A molecular switch regulating transcriptional repression and activation of PPAR\u03b3

Jinsai Shang,Patrick R Griffin,Laura A Solt,Sarah A Mosure,Jie Zheng,Jared Bass,Ashley Nichols,Douglas J Kojetin,Richard Brust

doi:10.1038/s41467-020-14750-x

Abstract

Nuclear receptor (NR) transcription factors use a conserved activation function-2 (AF-2) helix 12 mechanism for agonist-induced coactivator interaction and NR transcriptional activation. In contrast, ligand-induced corepressor-dependent NR repression appears to occur through structurally diverse mechanisms. We report two crystal structures of peroxisome proliferator-activated receptor gamma (PPARγ) in an inverse agonist/corepressor-bound transcriptionally repressive conformation. Helix 12 is displaced from the solvent-exposed active conformation and occupies the orthosteric ligand-binding pocket enabled by a conformational change that doubles the pocket volume. Paramagnetic relaxation enhancement (PRE) NMR and chemical crosslinking mass spectrometry confirm the repressive helix 12 conformation. PRE NMR also defines the mechanism of action of the corepressor-selective inverse agonist T0070907, and reveals that apo-helix 12 exchanges between transcriptionally active and repressive conformations—supporting a fundamental hypothesis in the NR field that helix 12 exchanges between transcriptionally active and repressive conformations.

Highlights

Nuclear receptor (NR) transcription factors use a conserved activation function-2 (AF-2) helix 12 mechanism for agonist-induced coactivator interaction and NR transcriptional activation
Consistent with the mammalian two-hybrid showing the ID2 sequence is necessary for PPARγ interaction, in a Time-resolved fluorescence resonance energy transfer (TR-FRET) biochemical peptide interaction assay (Fig. 1d) T0070907 enhances the binding of corepressor ID2 peptides to the PPARγ ligand-binding domain (LBD) and decreases binding of coactivator peptide
Other reported corepressorpeptide-bound NR crystal structures including ERRγ, FXR, GR, PPARα, PR, RARα, REV-ERBα, RORγ, and RXRα show structurally diverse, solvent-exposed conformations of the AF-2 helix 12 with antagonist or inverse agonist ligands bound to the orthosteric pocket that enable binding of corepressor peptide (Supplementary Fig. 15)[3,4,5,6,7,8,9,10,11,12,13,14]

Summary

Introduction

Nuclear receptor (NR) transcription factors use a conserved activation function-2 (AF-2) helix 12 mechanism for agonist-induced coactivator interaction and NR transcriptional activation. We report two crystal structures of peroxisome proliferator-activated receptor gamma (PPARγ) in an inverse agonist/corepressor-bound transcriptionally repressive conformation. PRE NMR defines the mechanism of action of the corepressor-selective inverse agonist T0070907, and reveals that apo-helix 12 exchanges between transcriptionally active and repressive conformations—supporting a fundamental hypothesis in the NR field that helix 12 exchanges between transcriptionally active and repressive conformations. We report two crystal structures of PPARγ bound to T0070907 and corepressor peptides that reveal a unique a transcriptionally repressive conformation compared with all other reported structures of corepressor-bound NRs. A comparative structural analysis to the transcriptionally active conformation and the apoPPARγ conformational ensemble using solution paramagnetic relaxation enhancement (PRE) NMR validates the unique repressive conformation and provides evidence that helix 12 in an apo-NR exchanges between transcriptionally active and repressive conformations

Methods

Results

Conclusion