2000 6, DOI: ( /S (00) )

4. Figure 3 Helix 1 of TR Is Required for Ligand Binding
In vitro translated TR and TR were incubated in the presence or absence of 10−5 M T3 and digested with trypsin. The trypsin-resistant fragments were analyzed by SDS–PAGE.
Molecular Cell 2000 6, DOI: ( /S (00) )

5. Figure 4
Effect of Mutations within the Hinge Region on the Ligand-Dependent Association between the TR Hinge with the Remainder of TR-LBD
(A) Primary and secondary structure of the TR hinge region with point mutations reported to abolish corepressor binding. Schematic representation of the deletion and mutation constructs of the TR hinge region.
(B) Mammalian two-hybrid assay; Gal fusions of the wild type and deletions of the TR hinge were cotransfected in HepG2 cells together with VP-TR and G5E1B-Luc reporter in the presence of T3 (right panel) or vehicle alone (left panel).
(C) Mammalian two-hybrid assay; Gal fusions of the wild type and point mutations of the TR hinge were cotransfected in HepG2 cells together with VP-TR and G5E1B-Luc reporter in the presence of T3 (right panel) or vehicle alone (left panel).
(D) Yeast two-hybrid assay of LexA fusion of wild type and mutated TR hinge constructs with B42 fusion of TR
Molecular Cell 2000 6, DOI: ( /S (00) )

6. Figure 5 Generality of Helix1–LBD Interaction
Mammalian two-hybrid assay of Gal4 fusions containing helix 1 of either TR (A), RAR (B), RXR (C), or ER (D) with VP16 fusions of their respective LBDs containing or lacking helix 1. HepG2 cells were cotransfected with G5E1B-Luc reporter, the appropriate combination of the expression vectors and ligands, or vehicle.
Molecular Cell 2000 6, DOI: ( /S (00) )

7. Figure 6
NCoR Enhances the Hinge–LBD Interaction in the Absence of Ligand
(A) HepG2 cells were cotransfected with Gal4-hinge and VP16-LBD fusions of TR (left panel), RAR (middle panel), and RXR (right panel) in the presence or absence of Gal-N-CoR RID construct (Seol et al. 1996) and treated with the corresponding ligands or vehicle alone.
(B) GST pulldown assay of GST-TR with in vitro translated TR Wild-type and mutant peptides of N-CoR IDI, top and bottom panels, respectively, were included where indicated at concentrations of 10 (+) and 100 (++) μM.
Molecular Cell 2000 6, DOI: ( /S (00) )

8. Figure 7
N-CoR Binding Induces the Appearance of a Smaller Protease-Resistant Fragment
(A) 35S-labeled in vitro translated TR was incubated with 1 mM of either wild-type or mutated N-CoR peptide in the absence or presence of 1 μM T3. The mixture was digested with trypsin for 7 min at room temperature and analyzed by SDS–PAGE.
(B) Full-length TR LBD204–461 (F) or a C-terminal truncated TR LBD (Δ15C) was incubated with peptides and/or T3 and digested with trypsin as above.
Molecular Cell 2000 6, DOI: ( /S (00) )

9. Figure 8
Model of the Allosteric Effects of Ligands and Corepressors on the Interaction between the Hinge and the LBD of Nuclear Hormone Receptors
The model is based on the released coordinates of the human TRβ LBD with coactivator peptide (Darimont et al. 1998). The hinge region including the helix 1, the helix 12, and the corepressor peptide are drawn as ribbons. The rest of TR LBD is represented as surface. The blue color indicates stable position; the red color indicates instability. Actual position of helix 12 in the apo-TR structure is not known. Ligand is visible in the holo-LBD. The drawing was created with Swiss PDB viewer (Guex and Peitsch 1997).
Molecular Cell 2000 6, DOI: ( /S (00) )