Folding requirements of the ligand-binding domain of the human mineralocorticoid receptor.

The effects of aldosterone are mediated by the mineralocorticoid receptor (MR), a ligand-dependent transcription factor. We investigated the structural determinants for ligand binding to the receptor using a series of human MR (hMR) deletion mutants. These proteins were produced in vitro in rabbit reticulocyte lysate and analyzed for their ability to bind agonists, antagonists, and the heat shock protein hsp90, which is a prerequisite for ligand binding to hMR. Studies on N terminus-truncated hMRs showed that the ligand-binding domain (LBD: amino acids 734–984) has a lower affinity for aldosterone than the entire receptor [dissociation constant (Kd) 2.9 vs. 0.47 nm] and does not interact with hsp90. Addition of the five- amino acid sequence (729–733) upstream from the LBD is necessary for interaction with hsp90, but a larger region is needed for high aldosterone affinity. Deletions at the C-terminal end of the hMR greatly reduced both agonist and antagonist binding: deletion of the last three amino acids ...