D58 Rar _VERIFIED_
Copyright: 2010 Sato et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The funders (CNRS, INSERM, Université de Strasbourg, ANR and the European Commission Structural Proteomics in Europe SPINE2-Complexes) had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Upon ligation to their cognate receptors, naturally-occurring vitamin A derivates mediate several physiological processes, such as vertebrate morphogenesis, cellular growth, differentiation or survival, as well as pathological conditions e.g premature birth, skin diseases or cancer development (reviewed in ). The atRA isomer binds exclusively to RARs whereas the 9-cis form of RA binds to both RARs and RXRs, (each of which exists as three isoforms α, β and γ) (reviewed in ).
The ability of RAR/RXR to modulate the expression of target genes results from a combinatorial, coordinated and sequentially orchestrated exchange between nuclear hormone receptors (NHRs) and their coregulators. A general model of RAR/RXR-mediated transcription proposes that unliganded RAR/RXR heterodimers are bound to regulatory elements of their target genes and interact with transcriptional repressor complexes such as NCOR/SMRT/SIN3 to recruit histone deacetylases that lead to repression of target gene transcription . Binding of agonist ligand to the nuclear receptor, triggers a conformational change in the ligand binding domain (LBD) with the repositioning of the C-terminal helix H12 creating a binding surface that allow coactivator to bind. Coactivator proteins such as CBP/p300, the p160 family, CARM1 or the Mediator contain one or more consensus LXXLL motifs that form an α-helix that fits into the hydrophobic cleft on the LBD to allow activation of target genes . Antagonist ligands that prevent the C-terminal helix H12 from adopting its active conformation facilitate the interactions with corepressors.
This new structure explains the RXR full antagonism activity of LG100754. The comparison with the previous RAR/RXR structures together with biochemical assays of the SRC-1 coactivator peptide recruitment to the heterodimer provides insight into the molecular mechanism of LG100754 action and explains its phantom effect.
The crystal packing induces an intermolecular interaction between the flipped H12 of the RXRα-LG100754 and the coactivator binding surface of a symmetry related RXR molecule (Figure 4). This packing interface is made not only by the cofactor binding site but also H11 of RAR, H6, LoopH6-H7, H7 and H11 of RXR (Figure 4). Total surface area buried between the packing interface is 2000 Å2. Such tetrameric assembly induced by the flipped H12 is also observed in the other crystal structures of RXR [PDB IDs: 1LBD, 1G5Y, 1H9U, 2Q60, 2GL8] but the orientation of the present tetramer is different from any other. The oligomeric state of this complex in solution was determined by Small Angle X-ray Scattering (SAXS) the values of the radius of gyration Rg and of the maximal dimension Dmax as structural parameters (Table 1 and Figure S1). We further compared them to those of other RAR/RXR heterodimer or RXR homodimer or tetramer  and to theoretical values calculated from crystallographic structures. The values of Rg and of Dmax parameters measured for the RARα-atRA/RXRα-LG100754 complex clearly indicate that the complex is dimeric in solution. The Rg is 5 Å smaller than that calculated from the tetrameric crystal structure (Figure S1). Furthermore, the best fit of the experimental data is obtained unambiguously with the dimeric model in which one monomer is in a closed conformation and the other one in an open conformation with helix H12 pointing to the solvent (Figure S1). The experimental Rg is 1 Å larger than that of the heterodimer fully bound to agonists indicating a less compact conformation and 1 Å smaller than the relaxed apo-form of RXR dimer (Table 1). The tetramer generated by the crystallographic symmetry is induced by the highly concentrated conditions during the crystallization process.
The binding mode of atRA (Figure 2B) to RARα in the present heterodimer is identical to that observed previously for RARα LBD . The size of the atRA is 278 Å3. The comparison of the volume of the ligand binding cavity is 418 and 503 Å3 and the retinoic acid occupies 66.5% and 55.3% of the pockets for RARα and RARα, respectively. The difference of the cavity size around 100 Å3 is due to the different residues of the two isotypes forming the ligand binding pocket (LBP).
