Stable apo-active complex of a g protein coupled receptor (GPCR) and a g protein

Abstract

Disclosed herein is a stable apo (ligand-free)-active complex of a G protein coupled receptor (GPCR) and a G protein complex, wherein the G protein complex is a cognate heterotrimeric G protein comprising an alpha subunit (Gα), a beta subunit (Gβ) and a gamma subunit (Gγ). The apo-active GPCR-G protein complex can be isolated from a cell membrane in a purified form and maintains its stability in the purified form.

Description

STABLE APO-ACTIVE COMPLEX OF A G PROTEIN COUPLED RECEPTOR (GPCR) AND A G PROTEIN TECHNICAL FIELD

[0001] Disclosed herein is a stable apo (ligand-free)-active complex of a G protein coupled receptor (GPCR) and a G protein, wherein the G protein is a cognate heterotrimeric G protein comprising an alpha subunit (Gα), a beta subunit (Gβ) and a gamma subunit (Gγ). The apo- active GPCR-G protein complex can be isolated from a cell membrane in a purified form and maintains its stability in the purified form. BACKGROUND OF THE INVENTION

[0002] G proteins bind guanine nucleotides and act as molecular switches in several signaling pathways by interconverting between a GDP-bound inactive and a GTP-bound active state. Heterotrimeric G proteins contain a helical domain (H-domain) and forms a complex with G beta (Gβ) and G gamma (Gγ) subunits. The guanine nucleotide exchange factors (GEFs) of Gα subunits are usually membrane-bound G protein coupled receptors (GPCRs). GPCRs bind to Gα at a site almost 30 Å away from the GDP binding region and allosterically trigger GDP release to activate them.

[0003] GPCRs constitute a very large family of proteins that control many physiological processes and are the targets of many effective drugs. Numerous classification schemes have been proposed and the superfamily was classically divided into at least three main classes (A, B, and C) with no detectable shared sequence homology between the classes. Class A is a rhodopsin-like family and the largest class by far accounting for nearly 85% of the GPCR genes. Class B1 GPCR is a secretin receptor family and includes, but is not limited to, amylin receptor (AMYR), calcitonin receptor (CTR), glucagon receptor (GCGR), glucose-dependent insulinotropic polypeptide receptor (GIPR), and glucagon-like peptide-1 receptor (GLP-1R). GPCRs that act as receptors for stimuli that have not yet been identified are known as orphan receptor. Review articles by Fredriksson and Stevens have additional information on different classes of GPCRs (Fredriksson et al. (2003) Mol. Pharm. 63, 1256-1272; Stevens et al. (2013) Nat. Rev. Drug Discov. 12, 25-34.) GPCRs are of considerable pharmacological importance. Itis estimated that about 30-50% of the targets of currently marketed drugs involve GPCRs (Salon et al. (2011) Pharm. Rev. 63, 901-937; Santos et. al. (2017) Nat. Rev. Drug Discov. 16, 19-34.)

[0004] G proteins can be activated by GPCRs. Ligand binding to a GPCR induces subtle changes in the receptor structure, allowing a productive interaction to occur with the G protein. This process is believed to comprise at least two stages: an initial docking interaction, possibly involving the Gβ and Gγ subunits, induces a conformational change in the extreme C-terminus of the Gα subunit. The C-terminus, which is the major receptor-binding region and determinant of receptor specificity, is then able to fully engage the receptor. This interaction triggers mutually induced conformational changes in both the G protein and receptor. In the G protein, these changes are propagated to the nucleotide-binding pocket, resulting in the release of GDP. In the receptor the conformational changes feedback to the ligand-binding pocket, reducing the dissociation rate of the ligand, which results in increased agonist binding affinity. This liganded GPCR-G protein ternary complex can be isolated in the biochemically stable form when a high affinity GPCR agonist is present, and the G protein maintained in the nucleotide-free state. Often, further complexing with a G protein-binding nanobody / antibody fragment, such as NB35 or mAb16, is needed to ultimately stabilize the GPCR-G protein ternary complex for biochemical, biophysical and structural studies (Maeda et al. (2018) Nat. Commun. 9, 3712.)

[0005] The lack of structural information has been an impediment to progress in characterizing GPCRs in part because the absence of a high affinity ligand or tool compound which has made the protein biochemistry for the structural efforts challenging.

[0006] GPCR-Gα fusion construct was first reviewed by Kobilka et al (1999) Trends Pharmacol. Sci., Vol. 20, Issue 9, 383-389. At the time, such constructs were only studied in whole cell functional assays. Biochemical isolation of GPCR-Gα fusion for structural study was not described until recently by Lees et al (2023) where an orphan receptor GPR61 was fused to a Gαs subunit and co-purified with Gβ / Gγ subunits. Less, et al. (2023) Nature Communications, 14:5938. However, GPR61 is an orphan receptor and has no broad applicability. Additionally, such design for isolating stable ligand-free apo-active GPCR-G protein complex for Class B1 GPCRs and other Class A GPCRs has not been reported. SUMMARY OF THE INVENTION

[0007] There is an unmet need for a stable complex of a GPCR and its cognate G protein for structural studies, wherein the GPCR is a class B1 GPCR, NPY2R (class A) or GPR75 (class A), and wherein the GPCR is in an apo-active state (i.e. the GPCR is maintained in an active conformation in the absence of a ligand binding to the GPCR). There is a strong need for a GPCR-G protein complex wherein the complex can be isolated from a cell membrane in a purified form and wherein the GPCR maintains its stability in an active conformation without the presence of a GPCR ligand or the need to be further complexed with and stabilized by a nanobody or an antibody fragment.

[0008] Disclosed herein is a complex of a GPCR and a cognate G protein, wherein the cognate G protein is a heterotrimeric G protein comprising an alpha (Gα) subunit, a beta (Gβ) subunit and a gamma (Gγ) subunit; wherein the GPCR is fused to the Gα subunit of the cognate G protein; wherein the Gα subunit comprises an amino acid sequence that contains one or more mutations compared to the amino acid sequence of the naturally-occurring parent heterotrimeric Gα subunit; wherein the mutations stabilize the GPCR-G protein complex in its nucleotide-free state; and wherein the GPCR maintains its stability in an active conformation without the presence of a GPCR ligand (apo-active state). BRIEF DESCRIPTION OF DRAWINGS

[0009] Fig. 1 shows a schematic of an apo-active GPCR-G Protein complex. In the complex, the N-terminus of the Gα subunit is directly fused to the C-terminus of the GPCR and the GPCR-Gα fusion is co-expressed with the Gβ and Gγ subunits and isolated in the nucleotide-free state and in the absence of a GPCR ligand.

[0010] Fig. 2 Shows a size exclusion chromatography (SEC) of apo-active GLP-1R-Gαs fusion, Gβ subunit and Gγ subunit.

[0011] Fig. 3 shows a size exclusion chromatography (SEC) of apo-active GIPR-Gαs fusion and CTR-Gαs fusion in complex with their Gβ subunit and Gγ subunit.

[0012] Fig. 4 shows a mass spectrometry of GIPR-Gαs fusion, Gβ subunit and Gγ subunit.

[0013] Fig. 5 shows a mass spectrometry of CTR-Gαs fusion, Gβ subunit and Gγ subunit.

[0014] Fig. 6 shows a size exclusion chromatography (SEC) of apo-active GCGR-Gαs fusion, Gβ subunit and Gγ subunit.

