Structural and functional characterization of G protein-coupled receptors with deep mutational scanning.
Structural and functional characterization of G protein-coupled receptors with deep mutational scanning.
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DOI：
10.7554/elife.54895
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发表时间：
2020-10-21
期刊：
影响因子：
7.7
通讯作者：
Kosuri S
中科院分区：
文献类型：
--
作者：
Jones EM;Lubock NB;Venkatakrishnan AJ;Wang J;Tseng AM;Paggi JM;Latorraca NR;Cancilla D;Satyadi M;Davis JE;Babu MM;Dror RO;Kosuri S
超过 800 个人类 G 蛋白偶联受体 (GPCR) 负责转导多种化学刺激以改变细胞状态，并且是最大类别的药物靶点。它们无数的结构构象和各种信号传导模式使得理解它们的结构和功能具有挑战性。在这里，我们开发了一个平台，利用 G 蛋白信号转导的条形码转录报告基因来表征人类细胞系中 GPCR 变体的大型文库。我们在四种浓度的激动剂异丙肾上腺素下测试了 7828 个可能的 β-2 肾上腺素受体 (β2AR) 单氨基酸取代中的 7800 个。我们鉴定了对 β2AR 信号传导特别重要的残基、可能导致功能丧失的人群突变以及调节基础活性的残基。使用无监督学习，我们识别了对信号传导至关重要的残基，包括所有主要结构基序和分子界面。我们还发现了一个跨越前两个细胞外环的先前未表征的结构锁存器，该结构锁存器在 A 类 GPCR 中高度保守，并且在受体的非活性和活性状态下都具有构象刚性。更广泛地说，通过将深度突变扫描与工程转录报告基因联系起来，我们建立了一种通用的方法来探索广泛类别药物受体的药物基因组学、结构和功能。
The >800 human G protein–coupled receptors (GPCRs) are responsible for transducing diverse chemical stimuli to alter cell state- and are the largest class of drug targets. Their myriad structural conformations and various modes of signaling make it challenging to understand their structure and function. Here, we developed a platform to characterize large libraries of GPCR variants in human cell lines with a barcoded transcriptional reporter of G protein signal transduction. We tested 7800 of 7828 possible single amino acid substitutions to the beta-2 adrenergic receptor (β2AR) at four concentrations of the agonist isoproterenol. We identified residues specifically important for β2AR signaling, mutations in the human population that are potentially loss of function, and residues that modulate basal activity. Using unsupervised learning, we identify residues critical for signaling, including all major structural motifs and molecular interfaces. We also find a previously uncharacterized structural latch spanning the first two extracellular loops that is highly conserved across Class A GPCRs and is conformationally rigid in both the inactive and active states of the receptor. More broadly, by linking deep mutational scanning with engineered transcriptional reporters, we establish a generalizable method for exploring pharmacogenomics, structure and function across broad classes of drug receptors.