Structural and functional determination of peptide versus small molecule ligand binding at the apelin receptor

We describe a structural and functional study of the G protein-coupled apelin receptor, which binds two endogenous peptide ligands, apelin and Elabela/Toddler (ELA), to regulate cardiovascular development and function. Characterisation of naturally occurring apelin receptor variants from the UK Genomics England 100,000 Genomes Project, and AlphaFold2 modelling, identifies T892.64 as important in the ELA binding site, and R1684.64 as forming extensive interactions with the C-termini of both peptides. Base editing to introduce an R/H1684.64 variant into human stem cell-derived cardiomyocytes demonstrates that this residue is critical for receptor binding and function. Additionally, we present an apelin receptor crystal structure bound to the G protein-biased, small molecule agonist, CMF-019, which reveals a deeper binding mode versus the endogenous peptides at lipophilic pockets between transmembrane helices associated with GPCR activation. Overall, the data provide proof-of-principle for using genetic variation to identify key sites regulating receptor-ligand engagement.