Volume 5, Issue 2, Pages 289-298 (February 2000) Crystal Structure of a Heterodimeric Complex of RAR and RXR Ligand-Binding Domains  William Bourguet, Valérie Vivat, Jean-Marie Wurtz, Pierre Chambon, Hinrich Gronemeyer, Dino Moras  Molecular Cell  Volume 5, Issue 2, Pages 289-298 (February 2000) DOI: 10.1016/S1097-2765(00)80424-4

Figure 1 The RARα–RXRαF318A LBD Heterodimer and Its Component Monomers (A) The dimeric arrangement of hRARα and mRXRαF318A LBDs is viewed perpendicular to the dimer axis. The secondary structural elements are labeled according to the nomenclature of other nuclear receptors (Wurtz et al. 1996). Helices (H) are represented as tubes; coil regions are colored in orange; β strands (s) in red. The dotted line in mRXRαF318A indicates the unmodeled region between H1 and H3. The ligands in hRARα (BMS614) and mRXRαF318A (oleic acid, OA) are represented in yellow. (B) Superposition of the hRARα/BMS614 complex from the heterodimer crystal structure and the hRARγ/T-RA monomer complex (Renaud et al. 1995). (C) Superposition of the mRXRαF318A/OA LBD from the heterodimer crystal structure and the hRXRα/9C-RA LBD monomer crystal structure (P. Egea et al., submitted). (D) Superposition of the hRARα/BMS614 and the mRXRαF318A/OA LBD complexes, both from the heterodimer crystal structure. Molecular Cell 2000 5, 289-298DOI: (10.1016/S1097-2765(00)80424-4)

Figure 2 Structural and Sequence Analysis of the Heterodimer Interface (A) Molecular surfaces of hRARα and mRXRαF318A LBDs showing the heterodimerization footprints colored in magenta (4.5 Å cutoff). Helices and loops constituting the interface are labeled. (B) CPK models of the interacting surfaces of hRARα and mRXRαF318A, showing the side chains of the residues from both protomers making intermolecular contacts. The color code is as follows: yellow for hydrophobic, red for negatively charged, blue for positively charged, and green for hydrophilic residues. (C) Superposition of the hRXRα homo- (orange) and the mRXRαF318A heterodimerization (blue) surfaces. The side chains involved in both interfaces are depicted and labeled (mouse RXRα numbering) as well as the secondary structural elements. (D) Sequence identity over the entire LBDs and among the dimerization surfaces as seen in the mRXRαF318A–hRARα heterodimer structure within key members of the NR family represented as the three separate groups of distinct evolutionary conservation (RAR, RXR, and steroid subgroups; Wurtz et al. 1996). The sequence identity in the LBD (yellow) encompasses all residues from helices H1 to H12, except the variable regions between helices H1 and H3 and helix H5 (Phe-202-Asp-223) and β sheet 1 (279–283 in hRARγ). A total of 25 residues are used to calculate the sequence identity (green) of the dimer interface according to the hRARα interface as observed in the heterodimer. Molecular Cell 2000 5, 289-298DOI: (10.1016/S1097-2765(00)80424-4)

