Ligand-binding specificity of RARs
RXR exclusively binds 9-cRA, whereas RAR binds both 9-cRA and a-tRA stereoisomers (Heyman et al., 1992; Levin et al., 1992). The comparison of the 9-cRA molecules as seen in the structures of RXRα holo-LBD and RARγ holo-LBD reveals two major differences. (i) A more pronounced bending angle in RXRα (70 versus 60° in RARγ) (Figure 2E). The intrinsic geometry of the RXRα ligand-binding pocket (LBP) selects the 9-cis isomer because the sharper bend imposed by the induced fit cannot be reached by the all-trans isomer due to its flexure limit (Klaholz et al., 1998). The slightly lower affinity of RXRα for 9-cRA (KdRXR9c = 1.5 nM) compared with RARγ (KdRAR9c = 0.8 nM and KdRARat = 0.2 nM) (Allegretto et al., 1993; Allenby et al., 1993) could be explained by a smaller number of hydrophobic contacts (67 for RXRα–9-cRA versus 83 and 94 for RARγ–9-cRA and RARγ–a-tRA, respectively, with a 4.2 Å distance cut-off), which reflects the lower occupancy of the cavity in RXRα compared with that of RARγ (Figure 2C and D). (ii) A different relative orientation of the β-ionone ring with a rotation of ∼90° around the C9–C10 bond in RXRα (Figure 2F). This is the consequence of the different orientation and location of the β-ionone binding sites, which are 9 Å apart in the proteins. In RARγ holo-LBD, the β-ionone ring points towards helix H12 and makes hydrophobic contacts with it, whereas in RXRα it points to the bottom of the LBD, away from helix H12 (Figure 1A).
The superimposition of the two complexes (Figure 1B) reveals an additional difference in the relative position of the bound ligands. While the carboxylate group of all bound retinoids is anchored by an interaction with the conserved arginine residue of helix H5, the isoprenic chain being similarly oriented, a 2.7 Å shift of the ligand towards the centre of the cavity is observed in RXRα when compared with the 9-cRA–RARγ complex (Figure 2F). The natural flexibility of the arginine side chain allows such movement as would permit the binding of a longer ligand by sliding along the tunnel (Figure 2B).
The effects of point mutations on hRXRα residues Phe313 and Leu436 revealed that their substitutions generate two classes of RXR proteins with altered ligand specificities and responsiveness: a first class exhibiting decreased activation by 9-cRA and at the same time an increased activation by synthetic ligands, and a second class still responsive to 9-cRA, but insensitive to synthetic ligands (Peet et al., 1998). Mutation of the corresponding conserved leucine residue (Leu525) in ERα also strongly affects the transcriptional potency of synthetic ligands (Ekena et al., 1997). The present structure accounts for their roles in RXRα ligand specificity and activation by 9-cRA: Phe313 at one entry of the binding pocket is in very close contact with the carboxylate end of the ligand, whereas Leu436 is part of the cavity's lid and participates to stabilize helix H12 in the agonist position.
Modelling studies were performed considering synthetic compounds with known agonistic or antagonistic activity towards RXR. Agonist HX600 and antagonist HX531 (Umemiya et al., 1997a,b) were docked in the RXRα LBP (Figure 3A and B). All these compounds carry a carboxylate moiety that establishes a similar set of anchoring ionic and hydrogen interactions to those observed in the RXRα LBD bound to 9-cRA (Figure 3A). Agonist HX600 occupies the LBP and maximizes the cavity occupancy ratio at the level of residues Leu433, Trp305 and Gln306. In contrast, for the antagonist HX531 the additional bulky NO2 group causes steric hindrance with the side chains of residues Gln306, Trp305 and Leu433 (Figure 3B); the latter two residues being involved in the stabilization of helix H12 agonist conformation (see below). Docking of HX531 in the model of RXR under its antagonist conformation as observed in the RXR–RAR heterodimer (Bourguet et al., 2000) also suggests the necessity of additional conformational adaptation of either the ligand or the protein.
Figure 4. The agonist conformation of transactivation helix H12 in the holo form. Docking of retinoid agonist (HX630) and antagonist (HX531) in hRXRα LBP. (A and B) Agonist (HX600) and antagonist (HX531) compounds docked in the LBP of hRXRα. Protein atoms are coloured in grey for carbon, blue for nitrogen, red for oxygen and yellow for sulfur. The oxygen and nitrogen atoms of docked compounds are depicted as red and blue spheres, respectively. 9-cRA is coloured in yellow, whereas docked ligands are colored in salmon. Cyan dotted lines represent the structurally conserved hydrogen interaction between the carboxylic moiety of ligands and residues Arg316 and Gln275 of the protein. Green dotted lines underline steric clashes through close interatomic contacts between ligand and protein atoms (the distance between consecutive dots is 0.5 Å). (C) Detailed stereoview of helix H12 contacts showing the exposed glutamic residues Glu453 and Glu456 involved in transactivation and the interactions stabilizing helix H12 in its agonist position. Helix H12 is depicted in red. 9-cRA ligand atoms are coloured in yellow for carbon and red for oxygen, respectively. Protein atoms are coloured in grey for carbon, blue for nitrogen, red for oxygen and yellow for sulfur. The protein backbone is coloured in blue. A water molecule is drawn as a red sphere and hydrogen bonds are depicted as green dotted lines. For the sake of clarity only a few side chains are labelled. (D) Schematic drawing of interactions stabilizing H12 in its agonist conformation. van der Waals interactions and hydrogen bonds are represented as dotted and continuous lines, respectively. A water molecule is referred to as w.
