Poster Presentation The 46th Lorne Conference on Protein Structure and Function 2021

A novel binding mechanism of H2 relaxin to the human Glucocorticoid Receptor (#311)

Shatabdi Chakraborty 1 2 , Thomas Dschietzig 3 , Ross Bathgate 1 4 , Paul Gooley 1 2
  1. Department of Biochemistry and Pharmacology, University of Melbourne, Parkville, VIC, Australia
  2. Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia
  3. Relaxera Pharmazeutische Gesellschaft mbH & Co. KG, Bensheim, Hesse, Germany
  4. Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC, Australia

Relaxin, a 6-kDa peptide, is an important regulatory hormone having vital physiological roles including in cardiovascular and renal functions, amongst others. The best characterized receptor is RXFP1, however, relaxin has been reported to directly bind to and activate the glucocorticoid receptor (GR), via its ligand-binding domain (GR-LBD) and to regulate the transcription of several genes, including cytokines IL-6 and TNF-α, as well as the relaxin gene itself. This association and activation by relaxin may have therapeutic implications as relaxin has been shown to protect liver transplants from ischemia damage via hepatocyte GR1. The focus of this work is to characterize the mechanism of relaxin binding by systematic and combined usage of molecular and structural biology techniques.

The initial challenge has been to obtain the receptor recombinantly in workable yields as GR-LBD expresses poorly even after introduction of F602S mutation2. We developed the GR-LBD “super-receptor” by additionally mutating Cys638 to Gly and the F602S/C638G double mutant gave much higher protein expression in the presence of the steroid, prednisolone, in an optimised media.

Current information on binding sites and ligand-induced conformational changes of GR-LBD are based on solved crystal structures of the receptor bound to steroidal agonist/antagonist. The effect of relaxin binding on thermal stability of GR-LBD was found to be stabilizing, similar to the steroid agonist, dexamethasone. Competition assays show two different affinities of relaxin for GR-LBD (5 nM and 500 nM)3 and we have used  hydrogen-deuterium exchange Mass Spectrometry to study the regions involved in GR-relaxin interaction. A combination of 19F NMR and ligand-based NMR techniques have been used to find the binding site of relaxin, study the dynamics in the LBD on relaxin binding and to examine its effect on binding of steroid ligands. With these tools, we plan to determine the unique mechanism of GR activation by relaxin.

  1. Kageyama S, et al. Recombinant relaxin protects liver transplants from ischemia damage by hepatocyte glucocorticoid receptor: From bench-to-bedside. Hepatology 68, 258-273 (2018).
  2. Bledsoe RK, et al. Crystal Structure of the Glucocorticoid Receptor Ligand Binding Domain Reveals a Novel Mode of Receptor Dimerization and Coactivator Recognition. Cell 110, 93-105 (2002).
  3. Dschietzig T, Bartsch C, Stangl V, Baumann G, Stangl K. Identification of the pregnancy hormone relaxin as glucocorticoid receptor agonist. FASEB, (2004).