Patent Publication Number: US-9422359-B2

Title: GPCR fusion protein containing an N-terminal autonomously folding stable domain, and crystals of the same

Description:
CROSS-REFERENCING 
     This application claims the benefit of U.S. provisional application Ser. Nos. 61/453,020, filed Mar. 15, 2011 and 61/507,425, filed Jul. 13, 2011, which are incorporated by reference in their entirety. 
    
    
     GOVERNMENT RIGHTS 
     This invention was made with Government support under contract GM083118 awarded by the National Institutes of Health. The Government has certain rights in this invention. 
    
    
     BACKGROUND 
     G protein-coupled receptor (GPCR) signaling plays a vital role in a number of physiological contexts including, but not limited to, metabolism, inflammation, neuronal function, and cardiovascular function. For instance, GPCRs include receptors for biogenic amines, e.g., dopamine, epinephrine, histamine, glutamate, acetylcholine, and serotonin; for purines such as ADP and ATP; for the vitamin niacin; for lipid mediators of inflammation such as prostaglandins, lipoxins, platelet activating factor, and leukotrienes; for peptide hormones such as calcitonin, follicle stimulating hormone, gonadotropin releasing hormone, ghrelin, motilin, neurokinin, and oxytocin; for non-hormone peptides such as beta-endorphin, dynorphin A, Leu-enkephalin, and Met-enkephalin; for the non-peptide hormone melatonin; for polypeptides such as C5a anaphylatoxin and chemokines; for proteases such as thrombin, trypsin, and factor Xa; and for sensory signal mediators, e.g., retinal photopigments and olfactory stimulatory molecules. GPCRs are of immense interest for drug development. 
     SUMMARY 
     A GPCR fusion protein is provided. In certain embodiments, the GPCR fusion protein comprises: a) a G-protein coupled receptor (GPCR); and b) an autonomously folding stable domain, where the autonomously folding stable domain is N-terminal to the GPCR and is heterologous to the GPCR. The GPCR fusion protein is characterized in that is crystallizable under lipidic cubic phase crystallization conditions. In certain embodiments, the GPCR fusion protein may be crystallizable in a complex with a G-protein or in a complex with an antibody that binds to the IC3 loop of the GPCR. 
     In particular embodiments, the GPCR fusion protein may further comprise an epitope tag N-terminal to the autonomously folding stable domain. In some cases, the GPCR fusion protein may further comprise a protease cleavage site between the epitope tag and the autonomously folding stable domain, thereby allowing the epitope tag to cleaved off. 
     In particular embodiments, the autonomously folding stable domain may comprises the amino acid sequence of lysozyme. In some cases, the GPCR fusion protein may also comprise a second autonomously folding stable domain between the TM5 and TM6 regions of the GPCR (i.e., in the IC3 loop of the GPCR). 
     In certain embodiments, the GPCR of the fusion protein may be active. The GPCR of the fusion protein may be naturally occurring or non-naturally occurring. 
     Also provided is a composition of matter comprising: a) a subject GPCR fusion protein; and b) a moiety complexed with the GPCR fusion protein. The moiety complexed with the GPCR fusion protein may be, for example, a G-protein or an antibody that is bound to the IC3 loop of the GPCR. The moiety may also be a ligand for the GPCR. 
     A nucleic acid encoding the subject GPCR fusion protein is also provided. In particular embodiments, the nucleic acid may encode, from 5′ to 3′: a) a signal sequence; b) an epitope tag; c) a protease cleavage site; d) an autonomously folding stable domain; and e) a GPCR. Also provided is a cell containing the nucleic acid. In particular cases, the fusion protein may be expressed in the cell, and disposed on the plasma membrane of the cell. 
     Also provided is a crystal comprising a crystalline form of the subject GPCR fusion protein. The crystal may further contain, for example, a G protein complexed with the GPCR fusion protein, a ligand for the GPCR, or an antibody that is bound to the IC3 loop of the GPCR. In particular embodiments, the crystallized GPCR fusion protein may comprise a second autonomously folding stable domain between the TM5 and TM6 regions of the GPCR. 
     Also provided is a method for producing the subject fusion protein. In some embodiments, this method may involve culturing the above-described cell to produce the GPCR fusion protein; and isolating the GPCR fusion protein from the cell. The may further comprises crystallizing the GPCR fusion protein to make crystals, e.g., using a bicelle crystallization method or a lipidic cubic phase crystallization method. Prior to crystallization, the isolated GPCR fusion protein may be combined with a moiety to which it complexes, e.g., the G protein to which it couples, a ligand or an antibody, for example, to produce a complexes. This method may further comprise obtaining atomic coordinates of the GPCR fusion protein from said crystal. 
     A method of determining a crystal structure is also provided. In certain cases this method comprises: receiving a subject GPCR fusion protein, crystallizing the fusion protein to produce a crystal; and obtaining atomic coordinates of the fusion protein from the crystal. Other embodiment include forwarding a subject GPCR fusion protein to a remote location, and receiving atomic coordinates for said GPCR fusion protein. 
     In particular embodiments, a composition comprising a fusion protein in crystalline form is provided in which the fusion protein comprises: a) a G-protein coupled receptor (GPCR); and b) a lysozyme domain, where the lysozyme domain is N-terminal to the GPCR. 
     In particular embodiments, the GPCR may comprise the amino acid sequence of a naturally occurring GPCR. In other embodiments, GPCR may comprise the amino acid sequence of a non-naturally occurring GPCR. 
     The domain, in certain cases, may comprise an amino acid sequence having at least 80% identity to the amino acid sequence of a wild-type lysozyme. For example, in certain cases, the domain may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of T4 lysozyme. 
     In particular embodiments, the GPCR may be a family A GPCR, a family B GPCR or a family C GCPR. In particular embodiments, the GPCR may be a receptor for a biogenic amine, a dopamine receptor, a seratonin receptor, an adrenergic receptor, a β2-adrenergic receptor, a melanocortin receptor subtype 4, a ghrelin receptor, a metabotropic glutamate receptor or a chemokine receptor. The crystallized GPCR fusion protein may comprise a second autonomously folding stable domain (e.g., another lysozyme domain) between the TM5 and TM6 regions of the GPCR. 
     In some embodiments, the fusion protein is bound to a ligand for the GPCR. In particular embodiments, the fusion protein may be co-crystallized with a G protein to which the GPCR couples (which may be composed of the Gα, β and γ subunits) or an antibody that binds the IC3 loop of the GPCR, for example. 
     In particular cases, a GPCR-G-protein complex may be crystallized in conjunction with an antibody that stabilizes the G-protein in the same way as the nanobody described below. Such an antibody may be from any species and, in certain cases, may be a single chain antibody. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  is a schematic illustration of a GPCR, showing the canonical transmembrane regions (TM1, TM2, TM3, TM4, TM5, TM6, and TM7), intracellular regions (IC1, IC2, and IC3), and extracellular regions (EC1, EC2, and EC3). 
         FIG. 2  is a schematic illustration of a subject fusion protein, showing an autonomously folding stable domain that is N-terminal to a GPCR. 
         FIG. 3  is a schematic illustration of the fusion protein encoded by a subject nucleic acid. The encoded fusion protein contains an autonomously folding stable domain that is N-terminal to a GPCR. The protein further contains a signal sequence, an epitope tag and a protease cleavage site. 
         FIG. 4  shows exemplary sequences that may be employed in place of the lysozyme sequences of  FIG. 5 . From top to bottom, SEQ ID NOS: 2-6. 
         FIG. 5  shows the amino acid sequence of an exemplary fusion protein. SEQ ID NO:1. The HA signal peptide is shown in unbolded italic letters; the FLAG epitope tag is shown in underlined letters; the TEV recognition sequence is marked with non-underlined bold letters and the cleavage site is shown in asterisk. The full length T4L is shown by bold underlined letters and the β 2 AR sequence from Asp29 to Gly365 is shown by bold, underlined, italicized letters. 
         FIG. 6  shows the amino acid sequences of further exemplary fusion proteins. SEQ ID NOS: 7-13. The HA signal peptide is shown in unbolded italic letters; the FLAG epitope tag is shown in underlined letters; the TEV recognition sequence is marked with non-underlined bold letters and the cleavage site is shown in asterisk. The full length T4L is shown by bold underlined letters and the GPCR sequence is shown by bold, underlined, italicized letters. 
         FIG. 7 . G protein cycle for the β 2 AR-Gs complex. a, Extracellular agonist binding to the β 2 AR leads to conformational rearrangements of the cytoplasmic ends of transmembrane segments that enable the G s  heterotrimer (α, β, and γ) to bind the receptor. GDP is released from the α subunit upon formation of R:G complex. The GTP binds to the nucleotide-free α subunit resulting in dissociation of the α and βγ subunits from the receptor. The subunits regulate their respective effector proteins adenylyl cyclase (AC) and Ca 2+  channels. The G s  heterotrimer reassembles from α and βγ subunits following hydrolysis of GTP to GDP in the α subunit. b, The purified nucleotide-free β 2 AR-Gs protein complex maintained in detergent micelles. The Gsα subunit consists of two domains, the Ras domain (αRas) and the α-helical domain (αAH). Both are involved in nucleotide binding. In the nucleotide-free state, the αAH domain has a variable position relative the αRas domain. 
         FIG. 8 . Overall structure of the β 2 AR Gs complex. a, Lattice packing of the complex shows alternating layers of receptor and G protein within the crystal. Abundant contacts are formed among proteins within the aqueous layers. b, The overall structure of the asymmetric unit contents shows the β 2 AR bound to an agonist (spheres) and engaged in extensive interactions with Gsα. Gαs together with Gβ and Gγ constitute the heterotrimeric G protein Gs. A Gs binding nanobody binds the G protein between the α and β subunits. The nanobody (Nb35) facilitates crystallization, as does T4 lysozyme fused to the amino terminus of the β 2 AR. c, The biological complex omitting crystallization aids, showing its location and orientation within a cell membrane. 
         FIG. 9 . Comparison of active and inactive β 2 AR structures. a, Side and cytoplasmic views of the β 2 AR-Gs structure compared to the inactive carazolol-bound β 2 AR structure (blue). Significant structural changes are seen for the intracellular domain of TM5 and TM6. TM5 is extended by two helical turns while TM6 is moved outward by 14 Å as measured at the α-carbons of Glu268 in the two structures. b, β 2 AR-Gs compared with the nanobody-stabilized active state β 2 AR-Nb80 structure  12 c and d, The positions of residues in the E/DRY and NPxxY motifs and other key residues of the β 2 AR-Gs and β 2 AR-Nb80 structures as seen from the cytoplasmic side. All residues occupy very similar positions except Arg131 which in the β 2 AR-Nb80 structure interacts with the nanobody. 
         FIG. 10 . Receptor-G protein interactions. a, b The α5-helix of Gαs docks into a cavity formed on the intracellular side of the receptor by the opening of transmembrane helices 5 and 6. a. Within the transmembrane core, the interactions are primarily non-polar. An exception involves packing of Tyr391 of the α5-helix against Arg131 of the conserved DRY sequence in TM3 (see also  FIG. 15 ). Arg131 also packs against Tyr of the conserved NPxxY sequence in TM7. b. As α5-helix exits the receptor it forms a network of polar interactions with TM5 and TM3. c, Receptor residues Thr68 and Asp 130 interact with the IL2 helix of the β 2 AR via Tyr141, positioning the helix so that Phe139 of the receptor docks into a hydrophobic pocket on the G protein surface, thereby structurally linking receptor-G protein interactions with the highly conserved DRY motif of the β 2 AR. 
         FIG. 11 . Conformational changes in Gαs. a, A comparison of G s in the β 2 AR-Gs complex with the GTPγS-bound Gαs (PDB ID: 1AZT). GTPγS is shown as spheres. The helical domain of Gαs (GαsAH) exhibits a dramatic displacement relative to its position in the GTPγS-bound state. b, The α5-helix of Gαs is rotated and displaced toward the β 2 AR, perturbing the β6-α5 loop which otherwise forms part of the GTPγS binding pocket. c, The β1-α1 loop (P-loop) and β6-α5 loop of Gαs interact with the phosphates and purine ring, respectively, of GTPγS in the GTPγS-G s structure. d, The β1-α1 and β6-α5 loops are rearranged in the nucleotide-free β 2 AR-Gs structure. 
         FIG. 12 . Proposed model for structural changes causing GDP release from the R:G complex. a, Alignment of the TM segments of β 2 AR in the β 2 AR-Gs structure and metarhodopsin II  24 (PDB ID: 3PQR) (purple) bound with the C-terminal peptide of transducin (blue). b, The C-terminal end of G sRas domain from the GTPγS bound Gs structure  22  (PDB ID: 1AZT) is aligned with the C-terminal peptide of transducin. The C-terminal end of the α5-helix was moved away from the rest of the G sRas domain to avoid clashes with the β 2 AR. c, Cartoon of the β 2 AR-G s peptide fusion construct used in the binding experiments (d). d, Competition binding experiments between [ 3 H]-DHA and full agonist isoproterenol. Top panel shows binding data (reproduced from Rasmussen et al., 2011) on β 2 AR reconstituted in HDL particles with and without Gs heterotrimer. The fraction of β 2 AR in the K i high  state for the β 2 AR with Gs is 0.55. Bottom panel shows binding to β 2 AR and a β 2 AR-Gαs peptide fusion expressed in Sf9 cell membranes. The fraction of β 2 AR in the K i high  state for the β 2 AR-Gαs peptide fusion is 0.68. e, Same view as (b) but with metarhodopsin II structure and the C-terminal peptide removed. f, Comparison of G sRas domains of the transducin peptide aligned GTPγS bound Gs structure and the nucleotide-free Gs heterotrimer of the β 2 AR-Gs complex. 
         FIG. 13 . Effect of nucleotide analogs, pH, and nanobodies on the stability of the R:G complex. a) Analytical gel filtration showing that nucleotides GDP and GTPγS (0.1 mM) cause dissociation of the R:G complex. b) The phosphates pyrophosphate and foscarnet (used at 5 mM) resemble the nucleotide phosphate groups, but do not cause disruption of the complex. When used as additives they improved crystal growth of both the T4L-β2AR:Gs complex (without nanobodies), T4L-β2AR:Gs:Nb37, and T4L-β2AR:Gs:Nb35. c) The pH limit was determined to guide the preparation of crystallization screens. For the same purpose the effect of ionic strength (data not shown) was determined using NaCl at various concentrations. The complex is stable in 20, 100, and 500 mM but dissociates at 2.5 M NaCl. d) Nanobody 35 (Nb35, broken line) binds to the R:G complex (solid line) to form the R:G:Nb35 complex (red solid line) which is insensitive to GTP° S treatment (solid line) in contrast to the treated R:G complex alone (broken line). Nb35 and Nb37 binds separate epitopes on the Gs heterotrimer to form a R:G:Nb35:Nb37 complex (solid line). Nb37 binding also prevents GTP° S from dissociating the R:G complex (data not shown). 
         FIG. 14 . Crystals of the T4L-β2AR:Gs:Nb35 complex in sponge-like mesophase 
         FIG. 15 . Views of electron density for residues in the R:G interface. a) The D/ERY motif at the cytoplasmic end of TM3. b) Packing interaction between Arg131 of the E/DRY motif and Tyr391 of C-terminal Gsα. c) The NPxxY in the cytoplasmic end of TM7. d) Interactions of Thr68 and Tyr141 with Asp130 of the E/DRY motif. Phe139 of IL2 is buried in a hydrophobic pocket in Gsα. e) The β1-α1 loop (P-loop) of Gsα involved in nucleotide binding. Electron density maps are 2Fo-Fc maps contoured at 1 sigma. 
         FIG. 16 . Flow-chart of the purification procedures for preparing R:G complex with Nb35 
         FIG. 17 . Purity and homogeneity of the R:G complex: a) Analytical SDS-PAGE/Coomassie blue stain of samples obtained at various stages of receptor-G protein purification. BI167107 agonist bound, dephosphorylated, and deglycosylated receptor is used in excess of Gs heterotrimer for optimal coupling efficiency with the functional fraction of the G protein. Functional purification of Gs is archived through its interaction with the immobilized receptor on the M1 resin while non-functional/non-binding Gs is not retained. b) A representative elution profile of one of four consecutive preparative size exclusion chromatography (SEC) runs with fractionation indicated in red. SEC fractions containing the R:G complex (within the indicated dashed lines) were pooled, spin concentrated, and analyzed for purity and homogeneity by SDS-PAGE/Coomassie blue (a, lane 6), gel filtration (c), and by anion exchange chromotography (d). d) Upper panel shows elution proÿle from an analytical ion exchange chromatography (IEC) run of β2AR-365:Gs complex that was treated with λ phosphatase prior to complex formation. Lower panel shows IEC of complex which was not dephosphorylated resulting in a heterogeneous preparation. Off-peak fractions from the preparative SEC (b) were used for analytical gel filtration experiments shown in  FIGS. 13 and 21 . 
         FIG. 18 . Purification of Nb35 and determination of R:G:Nb mixing ratios a) Preparative ion exchange chromatography following nickel affinity chromatography purification of Nb35. The nanobody eluted in two populations (shown in red) as a minor peak and a major homogeneous peak which was collected, spin concentrated, and used for crystallography following determination of proper mixing ratio with the R:G complex as shown in (b). b) The R:G complex was mixed with slight excess of Nb35 (1 to 1.2 molar ratio of R:G complex to Nb35) on the basis of their protein concentrations and verified by analytical gel filtration. 
         FIG. 19 . Formation of a stable R:G complex. A stable complex was achieved by the combined effects of: 1) binding a high affinity agonist to the receptor with an extremely slow dissociation rate (as described in Rasmussen et al., 2011); 2) formation of a nucleotide free complex in the presence of apyrase that hydrolyses released GDP preventing it from rebinding and causing a less stable R:G interaction; and 3) detergent exchange of DDM for MNG-3 that stabilizes the complex. 
         FIG. 20 . Stabilizing effect of MNG-3 on the R:G complexes a) Analytical gel filtration of R:G complexes purified in DDM (in black), MNG-3 (in blue), or two MNG-3 analogs (in red and green) following incubation for 48 hrs at 4° C. In contrast to DDM, the R:G complexes are stable in the MNG detergents. b) Effect of diluting unliganded purified β2AR in either DDM or MNG-3 below the critical micelle concentration (CMC) of the detergent. Functional activity of the receptor was determined by 3H-dihydro alprenolol (3H-DHA) saturation binding. Diluting 2AR maintained in DDM by 1000-fold below the CMC cause loss in 3H-DHA binding (black data points) after 20 sec. In contrast, β2AR in MNG-3 diluted 1000-fold below the CMC maintained full ability to bind 3H-DHA after 24 hrs. 
         FIG. 21 . Effect of alkylating and reducing agents on the stability and aggregation of the R:G complex. a) Disulfide-mediated aggregation of the R:G complex was observed by size exclusion chromatography (SEC) following incubation at 0° C. for 7 days in buffer containing 0.1 mM tris(2-carboxyethyl)phosphine (TCEP). b) Treatment of the complex with iodoacetamide (5 mM for 20 hrs at 20° C.) led to dissociation of the complex. Alkylating free cysteines with iodoacetic acid and cadmium chloride also led to dissociation. c) Disulfide-mediated aggregation of the complex could be prevented by higher concentrations of reducing agents. Shown are the effects of 0.1, 1, and 10 mM TCEP for 1 hr at 20° C., or 10 mM betamercaptoethanol (β-ME, 1 hr at 20° C.). Crystallization setups were performed using 1 to 5 mM TCEP, which was essential for optimal crystal growth. 
         FIG. 22 . a. shows a schematic diagram of T4L-β 2 AR-ΔICL3 fusion protein used for crystallography, in including the β 2 AR residues, the wild type β 2 AR sequence, the HA signal peptide, the FLAG tag, the TEV recognition site the M96T, M98T mutations, the cysteines involved in disulfide bonds, disulfide bond linkages, the N187E mutation, and the 2-Ala linker. b. shows a chematic diagram of all of the T4L-β 2 AR-ΔICL3 constructs that were generated and evaluated for expression of functional receptor protein in insect cells. SEQ ID NOS: 18-29. 
         FIG. 23 . a, b. Packing interactions mediated by T4L. Each T4L packs against three adjacent T4L-β 2 AR-ΔICL3 molecules and is involved in 4 packing interactions. The T4L and β 2 AR-Δ-ICL3 from the reference molecule are shown. The T4L and β 2 AR-Δ-ICL3 from the three adjacent molecules are shown. c-f. Close-up few of packing interactions 1-4. The residues involved in interactions are shown as spheres c. In interaction 1 the reference T4L packs against ECL2 of its fused β 2 AR-Δ-ICL3. d. In interaction 2 the reference T4L packs against T4L of an adjacent T4L-β 2 AR-Δ-ICL3. e. In interaction 3 the reference T4L packs against T4L, ECL2 and ECL3 of a second adjacent T4L-β 2 AR-Δ-ICL3. f. In interaction 4 the reference T4L packs against ICL3 and helix 8 of a third T4L-β 2 AR-Δ-ICL3. 
         FIG. 24 . a. The crystal structure of the β 2 AR-Gs complex. The T4L, β 2 AR and the G-protein heterotrimer are shown in grey, as is the stabilizing nanobody. There is no packing interaction between the T4L and its fused β 2 AR. b. The crystal structure of T4L-β 2 AR-ΔICL3. The T4L is shown in red and its fused β 2 AR-ΔICL3 is shown. In contrast to the β 2 AR-Gs complex structure, there are packing interactions between the T4L and its fused receptor β 2 AR-ΔICL3. 
         FIG. 25 . a. Saturation binding curves for antagonist dihydroalprenolol (DHA) binding to T4L-β 2 AR-ΔICL3 and the wild type β 2 AR365. b. Competition binding curves for agonist isopreterenol binding to T4L-β 2 AR-ΔICL3 and the wild type β 2 AR365. 
         FIG. 26 . 2Fo-Fc map around the 2-Ala linker between T4L and the β 2 AR. The main chain of the fusion junction is shown in sticks. The electron density is shown in green mesh (1σ). The T4L and β 2 AR-ΔICL3 are shown in grey, as is the 2-Ala linker. 
         FIG. 27 . a. The superposed structures of the T4L-β 2 AR-ΔICL3 and the β 2 AR-T4L (pdb 2RH1). The T4L-β 2 AR-ΔICL3 and the β 2 AR-T4L are shown in grey. b. The extracellular side view of the superposed structures. c. The intracellular side view of the superposed structures. d. ICL2 in the β 2 AR-Fab5 structure (pdb 2R4R). e. ICL2 in the β 2 AR-T4L structure (pdb 2RH1). f. ICL2 in the T4L-β 2 AR-ΔICL3 structure. g. ICL2 in the structure of β 2 AR stabilized by Nb80 (pdb 3P0G) and h. ICL2 in the β 2 AR-Gs structure (pdb 3SN6) 
         FIG. 28 . Shows a model of β2AR bound to salmeterol, a partial agonist that is used to treat asthma. The partial-active state is stabilized by nanobody 71. 
     