[0015] Fig. 7 shows a mass spectrometry of GCGR-Gαs fusion, Gβ subunit and Gγ subunit.

[0016] Fig. 8 shows a size exclusion chromatography (SEC) of apo-active NPY2R-Gαi fusion, Gβ subunit and Gγ subunit.

[0017] Fig. 9 shows a mass spectrometry of NPY2R-Gαi fusion, Gβ subunit and Gγ subunit.

[0018] Fig. 10 shows a size exclusion chromatography (SEC) of apo-active GPR75-Gαo fusion, Gβ subunit and Gγ subunit.

[0019] Fig. 11 shows a mass spectrometry of GPR75-Gαo fusion, Gβ subunit and Gγ subunit.

[0020] Fig. 12 shows the cryo-EM structures of AMY3R(CTR / RAMP3) in complex with their non-peptide-agonists (NPAs).

[0021] Fig. 13 shows the radioligand binding assay on the purified complex of GIPR-Gαs fusion, Gβ subunit and Gγ subunit.

[0022] Fig. 14 shows data demonstrating novel scaffolds are being enriched from a DNA- encoded library screening campaign using purified apo-active complex of NPY2R-Gαi fusion, Gβ subunit and Gγ subunit. DESCRIPTION OF THE INVENTION

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which the disclosure pertains.

[0024] Moreover, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one element is present, unless the context clearly requires that there be one and only one element. The indefinite article “a” or “an” thus usually means “at least one.”

[0025] As used herein, “about” means within a statistically meaningful range of a value or values such as, for example, a stated concentration, length, molecular weight, pH, sequence identity, time frame, temperature or volume. Such a value or range can be within an order of magnitude typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by “about” will depend upon the particular system under study, and can be readily appreciated by one of skill in the art.

[0026] As used herein, the term “apo” means ligand-free when used to describe a GPCR. For example, an apo-active GPCR means a GPCR in an active conformation when no ligand is bound to the GPCR.

[0027] Historically, it has been challenging to biochemically prepare a purified apo (ligand-free) GPCR due to the inherent instability of the receptor after detergent extraction from a cell membrane. While this inherent instability can be resolved by adding a high affinity ligand to the receptor prior to detergent extraction, the addition of the ligand would greatly limit the usability of the purified GPCR in structural or ligand screening studies since GPCR must be purified in a stable form in the absence of a ligand for such studies.

[0028] It has been found that a GPCR can be stabilized in an ‘apo-active’ state by engineering the GPCR to engage with a trimeric G protein complex to form a complex without the presence of a ligand. Specifically, a GPCR can be engineered, stabilized, and purified in the ‘apo-active’ state via a construct of the GPCR with its C-terminus fused to the N-terminus of its cognate Gα subunit containing dominant negative mutations that favor the Gα protein in the nucleotide-free state. By fusing the C-terminus of the GPCR to the N-terminus of the Gα subunit, the two proteins are placed in close proximity, and the receptor can be stabilized in an active state in the absence of a ligand. In one embodiment, Gβ and Gγ are co-expressed with the GPCR-Gα fusion to achieve a stable apo-active GPCR-G protein complex.

[0029] In one embodiment, the Gα subunit is mutated to favor stability in the nucleotide-free state (without GTP or GDP). The mutations are noted in the exemplified protein sequences.

[0030] In one embodiment, Gβ and Gγ subunits are co-expressed with the Gα subunit. In one embodiment, Gβ and Gγ subunits are co-purified with the Gα subunit during a purification process. In one embodiment, Gβ and Gγ subunits are co-expressed and co-purified with the Gα subunit to obtain a stable isolation of a GPCR-G protein complex in the apo-active (ligand-free) and G protein pre-coupled state. In one embodiment, the Gβ can be any subtype. In one embodiment, the Gβ is of the Gβ2 subtype. In one embodiment, the Gγ can be any subtype. In one embodiment, the Gγ is of the Gγ1 subtype.

[0031] In one embodiment, the fusion point between the GPCR and the Gα subunit is optimized. In one embodiment, the N-terminus of the Gα subunit is fused at 20-25 amino acids past helix 8 of the receptor as defined by the secondary structure prediction shown in gpcrdb.org. In oneembodiment, the region is further optimized to reduce proteolytic degradation during protein purification. This is indicated in the design of the optimized GCGR-Gs fusion.

[0032] For detergent extraction, LMNG / CHS and GDN are most suitable for the purification of the apo-active GPCR-G protein complex and the application of downstream applications such as cryo-EM and biophysical screens using Surface Plasmon Resonance, Affinity Selection Mass Spec, and DNA-encoded Library.

[0033] In one embodiment, provided herein is a mutant of a parent heterotrimeric G protein alpha subunit (Gα), wherein the mutant (i) is capable of binding to a GPCR; (ii) has an amino acid sequence that contains one or more mutations compared to the amino acid sequence of the parent heterotrimeric Gα subunit, wherein the mutations comprises amino acid substitutions as compared to the parent Gα subunit, and (iii) wherein the mutations stabilize the GPCR-G protein complex such that the GPCR is in the apo-active state and the G protein in the nucleotide-free state. In one embodiment, the mutations comprise dominant negative mutations that favor the G protein in the nucleotide-free state. In one embodiment, the mutant Gα subunit comprises amino acid substitutions as compared to the parent Gα subunit.

[0034] In one embodiment of the GPCR-G protein complex described herein, the Gα subunit comprises a mutant amino acid sequence that contains one or more dominant negative mutations.

[0035] In one embodiment of the GPCR-G protein complex described herein, the mutant Gα subunit comprises an amino acid sequence selected from: Gαs long variant: 1-394 of SEQ. ID No. 27 with the following mutations: S54N, G226A, E268A, N271K, K274D, R280K, T284D, I285T, A366S; Gαi: 1-354 of SEQ. ID No. 21 with the following mutations: S47N, G203A, E245A, A326S; Gαo: 1-354 of SEQ. ID No. 25 with the following mutations: S47N, G204A, E246A, A326S; and Gαq: 1-359 of SEQ. ID No. 28 with the following mutations: S53N, G208A, E250A, A253K, R256D, P262K, N266D, S267T, A331S.

[0036] In one embodiment, disclosed herein is a G protein coupled receptor (GPCR)-G protein complex,wherein the G protein is a cognate heterotrimeric G protein comprising an alpha (Gα) subunit, a beta (Gβ) subunit and a gamma (Gγ) subunit; wherein the GPCR is fused to the Gα subunit of the G protein; wherein the Gα subunit comprises an amino acid sequence that contains one or more mutations compared to the amino acid sequence of the naturally-occurring parent heterotrimeric Gα subunit; wherein the mutations stabilize the GPCR-G protein complex in its nucleotide-free state; and wherein the GPCR maintains its stability in an active conformation without the presence of a GPCR ligand (apo-active state).

[0037] In one embodiment of the GPCR-G protein complex described herein, the GPCR is a Class B1 GPCR, NPY2R or GPR75.

[0038] In one embodiment of the GPCR-G protein complex described herein, the C-terminus of the GPCR is fused to the N-terminus of an amino acid of the cognate Gα subunit.

[0039] In one embodiment of the GPCR-G protein complex described herein, the amino acid of the Gα subunit that is fused to the GPCR corresponds to an amino acid residue between 20 and 25 in the C-terminal region past helix 8 of the GPCR where helix 8 is defined as the last helical structure of a Class A and Class B1 GPCR.

