Relaxin family peptide receptor 1 (RXFP1) is a class A G protein-coupled receptor (GPCR) that is activated by the peptide hormone H2 relaxin. RXFP1 has garnered attention as a therapeutic target for the treatment of acute heart failure and fibrosis-related pathologies. However, the understanding of its unique relaxin-mediated activation mechanism has been lacking due to the challenges in obtaining sufficient quantities of receptor for the determination of high resolution RXFP1 structures & biophysical analysis that will inform rational drug development.

An expression-enhanced mutant of RXFP1 (RXFP1#35), which was developed by our lab using a mammalian cell-based directed evolution method, demonstrated a six-fold improved cell surface expression compared to wild-type while retaining signalling competency. Upon purification, the apo-state RXFP1#35 mutant demonstrated markedly improved protein yield compared to wild-type receptor thus enabling its utilization as a tool for downstream cryo-electron microscopy (cryo-EM) studies of active and inactive state RXFP1 structures. For the active state RXFP1 structure, we engineered a fusion construct, which fused a minimal Gαs protein (1)  to the C-terminus. We then developed a purification scheme to complex this protein with purified Gβ₁γ₂ subunit, H2 relaxin, and a nanobody, Nb35, that binds at the interface between the Gαs and Gβ₁ subunits and stabilises the active complex. Size exclusion chromatography and single-particle negative-stain electron microscopy two-dimensional averages revealed the successful formation and architecture of a stable relaxin: RXFP1-miniGαs:Gβ₁γ₂:Nb35 complex, that could be purified at a promising yield to proceed further with cryo-EM studies. RXFP1#35 was also engineered to enable inactive state structural investigation through the design of a novel fusion for complexing with an intracellular inactive-state stabilizing nanobody (2). Hence, we are utilizing the stabilized, high expressing RXFP1#35 construct as a tool towards determining active and inactive state relaxin-RXFP1 complexes to unravel its unique activation mechanism and facilitate new drug design.