Protein Expression And Purification Of Angiotensin Receptor In Complex With Peptide Ligands

Angiotensin II type 1 receptor（AT₁R）is a member of the class A G Protein-Coupled Receptors family（GPCRs）.AT₁R expresses in many tissues and mediates most of the physiological effects of angiotensin II in human body,playing an important role in the Renin-Angiotensin System（RAS）.It regulates the blood pressure and maintains the body fluid homeostasis.AT₁R is an important drug target for heart failure and hypertension.Clinically used angiotensin receptor blockers（ARBs）could lower the blood pressure by inhibiting AT₁R.Recent studies suggested that TRV027,a biased agonist of AT₁R,could block the G protein signaling pathway and play a role in vasodilation and lower the blood pressure.Additionally,it could also selectively activate theβ-arrestin signaling pathway for the cardiomyocyte antiapoptosis and the myocardial contractility enhancement.The GPCR biased ligands have shown great potentials for precise targeted drugs research.However,the molecular mechanisms of GPCR biased agonism are still elusive,which is expected to be elucidated by the structural studies on the GPCRs in complex with biased ligands.Besides monomers,AT₁R could also form heterodimers with the other subtype of angiotensin receptor AT₂R to regulate different physiological functions,which could be used as potential drug targets.Therefore,it is important to elucidate the molecular mechanism of GPCRs dimerization.The crystal structures of AT₁R have been determined with antagonist ZD7155and inverse agonist Olmesartan in inactive conformations,and with peptide ligands S1I8,AngⅡ,TRV023,TRV026 in active conformations.These crystal structures have revealed the conformational differences between the active and inactive AT₁R,and the characteristics of active AT₁R.However,it is still difficult to elucidate the molecular mechanism of AT₁R biased agonism from the current structural information.On one hand,the intracellular binding Nanobody used for structure determination might constrain the conformational changes in the intracellular side of AT₁R during activation,resulting in the intracellular conformations of the four active AT₁R crystal structures are identical.On the other hand,the crystal structures of AT₁R in complex with Gq biased agonists need to be further determined for better understanding of the AT₁R biased agonism.This thesis aims to elucidate the molecular mechanism of AT₁R biased agonism by the structural biology studies on the AT₁R and peptide ligands complexes.The major research methods and results in this thesis are listed as below:（1）Protein engineering for AT₁R:the wild-type AT₁R has low expression and poor stability.Using fusion protein insertions,site-directed mutations and protein terminal truncations,the AT₁R protein yeilds and stability were improved;（2）Protein expression and purification for AT₁R:Sf9 insect cells were used to get high yields of the AT₁R protein with better protein folding and post-translational modification.The AT₁R protein was purified in the detergent micelles to get good protein purity;（3）The monodispersity and thermostability of the AT₁R protein with eight peptide ligands,including unbiased,G protein-biased,andβ-arrestin-biased ligands,were analysized by analytic size exclusion chromatography and thermostability assays;（4）Crystallization trials were performed with the AT₁R proteins in complex with peptide ligands.Additionally,Gq protein fragments（GqαCT）andβ-arrestin protein fragments（Arr FL）were added in the crystallization trials to further stabilize the protein conformation of the active AT₁R.（5）The AT₁R and AT₂R proteins were coexpressed in the insect cells and were found to form heterodimers using His-pulldown assays.The proteins of AT₁R-AT₂R heterodimers were purified using MBP-pulldown assays.In this thesis,the AT₁R proteins were expressed and purified with eight peptide ligands for structure determination.Moreover,the AT₁R-AT₂R heterodimers were identified in the insect cell membranes using the His-pulldown assays,and purified using the MBP-pulldown assays.The results have thus paved the way to reveal the molecular mechanism and the structure-function relationship of the AT₁R-AT₂R heterodimer by the structural biology methods.