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Structure. 2016 Dec 6;24(12):2190-2197. doi: 10.1016/j.str.2016.09.015. Epub 2016 Nov 10.

β2-Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop.

Author information

1
Departments of Biological Sciences and Chemistry, Bridge Institute, The University of Southern California, South Vermont Avenue, MC 3303, Los Angeles, CA 90089, USA.
2
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
3
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA; Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address: wuthrich@scripps.edu.

Abstract

Fluorine-19 nuclear magnetic resonance (NMR) was used to study conformational equilibria at the intracellular tips of helices VI and VII in a variant β2-adrenergic receptor (β2AR) containing T4-lysozyme fused into the third intracellular loop (β2AR-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.

KEYWORDS:

19F NMR spectroscopy; G-protein-coupled receptors; fusion protein; integrative structural biology; membrane proteins; protein dynamics; solution NMR; solution nuclear magnetic resonance; structural biology; β2-adrenergic receptor

PMID:
27839952
PMCID:
PMC5144828
DOI:
10.1016/j.str.2016.09.015
[Indexed for MEDLINE]
Free PMC Article

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