Crucial role of the H11-H12 loop in stabilizing the active conformation ofthe 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 LED core in the agonist-associated conformat ion, allowing the binding of coactivators. We used alanine scanning mutagen esis 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 ac tivities of the wild-type receptor and interacted with coactivators. In con trast F956A had no receptor functions. Aldosterone bound to the mutant hMRs (L952A, K953A, V954A, E955A, P957A) with nearly the same affinity as to th e wild-type receptor and caused a receptor conformational change in these m utant hMRs as it does for the wild-type receptor. But the aldosterone-induc ed transcriptional activity of the mutant hMRs was lower (L952A, E955A, P95 7A) than that of the wild-type receptor or completely abolished (K953A, V95 4A) and their interaction with coactivators was impaired (E955A) or suppres sed (L952A, K953A, V954A, P957A). In the light of a hMR-LBD model based on the structure of the progesterone-associated receptor-LED, 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 contac ts that stabilize the active receptor conformation.