Structural Studies On The Bradykinin Receptor Transmembrane Signaling Complex As Drug Target

Bradykinin receptors are members of G protein-coupled receptor（GPCR）superfamily and play important roles in kallikrein-kinin system（KKS）.There are two subtypes B1R and B2R which bind kinins in the blood such as bradykinin,kallidin and so on.B2R is expressed ubiquitously in human tissues and mainly mediates downstream Gα_q signaling pathway,inducing the activation of phospholipase C（PLC）and calcium mobilization.The physiological effects of B2R are regulating blood circulation and renal functions,inducing inflammatory responses and pain.However,malfunction of B2R is closely related to the development of cardiovascular disease（CVD）,renal and respiratory diseases,cancers as well as hereditary angioedema（HAE）.B2R is a promising drug target in the treatment of inflammation,cardiovascular diseases and neuralgia.Though several drug candidates targeting B2R were in the clinical trials,the peptidic icatibant was the only one approved for sale,which could relieve the symptoms of HAE.Other candidates such as peptide agonist Labradimil used for cerebral tumor,peptide antagonist Deltibant used for pain and non-peptide antagonist Fasitibant used for osteoarthritis were failed in phaseⅡ/Ⅲclinical trials.Many difficulties hinder the drug discovery on B2R.Firstly,the traditional screening procedures are inefficient,costly and time-consuming.Secondly,important residues participating in ligand-binding and receptor activation could not be exactly identified in the absence of B2R structure.Thus,it’s necessary to solve the B2R structure,which could accelerate the discovery of novel drugs targeting B2R for the treatment of cardiovascular diseases,inflammation and neuralgia.The goal of this thesis is to solve the B2R structure by structural biology technologies,to reveal the molecular mechanisms from agonist binding,B2R activation,to G_q protein coupling,which could accelerate the structure-based drug discovery.To obtain high-quality protein for structural biology studies,screening of the N/C-terminal truncations,fusion partners,fusion sites and mutations were performed on B2R.Thermostability analysis results showed that antagonistic icatibant-bound B2R was ideal B2R-ligand complex for the crystallization trials in lipidic cubic phase（LCP）.Studies on B2R-G_q complex structure were performed simultaneously.To obtain stable complex,the sequences of B2R and Gα_q protein were engineered.Buffer optimization trials showed that LMNG/CHS could extract more B2R from membranes to reconstitute into detergent micelles than DDM/CHS.Besides,B2R in LMNG/CHS micelles was stable during cryo-EM grid preparation.Cryo-EM data collection was performed on an electron microscope operated at 300 k V.Finally,two cryo-EM structures,B2R-G_q-bradykinin structure at 3.0（?）resolution and B2R-G_q-kallidin structure at 2.9（?）resolution,were determined.From the solved structures,it is found that in the orthosteric ligand-binding pocket,agonists adopt S-shaped conformations.There are lots of aspartic acids and glutamates locating at the entrance of pocket,they could form electrostatic interactions with arginines and lysine in agonists to prevent agonist dissociation.In addition,two pairs of hydrogen bonds P3^B/P4^K-I213^ECL2 and G4^B/G5^K-R196^4.64 anchor agonists inside the ligand-binding pocket and increase the affinity between them.Phenylalanines at the tails of agonists trigger the conformational changes of several microswitches such as toggle switch,PIF motif,DRY motif and NPxx Y motif,which lead to the outward movement of TM6 at the cytoplasmic side as well as G_q coupling.G_q protein forms extensive interactions with TMs2-3,TMs5-7,ICLs2-3 and helix 8 of B2R,including hydrogen bonds,polar,electrostatic,and hydrophobic interactions,to stabilize the complex.These structures of B2R transmembrane signaling complex thus revealed the molecular mechanisms on agonist binding,B2R activation and B2R-G_q coupling,which could accelerate the progress of drug discovery targeting B2R with the structure-based drug design（SBDD）.