Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Structure and mechanism of H2 relaxin binding to its receptors (#119)

Shatabdi Chakraborty 1 2 , Thomas B Dschietzig 3 , Ross A D Bathgate 1 4 , Paul R 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 is a peptide hormone of the insulin superfamily with important physiological roles including collagen metabolism, anti-fibrotic actions and cardiovascular and renal functions. The primary relaxin receptor is RXFP1 and our group has been engaged in elucidating the unique mechanism of activation1. Unexpectedly relaxin has been reported to directly bind to and activate the glucocorticoid receptor (GR) to regulate the transcription of a number of genes, including its own expression2. It is therefore important to fully determine the atomic details of relaxin binding to RXFP1 as well as to the ligand-binding domain of the human glucocorticoid receptor (GR-LBD). As at high concentrations, typically used for biophysical studies, relaxin self-associates to form a dimer, two modified versions of human H2 relaxin- mini-relaxin (MR) and relaxin K2R have been developed which can be purified from a bacterial expression system as a monomer in high yield and shown to bind to and activate RXFP1 with the same activity as its native form. Most of the studies on GR-LBD have been hindered by the inability to obtain pure receptor recombinantly. By introducing F602S mutation, Bledsoe et al3 expressed GR-LBD in bacteria in the presence of high affinity ligands, however the yield was still poor. Hence, we developed the GR-LBD “super-receptor” by mutating Cys638 to Gly and the F602S/C638G double mutant gave much higher protein expression in presence of ligand prednisolone. We also found that both mini-relaxin and relaxin K2R can bind to the ligand bound receptor. However, it will be interesting to find out whether relaxin competes with the bound ligand and displaces it or binds to a different site on the receptor. With these molecular tools developed, we plan to determine the structures of relaxin bound to its receptors.

  1. Sethi A, et al. The complex binding mode of the peptide hormone H2 relaxin to its receptor RXFP1. Nat Commun 7, 1-12 (2016).
  2. Dschietzig T, Bartsch C, Baumann G, Stangl K. RXFP1-inactive relaxin activates human glucocorticoid receptor: further investigations into the relaxin-GR pathway. Regul Pept 154, 77-84 (2009).
  3. 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).