From Apo to Ligand-Bound:Unraveling PPARγ-LBDConformational Shifts via Advanced Molecular Dynamics

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor whose ligand-induced conformational changes, primarily driven by helix 12 (H12) repositioning, regulate transcriptional activity. However, the precise mechanism remains elusive. In this study, we performed classical molecular dynamics (cMD) simulations of the PPARγ ligand binding domain (LBD) in complex with two agonists (BRL, 3EA), a partial agonist (GW0072), and an antagonist (EKP), generating 3 μs trajectories for each system. To gain deeper insights, we integrated machine learning-assisted clustering with MD simulations, revealing a favorable trend in binding free energy (Δ<i>G</i>_b), suggesting enhanced complex stability. A case study on EKP demonstrated that, despite fitting within the binding site, it failed to induce rapid LBD or H12 rearrangements in the apo agonist-induced conformation. Additionally, we investigated the apo-state conformations of PPARγ-LBD influenced by agonist and antagonist ligands, utilizing cMD and Gaussian accelerated molecular dynamics (GaMD) over a cumulative 6 μs (3 μs cMD + 3 μs GaMD). Key residues known to modulate PPARγ function upon mutation were analyzed, and simulations confirmed the high stability of both apo and ligand-bound conformations. Notably, in the apo state, specific H12 residues interacted with other PPARγ-LBD regions, preventing disorder and abrupt transitions. These findings guided the selection of collective variables (CVs) for well-tempered metadynamics (WT-MetaD) simulations, which-in the apo-agonist state-captured the H12 shift from agonist- to antagonist-like conformations, consistent with resolved X-ray structures. Overall, this computational framework provides novel insights into PPARγ-LBD conformational dynamics and establishes a valuable approach for rationally assessing the effects of modulators on PPARγ activity.