[0040] In one embodiment of the GPCR-G protein complex described herein, the GPCR maintains its stability after the complex is extracted from a cell membrane in a purified form.

[0041] In one embodiment of the GPCR-G protein complex described herein, the fused GPCR-G protein (Gα subunit), the Gβ subunit, and the Gγ subunit form a monodispersed complex.

[0042] In one embodiment of the GPCR-G protein complex described herein, the GPCR is capable of engaging with the trimeric G protein without the presence of a GPCR ligand.

[0043] In one embodiment of the GPCR-G protein complex described herein, the GPCR is a glucose-dependent insulinotropic polypeptide receptor (GIPR).

[0044] In one embodiment of the GPCR-G protein complex described herein, the GPCR is a glucagon receptor (GCGR).

[0045] In one embodiment of the GPCR-G protein complex described herein, the GPCR is a glucagon-like peptide-1 receptor (GLP-1R).

[0046] In one embodiment of the GPCR-G protein complex described herein, the GPCR is a calcitonin receptor (CTR) alone or its heterodimeric complex with Receptor Activity Modifying Protein (RAMP) 1, 2 or 3, which are known as amylin receptors 1, 2 and 3.

[0047] In one embodiment of the GPCR-G protein complex described herein, the GPCR is a neuropeptide Y type 2 receptor (NPY2R).

[0048] In one embodiment of the GPCR-G protein complex described herein, the GPCR is an orphan GPCR known as GPR75.

[0049] In one embodiment of the GPCR-G protein complex described herein, the complex is used for screening GPCR ligands.

[0050] In one embodiment of the GPCR-G protein complex described herein, the complex is further complexed with a GPCR ligand to form a ternary complex, and the structure of the ternary complex can be determined to atomic resolution via cryo-electron microscopy (cryo-EM) and leveraged in structure-based drug design. EXAMPLES

[0051] The following examples are provided for illustration purposes only. The abbreviations used in the preparations and examples are as defined below unless otherwise defined in the specification. °C degree Celsius CHS cholesteryl hemisuccinate GLP-1R-Gs glucagon like peptide-1 receptor-Gαs fusion GCGR-Gs glucagon receptor-Gαs fusion GIPR-Gs glucose-dependent insulinotropic polypeptide (GIP) receptor-Gαs fusion GPCR-Gα G protein coupled receptor-Gα fusion CTR-Gs calcitonin receptor-Gαs fusion NPY2R-Gi neuropeptide Y type 2 receptor-Gαi fusion GPR75-Go GPR75 receptor-Gαo fusion h hour(s) IPA isopropyl alcohol HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acidLCMS liquid chromatography mass spectrometry LMNG Lauryl Maltose Neopentyl Glycol kDa kilodalton MALDI matrix-assisted laser desorption / ionization min minute(s) mg milligram mL milliliter mM millimolar rpm revolutions per minute SEC size exclusion chromatography µL microliter Example 1: Experimental Protocols and Parameters

[0052] The Baculovirus Expression protocol described below was used for the preparation of GPCR-G protein fusion proteins and protein complexes. Baculovirus Expression:

[0053] Spodoptera frugiperda (Sf9) were grown in Sf-900II SFM media (Thermo Fisher) at 27 °C and 140 rpm. Standard baculovirus expression using a modified version of the Bac-to-Bac system protocol (Thermo Fisher) in combination with the DH10EMBacY bacmid (Geneva Boiotech) was used to generate high-titer recombinant baculovirus for the GPCR-Gα fusion (GLP-1R-Gs, GCGR-Gs, GIPR-Gs, CTR-Gs, NPY2R-Gi, GPR75-Go), Gβ and Gγ subunits. Cells were grown to a density of 2 × 10^6 cells / mL and then infected with two separate baculoviruses, one containing the GPCR-Gα fusion and the other containing both Gβ and Gγ subunits in one vector. GPCR-Gα fusion and Gβ / Gγ were co-expressed by infecting Sf9 cells at a ratio of 1:0.5. After a 48-hour incubation at 27 °C, the cells were collected by centrifugation and stored at -80 °C for purification.

[0054] The experimental protocols described below were used for the isolation and purification of apo-active GPCR-G protein complex (comprising the GPCR-Gα fusion, Gβ and Gγ subunits).Flag Affinity Purification and Size Exclusion Chromatography:

[0055] 10 Liters of insect cell pellets from baculovirus expression were lysed and solubilized at the same time in the lysis buffer containing 10 mM HEPES pH 7.5, 50 mM NaCl, 5 mM CaCl2, 2mM MgCl2, 0.5mL of Halt Protease inhibitors (Thermo Scientific), 1:1000 dilution of Cocktail VII protease inhibitors (Calbiochem), 10 units of Apyrase (NEB) and a final concentration of 0.5% LMNG / 0.05% CHS (Lauryl Maltose Neopentyl Glycol and Cholesteryl Hemisuccinate from Anatrace). After mixing for 2 hours at 4 °C, the supernatant was collected by centrifugation at 16,000 × g for 30 min at 4 °C and then stirred with 2 mL of Flag resin (Genscript) overnight at 4 °C. After packing, the resin was washed with 20 CV (column volumes) of buffer F1 containing 10 mM HEPES pH 7.5, 100 mM NaCl, 2 mM MgCl2, 0.1% LMNG / 0.01% CHS and another 20 CV of buffer F2 containing 10 mM HEPES pH 7.5, 100 mM NaCl, and 0.01% LMNG / 0.001% CHS. The complex was eluted from the Flag resin with 20 mL of buffer F2 + 0.1 mg / mL Flag peptide by collecting two fractions of 10 mL elution with a 10 min incubation in between. The complex was concentrated to 500 µL using a 100 kDa MWCO Amicon Ultra Centrifugal Filter. Size exclusion chromatography in buffer F2 was carried out by loading the protein sample to a Superdex 200 Increase 10 / 300 GL column or Superose 6 Increase 10 / 300 GL column (Cytiva) to obtain a pure and monodisperse apo-active GPCR-G protein complex. Formation of Ligand-Bound GPCR-G Protein Ternary Complex via ‘Soaking’:

[0056] Pure and monodisperse apo-active GPCR-G protein complex in detergent solution was then complexed with receptor-specific ligands (peptide agonists or non-peptide agonists) by co- incubation (‘soaking’). Protein-ligand complex was then subjected to the state-of-the-art cryo- electron microscopy techniques to determine its atomic resolution structure. Cryo-electron Microscopy (cryo-EM) Techniques:

[0057] The liganded GPCR-G protein ternary complex was concentrated to the desired concentration (typically 3-4 mg / ml). Aliquots of 3.5 μl protein solution were applied to plasma- cleaned UltrAuFoil R1.2 / 1.3300 mesh grids (Quantifoil). Cryo-EM grids were prepared with a Vitrobot Mark IV (ThermoFisher Scientific), which were blotted for 3-5 s under blot force -2 at 95% humidity 4 °C and frozen in liquid nitrogen-cooled liquid ethane. Cryo-EM projections were acquired by a 200-keV Glacios electron microscope (ThermoFisher Scientific) fitted with aFalcon 4i direct electron detector (ThermoFisher Scientific) and an energy filter Selectris (ThermoFisher Scientific). Images of all datasets were recorded with a EPU system (ThermoFisher Scientific) with a physical pixel size of 0.877 Å and a defocus range of −0.8 to −1.6 μm. Data were collected with a dose rate of 11.9 e / Å2·s, and images were recorded during a 4.2-s exposure with 84-ms subframes (50 total frames). The raw movie stacks were gain- normalized and corrected for beam-induced motion using cryoSPARC (Punjani et al., 2017). CTF parameters were determined and refined from motion-corrected images using Patch CTF estimation in cryoSPARC. Prior particles picking micrographs were analyzed manually for good power spectrum, and the bad ones were discarded. Typically, about 5,000 to 10,000 movies were collected to produce a single particle reconstruction map at resolution 3.0A or better. For particularly difficult datasets (featuring low binding and low potency ligands), a mask isolating solely the GPCR receptor domain was employed for local refinement. This led to an ultimate enhancement in map quality, facilitating precise structural analysis.