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The transactivation helix H12 adopts a canonical agonist conformation upon 9-cRA binding
In RXRα, holo-LBD helix H12 leans over the ligand-binding cavity and seals it. It is packed against helices H3, H4, H5 and H11, and held in place mainly by a set of tight hydrophobic interactions involving residues Leu451, Met424, Met454 and Leu455 of helix H12 and several residues of the core LBD such as Cys269, Ala272 and Leu276 (H3), Arg302 (H4), Trp305 (H5), Leu433, Leu436, Phe437 and Lys440 (H11) (Figure 3C and D). Two additional interactions exist: (i) a water-mediated hydrogen bond between the indole NH atom of Trp305 of helix H5 and the backbone carbonyl group of Met454 of helix H12; and (ii) two hydrogen bonds between the carboxylate of Asp273 of helix H3 and the backbone amide groups of Phe450 and Leu451 of helix H12. This last stabilizing interaction involving a conserved acidic amino acid on helix H3 and backbone amide groups of helix H12 is also present in both ERα and progesterone receptor (PR) but not in RARγ holo-LBDs. Unlike RARγ and vitamin D3 receptor (VDR) (Rochel et al., 2000) holo-LBDs, but like in thyroid hormone receptor α (TRα) holo-LBD (Wagner et al., 1995), the potential salt bridge between Arg302 of helix H4 and Glu453 of the conserved motif Glu453MetLeuGlu456 that constitutes the core of the AF-2 AD is not formed. In RXRα, Arg302 points towards the solvent and is involved in a crystal packing contact. Note that residues Cys269 and Ala272 of helix H3 and Trp305 of helix H5, which participate in the correct positioning and stabilization of helix H12, are also involved in ligand binding, suggesting an indirect influence of the ligand on the fine positioning of H12. Globally, in RARγ, ERα and PR, but also in TRα and peroxisome proliferator-activated receptor γ (PPARγ) LBDs, H12 makes similar contacts with the homologous residues that form the H12 interaction groove (data not shown), in keeping with a high structural homology of the agonist conformation of all NRs. In this position, H12 residues contribute, as in TRβ (Darimont et al., 1998), ERα (Shiau et al., 1998) and PPARγ (Nolte et al., 1998), to the formation of the hydrophobic cleft that constitutes the coactivator binding site. Note that the amino acid residues that constitute the H12 interaction groove are much less conserved than those forming the coactivator binding site; with the exception of Leu276 none of them belongs to the highly conserved NR signature motif (Wurtz et al., 1996).
Ligand-induced conformational changes: comparison between RXRα apo- and holo-LBD structures
The elucidation of the structure of the RXRα holo-LBD allows us to investigate the effects of ligand binding on receptor conformation. It supports the concept of a canonical LBD ‘agonist structure’ valid for all NRs (Figure 4A). The volumes of apo and holo monomers, 39 110 and 35 870 Å3, respectively, show the effective compaction resulting from ligand binding. The volume of 35 870 Å3 is close to those of 37 410 and 39 370 Å3 found for its homologues RARγ and ERα holo-LBDs (32 and 31% amino acid identity, 51 and 48% amino acid similarity). The main conformational changes affect the N-terminal part of helix H3 and helices H11 and H12. Helix H6 and the two-stranded β-sheet undergo small structural adaptations, while all other helices remain essentially unaffected.
The apo form exhibits an additional helix (H2) in the region connecting helix H1 and helix H3. In the holo form this segment unfolds and the flexible loop region sticks to the protein. This different conformation of loop H1–H3 probably illustrates the dynamics of this region, which may act as a molecular spring accompanying the movement of helix H3, which undergoes a very large conformational change from residues Asp263 to Thr278. This N-terminal part of H3 rotates by ∼90° around its helical axis, while kinking at the level of residue Thr278 to pack against the LBP. The resulting global shift of the N-terminal end of H3 is ∼13 Å. This H3 movement, which is permitted by the displacement of helix H11 from its apo position, accompanies the binding of the ligand in an induced fit mechanism. It has three effects: (i) it brings residues Ile268, Cys269, Ala271, Ala272 and Gln275 of H3 into contact with the ligand; (ii) it seals the binding pocket at the level of the β-ionone ring region by bringing four residues into close van der Waals contact: Val265, Ala337, Val342 and Phe439 from helices H3, H6, H7 and H11, respectively (Figure 4B); and (iii) it positions residues Asp273, Cys269, Ala272 and Leu276 to establish the proper set of helix H12-stabilizing van der Waals interactions.
