Structural basis for hydroxycholesterols as natural ligands of orphan nuclear receptor RORgamma

Abstract

The retinoic acid-related orphan receptor gamma (RORgamma) has important roles in development and metabolic homeostasis. Although the biological functions of RORgamma have been studied extensively, no ligands for RORgamma have been identified, and no structure of RORgamma has been reported. In this study, we showed that hydroxycholesterols promote the recruitment of coactivators by RORgamma using biochemical assays. We also report the crystal structures of the RORgamma ligand-binding domain bound with hydroxycholesterols. The structures reveal the binding modes of various hydroxycholesterols in the RORgamma pocket, with the receptors all adopting the canonical active conformation. Mutations that disrupt the binding of hydroxycholesterols abolish the constitutive activity of RORgamma. Our observations suggest an important role for the endogenous hydroxycholesterols in modulating RORgamma-dependent biological processes.

Figure 1

The hydroxycholesterols promote RORγ/coactivator interaction. A, Chemical structures of cholesterol and various hydroxycholesterols; B, modulation of the interactions of RORs LBD with SRC1-2 coactivator LXXLL motif in response to 1 μm hydroxycholesterols by AlphaScreen assays.

Figure 2

The transcriptional properties of RORγ in response to cholesterol derivatives. A, Modulation of the interaction of RORγ LBD with various coactivator LXXLL motifs and corepressor motifs in response to 1 μm hydroxycholesterols as shown by AlphaScreen assays. Background reading with the RORγ LBD is fewer than 200 photon counts. The peptide sequences are listed in experimental procedures. B, Dose curve of hydroxycholesterols in promoting RORγ LBD/SRC1-2 binding by AlphaScreen assays.

Figure 3

The structures of the RORγ complexed with hydroxycholesterols. A, Ribbon representation of the RORγ LBD complexed with 25-HC. RORγ is in blue, and the SRC2-2 motif is in yellow. The bound 25-HC is shown in stick representation with carbon and oxygen atoms depicted in green and red, respectively. B–D, 2Fo-Fc electron density map (1.0σ) showing bound 20α-HC (B), 22R-HC (C), and 25-HC (D). All hydroxycholesterols are shown in stick representation with carbon and oxygen atoms depicted in green and red, respectively.

Figure 4

The structural determinants of the interaction of RORγ LBD with ligands. A–C, The interactions of RORγ residues with specific groups on the hydroxycholesterol ligands including cholesterol rings (A) and hydroxyl groups of 22R-HC (B) and 25-HC (C). The hydrophobic interactions between RORγ and the ligands are shown with dashed lines. The potential hydrogen bonds, if the corresponding hydrophobic residues (Ile397 and Leu324) are mutated to Asn, are indicated by arrows. D and E, Effects of mutations of key RORγ residues on its transcriptional activity in cell-based reporter gene assays. The LBDs of LXRα, RORγ, and RORγ mutants were fused to Gal4 DNA-binding domain (DBD). The cells were cotransfected with pG5Luc reporter, together with the plasmids encoding Gal4-LBD fusion proteins (D). The cells were cotransfected with the Pcp2/RORE-Luc reporter, together with the plasmids encoding full-length LXRα, RORγ, or RORγ mutants (E), and 1 μm ligands were used in both D and E.

Figure 5

Hydroxycholesterol treatment partially restores RORγ transcriptional activity in cells with reduced levels of hydroxycholesterols. Cos7 cells were transiently introduced with SULT2B1b cholesterol sulfotransferase. Then the cells were cotransfected with the Pcp2/RORE-Luc reporter, together with the plasmids encoding full-length RORγ. The ligands were added at the indicated concentrations.

Figure 6

Coactivators bind to RORγ via a charge clamp. A, The docking mode of SRC2-2 coactivator motif (yellow) on RORγ (red) coactivator binding site with charge clamp residues shown in stick representation. B and C, Effects of the charge clamp mutations on RORγ activity. The cells were cotransfected with pG5Luc reporter and the plasmids encoding Gal4-LBD fusion proteins (B) or the Pcp2/RORE-Luc reporter together with the plasmids encoding full-length RORγ or RORγ mutants (C), and 1 μm ligands were used in these assays.