Structure of the intact PPAR-gamma-RXR- nuclear receptor complex on DNA

Abstract

Nuclear receptors are multi-domain transcription factors that bind to DNA elements from which they regulate gene expression. The peroxisome proliferator-activated receptors (PPARs) form heterodimers with the retinoid X receptor (RXR), and PPAR-gamma has been intensively studied as a drug target because of its link to insulin sensitization. Previous structural studies have focused on isolated DNA or ligand-binding segments, with no demonstration of how multiple domains cooperate to modulate receptor properties. Here we present structures of intact PPAR-gamma and RXR-alpha as a heterodimer bound to DNA, ligands and coactivator peptides. PPAR-gamma and RXR-alpha form a non-symmetric complex, allowing the ligand-binding domain (LBD) of PPAR-gamma to contact multiple domains in both proteins. Three interfaces link PPAR-gamma and RXR-alpha, including some that are DNA dependent. The PPAR-gamma LBD cooperates with both DNA-binding domains (DBDs) to enhance response-element binding. The A/B segments are highly dynamic, lacking folded substructures despite their gene-activation properties.

Figure 1. Overall structure of the PPAR-γ–RXR-α complex on PPRE

a, b, Orthogonal views are shown in which RXR-α is blue and PPAR-γ is red. The ligands rosliglitazone and 9-cis-retinoic acid are shown in green, the Zn(II) ions are white, and the coactivator LXXLL peptides are in light blue and purple.

Figure 2. DNA-binding features of the complex

a, The sequence of PPRE element containing direct repeats of the canonical AGGTCA half-site separated by one base pair (DR1) together with the upstream sequence AAACT specificity element. b, The DBDs and their C-terminal extensions are shown for PPAR-γ (red) and RXR-α (blue). These domains form a polar, DNA-dependent head-to-tail interaction through side chains that converge over the spacer element.

Figure 3. Domain–domain interactions involving the PPAR-γ LBD

a, The PPAR-γ LBD is closely positioned to the PPAR-γ DBD. b, The PPAR-γ LBD (red) interfaces with both receptor DBDs to enhance their DNA association. RXR DBD is in blue. c, The LBD and DBD of RXR-α, do not interact. d, PPAR-γ LBD bisects both RXR-α domains. The proximity of the PPAR-γ ligand to both RXR-α domains can be seen. e, The side-chain interactions between PPAR-γ LBD and RXR-α DBD in the rosiglitazone complex. The PPAR-γ LBD residue Phe 347 lies at the centre of the interface.

Figure 4. Effect of different ligands on the PPAR-γ–RXR-α complex

a, Electron density (F_o −-F_c) omit maps around the PPAR-γ ligands rosiglitazone, BVT.13 and GW9662 from the their respective complexes. b, The three PPAR-γ ligands as a group occupy distinct portions of the receptor’s Y-shaped pocket. c, Comparison of the binding mode of rosiglitazone with GW9662 within the PPAR-γ pocket. GW9662 does not form a covalent bond with Cys 285. d, Superposition of crystal structures obtained with the different PPAR-γ ligands showing minor variations in overall domain organization. The colours are indicated for each complex.

Figure 5. The dynamic features of the receptors

H/D-Ex data for intact PPAR-γ. From top, in a ligand-free state, with rosiglitazone, with GW9662, and with all of rosiglitazone, RXR-α, 9-cis-retinoic acid and PPRE DNA (RXR, etc.). Each horizontal colour block represents an analysed peptic fragment. The deuteration level of each peptide at each time point is colour-coded as in the insert. Each block contains five time points (from top, 15, 50, 150, 500, and 1,500 s).