Abstract
Inverse agonists of the constitutively active human estrogen-related receptor alpha (ERRalpha, NR3B1) are of potential interest for several disease indications (e.g. breast cancer, metabolic diseases, or osteoporosis). ERRalpha is constitutively active, because its ligand binding pocket (LBP) is practically filled with side chains (in particular with Phe(328), which is replaced by Ala in ERRbeta and ERRgamma). We present here the crystal structure of the ligand binding domain of ERRalpha (containing the mutation C325S) in complex with the inverse agonist cyclohexylmethyl-(1-p-tolyl-1H-indol-3-ylmethyl)-amine (compound 1a), to a resolution of 2.3A(.) The structure reveals the dramatic multiple conformational changes in the LBP, which create the necessary space for the ligand. As a consequence of the new side chain conformation of Phe(328) (on helix H3), Phe(510)(H12) has to move away, and thus the activation helix H12 is displaced from its agonist position. This is a novel mechanism of H12 inactivation, different from ERRgamma, estrogen receptor (ER) alpha, and ERbeta. H12 binds (with a surprising binding mode) in the coactivator groove of its ligand binding domain, at a similar place as a coactivator peptide. This is in contrast to ERRgamma but resembles the situation for ERalpha (raloxifene or 4-hydroxytamoxifen complexes). Our results explain the novel molecular mechanism of an inverse agonist for ERRalpha and provide the basis for rational drug design to obtain isotype-specific inverse agonists of this potential new drug target. Despite a practically filled LBP, the finding that a suitable ligand can induce an opening of the cavity also has broad implications for other orphan nuclear hormone receptors (e.g. the NGFI-B subfamily).
Highlights
Developmental and physiological pathways [1]
The asymmetric unit contains a homodimer of ERR␣ complexes, i.e. ligand binding did not interfere with dimer formation
We report the first x-ray structure of ERR␣ ligand binding domain (LBD) in complex with an inverse agonist, cyclohexylmethyl(1-p-tolyl-1H-indol-3-ylmethyl)-amine, to a resolution of 2.3 Å
Summary
Developmental and physiological pathways [1]. the transcriptional activity of NRs is often regulated by specific ligands, several members of the superfamily have no known natural ligands and are referred to as orphan NRs [2]. It has been shown that ERR␣ efficiently binds to estrogen-response elements and that these receptors share common target genes [4] This observation was further supported by studies demonstrating cross-talk between the ER and ERR pathways (reviewed in Ref. 5). The importance of ERR␣ as a drug target has been recently reviewed [12], which further re-emphasizes the urge for new synthetic ligands in the ERR subfamily Despite their overall sequence similarity with the ERs, ERRs seem to regulate transcription in the absence of known natural agonist ligands. The ERR␥ structures are in contrast to selective ER modulator (SERM) complexes for ER␣, where binding of raloxifene or 4-hydroxytamoxifen revealed H12 in the coactivator groove. For ER LBD complexed with the full antagonist ICI 164,384 (ICI), H12 was not visible because of high mobility [24]
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