Structural Basis Of Sphingosine-1-phosphate Receptor 1 Activation And Biased Agonism

Sphingosine 1-phosphate（S1P）,a metabolic product of cell membrane sphingolipids,is generally generated in red blood cells,endothelial cells and platelets.It can regulate inflammation,cell migration,angiogenesis,neuronal and cardiac development and other physiological processes.Dysregulation of S1P signaling is associated with autoimmune diseases,cardiovascular diseases,organ fibrosis,neurodegenerative diseases,and cancer.S1P binds to S1P receptors（S1PRs,S1PR1-5）on cell membranes and activates intracellular signaling pathways,such as G protein signaling pathway andβ-arrestin signaling pathway,causing intracellular responses and involving in many critical cell processes.S1PRs belongs to the Class A G-protein-coupled receptor（GPCR）superfamily,in which S1PR1 is activated and coupled with G_i.S1P-S1PR1 signaling induces lymphocyte migration from secondary lymphoid organs into the lymphatic and blood circulation,therefore S1PR1 is an important target for the treatment of multiple sclerosis（MS）.FDA-approved drug modulators Fingolimod（FTY720）and Siponimod（BAF312）binding to S1PR1,initially act as an agonist to induce G_i signaling pathway,but unlike the endogenous ligand S1P,they effectively associate with S1PR1 viaβ-arrestin.This leads to the continuous internalization and degradation of S1PR1 in lymphocytes,resulting in the inability of lymphocytes to migrate out of the lymph nodes,thereby alleviating inflammatory symptoms.In this study,we resolved cryo-electron microscopy（cryo-EM）structures of G_i-bound S1PR1 in complex with S1P,and two therapeutic modulators,FTY720-p and BAF312 with the resolution of 3.4?,3.3?and 3.1?,respectively.The structures show that the endogenous ligand S1P and the drug modulators FTY720-P and BAF312 are all bound in the orthotropic pocket of S1PR1,which is positioned in the upper part of the receptor core.Through structural analysis and mutagenesis study,we discovered that the three ligands use different strategies or dynamic processes binding to the receptor.S1P and FTY720-p use the same binding mode that the polarized or charged head being positioned in the upper pocket and the hydrophobic tail being swayed into the hydrophobic lower pocket,whereas BAF312 stabilizes interactions with receptors mainly through extensive hydrophobic interactions formed in the lower part of the binding pocket.In order to study the activation mechanism of S1PR1 under agonists,we compared the S1PR1 structures in different states,and found that the activated S1PR1 structure with three ligands have conformational changes compared to inactivated S1PR1 structure,such as up-lift movement of the N-terminal helix cap,TM6 outward displacement,and W269^6.48turnover,which indicates that the intracellular cavity of the receptor opens upon activation,allowing theα5-helix of Gα_ito embed into the intracellular cavity.In the ligand-binding pocket,the most notable difference is the position and the orientation of W269^6.48.In the S1P-bound pocket,the indole ring flips W269^6.48 about 70°toward TM5compared to the antagonist-bound pocket.Interestingly,the side chains of W269^6.48 in the FTY720-p and BAF312-bound pockets are intermediate between S1P and ML056,suggesting that W269^6.48 behaves as a sensor for ligand property.Structural superimposition shows that L276^6.55 and L297^7.39 of S1PR1 are the key sites that can be specifically bound by BAF312,while BAF312 clashes with F263^6.55 and I284^7.39 of S1PR3.Further in combination with functional assays and molecular dynamics simulation studies,we reveal that theβ-arrestin-biased ligands direct a distinct activation path in S1PR1 through the extensive interplay between the PIF and the NPxx Y motifs.Specifically,the intermediate flipping of W269^6.48 and the retained interaction between F265^6.44 and N307^7.49 are the key features of theβ-arrestin bias.Study on ligands shape revealed that shorter and wider agonist favors theβ-arrestin pathway,and longer and thinner agonist favors the G_i pathway.In summary,this study resolved the cryo-EM structures of S1PR1-G_i bound to therapeutic modulators and endogenous ligand,and revealed the active conformation of S1PR1 and the key mechanism of biased agonism.And further identified ligand–receptor interactions accounting for the S1PRs subtype specificity of BAF312.These structural insights provide a rational basis for designing novel signaling-biased S1PR modulators.