Binding of the N-terminal region of coactivator TIF2 to the intrinsically disordered AF1 domain of the glucocorticoid receptor is accompanied by conformational reorganizations

Abstract

Control of gene transcription by glucocorticoid receptors (GRs) is important for many physiological processes. Like other steroid hormone receptors, the regulation of target genes by GR is mediated by two transactivation domains: activation function 1 (AF1) in the N-terminal domain and AF2 in the C-terminal ligand-binding domain (LBD). Full receptor activity requires both AF1 and -2 plus assorted coregulatory proteins. Crystal structures of the ligand-bound LBD have provided insight regarding how AF2 interacts with specific coactivators. However, despite its being the major activation domain of GRs, knowledge of AF1 structure/function has languished. This is mainly because of the highly disorganized structure of the GR N-terminal domain. This lack of AF1 structure is shared by all members of the steroid/nuclear receptor superfamily for which it has been examined and AF1 is thought to allow productive interactions with assorted cofactors via protein-induced changes in secondary/tertiary structures. To date, there are no reports of a classical coactivator altering the secondary/tertiary structure of the GR AF1 domain. Earlier, we reported an N-terminal fragment of the p160 coactivator TIF2, called TIF2.0, that binds the GR N-terminal domain and alters GR transcriptional activity. We therefore proposed that TIF2.0 binding to AF1 changes both its conformation and transcriptional activity. We now report that TIF2.0 interacts with the GR AF1 domain to increase the amount of α-helical structure in the complex. Furthermore, TIF2 coactivator activity is observed in the absence of the GR LBD in a manner that requires the AF1 domain. This contrasts with previous models where TIF2 receptor interaction domains binding to GR LBD somehow alter AF1 conformation. Our results establish for the first time that coactivators can modify the structure of the AF1 domain directly via the binding of a second region of the coactivator and suggest a molecular explanation for how coactivators increase the transcriptional activity of GR-agonist complexes.

FIGURE 1.

TIF2.0 purification. A, diagram of domains of WT TIF2 and TIF2.0. B, Coomassie Blue-stained gel of stages of TIF2.0/FLAG purification. The lanes are as follow: M, molecular weight marker; C3 and C4, anti-FLAG M2-agarose column elution fractions 3 and 4; lysate, crude E. coli lysate; Com, combined elution fractions C3 and C4; Ft1–Ft3, YM-100 filtrates of three aliquots of combined C3 and C4 fractions; R1 and R2, YM-100 retentates of the two most concentrated aliquots of combined C3 and C4 fractions, which constitute the final purified material.

FIGURE 2.

TIF2.0/FLAG is functionally active. A, schematic of domains of WT GR and GR AF1 (amino acids 77–262). B, TIF2.0 pulldown of GR. Crude TIF2.0/FLAG lysate or purified TIF2.0/FLAG, was immobilized on anti-FLAG M2-agarose beads before being treated with crude overexpressed WT GR or purified GR AF1. The inputs and eluants of the beads were then analyzed by Western blotting with anti-GR antibody (upper panel) or anti-TIF2 antibody (lower panel). DBD, DNA-binding domain. C, TIF2.0 binding to GR requires activation of GR. Overexpressed WT GR (lanes 1–12) was incubated at 0 °C with vehicle (EtOH (E)), 1 μm dexamethasone (D), or 1 μm RU486 (R) under one of three conditions: −, no added Na₂MoO₄; +, Na₂MoO₄ added after steroid addition; and *, Na₂MoO₄ added before steroid addition. TIF2.0/FLAG (crude bacterial lysate) was then added as indicated. The rest of the assay was conducted as described in B using anti-GR antibody in the final Western blotting. I, input overexpressed GR.

FIGURE 3.

GRN523 and HA/TIF2.0 compete for complex formation of GR AF1 and TIF2.0/FLAG. A, schematic of competing proteins HA/TIF2.0 and GRN523. B, competition of GR AF1 and TIF2.0/FLAG complex formation. Anti-FLAG M2-agarose beads were prebound with nothing (lane 3), the control protein FLAG/BAP (lane 4), or TIF2.0/FLAG (lanes 5–10) and then treated with low (2 μg) or high (4 μg) amounts of GR AF1 without or with competitor (GRN523 or HA/TIF2.0) as indicated. Samples were then analyzed as described in the legend for Fig. 2.

FIGURE 4.

TIF2.0/FLAG is functionally active as a competitor in intact cells. A mammalian two-hybrid assay was conducted in triplicate as described under “Experimental Procedures,” where the indicated competitors (WT TIF2, TIF2.0, or TIF2.0/FLAG) were examined for their ability to disrupt the capacity of GAL/NCoR-RID and VP16/GR (with or without the agonist Dex or the antagonist RU486) to induce the reporter gene FRLuc. The data for each steroid are plotted relative to the luciferase activity of the uncompeted sample ± S.E. (n = 2).

