G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signaling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β2 adrenergic receptor (β2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signaling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β2AR and nucleotide-free Gs heterotrimer. The principal interactions between the β2AR and Gs involve the amino and carboxyl terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β2AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an alpha helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the alpha helical domain of Gαs relative to the ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signaling by a GPCR.

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...

G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviors in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human β2 adrenergic receptor (β2AR) that exhibits G protein-like behavior, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive β2AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.

G-protein-coupled receptors (GPCRs) mediate most of our physiological responses to hormones, neurotransmitters and environmental stimulants, and so have great potential as therapeutic targets for a broad spectrum of diseases. They are also fascinating molecules from the perspective of membrane-protein structure and biology. Great progress has been made over the past three decades in understanding diverse GPCRs, from pharmacology to functional characterization in vivo. Recent high-resolution structural studies have provided insights into the molecular mechanisms of GPCR activation and constitutive activity.