The multimerization pathway of the glucocorticoid receptor

The glucocorticoid receptor (GR/NR3C1), a ligand-activated transcription factor belonging to the nuclear receptor (NR) superfamily, is vital for life. Like other NRs, GR features an intrinsically disordered N-terminal domain (NTD) followed by a highly conserved DNA-binding domain (DBD) and a hormone- or ligand-binding domain (LBD), separated by a long linker. While the receptor's quaternary structure is critical for its transcriptional activity, its precise multimeric organization has remained elusive. Here, we conducted a comprehensive crosslinking-mass spectrometry (XL-MS) analysis using cleavable crosslinkers (DSSO and DSBU) to investigate the quaternary structure of the GR DBD-linker-LBD tandem. Our results strongly support a model of non-canonical receptor multimerization, revealing that GR monomers arrange in a parallel fashion through interactions involving residues within the LBD's L1-3 loop (evidenced by XLs K576-K576’ and K579-K579’). These parallel, non-canonical dimers then further associate to form dimers-of-dimers, mediated by contacts within the H9/L9-10/H10 region (e.g., K695-K695’). This study provides unambiguous evidence for a novel GR multimerization mechanism and has important implications for the design of glucocorticoids with fewer side effects.