TR Surfaces and Conformations Required to Bind Nuclear Receptor Corepressor

0888-8809/02/$15.00/0 Printed in U.S.A. Molecular Endocrinology 16(2):271–286 Copyright © 2002 by The Endocrine Society TR Surfaces and Conformations Required to Bind Nuclear Receptor Corepressor ADHIRAI MARIMUTHU*, WEIJUN FENG, TETSUYA TAGAMI, HOA NGUYEN*, J. LARRY JAMESON, ROBERT J. FLETTERICK, JOHN D. BAXTER†, AND BRIAN L. WEST* Residues of the TR that are critical for binding the nuclear receptor corepressor (N-CoR) were identified by testing more than 100 separate mutations of the full-length human TR␤ that scan the surface of its ligand binding domain. The primary inferred interaction surface overlaps the surface described for binding of p160 coactivators, but differs by extending to a novel site underneath which helix 12 rests in the liganded TR, rather than including residues of helix 12. Nonconservative mutations of this surface diminished binding similarly to three isolated N-CoR receptor interaction domains (RIDs), but conservative mutations affected binding variably, consistent with a role for this surface in RID selectivity. The commonality of this surface in binding N-CoR was confirmed for the RXRs and ERs. Deletion of helix 12 increased N-CoR binding by the TR modestly, and by the RXR and ER to a much greater extent, indicating a competition between this helix and the corepressor that regulates the extent of corepressor binding by nuclear receptors. When helix 12 was deleted, N-CoR binding by the ER was stimulated by tamoxifen, and bind- ing by the TR was stimulated by Triac, indicating that helix 12 is not the only feature that regulates corepressor binding. Two additional mutationsensitive surfaces were found alongside helix 1, near the previously described CoR box, and above helix 11, nearby but separate from residues that help link receptor in dimers. Based on effects of selected mutations on T3 and coactivator binding, and on results of combined mutations of the three sites on corepressor binding, we propose that the second and third surfaces stabilize TR unliganded conformation(s) required for efficient N-CoR binding. In transfection assays mutations of all three surfaces impaired the corepressor-mediated functions of unliganded TR repression or activation. These detailed mapping results suggest approaches for selective modulation of corepressor interaction that include the shape of the molecular binding surface, the competitive occupancy by helix 12, pharmacological stimulation, and specific conformational stabilization. (Molecular Endocrinology 16: 271–286, 2002) N tion or repression, depending on the response element encountered. By contrast, corepressors mediate the functions of unliganded receptors to repress the basal activity of promoters that contain TR binding sites, but also to stimulate the basal activity of certain promoters such as that for TSH (3). Corepressors like nuclear receptor copressor (NCoR) (4), also called RIP13 (5), and silencing mediator of retinoid and thyroid receptor (SMRT) (6), also called TRAC (T3 receptor associating cofactor) (7), were identified by their binding to the TR and RAR, but binding of varying strength has been detected to a number of different nuclear receptors including the RXR (5), VDR (8), PPAR (9, 10), ER (11, 12), PR (13–15), and the orphan receptors RevErb (16, 17), chicken ovalbumin upstream promoter transcription factor 1 (18), DAX-1 (19), steroidogenic factor-1 (20), and RVR (17). To understand corepressor function it is important to determine how they interact with receptors. Central to UCLEAR HORMONE RECEPTORS comprise a family of related proteins that regulate transcription. Prototypical receptors bind both DNA and hormone and can cause both positive and negative changes in gene expression, depending upon the target gene promoter. These receptors are single polypeptide chains containing an amino-terminal domain, a centrally placed DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD) (1). Several coregulatory proteins interact with nuclear receptors and contribute to gene-regulatory effects (2). Coactivators are recruited to the receptors in response to agonist binding and can participate in gene activaAbbreviations: DBD, DNA-binding domain; GRIP, GRinteracting protein; GST, glutathione-S-transferase; h, human; LBD, ligand-binding domain; LUC, luciferase; N-CoR, nuclear receptor corepressor; RID, receptor interaction domain; SMRT, silencing mediator of retinoid and thyroid receptor; TK, thymidine kinase; TRE, thyroid response element; WT, wild type. 271 Metabolic Research Unit (A.M., W.F., H.N., J.D.B., B.L.W.) and Departments of Biochemistry and Biophysics and Cellular and Molecular Pharmacology (R.J.F.), University of California San Francisco, San Francisco, California 94143; Center for Endocrinology, Metabolism, and Molecular Medicine (J.L.J.), Northwestern University Medical School, Chicago, Illinois 60611; and Clinical Research Institute, Center for Endocrine and Metabolic Diseases (T.T.), Kyoto National Hospital, Kyoto 612-8555, Japan 272 Mol Endocrinol, February 2002, 16(2):271–286 Marimuthu et al. • TR Surfaces and N-CoR Binding Fig. 1. A TR LBD Surface (Site 1) That Overlaps the Coactivator-Binding Surface, but Extends Underneath Helix 12, Selectively Binds All Three RIDs of N-CoR A, Binding of a selection of [35S]-labeled hTR␤ mutants to mouse N-CoR protein fragments containing RIDs I (2,231/2,321, solid), II (2,034/2,114, shaded), or III (1,888/2,031, open) in GST pull-down assays. Binding is expressed as the percentage of binding observed for the WT hTR␤ in the same assay (typically 12% of the input for RIDs I and II and 30% for RID III). Results are the average ⫾ SD of duplicates and are representative of at least two experiments. B, As for panel A, except a selection of mostly conservative mutants was examined. C, Space-filling models of TR␤ LBD showing the inferred corepressor-binding surface. Residues that bind N-CoR normally after mutation are shaded gray. Mutation-sensitive residues are colored by residue type: hydrophobic, green; polar, orange; basic, blue; and acidic, red. Computer graphics prepared using MidasPlus (UCSF Computer Graphics Laboratory) (53). Left panel, View based on the TR␣ LBD structure (21), which contains the complete helix 12 (H12, line), but in which the terminal two amino acids (E460 andD461) present on WT hTR␤ are unstructured and therefore not visible. Right panel, Model of the natural F451X mutant, in which 11 residues (451–461) are absent and therefore show the portion of the corepressor-binding surface obscured by helix 12 in the ligand-bound state. The corepressor-binding surface is comprised of a cluster of hydrophobic residues (I280, V283, V284, I302, and C309) bordered by polar (T277 and T281) and charged (K288 and K306) residues. The TR mutations tested and found not to diminish binding to N-CoR include (with approximate locations): Marimuthu et al. • TR Surfaces and N-CoR Binding 273 ences between these (...truncated)