title: β2-Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop.
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.
authors.affiliation: Departments of Biological Sciences and Chemistry, Bridge Institute, The University of Southern California, South Vermont Avenue, MC 3303, Los Angeles, CA 90089, USA.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Eddy
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Didenko
authors.affiliation: Departments of Biological Sciences and Chemistry, Bridge Institute, The University of Southern California, South Vermont Avenue, MC 3303, Los Angeles, CA 90089, USA.
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Stevens
authors.affiliation: 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.
authors.forename: Kurt
authors.initials: K
authors.lastname: Wüthrich
mesh_terms: D019393:Arrestin / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D009113:Muramidase / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D019393:Arrestin; D018343:Receptors, Adrenergic, beta-2; D009113:Muramidase
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