Cryo-electron Microscopy Structure And Functional Studies Of Leukotriene B4 Receptor 1

G-protein coupled receptors（GPCRs）are a family of membrane proteins with seven transmembrane helices which play important roles in signaling transduction.GPCRs are widely expressed in various tissues,and can recognize a variety of signaling molecules such as hormones,neurotransmitters,and cellular metabolites,and in turn activate a variety of intracellular signaling cascades by binding to downstream effector proteins.In human body,GPCR signalings are involved in almost every physiological and pathological events,and therefore GPCRs are the most well-studied drug targets at present time.In this study,we use leukotriene B4 receptor1（BLT1）as a model to understand leukotriene signaling,and investigate the structure and function relationship of ligand recognition and downstream G-protien coupling.Leukotriene B4（LTB4）is an important inflammatory factor of the arachidonic acid metabolic pathway,which acts on the BLT1 on the cell membrane surface and exerts its physiological functions by participating in intracellular inflammatory and immune responses.BLT1 is a drug target for anti-inflammatory,anti-allergic,and immune disorder diseases.However,there are limited structural studies of BLT1,especially,the structure of receptor in an activated state is absent.Therefore,we set to resolve the structure of active BLT1 in complex with G_i protein by cryoelectron microscopy（Cryo-EM）and further elucidate the activation mechanism by structural analysis of BLT1,which also provides a rational basis for the design and development of anti-inflammatory small molecule drug targeting BLT1.The insect cell baculovirus expression system was used to obtain the target protein by co-expressing BLT1 with G protein in insect cells.In order to obtain a stable complex structure between the receptor and G protein,we use molecular biology,and genetic engineering techniques to modify and screen BLT1 and the corresponding G protein.The complexes were stabilized by screening the protein expression signal peptide,altering specific residues of receptor protein and the corresponding G protein,and introducing the Nano Bi T auxiliary technology.After obtaining the optimal conditions for the expression and purification,we collect cryo-EM data and analyze the receptor/G protein complex via single particle analysis（SPA）,and the final structure of the active human BLT1 in complex with G_i protein（LTB4/BLT1/G_i）was obtained at a resolution of 2.91（?）.The structure reveals the binding mode of the endogenous ligand LTB4 to BLT1.Combining molecular dynamics simulations,molecular docking and targeted mutagenesis techniques,we unveil that the ligand/receptor interaction is mainly mediated by a water-mediated hydrogen bond network interactions formed by the polar amino acid residues in the pocket of BLT1(H94^3.29,R156^4.64,E185^5.42,H238^6.52,and N268^7.36),LTB4 and water molecules.We also discover an allsteroic binding site in the receptor.Liquid chromatography-mass spectrometry（LC/MS）analysis shows that the composition of the allsteroic modulator was phosphatidylinositol（PI）18:0/16:1 and 18:0/18:1 The discovery of the allosteric binding site provides clues for the subsequent study of allsteroic modulator of GPCRs,as well as a theoretical basis for the design of allsteroic modulator.The binding pocket of BLT1 is widely open on the extracellular side,compared to other lipid receptors of known structures,explaining the poor specificity of the current anti-inflammatory class of drugs.Comparing with inactive BLT1 reveals that the conformation changes of M101^3.36 and I271^7.39 in the binding pocket are major determinant of downstream signaling,specifically,the displacement of M101^3.36 to the center of receptor is crucial for the receptor activation.Furthermore,analyzing the binding of BLT1 to G_i protein revealed the existence of multiple interactions between the receptor and G protein.Focusing on the BLT1/G_i binding interface revealed that F123^ICL2 was able to insert like a wedge into the hydrophobic groove formed by theαN terminus of G_iprotein,which form more stabilized the binding of BLT1 receptor to G protein.In summary,we resolved the structure of the active BLT1 in complex with G_iprotein by Cryo-electron microscopy,reveal the coupling information between the receptor and G_i protein,and unveil ligand recognition and activation mechanism through dynamic simulation,molecular docking,site directed mutation,reporter assay and other functional experiments,which provides a framework for understanding BLT1 signaling and new clues and a rational basis for the development of drugs targeting BLT1.