[0058] The Mass Spectrometry protocol and parameters described below were used for analyzing and characterizing the apo-active GPCR-G protein complex. MALDI Protocol:

[0059] 2-8 mg of matrix (Alpha-Cyano-4-hydroxycinnamic Acid) was dissolved to 20 mg / mL with mixing and heating in a mixture of 16.67% LCMS grade water (1 part), 33.33% LCMS grade IPA (2 parts), and 50% formic acid (3 parts). The solution was centrifuged at 10,000 rpm for 15 seconds to remove insoluble material. Then, 1 µL of protein sample (at 1 mg / mL minimum) was mixed with 9 µL of the matrix solution, and 0.2 µL of that mixture was transferred and spotted onto a MALDI plate. The sample was allowed to dry on the plate, and the dried sample was then washed with 0.2 µL of 1% formic acid. The excess liquid was removed carefully, and the MALDI plate was inserted into the MALDI instrument for MALDI- TOF analysis. Mass Spectrometry Parameters Using MALDI:

[0060] Mass Spec: Bruker Ultraflextreme MALDI- TOF and TOF / TOF MALDI MTP target plate: Bruker Daltonics, Part No:8280784 Chemicals / Materials: LCMS Grade Isopropanol (CAS 67-63-0)LCMS Grade Water (CAS: 7732-18-5) LCMS Grade Formic Acid (CAS: 64-18-6) Alpha-Cyano-4-hydroxycinnamic Acid (CAS:28166-41-8) GPCR-Gα Fusion Radioligand Binding Method (as exemplified using the purified GIPR- Gs fusion in complex with Gβ and Gγ subunits): GIP(1-42)OH binding assay using FLAG capture

[0061] GIP(1-42)OH competitive binding using FLAG capture was designed following the protocol for assays using wheatgerm agglutinin (WGA) coated-polyvinyltoluene (PVT) scintillation proximity assay (SPA) beads and membrane preparations from cells expressing the GPCR of interest (Nat Metab, 2023), with the differences being that the GPCR protein is now FLAG-tagged and captured using mouse anti-FLAG antibody and anti-mouse IgG antibody coated PVT SPA beads. Additionally, for purified ‘apo-active’ FLAG-tagged GIPR-Gs + Gβ and Gγ complex, the assay was run in assay buffer containing added detergent, 0.01% lauryl maltose neopentyl glycol (LMNG) and 0.001% cholesteryl hemisuccinate tris salt (CHS). For Human ‘apo-active’ FLAG-tagged GIPR-Gs Fusion, Gβ and Gγ Complex:

[0062] The assays were performed with concentration-response curves generated by direct dilution (LabCyte Echo 555) in a 96 well white plate with a clear bottom (Costar 3632) and a final volume of 200 µL using assay buffer consisting of 2.5 mM MgCl2, 1.0 mM CaCl2, 0.003% v / v Tween-20, 0.01% LMNG, 0.001% CHS, 0.1 % w / v bacitracin (USB Cat# 11805), 0.1% w / v fatty acid free, globulin free human serum albumin (Sigma Cat# A3782) in 25 mM HEPES, pH 7.4 with KOH. This assay buffer was used for each step. Human [125I]GIP(1-42)OH (PerkinElmer Cat# NEX-402), 50 µL in assay buffer, (final approximately 0.06 nM) was added to 50 µL competing peptide in assay buffer containing monoclonal anti-FLAG M2 IgG antibody (5 µg / mL final, Sigma Cat# F3165) followed by the addition of 100 µL of assay buffer containing human ‘apo-active’ FLAG-tagged GIPR-Gs fusion, Gβ and Gγ complex (1 nM final) and 0.5 mg anti-mouse IgG coated PVT SPA beads (PerkinElmer RPNQ0017), then incubated for ~16 hours at 25 °C. The assay plate was then centrifuged at 1000 rpm for 5 min (Beckman Avanti J-15R tabletop centrifuge) followed by counting in a 1450 MicroBeta TriLux (PerkinElmer). Nonspecific binding was defined using 100 nM unlabeled GIP(1-42)NH2. Dataanalysis was performed using the four-parameter log(inhibitor) versus response with variable slope equation in GraphPad Prism. For Human FLAG-tagged GIPR Expressed in HEK-293T Cells:

[0063] The assays were performed identical to the assay for human ‘apo-active’ FLAG-tagged GIPR-Gs fusion, Gβ and Gγ complex, with the exception that the assay buffer did not contain the detergents LMNG or CHS. Additionally, membrane protein, 10 µg, from HEK-293T cells expressing the human FLAG-tagged GIPR (Multispan, MultiscreenTM Cat# MC1290) was added in place of the human ‘apo-active’ FLAG-tagged GIPR-Gs fusion, Gβ and Gγ complex. (El K, Douros JD, Willard FS, Novikoff A, Sargsyan A, Perez-Tilve D, Wainscott DB, Yang B, Chen A, Wothe D, Coupland C, Tschöp MH, Finan B, D'Alessio DA, Sloop KW, Müller TD, Campbell JE. The incretin co-agonist tirzepatide requires GIPR for hormone secretion from human islets. Nat Metab. 2023 Jun;5(6):945-954. doi: 10.1038 / s42255-023-00811-0. Epub 2023 Jun 5. PMID: 37277609; PMCID: PMC10290954.) DNA-Encoded Library Screen (as exemplified using the apo-active NPY2R-G protein complex):

[0064] A purified apo-active GPCR-G protein complex consisting of NPY2R-Gαi fusion, Gβ and Gγ subunits was used in a screening campaign utilizing DNA-encoded libraries (DEL). The biotinylated protein was supported through the Avi-Tag with streptavidin beads and incubated with the Lilly small molecule DEL pool. After three rounds of incubation / washings, protein was denatured, and the on-DNA binders were identified by PCR and sequencing, and then deconvoluted to afford the chemical structures for five well-enriched scaffolds. Example 2: GLP-1R-Gαs Fusion and Complex Proteins

[0065] Apo-active GLP-1R-Gαs fusion and complex proteins were prepared using the Baculovirus Expression Protocol and isolated and purified using the Flag Affinity Purification and Size Exclusion Chromatography Protocol as described above.

