Tian He, Gunnison Kathryn M, Kazmi Manija A, Sakmar Thomas P, Huber Thomas
Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
Tri-Institutional PhD Program in Chemical Biology, 1300 York Avenue, Box 194, New York, NY 10065, USA.
iScience. 2022 Mar 11;25(4):104060. doi: 10.1016/j.isci.2022.104060. eCollection 2022 Apr 15.
The photoreceptor rhodopsin (Rho) becomes active when a tethered inverse agonist ligand (11CR) is photoconverted to an agonist (ATR). The ligand-binding pocket of inactive rhodopsin is completely enclosed, whereas active rhodopsin displays pores accessible from the lipid bilayer. Stabilization of active rhodopsin impedes 11CR binding and photoreceptor dark adaptation. Here, we used genetic code expansion and bioorthogonal labeling to engineer Rho mutants that serve as FRET sensors for measuring 11CR binding kinetics and energetics. We found that mutations that alter a channel between transmembrane helices 5 and 6 (TM5/6) dramatically affect 11CR binding kinetics but not agonist release kinetics. Our data provide direct experimental evidence for 11CR entry between TM5/6 in Rho that involves dynamic allosteric control of the ligand entry channel. Our findings provide a conceptual framework for understanding the function of G protein-coupled receptors with hydrophobic ligands that are hypothesized to enter their binding pockets through transmembrane pores.
当一个 tethered 反向激动剂配体（11CR）经光转化为激动剂（ATR）时，光感受器视紫红质（Rho）变得活跃。无活性视紫红质的配体结合口袋完全封闭，而活性视紫红质则显示出可从脂质双层进入的孔隙。活性视紫红质的稳定化会阻碍 11CR 的结合以及光感受器的暗适应。在这里，我们利用遗传密码扩展和生物正交标记技术构建了 Rho 突变体，作为用于测量 11CR 结合动力学和能量学的荧光共振能量转移（FRET）传感器。我们发现，改变跨膜螺旋 5 和 6（TM5/6）之间通道的突变会显著影响 11CR 的结合动力学，但不影响激动剂释放动力学。我们的数据为 11CR 在 Rho 的 TM5/6 之间进入提供了直接实验证据，这涉及配体进入通道的动态变构控制。我们的研究结果为理解具有疏水配体的 G 蛋白偶联受体的功能提供了一个概念框架，这些疏水配体被假定通过跨膜孔进入其结合口袋。
