Abstract

Both synthetic and endogenous glucocorticoids are important pharmaceutical drugs known to bind to the ligand binding domain (LBD) of glucocorticoid receptor (GR), a member of the nuclear receptor (NR) superfamily. Ligand binding induces conformational changes within GR, resulting in subsequent DNA binding and differential co-regulator recruitment, ultimately activating or repressing target gene expression. One of the most crucial co-regulators is peroxisome proliferator-activated gamma coactivator 1-lower case Greek alpha (PGC1lower case Greek alpha), which acts to regulate energy metabolism by directly interacting with GR to modulate gene expression. However, the mechanisms through which PGC1lower case Greek alpha senses GR conformation to drive transcription are not completely known. Here, an ancestral variant of the GR (AncGR2) LBD was utilized as a tool to produce stable protein for biochemical and structural studies. PGC1α is found to interact more tightly and form a more stable complex with AncGR2 LBD than Tif2. We report the first high resolution X-ray crystal structures of AncGR2 LBD in complex with PGC1α and dexamethasone or hydrocortisone. Structural analyses reveal how distinct steroid drugs bind to GR with different affinities by unique hydrogen bonds and hydrophobic interactions. Important charge clamps are formed between the activation function-2 (AF-2) and PGC1lower case Greek alpha to mediate their specific interactions. These interactions lead to a high level of protection from hydrogen-deuterium exchange at the coregulator interaction site and strong intramolecular allosteric communication to ligand binding site. This is the first structure detailing the GR- PGC1lower case Greek alpha interaction providing a foundation for future design of specific therapeutic agents targeting these critical metabolic regulators.