[0066] The GLP-1R-Gαs fusion protein has the following components and SEQ ID No. 6. Detailed sequences are provided in the Sequences section.(a). CD8 signal sequence: MALPVTALLLPLALLLHAARPAAASGI (SEQ ID No. 1); (b). FLAG tag: DYKDDDDK (SEQ ID No. 2); (c). GLP-1 Receptor (R24 to S452): (SEQ ID No. 3); (d). Gαi (2-18): GCTLSAEDKAAVERSKM (SEQ ID No. 4); and (e). Gαs long variant (26 to 394, with mutations S54N, G226A, E268A, N271K, K274D, R280K, T284D, I285T, A366S): (SEQ ID No. 5).

[0067] Using the SEC method described above and the size exclusion column S610 / 300 GL from Cytiva, the GLP-1R-Gαs fusion and complex proteins were analyzed and the results are shown in Fig. 2. This figure indicates a purified GLP-1R-Gαs fusion protein together with the Gβ and Gγ subunits. The peak fraction as indicated by the arrow is the monodispersed species of the complex containing GLP-1R-Gαs fusion, Gβ subunit and Gγ subunit.

[0068] From the SEC data in Fig. 2, the GLP-1R-Gαs fusion is about 96 kDa in molecular weight. The Gβ subunit is about 37 kDa in molecular weight. The Gγ subunit is about 9 kDa in molecular weight. The Gγ subunit is not stained in this tryptophan-florescence protein gel. Example 3: GIPR-Gαs Fusion and Complex Proteins

[0069] Apo-active GIPR-Gαs fusion and complex Proteins were prepared using the Baculovirus Expression Protocol and isolated and purified using the Flag Affinity Purification and Size Exclusion Chromatography Protocol as described above.

[0070] The GIPR-Gαs fusion protein has the following components and SEQ ID No. 10. Detailed sequences are provided in the Sequences section. (a). HA signal sequence: MKTIIALSYIFCLVFA (SEQ ID No. 7); (b). FLAG tag: DYKDDDDA (SEQ ID No. 8); (c). GIPR Receptor (L19 to S443): (SEQ ID No. 9); (d). Gαi (2-18): GCTLSAEDKAAVERSKM (SEQ ID No. 4); and (e). Gαs long variant (26 to 394, with mutations S54N, G226A, E268A, N271K, K274D, R280K, T284D, I285T, A366S): (SEQ ID No. 5).

[0071] Using the SEC method described above, the GIPR-Gαs fusion and complex proteins were analyzed and the results are shown in Fig. 3. This figure indicates a purified GIPR-Gαs fusionprotein together with the Gβ and Gγ subunits. The GIPR-Gαs fusion is about 96 kDa in molecular weight. The Gβ subunit is about 37 kDa and the Gγ subunit is about 9 kDa in molecular weight. The Gγ subunit is not stained in this tryptophan-florescence protein gel.

[0072] Also shown in Fig. 3 is the SEC data which indicates the Superdex S20010 / 300 GL from Cytiva. The SEC peak fraction (noted as bracketed portion) is the monodisperse portion and contained the purified complex as shown in the ‘Final’ lane.

[0073] Mass data by MALDI for the GIPR-Gαs complex is shown in Fig. 4. The GIPR-Gαs complex is identified at their expected molecular weights: GIPR-Gαs fusion at ~96 kDa (including glycosylation), Gβ at ~37 kDa, and Gγ at ~9.6 kDa. Example 4: CTR-Gαs Fusion and Complex Proteins

[0074] Apo-active CTR-Gαs fusion and complex Proteins were prepared using the Baculovirus Expression Protocol and isolated and purified using the Flag Affinity Purification and Size Exclusion Chromatography Protocol as described above.

[0075] The CTR-Gαs fusion protein has the following components and SEQ ID No. 16. Detailed sequences are provided in the Sequences section. (a). HA signal sequence: MKTIIALSYIFCLVFA (SEQ ID No. 7) (b). FLAG tag: DYKDDDDK (SEQ ID No. 2) (c). CTR (Calcitonin Receptor) (A25 to I442): (SEQ ID No. 15) (d). Gαi (2-18): GCTLSAEDKAAVERSKM (SEQ ID No. 4) (e). Gαs long variant (26 to 394, with mutations S54N, G226A, E268A, N271K, K274D, R280K, T284D, I285T, A366S): (SEQ ID No. 5).

[0076] Using the SEC method described above, the CTR-Gαs fusion and complex proteins were analyzed and the results are shown in Fig. 3. This figure indicates a purified CTR-Gαs fusion protein together with the Gβ and Gγ subunits. The CTR-Gαs fusion is about 96 kDa in molecular weight. The Gβ subunit is about 37 kDa and the Gγ subunit is about 9 kDa in molecular weight. The Gγ subunit is not stained in this tryptophan-florescence protein gel.

[0077] Also shown in Fig. 3 is the SEC data which indicates the Superdex 20010 / 300 GL from Cytiva. The Superdex 200 peak fraction (noted as bracketed portion) is the monodisperse portion and contained the purified complex as shown in the ‘Final’ lane.

[0078] Mass data by MALDI for the CTR-Gαs complex is shown in Fig. 5. The CTR-Gαs complex is identified at their expected molecular weights: CTR-Gαs fusion at ~98 kDa (including glycosylation), Gβ at ~37 kDa, and Gγ at ~9.6 kDa. Example 5: GCGR-Gαs Fusion and Complex Proteins

[0079] Apo-active GCGR-Gαs fusion and complex Proteins were prepared using the Baculovirus Expression Protocol and isolated and purified using the Flag Affinity Purification and Size Exclusion Chromatography Protocol as described above.

[0080] The GCGR-Gαs fusion protein has the following components and SEQ ID No. 14. Detailed sequences are provided in the Sequences section. (a). HA signal sequence: MKTIIALSYIFCLVFA (SEQ ID No. 7) (b). FLAG tag: DYKDDDDGGS (SEQ ID No. 11) (c). GCGR (glucagon receptor) (P24 to L424): (SEQ ID No. 12) (d). CTR (calcitonin receptor) (R416 to I442): RWGRRPSNRSARAAAAAAEAGDIPIYI (SEQ ID No. 13) (e). Gαi (2-18): GCTLSAEDKAAVERSKM (SEQ ID No. 4) (f). Gαs long variant (26 to 394, with mutations S54N, G226A, E268A, N271K, K274D, R280K, T284D, I285T, A366S): (SEQ ID No. 5).

[0081] The SEC column used for analyzing the GCGR-Gαs fusion and complex proteins is the S610 / 300 GL column from Cytiva and the results are shown in Fig. 6. The left hand side of this figure demonstrates that the “old” linker between GCGR and Gαs was sub-optimal since the GCGR-Gαs fusion protein was easily proteolyzed (cleaved by endogenous proteases). After linker optimization (“new” linker), no proteolysis in the GCGR-Gαs fusion protein was observed and the complex (GCGR-Gαs + Gβ + Gγ) is monodisperse and stable as indicated by the sharp peak (indicated by the arrow) on the right hand side of Fig. 6. The Gγ subunit is not stained in this tryptophan-florescence protein gel. The protein sequence of the GCGR-Gαs fusion with theoptimized linker utilizes a portion of the calcitonin receptor C-terminal region and is exemplified in the protein sequence.

[0082] The complex of GCGR-Gαs is identified in Fig. 7 at their expected molecular weights using MALDI: GCGR-Gαs fusion at ~100 kDa (including glycosylation), Gβ at ~37 kDa, and Gγ at ~9.6 kDa. Example 6: NPY2R-Gαi Fusion and Complex Proteins

[0083] Apo-active NPY2R-Gαi fusion and complex Proteins were prepared using the Baculovirus Expression Protocol and isolated and purified using the Flag Affinity Purification and Size Exclusion Chromatography Protocol as described above.

