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Science 318 (5854): 1258-1265

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

High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein–Coupled Receptor

Vadim Cherezov1,*, Daniel M. Rosenbaum2,*, Michael A. Hanson1, Søren G. F. Rasmussen2, Foon Sun Thian2, Tong Sun Kobilka2, Hee-Jung Choi2,3, Peter Kuhn4, William I. Weis2,3, Brian K. Kobilka2{dagger}, and Raymond C. Stevens1{dagger}

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


Figure 1
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Fig. 1. . Overall fold of the β2AR-T4L fusion with its predicted orientation in the plasma membrane and key intramolecular interactions. (A) Stereoview of the overall fold of β2AR-T4L. The receptor and T4L are colored gray and green, respectively. Carazolol is shown in blue; the lipid molecules bound to the receptor are in yellow. (B) The receptor is aligned to a rhodopsin model that was positioned in a lipid membrane (boundaries indicated by horizontal black lines) as found in the Orientations of Proteins in Membranes database (74). T4L is fused internally into the third intracellular loop of β2AR and maintains minimal intramolecular packing interactions by tilting away from the receptor. (C) Specific intramolecular interactions between β2AR and T4L.

 

Figure 2
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Fig. 2. . Crystal-packing interactions in the lipidic mesophase–crystallized β2AR-T4L. (A) There are four main contact areas, two of which are mediated by T4L in the plane of the membrane with itself through a two-fold symmetry axis and translation. The third interaction is normal to the membrane plane between T4L and lumen-exposed loops of β2AR. The fourth interaction is generated by the two-fold symmetry axis, packing one receptor to another in the plane of the membrane. (B) The receptor crystal-packing interface is composed mainly of lipids, with two cholesterol molecules and two palmitic acid molecules forming the majority of the interactions. A network of ionic charge interactions exists on the cytoplasmic end of the interface, forming the only interreceptor protein contacts. (C)Comparison between β2AR-T4L and rhodopsin (PDB ID code 2I35) parallel receptor association interface. Helices I (blue) and VIII (magenta) are highlighted in both structures. Only one monomer is shown for each receptor representation, along with helices I' and VIII' only from the opposing symmetry-related molecule. The rhodopsin interface is twisted relative to β2AR-T4L, resulting in a substantial offset from the parallel orientation required for a physiological dimer interface. β2AR-T4L–associated monomers are in a highly parallel orientation.

 

Figure 3
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Fig. 3. . Surface representation of β2AR colored by calculated charge from red (–10 kbT/ec) to blue (+10 kbT/ec) using a dielectric constant of 70. (A) Three main areas of interest are indicated. The binding-site cleft is negatively charged, as is a groove between helices III, IV, and V. The third region is an overall positive charge in the region of the ionic lock and Asp-Arg-Tyr motif on the cytoplasmic face. The overall result is a highly polarized molecule that may use its negative charge to facilitate binding of catecholamine ligands. The presence of a negative charge in the groove between helices III, IV, and V is unexpected, as it is in the middle of the lipid membrane. This charge may be partially derived from the presence of an unpaired glutamate at position 1223.41. The effective charge in this region is likely greater than shown here because of its location in the low-dielectric environment of the lipid membrane. (B) View rotated 90° from (A), showing the negatively charged binding-site cleft (top) and the positively charged cytoplasmic face (bottom). Poisson-Boltzmann electrostatics were calculated using APBS (52) as implemented in PyMOL (75). PyMOL was used exclusively in the preparation of all figures.

 

Figure 4
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Fig. 4. . Comparison of the extracellular sides of β2AR-T4L and rhodopsin. (A) The N terminus is missing from the experimental density in the β2AR-T4L structure and is not shown. ECL2 is shown in green and contains a short α helix and two disulfide bonds (yellow). The intraloop disulfide bond constrains the tip of ECL2, which interacts with ECL1. The second disulfide bond links ECL2 with helix III. There is one interaction between ECL2 and carazolol (blue) through Phe1935.32. The entire loop is held out of the ligand-binding site by a combination of the rigid helical segment and the two disulfide bonds. (B) In contrast, ECL2 (green) in rhodopsin assumes a lower position in the structure that occludes direct access to the retinal-binding site and forms a small β sheet in combination with the N-terminal region (magenta) directly above the bound retinal (pink).

 

Figure 5
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Fig. 5. . Ligand-binding characterization and comparison to rhodopsin. (A) View looking down on the plane of the membrane from the extracellular surface, showing a detailed representation of the carazolol-binding site in β2AR-T4L. Carazolol is shown as sticks with carbon atoms colored yellow. β2AR-T4L residues contributing to carazolol binding are shown in green and labeled. Electron density is contoured at 5{sigma} from an FobsFcalc omit map calculated without the contribution of carazolol. Abbreviations: D, Asp; F, Phe; N, Asn; S, Ser; W, Trp; Y, Tyr. (B) Binding orientation comparison between 11-cis-retinal in rhodopsin and carazolol in β2AR-T4L. Van der Waals surfaces for carazolol and retinal are represented as dots to accentuate the close-packing interactions. Retinal in the 11-cis conformation (pink) binds deep in the active site of rhodopsin as compared to carazolol (blue), packing its β-ionone ring between Tyr2686.51 and Phe2125.47 (cyan) and blocking movement of Trp2656.48 (magenta) into the space. The β-ionone ring of all-trans-retinal in activated rhodopsin would not block Trp2656.48 from rotating into the space, allowing a rotameric shift into its proposed active form. (C) Four residues are involved in the toggle switch mechanism of β2AR-T4L. Phe2906.52 (magenta) is sandwiched between Phe2085.47 (tan) and Phe2896.51 (tan), forming a ring-face aromatic interaction. Like rhodopsin, an activation step is thought to occur by a rotameric change of Trp2866.48 (magenta), which would displace Phe2906.52. Carazolol is shown to interact extensively with the sandwich motif; however, few interactions are seen with Trp2866.48. The 6.52 position in β2AR-T4L is occupied by Phe2906.52, as opposed to Ala2696.52 in rhodopsin, where the β-ionone ring replaces an aromatic protein side chain in forming the sandwich interactions. The aromatic character of the sandwich is otherwise maintained by Phe2896.51 and Phe2085.47 in β2AR-T4L.

 

Figure 6
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Fig. 6. . Comparison of β2AR-T4L helical orientations with those of rhodopsin (PDB ID code 1U19). (A) β2AR-T4L is rendered as a ribbon trace colored with a blue-to-red spectrum corresponding to observed distances between Cα positions in the two structures [root mean square deviation (RMSD) 2.7 A between all residues in the transmembrane region]. Helix II shows very little movement, whereas the entire lengths of helices III, IV, and V shift substantially. Helix VIII and loops were not included in the comparison and are colored tan. (B) Movements of helices I and V of rhodopsin (gray) are shown relative to β2AR-T4L. (C) Movements of helices III, IV, and VI. (D) Ligand-binding site representation. Carazolol is shown with yellow carbons. Entire helices are assigned a single designation on the basis of their divergence from the rhodopsin position in the area of the ligand-binding site as shown. Helix I is highly divergent; helices II and VI are similar to rhodopsin. Helices IV and VII are moderately constant. Helices III and V are moderately divergent.

 


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