The present structure confirms the crucial role of helix H11 (Bourguet et al., 1995; Wurtz et al., 1996; Vivat et al., 1997). In the absence of the ligand, it stabilizes the apo form by filling the pocket with hydrophobic residues. In the holo form, it is displaced by helix H3, moves away and rotates by 180° around its own axis, generating a proper binding site and helping the repositioning of helix H12 (Figure 4C and D). In both structures, helix H11 remains unchanged to position His435. While in the RXRα apo-LBD, helix H11 is kinked at position Leu436 and then extends to position Asp444, thereby filling the unoccupied binding pocket, in the RXRα holo-LBD, helix H11 adopts a regular α-helical conformation in the continuity of H10 until position Ile442. In RXRα apo-LBD, the LBP cavity is filled with several side chains from helix H11 (Leu441, Phe437 and Phe438) that occupy the β-ionone ring position, while two hydrophobic side chains from the same helix (Leu436 and Phe439) are exposed to the solvent. In the RXRα holo-LBD, 9-cRA occupies the binding pocket, and the side chains of Leu441, Phe437 and Phe438 are outside, exposed to the solvent, while the side chains of Leu436 and Phe439 are internalized and form the lower part of the binding pocket. In conclusion, H11 exposes one face towards the solvent in the apo state and the other face in the holo state. The movements of helix H11 emphasize the role of solvation versus desolvation processes in the structural transition. These entropic effects are expected to be general, as suggested by structural and mutational studies on the ER.
The most striking conformational change affects helix H12, which is completely repositioned upon ligand binding. In the apo form, H12 protrudes from the protein core and is exposed to the solvent, whereas in the holo form it rotates and folds back towards the LBP, thus inducing the compaction of the LBD. Comparison with the RXRα apo-LBD structure reveals that upon ligand binding some key residues of the AF-2 AD core, such as Phe450, Glu453 and Glu456 involved in coactivator interaction and transactivation are exposed to the solvent. In the RXRα apo structure, residue Glu453 is hydrogen bonded to the backbone carbonyl group of residue Pro461 and the side chain of residue Asn262 on loop H2–H3, whereas residue Phe450 is close to residue Pro264 on helix H3. Thus, ligand binding, acting first through the rearrangement of helix H3, induces the repositioning of helix H12 by expelling helix H11. It must be emphasized that the ligand plays an indirect role in the stabilization of H12 through a set of hydrophobic interactions that stabilize the compact core of the LBD (Figures 3A, B and 4B).
We have shown here that RXR, a unique ubiquitous heterodimeric partner for several NR signalling pathways, exhibits a canonical holo-LBD conformation. Like its heterodimerization partners, e.g. RAR, VDR, PPAR and TR, the liganded RXRα LBD crystallizes as a monomer. The present structure unequivocally supports the existence of a structural switch with a ligand-induced transition that triggers the repositioning of helix H12 in an ‘agonist conformation’ and generates an ‘activated’ LBD able to interact subsequently with coactivators. This agonistic conformation of the 9-cRA RXR complex raises the question of RXR subordination, that is why are agonist-liganded RXRs transcriptionally inactive in RXR–RAR heterodimers, unless RAR is itself liganded (Vivat et al., 1997; Dilworth et al., 1999). Further crystallographic studies may reveal the underlying mechanism. Our present results also show how ligand binding promotes protein rearrangements mainly through helix rotations and translations, and suggest that the correlated desolvation processes could be important for the establishment of proper protein–protein interactions.
The specificity of RXR for the 9-cRA stereoisomer results from a different shape and size of its LBP, as compared with that of RAR. The RXR LBP exhibits a more pronounced kink, which prevents binding to a-tRA, the binding site of the β-ionone moiety being rotated by ∼90° towards the bottom core of the protein. As a consequence, and similar to some steroid receptors (see above), the ligand does not contact helix H12. The structure shows that the size of the pocket could accommodate slightly bulkier ligands at the level of the C18 and C19 methyl groups of 9-cRA.
The comparison of RXRα apo- and holo-LBD structures suggests that the molecular mechanism that leads to the holo structure relies upon a dynamic equilibrium of the LBD between an apo form where the ligand binding pocket is partly filled by the hydrophobic end of H11 and a holo form where H11 extends its helical structure leaving the LBP unoccupied and accessible for ligand binding. The bending of H3 may favour the dynamics of the equilibrium. Upon ligand binding, through a path similar to that described for RARγ (Blondel et al., 1999), helix H11 would assume its stable straight conformation, while helix H3 clamps the ligand and locks it in. The subsequent reorganization of H12, which seals the LBP in the agonist conformation, is most likely to be entropy driven. Indeed, solvent exclusion seems to play an important role in the various steps. The essentially hydrophobic ligand pocket results from the concerted and cooperative motions of helices H3 and H11, which clamp the ligand. H3 rotates and bends back towards the domain core, bringing together residues essential for ligand binding and H12 positioning. H11 rotates by half a turn around its axis and is tilted away to open the cavity for ligand binding. This movement brings hydrophobic residues previously exposed to the solvent inside the pocket towards the ligand, and concomitantly exposes others that could be subsequently implied in contacts with coactivators.