[0084] The NPY2R-Gαi fusion protein has the following components and SEQ ID No. 22. Detailed sequences are provided in the Sequences section. (a). HA signal sequence: MKTIIALSYIFCLVFA (SEQ ID No. 7) (b). FLAG tag: DYKDDDDK (SEQ ID No. 2) (c). BRIL fusion protein: (SEQ ID No. 17) (d). TEV protease cleavage site: ENLYFQG (SEQ ID No. 18) (e). NPY2R (NPY2 Receptor) (G2 to F357): (SEQ ID No. 19) (f). AVI tag : GLNDIFEAQKIEWHE (SEQ ID No. 20) (g). Gαi (1-354, with mutations S47N, G203A, E245A, A326S): (SEQ ID No. 21) The SEC column used is the S610 / 300 GL from Cytiva and the SEC data is shown in Fig. 8. The arrow indicates the monodispersed region of the complex, which contains NPY2R- Gαi fusion, Gβ, and Gγ subunits. To enable biotinylation, an AVI tag was inserted between NPY2R and Gαi as exemplified in the protein sequence.

[0085] Mass data by MALDI for the NPY2R-Gαi complex is shown in Fig. 9. The NPY2R-Gαi complex is identified at their expected molecular weights: NPY2R-Gαi fusion at ~95 kDa (including glycosylation and N-term BRIL fusion), Gβ at ~37 kDa, and Gγ at ~9.6 kDa. Example 7: GPR75-Gαo Fusion and Complex Proteins

[0086] Apo-active GPR75-Gαo fusion and complex Proteins were prepared using the Baculovirus Expression Protocol and isolated and purified using the Flag Affinity Purification and Size Exclusion Chromatography Protocol as described above.

[0087] The GPR75-Gαo fusion protein has the following components and SEQ ID No. 26. Detailed sequences are provided in the Sequences section. (a). HA signal sequence: MKTIIALSYIFCLVFA (SEQ ID No. 7) (b). FLAG tag: DYKDDDDAL (SEQ ID No. 23) (c). GPR75 (GPR75 Receptor) (N2 to S423, with deletion of residues 240-309): (SEQ ID No. 24) (d). Gαo (1-354, with mutations S47N, G204A, E246A, A326S): (SEQ ID No. 25).

[0088] The SEC column used is the S610 / 300 GL from Cytiva and the SEC data is shown in Fig. 10. The arrow indicates the monodispersed region of the complex, which contains GPR75- Go fusion, Gβ, and Gγ subunits. The Gγ subunit is visible in this Sypro-stained protein gel.

[0089] Mass data by MALDI for the GPR75-Gαo complex is shown in Fig. 11. The GPR75- Gαo complex is identified at their expected molecular weights: GPR75-Gαo fusion at ~81 kDa, Gβ at ~37 kDa, and Gγ at ~9.6 kDa. Example 8: Cryo-EM Studies of Liganded GPCR-Gα Fusion in Ternary Complex

[0090] Cryo-EM structures of AMY3R (CTR / RAMP3) in complex with their non-peptide agonists (NPAs) are exemplified in Fig. 12. Protein-ligand complexes were prepared by incubating (‘soaking’) the purified apo-active GPCR-G protein complex with receptor-specific NPA ligands. Example 9: Radioligand Binding Study on Purified GPCR-Gα Fusion with Endogenous Ligand

[0091] Purified GPCR-G protein complex binds endogenous ligand at the expected affinity when compared to the membrane-embedded receptor. This is exemplified in Fig. 13 where a radioligand binding assay was used to compare the affinity of gastric inhibitory peptide (GIP) on purified GIPR-Gs fusion complex and on GIPR expressed in the membrane of HEK293 cells.Example 10: Use of Purified Apo-Active GPCR-Gα Fusion and Complex Proteins in Ligand Discovery