    
    
     Certain of the figures described above are shown in color in U.S. provisional application Ser. Nos. 61/453,020, filed Mar. 15, 2011 and 61/507,425, filed Jul. 13, 2011. Those color figures, the brief description of those figures, and all references to color figures in those applications are incorporated by reference herein. 
     DEFINITIONS 
     Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., D ICTIONARY OF  M ICROBIOLOGY AND  M OLECULAR  B IOLOGY , 2 D  E D ., John Wiley and Sons, New York (1994), and Hale &amp; Marham, T HE  H ARPER  C OLLINS  D ICTIONARY OF  B IOLOGY , Harper Perennial, N.Y. (1991) provide one of skill with general dictionaries of many of the terms used in this disclosure. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. 
     All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference. 
     Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. 
     The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole. 
     “G-protein coupled receptors”, or “GPCRs” are polypeptides that share a common structural motif, having seven regions of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans a membrane. As illustrated in  FIG. 1 , each span is identified by number, i.e., transmembrane-1 (TM1), transmembrane-2 (TM2), etc. The transmembrane helices are joined by regions of amino acids between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or “extracellular” side, of the cell membrane, referred to as “extracellular” regions 1, 2 and 3 (EC1, EC2 and EC3), respectively. The transmembrane helices are also joined by regions of amino acids between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or “intracellular” side, of the cell membrane, referred to as “intracellular” regions 1, 2 and 3 (IC1, IC2 and IC3), respectively. The “carboxy” (“C”) terminus of the receptor lies in the intracellular space within the cell, and the “amino” (“N”) terminus of the receptor lies in the extracellular space outside of the cell. GPCR structure and classification is generally well known in the art, and further discussion of GPCRs may be found in Probst, DNA Cell Biol. 1992 11:1-20; Marchese et al Genomics 23: 609-618, 1994; and the following books: Jürgen Wess (Ed) Structure-Function Analysis of G Protein-Coupled Receptors published by Wiley-Liss (1st edition; Oct. 15, 1999); Kevin R. Lynch (Ed) Identification and Expression of G Protein-Coupled Receptors published by John Wiley &amp; Sons (March 1998) and Tatsuya Haga (Ed), G Protein-Coupled Receptors, published by CRC Press (Sep. 24, 1999); and Steve Watson (Ed) G-Protein Linked Receptor Factsbook, published by Academic Press (1st edition; 1994). A schematic representation of a typical GPCR is shown in  FIG. 1 . 
     The term “naturally-occurring” in reference to a GPCR means a GPCR that is naturally produced (for example and not limitation, by a mammal or by a human). Such GPCRs are found in nature. The term “non-naturally occurring” in reference to a GPCR means a GPCR that is not naturally-occurring. Wild-type GPCRs that have been made constitutively active through mutation, and variants of naturally-occurring GPCRs, e.g., epitope-tagged GPCR and GPCRs lacking their native N-terminus are examples of non-naturally occurring GPCRs. Non-naturally occurring versions of a naturally occurring GPCR are activated by the same ligand as the naturally-occurring GPCR. 
     The term “ligand” means a molecule that specifically binds to a GPCR. A ligand may be, for example a polypeptide, a lipid, a small molecule, an antibody. A “native ligand” is a ligand that is an endogenous, natural ligand for a native GPCR. A ligand may be a GPCR “antagonist”, “agonist”, “partial agonist” or “inverse agonist”, or the like. 
     A “modulator” is a ligand that increases or decreases a GPCR intracellular response when it is in contact with, e.g., binds, to a GPCR that is expressed in a cell. This term includes agonists, including partial agonists and inverse agonists, and antagonists. 
     A “deletion” is defined as a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent as compared to an amino acid sequence or nucleotide sequence of a parental GPCR polypeptide or nucleic acid. In the context of a GPCR or a fragment thereof, a deletion can involve deletion of about 2, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids. A GPCR or a fragment thereof may contain more than one deletion. 
     An “insertion” or “addition” is that change in an amino acid or nucleotide sequence which has resulted in the addition of one or more amino acid or nucleotide residues, respectively, as compared to an amino acid sequence or nucleotide sequence of a parental GPCR. “Insertion” generally refers to addition to one or more amino acid residues within an amino acid sequence of a polypeptide, while “addition” can be an insertion or refer to amino acid residues added at an N- or C-terminus, or both termini. In the context of a GPCR or fragment thereof, an insertion or addition is usually of about 1, about 3, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids. A GPCR or fragment thereof may contain more than one insertion. Reference to particular GPCR or group of GPCRs by name, e.g., reference to the serotonin or histamine receptor, is intended to refer to the wild type receptor as well as active variants of that receptor that can bind to the same ligand as the wild type receptor and/or transduce a signal in the same way as the wild type receptor. 
     A “substitution” results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental GPCR or a fragment thereof. It is understood that a GPCR or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on GPCR activity. By conservative substitutions is intended combinations such as gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. 
     The term “biologically active”, with respect to a GPCR, refers to a GPCR having a biochemical function (e.g., a binding function, a signal transduction function, or an ability to change conformation as a result of ligand binding) of a naturally occurring GPCR. 
     As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations. Reference to an “amount” of a GPCR in these contexts is not intended to require quantitative assessment, and may be either qualitative or quantitative, unless specifically indicated otherwise. 
     The terms “polypeptide” and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. 
     The term “fusion protein” or grammatical equivalents thereof is meant a protein composed of a plurality of polypeptide components, that while typically unjoined in their native state, are joined by their respective amino and carboxyl termini through a peptide linkage to form a single continuous polypeptide. Fusion proteins may be a combination of two, three or even four or more different proteins. The term polypeptide includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; fusion proteins with detectable fusion partners, e.g., fusion proteins including as a fusion partner a fluorescent protein, β-galactosidase, luciferase, etc.; and the like. 
     The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular. 
     The terms “antibodies” and “immunoglobulin” include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The antibodies may be detectably labeled, e.g., with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like. The antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like. The antibodies may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like. Also encompassed by the terms are Fab′, Fv, F(ab′) 2 , and or other antibody fragments that retain specific binding to antigen. 
     Antibodies may exist in a variety of other forms including, for example, Fv, Fab, and (Fab′) 2 , as well as bi-functional (i.e. bi-specific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), which are incorporated herein by reference). (See, generally, Hood et al., “Immunology”, Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986),). This term also encompasses so-called “phage display” antibodies. 
     A “monovalent” antibody is an antibody that has a single antigen binding region. Fab fragments, scFv antibodies, and phage display antibodies are types of monovalent antibodies, although others are known. A “Fab” fragment of an antibody has a single binding region, and may be made by papain digestion of a full length monoclonal antibody. A single chain variable (or “scFv”) fragment of an antibody is an antibody fragment containing the variable regions of the heavy and light chains of immunoglobulins, linked together with a short flexible linker. 
     As used herein the term “isolated,” when used in the context of an isolated compound, refers to a compound of interest that is in an environment different from that in which the compound naturally occurs. “Isolated” is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified. 
     As used herein, the term “substantially pure” refers to a compound that is removed from its natural environment and is at least 60% free, at least 75% free, or at least 90% free from other components with which it is naturally associated. 
     A “coding sequence” or a sequence that “encodes” a selected polypeptide, is a nucleic acid molecule which can be transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, for example, in a host cell when placed under the control of appropriate regulatory sequences (or “control elements”). The boundaries of the coding sequence are typically determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and synthetic DNA sequences. A transcription termination sequence may be located 3′ to the coding sequence. Other “control elements” may also be associated with a coding sequence. A DNA sequence encoding a polypeptide can be optimized for expression in a selected cell by using the codons preferred by the selected cell to represent the DNA copy of the desired polypeptide coding sequence. 
     “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. In the case of a promoter, a promoter that is operably linked to a coding sequence will effect the expression of a coding sequence. The promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence. 
     By “nucleic acid construct” it is meant a nucleic acid sequence that has been constructed to comprise one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, and the like. 
     A “vector” is capable of transferring gene sequences to a host cell. Typically, “vector construct,” “expression vector,” and “gene transfer vector,” mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to host cells, which can be accomplished by genomic integration of all or a portion of the vector, or transient or inheritable maintenance of the vector as an extrachromosomal element. Thus, the term includes cloning, and expression vehicles, as well as integrating vectors. 
     An “expression cassette” comprises any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a promoter of the expression cassette. Such cassettes can be constructed into a “vector,” “vector construct,” “expression vector,” or “gene transfer vector,” in order to transfer the expression cassette into a host cell. Thus, the term includes cloning and expression vehicles, as well as viral vectors. 
     A first polynucleotide is “derived from” or “corresponds to” a second polynucleotide if it has the same or substantially the same nucleotide sequence as a region of the second polynucleotide, its cDNA, complements thereof, or if it displays sequence identity as described above. 
     A first polypeptide is “derived from” or “corresponds to” a second polypeptide if it is (i) encoded by a first polynucleotide derived from a second polynucleotide, or (ii) displays sequence identity to the second polypeptides as described above. 
     The term “autonomously folding stable domain” is intended to exclude the amino acid sequence of a reporter protein, e.g., an optically detectable protein such as a fluorescent protein (e.g., GFP, CFP or YFP) or luciferase, and also excludes amino acid sequences that are at least 90% identical to the extracellular of a naturally occurring GPCR. 
     The term “active form” or “native state” of a protein is a protein that is folded in a way so as to be active. A GPCR is in its active form if it can bind ligand, alter conformation in response to ligand binding, and/or transduce a signal which may or may not be induced by ligand binding. An active or native protein is not denatured. 
     The term “stable domain” is a polypeptide domain that, when folded in its active form, is stable, i.e., does not readily become inactive or denatured. 
     The term “folds autonomously” indicates a protein that folds into its active form in a cell, without biochemical denaturation and renaturation of the protein, and without chaperones. 
     The term “naturally-occurring” refers to an object that is found in nature. 
     The term “non-naturally-occurring” refers to an object that is not found in nature. 
     The term “heterologous”, in the context of two things that are heterologous to one another, refers to two things that do not exist in the same arrangement in nature. 
     The term “signal sequence” or “signal peptide” refers to a sequence of amino acids at the N-terminal portion of a protein, which facilitates the secretion of the mature form of the protein through the plasma membrane. The mature form of the protein lacks the signal sequence which is cleaved off during the secretion process. 
     DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS 
     In the following description, the fusion protein is described first, followed by a discussion of the crystallization method in which the fusion protein may be employed. 
     Fusion Proteins 
     As noted above, a subject fusion protein comprise: a) GPCR; and b) an autonomously folding stable domain, where the autonomously folding stable domain is N-terminal to the GPCR and is heterologous to the GPCR. The autonomously folding stable domain is believed to provide a polar surface for crystal lattice contacts on the extracellular surface of the protein, thereby allowing the fusion protein to be crystallized. In particular embodiments, the protein is characterized in that is crystallizable under lipidic cubic phase crystallization conditions, although other crystallization conditions may be employed. A polar surface for crystal lattice contacts on the extracellular surface of the protein provides several options for crystallizing the fusion protein. In one embodiment, the fusion protein may be crystallized as a complex with the G-protein to which the GPCR couples. In another embodiment, the protein may be crystallized as a complex with an monovalent antibody that binds to the IC3 loop of the GPCR, as described in published US patent application US20090148510 and by Rasmusson et al (Nature 2007 450: 383-388), which publications are incorporated by reference for disclosure of those methods. In another embodiment, the third intracellular loop of the GPCR may contain another autonomously folding stable domain (which may be the same as or different to the autonomously folding stable domain at the N-terminal end of the protein) as described in Rosenbaum et al (Science 2007 318: 1266-73) and published U.S. patent application US20090118474, which publications are incorporated by reference for disclosure of those methods 
     In very general terms, such a fusion protein may be made by substituting the N-terminal extracellular region of a GPCR with an autonomously folding stable protein that is globular and readily crystallizable, e.g., lysozyme, chitinase, glucose isomerase, xylanase, trypsin inhibitor, crambin or ribonuclease, for example. During crystallization, the autonomously folding stable domain is thought to provides a polar surface for crystal lattice contacts on the extracellular surface of the protein, thereby facilitating crystallization of the protein. 
     As will be described in greater detail below, the GPCR fusion protein may be produced using a nucleic acid encoding a longer protein that, in order from N- to C-terminus, contains a signal peptide, an epitope tag and a protease cleavage site and the GPCR fusion protein. The longer protein is produced in the cell. During secretion, the signal peptide is cleaved from the protein and the resulting protein can be purified using the epitope tag. The epitope tag can be cleaved from the GPCR fusion protein prior use. Various signal peptides, epitope tags and protease cleavage sites and methods for their use are known in the art. 
     GPCRs 
     Any known GPCR is suitable for use in the subject method. A disclosure of the sequences and phylogenetic relationships between 277 GPCRs is provided in Joost et al. (Genome Biol. 2002 3:RESEARCH0063, the entire contents of which is incorporated by reference) and, as such, at least 277 GPCRs are suitable for the subject methods. A more recent disclosure of the sequences and phylogenetic relationships between 367 human and 392 mouse GPCRs is provided in Vassilatis et al. (Proc Natl Acad Sci 2003 100:4903-8 and www.primalinc.com, each of which is hereby incorporated by reference in its entirely) and, as such, at least 367 human and at least 392 mouse GPCRs are suitable for the subject methods. GPCR families are also described in Fredriksson et al (Mol. Pharmacol. 2003 63, 1256-72). 
     The methods may be used, by way of exemplification, for purinergic receptors, vitamin receptors, lipid receptors, peptide hormone receptors, non-hormone peptide receptors, non-peptide hormone receptors, polypeptide receptors, protease receptors, receptors for sensory signal mediator, and biogenic amine receptors not including β2-adrenergic receptor. In certain embodiments, said biogenic amine receptor does not include an adrenoreceptor. α-type adrenoreceptors (e.g. α 1A , α 1B  or α 1C  adrenoreceptors), and β-type adrenoreceptors (e.g. β 1 , β 2 , or β 3  adrenoreceptors) are discussed in Singh et al., J. Cell Phys. 189:257-265, 2001. 
     It is recognized that both native (naturally occurring) and altered native (non-naturally occurring) GPCRs may be used in the subject methods. In certain embodiments, therefore, an altered native GPCR (e.g. a native GPCR that is altered by an amino acid substitution, deletion and/or insertion) such that it binds the same ligand as a corresponding native GPCR, and/or couples to a G-protein as a result of the binding. In certain cases, a GPCR employed herein may have an amino acid sequence that is at least 80% identical to, e.g., at least 90% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical, to at least the heptahelical domain of a naturally occurring GPCR. A GPCR employed herein may optionally contain the C-terminal domain of a GPCR. In certain embodiments, a native GPCR may be “trimmed back” from its N-terminus and/or its C-terminus to leave its heptahelical domain, prior to use. 
