FRI162 Ligand-dependent Photocrosslinking of Mineralocorticoid Receptors Using Site-directed Incorporation of Unnatural Amino Acids

Abstract

Abstract Disclosure: B. Almeida-Prieto: None. A.J. Gonzalez-Hernandez: None. T. Giraldez: None. D. Alvarez de la Rosa: None. Steroid receptors dimerize prior DNA binding as a key step for translational activation of target genes. It appears that several domains of the receptors play a role in the process. In addition, current evidence suggests that the active conformation of steroid receptors may not always be a dimer, but involves different quaternary structures, characteristic for each receptor. Recent data obtained by our group suggests that agonist-bound mineralocorticoid receptors (MR) adopt a tetrameric form in the nucleoplasm, forming higher order oligomers when bound to hormone response elements in the chromatin. The formation of such complexes would likely requires more than one oligomerization interface. To generate a tool for investigating inter-subunit contacts, we incorporated photoactivatable crosslinkers into specific sites in MR using a genetically-encoded unnatural amino acid (p-Benzoyl-L-phenylalanine, BzF). We created a library of 26 amber stop codon mutants substituting conserved A, F, W and Y residues distributed along the N-terminal domain (NTD), DNA-binding domain (DBD) and ligand-binding domain (LBD) of the mouse MR coding sequence. Site-directed incorporation of BzF into MR was performed by co-expressing an orthogonal pair of BzF-specific aminoacyl-tRNA synthetase and an amber-suppressor tRNA with the mutant receptors. Characterization of the library showed that 18 mutants produced functional receptors after BzF incorporation. Using an in silico approach we identified position W914 in helix 10 of the LBD as a potential candidate for photocrosslinking. Treatment of cells expressing W914BzF with UV light selectively increased aldosterone-bound, but not cortisol-bound, MR transcriptional activity by approximately 50%. Increased activity was still apparent after hormone washout, suggesting that BzF-induced photocrosslinking at position 914 contributes to stabilizing an active conformation and identifying H10 as a putative region for ligand-specific oligomerization interface. Our results support the strong potential of this methodology for studying this new organization model proposed for MR and other steroid receptors, along with the characterization of functional protein-protein interactions. Presentation Date: Friday, June 16, 2023