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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., Structural studies of nuclear receptor transcription factors revealed that nearly all nuclear receptors share a conserved helix 12 dependent transcriptional activation mechanism. Here the authors present two crystal structures of peroxisome proliferator-activated receptor gamma (PPARγ) in an inverse agonist/corepressor-bound transcriptionally repressive conformation, where helix 12 is located within the orthosteric ligand-binding pocket instead, and discuss mechanistic implications.

Small chemical modifications can have significant effects on ligand efficacy and receptor activity, but the underlying structural mechanisms can be difficult to predict from static crystal structures alone. Here we show how a simple phenyl-to-pyridyl substitution between two common covalent orthosteric ligands targeting peroxisome proliferator-activated receptor (PPAR) gamma converts a transcriptionally neutral antagonist (GW9662) into a repressive inverse agonist (T0070907) relative to basal cellular activity. X-ray crystallography, molecular dynamics simulations, and mutagenesis coupled to activity assays reveal a water-mediated hydrogen bond network linking the T0070907 pyridyl group to Arg288 that is essential for corepressor-selective inverse agonism. NMR spectroscopy reveals that PPARγ exchanges between two long-lived conformations when bound to T0070907 but not GW9662, including a conformation that prepopulates a corepressor-bound state, priming PPARγ for high affinity corepressor binding. Our findings demonstrate that ligand engagement of Arg288 may provide routes for developing corepressor-selective repressive PPARγ ligands., Peroxisome proliferator-activated receptor gamma (PPARγ) is a target for insulin sensitizing drugs. Here the authors combine NMR, X-ray crystallography and MD simulations and report a structural mechanism for eliciting PPARγ inverse agonism, where coactivator binding is inhibited and corepressor binding promoted, which causes PPARγ repression.

Crystal structures of peroxisome proliferator-activated receptor gamma (PPARγ) have revealed overlapping binding modes for synthetic and natural/endogenous ligands, indicating competition for the orthosteric pocket. Here we show that cobinding of a synthetic ligand to the orthosteric pocket can push natural and endogenous PPARγ ligands (fatty acids) out of the orthosteric pocket towards an alternate ligand-binding site near the functionally important omega (Ω)-loop. X-ray crystallography, NMR spectroscopy, all-atom molecular dynamics simulations, and mutagenesis coupled to quantitative biochemical functional and cellular assays reveal that synthetic ligand and fatty acid cobinding can form a ‘ligand link’ to the Ω-loop and synergistically affect the structure and function of PPARγ. These findings contribute to a growing body of evidence indicating ligand binding to nuclear receptors can be more complex than the classical one-for-one orthosteric exchange of a natural or endogenous ligand with a synthetic ligand.

Small chemical modifications can have significant effects on ligand efficacy and receptor activity, but the underlying structural mechanisms can be difficult to predict from static crystal structures alone. Here we show how a simple phenyl-to-pyridyl substitution between two common covalent orthosteric ligands targeting peroxisome proliferator-activated receptor gamma (PPARγ) converts a transcriptionally neutral antagonist (GW9662) into an inverse agonist (T0070907). X-ray crystallography, molecular dynamics simulations, and mutagenesis coupled to activity assays reveal a water-mediated hydrogen bond network linking the T0070907 pyridyl group to Arg288 that is essential for inverse agonism. NMR spectroscopy reveals that PPARγ exchanges between two long-lived conformations when bound to T0070907 but not GW9662, including a conformation that prepopulates a corepressor-bound state, priming PPARγ for high affinity corepressor binding. Our findings demonstrate that ligand engagement of Arg288 may provide new routes for developing PPARγ inverse agonist.