Figure 3 The Binding Mode of BMS614 and Its Consequences on H11 and H12 Integrity and Positioning (A) BMS614 interactions with the RARα LBD. Interactions between the protein and the ligand (4.2 Å cutoff) are depicted as broken lines. Residue names are colored as a function of the RARα structural element from which they originate. Boxed amino acids correspond to those residues that determine the RARα, β, or γ isotype selectivity of synthetic retinoids. W indicates a water molecule. (B) Stereo view of the 2.5 Å resolution (2Fo-Fc) map contoured at 1.0 standard deviation, showing BMS614 in its RARα LBD binding site. Thin lines correspond to the protein Cα trace, and the ligand-contacting side chains (carbons, yellow; oxygen, red; nitrogen, blue; sulphur, green) and the ligand (light green) are represented by thick lines. (C) Superposition of the ligand-binding sites of the RARα/BMS614 (green) and the RARγ/T-RA (orange) complexes illustrating the steric clash between the BMS614 and I412 of holo-H12, which prevents the positioning of the AF-2 AD core in the agonist orientation, and the increased size of L11–12, which permits H12 to be positioned in the antagonist cleft. (D) Structural sequence alignment showing the amino acid residues forming the groove that interacts with the NR box of coactivators in ERα (Shiau et al. 1998) and TRβ1 (Darimont et al. 1998) (asterisks) together with the interacting residues at the H12/LBD interface in the antagonist RARα/BMS614, RXRαF318A/OA, ERα/OHT (Shiau et al. 1998) complexes (yellow dots). Sequences corresponding to the secondary structure elements found in the hRARα (H3, L3–4, H4, and H12) or in the hTRβ (H3, H4, H5, and H12) LBDs are indicated. Conserved residues in the consensus sequence are indicated above the alignment by the single-letter code for invariant or highly conserved amino acids (h, hydrophobic; φ, aromatic). Molecular Cell 2000 5, 289-298DOI: (10.1016/S1097-2765(00)80424-4)

Figure 4 The mRXRαF318A Ligand-Binding Pocket (A) Oleic acid (OA) interactions with the mRXRαF318A LBD. Interactions between the protein and the ligand (4.2 Å cutoff) are depicted as broken lines. Residue names are colored as a function of the mRXRαF318A structural element from which they originate. W indicates a water molecule. (B) Stereo view of the 2.5 Å resolution (2Fo-Fc) map contoured at 1.0 standard deviation, with OA docked into its binding site. The protein Cα trace is represented in gray, the contacting side chains (carbons, yellow; oxygen, red; nitrogen, blue; sulphur, green) and the ligand (blue) by thick lines. (C) Superposition of the ligand-binding sites of the mRXRαF318A/OA (blue) and the hRXRα/9C-RA (pink) LBDs. The gray arrows highlight the different ligand-specific induced conformations in both complexes. Helices (ribbons) and loops (rods) are indicated, as well as some residues (mouse RXRα numbering) discussed in the text. Molecular Cell 2000 5, 289-298DOI: (10.1016/S1097-2765(00)80424-4)

Figure 5 C16–C18 Fatty Acids Induce Transcriptional Activation and Promote the Recruitment of the Coactivator TIF2 by mRXRαF318A (A) Transcriptional activation of the DR1-tk-CAT reporter gene by full-length wild-type and F318A mutant mRXRα in transient transactivation assays. Transfected Cos1 cells were exposed to the indicated ligands for 24 hr. Addition of the RXR agonist SR11237 (1 μM) induces transcriptional activity of the wild-type mRXRα and further stimulates the transactivation by mRXRαF318A (lanes 1–4). Increasing concentrations (10−6, 10−5, and 10−4 M) of oleic acid do not affect SR11237-mRXRα induced transcription (lanes 5–7). The inhibition of the seemingly “constitutive” activity of mRXRαF318A by the RXR antagonist HX531 (lane 9) is relieved by adding inceasing concentrations of the indicated fatty acid ligands, but no effect is seen with the wild-type receptor (lanes 11–27). (B and C) Pulldown assays. Purified His-mRXRαF318A LBD was incubated, in the presence of 5 μM SR11237 ([B], lanes 3–12) or oleic acid (lanes 13–22), with increasing quantities of in vitro translated [35S]methionine TIF2.1 or TIF2.1m123 as indicated. Purified His-hRARα/BMS614-mRXRαF318A LBD heterodimer ([C], lanes 1–10) was incubated with in vitro translated [35S]methionine TIF2.1 or TIF2.1m123 in the presence of increasing concentrations of oleic acid (5.10−8 to 5.10−5 M). In lanes 11 and 12, additional BMS614 was added during incubation. Input corresponds to 100% of the TIF2 used in the assays. Molecular Cell 2000 5, 289-298DOI: (10.1016/S1097-2765(00)80424-4)