A Ligand-mediated Hydrogen Bond Network Required for the Activation of the Mineralocorticoid Receptor

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 Asn 770 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 Thr 945 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.

Crystallization of protein–ligand complexes

“…In similar expression studies with MR, the equivalent residue, Cys808, was also mutated to serine (C808S), producing an even more dramatic increase in expression of soluble GST MR LBD in Escherichia coli (Bledsoe et al, 2005;Figs. 5 and 6).…”

“…The MR C808S mutant expressed well in the presence of potent compounds and its structure has been determined in complex with several ligands (Bledsoe et al, 2005).…”

A New Mode of Mineralocorticoid Receptor Antagonism by a Potent and Selective Nonsteroidal Molecule

“…Mode-The crystal structures of the LBD of MR and MR S810L complexed with steroidal agonists have been recently solved, allowing their anchoring mode to be studied in detail (28,30,35,36). The nonsteroidal nature of BR-4628 raised the question of its binding mode within the MR binding pocket.…”

30 YEARS OF THE MINERALOCORTICOID RECEPTOR: Evolution of the mineralocorticoid receptor: sequence, structure and function

“…In a second pathway, progesterone is hydroxylated at C17 and then hydroxylated at C21 to form 11-deoxycortisol, which is hydroxylated at C11 to form cortisol (Baker 2011, Rossier et al 2015. Fagart et al 2005, Edman et al 2015 and GR (Bledsoe et al 2002, He et al 2014 and functional studies of human MR mutants (Fagart et al 1998, Geller et al 2000, Bledsoe et al 2005, Shibata et al 2013, Jimenez-Canino et al 2016, Mani 2016 to investigate the evolution of selectivity for Aldo and other corticosteroids by the MR in terrestrial vertebrates (Baker et al 2013) and ray-finned fish (Prunet et al 2006, Bury & Sturm 2007, Arterbery et al 2011, Sugimoto et al 2016, as well as the basis for binding of some glucocorticoids by human MR and other vertebrate MRs , Mani et al 2016. Together, these data provide molecular and structural fingerprints for investigating the evolution of selectivity for 3-ketosteroids by the MR in vertebrates.…”

“…To gain a deeper understanding of the evolution of the MR, we have taken advantage of the sequencing, in the last five years, of a cornucopia of genomes from vertebrates at key evolutionary transitions, including lamprey, a jawless fish, elephant shark, a basal jawed fish and coelacanth, which, along with lungfish, belongs to the lobe-finned fish clade, forerunners of terrestrial vertebrates, to investigate regions of conservation and divergence among and between MRs and GRs. We use these sequence analyses, the crystal structures human MR (Bledsoe et al 2005, Figure 3 Pathway for the synthesis of aldosterone from progesterone and cortisol from 17α-OH-Progesterone. Progesterone is hydroxylated at C21 to form 11-deoxycorticosterone, which is hydroxylated at C11 to form corticosterone.…”