Conformation of the Mineralocorticoid Receptor N-terminal Domain: Evidence for Induced and Stable Structure

Abstract

The mineralocorticoid receptor (MR) binds the steroid hormones aldosterone and cortisol and has an important physiological role in the control of salt homeostasis. Regions of the protein important for gene regulation have been mapped to the amino-terminal domain (NTD) and termed activation function (AF)1a, AF1b, and middle domain (MD). In the present study, we used a combination of biophysical and biochemical techniques to investigate the folding and function of the MR-NTD transactivation functions. We demonstrate that MR-AF1a and MR-MD have relatively little stable secondary structure but have the propensity to form α-helical conformation. Induced folding of the MR-MD enhanced protein-protein binding with a number of coregulatory proteins, including the coactivator cAMP response element-binding protein-binding protein and the corepressors SMRT and RIP140. By contrast, the MR-AF1b domain appeared to have a more stable conformation consisting predominantly of β-secondary structure. Furthermore, MR-AF1b specifically interacted with the TATA-binding protein, via an LxxLL-like motif, in the absence of induced folding. Together, these data suggest that the MR-NTD contains a complex transactivation system made up of distinct structural and functional domains. The results are discussed in the context of the induced folding paradigm for steroid receptor NTDs.

Regulation of the Structurally Dynamic N-terminal Domain of Progesterone Receptor by Protein-induced Folding

“…4). The affinity and kinetics are similar to that reported for TBP C binding with NTDs of GR, MR, and ER␣ (31,33,36). The onand off-rates are consistent with a two-step model that involves a fast initial binding event and a slower dissociation once the complex is formed due to structural rearrangements that form a more stable conformation (31,33).…”

“…In previous studies, conditional folding of the NTD of GR and MR induced by osmolytes or TBP was shown to facilitate binding of co-activators such as SRC-1, -2, and -3, CBP and co-repressors SMRT and RIP140 (31,33). Conditional folding by TBP also enhanced the ability of SRC-1 to stimulate GR or MR NTD-dependent transcriptional activity.…”

“…The modular nature of the NTD with the potential to fold into multiple distinct active conformations is well exemplified by studies of the MR. Three regions of the MR NTD capable of mediating transactivation of reporter genes were identified, including two unstructured modules that undergo folding by osmolytes and binding of co-regulatory proteins such as CBP, SRCs, RIP140, and SMRT and a third region of pre-formed stable secondary structure (␤-sheet) that binds TBP without folding (31). Previous studies of truncation and deletion mutations of NTD of PR also illustrated that distinct regions of the NTD were required for co-activator enhancement of PR activity when bound to hormone agonist or antagonist suggesting the NTD has the flexibility to utilize distinct regions for different activities (52).…”

“…However, a core C-terminal domain of human TBP consisting of aa 159 -339 (TBP C ) was reported to interact with the NTDs of several SRs and to promote a more compact tertiary structure with increased ␣-helical content (14,(33)(34)(35)(36). In the case of glucocorticoid (GR) and mineralocorticoid receptors (MR), TBP C binding was also observed to be associated with an enhancement of NTD-dependent transcriptional activity (30,31,33). Coupled binding and folding events leading to disorderto-order transitions in the NTDs of the steroid receptors have been studied using isolated NTD peptides or a minimal AF1 subregion that may not represent the entire SR signaling spectrum.…”

An androgen receptor N-terminal domain antagonist for treating prostate cancer

“…Consistent with the properties of IDPs, AR interacts with more than 160 proteins (22), and proteinprotein interactions with the activation function-1 (AF1) region in the NTD are essential for AR transcriptional activity (23)(24)(25)(26). AR NTD has less than 15% homology with other steroid receptors that also have predominantly intrinsically disordered NTDs (27)(28)(29)(30)(31). Malleability of intrinsically disordered NTDs of these transcription factors is crucial for their function that requires interactions with many binding partners.…”