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 (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) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signaling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs1, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human β2 adrenergic receptor (β2AR) as a guide, we designed a β2AR agonist that can be covalently tethered to a specific site on the receptor through a disulfide bond. The covalent β2AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound β2AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method2, and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper3) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 μs) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.

Summary G protein-coupled receptors (GPCRs) represent the largest family of membrane receptors1 that instigate signaling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely GDP dissociation from the G protein α-subunit, is the key step toward G protein activation and initiation of downstream signaling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism we applied peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS) to probe changes in the structure of the heterotrimeric G protein Gs (the stimulatory G protein for adenylyl cyclase) upon formation of a complex with agonist-bound β2 adrenergic receptor (β2AR). Our studies reveal structural links between the receptor binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange (HX) than would be predicted from the crystal structure of the β2AR-Gs complex. Together with x-ray crystallographic and electron microscopic data of the β2AR-Gs complex (ref 2 and Westfield et al, manuscript submitted), we provide a rationale for a mechanism of nucleotide exchange whereby the receptor perturbs the structure of the amino-terminal region of α-subunit of Gs and consequently alters the ‘P-loop’ that binds the β-phosphate in GDP. As with the ras-family of small molecular weight G proteins, P-loop stabilization and β-phosphate coordination are key determinants of GDP (and GTP) binding affinity.

G protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other1. Upon activation by extracellular agonists, these seven transmembrane domain (7TM)-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades1. Crystallographic evidence from a prototypic GPCR, the β2-adrenergic receptor (β2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide binding pocket of the G protein α-subunit to catalyze GDP release, the key step required for GTP binding and activation of G proteins2. The structure also offers hints on how G protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G protein coupling to β2AR stabilizes a ‘closed’ receptor conformation characterized by restricted access to and egress from the hormone binding site. Surprisingly, the effects of G protein on the hormone binding site can be observed in the absence of a bound agonist, where G protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G protein-mediated enhancement of agonist affinity, which has been observed for many GPCR-G protein pairs. Our studies also suggest that in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G protein-mediated enhancement of agonist affinity.