     As such, the following GPCRs (native or altered) find particular use as parental GPCRs in the subject methods: cholinergic receptor, muscarinic 3; melanin-concentrating hormone receptor 2; cholinergic receptor, muscarinic 4; niacin receptor; histamine 4 receptor; ghrelin receptor; CXCR3 chemokine receptor; motilin receptor; 5-hydroxytryptamine (serotonin) receptor 2A; 5-hydroxytryptamine (serotonin) receptor 2B; 5-hydroxytryptamine (serotonin) receptor 2C; dopamine receptor D3; dopamine receptor D4; dopamine receptor D1; histamine receptor H2; histamine receptor H3; galanin receptor 1; neuropeptide Y receptor Y1; angiotensin II receptor 1; neurotensin receptor 1; melanocortin 4 receptor; glucagon-like peptide 1 receptor; adenosine A1 receptor; cannabinoid receptor 1; and melanin-concentrating hormone receptor 1. 
     In particular embodiments, the GPCR may belong to one of the following GPCR families: amine, peptide, glycoprotein hormone, opsin, olfactory, prostanoid, nucleotide-like, cannabinoid, platelet activating factor, gonadotropin-releasing hormone, thyrotropin-releasing hormone or melatonin families, as defined by Lapinsh et al (Classification of G-protein coupled receptors by alignment-independent extraction of principle chemical properties of primary amino acid sequences. Prot. Sci. 2002 11:795-805). The subject GPCR may be a family A GPCR (rhodopsin-like), family B GPCR (secretin-like, which includes the PTH and glucagon receptors), or a family C GPCR (glutamate receptor-like, which includes the GABA glutamate receptors), or an “other” family GPCR (which includes adhesion, frizzled, taste type-2, and unclassified family members). 
     In the subject methods, the N-terminal extracellular region N-terminal to the TM1 region of a GPCR is usually identified, and replaced with an autonomously folding stable domain to produce a fusion protein. A schematic representation of the prototypical structure of a GPCR is provided in  FIG. 1 , where these regions, in the context of the entire structure of a GPCR, may be seen. A schematic representation of a subject fusion protein is shown in  FIG. 2 . 
     The N-terminal extracellular region is readily discernable by one of skill in the art using, for example, a program for identifying transmembrane regions: once transmembrane region TM1 is identified, the N-terminal extracellular region will be apparent. The N-terminal extracellular region may also be identified using such methods as pairwise or multiple sequence alignment (e.g. using the GAP or BESTFIT of the University of Wisconsin&#39;s GCG program, or CLUSTAL alignment programs, Higgins et al., Gene. 1988 73:237-44), using a target GPCR and, for example, GPCRs of known structure. 
     Suitable programs for identifying transmembrane regions include those described by Moller et al., (Bioinformatics, 17:646-653, 2001). A particularly suitable program is called “TMHMM” Krogh et al., (Journal of Molecular Biology, 305:567-580, 2001). To use these programs via a user interface, a sequence corresponding to a GPCR or a fragment thereof is entered into the user interface and the program run. Such programs are currently available over the world wide web, for example at the website of the Center for Biological Sequence Analysis at cbs.dtu.dk/services/. The output of these programs may be variable in terms its format, however they usually indicate transmembrane regions of a GPCR using amino acid coordinates of a GPCR. 
     When TM regions of a GPCR polypeptide are determined using TMHMM, the prototypical GPCR profile is usually obtained: an N-terminus that is extracellular, followed by a segment comprising seven TM regions, and further followed by a C-terminus that is intracellular. TM numbering for this prototypical GPCR profile begins with the most N-terminally disposed TM region (TM1) and concludes with the most C-terminally disposed TM region (TM7). 
     In certain cases, once the N-terminal extracellular region is identified in a GPCR, a suitable region of amino acids is chosen for substitution with the amino acid sequence of the autonomously folding stable domain. In certain embodiments, the C-terminus of the autonomously folding stable domain is linked to the amino acid that is within 50 residues (e.g., e.g., 1-5, 1-10, 1-20, 1-30, 1-40, etc. residues) N-terminal to the N-terminal amino acid of the TM 1 region of the GPCR, although linkages outside of this region are envisioned. In one exemplary embodiment, amino acids that are at the N-terminal end of the TM1 region (i.e., within what would be referred to as the TM1 region) may be replaced in addition the amino acids that are N-terminal to the TM region. In particular embodiments, this junction may be optimized to provide for maximal expression and receptor activity. 
     In addition to substituting N-terminal extracellular region of a GPCR with a autonomously folding stable domain, as described above, in certain cases, the intracellular C-terminal region of the GPCR (which may C-terminal to the cysteine palmitoylation site that is approximately 10 to 25 amino acid residues downstream of a conserved NPXXY motif), may be deleted. In certain cases, the 20-30 amino acids immediately C-terminal to the cysteine palmitoylation site are not deleted. In particular embodiments, this position may be optimized to provide for maximal expression and receptor activity. 
     Autonomously Folding Stable Domains 
     In particular embodiments, the autonomously folding stable domain is a polypeptide than can fold autonomously in a variety of cellular expression hosts, and is resistant to chemical and thermal denaturation. In particular embodiments, the autonomously folding stable domains may be derived from a protein that is known to be highly crystallizable in a variety of space groups and crystal packing arrangements. In certain cases, the stable, folded protein insertion may also shield the fusion protein from proteolysis, and may itself be protease resistant. Lysozyme is one such polypeptide, however many others are known. 
     In certain embodiments, a autonomously folding stable domain of a subject fusion protein may be a soluble, stable protein (e.g., a protein displaying resistance to thermal and chemical denaturation) that folds autonomously of the GPCR portion of the fusion protein, in a cell. In certain cases, the stable, autonomously folding stable domain may have no cysteine residues (or may be engineered to have no cysteine residues) in order to avoid potential disulphide bonds between the autonomously folding stable domain and a GPCR portion of the fusion protein, or internal disulphide bonds. Autonomously folding stable domains are conformationally restrained, and are resistant to protease cleavage. 
     In certain cases, the autonomously folding stable domain may contain most or all of the amino acid sequence of a polypeptide that is readily crystallized. Such proteins may be characterized by a large number of deposits in the protein data bank (www.rcsb.org) in a variety of space groups and crystal packing arrangements. While examples that employ lysozyme as stable, folded protein insertion are discussed below, the general principles may be used to employ any of a number of polypeptides that have the characteristics discussed above. Autonomously folding stable domain candidates include those containing the amino acid sequence of proteins that are readily crystallized including, but not limited to: lysozyme, chitinase, glucose isomerase, xylanase, trypsin inhibitor, crambin, ribonuclease. Other suitable polypeptides may be found at the BMCD database (Gilliland et al 1994. The Biological Macromolecule Crystallization Database, Version 3.0: New Features, Data, and the NASA Archive for Protein Crystal Growth Data. Acta Crystallogr. D50 408-413), as published to the world wide web. 
     In certain embodiments, the autonomously folding stable domain used may be at least 80% identical (e.g., at least 85% identical, at least 90% identical, at least 95% identical or at least 98% identical to a wild type protein. Many suitable wild type proteins, including non-naturally occurring variants thereof, are readily crystalizable. 
     In one embodiment, the autonomously folding stable domain may be of the lysozyme superfamily, which share a common structure and are readily crystallized. Such proteins are described in, e.g., Wohlkönig et al (Structural Relationships in the Lysozyme Superfamily: Significant Evidence for Glycoside Hydrolase Signature Motifs. PLoS ONE 2010 5: e15388). 
     As noted above, one such autonomously folding stable domain that may be employed in a subject fusion protein is lysozyme. Lysozyme is a highly crystallizable protein (see, e.g., Strynadka et al Lysozyme: a model enzyme in protein crystallography EXS 1996 75: 185-222) and at present over 200 atomic coordinates for various lysozymes, including many wild-type lysozymes and variants thereof, including lysozymes from phage T4, human, swan, rainbow trout, guinea fowl, soft-shelled turtle, tapes japonica, nurse shark, mouse sperm, dog and phage P1, as well as man-made variants thereof, have been deposited in NCBI&#39;s structure database. A subject fusion protein may contain any of a wide variety of lysozyme sequences. See, e.g., Strynadka et al ( Lysozyme: a model enzyme in protein crystallography  (EXS. 1996; 75:185-222), Evrard et al ( Crystal structure of the lysozyme from bacteriophage lambda and its relationship with V and C - type lysozymes ) J. Mol. Biol. 1998 276:151-64), Forsythe et al ( Crystallization of chicken egg - white lysozyme from ammonium sulfate . Acta Crystallogr D Biol Crystallogr. 1997 53:795-7), Remington et al ( Structure of the Lysozyme from Bacteriophage T 4 : An Electron Density Map at  2.4 A Resolution ), Lyne et al ( Preliminary crystallographic examination of a novel fungal lysozyme from Chalaropsis . J Biol Chem. 1990 265:6928-30), Marana et al. ( Crystallization, data collection and phasing of two digestive lysozymes from Musca domestica . Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 62:750-2), Harada et al ( Preliminary X - ray crystallographic study of lysozyme produced by Streptomyces globisporus . J Mol Biol. 1989 207:851-2) and Yao et al ( Crystallization and preliminary X - ray structure analysis of pigeon egg - white lysozyme ). J. Biochem. 1992 111:1-3). 
     The length of the autonomously folding stable domain may be in the range of 50-500 amino acids, e.g., 80-200 amino acids in length, although autonomously folding stable domain having lengths outside of this range are also envisioned. 
     As noted above, the autonomously folding stable domain is not fluorescent or light-emitting. As such, the autonomously folding stable domain is not CFP, GFP, YFP, luciferase, or other light emitting, fluorescent variants thereof. In certain cases, a autonomously folding stable domain does not contain a flexible linker (e.g., a flexible polyglycine linker) or other such conformationally unrestrained regions. In certain cases, the autonomously folding stable domain contains a sequence of amino acids from a protein that has a crystal structure that has been solved. In certain cases, the stable, folded protein insertion should not have highly flexible loop region characterized by high cyrstallographic temperature factors (i.e., high B-factors). 
     An exemplary amino acid sequence for exemplary lysozyme fusion protein is set forth in  FIG. 5 , and the amino acid sequences of exemplary alternative additions (which may be substituted into any of the sequences of  FIG. 5  in place of the lysozyme sequence) are shown in  FIG. 4 . These sequences include the sequences of trypsin inhibitor, calbindin, barnase, xylanase, glucokinase or a cytochrome, e.g., cytochrome a, b or c, although other sequences can be readily used. In particular embodiments, any of the proteins listed in table 1 of Papandreou et al (Eur. J. Biochem. 271, 4762-4768 (2004) FEBS 2004) or any of the 674 globular proteins listed by Wang and Yuan (Proteins 2000 38, 165-175) (which publications are incorporated by reference for disclosure of individual proteins), including orthologs from other species and variants proteins that are at least 80% identical to the listed proteins. Exemplary sequences include those of apolipophorin-III, staphylococcal nuclease, RNAse sa, uteroglobin, xylanase II, glutaredoxin, myohemerythin, bacillus 1-3, 1-4-β-glucanase, orotate phosphoribosyltransferase, cytochrome b562, serine esterase, fructose permease, subunit IIb, fibritin, legume lectin, chloramphenicol acetyltransferase, cytochrome c oxidase, adenovirus fibre, flavodoxin, phospholipase a2, stnv coat protein, signal transduction protein, lysin, pseudoazurin, cutinase, retinoid-x receptor α, transthyretin, dihydropteridin reductase, cytochrome c3, picornavirus, ch-p21 ras, interleukin-10, cellular retinoic-acid-binding protein, retroviral integrase, catalytic domain, oncomodulin, 2 (hiv-2) protease, glutamate receptor ligand binding core, calcium-binding protein, histidine-containing phosphocarrier, cellulase e2, parvalbumin, ubiquitin, triosephosphate isomerase, myoglobin, 2fe-2s ferredoxin, endonuclease, glycera globin, lysozyme, goose, uracil-dna glycosylase, lamprey globin, lysozyme, chicken, lumazine synthase, hemoglobin (horse), profilin, hypothetical protein ybea, hemoglobin (human), ribosomal protein, d-tyr trnatyr deacylase, erythrocruorin, integrase, coagulation factor x, leukemia inhibitory factor, glycosylasparaginase, carboxypeptidase inhibitor, mitochondrial cytochrome c, astacin, mhc class II p41 invariantchain fragment, cytochrome c2, diphtheria toxin, methylamine dehydrogenase, phospholipase, nadh oxidase, ovomucoid iii domain, dna-binding protein, signal transduction protein, ldl receptor, pheromone, ferredoxin ii, peptostreptococcus, anti-platelet protein, phosphatidylinositol 3-kinase, ferredoxin ii, desulfovibrio gigas, crambin, α-spectrin, sh3 domain, 1c0ba ribonuclease a, heat-stable enterotoxin b, signal transduction protein, c-src tyrosine kinase, tgf-β3, seed storage protein 7 s vicillin, prion protein domain, rubredoxin, clostridium pasteurianum, immunoglobulin, abrin a-chain, rubredoxin, archaeon pyrococcus furiosus, cd2, first domain, platelet factor 4, fasciculin, macromycin, chemokine (growth factor), plasminogen, cohesin-2 domain, (pro)cathepsin b, ectothiorhodospira vacuolata, glucose-specific factor iii, actinidin, hipip, allochromatium vinosum, staphylococcal nuclease, chymotrypsin inhibitor CI-2, collagen type VI, dna-binding protein, fk-506 binding, and factor IX. 
     The amino acid sequences of a variety of exemplary GPCR fusion proteins that can be employed herein are set forth in  FIG. 6 . Given these sequences, suitable fusion proteins could be designed using other GPCR. 
     Nucleic Acids 
     A nucleic acid comprising a nucleotide sequence encoding a subject fusion protein is also provided. A subject nucleic acid may be produced by any method. Since the genetic code and recombinant techniques for manipulating nucleic acid are known, the design and production of nucleic acids encoding a subject fusion protein is well within the skill of an artisan. In certain embodiments, standard recombinant DNA technology (Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley &amp; Sons, 1995; Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.) methods are used. 
     For example, site directed mutagenesis and subcloning may be used to introduce/delete/substitute nucleic acid residues in a polynucleotide encoding GPCR. In other embodiments, PCR may be used. Nucleic acids encoding a polypeptide of interest may also be made by chemical synthesis entirely from oligonucleotides (e.g., Cello et al., Science (2002) 297:1016-8). 
     In certain embodiments, the codons of the nucleic acids encoding polypeptides of interest are optimized for expression in cells of a particular species, particularly a mammalian, e.g., human, species. Vectors comprising a subject nucleic acid are also provided. A vector may contain a subject nucleic acid, operably linked to a promoter. 
     A host cell (e.g., a host bacterial, mammalian, insect, plant or yeast cell) comprising a subject nucleic acid is also provided as well a culture of subject cells. The culture of cells may contain growth medium, as well as a population of the cells. The cells may be employed to make the subject fusion protein in a method that includes culturing the cells to provide for production of the fusion protein. In many embodiments, the fusion protein is directed to the plasma membrane of the cell, and is folded into its active form by the cell. 
     The native form of a subject fusion protein may be isolated from a subject cell by conventional technology, e.g., by precipitation, centrifugation, affinity, filtration or any other method known in the art. For example, affinity chromatography (Tilbeurgh et al., (1984) FEBS Lett. 16:215); ion-exchange chromatographic methods (Goyal et al., (1991) Biores. Technol. 36:37; Fliess et al., (1983) Eur. J. Appl. Microbiol. Biotechnol. 17:314; Bhikhabhai et al., (1984) J. Appl. Biochem. 6:336; and Ellouz et al., (1987) Chromatography 396:307), including ion-exchange using materials with high resolution power (Medve et al., (1998) J. Chromatography A 808:153; hydrophobic interaction chromatography (Tomaz and Queiroz, (1999) J. Chromatography A 865:123; two-phase partitioning (Brumbauer, et al., (1999) Bioseparation 7:287); ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; or size exclusion chromatography using, e.g., Sephadex G-75, may be employed. 
     In particular embodiments, the GPCR, e.g., the N- or C-terminus of the GPCR or an external loop of the GPCR, may be tagged with an affinity moiety, e.g., a his tag, GST, MBP, flag tag, or other antibody binding site, in order to facilitate purification of the GPCR fusion protein by affinity methods. Before crystallization, a subject fusion protein may be assayed to determine if the fusion protein is active, e.g., can bind ligand and change in conformation upon ligand binding, and if the fusion protein is resistant to protease cleavage. Such assays are well known in the art. 
     In particular embodiments and illustrated in  FIG. 3 , the protein encoded by the nucleic acid contains, from N-terminus to C-terminus: a) a signal sequence; b) an affinity, e.g., epitope, tag; c) a protease cleavage site; d) an autonomously folding stable domain; and e) a GPCR. During secretion, the signal peptide is cleaved from the protein and the resulting protein can be purified using the affinity tag. The affinity tag can be cleaved from the GPCR fusion protein prior use. 
     Crystallization Methods 
     Prior to crystallization, the isolated fusion protein may optionally be combined with a variety of moieties (e.g., an antibody (see, e.g., US20090148510, Rasmusson et al Nature 2007 450: 383-388 and Day et al Nature Methods 2007 4:927-9), a modulator (such as an agonist, an antagonist, a native ligand, etc., as described in, e.g., Rosenbaum Science. 2007 318:1266-73 etc), another GPCR, the G protein to which the GPCR couples or another protein, e.g., Gs, Gi, or Gq), that bind to the GPCR, to produce a complex. The complex is then crystallized and the atomic coordinates of the complex can be obtained. 
