Conformational diversity of the helix 12 of PPARgamma receptor and functional implications

Nuclear Hormone Receptors are transcription regulators modulated by ligand binding. Structurally, they are composed by three domains: a variable N-terminal domain, a highly conserved DNA-binding domain (DBD) and a less conserved C-terminal domain, known as ligand binding domain (DBD). Several studies have revealed that the dynamic properties of the C-terminal helix (H12) of the LBD are one of the most important aspects governing NR activity. When bound to agonist ligands, the H12 is stabilized in a conformation that promotes the binding of coactivator proteins. On the other side, in the absence of ligand, H12 is flexible and can adopt various conformations. To provide a detailed picture of H12 conformational equilibrium, molecular dynamics simulations of the LBD of PPARgamma were performed in different conditions: in the presence or absence of ligand and of corregulators peptides. Free-energy profiles of the conformational variability of the H12 were obtained from ABF calculations. Our results demonstrate that, without ligand, multiple conformations of the H12 are accessible, being the agonist one the most stable. Ligand binding strongly stabilizes the agonist H12 conformation relative to other structures, promoting a conformational selection. Similar effects in the free-energy surface are observed with coactivator association. On the other side, the presence of corepressor peptides stabilizes conformations not allowed in the previous systems and, therefore, induces a conformational transition in the protein. The mechanisms of dissociation of Rosiglitazone, a strong PPARgamma agonist, were also studied in this work. Two pathways for ligand dissociation were obtained, both involving rearrangements in the helices H2, H2', H3 and in the Omega-loop. These mechanisms are consistent with previous results and show that ligand escape does not require the displacement of helix 12 (AU)