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...

Chemokine receptors are critical regulators of cell migration in the context of immune surveillance, inflammation and development. The G protein-coupled chemokine receptor, CXCR4, is specifically implicated in cancer metastasis and HIV-1 infection. Here we report five independent crystal structures of CXCR4 bound to an antagonist small molecule IT1t and a cyclic peptide CVX15 at 2.5–3.2 Å resolution. All structures reveal a consistent homodimer with an interface involving helices V and VI that may be involved in regulating signaling. The location and shape of the ligand binding sites differ from other G protein-coupled receptors and are closer to the extracellular surface. These structures provide new clues about the interactions between CXCR4 and its natural ligand CXCL12 and with the HIV-1 glycoprotein gp120.

The adenosine class of G protein-coupled receptors mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A 2A adenosine receptor in complex with a high affinity subtypeselective antagonist, ZM241385, to 2.6 Å resolution. Four disulfide bridges in the extracellular domain combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures defines a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extended conformation perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core in ligand recognition by this class of GPCRs, and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.

Dopamine modulates movement, cognitive, and emotional functions of the brain through activation of dopamine receptors that belong to the G protein-coupled receptor (GPCR) superfamily. Here we present the crystal structure of the human dopamine D3 receptor (D3R) in complex with the small molecule D2R/D3R-specific antagonist eticlopride at 3.15 Å resolution. Docking of R-22, a D3R-selective antagonist to the D3R structure reveals an extracellular extension of the eticlopride binding site that comprises a connected second binding pocket for the aryl amide of R-22.Dopamine is an essential neurotransmitter in the central nervous system and exerts its effects through activation of five distinct dopamine receptor subtypes that belong to the G proteincoupled receptor (GPCR) superfamily. The receptors have been classified into two subfamilies, D1-like and D2-like, on the basis of their sequence and pharmacological similarities (1). The D1-like receptors (D1R and D5R) couple to stimulatory G-protein alpha subunits (G s/olf ), activating adenyl cyclase, whereas D2-like receptors (D2R, D3R and D4R) couple to inhibitory G-protein alpha subunits (G i/o ), inhibiting adenyl cyclase. The high degree of sequence identity (2-3) within the transmembrane helices between D2R and D3R

The beta2-adrenergic receptor (beta2AR) is a well-studied prototype for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the beta2AR and to facilitate its crystallization, we engineered a beta2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR ("beta2AR-T4L") and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context of the reported high-resolution structure of beta2AR-T4L provides insights into inverse-agonist binding and the structural changes required to accommodate catecholamine agonists. Amino acids known to regulate receptor function are linked through packing interactions and a network of hydrogen bonds, suggesting a conformational pathway from the ligand-binding pocket to regions that interact with G proteins.

Pharmacological responses of G protein-coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR structures. We re-engineered the human A2A adenosine receptor by replacing its third intracellular loop with apo-cytochrome b562RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure identified about 57 ordered waters inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved Asp2.50. Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured waters, sodium ions and lipids/cholesterol in GPCR stabilization and function.

Activation of G protein-coupled receptors upon agonist binding is a critical step in the signaling cascade for this family of cell surface proteins. Here we report the crystal structure of the A2A adenosine receptor (A2AAR) bound to an agonist UK-432097 at 2.7 angstrom resolution. Relative to inactive, antagonist-bound A2AAR, the agonist-bound structure displays an outward tilt and rotation of the cytoplasmic half of helix VI, a movement of helix V and an axial shift of helix III, resembling the changes associated with the active-state opsin structure. Additionally, a seesaw movement of helix VII and a shift of extracellular loop 3 are likely specific to A2AAR and its ligand. The results define the molecule UK-432097 as a “conformationally selective agonist” capable of receptor stabilization in a specific active state configuration.

The role of cholesterol in eukaryotic membrane protein function has been attributed primarily to an influence on membrane fluidity and curvature. We present the 2.8 A resolution crystal structure of a thermally stabilized human beta(2)-adrenergic receptor bound to cholesterol and the partial inverse agonist timolol. The receptors pack as monomers in an antiparallel association with two distinct cholesterol molecules bound per receptor, but not in the packing interface, thereby indicating a structurally relevant cholesterol-binding site between helices I, II, III, and IV. Thermal stability analysis using isothermal denaturation confirms that a cholesterol analog significantly enhances the stability of the receptor. A consensus motif is defined that predicts cholesterol binding for 44% of human class A receptors, suggesting that specific sterol binding is important to the structure and stability of other G protein-coupled receptors, and that this site may provide a target for therapeutic discovery.