     A subject fusion protein may be crystallized using any of a variety of crystallization methods, many of which are reviewed in Caffrey Membrane protein crystallization. J Struct. Biol. 2003 142:108-32, including those that employ detergent micelles, bicelles and lipidic cubic phase (LCP). In general terms, the methods are lipid-based methods that include adding lipid to the fusion protein prior to crystallization. Such methods have previously been used to crystallize other membrane proteins. Many of these methods, including the lipidic cubic phase crystallization method and the bicelle crystallization method, exploit the spontaneous self-assembling properties of lipids and detergent as vesicles (vesicle-fusion method), discoidal micelles (bicelle method), and liquid crystals or mesophases (in meso or cubic-phase method). Lipidic cubic phases crystallization methods are described in, for example: Landau et al,  Lipidic cubic phases: a novel concept for the crystallization of membrane proteins . Proc. Natl. Acad. Sci. 1996 93:14532-5; Gouaux, It&#39;s not just a phase: crystallization and X-ray structure determination of bacteriorhodopsin in lipidic cubic phases. Structure. 1998 6:5-10; Rummel et al,  Lipidic Cubic Phases: New Matrices for the Three - Dimensional Crystallization of Membrane Proteins . J. Struct. Biol. 1998 121:82-91; and Nollert et al  Lipidic cubic phases as matrices for membrane protein crystallization  Methods. 2004 34:348-53, which publications are incorporated by reference for disclosure of those methods. Bicelle crystallization methods are described in, for example: Faham et al  Crystallization of bacteriorhodopsin from bicelle formulations at room temperature . Protein Sci. 2005 14:836-40. 2005 and Faham et al,  Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure . J Mol Biol. 2002 Feb. 8; 316(1):1-6, which publications are incorporated by reference for disclosure of those methods. 
     Computer Models and Computer Systems 
     In certain embodiments, the above-described computer readable medium may further comprise programming for displaying a molecular model of a GPCR or a complex of the same crystallized by the instant method, programming for identifying a compound that binds to the GPCR and/or a database of structures of known test compounds, for example. A computer system comprising the computer-readable medium is also provided. The model may be displayed to a user via a display, e.g., a computer monitor, for example. 
     The atomic coordinates may be employed in conjunction with a modeling program to provide a model of the a GPCR or a complex of the same. As used herein, the term “model” refers to a representation in a tangible medium of the three dimensional structure of the a GPCR or a complex of the same. For example, a model can be a representation of the three dimensional structure in an electronic file, on a display, e.g., a computer screen, on a piece of paper (i.e., on a two dimensional medium), and/or as a ball-and-stick figure. Physical three-dimensional models are tangible and include, but are not limited to, stick models and space-filling models. The phrase “imaging the model on a computer screen” refers to the ability to express (or represent) and manipulate the model on a computer screen using appropriate computer hardware and software technology known to those skilled in the art. Such technology is available from a variety of sources including, for example, Evans and Sutherland, Salt Lake City, Utah, and Biosym Technologies, San Diego, Calif. The phrase “providing a picture of the model” refers to the ability to generate a “hard copy” of the model. Hard copies include both motion and still pictures. Computer screen images and pictures of the model can be visualized in a number of formats including space-filling representations, backbone traces, ribbon diagrams, and electron density maps. Exemplary modeling programs include, but are not limited to PYMOL, GRASP, or O software, for example. 
     In another embodiment, the invention provides a computer system having a memory comprising the above-described atomic coordinates; and a processor in communication with the memory, wherein the processor generates a molecular model having a three dimensional structure representative of a GPCR or a complex of the same. The processor can be adapted for identifying a candidate compound having a structure that is capable of binding to the a GPCR or a complex of the same, for example. 
     In the present disclosure, the processor may execute a modeling program which accesses data representative of the GPCR structure. In addition, the processor also can execute another program, a compound modeling program, which uses the three-dimensional model of the GPCR or a complex of the same to identify compounds having a chemical structure that binds to the GPCR or a complex of the same. In one embodiment the compound identification program and the structure modeling program are the same program. In another embodiment, the compound identification program and the structure modeling program are different programs, which programs may be stored on the same or different storage medium. 
     A number of exemplary public and commercial sources of libraries of compound structures are available, for example the Cambridge Structural Database (CSD), the Chemical Directory (ACD) from the company MDL (US), ZINC (Irwin and Shoichet, J. Chem. Inf Model. (2005) 45:177-82) as well as various electronic catalogues of publicly available compounds such as the National Cancer Institute (NCI, US) catalogue, ComGenex catalogue (Budapest, Hungary), and Asinex (Moscow, Russia). Such libraries may be used to allow computer-based docking of many compounds in order to identify those with potential to interact with the GPCR using the atomic coordinates described herein. 
     In certain cases, the method may further comprise a testing a compound to determine if it binds and/or modulates the GPCR or a complex of the same, using the atomic coordinates provided herein. In some embodiments, the method may further comprise obtaining the compound (e.g., purchasing or synthesizing the compound) and testing the compound to determine if it modulates (e.g., activates or inhibits) the GPCR, e.g., acts an agonist, antagonist or inverse agonist of the GPCR). 
     In some embodiments, the method employs a docking program that computationally tests known compounds for binding to the GPCR or complex of the same. Structural databases of known compounds are known in the art. In certain cases, compounds that are known to bind and modulate the GPCR or complex of the same may be computationally tested for binding to GPCR or complex of the same, e.g., in order to identify a binding site and/or facilitate the identification of active variants of an existing compound. Such compounds include compounds that are known to be agonists of the GPCR. In other cases, the method may include designing a compound that binds to the GPCR, either de novo, or by modifying an existing compound that is known to bind to the GPCR. 
     A method that comprises receiving a set of atomic coordinates for the GPCR or complex of the same; and identifying a compound that binds to said GPCR or complex of the same using the coordinates is also provided, as is a method comprising: forwarding to a remote location a set of atomic coordinates for the GPCR or complex of the same; and receiving the identity of a compound that binds to the GPCR or complex of the same. 
     In certain embodiments, a computer system comprising a memory comprising the atomic coordinates of a GPCR or complex of the same is provided. The atomic coordinates are useful as models for rationally identifying compounds that bind to the GPCR or complex of the same. Such compounds may be designed either de novo, or by modification of a known compound, for example. In other cases, binding compounds may be identified by testing known compounds to determine if the “dock” with a molecular model of the GPCR. Such docking methods are generally well known in the art. 
     The structure data provided can be used in conjunction with computer-modeling techniques to develop models of ligand-binding sites on the GPCR or complex of the same selected by analysis of the crystal structure data. The site models characterize the three-dimensional topography of site surface, as well as factors including van der Waals contacts, electrostatic interactions, and hydrogen-bonding opportunities. Computer simulation techniques are then used to map interaction positions for functional groups including but not limited to protons, hydroxyl groups, amine groups, divalent cations, aromatic and aliphatic functional groups, amide groups, alcohol groups, etc. that are designed to interact with the model site. These groups may be designed into a candidate compound with the expectation that the candidate compound will specifically bind to the site. 
     The ability of a candidate compound to bind to a GPCR can be analyzed prior to actual synthesis using computer modeling techniques. Only those candidates that are indicated by computer modeling to bind the target with sufficient binding energy (i.e., binding energy corresponding to a dissociation constant with the target on the order of 10 −2  M or tighter) may be synthesized and tested for their ability to bind to and modulate the GPCR. Such assays are known to those of skill in the art. The computational evaluation step thus avoids the unnecessary synthesis of compounds that are unlikely to bind the GPCR with adequate affinity. 
     A candidate compound may be computationally identified by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with individual binding target sites on the GPCR. One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with the GPCR, and more particularly with target sites on the GPCR. The process may begin by visual inspection of, for example a target site on a computer screen, based on the coordinates, or a subset of those coordinates. Selected fragments or chemical entities may then be positioned in a variety of orientations or “docked” within a target site of the GPCR as defined from analysis of the crystal structure data. Docking may be accomplished using software such as Quanta (Molecular Simulations, Inc., San Diego, Calif.) and Sybyl (Tripos, Inc. St. Louis, Mo.) followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields such as CHARMM (Molecular Simulations, Inc., San Diego, Calif.) and AMBER (University of California at San Francisco). 
     Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include but are not limited to: GRID (Goodford, P. J., “A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules,” J. Med. Chem., 28, pp. 849-857 (1985)); GRID is available from Oxford University, Oxford, UK; MCSS (Miranker, A. and M. Karplus, “Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method,” Proteins: Structure, Function and Genetics, 11, pp. 29-34 (1991)); MCSS is available from Molecular Simulations, Inc., San Diego, Calif.; AUTODOCK (Goodsell, D. S. and A. J. Olsen, “Automated Docking of Substrates to Proteins by Simulated Annealing,” Proteins: Structure, Function, and Genetics, 8, pp. 195-202 (1990)); AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.; DOCK (Kunts, I. D., et al. “A Geometric Approach to Macromolecule-Ligand Interactions,” J. Mol. Biol., 161, pp. 269-288 (1982)); DOCK is available from University of California, San Francisco, Calif.; CERIUS II (available from Molecular Simulations, Inc., San Diego, Calif.); and Flexx (Raret, et al. J. Mol. Biol. 261, pp. 470-489 (1996)). 
     Also provided is a method of determining a crystal structure. This method may comprise receiving an above described fusion protein, crystallizing the fusion protein to produce a crystal; and obtaining atomic coordinates of the fusion protein from the crystal. The fusion protein may be received from a remote location (e.g., a different laboratory in the same building or campus, or from a different campus or city), and, in certain embodiments, the method may also comprise transmitting the atomic coordinates, e.g., by mail, e-mail or using the internet, to the remote location or to a third party. 
     In other embodiments, the method may comprise forwarding a fusion protein to a remote location where the protein may be crystallized and analyzed, and receiving the atomic coordinates of the fusion protein. 
     In some embodiments a method for displaying the three dimensional structure of a GPCR on a computer system is provided. This method may comprise: a) accessing a file containing atomic coordinates of a GPCR using a computer system that comprises a modeling program, wherein the atomic coordinates are produced by subjecting crystals of a GPCR fusion protein to X-ray diffraction analysis, wherein the GPCR fusion protein is described above, b) modeling the atomic coordinates on the computer system using the modeling program to produce a model of the three dimensional structure of at least a portion of the GPCR by; and c) displaying the model of the three dimensional structure on the computer system. The crystals also contain a ligand for the GPCR, and the method further comprises identifying the binding site for the ligand in the GPCR using the model. This method may further comprises identifying the amino acids in the binding site. This method may further comprise determining whether a test compound docks with the binding site using the model. This method may further comprise analyzing the packing between the test compound and surrounding amino acids in said binding site. In some embodiments, the analyzing may comprise calculating polar contacts between the ligand and the model. 
     In particular embodiments, a method for analyzing the three dimensional structure of a GPCR on a computer system is provided. This method may involve: a) accessing a file containing atomic coordinates of a GPCR using a computer system that comprises a modeling program, wherein the atomic coordinates are produced by subjecting crystals of a GPCR fusion protein to X-ray diffraction analysis, wherein the GPCR fusion protein is described above, b) modeling the atomic coordinates on the computer system using the modeling program to produce a model of the three dimensional structure of at least a portion of the GPCR.; and c) displaying the model of the three dimensional structure on the computer system. In certain cases, the crystals contain a ligand for the GPCR (e.g., a known inhibitor, natural ligand or agonist, etc.), and the method further comprises identifying the binding site for the ligand in the GPCR using the model. The analyzing step may comprise identifying amino acids that form polar contacts between the ligand and amino acids in the binding site, using the model. This method may further comprise determining whether a test compound, e.g., a candidate pharmaceutical, docks with the binding site using the model. The method may comprise analyzing the packing of the test compound and amino acids in the binding site, using the model. This method may further comprise making the modulator and testing it on the GPCR in the presence of a ligand for the GPCR. 
     In order to further illustrate the present invention, the following specific examples are given with the understanding that they are being offered to illustrate the present invention and should not be construed in any way as limiting its scope. 
     Materials, Methods and Results I 
     Molecular Biology for the Generation of N-T4L Fused β2AR Construct FLAAT 
     The previously generated construct β 2 AR365 was used as the template for further modification to generate the N-T4L fused β 2 AR construct FLAAT. In this β 2 AR365 template construct, the coding sequence of human β 2 AR encompassing Gly2 to Gly365 was cloned into the pFastbac1 Sf9 expression vector (Invitrogen). The HA signal peptide followed by FLAG epitope tag and tobacco etch virus (TEV) protease recognition sequence was directly added to the N-terminus of the receptor for expression and purification purpose. A point mutation of N187E was also introduced to the construct to disrupt this unwanted glycosylation site. 
     The DNA cassette encoding the full length T4L lysozyme (WT*, C54T, C97A) with 2 additional alanines attached at the C-terminus was made and amplified by PCR using previously described construct β 2 AR-T4L (Rasmussen et al. Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature. 2007 450:383 and Cherezov et al High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science. 2007 318:1258-65) as the template and synthetic oligonucleotides as primers. This cassette was inserted into the β 2 AR365 construct between the end of the TEV protease recognition sequence and Asp29 of the receptor by using the Quickchange multi protocol (Stratagene). Two point mutations M96T, M98T were also introduced into the construct based on the Quickchange multi protocol using synthetic oligonucleotides as mutation primers. The protein sequence of the entire fusion FLAAT is shown in  FIG. 5 . 
     The entire FLAAT gene described above was further cloned into the Best-Bac Sf9 expression vector pv11393 (expressionsystems) using the restriction enzyme digestion site XbaI and EcoRI. The final construct was confirmed by NDA sequencing. 
     Expression and Purification of FLAAT from Baculovirus-Infected Sf9 Cells 
     Recombinant baculovirus was made from pv11393-FLAAT using Best-Bac expression system, as described by the system protocol (expressionsystem). FLAAT was expressed by Sf9 cells that were infected by this baculovirus with 1:50 dilution at the cell density of 4 million/ml. 1 μM of receptor antagonist alprenolol was included to enhance the receptor stability and yield. The infected cells were harvested after 48 hs of incubation at 27° C. 
     The harvested cells were lysed by vigorous stifling in 10 times volume of lysis buffer (10 mM TRIS-Cl pH 7.5, 2 mM EDTA) complemented with protease inhibitor Leupeptin (2.5 μg/ml final concentration, Sigma) and Benzamindine (160 μg/ml final concentration, Sigma) for 15 minutes. The FLAAT protein was extracted from the cell membrane by thorough homogenization using solubilization buffer (100 mM NaCl, 20 mM TRIS-Cl, pH 7.5, 1% Dodecylmaltoside) complemented with Leupeptin and Benzamindine (2.5 μg/ml and 160 μg/ml final concentration, respectively). 10 ml of solubilization buffer was used for each gram of cell pellet. The Dodecylmaltoside (DDM)-solubilized FLAAT bearing the FLAG epitope was then purified by M1 antibody affinity chromatography (Sigma). Extensive washing using HLS buffer (100 mM NaCl, 20 mM HEPES pH 7.5, 0.1% DDM) was performed to get rid of alprenolol. The protein was then eluted with HLS buffer complemented with 5 mM EDTA, 200 μg free FLAG peptide and saturating concentration of cholesterol hemisuccinate. 
