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. 1999 Dec 15;13(24):3198-208.
doi: 10.1101/gad.13.24.3198.

Molecular determinants of nuclear receptor-corepressor interaction

V Perissi  1 , L M Staszewski, E M McInerney, R Kurokawa, A Krones, D W Rose, M H Lambert, M V Milburn, C K Glass, M G Rosenfeld
Affiliations

Molecular determinants of nuclear receptor-corepressor interaction

V Perissi et al. Genes Dev. .

Abstract

Retinoic acid and thyroid hormone receptors can act alternatively as ligand-independent repressors or ligand-dependent activators, based on an exchange of N-CoR or SMRT-containing corepressor complexes for coactivator complexes in response to ligands. We provide evidence that the molecular basis of N-CoR recruitment is similar to that of coactivator recruitment, involving cooperative binding of two helical interaction motifs within the N-CoR carboxyl terminus to both subunits of a RAR-RXR heterodimer. The N-CoR and SMRT nuclear receptor interaction motifs exhibit a consensus sequence of LXX I/H I XXX I/L, representing an extended helix compared to the coactivator LXXLL helix, which is able to interact with specific residues in the same receptor pocket required for coactivator binding. We propose a model in which discrimination of the different lengths of the coactivator and corepressor interaction helices by the nuclear receptor AF2 motif provides the molecular basis for the exchange of coactivators for corepressors, with ligand-dependent formation of the charge clamp that stabilizes LXXLL binding sterically inhibiting interaction of the extended corepressor helix.

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Figures

Figure 1
Figure 1
Characterization of the amino-terminal interaction domain of N-CoR. (A) Schematic representation of murine N-CoR showing the location of the repressor domains (RDI, RDII, RDIII) and the nuclear receptor interaction domains (ID-N, ID-C). (B) ChIP assay of N-CoR binding to the βRAR promoter. The experiments reproducibly revealed the presence of N-CoR on the βRAR promoter in the absence of ligand, but not in the presence of RA; no detectable precipitation of promoter was observed with control preimmune IgG (ct IgG). (C) GST pull-down assay testing binding of overlapping fragments of ID-N to T3Rβ. Sequences spanning amino acids 2040–2090 are required for effective interaction. (D) Mapping of the critical residues in the 2040–2090 interaction domain. Cluster mutation of the indicated five adjacent amino acids to alanine residues was performed across the interval. GST pull-down analysis revealed that residues spanning amino acids 2060–2080 were quantitatively the most critical for interaction, with some contribution from amino acids 2080–2090.
Figure 2
Figure 2
Identification of a putative corepressor extended helix interaction motif. (A) Alignment of critical regions of N-CoR and SMRT ID-N and ID-C motifs reveals a conserved LXX I/H IXXX I/L extended helix compared to that of the LXXLL motif of SRC1 or the AF2 domain of RXR. Clustered mutation of these residues in ID-C or in a region (1954–2215) encompassing ID-N resulted in loss of interactions in GST pull-down assays or a mammalian two-hybrid assays, confirming the critical importance of L1, I5, and I9 residues. (B) ABCD analysis of N-CoR binding to RXR/RAR heterodimers on a DR+5 element. An N-CoR interaction region spanning amino acids 2053–2453 was bound effectively in the absence, but not in the presence, of RA; mutation of the three conserved L and I residues in either ID-N or in ID-C markedly diminished or abolished N-CoR interaction with the DNA-bound receptor heterodimer. (C) Synthetic peptides used for competition studies. (D) Peptide competition of NCoR binding by T3R: Addition of ID-C peptide gives clear competition at 50 μm, the RXR AF2 peptide does not compete even at higher concentration; the SRC1–LXD2 peptide gives a detectable slight competition at high concentrations. (E) Peptide competition of GAL4/T3R carboxyl terminus fusion protein-dependent inhibition of UAS × 3/tk–lacZ reporter in single cell nuclear microinjection assays in Rat-1 cells. The nuclear receptor interaction domain (amino acids 570–843 and 626–783) of SRC1 was expressed as a GAL4 fusion protein under control of the CMV promoter.
Figure 3
Figure 3
Analysis of ID-C and ID-N amino acid determinants of NR binding to N-CoR. (A) Amino acid mutations introduced in the context of GST or GAL4 fusion proteins with a summary of their effects on receptor interaction. (B) GST pull-down assay of 35S-labeled T3R by the GST/ID-C(2268–2289) fusion protein. (C) Mammalian two-hybrid assay using GAL4 ID-C fusion proteins in the presence or absence of VP16/RAR with an UAS × 3/p36 luciferase reporter. Results are average of triplicate determinations ±s.e.m., with similar results in three independent assays. (D) Schematic diagram of mutations introduced in the ID-N region in the context of GAL4 N-CoR(1954–2215). (E) Ability of GAL4/NCoR mutants to recruit RAR, thereby activating the UAS × 3/p36 reporter.
Figure 4
Figure 4
NR determinants of N-CoR binding. (A) The position of a series of mutations introduced into T3Rβ, involving residues in helixes 1, 3, 5, 6, and 11, is imposed on the known structure of the T3Rα LBD (Wagner et al. 1995), with the ligand removed and the position of AF2 rotated. The effect of the mutations on N-CoR binding is listed. (B) Analysis of these mutations in GST pull-down assays using GST–N-CoR(2040–2300) and 35S-labeled T3Rβ. (C) Similar analysis of the effect of T3Rβ mutations on GST–SRC(631–763) binding. (D) Repressor function of mutated T3R in a single cell nuclear microinjection assay was performed in Rat-1 cells using a GAL4/T3R carboxy-terminal fusion protein and a UAS × 3/tk–lacZ reporter.
Figure 5
Figure 5
Model of corepressor/NR interaction. (A) Helical plots of IDN and IDC core motifs. (B) Schematic diagram of point mutations designed to disrupt the amino-terminal helical extension (Mut1, Mut2, Mut3) of ID-N or to convert residues 5–9 into an LXXLL consensus (Mut4). (C) Mammalian two-hybrid assay of the interaction between wild-type and mutant GAL4 N-CoR(1954–2215) and VP16/RAR using a UAS × 3/p36 luciferase reporter. (D) Ribbon diagram of the corepressor extended helix (in red) predicted to contact the hydrophobic (coactivator) binding pocket formed by helices 3, 5, and 6. An idealized helix [sequence (A5)LAAIIAAALRL] was built and transformed it into the coactivator binding site by superimposing the LAAII residues onto the corresponding LXXLL residues of the coactivator peptide using the PPARγ/SRC-1/BRL49653 complex as a model (Nolte et al. 1998). This idealized helix position was then transformed onto T3Rβ by superimposing PPARγ onto T3Rβ. The carboxy-terminal end of the helix is pointed at helix 1 and the amino-terminal end of the helix is sterically blocked by the AF-2 helix (in yellow) position. The binding of the shorter coactivator helix of GRIP-1 to the same pocket is represented in green. Below is shown an expanded view of AF-2 (yellow), corepressor (red), and coactivator (green) helices. (E) Model of the ligand-dependent exchange of corepressor for coactivator. The two related N-CoR interaction helices are suggested to cooperatively be recruited into the helix 3, 5, 6 binding pocket of RXR/RAR or RXR/T3R heterodimers on DNA, with no requirement for the conserved glutamic acid residues of the AF2 helix. Ligand binding induces exchange for coactivators, which contain the short LXXLL helical motifs, requiring the conserved glutamic acid residue of the AF-2 helix for effective orientation and positioning into the receptor binding pocket.
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