FIGURE 5.

Analysis of the binding of GR AF1 to TIF2.0 by SPR. Measurement of the binding of TIF2.0 to AF1 by SPR was carried out on a Biacore X-100. AF1 was immobilized to the Fc2 channel of CM5 as the ligand by amine coupling. The Fc1 channel was treated similarly but without AF1 protein (control). TIF2.0/FLAG was used as analyte to flow through both Fc1 and Fc2 channels for binding. A, the adjusted (Fc2 - Fc1) sensorgrams from a series of TIF2.0/FLAG concentrations (shown on the right) are plotted. B, the binding affinity of the AF1-TIF2.0 interaction as calculated by steady-state fitting.

FIGURE 6.

Solution properties of AF1 domain using HDX mass spectrometry. HDX protection map of GR-AF1. The bars below the sequence represent the peptide fragments resolved by mass spectrometry, and the color of the bars represents the relative deuterium/hydrogen exchange (color code at top). Each bar has six segments showing six different time points (see inset).

FIGURE 7.

Differential HDX map comparing protection of GR AF1 and TIF2.0-bound GR AF1. The bars below the sequence represent the peptide fragments resolved by mass spectrometry, and the color of the bars represents the relative change in HDX protection. All peptides resolved showed no significant change in protection (color code is in lower right corner; ns, no significant change in protection) between GR AF1 and TIF2.0-bound GR AF1.

FIGURE 8.

Far-UV CD spectra of GR AF1.TIF2.0 complex. A, far-UV CD spectra of the GR AF1·TIF2.0 complex (experimental) at a constant AF1 concentration and varying concentrations of TIF2.0/FLAG (as indicated). B, far-UV CD spectra of GR AF1 + TIF2.0 (theoretical sum) at a constant AF1 concentration and varying concentrations of TIF2.0/FLAG (as indicated). C, a comparison of changes in the ellipticity at 222 nm for experimental and theoretical sums with respect to the AF1:TIF2.0 ratio. Each spectrum presented is the result of five spectra that were averaged, corrected for the contribution of the buffer, and smoothed.

FIGURE 9.

GR AF1 adopts higher helical content when complexed with TIF2.0. A, far-UV CD spectra showing AF1 after subtracting the contribution of TIF2.0/FLAG in each case. B, a plot ellipticity at 222 nm of AF1·TIF2.0 minus TIF2.0 with respect to the AF1:TIF2.0 ratio. A summary of percent secondary structural elements in AF1 with and without TIF2.0 binding is shown in the box.

FIGURE 10.

Far-UV CD spectra of TIF2.0/FLAG at various concentrations. A, spectra plotted at each concentration of TIF2.0 (as indicated). B, the data from each concentration are converted to a molar scale. Each spectrum presented is the result of five spectra that were averaged, corrected for the contribution of the buffer, and smoothed.

FIGURE 11.

TIF2.0 interaction with GR AF1 forms a stable complex. TIF2.0/FLAG alone or in a mixture with AF1 was incubated in a temperature-regulated CD cell, and absorbance at 222 nm was monitored as the temperature in the cell was raised from 20 to 90 °C at a controlled rate of 1 °C/min. T_m is the temperature at which half of the sample persists in native conformation and half has unfolded and lost absorbance at 222 nm.

FIGURE 12.

Limited proteolysis of AF1, TIF2.0/FLAG, and the AF1·TIF2.0/FLAG complex. A, Coomassie Blue-stained gel showing product of proteolytic digestion. MW, molecular weight markers; lanes 1–3, undigested AF1, TIF2.0, and AF1:TIF2.0 mixture, respectively; lanes 4–6, trypsin-digested AF1, TIF2.0, and AF1:TIF2.0 mixture, respectively; lanes 7–9, chymotrypsin-digested AF1, TIF2.0, and AF1·TIF2.0 complex, respectively. B, immunoreactions with an antibody raised against amino acids 150–175 in the human GR AF1 showing products of trypsin and chymotrypsin digestions. Numbering of the lanes is same as in A.

FIGURE 13.

TIF2 increases GRE-mediated AF1 activity as assessed by SEAP-based promoter-reporter assay in CV-1 cells. A, schematic representation of the GR constructs used in the transient transfection experiments. B, test of GR AF1 activity in the absence of the LBD. CV-1 cells were cotransfected with pECFP/GR500, or pECFP/GR500(ΔAF1) ± pSG5/TIF2 (as described under “Experimental Procedures”). The medium was assayed for SEAP activity 24 h later. The levels of significance were evaluated by a two-tailed paired Student's t test; *, p value of <0.05 was considered significant. RLU, relative light units; DBD, DNA-binding domain.