     The eluted FLAAT was further purified by affinity chromatography using Sepharose attached with Alprenolol as previously described (Cherezov et al High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 2007 318:1258-65) in order to selectively isolate functional FLAAT from non-functional protein. HHS buffer (350 mM NaCl, 20 mM HEPES pH 7.5, 0.1% DDM) complemented with 300 μM alprenolol and saturating concentration of cholesterol hemisuccinate was used to elute the protein. The eluted FLAAT bound with Alprenolol was then re-applied to M1 resin, allowing either washing off Alprenolol or exchanging Alprenolol with different ligand (for example, full agonist BI167107). Unliganded FLAAT or FLAAT bound with BI167107 was then eluted from M1 resin with HLS buffer complemented with 5 mM EDTA, 200 mg/ml free FLAG peptide and saturating concentration of cholesterol hemisuccinate. The FLAG epitope tag of FLAAT was removed by the treatment of tobacco etch virus (TEV) protease (invitrogen) for 3 hs at room temperature or overnight at 4° C. The purity of the final FLAAT is more than 90% according to the result of SDS-PAGE electrophoresis. 
     Crystallization of the FLAAT-BI167107-NB80 Ternary Complex 
     Nanobody80 (NB80) was expressed and purified as previously described (Rasmussen Structure of a nanobody-stabilized active state of the β(2) adrenoceptor. Nature. 2011 469:175-80.). The untagged FLAAT bound with high affinity agonist BI167107 was purified as described above. The purified FLAAT-BI167107 and NB80 was mixed with a 1:2 molar ratio. The FLAAT-BI167107-NB80 ternary complex was then isolated from free NB80 by size exclusion chromatography (SEC) using sephacryl S-200 column (GE health care life sciences) equilibrated in 100 mM NaCl, 10 mM HEPES pH 7.5, 0.1% DDM and 10 μM BI167107. The same buffer was used as the running buffer for SEC. 
     The FLAAT-BI167107-NB80 complex after SEC was concentrated to a final concentration of 60 mg/ml using vivaspin concentrator (Sartorius-Stedim). The complex was crystallized using lipid cubic phase (LCP) method as previously described (Rosenbaum et al, GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science. 2007 318: 1266-73.). The protein complex was firstly mixed with lipid moloolein with a 1:1.5 mass ratio in room temperature. 0.1 μl of the protein-lipid mixture drop was put in each well of a 24-well glass sandwich plate. The drop was then overlaid with 0.80 of precipitant and the well was sealed by glass coverslip. By using this method, the FLAAT-BI167107-NB80 ternary complex was crystallized in 31%-35% PEG400 (v/v) and 0.1M Tris-Cl, pH8.0 after 4 days of incubation in 20° C. 
     Materials and Methods II 
     Expression and Purification of β2AR, Gs Heterotrimer, and Nanobody-35 
     An N-terminally fused T4 lysozyme-β2AR construct β2AR truncated in position 365 (T4L-β2AR, described in detail below) was expressed in Sf9 insect cell cultures infected with recombinant baculovirus (BestBac, Expression Systems), and solubilized in n-Dodecyl-β-D-maltoside (DDM) according to methods described previously Kobilka (Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor. Anal Biochem 1995 231, 269-271; see  FIG. 16  for purification overview). A β2AR construct truncated after residue 365 (β2AR-365) was used for the majority of the analytical experiments and for deuterium exchange experiments. M1 Flag affinity chromatography (Sigma) served as the initial purification step followed by alprenolol-Sepharose chromatography for selection of functional receptor. A subsequent M1 Flag affinity chromatography step was used to exchange receptor-bound alprenolol for high-affinity agonist BI-167107. The agonist-bound receptor was eluted, dialyzed against buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 0.1% DDM and 10 μM BI-167107), treated with lambda phosphatase (New England Biolabs), and concentrated to approximately 50 mg ml −1  with a 50 kDa molecular weight cut off (MWCO) Millipore concentrator. Prior to spin concentration, the β2AR-365 construct, but not T4L-β2AR, was treated with PNGaseF (New England Biolabs) to remove amino-terminal N-linked glycosylation. The purified receptor was routinely analyzed by SDS-PAGE/Coomassie brilliant blue staining (see  FIG. 17 a   ). 
     Bovine Gαs short, His6-bovine Gβ1, and bovine Gγ2 were expressed in HighFive insect cells (Invitrogen) grown in Insect Xpress serum-free media (Lonza). Cultures were grown to a density of 1.5 million cells per ml and then infected with three separate  Autographa californica  nuclear polyhedrosis virus each containing the gene for one of the G protein subunits at a 1:1 multiplicity of infection (the viruses were a generous gift from Dr. Alfred Gilman). After 40-48 hours of incubation the infected cells were harvested by centrifugation and resuspended in 75 ml lysis buffer (50 mM HEPES, pH 8.0, 65 mM NaCl, 1.1 mM MgCl 2 , 1 mM EDTA, 1×PTT (35 μg/ml phenylmethanesulfonyl fluoride, 32 μg/ml tosyl phenylalanyl chloromethyl ketone, 32 μg/ml tosyl lysyl chloromethyl ketone), 1× LS (3.2 μg/ml leupeptin and 3.2 μg/ml soybean trypsin inhibitor), 5 mM β-mercaptoethanol (β-ME), and 10 μM GDP) per liter of culture volume. The suspension was pressurized with 600 psi N 2  for 40 minutes in a nitrogen cavitation bomb (Parr Instrument Company). After depressurization, the lysate was centrifuged to remove nuclei and unlysed cells, and then ultracentrifuged at 180,000×g for 40 minutes. The pelleted membranes were resuspended in 30 ml wash buffer (50 mM HEPES, pH 8.0, 50 mM NaCl, 100 μM MgCl 2 , 1×PTT, 1× LS, 5 mM β-ME, 10 μM GDP) per liter culture volume using a Dounce homogenizer and centrifuged again at 180,000×g for 40 minutes. The washed pellet was resuspended in a minimal volume of wash buffer and flash frozen with liquid nitrogen. 
     The frozen membranes were thawed and diluted to a total protein concentration of 5 mg/ml with fresh wash buffer. Sodium cholate detergent was added to the suspension at a final concentration of 1.0%, MgCl 2  was added to a final concentration of 5 mM, and 0.05 mg of purified protein phosphatase 5 (prepared in house) was added per liter of culture volume. The sample was stirred on ice for 40 minutes, and then centrifuged at 180,000×g for 40 minutes to remove insoluble debris. The supernatant was diluted 5-fold with Ni-NTA load buffer (20 mM HEPES, pH 8.0, 363 mM NaCl, 1.25 mM MgCl 2 , 6.25 mM imidazole, 0.2% Anzergent 3-12, 1×PTT, 1× LS, 5 mM β-ME, 10 μM GDP), taking care to add the buffer slowly to avoid dropping the cholate concentration below its critical micelle concentration too quickly. 3 ml of Ni-NTA resin (Qiagen) pre-equlibrated in Ni-NTA wash buffer 1 (20 mM HEPES, pH 8.0, 300 mM NaCl, 2 mM MgCl 2 , 5 mM imidazole, 0.2% Cholate, 0.15% Anzergent 3-12, 1×PTT, 1×LS, 5 mM β-ME, 10 μM GDP) per liter culture volume was added and the sample was stirred on ice for 20 minutes. The resin was collected into a gravity column and washed with 4× column volumes of Ni-NTA wash buffer 1, Ni-NTA wash buffer 2 (20 mM HEPES, pH 8.0, 50 mM NaCl, 1 mM MgCl 2 , 10 mM imidazole, 0.15% Anzergent 3-12, 0.1% DDM, 1×PTT, 1× LS, 5 mM β-ME, 10 μM GDP), and Ni-NTA wash buffer 3 (20 mM HEPES, pH 8.0, 50 mM NaCl, 1 mM MgCl 2 , 5 mM imidazole, 0.1% DDM, 1×PTT, 1× LS, 5 mM β-ME, 10 μM GDP). The protein was eluted with Ni-NTA elution buffer (20 mM HEPES, pH 8.0, 40 mM NaCl, 1 mM MgCl2, 200 mM imidazole, 0.1% DDM, 1×PTT, 1× LS, 5 mM β-ME, 10 μM GDP). Protein-containing fractions were pooled and MnCl 2  was added to a final concentration of 100 μM. Fifty μg of purified lambda protein phosphatase (prepared in house) was added per liter of culture volume and the elute was incubated on ice with stifling for 30 minutes. The eluate was passed through a 0.22 μm filter and loaded directly onto a MonoQ HR 16/10 column (GE Healthcare) equilibrated in MonoQ buffer A (20 mM HEPES, pH 8.0, 50 mM NaCl, 100 μM MgCl 2 , 0.1% DDM, 5 mM β-ME, 1×PTT). The column was washed with 150 ml buffer A at 5 ml/min and bound proteins were eluted over 350 ml with a linear gradient up to 28% MonoQ buffer B (same as buffer A except with 1 M NaCl). Fractions were collected in tubes spotted with enough GDP to make a final concentration of 10 μM. The Gs containing fractions were concentrated to 2 ml using a stirred ultrafiltration cell (Amicon) with a 10 kDa NMWL regenerated cellulose membrane (Millipore). The concentrated sample was run on a Superdex 200 prep grade XK 16/70 column (GE Healthcare) equilibrated in 5200 buffer (20 mM HEPES, pH 8.0, 100 mM NaCl, 1.1 mM MgCl 2 , 1 mM EDTA, 0.012% DDM, 100 μM TCEP, 2 μM GDP). The fractions containing pure Gs were pooled, glycerol was added to 10% final concentration, and then the protein was concentrated to at least 10 mg/ml using a 30 kDa MWCO centrifugal ultrafiltration device (Millipore). The concentrated sample was then aliquoted, flash frozen, and stored at −80°. A typical yield of final, purified Gs heterotrimer from 8 liters of cell culture volume was 6 mg. 
     Nanobody-35 (Nb35) was expressed in the periplasm of  E. coli  strain WK6, extracted, and purified by nickel affinity chromatography according to previously described methods (Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature 2011 469, 175-180) followed by ion-exchange chromatography ( FIG. 18 a   ) using a Mono S 10/100 GL column (GE Healthcare). Selected Nb35 fractions were dialysis against buffer (10 mM HEPES, pH 7.5, 100 mM NaCl) and concentrated to approximately 65 mg ml-1 with a 10 kDa MWCO Millipore concentrator. 
     Complex Formation, Stabilization and Purification 
     Formation of a stable complex (see  FIG. 19 ) was accomplished by mixing Gs heterotrimer at approximately 100 μM concentration with BI-167107 bound T4L-β 2 AR (or β2AR-365) in molar excess (approximately 130 μM) in 2 ml buffer (10 mM HEPES, pH 7.5, 100 mM NaCl, 0.1% DDM, 1 mM EDTA, 3 mM MgCl 2 , 10 μM BI-167107) and incubating for 3 hrs at room temperature. BI-167107, which was identified from screening and characterizing approximately 50 different β 2 AR agonists, has a dissociation half-time of approximately 30 hrs providing higher degree of stabilization to the active G protein-bound receptor than other full agonists such as isoproterenol (Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature 2011 469, 175-180). To maintain the high-affinity nucleotide-free state of the complex, apyrase (25 mU/ml, NEB) was added after 90 min to hydrolyze residual GDP released from G supon binding to the receptor. GMP resulting from hydrolysis of GDP by apyrase has very poor affinity for the G protein in the complex. Rebinding of GDP can cause dissociation of the R:G complex ( FIG. 13A ). 
     The R:G complex in DDM shows significant dissociation after 48 hours at 4° C. ( FIG. 20A ). Over 50 amphiphiles were screened and identified MNG-3 (Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature 2011 469, 175-180; Chae, P. S. et al. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nat Methods 7, 1003-1008; NG-310, Affymetrix-Anatrace) and its closely related analogs as detergents that substantially stabilize the complex ( FIGS. 20A  and B). The complex was exchanged into MNG-3 by adding the R:G mixture (2 ml) to 8 ml buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 10 μM BI-167107) containing 1% MNG-3 for 1 hr at room temperature. 
     At this stage the mixture contains the R:G complex, non-functional Gs, and an excess of β 2 AR. To separate functional R:G complex from non-functional Gs, and to complete the detergent exchange, the R:G complex was immobilized on M1 Flag resin and washed in buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 10 μM BI-167107, and 3 mM CaCl 2 ) containing 0.2% MNG-3. To prevent cysteine bridge-mediated aggregation of R:G complexes, 100 μM TCEP was added to the eluted protein prior to concentrating it with a 50 kDa MWCO Millipore concentrator. Of note, it was discovered later that crystal growth improved at even higher TCEP concentrations (above 1 mM) compared to 100 μM TCEP, and that the integrity of the R:G complex in MNG-3 was stable to 10 mM TCEP as measured by gel filtration analysis ( FIG. 21C ). In contrast, DDM-solubilized β 2 AR loses its ability to bind the high-affinity antagonist  3 H-dihydroalprenolol in 10 mM TCEP (data not shown), probably due to disruption of extracellular disulfide bonds. Iodoacetamide could not be used to block reactive cysteines on G s  alpha and beta subunits as it caused dissociation of the R:G complex ( figure S9   b ). The final size exclusion chromatography procedure to separate excess free receptor from the R:G complex ( FIG. 17 b   ) was performed on a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with buffer containing 0.02% MNG-3, 10 mM HEPES pH 7.5, 100 mM NaCl, 10 μM BI-167107, and 100 μM TCEP. Peak fractions were pooled ( FIG. 17 b   ) and concentrated to approximately 90 mg ml −1  with a 100 kDa MWCO Viva-spin concentrator and analyzed by SDS-PAGE/Coomassie brilliant blue staining ( FIG. 17 a   ) and gel filtration ( FIG. 17 c   ). To confirm a pure, homogeneous, and dephosphorylated preparation, the R:G complex was routinely analyzed by ion exchange chromatography ( FIG. 17 d   ). 
     Protein Engineering 
     To increase the probability of obtaining crystals of the R:G complex two strategies were used to increase the polar surface area on the extracellular side of the receptor. The first approach, to generate extracellular binding antibodies, was not successful. The second approach was to replace the flexible and presumably unstructured N-terminus with the globular protein T4 lysozyme (T4L) used previously to crystallize and solve the carazolol-bound receptor (Rosenbaum, D. M. et al. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science 2007 318, 1266-1273). The construct used here (T4L-β 2 AR) contained the cleavable signal sequence followed by the M1 Flag epitope (DYKDDDDA; SEQ ID NO: 14), the TEV protease recognition sequence (ENLYFQG; SEQ ID NO: 15), bacteriophage T4 lysozyme from N2 through Y161 including C54T and C97A mutations, and a two residue alanine linker fused to the human β 2 AR sequence D29 through G365. The PNGaseF-inaccessible glycosylation site of the β 2 AR at N187 was mutated to Glu. M96 and M98 in the first extracellular loop were each replaced by Thr to increase the otherwise low expression level of T4L-β 2 AR. The threonine mutations did not affect ligand binding affinity for  3 H-dihydro-alprenolol, but caused a small, approximately two-fold decrease in affinity for isoproterenol. 
     The β 2 AR-Gs peptide fusion construct used for [ 3 H]-DHA competition binding with isoproterenol was constructed from the receptor truncated at position 365 and fused to the last 21 amino acids of the Gαs subunit (amino acids 374-394, except for C379A). A Gly-Ser is inserted between the receptor and the peptide. Also an extended TEV protease site (SENLYFQGS; SEQ ID NO: 16) was introduced in the β 2 AR between G360 and G361. 
     Stabilization of Gs with Nanobodies 
     From negative stain EM imaging, we observed that the alpha helical domain of Gαs was flexible and therefore possibly responsible for poor crystal quality. Targeted stabilization of this domain was addressed by immunizing two llamas ( Llama glama ) with the bis(sulfosuccinimidyl)glutarate (BS2G, Pierce) cross-linked β 2 AR-Gs-BI-167107 ternary complex. Peripheral blood lymphocytes were isolated from the immunized animals to extract total RNA, prepare cDNA and construct a Nanobody phage display library according to published methods. Nb35 and Nb37 were enriched by two rounds of biopanning on the β 2 AR-Gs-BI-167107 ternary complex embedded in biotinylated high-density lipoprotein particles (Whorton, et al. Proc Natl Acad Sci USA 2007 104, 7682-7687). Nb35 and Nb37 were selected for further characterization because they bind the β 2 AR-Gs-BI-167107 ternary complex but not the free receptor in an ELISA assay. Nanobody binding to the R:G complex was confirmed by size exclusion chromatography ( FIG. 13 d   ), and it was noted that both nanobodies protected the complex from dissociation by GTPγS, suggestive of a stabilizing Gs:Nb interaction ( FIG. 13 d   ). 
     Crystallization 
     BI-167107 bound T4L-β 2 AR:Gs complex and Nb35 were mixed in 1:1.2 molar ratio. The small molar excess of Nb35 was verified by analytical gel filtration (see  FIG. 15 b   ). The mixture incubated for 1 hr at room temperature prior to mixing with 7.7 MAG containing 10% cholesterol (C8667, Sigma) in 1:1 protein to lipid ratio (w/w) using the twin-syringe mixing method reported previously. The concentration of R:G:Nb complex in 7.7 MAG was approximately 25 mg ml −1 . The detergent MNG-3 may stabilize the T4L-β 2 AR-Gs complex during its incorporation into the lipid cubic phase. This may be due to the high affinity of MNG-3 for the receptor. The β 2 AR in MNG-3 maintains its structural integrity even when diluted below the CMC of the detergent, in contrast to β 2 AR in DDM, which rapidly loses binding activity ( FIG. 20 b   ). Moreover, MNG-3 improved crystal size and quality, as previously reported. The protein:lipid mixture was delivered through an LCP dispensing robot (Gryphon, Art Robbins Instruments) in 40 nl drops to either 24-well or 96-well glass sandwich plates and overlaid en-bloc with 0.8 μl precipitant solution. Multiple crystallization leads were initially identified using in-house screens partly based on reagents from the StockOptions Salt kit (Hampton Research). Crystals for data collection were grown in 18 to 22% PEG 400, 100 mM MES pH 6.5 ( FIG. 13 c   ), 350 to 450 mM potassium nitrate, 10 mM foscarnet ( FIG. 13 b   ), 1 mM TCEP ( FIG. 21 c   ), and 10 μM BI-167107 Crystals reached full size within 3-4 days at 20° C. and were picked from a sponge-like mesophase and flash-frozen in liquid nitrogen without additional cryo-protectant. 
     Microcrystallography Data Collection and Processing. 
     Diffraction data were measured at the Advanced Photon Source beamline 23 ID-B. Hundreds of crystals were screened, and a final dataset was compiled using diffraction wedges of typically 10 degrees from 20 strongly diffracting crystals. All data reduction was performed using HKL2000 (Otwinowski. &amp; Minor, W. Processing of x-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997 276, 307-326). Although in many cases diffraction to beyond 3 Å was seen in initial frames, radiation damage and anisotropic diffraction resulted in low completeness in higher resolution shells. Analysis of the final dataset by the UCLA diffraction anisotropy server  31  indicated that diffraction along the a* axis was superior to that in other directions. On the basis of an F/σ (F) cutoff of 3 along each reciprocal space axis, reflections were subjected to an anisotropic truncation with resolution limits of 2.9, 3.2, and 3.2 Angstroms along a*, b*, and c* prior to use in refinement. The structure is reported to an overall resolution of 3.2 Å. Despite the low completeness in the highest resolution shells (Table 3) inclusion of these reflections gave substantial improvements in map quality and lower Rfree during refinement. 
     Structure Solution and Refinement 
