Structural and biochemical basis for selective repression of the orphan nuclear receptor liver receptor homolog 1 by small heterodimer partner

Abstract

The functional interaction between the orphan nuclear receptors small heterodimer partner (SHP) and liver receptor homolog 1 (LRH-1), where SHP binds to LRH-1 and represses its constitutive transcriptional activity, is crucial for regulating genes involved in cholesterol homeostasis. Here, we report structural and biochemical analyses of the LRH-1/SHP interaction. The crystal structure and modeling studies of the LRH-1 ligand-binding domain bound to either of the two LXXLL-related motifs of SHP show that the receptor undergoes conformational changes to accommodate the SHP docking and reveal key residues that determine the potency and selectivity of SHP binding. Through a combination of mutagenesis and binding studies, we demonstrate that only the second SHP LXXLL motif is required for repressing LRH-1, and this motif displays a strong preference for binding to LRH-1 over the closely related receptor steroidogeneic factor 1 (SF-1). Structural comparisons indicate that this binding selectivity is determined by residues flanking the core LXXLL motifs. These results establish a structural model for understanding how SHP interacts with LRH-1 to regulate cholesterol homeostasis and provide new insights into how nuclear receptor/coregulator selectivity is achieved.

Fig. 1.

Binding of the SHP LXXLL-related motifs to LRH-1. (A) A schematic representation showing the SHP protein and its two receptor-interacting motifs (ID1 and ID2), which were used in cocrystallization with LRH-1 and PPARγ. The sequences were adopted from Johansson et al. (17). (B) Binding of various LXXLL motifs to the purified LRH-1-1 LBD as measured by AlphaScreen assays. The background reading of either LRH-1 or the peptides alone is <200. (C) Binding affinity of LRH-1 with the SHP ID1 and ID2 motifs and coactivator LXXLL motifs is determined by IC₅₀ values from peptide competitions of the binding of the TIF2 third motif to LRH-1 as measured by AlphaScreen assays. (D) The relative binding affinity of the SHP motifs to various purified receptor LBDs is measured by AlphaScreen assays by using 500 nM SHP ID1 and ID2 peptides to compete off the binding of the TIF2 third motif to the receptors. A 0% inhibition is the reading in the absence of any competing peptide where 100% inhibition represents the reading in the presence of 100 μM TIF2 peptide. GR, glucocorticoid receptor; MR, mineralocorticoid receptor; AR, androgen receptor, ER, estrogen receptor; PR, progesterone receptor; TR, thyroid hormone receptor.

Fig. 2.

Structure of LRH-1 bound to the SHP LXXLL motifs. (A and B) Two 90-degree views of the LRH-1/SHP ID1 complex in ribbon representation. LRH-1 is colored in gold with its charged clamp residues colored in red (AF-2) and blue (end of H3). The SHP ID1 peptide is in yellow, and the LRH-1 ligand-binding pocket is represented by a pink surface. (C) The overall structure of the PPARγ/rosiglitazone/SHP ID2 ternary complex. PPARγ is colored in gold with its charged clamp residues colored in red (AF-2) and blue (end of H3). The SHP is in yellow, and rosiglitazone is shown in space filling. (D) Docking of the SHP ID2 motif (green) on the LRH-1 coactivator-binding site is shown in surface representation. (E) A schematic representation depicting the intermolecular interactions of the SHP ID1 and ID2 motifs with LRH-1.

Fig. 3.

Conformational changes of LRH-1 induced by SHP binding. (A) Superposition of the apo-LRH-1 (blue) with the LRH-1/SHP complex, where LRH-1 is colored in red and SHP in yellow. (B–D) Conformational changes of the side chain in the LRH-1 residues involved in binding of the SHP ID1 motif. The apo-LRH-1 is shown in green, the SHP-bound LRH-1 is in blue, and the SHP is in yellow. The electron density is shown in purple at 1σ contoured level. (E) Docking mode of the SHP ID1 motif on to the surface of the LRH-1 coactivator-binding site. (F) Surface topology of the cofactor-binding site of the LRH-1/SHP complex as distorted by the side chains of the apo-LRH-1 (green).

Fig. 4.

Determinants and functional correlation of the LRH-1/SHP interactions. (A) Effects of alanine scanning mutations of the SHP ID1 and ID2 motifs on their binding to LRH-1 as determined by AlphaScreen. IC₅₀ ratios are determined between the values of mutated peptides and the IC₅₀ values for the wild-type SHP ID1 and ID2 motifs. (B) Effects of the mutations of the SHP ID1 and ID2 motifs on repression of LRH-1 in cell-based reporter assays. The amount of the expressing plasmids of SHP and LRH-1 is 2 ng and 40 ng, respectively (1:20 ratio). The luciferase activity for the transfections performed with LRH-1 alone was normalized to 100%. (C) Direct comparison of repression of LRH-1 and SF-1 by SHP and Dax-1 in cell-based reporter assays. The amount of the mouse SHP, mouse Dax-1, and the SHP SWAP expression vector is either 40 ng (1:1 ratio to LRH-1 or SF-1) or 2 ng in the repression assays (1:20 ratio to LRH-1 or SF-1). SWAP represents the SHP mutant containing K122Y/K123S/I124L/E126T/E127S/P128S that replace the SHP ID2 motif with the Dax-1 third motif. The luciferase activities for the transfections performed with LRH-1 and SF-1 alone were normalized to 100%. (D) Binding affinity of the SHP motifs and the Dax-1 motifs to SF-1 as determined by peptide competition experiments by using AlphaScreen. The IC₅₀ of the second and the third motifs of Dax-1 is ≈500 nM vs. 10–40 μM for the two SHP motifs. (E) No binding of the biotinylated SHP ID2 motifs to SF-1 as compared with the binding of the TIF2 and SHP ID1 motif to SF-1.

Fig. 5.

Basis for the SHP selectivity between LRH-1 and SF-1. (A) An overlap of the LRH-1 (blue) with the SF-1 (yellow) showing the difference of their coactivator-binding site and the docking mode of the SHP helix. The SHP ID1 motif in the LRH-1 and SF-1 is shown in pink and red, respectively. Key differences in their residues, the AF-2 helix and the shift of the SHP helix are noted. (B–D) Surface topology of the LRH-1 (blue) and SF-1 (yellow) coactivator-binding site showing a narrow but deep site in SF-1 (C), and a wide but shallow site in LRH-1 (D). The pocket for adopting the SHP LXXLL residues are noted in red, and key SF-1 and LRH-1 residues are noted in black. The differences between the coactivator-binding site of LRH-1 and SF-1 are further illustrated by an overlay of their surfaces in B, which shows that the center of the coactivator-binding site is mostly covered by LRH-1 (blue), whereas the flanking ridge is mostly covered by SF-1 (yellow). (E) The binding affinity (IC₅₀) of various SHP and Dax LXXLL motifs to LRH-1 and SF-1 as determined by peptide competitions. The residues that affect the binding affinity to SF-1 are in red. (F) A model for the SHP selectivity between LRH-1 and SF-1. The SF-1/SHP model is shown in yellow/red solid lines and the LRH-1/SHP is shown in blue/pink dashed lines. The 1.0-Å shift of the SHP helix into the SF-1 pocket (yellow) is also indicated.