The rexinoid antagonist LG100754 is buried in the LBP of RXRα formed by residues located on helices 3, 5, 7, 11 and the β-turn (Figure 5). The interactions are mainly hydrophobic with 80 Van Der Waals (VDW) contacts with the LBP at 4.2 Å cutoff. The carboxylate group makes an anchoring salt bridge with Arg321 [hArg316] (H5) and hydrogen bond with amino group of Ala332 [hAla327] (LoopH5-H6) in the hydrophobic pocket, similarly as observed with the carboxylate of 9-cisRA in the RXRα complex . One water molecule makes a hydrogen bond network between the carboxyl group of LG100754 and the amino group of Leu314 [hLeu309]. The tetrahydronaphatalene moiety of LG100754 interacts with residues of H3, H5, H7 and H11 through VDW contacts and notably with Trp310 [hTrp305] (H5) (Figure 5). Compared to the 9-cisRA-bound RXR, the carboxylate and tetrahydronaphatalene group of LG100754 are located at the places which correspond to that of the carboxylate and β-ionone group of 9-cisRA. The propoxy group is pointing towards H11 and interacts with this helix through VDW contacts notably with Leu441 [hLeu436] which is repositioned (Figure 6A). The electron density map of the end of the propoxy group is poor because of its flexibility (see Figure 2B). A remarkable feature is the solvent accessibility of this LBP because of the flip of H12 to the solvent. According to crystallographic symmetry, this accessible region of the LBP is covered by LoopH11-H12 (mainly Phe443 [hPhe338] and Asp449 [hAsp444]) of another RXRα symmetry related molecule (Figure 4). The active agonistic conformation of H12 of RXRα is prevented by the long-tailed propoxy group of LG100754 which induces a steric hindrance with Leu456 [hLeu451], and consequently the coactivator peptide binding as shown for the superimposition of RXR-LG100754 and RXR-9cis RA (Figure 6A). Oleic acid, a neutral RXR ligand, has been crystallized in an RXR agonist conformation in RXR homodimer  and in an RXR antagonist conformation in RAR/RXR heterodimer . Superposition of RXRα bound to LG100754 and to oleic acid in RXR antagonist conformation shows two different antagonist conformations. Indeed, the propoxy group of LG100754 induces a steric hindrance with Leu446 [hLeu441] in the LoopH11-H12 as observed in the RXR-oleic acid antagonist conformation, precluding H12 binding to the coactivator cleft (Figure 6B). This new structural information is in agreement with the inability of RXRα-LG100754 homodimer to bind to any coactivator or corepressor . Phe442 [hPhe437] and Phe443 [hPhe438] in H11 of RXRα which are known to play important roles in the transition of the apo to agonist conformation , flip out to the solvent region in the present antagonistic structure (Figure 6).
(A) Close-up view showing the superposition of RXRα LBP bound to LG100754 (in cyan) and to 9-cisRA (in red). LG100754 and 9-cisRA are shown by stick representation in yellow and magenta, respectively, with oxygen atoms in red. The propoxy group of LG100754 induces a steric hindrance (solid arrow) with Leu456 (H12). Residues involved in the transition agonist to antagonist transition (dotted arrow) conformation are labelled. (B) Close up view of the superposition of RXRα LBP bound to LG100754 (in cyan) and to oleic acid in antagonist conformation (in orange). Oleic acid is show by stick representation in magenta. The propoxy group of LG100754 induces a steric hindrance (solid arrow) with Leu446 as shown by an arrow.
In contrast, the structural basis of the antagonism of UVI3003 should be similar to that of LG100754. The crystal structure of the complex of RXR and the partial agonist UVI3002  (Figure 1) reveals that the alkyl ether group of UVI3002 is located at the same position as the propoxy group of LG100754 but its length do not prevent the agonist conformation. Therefore, UVI3003 which has a longer alkyl group than UVI3002 should similarly prevent H12 associating to the LBD and the RXR complex should adopt an antagonistic conformation as in RXR-LG100754. In agreement with this molecular mechanism of antagonism, analogues of LG100754 with shorter groups such as ethyl or methyl groups instead of the propoxy group act either as partial agonist or full agonist for RXR, respectively .
A recent NMR study on the effect of RXR antagonists on the conformation of H12 in the RXR homodimer and in the permissive PPAR/RXR with PPAR bound to an agonist ligand reveals similar features, namely the rexinoid antagonist is unable to stabilize a compact state and therefore prevents the coactivator from binding to RXR .
To quantify the recruitment of the SRC-1 NR2 peptide to RARα, RXRα and RARα/RXRα, Isothermal Titration Calorimetry (ITC) was used, thus providing the full thermodynamic profile of SRC-1 binding. The similar SRC-1 NR2 peptide (25 residues) to the one used in ESI-MS was used in the ITC experiments. Representative titrations for SRC-1 NR2 binding are shown in Figure S3. In the presence of LG100754, the RARα monomer binds the SRC-1 NR2 peptide with an affinity similar to that measured in presence of an RAR agonist ligand (Figure 9 and Table S2). Therefore, the LG100754 ligand stabilizes the agonist conformation of RAR that renders accessible the binding surface for coactivator recruitment. Docking of L100754 in the ligand binding pocket of hRARα reveals that the ligand easily adapts to fit the RARα agonist conformation without significant steric clashes (Figure S4). No significant interaction of the SRC-1 NR2 peptide with RXRα LBD was observed (Figure S3) in agreement with our ESI-MS data and with the literature . In RAR/RXR LBDs bound to LG100754, SRC-1 NR2 binds to the heterodimer with a stoechiometry of one peptide per heterodimer and with an affinity similar to the one for the RARα monomer. Together these data demonstrate (Figure 9) that the LG100754 compound is able to stabilize an agonist conformation when bound to RAR while in RXR it inhibits the interaction with the coactivator SRC-1 peptide. LG100754 has also been shown to be able to dissociate corepressors from RAR  and to prevent their binding to RXR . 041b061a72