Structural basis for ligand-independent activation of the orphan nuclear receptor LRH-1

The orphan nuclear receptors SF-1 and LRH-1 are constitutively active, but it remains uncertain whether their activation is hormone dependent. We report the crystal structure of the LRH-1 ligand binding domain to 2.4 A resolution and find the receptor to be a monomer that adopts an active conformation with a large but empty hydrophobic pocket. Adding bulky side chains into this pocket resulted in full or greater activity suggesting that, while LRH-1 could accommodate potential ligands, these are dispensable for basal activity. Constitutive LRH-1 activity appears to be conferred by a distinct structural element consisting of an extended helix 2 that provides an additional layer to the canonical LBD fold. Mutating the conserved arginine in helix 2 reduced LRH-1 receptor activity and coregulator recruitment, consistent with the partial loss-of-function phenotype exhibited by an analogous SF-1 human mutant. These findings illustrate an alternative structural strategy for nuclear receptor stabilization in the absence of ligand binding.     

Dynamic stabilization of nuclear receptor ligand binding domains by hormone or corepressor binding

We have developed a novel assembly assay to examine structural changes in the ligand binding domain (LBD) of the thyroid hormone receptor (TR). Fragments including the first helix of the TR LBD interact only weakly with the remainder of the LBD in the absence of hormone, but this interaction is strongly enhanced by the addition of either hormone or the corepressor NCoR. Since neither the ligand nor the corepressor shows direct interaction with this helix, we propose that both exert their effects by stabilizing the overall structure of the LBD. Current models of activation of nuclear hormone receptors focus on a ligand-induced allosteric shift in the position of the C-terminal helix 12 that generates the coactivator binding site. Our results suggest that ligand binding also has more global effects that dynamically alter the structure of the receptor LBD.     

Modulation of retinoic acid receptor alpha activity by lysine methylation in the DNA binding domain

Metabolic labeling and detection with a methylated lysine-specific antibody confirm lysine methylation of RAR alpha in mammalian cells. We previously reported Lys (347) trimethylation of mouse retinoic acid receptor alpha (RAR alpha) in the ligand binding domain (LBD) that affected ligand sensitivity of the dissected LBD. Here we report two monomethylated residues, Lys (109) and Lys (171) identified by LC-ESI-MS/MS in the DNA binding domain (DBD) and the hinge region, which affect retinoic acid (RA) sensitivity, coregulator interaction and heterodimerization with retinoid X receptor (RXR) in the context of the full-length protein. Constitutive negative mutation at Lys (109), but not Lys (171), reduces RA-dependent activation. Methylation at Lys (109) plays a more dominant role than trimethylation at Lys (347) in terms of RA activation of the full-length receptor. Lys (109) is located in a homologous sequence (CEGC K GFFRRS) of the DBD in RARs and is conserved in the nuclear receptor superfamily even across the species boundary. This study uncovers a potential role for monomethylation at Lys (109) in coordinating the synergy between DBD and LBD for ligand-dependent activation of RAR alpha.     

Constitutive activity and ligand-dependent activation of the nuclear receptor CAR-insights from molecular dynamics simulations

The constitutive androstane receptor (CAR) possesses, unlike most other nuclear receptors, a pronounced basal activity in vitro whose structural basis is still not fully understood. Using comparative molecular dynamics simulations of CAR X-ray crystal structures, we evaluated the molecular basis for constitutive activity and ligand-dependent receptor activation. Our results suggest that a combination of van der Waals interactions and hydrogen bonds is required to maintain the activation helix in the active conformation also in absence of a ligand. Furthermore, we identified conformational rearrangements within the ligand-binding pocket upon agonist binding and an influence of CAR inducers pregnanedione and CITCO on the helical conformation of the activation helix. Based on the results a model for ligand-dependent CAR activation is suggested.     

New insights into receptor ligand binding domains from a novel assembly assay

Previous studies have demonstrated that hormone binding stabilizes the ligand binding domain (LBD) of the nuclear hormone receptors against proteolysis. We have confirmed and extended this observation using a newly developed assembly assay. In this assay, the LBD is divided into two parts, of which one includes the first helix of this domain and the other corresponds to the remainder of the LBD. Several independent criteria demonstrate that these two fragments can assemble into a functional LBD in the presence of a ligand, but not in its absence, and that this is a reflection of the stabilizing effect of ligand. We have also used this assay to demonstrate that binding of the nuclear receptor corepressor NCoR can directly stabilize the LBD. Overall, these results highlight the dynamic nature of the LBD and suggest that current models for activation based solely on allosteric effects on the C-terminal helix may be too limited.