Hormone binding and co-regulator binding to the glucocorticoid receptor are allosterically coupled

Abstract

The glucocorticoid receptor initiates the cellular response to glucocorticoid steroid hormones in vertebrates. Co-regulator proteins dock to the receptor in response to hormone binding and potentiate the transcriptional activity of the receptor by modifying DNA and recruiting essential transcription factors like RNA polymerase II. Hormones and co-regulators bind at distinct sites in the ligand binding domain yet function cooperatively to mediate transcriptional control. This study reveals and quantifies energetic coupling between two binding sites using purified components. Using a library of peptides taken from co-regulator proteins, we determine the pattern of co-regulator binding to the glucocorticoid receptor ligand binding domain. We show that peptides from co-regulators differ in their effects on hormone binding and kinetics. Peptides from DAX1 and SRC1 bind with similar affinity, but DAX1 binding is coupled to hormone binding, and SRC1 is not. Mechanistic details of co-regulator binding and coupling to the hormone binding pocket are uncovered by analysis of properties endowed by mutation of a key residue in the allosteric network connecting the sites.

FIGURE 1.

A, dexamethasone (left) and dexamethasone-fluorescein (right) are shown. B, schematics depicting the interactions observable by fluorescence polarization with dex-fl are shown. dex-fl (ring structure) binds GR (large shape) yielding high polarization. Co-regulator peptide (gray oval) binds GR·dex-fl, and binding is affected through allostery, further increasing dex-fl polarization.

FIGURE 2.

GR·dex-fl binding kinetics. A, 1 μm GR_SD (F602S, C638D), 20 nm dex-fl association is monitored by fluorescence polarization. B, 1 μm GR_SD, 20 nm dex-fl dissociation induced by the addition of 50 μm unlabeled dex is shown. C, 1 μm GR_M752I, 20 nm dex-fl association is shown. D, 1 μm GR_M752I, 20 nm dex-fl dissociation. All data fit to one-phase exponential models to yield half-times.

FIGURE 3.

NR box binding is coupled to ligand binding kinetics. A, 1 μm GR_SD, 20 nm dex-fl association in the presence of 10 μm SRC2-3 (circles), DAX1-3 (squares), and no NR box (gray circles) is shown. Base-line-subtracted data fit to one-phase exponential model. Error bars are omitted for clarity. B, half-times of GR_SD·dex-fl association in the presence of 10 μm NR box are shown. C, 1 μm GR_SD, 20 nm dex-fl dissociation in the presence of 10 μm SRC2-3 (circles) and DAX1-3 (squares) is shown. D, half times of GR_SD·dex-fl dissociation in the presence of 10 μm NR box are shown.

FIGURE 4.

GR_M752I·dex-fl binding kinetics in the presence of NR box peptides. A, 1 μm GR_M752I, 20 nm dex-fl association in the presence of 10 μm SRC2-3 (circles) and DAX1-3 (squares) is shown. Base-line-subtracted data fit to a one-phase exponential model. B, half-times of GR_M752I·dex-fl association in the presence of 10 μm NR box are shown. C, 1 μm GR_M752I, 20 nm dex-fl dissociation in the presence of 10 μm SRC2-3 (circles) and DAX1-3 (squares) is shown. D, half-times of GR_M752I·dex-fl dissociation in the presence of 10 μm NR box are shown.

FIGURE 5.

GR binding to SRC3-3-fluor with and without dex. A, GR_SD titrated against 20 nm SRC3-3 conjugated to Alexa-Fluor 555 in the presence (circles) or absence (squares) of 10 μm dex is shown. B, GR_M752I titrated against 20 nm SRC3-3 conjugated to Alexa-Fluor 555 in the presence (circles) or absence (squares) of 10 μm dex is shown.

FIGURE 6.

GR binding to NR box peptides by allosteric FP reveals six binding modes. NR box peptides titrated against 500 nm GR_SD, 20 nm dex-fl are shown. A, SRC3-3, high plateau saturation is shown. B, SRC1-3, low-plateau saturation is shown. C, SHP-1, non-saturable binding is shown. D, SRC2-2, weak binding is shown. E, Hsp90-2, no binding is shown. F, SMRT-2, corepressor binding is shown. All data fit to one-site saturation binding model. Residuals shown in the inset graphs.

FIGURE 7.

Hierarchical clustering of NR box peptides. NR box peptides were clustered using half-times shown in Figs. 3 and 4.

FIGURE 8.

Core sequences of GR binders. GR binding NR box core sequences with conserved residues are highlighted. LXXLL Leu residues are in black, and GR-specific conserved positions are in gray.

FIGURE 9.

GR binding to SRC1-3-fluor. GR_SD (open circles) and GR_M752I (closed squares) were titrated against 20 nm SRC1-3 conjugated to Alexa-Fluor 555 in the presence of 10 μm dex. Data are fit to a one-site saturation binding model.

FIGURE 10.

Sequence alignment of NR AF-2 regions. NR LBD crystal structures were aligned in PyMOL. Residues conserved across the NR superfamily are in light gray. Residues conserved among SRs are in dark gray. Residues conserved among non-SRs are in black. The Protein Data Bank codes of structures used for the alignment are GR (3BQD), mineralocorticoid receptor (MR) (2ABI), progesterone receptor (PR) (3D90), androgen receptor (1T7R), ER (2OCF), vitamin D receptor (VDR) (2ZL9), thyroid hormone receptor (TR) (3HZF), LRH-1 (1YOK).