High-Resolution Crystal Structure of an Engineered Human β ₂ -Adrenergic G Protein–Coupled Receptor

Abstract

G protein-coupled receptors comprise the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human β 2 -adrenergic receptor-T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 Å resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the β 2 -adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.* To whom correspondence should be addressed: stevens@scripps.edu; kobilka@stanford.edu $ These authors contributed equally Author Contributions: RCS and BKK independently pushed the GPCR structural biology projects for more than 15 years. BKK managed the protein design, production and purification. RCS managed novel crystallization and data collection methods development and experiments. VC developed novel methods for, and performed LCP crystallization, LCP crystal mounting, LCP data collection, model refinement, analyzed the results, and was involved in manuscript preparation. DMR supplied protein materials for all crystallization trials, grew and collected data from the bicelle crystals, collected, processed and refined the 3.5 Å LCP structure, refined the 2.4 Å structure, analyzed the results, and was involved in manuscript preparation. MAH designed the blind crystal screening protocol and collected the 2.4 Å data set, processed the 2.4 Å data, solved the structure by MR at 3.5 Å and 2.4 Å resolution, wrote the initial draft of the manuscript and created all figures. SGFR assisted with the final stages of β 2 AR-T4L purification. FST expressed β 2 AR-T4L in insect cells and, together with TSK, performed the initial stage of β 2 AR purification. HJC assisted with the refinement. PK assisted in developing novel methods to screen the transparent crystals, data collection, refinement, and was involved in manuscript preparation. WIW assisted with low resolution data collection and processing, solved the β 2 AR-T4L molecular replacement problem at 3.5 Å, participated in the 2.4 Å refinement process, and participated in structure analysis and manuscript preparation. BKK additionally assisted with β 2 AR-T4L purification, β 2 AR-T4L 3.5 Å synchrotron data collection, structure analysis and manuscript preparation. BKK and DMR designed the β 2 AR-T4L fusion protein strategy. RCS additionally assisted with β 2 AR-T4L crystallization, 2.4 Å data collection, structure solution, refinem...

Light on the structure of thromboxane A2 receptor heterodimers

“…A similar architecture was also found for the opsin apoprotein [28]. With respect to the b 2 -AR, crystal packing in the presence of cholesterol shows a significant involvement of the hydrophobic molecule in the intermonomer interface [24]. In this framework, protein-protein contacts are minimal and include a pair of salt bridges between K60 (1.59) (the first number in parenthesis refers to the helix and the following indicate the position of the helical residue relative to the most highly conserved residue within that helix, here denoted as 50, according to the nomenclature proposed by Ballesteros and Weinstein [30]) and E338 from H8 [24].…”

“…System's dependent variety in the dimer architecture emerges also from results of in vitro experiments. In this respect, the highest resolution information available thus far comes from X-ray crystallography and atomic force microscopy (AFM) measurements on rhodopsin, opsin, b 2 -AR and CXCR4 chemokine receptor, as well as from cysteine crosslinking experiments on D 2 receptor (D 2 R), and disulphide trapping experiments on 5HT 1c receptor [22][23][24][25][26][27][28][29]. With regard to rhodopsin, the geometrical constraints from AFM measurements led to the proposal of a semi-empirical model of a higher order rhodopsin structure [22].…”

Regulation of neuronal chloride homeostasis by neuromodulators

“…Crystal structure of human adrenergic GPCR (PDB 2RH1, in blue; Cherezov et al . 2007) is used in this cartoon. Chimera 1.10.1 was used for molecular graphics rendering.…”

The structure of active opsin as a basis for identification of GPCR agonists by dynamic homology modelling and virtual screening assays

“…Around 45% of all drugs on the market modulate the activity of GPCRs [2]. Despite their pharmaceutical importance, only seven GPCRs are available as crystal structures up to now [3][4][5][6][7][8]. To overcome the lack in structural information on other GPCRs for virtual ligand screening, homology modelling is usually applied [9,10].…”

“…We modelled the activated state in its apo form and in complex with an agonist (epinephrine) and an inverse agonist (carazolol). For comparison, we also introduced those ligands into the inactive state crystal structure of B2AR (PDB ID 2RH1) [5] …”