     The structure was solved by molecular replacement using Phaser. In order, the search models used were: the β and γ subunits from a Gi heterotrimer (PDB ID: 1GP2), the Gs alpha ras-like domain (PDB ID: 1AZT), the active-state β2 adrenergic receptor (PDB ID: 3P0G), a β 2 AR binding nanobody (PDB ID: 3P0G), T4 lysozyme (PDB ID: 2RH1), and the Gs alpha helical domain (PDB ID: 1AZT). Following the determination of the initial structure by molecular replacement, rigid body refinement and simulated annealing were performed in Phenix and BUSTER, followed by restrained refinement and manual rebuilding in Coot. After iterative refinement and manual adjustments, the structure was refined in CNS using the DEN method. Although the resolution of this structure exceeds that for which DEN is typically most useful, the presence of several poorly resolved regions indicated that the incorporation of additional information to guide refinement could provide better results. The DEN reference models used were those used for molecular replacement, with the exception of NB35, which was well ordered and for which no higher resolution structure is available. Side chains were omitted from 52 residues for which there was no electron density past Cβ below a low contour level of 0.7σ in a 2Fo-Fc map. Figures were prepared using PyMOL (The PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC.). MolProbity was used to determine Ramachandran statistics. 
     Competition Binding 
     Membranes expressing the β 2 AR or the β 2 AR-Gs peptide fusion were prepared from baculovirus-infected Sf9 cells and [ 3 H]-dihydroalprenolol ([ 3 H]-DHA) binding performed as previously described (Swaminath et al Mol Pharmacol 2002 61, 65-72). For competition binding, membranes were incubated with [ 3 H]-DHA (1.1 nM final) and increasing concentrations of (−)-isoproterenol (ISO) for 1 hr before harvesting onto GF/B filters. Competition data were fitted to a two-site binding model and ISO high and low Ki&#39;s and fractions calculated using GraphPad prism. 
     Results II 
     Crystallization of the β2AR-Gs Complex 
     One challenge for crystallogenesis was to prepare a stable β 2 AR-Gs complex in detergent solution. The β 2 AR and Gs couple efficiently in lipid bilayers, but not in detergents used to solubilize and purify these proteins. We found that a relatively stable β 2 AR-Gs complex could be prepared by mixing purified GDP-Gs (approximately 100 μM final concentration) with a molar excess of purified β 2 AR bound to a high affinity agonist (BI-167107, Boehringer Ingelheim) in dodecylmaltoside solution. Apyrase, a non-selective purine pyrophosphatase, was added to hydrolyze GDP released from Gs on forming a complex with the β 2 AR. The complex was subsequently purified by sequential antibody affinity chromatography and size exclusion chromatography. The stability of the complex was enhanced by exchanging it into a recently developed maltose neopentyl glycol detergent (NG-310, Anatrace). The complex could be incubated at room temperature for 24 hrs without any noticeable degradation; however, initial efforts to crystallize the complex using sparse matrix screens in detergent micelles, bicelles and lipidic cubic phase (LCP) failed. 
     To further assess the quality of the complex, the protein was analyzed by single particle electron microscopy (EM). The results confirmed that the complex was monodispersed, and revealed two potential problems for obtaining diffraction of quality crystals. First, the detergent used to stabilize the complex formed a large micelle, leaving little polar surface on the extracellular side of the β 2 AR-Gs complex for the formation of crystal lattice contacts. The initial approach to this problem, which was to generate antibodies to the extracellular surface, was not successful. As an alternative approach, we replaced the amino terminus of the β 2 AR with T4 lysozyme (T4L). Several different amino-terminal fusion proteins were prepared and single particle EM was used to identify a fusion with a relatively fixed orientation of T4L in relation to the β 2 AR. 
     The second problem revealed by single particle EM analysis was increased variability in the positioning of the α-helical component of the Gαs subunit. Gαs consists of two domains, the ras-like GTPase domain (GαsRas), which interacts with the β 2 AR and the Gβ subunit, and the α-helical domain (GαsAH). The interface of the two Gαs subdomains forms the nucleotide-binding pocket ( FIG. 7 ), and EM 2D averages and 3D reconstructions show that in the absence of guanine nucleotide, GαsAH has a variable position relative to the complex of T4L-β 2 AR-GαsRAS-Gβγ ( FIG. 7 b   ). 
     The variable position of GαsAH was attributed to the empty nucleotide-binding pocket. However, both GDP and nonhydrolyzable GTP analogs disrupt the β 2 AR-Gs complex ( FIG. 13 ). The addition of pyrophosphate and its analog phosphonoformate (foscarnet) led to a significant increase in stabilization of GαsAH as determined by EM analysis of the detergent solubilized complex. Crystallization trials were carried out in Lipidic Cubic Phase (LCP) using a modified monolein designed to accommodate the large hydrophilic component of the T4L-β2AR-Gs complex (Misquitta, L. V. et al. Membrane protein crystallization in lipidic mesophases with tailored bilayers. Structure 2004 12, 2113-2124). Although we were able to obtain small crystals that diffracted to 7 Å, we were unable to improve their quality through the use of additives and other modifications. 
     In an effort to generate an antibody that would further stabilize the complex and facilitate crystallogenesis, β2AR and the Gs heterotrimer were crosslinked with a small, homobifunctional amine-reactive crosslinker and used this stabilized complex to immunized llamas. Llamas and other camelids produce antibodies devoid of light chains. The single domain antigen binding fragments of these heavy chain only antibodies, known as nanobodies, are small (15 kDa), rigid and are easily cloned and expressed in  E. coli . A nanobody (Nb35) was obtained that binds to the complex and prevents dissociation of the complex by GTPγS ( FIG. 13 ). The T4L-β2AR-Gs-Nb35 complex was used to obtain crystals that grew to 250 microns ( FIG. 14 ) in LCP (monoolein 7.7) and diffracted to 2.9 Å. A 3.2 Å data set was obtained from 20 crystals and the structure was determined by molecular replacement. 
     The β 2 AR-Gs complex crystallized in space group P2 1 , with a single complex in each asymmetric unit.  FIG. 8 a    shows the crystallographic packing interactions. Complexes are arrayed in alternating aqueous and lipidic layers with lattice contacts formed almost exclusively between soluble components of the complex, leaving receptor molecules suspended between G protein layers and widely separated from one another in the plane of the membrane. Extensive lattice contacts were formed among all the soluble proteins, likely accounting for the strong overall diffraction and remarkably clear electron density for the G protein. Nb35 and T4L facilitated crystal formation. Nb35 packs at the interface of Gβ and Gα subunits with complementarity determining region (CDR) 1 interacting primarily with Gβ and a long CDR3 loop interacting with both Gβ and Gα subunits. The framework regions of Nb35 from one complex also interact with Gα subunits from two adjacent complexes. T4L forms relatively sparse interactions with the amino terminus of the receptor, but packs against the amino terminus of the Gβ subunit of one complex, the carboxyl terminus of the Gβ subunit of another complex, and the Gβ subunit of yet another complex.  FIG. 8 b    shows the structure of the complete complex including T4L and Nb35, and  FIG. 8 c    shows the β 2 AR-Gs complex alone. 
     Structure of the Active-State β2AR 
     The β 2 AR-Gs structure provides the first high-resolution insight into the mechanism of signal transduction across the plasma membrane by a GPCR, and the structural basis for the functional properties of the ternary complex.  FIG. 9 a    compares the structures of the agonist-bound receptor in the β 2 AR-Gs complex and the inactive carazolol-bound β 2 AR. The largest difference between the inactive and active structures is a 14 Å outward movement of TM6 when measured at the Cα carbon of E268. There is a smaller outward movement and extension of the cytoplasmic end of the TM5 helix by 7 residues. A stretch of 26 amino acids in the third intracellular loop (ICL3) is disordered. Another notable difference between inactive and active structures is the second intracellular loop (ICL2), which forms an extended loop in the inactive β 2 AR structure and an α-helix in the β 2 AR-Gs complex. This helix is also observed in the β 2 AR-Nb80 structure ( FIG. 9 b   ); however, it may not be a feature that is unique to the active state, since it is also observed in the inactive structure of the highly homologous avian β 1 AR. 
     The quality of the electron density maps for the β 2 AR is highest at this β 2 AR-GαsRas interface, and much weaker for the extracellular half, possibly due to the lack of crystal lattice contacts with the extracellular surface ( FIG. 8 a   ). As a result, we cannot confidently model the high-affinity agonist (BI-167107) in the ligand-binding pocket. However, the overall structure of the β 2 AR in the T4L-β 2 AR-Gs complex is very similar to our recent active-state structure of β 2 AR stabilized by a G protein mimetic nanobody (Nb80). These structures deviate primarily at the cytoplasmic ends of TMs 5 and 6 ( FIG. 9 b   ), possibly due to the presence of T4L that replaces ICL3 in the β 2 AR-Nb80 structure. Nonetheless, the β 2 AR-Nb80 complex exhibits the same high affinity for the agonist isoproterenol as does the β 2 AR-Gs complex, consistent with high structural homology around the ligand binding pocket. The electron density maps for the β 2 AR-Nb80 crystals provide a more reliable view of the conformational rearrangements of amino acids around the ligand-binding pocket and between the ligand-binding pocket and the Gs-coupling interface. 
       FIG. 9 c    shows the position of the highly conserved sequence motifs including D/ERY and NPxxY in the β 2 AR-Gs complex compared with the β 2 AR-Nb80 complex (see also  Fig. S3 ). These conserved sequences have been proposed to be important for activation or for maintaining the receptor in the inactive state. The positions of these amino acids are essentially identical in these two structures demonstrating that Nb80 is a very good G protein surrogate. Only Arg131 differs between these two structures. In the β 2 AR-Nb80 structure Arg131 interacts with Nb80, whereas in the β 2 AR-Gs structure Arg131 packs against Tyr391 of Gαs ( FIG. 15 ). 
     The active state of the β 2 AR is stabilized by extensive interactions with (GαsRas) ( FIG. 10 ). There are no direct interactions with Gβ or Gγ subunits. The total buried surface of the β 2 AR-G sRas interface is 2576 Å 2  (1300 Å 2  for G sRas and 1276 Å 2  for the β 2 AR). This interface is formed by ICL2, TM5 and TM6 of the β 2 AR, and by α5-helix, the αN-β1 junction, the top of the β3-strand, and the α4-helix of GαsRas (see Table 1 below for specific interactions). The β 2 AR sequences involved in this interaction have been shown to play a role in G protein coupling; however, there is no clear consensus sequence for Gs-coupling specificity when these segments are aligned with other GPCRs. Perhaps this is not surprising considering that the β 2 AR also couples to Gi and that many GPCRs couple to more than one G protein isoform. The structural basis for G protein coupling specificity must therefore involve more subtle features of the secondary and tertiary structure. Nevertheless, a noteworthy interaction involves Phe139, which is located at the beginning of the ICL2 helix and sits in a hydrophobic pocket formed by Gαs His41 at the beginning of the β1-strand, Val213 at the start of the β3-strand and Phe376, Arg380 and Ile383 in the α5-helix ( FIG. 4 c   ). The β 2 AR mutant F139A displays severely impaired coupling to Gs. The residue corresponding to Phe139 is a Phe or Leu on almost all Gs coupled receptors, but is more variable in GPCRs known to couple to other G proteins. Of interest, the ICL2 helix is stabilized by an interaction between Asp130 of the conserved DRY sequence and Tyr141 in the middle of the ICL2 helix ( FIG. 10 c   ). Tyr141 has been shown to be a substrate for the insulin receptor tyrosine kinase; however, the functional significance of this phosphorylation is currently unknown. 
     Structure of Activated Gs 
     One surprising observation in the β 2 AR-Gs complex is the large displacement of the GαsAH relative to GαsRas (an approximately 127° rotation about the junction between the domains) ( FIG. 11 a   ). In the crystal structure of Gαs, the nucleotide-binding pocket is formed by the interface between GαsRas and GαsAH. Guanine nucleotide binding stabilizes the interaction between these two domains. The loss of this stabilizing effect of guanine nucleotide binding is consistent with the high flexibility observed for GαsAH in single particle EM analysis of the detergent solubilized complex. It is also in agreement with the increase in deuterium exchange at the interface between these two domains upon formation of the complex. Recently Hamm, Hubbell and colleagues, using double electron-electron resonance (DEER) spectroscopy, documented large (up to 20 Å) changes in distance between nitroxide probes positioned on the Ras and α-helical domains of Gi upon formation of a complex with light-activated rhodopsin. Therefore, it is perhaps not surprising that G sAH is displaced relative to GαsRas; however, its location in this crystal structure most likely reflects only one of an ensemble of conformations that it can adopt under physiological conditions, but has been stabilized by crystal packing interactions. 
     The conformational links between the β 2 AR and the nucleotide-binding pocket primarily involve the amino and carboxyl terminal helices of Gαs ( FIG. 10 ).  FIG. 11 b    focuses on the region of GαsRas that undergoes the largest conformational change when comparing the structure of GαsRas from the Gs-β 2 AR complex with that from the Gαs-GTPγS complex. The largest difference is observed for the α5-helix, which is displaced 6 Å towards the receptor and rotated as the carboxyl terminal end projects into transmembrane core of the β 2 AR. Associated with this movement, the β6-α5 loop, which interacts with the guanine ring in the Gαs-GTPγS structure, is displaced outward, away from the nucleotide-binding pocket ( FIG. 11 b - d   ). The movement of α5-helix is also associated with changes in interactions between this helix and the β6-strand, the αN-β1 loop, and the α1-helix. The β1-strand forms another link between the β 2 AR and the nucleotide-binding pocket. The C-terminal end of this strand changes conformation around Gly47, and there are further changes in the β1-α1 loop (P-loop) that coordinates the γ-phosphate in the GTP-bound form ( FIG. 11   b - d ). The observations in the crystal structure are in agreement with deuterium exchange experiments where there is enhanced deuterium exchange in the β1-strand and the amino terminal end of the α5-helix upon formation of the nucleotide-free β 2 AR-Gs complex. The DXMS studies provide additional insights into the dynamic nature of these conformational changes in Gs upon complex formation. 
     The structure of a GDP-bound Gs heterotrimer has not been determined in this study, so it is not possible to directly compare the Gαs-Gβγ interface before and after formation of the β 2 AR-Gs complex. Based on the structure of the GDP-bound Gi heterotrimer, large changes in interactions between GαsRas and Gβγ upon formation of the complex with β 2 AR are not observed. This is also consistent with deuterium exchange studies. It should be noted that Nb35 binds at the interface between GαsRas and Gβ ( FIG. 8 b   ). Therefore, we cannot exclude the possibility that Nb35 may influence the relative orientation of the GαsRas-Gβγ interface in the crystal structure. However, single particle EM studies provide evidence that Nb35 does not disrupt interactions between GαsAH and GαsRas. 
     Assembly of the β2AR-Gs Complex 
     Clues to the initial stages of complex formation may come from the recent active state structures of rhodopsin.  FIGS. 12 a  and  b    compare the active-state structure of β 2 AR in the β 2 AR-Gs complex with the recent structure of metarhodopsin II bound to the transducin peptide. The conformational changes in TM5 and TM6 are smaller in metarhodopsin II, and the position of the carboxyl terminal alpha helix of transducin is tilted by approximately 30° relative to the position of the homologous region of Gs. These may represent fundamental differences in the receptor-G protein interactions between these two proteins, but given the strong conservation of the G-protein binding pocket, the changes more likely reflect the extensive contacts formed with the intact G protein. The position of the transducin peptide in metarhodopsin II may represent the initial interaction between a GDP-bound G protein and a GPCR. We have attempted to reproduce a similar complex between the β 2 AR and a synthetic peptide representing the carboxyl terminal 20 amino acids of Gs, but did not observe any effect of this peptide on receptor function, possibly due to the solubility and behavior of the peptide in solution. However, when the carboxyl terminal 20 amino acids of Gs are fused to the carboxyl terminus of the β 2 AR ( FIG. 12 c   ), we observe a 27-fold increase in agonist affinity ( FIG. 12 d   ). This effect is only 3.5-fold smaller than the effect we observe on agonist binding affinity in the β 2 AR-Gs complex, and demonstrates that there is a functional interaction between the peptide and receptor that may represent an initial stage in β 2 AR-Gs complex formation.  FIG. 12   e, f  presents a possible sequence of interactions of β 2 AR and Gs when forming the nucleotide free complex. The initial interaction of the β 2 AR with Gs would require an outward movement of the carboxyl terminus of the α5-helix away from the β6-strand to permit interactions with the β 2 AR similar to those observed in metarhodopsin II. The dynamic character of the carboxyl terminal end of α5 is supported by deuterium exchange studies and the relatively loose packing of α5 with the rest of GαsRas in the structure of Gαs alone. The subsequent formation of more extensive interactions between the β 2 AR ICL 2 and the amino terminus of Gαs requires a rotation of GαsRas relative to the receptor and would be associated with further conformational changes in both β 2 AR and GαsRas ( FIG. 12 f   ). This binding model is in agreement with deuterium exchange experiments. 
     The coordinates and structure factors for the β 2 AR-Gs complex are deposited in the Protein Data Bank as accession number 3SN6, which is incorporated by reference herein. 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Potential intermolecular interaction within the R:G interface 
               
