Jules Stein Eye Institute, University of California, Los Angeles, CA 90095.
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095.
Proc Natl Acad Sci U S A. 2020 Dec 15;117(50):31824-31831. doi: 10.1073/pnas.2013904117. Epub 2020 Nov 30.
The β adrenergic receptor (βAR) is an archetypal G protein coupled receptor (GPCR). One structural signature of GPCR activation is a large-scale movement (ca. 6 to 14 Å) of transmembrane helix 6 (TM6) to a conformation which binds and activates a cognate G protein. The βAR exhibits a low level of agonist-independent G protein activation. The structural origin of this basal activity and its suppression by inverse agonists is unknown but could involve a unique receptor conformation that promotes G protein activation. Alternatively, a conformational selection model proposes that a minor population of the canonical active receptor conformation exists in equilibrium with inactive forms, thus giving rise to basal activity of the ligand-free receptor. Previous spin-labeling and fluorescence resonance energy transfer experiments designed to monitor the positional distribution of TM6 did not detect the presence of the active conformation of ligand-free βAR. Here we employ spin-labeling and pressure-resolved double electron-electron resonance spectroscopy to reveal the presence of a minor population of unliganded receptor, with the signature outward TM6 displacement, in equilibrium with inactive conformations. Binding of inverse agonists suppresses this population. These results provide direct structural evidence in favor of a conformational selection model for basal activity in βAR and provide a mechanism for inverse agonism. In addition, they emphasize 1) the importance of minor populations in GPCR catalytic function; 2) the use of spin-labeling and variable-pressure electron paramagnetic resonance to reveal them in a membrane protein; and 3) the quantitative evaluation of their thermodynamic properties relative to the inactive forms, including free energy, partial molar volume, and compressibility.
β肾上腺素能受体（βAR）是典型的 G 蛋白偶联受体（GPCR）。GPCR 激活的一个结构特征是跨膜螺旋 6（TM6）的大规模运动（约 6 至 14Å），使其构象与同源 G 蛋白结合并激活。βAR 表现出低水平的激动剂非依赖性 G 蛋白激活。这种基础活性的结构起源及其被反向激动剂抑制的机制尚不清楚，但可能涉及促进 G 蛋白激活的独特受体构象。或者，构象选择模型提出，在配体自由受体中，存在与无活性形式处于平衡的少量规范活性受体构象，从而导致配体自由受体的基础活性。先前设计用于监测 TM6 位置分布的自旋标记和荧光共振能量转移实验并未检测到配体自由βAR 的活性构象的存在。在这里，我们采用自旋标记和压力分辨双电子电子共振波谱法来揭示配体自由受体的小部分群体的存在，其特征是向外 TM6 位移，与无活性构象处于平衡状态。反向激动剂的结合抑制了这种群体。这些结果提供了直接的结构证据，支持βAR 基础活性的构象选择模型，并提供了反向激动作用的机制。此外，它们强调了 1）小部分群体在 GPCR 催化功能中的重要性；2）使用自旋标记和可变压力电子顺磁共振在膜蛋白中揭示它们；3）相对于无活性形式，包括自由能、偏摩尔体积和压缩性，对其热力学性质进行定量评估。
