Structural Insights Into Ligand Efficacy And G-Protein Coupling Selectivity Of β2 Adrenergic Receptor

G protein-coupled receptors(GPCRs)are an important class of cell membrane receptors in the human body which can recognize a variety of extracellular physical and chemical signals and transmit the signals into cells by coupling intracellular signal transduction proteins.β2 adrenergic receptor(β2AR)is a typical GPCR which mainly recognizes adrenergic ligands in the human body and couples intracellular G proteins to regulate cell activities.Previous studies have shown that β2AR is widely distributed in the smooth muscle of the lung,myocardium and other tissues.The beta-agonists can activate β2AR on the smooth muscle of bronchia and relax the airway,so they can be used as drugs for the treatment of asthma and chronic obstructive pulmonary disease.Agonists with different efficacy have different pharmacological effects on β2AR.β2AR has a weaker response upon partial agonists than full agonists at the maximum ligand concentration.In addition,full agonists of β2AR can induce more desensitization than partial agonists.However,the structural mechanism of the efficacy of β2AR agonists remains obscure.While GPCRs can couple to different types of G proteins,β2AR could mainly couple to intracellular Gs protein to transduce signals and can also couple to Gi or Gq proteins to induce different signal transduction.The study on the G protein coupling selectivity of β2AR could help to explain the activation mechanism of the important receptor and provides a structural basis for drug development.Here we took the advantage of cryo-electron microscopy combined with cell experiments to study the mechanism of β2AR agonists with different efficacy and the G protein coupling selectivity during activation.The first part of this dissertation is the study on β2AR agonists with different efficacy.We solved the three-dimensional structures of two β2AR-Gs protein complex at an overall resolution of 3.58 A(bound with the full agonist isoproterenol)and 3.33?(bound with the partial agonist salbutamol)respectively.Isoproterenol forms stronger hydrogen bond network and hydrophobic interaction with β2AR compared with salbutamol.Conformational changes upon ligand binding are transduced to the cytoplasmic side of the β2AR receptor through multiple molecular switches.Structural analysis and cell experiments suggest that the stronger interactions of isoproterenol with β2AR make the receptor in a more activated state,and thus couple with Gs protein more strongly.We think this might be the structural basis for isoproterenol as a full agonist of β2AR.The binding interface between β2AR and Gs protein is relatively large,which can be divided into receptor core of β2AR-Gs protein interface and intracellular loops of β2AR-Gs protein interface.We found that β2AR was more closely bound to Gs protein under the action of partial agonist salbutamol,which may hamper the phosphorylation of ICL3 of β2AR，resulting in a weaker desensitization effect of β2AR.In the second part of this dissertation,we solved the structures of the β2AR-Gi andβ2AR-Gq protein complexes bound with isoproterenol using the optimized complex assembly method.The binding mode of Gi protein or Gq protein to β2AR is quite different from that of Gs protein.We observed the rotation of Gi/Gq protein towards the TM5 helix of β2AR with the a5-helix of Ga protein as the axis.This rotation leads to the lack of interaction between the ICL3 of β2AR and Gi/Gq.The binding mode between Gi or Gq protein with ICL2 of β2AR is similar but has a remarkable angular difference from the binding position of Gs protein.The diversity of interactions bewteen ICL2 of β2AR and G protein may be the reason for the difference in the binding mode of G protein with β2AR.The α5-helix of G protein plays an important role in the interaction of G protein and β2AR.We found that there was a weaker polar interaction between the a5-helix of Gi or Gq protein with β2AR due to their carboxy-terminal sequence diversity with Gs protein.This may be the structural basis for the stronger coupling of β2AR with Gs than Gi or Gq proteins.This dissertation elucidates the structural basis of β2AR agonists with different efficacy and the G protein selectivity mechanism of β2AR,which deepens our understanding of the activation process of β2AR and contributes to drug development targeting β2AR.