               
                   
               
             
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Data collection and refinement statistics 
               
               
                 Data collection* 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Number of crystals 
                 20 
               
               
                 Space group 
                 P 2 1   
               
               
                 Cell dimensions 
                   
               
               
                 a, b, c (Å) 
                 119.3, 64.6, 131.2 
               
               
                 α, β, γ (°) 
                 90.0, 91.7, 90.0 
               
               
                 Resolution (Å) 
                   41-3.2 (3.26-3.20) 
               
               
                 R merge  (%) 
                 15.6 (553)  
               
               
                 &lt;I&gt;/&lt;σI&gt; 
                 10.8 (1.8)  
               
               
                 Completeness (%) 
                 91.2 (53.9) 
               
               
                 Redundancy 
                 6.5 (5.0) 
               
               
                 Refinement 
                   
               
               
                 Resolution (Å) 
                 41-3.2 
               
               
                 No. reflections 
                 31075 (1557 in test set) 
               
               
                 R work /R free  (%) 
                 22.5/27.7 
               
               
                 No. atoms 
                 10277 
               
               
                 No. protein residues 
                 1318 
               
               
                 Anisotropic B tensor 
                 B 11  = −7.0/B 22  = 4.7/B 33  = 2.3/B 13  = 2.1 
               
               
                 Unmodelled sequences* 
                   
               
               
                 β 2  adrenergic receptor 
                 29 b , 176-178, 240-264, 342-365 
               
               
                 G s α, ras domain 
                 1-8, 60-88, 203-204, 256-262 
               
               
                 G s γ 
                 1-4, 63-68 
               
               
                 T4 lysozyme 
                 161 c   
               
               
                 Average B-factors (Å 2 ) 
                   
               
               
                 β 2  adrenergic receptor 
                 133.5 
               
               
                 G s α, ras domain 
                 82.8 
               
               
                 G s α, helical domain 
                 123.0 
               
               
                 G s β 
                 64.2 
               
               
                 G s γ 
                 85.2 
               
               
                 Nanobody 35 
                 60.7 
               
               
                 T4 lysozyme 
                 113.7 
               
               
                 R.m.s. deviation from ideality 
                   
               
               
                 Bond length (Å) 
                 0.007 
               
               
                 Bond angles (°) 
                 0.72 
               
               
                 Ramachandran statistics d   
                   
               
               
                 Favored regions (%) 
                 95.8 
               
               
                 Allowed regions (%) 
                 4.2 
               
               
                 Outliers (%) 
                 0 
               
               
                   
               
               
                 *Highest shell statistics are in parentheses. 
               
               
                   a These regions were omitted from the model due to poorly resolved electron density. Unmodelled purification tags are not included in these residue ranges. 
               
               
                   b Residues 1-28 of the β2AR were omitted from the construct and T4L was fused to the amino terminus of transmembrane helix 1 to facilitate crystallization. 
               
               
                   c Residue 1 of T4L was omitted from the construct 
               
               
                   d As defined by MolProbity 3B . 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Data collection statistics by resolution shell 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Resolution 
                   
                   
                   
               
               
                   
                 Shell (Å) 
                 &lt;I&gt;/&lt;σI&gt; 
                 R merge  (%) 
                 Completeness (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                     41-8.67 
                 18.8 
                 06.6 
                 97.1 
               
               
                   
                 8.67-6.89 
                 16.9 
                 09.2 
                 99.5 
               
               
                   
                 6.89-6.02 
                 14.4 
                 13.0 
                 99.7 
               
               
                   
                 6.02-5.47 
                 12.8 
                 16.7 
                 99.9 
               
               
                   
                 5.47-5.08 
                 13.4 
                 15.9 
                 99.9 
               
               
                   
                 5.08-4.78 
                 13.4 
                 16.9 
                 99.8 
               
               
                   
                 4.78-4.54 
                 12.2 
                 18.2 
                 99.6 
               
               
                   
                 4.54-4.34 
                 11.6 
                 20.1 
                 99.8 
               
               
                   
                 4.34-4.18 
                 9.5 
                 22.9 
                 99.4 
               
               
                   
                 4.18-4.03 
                 7.7 
                 26.2 
                 99.1 
               
               
                   
                 4.03-3.91 
                 6.6 
                 27.9 
                 98.7 
               
               
                   
                 3.91-3.79 
                 5.3 
                 30.2 
                 98.7 
               
               
                   
                 3.79-3.69 
                 3.8 
                 36.6 
                 96.7 
               
               
                   
                 3.69-3.60 
                 4.6 
                 36.9 
                 94.6 
               
               
                   
                 3.60-3.52 
                 2.3 
                 45.7 
                 90.3 
               
               
                   
                 3.52-3.45 
                 2.2 
                 47.9 
                 86.3 
               
               
                   
                 3.45-3.38 
                 2.4 
                 45.6 
                 80.5 
               
               
                   
                 3.38-3.31 
                 2.1 
                 47.3 
                 69 
               
               
                   
                 3.31-3.26 
                 2.2 
                 49.8 
                 59.4 
               
               
                   
                 3.26-3.20 
                 1.8 
                 55.3 
                 53.9 
               
               
                   
                 Overall 
                 10.8 
                 15.6 
                 91.2 
               
               
                   
               
            
           
         
       
     
     Materials and Methods III 
     Generation of N-T4L Fused β2AR Constructs 
     The human β 2 AR in the pFastbac1 Sf9 expression vector truncated at amino acid 365 in the cytoplasmic tail (β 2 AR365) was used as the starting template for generating the N-T4L fused β 2 AR constructs. The HA signal peptide followed by FLAG epitope tag and tobacco etch virus (TEV) protease recognition sequence were added to the N-terminus of the receptor to facilitate expression and purification. A point mutation of N187E was also introduced in the second extracellular loop to remove a glycosylation site ( FIG. 22 ). 
     DNA cassettes encoding two different versions of T4L lysozyme (full length or with truncated C-terminus) with different numbers of additional alanines attached to the C-terminus were generated and amplified by PCR using the original β 2 AR-T4L  3  as the template and synthetic oligonucleotides as primers. These different cassettes were inserted into the β 2 AR365 construct between the end of the TEV protease recognition sequence and Asp29, Glu30 or Val31 of the receptor as shown in ( FIG. 22 ) by using the Quickchange multi protocol (Stratagene). Two point mutations M96T, M98T were also introduced into the β 2 AR sequence. Residues from Ser235 to Lys263 in the third intracellular loop were deleted with the Quickchange multi protocol using synthetic oligonucleotides as mutation primers. All the constructs were confirmed by DNA sequencing. The protein sequence of T4L-β 2 AR-Δ-ICL3 is shown below: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 17) 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   DTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNTNGVITKDEAEKLFNQ 
                 
               
               
                   
               
               
                 
                   DVDAAVRGILRNAKLKPVYDSLDAVRRAALINMVFQMGETGVAGFTNSLR 
                 
               
               
                   
               
               
                 
                   MLQQKRWDEAAVNLAKSRWYNQTPNRAKRVITTFRTGTWDAY 
                   AA DEVWVV   
                 
               
               
                   
               
               
                 
                   
                     GMGIVMSLIVLAIVF 
                     GNVLVITAIAKFERLQTVTNYFITSLACADLVMGL 
                   
                 
               
               
                   
               
               
                 
                   
                     AVVPFGAAHILTKTWTFGNF 
                     WCEFWTSIDVLCVTASIETLCVIAVDRYFA 
                   
                 
               
               
                   
               
               
                 
                   
                     ITSPFKYQSLLTKNKARVIILMVWIV 
                     SGLTSFLPIQMHWYRATHQEAINC 
                   
                 
               
               
                   
               
               
                 
                   
                     YAEETCCDFFTNQAYAIASSIVSFYVPLVIMV 
                     FVYSRVFQEAKROLOKID 
                   
                 
               
               
                   
               
               
                 
                   
                     KFCLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQ 
                     DNLIRKEVYILLN 
                   
                 
               
               
                   
               
               
                 
                   
                     WIGYVNSGFNPLIYCRSPDFRIAFQELLCLRRSSLKAYGNGYSS 
                     NGNTGE 
                   
                 
               
               
                   
               
               
                     QSG   . 
               
