Crystal structures of PPARγ LBD in the transcriptionally active and repressive conformations.

The active LBD (PDB 6ONJ) is stabilized by agonist (rosiglitazone) and coactivator peptide (TRAP220/MED1), whereas the repressive LBD (PDB 6ONI) is stabilized by covalent inverse agonist (T0070907) and corepressor peptide (NCoR1).

Compounds used in the study and relative affinities in a ligand displacement assay.

(A) Chemical structures of the compounds. (B) TR-FRET ligand displacement data for the compounds (n=3; mean ± s.d.).

Ligand cobinding functional profiling in TR-FRET coregulator interaction assays.

(A) TR-FRET coregulator interaction assays performed using PPARγ LBD protein with or without preincubation of GW9662 or T0070907 to determine how the non-covalent synthetic ligands influence recruitment of peptides derived from NCoR1 corepressor protein and TRAP220/MED1 coactivator protein fit to a sigmoidal dose response equation or biphasic dose response equation for select cases where a biphasic response is observed (n=3; mean ± s.d.). (B) IC50 and EC50 values extracted from the TR-FRET coregulator interaction data. For curves showing a biphasic response, the higher affinity value is displayed; no value is displayed in cases where the dose response is flat. Error bars when present represent the fitted errors; some fits did not converge to a well-fitted error (see Source Data 1).

NMR implicates covalent inhibitor-induced stabilization of a repressive LBD conformation in the mechanism of weakening non-covalent synthetic ligand cobinding.

Overlays of 2D [1H,15N]-TROSY-HSQC NMR data of 15N-labeled PPARγ LBD preincubated with covalent inhibitor, (A) GW9662 or (B) T0070907, in the absence or presence of the indicated non-covalent synthetic ligands added at 2 molar equivalents. (C) Overlays of 2D [1H,15N]-TROSY-HSQC NMR data of 15N-labeled PPARγ LBD in the presence of non-covalent synthetic ligands (singly bound state) compared to the cobound states with a covalent inhibitor.

Non-covalent synthetic ligands adopt orthosteric binding modes when cobound with a covalent inhibitor.

(A) Ligand cobinding modes in crystal structures of PPARγ LBD. (B) Comparison of the non-covalent synthetic ligand orthosteric binding mode (singly bound) and ligand cobinding mode with a covalent inhibitor (transparent sticks). Differences between these binding modes are indicated with a black arrow. (C) Structural clashes observed between the covalent inhibitor orthosteric binding mode (transparent sticks) and the non-covalent synthetic ligand binding mode. PDB codes for crystal structures used in the overlays are listed in the Methods section.

Covalent inhibitors adopt different binding modes to accommodate a cobound non-covalent synthetic ligand.

(A) Structural overlay showing the orthosteric binding modes of GW9662 and T0070907 in crystal structures of PPARγ LBD in active and repressive conformations. (B) Comparison of the covalent binding modes when singly bound (orthosteric) and cobound to a non-covalent synthetic ligand. Black arrows indicate the conformational differences between the orthosteric binding modes vs. cobinding modes. PDB codes for crystal structures used in the overlays are listed in the Methods section.