Structure-function analysis of the Rev-erbA and RVR ligand-binding domainsreveals a large hydrophobic surface that mediates corepressor binding and a ligand cavity occupied by side chains

Rev-erbA/RVR are closely related orphan nuclear receptors (NRs) functioning as dominant transcriptional silencers through an association with the nucl ear receptor corepressor N-CoR. In contrast with ligand-regulated NRs, Rev- erbA/RVR lack the ligand-binding domain (LBD) C-terminal activation helix, H12. In the case of retinoid acid receptor and thyroid hormone receptor, li gand binding is thought to reposition H12, causing corepressor dissociation and coactivator recruitment, thus leading to transcriptional activation. H ere we present homology models of the Rev-erbA/RVR LBDs, which show that th e putative ligand cavity is occupied by side chains, suggesting the absence of endogenous ligands. Modeling also revealed a very hydrophobic surface d ue to the absence of H12, exposing residues from H3, loop 3-4, H4, and H11. Mutation of specific residues from this surface severely impaired the in v itro and in vivo interaction of the Rev-erbA/RVR LED with the receptor-inte racting domain of the corepressors N-CoR or its splice variant RIP13 Delta 1, reinforcing the view of the physical association of N-CoR with a LED sur face encompassing H3-H4 and H11. Furthermore, mutations in the LED surface significantly reduced the ability of Rev-erbA and RVR to function as repres sors of transcription. Interestingly, a hydrophobic surface comprised of H3 -H4 and H12 in liganded NRs mediates the interaction with coactivators. Hen ce, it appears that corepressors and coactivators bind to overlapping surfa ces of NR LBDs, the conformational change associated with H12 upon ligand b inding resulting in a switch from a corepressor- to a coactivator-binding s urface.