            
           
         
       
     
     The HA signal peptide is shown in italic letters; the FLAG epitope tag is shown in letters with underscore; the TEV recognition sequence is marked with a box and the cleavage site is shown with an asterisk; the full length T4L is shown in bold; the β 2 AR sequence from Asp29 to Gly365 excluding Ser235 to K263 is shown in bold underline, the 2-Ala linker is underlined). 
     The entire T4L-β 2 AR-Δ-ICL3 gene described above was further cloned into the Best-Bac Sf9 expression vector pv11393 (expression systems) using the restriction enzyme digestion site XbaI and EcoRI. This version of T4L-β 2 AR-Δ-ICL3 construct was also confirmed by DNA sequencing. 
     Whole Cell Binding to Assess the Expression Yield of Each Construct. 
     Recombinant baculovirus was made from the pFastbac1 Sf9 expression vector for each of the constructs illustrated in  FIG. 22  using the Invitrogen protocol. Sf9 cells at a density of 4 million/ml were infected with second passage virus at different ratios of virus stock to cell culture (1:20, 1:50, and 1:100). After 48 hours, 5 μl of the infected cells were incubated with 10 nM of [ 3 H]-dihydroalprenolol (DHA) in 500 μl of binding buffer (75 mM Tris, 12.5 mM MgCl2, 1 mM EDTA, pH 7.4, supplemented with 5 mg/ml BSA). Cells were harvested and washed with cold binding buffer using a Brandel harvester. Bound [ 3 H]DHA was measured with scintillation counter (Beckman). Non-specific binding of [ 3 H]DHA was assessed by including 10 μM of alprenolol (Sigma) in the same binding reaction. The expression level of each construct was determined using the specific activity of the bound [ 3 H]DHA. Each experiment was performed in triplicate. 
     Saturation and Competition Binding Assays. 
     Membranes from Sf9 cells expressing either wild-type β2AR or T4L-β 2 AR-Δ-ICL3 were prepared based on a previously describe protocol 12 . In each reaction for the saturation binding assay, membranes containing approximately 0.2 pmol receptor were incubated with concentrations of [ 3 H]DHA ranging from 5 pM to 10 nM in 500 μl of buffer (75 mM Tris, 12.5 mM MgCl2, 1 mM EDTA, pH 7.4, supplemented with 0.5 mg/ml BSA) at room temperature with shaking at 230 rpm for 1 hour. Membranes were isolated from free [ 3 H]DHA using a Brandel harvester and washed three times with cold buffer. The amount of receptor bound [ 3 H]DHA was measured using a scintillation counter (Beckman). Non-specific binding of the [ 3 H]DHA in each reaction was assessed by including 1 μM alprenolol (Sigma) in the same reaction. In each reaction for the competition binding assay, membrane containing approximately 0.2 pmol receptor was incubated with 1 nM [ 3 H]DHA and different concentrations of (−)-isoproterenol (Sigma) ranging from 1 nM to 1 mM. Membranes were harvested and washed three times with cold buffer. The bound [ 3 H]DHA was counted as described above. Non-specific [ 3 H]DHA was assessed by replacing (−)-isoproterenol with 1 μM alprenolol. All the binding data was analyzed by non-linear regression method using Graphpad Prism. Each experiment was performed in triplicate. 
     Expression and Purification of T4L-β 2 AR-Δ-ICL3 from Baculovirus-Infected Sf9 Cells 
     Recombinant baculovirus was made from pv11393-T4L-β 2 AR-Δ-ICL3 using Best-Bac expression system, as described by the system protocol (Expression Systems). T4L-β 2 AR-Δ-ICL3 was expressed by infecting Sf9 cells at a density of 4 million/ml with a second passage baculovirus stock at a virus to cell ratio of 1:50. 1 μM of the antagonist alprenolol was included to enhance the receptor stability and yield. The infected cells were harvested after 48 hs of incubation at 27° C. 
     Cell pellets were lysed by vigorous stirring in lysis buffer (10 mM TRIS-Cl pH 7.5, 2 mM EDTA, 10 ml of buffer per gram of cell pellet) supplemented with protease inhibitor Leupeptin (2.5 μg/ml final concentration, Sigma) and Benzamindine (160 μg/ml final concentration, Sigma) for 15 minutes. The T4L-β 2 AR-Δ-ICL3 protein was extracted from the cell membrane by dounce homogenization in solubilization buffer (100 mM NaCl, 20 mM TRIS-Cl, pH 7.5, 1% Dodecylmaltoside) supplemented with Leupeptin and Benzamindine (2.5 μg/ml and 160 μg/ml final concentration, respectively). 10 ml of solubilization buffer was used for each gram of cell pellet. The Dodecylmaltoside (DDM)-solubilized T4L-β 2 AR-Δ-ICL3 bearing the FLAG epitope was then purified by M1 antibody affinity chromatography (Sigma). Extensive washing using HLS buffer (100 mM NaCl, 20 mM HEPES pH 7.5, 0.1% DDM) was performed to get rid of alprenolol. The protein was then eluted with HLS buffer supplemented with 5 mM EDTA, 200 μg/ml free FLAG peptide and a saturating concentration of cholesterol hemisuccinate. 
     The eluted T4L-β 2 AR-Δ-ICL3 was further purified by affinity chromatography using alprenolol-Sepharose as previously described  3  in order to isolate functional T4L-β 2 AR-Δ-ICL3 from non-functional protein. HHS buffer (350 mM NaCl, 20 mM HEPES pH 7.5, 0.1% DDM) supplemented with 300 μM alprenolol and a saturating concentration of cholesterol hemisuccinate was used to elute the protein. The eluted T4L-β 2 AR-Δ-ICL3 bound with alprenolol was then re-applied to M1 resin, allowing exchanging alprenolol with carazolol in HHS buffer supplemented with 30 nM carazolol. T4L-β 2 AR-Δ-ICL3 bound with carazolol was then eluted from M1 resin with HHS buffer supplemented with 5 mM EDTA, 200 μg/ml free FLAG peptide and saturating concentration of cholesterol hemisuccinate. The FLAG epitope tag of T4L-β 2 AR-Δ-ICL3 was removed by the treatment of tobacco etch virus (TEV) protease (invitrogen) for 3 hs at room temperature or overnight at 4° C. The untagged T4L-β 2 AR-Δ-ICL3-cazazolol complex was then further purified by chromatography (SEC) using S200 column (GE healthcare) equilibrated in 100 mM NaCl, 10 mM HEPES pH 7.5, 0.1% DDM and 1 nM carazolol. The same buffer was used as the running buffer for SEC. The purity of the final T4L-β 2 AR-ΔICL3 is more than 90% according to the result of SDS-PAGE electrophoresis. 
     Crystallization of the T4L-β 2 AR-ΔICL3-Carazolo Complex 
     The purified T4L-β 2 AR-Δ-ICL3-carazolol complex was concentrated to a final concentration of 60 mg/ml using centricon Vivaspin (GE healthcare). The complex was crystallized using the lipid cubic phase (LCP) method as previously described 3 . The protein complex was mixed with lipid moloolein with a 1:1.5 mass ratio at room temperature. 0.03 μl of the protein-lipid mixture drop was deposited in each well of a 96-well glass sandwich plate (Molecular Dimensions). The drop was then overlaid with 0.65 μl of precipitant and the well was sealed by glass coverslip. By using this method, the T4L-β 2 AR-Δ-ICL3-carazolol complex was crystallized in 37% PEG300 (v/v), 0.1M Bis-Tris propane, pH 6.5, 0.1 M ammonium phosphate after 2 days of incubation in 20° C. 
     Data Collection and Structure Determination 
     The crystals were harvested and frozen in liquid nitrogen directly without using additional cryo-protectant. Diffraction data from 15 different crystals was collected using the GM/CA-CAT minibeam at 23-ID-D, Advance Photon Source, Argonne National Labs. The data was processed with HKL2000 and the structure was solved by molecular replacement using Molrep. Further model rebuilding was performed by using coot and the structure was refined with Phenix. The validation of the final structural model was performed using Molprobity. Data processing and refinement statistics are shown in Table 4. 
     Results III 
     T4 lysozyme was fused to the N-terminus of the β 2  adrenergic receptor (β 2 AR), a G-protein coupled receptor (GPCR) for catecholamines. The N-terminally fused T4L is sufficiently rigid relative to the receptor to facilitate crystallogenesis without thermostabilizing mutations or the use of a stabilizing antibody, G protein, or protein fused to the 3rd intracellular loop. This approach adds to the protein engineering strategies that enable crystallographic studies of GPCRs alone or in complex with a signaling partner. 
     The N terminus of the β 2 AR was replaced with T4 lysozyme to produce a T4L-GPCR fusion. To have a T4L-β 2 AR construct suitable for crystallization, the link between T4L and the receptor should be relatively short and rigid, yet not interfere with receptor function. Several different constructs were generated and examined for expression levels and binding properties ( FIG. 22 ). In an effort to generate a rigid interaction between T4L and the β 2 AR, we removed the relatively flexible C-terminus of the T4L and attempted to fuse the remaining C terminal helix of T4L with the extracellular end of TM1 of the β 2 AR. None of these constructs gave sufficient amounts of functional receptor. 
     In the second approach, we fused the carboxyl terminus of T4L to D29, the first amino acid of the extracellular helical extension of TM 1. Four constructs were generated and examined: direct fusion of T4L to D29, and the inclusion of 1-3 Ala residues between T4L and the β 2 AR ( FIG. 22 ). The highest level of expression was obtained from the fusion with a 2-Ala linker. The fusion protein had normal pharmacology and G protein coupling. To improve expression, two additional point mutations M96T and M98T were made in the β 2 AR component of the fusion protein. We have previously observed that mutation of these residues, which are located in the first extracellular loop and face away from the protein, had no effect on receptor function, but enhanced expression by up to two-fold. We were able to produce 1.5 mg of pure, functional protein from 1 liter of Sf9 cells. 
     This version of T4L-β 2 AR was recently used to obtain the crystal structure of the β 2 AR-Gs complex. However, in this structure most of the lattice contacts in this crystal are mediated by Gs, and the N terminal fused T4L does not pack against the extracellular surface of its fused β 2 AR ( FIG. 24 ). The lack of interactions between T4L and the extracellular surface of the β 2 AR in the β 2 AR-Gs complex suggested that T4L fused to the N terminus of the β 2 AR might not be sufficiently constrained to facilitate crystallogenesis in the absence of the cytoplasmic G protein. The amino terminal T4L facilitated crystallogenesis in the absence of a soluble protein bound or fused to the third intracellular loop. Additional modifications were made to minimize unstructured sequence in the third intracellular loop and carboxyl terminus ( FIG. 22 ). The C-terminus was truncated after amino acid 365. The 3 rd  intracellular loop (ICL3) of β 2 AR is another flexible region and it is subject to proteolysis. This loop was truncated in the fusion protein by removing residues 235 to 263. The final construct T4L-β 2 AR-Δ-ICL3 is illustrated in  FIG. 22 . 
     To determine the functional integrity of T4L-β 2 AR-Δ-ICL3, agonist and antagonist binding affinities were determined. The ligand binding pocket is formed by amino acids from four transmembrane domains and is therefore very sensitive to any perturbation of the receptor structure. T4L-β 2 AR-Δ-ICL3 exhibits ligand binding affinities for the antagonist [3H]-Dihydroalprenolol and the agonist isopreterenol that are comparable to those of the wild type receptor ( FIG. 25 ). 
     Purified T4L-β 2 AR-Δ-ICL3 bound to the inverse agonist carazolol crystallized as small rods in lipid cubic phase (37% PEG300 (v/v), 0.1M Bis-Tris propane, pH 6.5, 0.1 M ammonium phosphate). Crystals diffracted to a resolution of 3.3 Å; however, due to radiation damage, our dataset was limited to 4.0 (Table 4). Nevertheless, the dataset allowed us to solve the structure by molecular replacement. The interaction between the β 2 AR and T4L is sufficiently rigid to detect electron density for the 2 Ala link between these two proteins ( FIG. 26 ). This link was not detectable in the electron density map of the β 2 AR-Gs structure ( FIG. 24 ). In the T4L-β 2 AR-Δ-ICL3 crystal lattice, the packing interactions are primarily mediated by T4L and there are no contacts between adjacent receptors ( FIG. 23 ), indicating the important role of the T4L in facilitating GPCR crystallization. Each T4L has four packing interactions: 1-against ECL1 and ECL2 of its fused β 2 AR-Δ-ICL3, 2-against T4L of one adjacent T4L-β 2 AR-Δ-ICL3, 3-against T4L, ECL2 and ECL3 of a second T4L-β 2 AR-Δ-ICL3, and 4-against ICL3 and Helix 8 of a third T4L-β 2 AR-Δ-ICL3 ( FIG. 23 ). 
     The structures of the β 2 AR in T4L-β 2 AR-Δ-ICL3 and β2AR-T4L (pdb 2RH1) are very similar to each other ( FIG. 27 ), with an overall root mean square deviation of 0.48 Å. Only minor differences can be observed in these two structures, presumably due to different crystal packing patterns. The similarity of the structures determined independently through different strategies further validates the fusion protein approach, demonstrating that structural distortions due to protein engineering or crystal packing are unlikely. 
     Of interest, ICL2 in the two inactive structures of β 2 AR-Fab5 and β 2 AR-T4L is in an extended loop while it is an alpha helix in both active structures: the β 2 AR-Gs complex and the β 2 AR stabilized by Nb80. In both of the inactive structures (β 2 AR-Fab5 and β 2 AR-T4L), ICL2 participates in lattice contacts that may influence its conformation. However, in the T4L-β2AR-Δ-ICL3 structure ICL2 is not involved in packing interactions, yet is an extended loop is nearly identical to that observed in the other inactive state β 2 AR structures ( FIG. 27 ). Thus, this extended loop structure may reflect an inactive state. 
     In conclusion, fusion of T4L to the amino terminus of a GPCR can facilitate crystallogenesis. This approach can also facilitate the formation of crystals of a GPCR in complex with a cytoplasmic signaling protein. 
       FIG. 28  illustrates shows the structure of T4L-β2AR fusion bound to salmeterol, a partial agonist used to treat asthma. In this structure, the partial-active state is stabilized by a nanobody (nanobody 71). This structure was obtained using similar methods to those described above. 
     
       
         
           
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                   
                 Data collection 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Space group 
                 P2 1 2 1 2 1   
               
               
                   
                 Unit cell dimensions 
                   
               
               
                   
                 a, b, c (Å) 
                 51.4, 71.4, 161.4 
               
               
                   
                 Resolution (Å) 
                 50-4.0 (4.07-4.00)* 
               
               
                   
                 R merge   
                 0.199 (0.799) 
               
               
                   
                 &lt;I/σI&gt; 
                 8.4 (1.5) 
               
               
                   
                 Completeness (%) 
                 84.3 (71.2) 
               
               
                   
                 Multiplicity 
                 4.7 (3.7) 
               
               
                   
                 Refinement 
                   
               
               
                   
                 Resolution (Å) 
                 30-3.99 
               
               
                   
                 No. reflections work/free 
                 4547/691  
               
               
                   
                 R work /R free   
                 0.267/0.293 
               
               
                   
                 No. atoms 
                 3623 
               
               
                   
                 Average B values (Å 2 ) 
                   
               
               
                   
                 Receptor 
                 197 
               
               
                   
                 T4L 
                 177 
               
               
                   
                 Carazolol 
                 160 
               
               
                   
                 Overall anisotropic B (Å 2 ) 
                   
               
               
                   
                 B11/B22/B33 
                 −21.2/59.3/−38.0 
               
               
                   
                 R.m.s deviations 
                   
               
               
                   
                 Bond lengths (Å) 
                 0.004 
               
               
                   
                 Bond angles (°) 
                 0.6764 
               
               
                   
                 Ramachandran plot** 
                   
               
               
                   
                 % favored 
                 96.4 
               
               
                   
                 allowed 
                 3.6 
               
               
                   
                 generously allowed 
                 0.0 
               
               
                   
                 disallowed 
                 0.0 
               
               
                   
               
               
                 *High resolution shell in parenthesis. 
               
               
                 **As defined by Molprobity 
               
               
                 R merge  = Σ hkI  Σ i |I i  − &lt;I&gt;/Σ hki Σ i I i