Molecular dynamics simulations reveal multiple pathways of ligand dissociation from thyroid hormone receptors

Nuclear receptor (NR) ligands occupy a pocket that lies within the core of the NR ligand-binding domain (LBD), and most NR LBDs lack obvious entry/exit routes upon the protein surface. Thus, significant NR conformational rearrangements must accompany ligand binding and release. The precise nature of these processes, however, remains poorly understood. Here, we utilize locally enhanced sampling (LES) molecular dynamics computer simulations to predict molecular motions of x-ray structures of thyroid hormone receptor (TR) LBDs and determine events that permit ligand escape. We find that the natural ligand 3,5,3′-triiodo-L-thyronine (T₃) dissociates from the TRα1 LBD along three competing pathways generated through i), opening of helix (H) 12; ii), separation of H8 and H11 and the Ω-loop between H2 and H3; and iii), opening of H2 and H3, and the intervening β-strand. Similar pathways are involved in dissociation of T₃ and the TRβ-selective ligand GC24 from TRβ; the TR agonist IH5 from the α- and β-TR forms; and Triac from two natural human TRβ mutants, A317T and A234T, but are detected with different frequencies in simulations performed with the different structures. Path I was previously suggested to represent a major pathway for NR ligand dissociation. We propose here that Paths II and III are also likely ligand escape routes for TRs and other NRs. We also propose that different escape paths are preferred in different situations, implying that it will be possible to design NR ligands that only associate stably with their cognate receptors in specific cellular contexts.