Molecular Evolution of the Switch for Progesterone and Spironolactone from Mineralocorticoid Receptor Agonist to Antagonist
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Molecular evolution of the switch for progesterone and spironolactone from mineralocorticoid receptor agonist to antagonist Peter J. Fullera,b,1, Yi-Zhou Yaoa,b, Ruitao Jinc, Sitong Hec, Beatriz Martín-Fernándeza,b,2, Morag J. Younga,b, and Brian J. Smithc aCentre for Endocrinology and Metabolism, Hudson Institute of Medical Research, Clayton, VIC 3168, Australia; bDepartment of Molecular Translational Science, Monash University, Clayton, VIC 3168, Australia; and cLa Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia Edited by Huda Akil, University of Michigan, Ann Arbor, MI, and approved July 29, 2019 (received for review February 25, 2019) The mineralocorticoid receptor (MR) is highly conserved across In the present study, we demonstrate that a single residue change vertebrate evolution. In terrestrial vertebrates, the MR mediates is responsible for the switch from an agonist response to progesterone sodium homeostasis by aldosterone and also acts as a receptor for for zebrafish MR (zMR) to antagonist for human MR (hMR). This cortisol. Although the MR is present in fish, they lack aldosterone. change in the MR ligand-binding domain (LBD) is surprisingly dis- The MR binds progesterone and spironolactone as antagonists in tant from the ligand-binding site and has not previously been asso- human MR but as agonists in zebrafish MR. We have defined the ciated with agonist to antagonist switching in the MR. molecular basis of these divergent responses using MR chimeras between the zebrafish and human MR coupled with reciprocal Results site-directed mutagenesis and molecular dynamic (MD) simulation LBD Mediates the Agonist Response of the Zebrafish MR to based on the crystal structures of the MR ligand-binding domain. Spironolactone. Previous studies using full-length MR in trans- Substitution of a leucine by threonine in helix 8 of the ligand-binding activation assays demonstrated that spironolactone and progesterone domain of the zebrafish MR confers the antagonist response. This were agonists for fish MR (2, 5, 6); to formally demonstrate that the leucine is conserved across fish species, whereas threonine (serine in difference is a property of the LBD, the zMR LBD, amino acids 670 rodents) is conserved in terrestrial vertebrate MR. MD identified an to 970, was substituted for the equivalent region (amino acids 672 to interaction of the leucine in helix 8 with a highly conserved leucine in 984) in hMR (Fig. 1A). This initial chimera (zMR LBD) demon- helix 1 that stabilizes the agonist conformation including the inter- B action between helices 3 and 5, an interaction which has previously strated the same robust agonist response to spironolactone (Fig. 1 ) been characterized. This switch in the MR coincides with the evolu- as for full-length zMR (6). Antagonism of the aldosterone re- tion of terrestrial vertebrates and of aldosterone synthesis. It was sponse by spironolactone at the hMR demonstrates that there is perhaps mandatory if the appearance of aldosterone as a specific a loss of receptor activation by spironolactone, not of ligand mediator of the homeostatic salt retention was to be tolerated. The binding. The response of hMR to spironolactone alone is 4% conformational changes also provide insights into the structural basis that of aldosterone, while for the zMR LBD chimera it is 46% of agonism versus antagonism in steroid receptors with potential (Fig. 1B). implications for drug design in this important therapeutic target. Significance aldosterone | cortisol | mineralocorticoid receptor | sodium homeostasis | progesterone The mineralocorticoid receptor (MR), the receptor for aldoste- rone, appears in evolution well before the appearance of ter- he mineralocorticoid receptor (MR) is a member of the restrial vertebrates, yet aldosterone emerges in vertebrates Tnuclear receptor superfamily of ligand-dependent transcrip- only with terrestrial life. Curiously, in fish, the MR sees pro- tion factors; in vertebrates, it diverged with the glucocorticoid gesterone and spironolactone as agonists, whereas in terres- receptor (GR) by a gene duplication from the ancestral corticoid trial species, they are antagonist at the MR. We have identified > receptor 450 million years ago (1). MR has been highly con- a unique single amino acid difference between the fish MR and served across vertebrate evolution (2). In terrestrial vertebrates, the other vertebrate MR that mediates this switch, agonist to including humans, MR mediates the regulation of sodium homeo- antagonist. This striking evolutionary event was perhaps stasis by aldosterone. The MR also binds