Eddy Matthew T, Didenko Tatiana, Stevens Raymond C, Wüthrich Kurt
Departments of Biological Sciences and Chemistry, Bridge Institute, The University of Southern California, South Vermont Avenue, MC 3303, Los Angeles, CA 90089, USA.
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Structure. 2016 Dec 6;24(12):2190-2197. doi: 10.1016/j.str.2016.09.015. Epub 2016 Nov 10.
Fluorine-19 nuclear magnetic resonance (NMR) was used to study conformational equilibria at the intracellular tips of helices VI and VII in a variant β-adrenergic receptor (βAR) containing T4-lysozyme fused into the third intracellular loop (βAR-T4L), a G protein-coupled receptor (GPCR) modification widely used in crystal structure determination. G-protein signaling at helix VI showed nearly complete population of an active-like state for all ligand efficacies in the absence of an intracellular protein. For arrestin signaling at helix VII, a native-like equilibrium was observed, except for complexes with ligands devoid of a hydrophobic moiety at the ethanolamine end. These data confirm that response of G-protein and arrestin signaling to ligand efficacy is not coupled, and presents evidence for long-range effects between fusion protein and orthosteric binding cavity, which are suppressed by voluminous bound ligands. Solution NMR thus provides complementary information, which should be considered in functional interpretations of GPCR crystal structures obtained with ICL3 fusions.
利用氟 - 19核磁共振（NMR）研究了一种变异的β - 肾上腺素能受体（βAR）中螺旋VI和VII细胞内末端的构象平衡，该受体在第三个细胞内环中融合了T4 - 溶菌酶（βAR - T4L），这是一种在晶体结构测定中广泛使用的G蛋白偶联受体（GPCR）修饰。在没有细胞内蛋白的情况下，对于所有配体效力，螺旋VI处的G蛋白信号显示出几乎完全处于活性样状态。对于螺旋VII处的阻遏蛋白信号，观察到类似天然的平衡，除了与乙醇胺末端缺乏疏水部分的配体形成的复合物。这些数据证实了G蛋白和阻遏蛋白信号对配体效力的反应不相关联，并提供了融合蛋白与正构结合腔之间存在长程效应的证据，这种效应被大量结合的配体所抑制。因此，溶液核磁共振提供了补充信息，在对通过ICL3融合获得的GPCR晶体结构进行功能解释时应予以考虑。
