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

Previous studies have demonstrated that hormone binding stabilizes the liga nd binding domain (LBD) of the nuclear hormone receptors against proteolysi s. We have confirmed and extended this observation using a newly developed assembly assay. In this assay, the LED is divided into two parts, of which one includes the first helix of this domain and the other corresponds to th e remainder of the LED. Several independent criteria demonstrate that these two fragments can assemble into a functional LED in the presence of a liga nd, but not in its absence, and that this is a reflection of the stabilizin g effect of ligand. We have also used this assay to demonstrate that bindin g of the nuclear receptor corepressor NCoR can directly stabilize the IBD. Overall, these results highlight the dynamic nature of the LED and suggest that current models for activation based solely on allosteric effects on th e C-terminal helix may be too limited. (C) 2001 Elsevier Science Ltd. All r ights reserved.