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.

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G protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G proteinindependent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly, in which rhodopsin uses distinct structural elements, including TM7 and Helix 8 to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms § Correspondence to H. Eric Xu: Eric.Xu@vai.org. * These authors contributed equally.Contributions: Y.K. initiated the project, developed the expression and purification methods for rhodopsin-arrestin complex, and bulk-purified expression constructs and proteins used in LCP crystallization for the SFX method; X.E.Z. collected the synchrotron data, helped with the SFX data collection, processed the data, and solved the structures; X.G. expressed and purified rhodopsinarrestin complexes, characterized their binding and thermal stability, discovered the initial crystallization conditions with 9.7 MAG, prepared most crystals for synchrotron data collection, prepared all crystals for the final data collection by SFX, helped with SFX data collection, and established the initial cross-linking method for the rhodopsin-arrestin complex; Y.H. designed and performed Tango assays and disulfide bond cross-linking experiments; C.Z. developed the mammalian expression methods; P.W.dW helped with XFEL data processing and performed computational experiments; J.K., M.H.E.T., K. M. S-P., K. P., J. M., Y.J., X.Y.Z., and Q.C. performed cell culture, mutagenesis, protein purification, rhodopsin-arrestin binding experiments; W.L. and A.I. grew crystals and collected synchrotron data at APS and SFX data at LCLS, G.W.H. and Q.X. determined and validated the structure. Z.Z. and V.K. constructed the full model, the phosphorylated rhodopsin-arrestin model, and help writing the paper; D.W., S.L., D.J., C.K., Sh.B., and N.A. Z. helped with XFEL data collection and initial data analysis; S.B., M.M., and G.J.W. set up the XFEL experiment, performed the data collection, and commented on the paper. A.B., T.W., C.G., O.Y., and H.C. helped with XFEL data collection and data analysis, processed the data and helped with structure validation. G.M. W., B.P., and P.G. performed HDX experiments and helped with manuscript writing. J.L. helped initiate this collaborative project and with writing the paper. M.W. collected the 7.7 Å dataset at Swiss Light Source. A.M.,...

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.

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