【作者】 王娜；

【导师】 何元政；

【作者基本信息】 哈尔滨工业大学 ， 生物学， 2023， 博士

【摘要】 G蛋白偶联受体（G Protein-Coupled Receptors,GPCRs）是位于细胞膜表面一类重要的蛋白家族,具有七次跨膜结构。GPCRs广泛地表达于人体的各个组织,它能够识别激素、神经递质、细胞代谢产物等多种信号分子,从而与下游效应蛋白结合引发多种细胞内信号级联反应。在体内,由GPCRs引起的信号级联反应几乎参与所有生理和病理过程,因此GPCRs是目前研究最多的药物靶点。在本课题研究中,主要以白三烯B4受体1（Leukotriene B4 receptor 1,BLT1）为研究对象,分别展开对其与配体识别、偶联下游G蛋白分子的结构和功能的研究。白三烯B4（Leukotriene B4,LTB4）是花生四烯酸代谢途径产生的一种重要炎症因子,可作用于细胞膜表面的BLT1亚型,参与细胞内炎症和免疫反应而发挥其生理功能。BLT1亚型属于A类GPCR,其不仅在粒细胞和巨噬细胞有高表达,也在嗜酸性粒细胞、T细胞、树突细胞等中有表达。BLT1是抗炎、抗过敏、以及免疫疾病相关药物的作用靶点。但目前对于BLT1结构研究较少,特别是对激活状态下受体的三维结构始终未获成功。因此,希望通过借助冷冻电镜技术解析LTB4激活状态下BLT1与G_i蛋白的复合物结构,针对BLT1的结构分析研究并进一步阐明其激活机制,同时也期望为靶向BLT1的小分子抗炎类药物设计和开发提供相关信息。本课题采用昆虫细胞杆状病毒表达系统,通过将BLT1与G蛋白的杆状病毒在昆虫细胞中共表达,从而获得目的蛋白复合物。为了得到受体与G蛋白之间稳定的复合物结构,课题借助分子生物学,基因工程等技术对BLT1以及相应G蛋白进行克隆改造与筛选。通过筛选受体蛋白表达的信号肽、受体蛋白突变体和改造G蛋白、引入NanoBiT辅助技术等方面来稳定复合物。在确定蛋白纯化和表达的最佳条件后,通过冷冻电镜技术对复合物蛋白进行数据收集和分析,最终获得了激活状态的人源BLT1与G_i蛋白复合物的三维结构,分辨率达2.91（?）。该结构揭示了内源性配体LTB4与BLT1之间的作用方式,结合分子动力学模拟、分子对接和定点突变技术验证配体LTB4通过水分子与BLT1的口袋中的极性氨基酸残基(H94^3.29、R156^4.64、E185^5.42、H238^6.52、R267^7.35和N268^7.36等位点)之间形成紧密的氢键网络。在复合物结构中,发现一个别构调节剂结合位点。借助液相色谱-质谱联用技术分析,实验结果表明:别调节剂结合位点的物质组成成分为磷脂酰肌醇（PI）18:0/16:1和18:0/18:1,该位点的发现有望为后续研究GPCRs别构调节剂提供线索,同时也可为设计别构调剂提供理论基础。与其他已知结构的磷脂类受体相比,BLT1的配体结合口袋上方是呈广泛开口的状态,解释了目前靶向BLT1抗炎类药物专一性较差的原因。通过与非激活状态下的BLT1结构比较发现,配体结合口袋中的M101^3.36与I271^7.39氨基酸残基位点位移变化可引起下游信号传导,且M101^3.36位移变化是受体被激活的关键。此外,对BLT1与G_i蛋白结合界面分析发现,受体与G蛋白之间存在多种极性相互作用。聚焦BLT1/G_i结合界面发现,F123^ICL2能够像楔子一样插入到由G_i蛋白的αN端形成的疏水凹槽中,这种形式更加稳定了BLT1与G蛋白的结合。综上所述,课题通过冷冻电镜技术解析激活状态下的BLT1与G蛋白结合的复合物结构,揭示了BLT1与下游G蛋白间的信号传导关系;结合分子动态模拟,分子对接,定点突变,报告基因等功能实验阐明其配体识别及激活机制,为进一步深入理解BLT1的生物学功能及其下游信号传导机制提供基础,也为靶向BLT1的药物研发提供新的线索及理论基础。更多还原

【Abstract】 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.更多还原