Mechanistic insights from the atomic-level quaternary structure of short-lived GPCR oligomers in live cells

The functional significance of the interactions between proteins in living cells to form short-lived quaternary structures cannot be overemphasized. Yet, quaternary structure information is not captured by current methods, neither can those methods determine structure within living cells. The dynamic versatility, abundance, and functional diversity of G protein-coupled receptors (GPCRs) pose myriad challenges to existing technologies but also present these proteins as the ideal testbed for new technologies to investigate the complex inter-regulation of receptor-ligand, receptor-receptor, and receptor-downstream effector interfaces in living cells. Here, we present development and use of a novel method capable of overcoming existing challenges by combining distributions (or spectrograms) of FRET efficiencies from populations of fluorescently tagged proteins associating into oligomeric complexes in live cells with diffusion-like trajectories of FRET donors and acceptors obtained from coarse-grained molecular dynamics (CGMD) simulations. Our approach provides an atom-level picture of the binding interfaces within oligomers of the human secretin receptor (hSecR) in live cells and allows for extraction of mechanistic insights into the function of GPCRs oligomerization. This FRET-MD spectrometry approach is a robust platform for investigating protein-protein binding mechanisms and opens a new avenue for investigating stable as well as fleeting quaternary structures of any membrane proteins in living cells.

活细胞内蛋白质相互作用形成瞬时蛋白质四级结构（quaternary structure）的功能重要性无论如何强调都不为过。然而，现有方法无法捕获四级结构信息，也无法在活细胞内解析蛋白质结构。G蛋白偶联受体（G protein-coupled receptors, GPCRs）所具备的动态可塑性、丰度及功能多样性，既为现有技术带来了诸多挑战，也使其成为新技术的理想测试平台，用于研究活细胞内受体-配体、受体-受体以及受体-下游效应分子界面间的复杂调控机制。本文介绍了一种可克服现有挑战的新型方法的开发与应用：该方法将活细胞内结合为寡聚复合物的荧光标记蛋白质群体的荧光共振能量转移（Fluorescence Resonance Energy Transfer, FRET）效率分布（或频谱图），与通过粗粒度分子动力学（coarse-grained molecular dynamics, CGMD）模拟获得的FRET供体与受体的类扩散轨迹相结合。我们的方法能够解析活细胞内人促胰液素受体（human secretin receptor, hSecR）寡聚体内部结合界面的原子级图景，并可提取GPCRs寡聚化功能相关的机制性见解。这种FRET-MD光谱法是研究蛋白质-蛋白质结合机制的稳健平台，同时为研究活细胞内任意膜蛋白的稳定及瞬时四级结构开辟了全新途径。