cortisol and the less potent mandatory if the appearance of aldosterone as a specific me- deoxycorticosterone (DOC), which is 21-hydroxyprogesterone. In diator of the homeostatic salt retention required for terrestrial most tetrapods (including human, rodent, alligator, and Xenopus), life was to be tolerated. The conformational changes also progesterone is an antagonist of MR, as is its derivative provide insights into the structural basis of agonism versus spironolactone (3). Although the MR is present in teleosts that lack antagonism in steroid receptors. aldosterone synthesis (4), the teleost MR responds to aldosterone, as well as to cortisol and DOC (2, 5, 6) that have been proposed as Author contributions: P.J.F. and B.J.S. designed research; Y.-Z.Y., R.J., S.H., and B.M.-F. the “physiological” mineralocorticoids for the fish MR (5, 6). performed research; P.J.F., Y.-Z.Y., R.J., S.H., M.J.Y., and B.J.S. analyzed data; P.J.F. and Characterization of various fish MR found that, in contrast to ter- B.J.S. wrote the paper; and P.J.F. led the team. restrial animals, teleost MR were activated by both progesterone The authors declare no conflict of interest. and spironolactone. These findings suggest that progesterone may This article is a PNAS Direct Submission. be a physiological agonist for the MR in teleosts. The evolutionary Published under the PNAS license. adaptability of proteins is brought into sharp focus by divergent 1To whom correspondence may be addressed. Email: [email protected]. responses to the same ligands (progesterone and spironolactone) in 2Present address: Department of Molecular Biology (Cell Biology), Faculty of Biology, such closely related receptors, while responses to other ligands are Universidad de León, 24071 León, Spain. essentially equivalent. Understanding the structural basis of agonism This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. versus antagonism in steroid receptors is also of considerable ther- 1073/pnas.1903172116/-/DCSupplemental. apeutic importance (7). Published online August 22, 2019. 18578–18583 | PNAS | September 10, 2019 | vol. 116 | no. 37 www.pnas.org/cgi/doi/10.1073/pnas.1903172116 Downloaded by guest on September 29, 2021 N-terminal DBD LBD y A B ti 1.4 1 603 669 732 984 vitcAe 1.2 hMR 1 1 s 601 667 718 970 arefic 0.8 37% 97% 77% zMR 0.6 uLev LBD Chimeras 0.4 ** 672 785 845 880 933 984 ital hMR 0.2 hMR zMR e ** 670 771 831 866 970919 R 0 VASA+SVASA+S C Z Z ZZ y t HMR zMRLBD i 1.4 vit SvsA% 4±0.34 46±13 c 1.2 Ae 1 s * aref 0.8 * E i ** ** c 0.6 1.6 u y tiv L 0.4 ** 1.4 itcAes ev i taleR 0.2 1.2 ** ** 0 1 a VA SA+S VA SA+SVASA+SVA SA+S r ef i 0.8 HHHH ZZZZ HZHH ZHZZ c uL 0.6 SvsA% 4±0.3 53±6.6 58±6.7 6±0.4 ev * i 0.4 ta * ytivitcAesareficuLevi D 1.6 leR 0.2 ** ** ** * 1.4 0 1.2 VASA+SVASA+SVASA+SVASA+SVASA+SVASA+S 1 * hMR hMR-T870L zMRLBD zMRLBD- zMR zMR-L856T 0.8 ** L856T 0.6 ** 0.4 SvsA% 5±3.1 55±26.3 80±42.6 5±8.6 95±75.7 6±5.3 ** MEDICAL SCIENCES t 0.2 ** aleR ** ** ** ** 0 VASA+SVASA+SVA SA+SVA SA+SVA SA+SVA SA+S HHHH ZZZZ HhzHH HzhHH ZhzZZ ZzhZZ SvsA% 4±0.4 84±4.1 51±2.3 3±0.9 44±14.1 3±0.3 Fig. 1. Identification of the critical amino acid switch that determines agonism versus antagonism for spironolactone in the MR. (A) Schematic representation of the comparison of the hMR and zMR. The MR is divided into three principal domains: the N-terminal domain, the DNA-binding domain (DBD), and the ligand-binding domain (LBD). The amino acid numbers and the percentage amino acid identity are shown (6, 24). The architecture of the LBD chimeras is shown below with the amino acid numbers of the breakpoints shown above (hMR) and below (zMR). (B) Transactivation responses of hMR and a chimera of hMR N terminus and DBD with the zMR LBD (zMRLBD) to aldosterone and spironolactone. CV-1 cells were transiently transfected with 250 ng of pRShMR or pRSzMRLBD together with 250 ng of MMTV-LUC reporter gene and 50 ng of pREN-LUC. The cells were treated with vehicle (V), 10 nM aldosterone (A), 1 μM spironolactone (S), or aldosterone plus spironolactone (A+S). Corrected luciferase activity is expressed relative to the response of the hMR to 10 nM aldosterone (mean ± SEM) derived from two in- dependent experiments with treatment groups aldosterone and aldosterone plus spironolactone being greater than vehicle alone (P < 0.05). Spironolactone and spironolactone plus aldosterone were less than aldosterone alone for hMR (**P < 0.0001) but not for zMR LBD. The activation with spironolactone alone as a percentage of that for aldosterone (S vs. A) for each MR is shown below the graph and significantly differs between the MR (P < 0.05). (C) Transactivation responses of hMR:zMR LBD chimeras to aldosterone and spironolactone highlighting the critical role of the second region in reciprocal chimeras.