Relaxin, a 6-kDa peptide, is an important regulatory hormone having vital physiological roles in inflammatory and cardiovascular functions, amongst others. The best characterized receptor is RXFP1, however, relaxin has been reported to directly bind to and activate the human glucocorticoid receptor (GR). The interaction takes place via its ligand-binding domain (GR-LBD) to regulate the transcription of several inflammatory genes such as cytokines IL-6 and TNF-α, as well as the relaxin gene itself. This association has therapeutic implications as relaxin has been shown to protect liver transplants from ischemia damage via hepatocyte GR [1]. The focus of this work is to characterize this mechanism of relaxin binding by systematic usage of molecular and solution-based structural biology techniques.
The initial challenge has been to obtain the receptor recombinantly in workable yields as wild type GR-LBD expresses poorly and has weak solubility [2]. We developed the GR-LBD “super-receptor” which gave multiple fold higher protein expression in bacterial system in the presence of steroid agonists and in an optimised expression medium.
We found the effect of relaxin binding on thermal stability of GR-LBD to be stabilizing, like the steroid agonist dexamethasone. Fluorescence based competition assays were performed to show two different affinities of relaxin for GR-LBD (5 nM and 500 nM) [3]. Next, we have used hydrogen-deuterium exchange Mass Spectrometry (HDX-MS) to identify the residues involved in GR-relaxin interaction and associated conformational changes. A combination of solution NMR techniques like 19F NMR and SOFAST HMQC have been used to locate the binding site of relaxin, study the ligand-induced changes of the LBD on relaxin binding and to determine whether it acts as a receptor agonist or antagonist. With this work, we have been able to explore a unique and previously unknown binding mechanism of H2 relaxin.