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.
Highlights
The first localized step in translating a glucocorticoid signal toward a genomic outcome is the binding of GC to the glu
The surface of the nuclear receptor ligand binding domain (LBD) typically contains at least two protein-protein interaction sites; they are a dimerization surface, more prevalent in the class II receptors that are regulated by forming heterodimers with retinoid X receptor [33], and a co-regulator protein docking site, termed activation function-2 (AF-2)
GR Ligand Binding Pocket and AF-2 Are Allosterically Coupled—An integral step in signal transduction through nuclear receptors (NRs) is the formation of the AF-2 co-regulator binding surface in response to hormone binding
Summary
Co-regulators are essential players in the transduction of hormone signals through nuclear receptors [37,38,39]. The intrinsic functionality of co-regulators is varied, with many implicated in chromatin remodeling and recruitment of the basal transcription machinery [44, 45] As their docking to nuclear receptors typically follows ligand binding and subsequent localization to the nucleus, co-regulators represent a vital, cell-specific layer of control that can be employed to modify the cellular response to hormone. The most well characterized co-activators, the p160 family (40 – 42, 47– 49), each contain at least three NR boxes that share the LXXLL core but differ in the residues at the ϩ2 and ϩ3 positions in addition to the flanking sequence These residues have been shown to be key determinants of selectivity between coregulators and the wide array of NRs with which they interact [50]. Together these results represent a first step in the development of a quantitative framework for the description of coupling between the co-regulator docking site and the ligand binding site
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