[0092] Purified apo-active GPCR-Gα fusion complex protein can be leveraged in a screening campaign to discover new small molecule binders. This is exemplified in Fig. 14 where the purified apo-active complex of NPY2R-Gαi fusion, Gβ, and Gγ was used in a DNA-encoded library screening campaign, and five unique small molecule scaffolds were enriched.SEQUENCES NCBI reference protein sequences for the human proteins described herein: GLP-1 receptor: NP_002053.3 GIP receptor: NP_000155.1 GCG / glucagon receptor: NP_000151.1 CTR / calcitonin receptor: NP_001733 NPY2R: NP_000901.1 GPR75: NP_006785 Gαs long variant: AAM12611 Gαi: NP_002060 Gαo: NP_066268 Gβ2: CAA28207 Gγ1: AAM12584 SEQ ID No. 1 MALPVTALLLPLALLLHAARPAAASGI SEQ ID No. 2 DYKDDDDK SEQ ID No. 3 RPQGATVSLWETVQKWREYRRQCQRSLTEDPPPATDLFCNRTFDEYACWPDGEPGSFVNVSCPWYLPWAS SVPQGHVYRFCTAEGLWLQKDNSSLPWRDLSECEESKRGERSSPEEQLLFLYIIYTVGYALSFSALVIAS AILLGFRHLHCTRNYIHLNLFASFILRALSVFIKDAALKWMYSTAAQQHQWDGLLSYQDSLSCRLVFLLM QYCVAANYYWLLVEGVYLYTLLAFSVLSEQWIFRLYVSIGWGVPLLFVVPWGIVKYLYEDEGCWTRNSNM NYWLIIRLPILFAIGVNFLIFVRVICIVVSKLKANLMCKTDIKCRLAKSTLTLIPLLGTHEVIFAFVMDE HARGTLRFIKLFTELSFTSFQGLMVAILYCFVNNEVQLEFRKSWE RWRLEHLHIQ RDSSMKPLKCPTSSLSSGATAGSS SEQ ID No. 4 GCTLSAEDKAAVERSKMSEQ ID No. 5 IEKQLQKDKQVYRATHRLLLLGAGESGKNTIVKQMRILHVNGFNGEGGEEDPQAARSNSDGEKATKVQDI KNNLKEAIETIVAAMSNLVPPVELANPENQFRVDYILSVMNVPDFDFPPEFYEHAKALWEDEGVRACYER SNEYQLIDCAQYFLDKIDVIKQADYVPSDQDLLRCRVLTSGIFETKFQVDKVNFHMFDVGAQRDERRKWI QCFNDVTAIIFVVASSSYNMVIREDNQTNRLQAALKLFDSIWNNKWLRDTSVILFLNKQDLLAEKVLAGK SKIEDYFPEFARYTTPEDATPEPGEDPRVTRAKYFIRDEFLRISTASGDGRHYCYPHFTCSVDTENIRRV FNDCRDIIQRMHLRQYELL SEQ ID No. 6 MALPVTALLL PLALLLHAAR PAAASGIDYK DDDDKRPQGA TVSLWETVQK WREYRRQCQR SLTEDPPPAT DLFCNRTFDE YACWPDGEPG SFVNVSCPWY LPWASSVPQG HVYRFCTAEG LWLQKDNSSL PWRDLSECEE SKRGERSSPE EQLLFLYIIY TVGYALSFSA LVIASAILLG FRHLHCTRNY IHLNLFASFI LRALSVFIKD AALKWMYSTA AQQHQWDGLL SYQDSLSCRL VFLLMQYCVA ANYYWLLVEG VYLYTLLAFS VLSEQWIFRL YVSIGWGVPL LFVVPWGIVK YLYEDEGCWT RNSNMNYWLI IRLPILFAIG VNFLIFVRVI CIVVSKLKAN LMCKTDIKCR LAKSTLTLIP LLGTHEVIFA FVMDEHARGT LRFIKLFTEL SFTSFQGLMV AILYCFVNNE VQLEFRKSWE RWRLEHLHIQ RDSSMKPLKC PTSSLSSGAT AGSSGCTLSA EDKAAVERSK MIEKQLQKDK QVYRATHRLL LLGAGESGKN TIVKQMRILH VNGFNGEGGE EDPQAARSNS DGEKATKVQD IKNNLKEAIE TIVAAMSNLV PPVELANPEN QFRVDYILSV MNVPDFDFPP EFYEHAKALW EDEGVRACYE RSNEYQLIDC AQYFLDKIDV IKQADYVPSD QDLLRCRVLT SGIFETKFQV DKVNFHMFDV GAQRDERRKW IQCFNDVTAI IFVVASSSYN MVIREDNQTN RLQAALKLFD SIWNNKWLRD TSVILFLNKQ DLLAEKVLAG KSKIEDYFPE FARYTTPEDA TPEPGEDPRV TRAKYFIRDE FLRISTASGD GRHYCYPHFT CSVDTENIRR VFNDCRDIIQ RMHLRQYELL SEQ ID No. 7 MKTIIALSYIFCLVFA SEQ ID No. 8 DYKDDDDA SEQ ID No. 9LLQRAETGSKGQTAGELYQRWERYRRECQETLAAAEPPSGLACNGSFDMYVCWDYAAPNATARASCPWYL PWHHHVAAGFVLRQCGSDGQWGLWRDHTQCENPEKNEAFLDQRLILERLQVMYTVGYSLSLATLLLALLI LSLFRRLHCTRNYIHINLFTSFMLRAAAILSRDRLLPRPGPYLGDQALALWNQALAACRTAQIVTQYCVG ANYTWLLVEGVYLHSLLVLVGGSEEGHFRYYLLLGWGAPALFVIPWVIVRYLYENTQCWERNEVKAIWWI IRTPILMTILINFLIFIRILGILLSKLRTRQMRCRDYRLRLARSTLTLVPLLGVHEVVFAPVTEEQARGA LRFAKLGFEIFLSSFQGFLVSVLYCFINKEVQSEIRRGWHHCRLRRSLGEEQRQLPERAFRALPSG SGPGEVPTS SEQ ID No. 10 MKTIIALSYI FCLVFADYKD DDDALLQRAE TGSKGQTAGE LYQRWERYRR ECQETLAAAE PPSGLACNGS FDMYVCWDYA APNATARASC PWYLPWHHHV AAGFVLRQCG SDGQWGLWRD HTQCENPEKN EAFLDQRLIL ERLQVMYTVG YSLSLATLLL ALLILSLFRR LHCTRNYIHI NLFTSFMLRA AAILSRDRLL PRPGPYLGDQ ALALWNQALA ACRTAQIVTQ YCVGANYTWL LVEGVYLHSL LVLVGGSEEG HFRYYLLLGW GAPALFVIPW VIVRYLYENT QCWERNEVKA IWWIIRTPIL MTILINFLIF IRILGILLSK LRTRQMRCRD YRLRLARSTL TLVPLLGVHE VVFAPVTEEQ ARGALRFAKL GFEIFLSSFQ GFLVSVLYCF INKEVQSEIR RGWHHCRLRR SLGEEQRQLP ERAFRALPSG SGPGEVPTSG CTLSAEDKAA VERSKMIEKQ LQKDKQVYRA THRLLLLGAG ESGKNTIVKQ MRILHVNGFN GEGGEEDPQA ARSNSDGEKA TKVQDIKNNL KEAIETIVAA MSNLVPPVEL ANPENQFRVD YILSVMNVPD FDFPPEFYEH AKALWEDEGV RACYERSNEY QLIDCAQYFL DKIDVIKQAD YVPSDQDLLR CRVLTSGIFE TKFQVDKVNF HMFDVGAQRD ERRKWIQCFN DVTAIIFVVA SSSYNMVIRE DNQTNRLQAA LKLFDSIWNN KWLRDTSVIL FLNKQDLLAE KVLAGKSKIE DYFPEFARYT TPEDATPEPG EDPRVTRAKY FIRDEFLRIS TASGDGRHYC YPHFTCSVDT ENIRRVFNDC RDIIQRMHLR QYELL* SEQ ID No. 11 DYKDDDDGGS SEQ ID No. 12 PSAQVMDFLFEKWKLYGDQCHHNLSLLPPPTELVCNRTFDKYSCWPDTPANTTANISCPWYLPWHHKVQH RFVFKRCGPDGQWVRGPRGQPWRDASQCQMDGEEIEVQKEVAKMYSSFQVMYTVGYSLSLGALLLALAIL GGLSKLHCTRNAIHANLFASFVLKASSVLVIDGLLRTRYSQKIGDDLSVSTWLSDGAVAGCRVAAVFMQY GIVANYCWLLVEGLYLHNLLGLATLPERSFFSLYLGIGWGAPMLFVVPWAVVKCLFENVQCWTSNDNMGF