Relaxin is a peptide hormone originally discovered for its role in pregnancy but has since been shown to have diverse physiological actions, including cardio protection, increased lung perfusion, gas exchange and renal blood flow. Therapeutically, relaxin recently showed positive effects when used as treatment of acute heart failure. Relaxin’s actions are mediated by its receptor, RXFP1, an atypical class A G protein-coupled receptor (GPCR), possessing a unique extracellular domain (ectodomain). Relaxin binds to the ectodomain, triggering a series of complex conformational events that lead to RXFP1 activation. The molecular mechanics of this are not well understood as we do not have an atomic-resolution structure of relaxin bound to RXFP1. Structures of both a relaxin and non-relaxin bound RXFP1 will allow us to understand the unique activation mechanism of RXFP1, facilitating rational drug design. Structural studies of RXFP1, like all GPCRs, are difficult because of low receptor expression in recombinant systems, and low stability of the protein when purified in detergents.
Using a multifaceted approach incorporating protein engineering and novel purifications regimes, we hope to overcome the difficulties associated with RXPF1 structural biology.
Here we engineered RXFP1 for cryo-electron microscopy (EM). Cryo-EM requires the sample to be homogenous and detergent solubilized, however, WT RXFP1 is unstable in detergent. This was overcome by replacing a flexible region of RXFP1, intracellular loop 3, with a novel fusion partner, monomeric ultra-stable GFP (muGFP) to improve RXFP1’s detergent stability. Furthermore, muGFP will aid in the orientation of the RXFP1-muGFP complex in cryo-EM. RXFP1-muGFP was shown to have similar expression levels and binding when compared to WT RXFP1. However, RXFP1-muGFP was unable to activate and trigger downstream signaling pathways.
We were able to purify RXFP1-muGFP in detergent using FLAG affinity purification and size exclusion chromatography, though yield was too low for structural studies. To overcome this, we developed a novel form of directed evolution in mammalian cells using lentivirus to evolve RXFP1 mutants which highly express. Introducing these mutations into RXFP1-muGFP will improve purification yield and facilitate structure determination using cryo-EM.