Structural basis of allosteric and bitopic ligands binding in sphingosine-1-phosphate receptors 2 and 3

Allosteric ligands of G protein-coupled receptors are believed to be superior to typical orthosteric ligands as they generally provide better subtype selectivity, spatiotemporal sensitivity and potentially biased signaling properties However, the discovery process of allosteric ligands is very challenging, partially due to the lack of structural information. Here, we report two crystal structures of human sphingosine-1-phosphate receptor 3 (S1P3) in complex with one bitopic ligand SPM-242 as well as allosteric ligands Cpd-32 or CYM52581 bound simultaneously. To further reveal the inhibition mechanism of antagonists and ligand subtype selectivity, two cryo-electron microscopy structures of S1P2 and S1P3 in complex with heterotrimeric Gi protein were solved. The S1P3 complexes (S1P3–SPM-242–Cpd-32 and S1P3–SPM-242–CYM52581) reveal an allosteric site that lies outside of the helical bundle in S1P3 receptor, which is a new site recognized by allosteric modulators in class A GPCR. Structural comparison further explains the selectivity of bitopic antagonist SPM-242 and allosteric antagonists CYM52581 on S1P3 over S1P1. These structural studies, together with functional assays, provide structural insights into the bitopic and allosteric antagonism of class A GPCRs, which will further facilitate the design of selective drugs targeting these receptors.