Structural Studies On Angiotensin Ⅱ Receptor Biased Signaling Modulation

AngiotensinⅡreceptors,members of class A G protein-coupled receptor（GPCR）superfamily,are key components of Renin-Angiotensin-Aldosterone System（RAAS）.AngiotensinⅡmainly binds with angiotensinⅡtype 1 receptor（AT₁R）to mediates vasoconstriction,aldosterone release,cell differentiation and other biological functions.In human body,the Gq pathway of AT1R is responsible for the vasoconstriction and hypertension,while the GRK/β-arrestin pathway would increase the cardiovascular contractility and performance,suggesting biased agonists towards the GRK/β-arrestin pathway of AT1R may potentially be developed into clinical drugs to treat congestive heart-failure and relevant diseases.Structures of AT1R bound with small-molecular antagonists presented the binding pockets of sartan drugs.Crystal structures of AT1R in complex with different peptide agonists elucidated the structural characteristics of the partial-active state of the receptor.Spectroscopic and computational studies of AT1R indicated that different peptide agonists would induce diverse conformations of the receptor,resulting preferences towards Gq or GRK/β-arrestin signaling pathway.Nevertheless,the high-resolution structures of AT1R coupled to the signaling proteins haven’t been determined yet,hindering us from understanding the structural determinants for the biased signaling properties and designing biased modulation molecules rationally.This study mainly focuses on the Gq signaling pathway of AT1R.Through molecular cloning,protein expression and purification optimization,single-chain antibody assembly and nanodics assembly,we assembled the AT₁R-G_q-Sar¹-AngⅡcomplex in vitro and prepared the cryogenic electron microscopy（cryo-EM）sample of AT1R-Gq-Sar¹-AngⅡcomplex in high integrity and homogeneity.Using single-particle cryo-EM,the 2.9（?）AT1R-Gq-Sar¹-AngⅡstructure was determined for the first time.In this structure,the Sar¹-AngⅡwas observed to adopt an extensive conformation to interact with the receptor.Two of the four polar interactions in Sar¹-AngⅡbinding pocket,R2-D263^6.58 and R2-D281^7.32,are dismissed in the G_q-biased agonists binding pockets revealed by the molecular dynamics simulations and bioluminescence resonance energy transfer（BRET）studies.Besides,the heterotrimeric Gq coupling further rearranges the conformation of the receptor to achieve a full-active state.Moreover,the major hydrogen bond networks（MHN）,identified and concluded from the AT1R structures in different states,are believed to be the main maintainer of receptor conformations and mutations in this region could confer biased signaling.Finally,we proposed a signaling model of AT1R to illustrate its biased signaling pathway.Integrating the structure of AT1R-Gq complex with extensive computational and functional information,this work explores the binding characteristics of Gq biased agonists TRV055/TRV056,explains the structural basis of the biased signaling property for N111^3.35A and N294^7.45A mutants,and proposes the biased signaling model of AT₁R to shed light on the structure-based biased molecule development.