Crucial role of the H11-H12 loop in stabilizing the active conformation of the human mineralocorticoid receptor.
The crystal structures of ligand-free and agonist-associated ligand-binding domain (LBD) of nuclear receptors (NRs) reveal that the amphipathic helix H12 is folded back toward the LBD core in the agonist-associated conformation, allowing the binding of coactivators. We used alanine scanning mutagenesis to explore the role of the residues of the loop connecting H11 and H12 in the activation of the human mineralocorticoid receptor (hMR), a member of the NRs family. H950A retained the ligand binding and transcriptional activities of the wild-type receptor and interacted with coactivators. In contrast F956A had no receptor functions. Aldosterone bound to the mutant hMRs (L952A, K953A, V954A, E955A, P957A) with nearly the same affinity as to the wild-type receptor and caused a receptor conformational change in these mutant hMRs as it does for the wild-type receptor. But the aldosterone-induced transcriptional activity of the mutant hMRs was lower (L952A, E955A, P957A) than that of the wild-type receptor or completely abolished (K953A, V954A) and their interaction with coactivators was impaired (E955A) or suppressed (L952A, K953A, V954A, P957A). In the light of a hMR-LBD model based on the structure of the progesterone-associated receptor-LBD, we propose that the integrity of the H11-H12 loop is crucial for folding the receptor into a ligand-binding competent state and for establishing the network of contacts that stabilize the active receptor conformation.
Hellal-Levy
C
C
Faculté de médecine Xavier Bichat, Institut fédératif de recherche 02, Paris, France.
Fagart
J
J
Souque
A
A
Wurtz
J M
JM
Moras
D
D
Rafestin-Oblin
M E
ME
D048868:Adaptor Proteins, Signal Transducing; D000450:Aldosterone / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D051548:Histone Acetyltransferases; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D056920:Nuclear Receptor Coactivator 1; D000078723:Nuclear Receptor Interacting Protein 1; D011374:Progesterone / Q000494:pharmacology; D011487:Protein Conformation; D018161:Receptors, Mineralocorticoid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013256:Steroids / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D018841:HSP90 Heat-Shock Proteins; D009687:Nuclear Proteins; D000078723:Nuclear Receptor Interacting Protein 1; D018161:Receptors, Mineralocorticoid; D013256:Steroids; D015534:Trans-Activators; D014157:Transcription Factors; C093517:transcriptional intermediary factor 1; D000450:Aldosterone; D011374:Progesterone; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1