Structure-Function Analysis of the Rev-erbA and RVR Ligand-Binding Domains Reveals a Large Hydrophobic Surface That Mediates Corepressor Binding and a Ligand Cavity Occupied by Side Chains

Structure-Function Analysis of the Rev-erbA and RVR Ligand-Binding Domains Reveals a Large Hydrophobic Surface That Mediates Corepressor Binding and a Ligand Cavity Occupied by Side Chains Jean-Paul Renaud, Jonathan M. Harris, Michael Downes, Les J. Burke, and George E .O. Muscat Centre Nationale de la Recherche Scientifique UPR9004 Laboratoire de Biologie et Genomic Structurales (J.P.R.) Institut de Génétique et Biologie Moléculaire et Cellulaire F-67404 Illkirch, France University of Queensland (J.M.H., L.J.B., M.D., G.E.O.M.) Institute for Molecular Bioscience (I.M.B.) Centre for Molecular and Cellular Biology Ritchie Research Laboratories B402A St. Lucia, 4072 Queensland, Australia Rev-erbA/RVR are closely related orphan nuclear receptors (NRs) functioning as dominant transcriptional silencers through an association with the nuclear 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, ligand binding is thought to reposition H12, causing corepressor dissociation and coactivator recruitment, thus leading to transcriptional activation. Here we present homology models of the Rev-erbA/RVR LBDs, which show that the putative ligand cavity is occupied by side chains, suggesting the absence of endogenous ligands. Modeling also revealed a very hydrophobic surface due 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 vitro and in vivo interaction of the Rev-erbA/RVR LBD with the receptor-interacting domain of the corepressors N-CoR or its splice variant RIP13⌬1, reinforcing the view of the physical association of N-CoR with a LBD surface encompassing H3-H4 and H11. Furthermore, mutations in the LBD surface significantly reduced the ability of Rev-erbA and RVR to function as repressors of transcription. Interestingly, a hydrophobic surface comprised of H3-H4 and H12 in liganded NRs mediates the interaction with coactivators. Hence, it appears that corepressors and coactivators bind to overlapping surfaces of NR LBDs, the conformational change associated with H12 upon ligand binding resulting in a switch from a corepressor- to a coactivator-binding surface. (Molecular Endocrinology 14: 700–717, 2000) INTRODUCTION Members of the nuclear receptor (NR) superfamily bind specific DNA elements and can function as ligand-regulated transcription factors (1, 2). This group includes the orphan receptors, which have no known ligands in the classical sense and appear to be the ancient progenitors of this receptor superfamily (3). The Rev-erb family of proteins, Rev-erbA/ear-1 (4–7) and RVR/Rev-erbB/BD73 (8–10), are orphan members of this superfamily. These two proteins are highly conserved in the DNA-binding domain (DBD) (95%) and the ligand-binding domain (LBD) (70%); however, major differences between the two isoforms occur within the hypervariable A/B and D regions of the proteins. Rev-erbA and RVR bind as monomers to the nuclear receptor half-site motif, RGGTCA flanked 5⬘ by an AT-rich sequence [(A/T)6RGGTCA], and as dimers to a novel direct repeat motif separated by 2 bp [Rev-DR2, (A/T)4 AGGTCA CT AGGTCA] (11, 12). The Rev-erb family members function as ligand-independent dom- 0888-8809/00/$3.00/0 Molecular Endocrinology 14(5): 700–717 Copyright © 2000 by The Endocrine Society Printed in U.S.A. 700 Structure-Function Analysis of Rev-erbA and RVR inant transcriptional repressors (13–15) and the LBDs of Rev-erbA and RVR encode active transcriptional silencers (14, 15). Furthermore, we demonstrated that efficient repression (of GAL4VP16-mediated transactivation) is independently mediated by approximately 35 amino acids (aa) between aa 455–488 in the LBD of Rev-erbA and aa 416-449 of RVR (14, 15). This repression domain contains the LBD-specific signature motif (F/W)AKXXXXFXXLXXXDQXXLL (16) and spans H3–H5. Ligand-independent repression of transcription by Rev-erbA and RVR is mediated by the nuclear receptor corepressor N-CoR and its variants RIP13a and RIP13⌬1 (17–22). Detailed analysis of the corepressors identified a C-terminal receptor interaction domain (RID) that consists of two interaction domains, ID-I and ID-II, that efficiently interact with the ligandregulated and orphan nuclear receptors (20–22). Furthermore, it has been demonstrated that RVR and Rev-erbA interact very efficiently with the RID from N-CoR and RIP13a, although they preferentially interact with the RID from RIP13⌬1 (20). Moreover, it was demonstrated that the LBD of RVR and Rev-erbA was necessary for the interaction with corepressors (20). The Rev-erbA LBD regions interacting with N-CoR have also been identified. Initially Lazar and colleagues delineated two regions in Rev-erbA by in vitro studies, domain X (aa 407–418) and domain Y (aa 602–614) (21), that were shown to be necessary for the efficient interaction with the corepressor, N-CoR. These regions correspond to the extra domain and H11, respectively (see Fig. 1). However, in a follow-up study by the same group, they suggested that the X domain was not required for corepressor binding in vivo (23). In a later study (24), we demonstrated that corepressor interaction region 2 (CIR-2)/Y-domain in H11 of ReverbA and RVR was necessary for the interaction with the corepressor, N-CoR, and the variant, RIP13⌬1. Furthermore, we also defined a novel domain (CIR-1), which corresponds to H3 in both Rev-erbA and RVR, that was necessary for the efficient interaction of both orphan nuclear receptors with both corepressors, NCoR and RIP13⌬1. Moreover, we used mutagenesis to demonstrate that CIR-1/H3 and CIR-2/Y-domain/H11 in both receptors are necessary for 1) the interaction with the corepressors N-CoR and RIP13⌬1, and 2) transcriptional repression of a physiological target, the human (h) Rev-erbA promoter (24). F439 in Rev-erbA and F402 in RVR were critical residues in supporting corepressor interaction. This suggested that a minimal region containing CIR-1 and CIR-2, in H3 and H11, respectively, was necessary for corepressor binding and transcriptional repression. It was hypothesized that CIR-1 and CIR-2 are juxtaposed in the putative three-dimensional tertiary structure of these orphan receptors and probably form a single contact interface that interacts with the nuclear receptor corepressors. However, further experimentation was required to verify this hypothesis. To resolve these contradictions in the structure and specificity of the orphan nuclear receptor-corepressor interaction, three dimensional 701 analysis of Rev-erbA and RVR was required. The crystal structure of several NR LBDs has already been determined and reviewed recently (Ref. 25 and references therein). Furthermore, the structure and specificity of ligand-activated NR-coactivator interactions have been resolved. These studies s (...truncated)