Abstract
The nuclear receptor retinoid acid receptor-related orphan receptor γt (RORγt) is a master regulator of the Th17/IL-17 pathway that plays crucial roles in the pathogenesis of autoimmunity. RORγt has recently emerged as a highly promising target for treatment of a number of autoimmune diseases. Through high-throughput screening, we previously identified several classes of inverse agonists for RORγt. Here, we report the crystal structures for the ligand-binding domain of RORγt in both apo and ligand-bound states. We show that apo RORγt adopts an active conformation capable of recruiting coactivator peptides and present a detailed analysis of the structural determinants that stabilize helix 12 (H12) of RORγt in the active state in the absence of a ligand. The structures of ligand-bound RORγt reveal that binding of the inverse agonists disrupts critical interactions that stabilize H12. This destabilizing effect is supported by ab initio calculations and experimentally by a normalized crystallographic B-factor analysis. Of note, the H12 destabilization in the active state shifts the conformational equilibrium of RORγt toward an inactive state, which underlies the molecular mechanism of action for the inverse agonists reported here. Our findings highlight that nuclear receptor structure and function are dictated by a dynamic conformational equilibrium and that subtle changes in ligand structures can shift this equilibrium in opposite directions, leading to a functional switch from agonists to inverse agonists.
Highlights
The nuclear receptor retinoid acid receptor-related orphan receptor ␥t (ROR␥t) is a master regulator of the Th17/IL-17 pathway that plays crucial roles in the pathogenesis of autoimmunity
When examining the published crystal structure (PDB code 3L0L), we noticed that the C␣–C␣ distance is only 8 Å between the first resolved N-terminal residue of the steroid receptor coactivator-2 (SRC2) peptide and the C-terminal residue (Ser-507) of the ROR␥t-ligand-binding domains (LBD)
The interaction energy of the HYF triplet is reduced to only Ϫ1.4 kcal/mol as calculated using the same Density functional theory (DFT) protocol, which amounts to a loss of 11.5 kcal/mol stabilizing energy for helix 12 (H12) compared with the apo structure
Summary
The nuclear receptor retinoid acid receptor-related orphan receptor ␥t (ROR␥t) is a master regulator of the Th17/IL-17 pathway that plays crucial roles in the pathogenesis of autoimmunity. We report the crystal structures for the ligand-binding domain of ROR␥t in both apo and ligand-bound states. The structures of ligand-bound ROR␥t reveal that binding of the inverse agonists disrupts critical interactions that stabilize H12. This destabilizing effect is supported by ab initio calculations and experimentally by a normalized crystallographic B-factor analysis. Two monoclonal antibodies (mAb) targeting IL-17A, secukinumab (Cosentyx) and ixekizumab (Taltz), have been approved recently for the treatment of moderate to severe plaque psoriasis. We report the crystal structures of ROR␥t LBD in both apo and ligand-bound states with two novel classes of synthetic ligands. Subtle changes in ligand structures can shift the equilibrium in opposite directions and lead to a functional switch from an agonist to an inverse agonist
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