Abstract
The repressive states of nuclear receptors (i.e., apo or bound to antagonists or inverse agonists) are poorly defined, despite the fact that nuclear receptors are a major drug target. Most ligand bound structures of nuclear receptors, including peroxisome proliferator-activated receptor γ (PPARγ), are similar to the apo structure. Here we use NMR, accelerated molecular dynamics and hydrogen-deuterium exchange mass spectrometry to define the PPARγ structural ensemble. We find that the helix 3 charge clamp positioning varies widely in apo and is stabilized by efficacious ligand binding. We also reveal a previously undescribed mechanism for inverse agonism involving an omega loop to helix switch which induces disruption of a tripartite salt-bridge network. We demonstrate that ligand binding can induce multiple structurally distinct repressive states. One state recruits peptides from two different corepressors, while another recruits just one, providing structural evidence of ligand bias in a nuclear receptor.
Highlights
The repressive states of nuclear receptors are poorly defined, despite the fact that nuclear receptors are a major drug target
The relative area of peaks within a nuclear magnetic resonance (NMR) spectrum can correspond to the relative populations of structural states that compose the overall ensemble
Biophysical work in myoglobin demonstrated that proteins exhibit complex dynamics on a variety of timescales[63], which has been observed in many other systems with many methods[34]
Summary
The repressive states of nuclear receptors (i.e., apo or bound to antagonists or inverse agonists) are poorly defined, despite the fact that nuclear receptors are a major drug target. Models of drug induced changes to the coregulator binding surface are based on the over 800 nuclear receptor structures[14], simulations[15], nuclear magnetic resonance (NMR)[16,17], fluorescence anisotropy[18,19,20], and hydrogen deuterium exchange mass spectrometry (HDX-MS)[21,22] These reports support the idea that the position and/or dynamics of the c-terminal helix in most nuclear receptors (helix 12) is an important determinant of activity. The lack of such drug-induced changes for other receptors and solution state data has led to the dynamic stabilization model[8], which posits that activation involves reduction of helix 12 movement and/or an increase in helical integrity Consistent with this model, protein NMR shows that agonist binding diminishes intermediate exchange (i.e., μs–ms dynamics) throughout the LBD16,17. These two states are different from the agonist bound helix 12 state[24], which is the only currently well-defined state
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