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Science 318 (5854): 1266-1273

Copyright © 2007 by the American Association for the Advancement of Science

GPCR Engineering Yields High-Resolution Structural Insights into β2-Adrenergic Receptor Function

Daniel M. Rosenbaum1,*, Vadim Cherezov2,*, Michael A. Hanson2, Søren G. F. Rasmussen1, Foon Sun Thian1, Tong Sun Kobilka1, Hee-Jung Choi1,3, Xiao-Jie Yao1, William I. Weis1,3, Raymond C. Stevens2{dagger}, and Brian K. Kobilka1{dagger}

1 Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA.
2 Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
3 Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.

Figure 1
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Fig. 1. . Design and optimization of the β2AR-T4L fusion protein. (A) The sequence of the region of the β2AR targeted for insertion of a crystallizable domain is shown, and the positions of the junctions between the receptor and T4L (red) for various constructs are indicated. The sequences that were initially replaced or removed are faded. Red lines are shown after every tenth residue. ECL, extracellular loop. (B) Immunofluorescence images of HEK293 cells expressing selected fusion constructs. (Left) M1 anti-FLAG signal corresponding to antibody bound to the N terminus of the receptor. (Right) Same signal merged with blue emission from 4',6'-diamidino-2-phenylindole (nuclear staining for all cells). Plasma membrane staining is observed in the positive control, D3, and D1, whereas C3 and D5 are retained in the endoplasmic reticulum.


Figure 2
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Fig. 2. . Functional characterization of β2AR-T4L. (A) Affinity competition curves for adrenergic ligands binding to β2AR-T4L and WT β2AR. Binding experiments on membranes isolated from Sf9 insect cells expressing the receptors were performed as described (13). (B) β2AR-T4L is still able to undergo ligand-induced conformational changes. Bimane fluorescence spectra (excitation at 350 nm) of detergent-solubilized β2AR-T4L and WT β2AR truncated at 365, labeled under conditions that selectively modify Cys2656.27 (13), were measured after incubating the unliganded receptors with compounds for 15 min at room temperature. The cartoon illustrates that the observed changes in fluorescence can be interpreted as a movement of the bimane probe from a more buried, hydrophobic environment to a more polar, solvent-exposed position. cps, counts per second.


Figure 3
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Fig. 3. . (A) Side-by-side comparison of the crystal structures of the β2AR-T4L fusion protein and the complex between β2AR365 and a Fab fragment. The receptor component of the fusion protein is shown in blue (with modeled carazolol as red spheres), whereas the receptor bound to Fab5 is yellow. (B) Differences in the environment surrounding Phe2646.26 (shown as spheres) for the two proteins. (C) Analogous interactions to the ionic lock between the E(D)RY motif and Glu2476.30 seen in rhodopsin (right panel, purple) are broken in both structures of the β2AR (left panel, blue and yellow as above). PyMOL (43) was used for the preparation of all figures.


Figure 4
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Fig. 4. . Schematic representation of the interactions between β2AR-T4L and carazolol at the ligand binding pocket. The residues shown here have at least one atom within 4 A of the ligand in the 2.4 A resolution crystal structure.


Figure 5
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Fig. 5. . Ligand binding pocket of β2AR-T4L with carazolol bound. (A) Residues within 4 A of the ligand are shown as sticks, with the exception of A200, N293, F289, and Y308. Residues that form polar contacts with the ligand (distance cutoff: 3.5 A) are shown in green, other residues are gray, and carazolol is yellow (in all panels, oxygens are red and nitrogens are blue). (B) Same as (A), except that the ligand is oriented with its amine facing out of the page. W109 is not shown. (C) Packing interactions between carazolol and all residues within 5 A of the ligand. The view is from the extracellular side of the membrane. Carazolol is shown as yellow spheres, and receptor residues are shown as sticks within van der Waals dot surfaces. Val1143.33, Phe1935.32, and Phe2906.52 are red, and all other residues are gray. (D) Model of (–)-isoproterenol (magenta sticks) in the ligand binding pocket observed in the crystal structure. A model of the agonist with optimal bond lengths and angles was obtained from the PRODRG server (44), and the dihedral angles were adjusted to the values observed in the homologous atoms of bound carazolol (16 to 22 in Fig. 4). The one remaining unaccounted dihedral in (–)-isoproterenol was adjusted in order to place the catechol ring in the same plane as the C16–C15–O14 plane in carazolol. Residues known to specifically interact with agonists are shown as green sticks.


Figure 6
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Fig. 6. . Packing interactions in the β2AR that are likely to be modulated during the activation process. (A) (Left) Residues previously demonstrated to be CAMs (16, 25, 3739, 45) or UCMs (26, 41, 4649) are shown as van der Waals spheres mapped onto a backbone cartoon of the β2AR-T4L structure. (Right) Residues that are found within 4 A of the CAMs Leu1243.43 and Leu2726.34 are shown as yellow spheres or dot surfaces. A vertical cross section through the structure illustrates that these surrounding residues connect the CAMs on helices III and VI with the UCMs on helix VII through packing interactions. (B) In both β2AR-T4L (blue) and rhodopsin (purple), a network of ordered water molecules is found at the interface between the transmembrane helices at their cytoplasmic ends. (C) Network of hydrogen bonding interactions between water molecules and β2AR-T4L residues (side chains shown as blue sticks), notably three UCMs on helix VII (orange) and one on helix II (yellow). In (B) and (C), only Ballesteros-Weinstein numbers (9) are used to identify amino acids.


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