Abstract
Ligand binding is the first step in hormone regulation of mineralocorticoid receptor (MR) activity. Here, we report multiple crystal structures of MR (NR3C2) bound to both agonist and antagonists. These structures combined with mutagenesis studies reveal that maximal receptor activation involves an intricate ligand-mediated hydrogen bond network with Asn770 which serves dual roles: stabilization of the loop preceding the C-terminal activation function-2 helix and direct contact with the hormone ligand. In addition, most activating ligands hydrogen bond to Thr945 on helix 10. Structural characterization of the naturally occurring S810L mutant explains how stabilization of a helix 3/helix 5 interaction can circumvent the requirement for this hydrogen bond network. Taken together, these results explain the potency of MR activation by aldosterone, the weak activation induced by progesterone and the antihypertensive agent spironolactone, and the binding selectivity of cortisol over cortisone.
Highlights
Ligand binding is the first step in hormone regulation of mineralocorticoid receptor (MR) activity
MR Interactions with Potent Agonists—Previous work on the steroid receptors progesterone receptors (PR), androgen receptor (AR), and glucocorticoid receptor (GR) has demonstrated that receptor expression in Escherichia coli can be accomplished in the presence of high affinity ligands (9, 10, 26 –28)
The MR ligand binding domain (LBD) shows the three-layered ␣-helical fold observed in other nuclear receptor LBDs, with aldosterone bound in a fully enclosed pocket contacting residues in helices 3, 4, 5, 6, 7, and 11, and the -turn (Fig. 3A)
Summary
Ligand binding is the first step in hormone regulation of mineralocorticoid receptor (MR) activity. We report multiple crystal structures of MR (NR3C2) bound to both agonist and antagonists These structures combined with mutagenesis studies reveal that maximal receptor activation involves an intricate ligand-mediated hydrogen bond network with Asn770 which serves dual roles: stabilization of the loop preceding the C-terminal activation function-2 helix and direct contact with the hormone ligand. Structural characterization of the naturally occurring S810L mutant explains how stabilization of a helix 3/helix 5 interaction can circumvent the requirement for this hydrogen bond network. Taken together, these results explain the potency of MR activation by aldosterone, the weak activation induced by progesterone and the antihypertensive agent spironolactone, and the binding selectivity of cortisol over cortisone. Understanding the ligand requirements that lead to the proper positioning of the AF-2 and activation of MR is fundamental to designing drugs that can modulate receptor activation
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