WWILRFPVFLAILINFFIFVRIVQLLVAKLRARQMHHTDYKFRLAKSTLTLIPLLGVHEVVFAFVTDEHA QGTLRSAKLFFDLFLSSFQGLLVAVLYCFLNKEVQSELRRRWHRWRLGKVLSEQ ID No. 13 RWGRRPSNRSARAAAAAAEAGDIPIYI SEQ ID No. 14 MKTIIALSYI FCLVFADYKD DDDGGGSPSA QVMDFLFEKW KLYGDQCHHN LSLLPPPTEL VCNRTFDKYS CWPDTPANTT ANISCPWYLP WHHKVQHRFV FKRCGPDGQW VRGPRGQPWR DASQCQMDGE EIEVQKEVAK MYSSFQVMYT VGYSLSLGAL LLALAILGGL SKLHCTRNAI HANLFASFVL KASSVLVIDG LLRTRYSQKI GDDLSVSTWL SDGAVAGCRV AAVFMQYGIV ANYCWLLVEG LYLHNLLGLA TLPERSFFSL YLGIGWGAPM LFVVPWAVVK CLFENVQCWT SNDNMGFWWI LRFPVFLAIL INFFIFVRIV QLLVAKLRAR QMHHTDYKFR LAKSTLTLIP LLGVHEVVFA FVTDEHAQGT LRSAKLFFDL FLSSFQGLLV AVLYCFLNKE VQSELRRRWH RWRLGKVLRW GRRPSNRSAR AAAAAAEAGD IPIYIGCTLS AEDKAAVERS KMIEKQLQKD KQVYRATHRL LLLGAGESGK NTIVKQMRIL HVNGFNGEGG EEDPQAARSN SDGEKATKVQ DIKNNLKEAI ETIVAAMSNL VPPVELANPE NQFRVDYILS VMNVPDFDFP PEFYEHAKAL WEDEGVRACY ERSNEYQLID CAQYFLDKID VIKQADYVPS DQDLLRCRVL TSGIFETKFQ VDKVNFHMFD VGAQRDERRK WIQCFNDVTA IIFVVASSSY NMVIREDNQT NRLQAALKLF DSIWNNKWLR DTSVILFLNK QDLLAEKVLA GKSKIEDYFP EFARYTTPED ATPEPGEDPR VTRAKYFIRD EFLRISTASG DGRHYCYPHF TCSVDTENIR RVFNDCRDII QRMHLRQYEL L* SEQ ID No. 15 AFSNQTYPTIEPKPFLYVVGRKKMMDAQYKCYDRMQQLPAYQGEGPYCNRTWDGWLCWDDTPAGVLSYQF CPDYFPDFDPSEKVTKYCDEKGVWFKHPENNRTWSNYTMCNAFTPEKLKNAYVLYYLAIVGHSLSIFTLV ISLGIFVFFRSLGCQRVTLHKNMFLTYILNSMIIIIHLVEVVPNGELVRRDPVSCKILHFFHQYMMACNY FWMLCEGIYLHTLIVVAVFTEKQRLRWYYLLGWGFPLVPTTIHAITRAVYFNDNCWLSVETHLLYIIHGP VMAALVVNFFFLLNIVRVLVTKMRETHEAESHMYLKAVKATMILVPLLGIQFVVFPWRPSNKMLGKIYDY VMHSLIHFQGFFVATIYCFCNNEVQTTVKRQWAQFKIQWNQRWGRRPSNRSARAAAAAAEAGDIPIYI SEQ ID No. 16 MKTIIALSYI FCLVFADYKD DDDKAFSNQT YPTIEPKPFL YVVGRKKMMD AQYKCYDRMQ QLPAYQGEGP YCNRTWDGWL CWDDTPAGVL SYQFCPDYFP DFDPSEKVTK YCDEKGVWFK HPENNRTWSN YTMCNAFTPE KLKNAYVLYY LAIVGHSLSI FTLVISLGIF VFFRSLGCQR VTLHKNMFLT YILNSMIIII HLVEVVPNGE LVRRDPVSCK ILHFFHQYMM ACNYFWMLCEGIYLHTLIVV AVFTEKQRLR WYYLLGWGFP LVPTTIHAIT RAVYFNDNCW LSVETHLLYI IHGPVMAALV VNFFFLLNIV RVLVTKMRET HEAESHMYLK AVKATMILVP LLGIQFVVFP WRPSNKMLGK IYDYVMHSLI HFQGFFVATI YCFCNNEVQT TVKRQWAQFK IQWNQRWGRR PSNRSARAAA AAAEAGDIPI YIGCTLSAED KAAVERSKMI EKQLQKDKQV YRATHRLLLL GAGESGKNTI VKQMRILHVN GFNGEGGEED PQAARSNSDG EKATKVQDIK NNLKEAIETI VAAMSNLVPP VELANPENQF RVDYILSVMN VPDFDFPPEF YEHAKALWED EGVRACYERS NEYQLIDCAQ YFLDKIDVIK QADYVPSDQD LLRCRVLTSG IFETKFQVDK VNFHMFDVGA QRDERRKWIQ CFNDVTAIIF VVASSSYNMV IREDNQTNRL QAALKLFDSI WNNKWLRDTS VILFLNKQDL LAEKVLAGKS KIEDYFPEFA RYTTPEDATP EPGEDPRVTR AKYFIRDEFL RISTASGDGR HYCYPHFTCS VDTENIRRVF NDCRDIIQRM HLRQYELL SEQ ID No. 17 ADLEDNWETLNDNLKVIEKADNAAQVKDALTKMRAAALDAQKATPPKLEDKSPDSP EMKDFRHGFDILVGQIDDALKLANEGKVKEAQAAAEQLKTTRNAYIQKYL SEQ ID No. 18 ENLYFQG SEQ ID No. 19 GPIGAEADENQTVEEMKVEQYGPQTTPRGELVPDPEPELIDSTKLIEVQVVLI LAYCSIILLGVIGNSLVIHVVIKFKSMRTVTNFFIANLAVADLLVNTLCLPFTLTYTLMGEWKMGPVLCH LVPYAQGLAVQVSTITLTVIALDRHRCIVYHLESKISKRISFLIIGLAWGISALLASPLAIFREYSLIEI IPDFEIVACTEKWPGEEKSIYGTVYSLSSLLILYVLPLGIISFSYTRIWSKLKNHVSPGAANDHYHQRRQ KTTKMLVCVVVVFAVSWLPLHAFQLAVDIDSQVLDLKEYKLIFTVFHIIAMCSTFANPLLYGWMNSNYRK AFLSAFRCEQRLDAIHSEVSVTF SEQ ID No. 20 GLNDIFEAQKIEWHE SEQ ID No. 21 MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKNTIVKQMKIIHEAGYSEEECKQYK AVVYSNTIQSIIAIIRAMGRLKIDFGDSARADDARQLFVLAGAAEEGFMTAELAGVIKRLWKDSGVQACF NRSREYQLNDSAAYYLNDLDRIAQPNYIPTQQDVLRTRVKTTGIVETHFTFKDLHFKMFDVGAQRSERKK WIHCFEGVTAIIFCVALSDYDLVLAEDEEMNRMHASMKLFDSICNNKWFTDTSIILFLNKKDLFEEKIKKSPLTICYPEYAGSNTYEEAAAYIQCQFEDLNKRKDTKEIYTHFTCSTDTKNVQFVFDAVTDVIIKNNLKD CGLF SEQ ID No. 22 MKTIIALSYI FCLVFADYKD DDDKADLEDN WETLNDNLKV IEKADNAAQV KDALTKMRAA ALDAQKATPP KLEDKSPDSP EMKDFRHGFD ILVGQIDDAL KLANEGKVKE AQAAAEQLKT TRNAYIQKYL ENLYFQGGPI GAEADENQTV EEMKVEQYGP QTTPRGELVP DPEPELIDST KLIEVQVVLI LAYCSIILLG VIGNSLVIHV VIKFKSMRTV TNFFIANLAV ADLLVNTLCL PFTLTYTLMG EWKMGPVLCH LVPYAQGLAV QVSTITLTVI ALDRHRCIVY HLESKISKRI SFLIIGLAWG ISALLASPLA IFREYSLIEI IPDFEIVACT EKWPGEEKSI YGTVYSLSSL LILYVLPLGI ISFSYTRIWS KLKNHVSPGA ANDHYHQRRQ KTTKMLVCVV VVFAVSWLPL HAFQLAVDID SQVLDLKEYK LIFTVFHIIA MCSTFANPLL YGWMNSNYRK AFLSAFRCEQ RLDAIHSEVS VTFGLNDIFE AQKIEWHEMG CTLSAEDKAA VERSKMIDRN LREDGEKAAR EVKLLLLGAG ESGKNTIVKQ MKIIHEAGYS EEECKQYKAV VYSNTIQSII AIIRAMGRLK IDFGDSARAD DARQLFVLAG AAEEGFMTAE LAGVIKRLWK DSGVQACFNR SREYQLNDSA AYYLNDLDRI AQPNYIPTQQ DVLRTRVKTT GIVETHFTFK DLHFKMFDVG AQRSERKKWI HCFEGVTAII FCVALSDYDL VLAEDEEMNR MHASMKLFDS ICNNKWFTDT SIILFLNKKD LFEEKIKKSP LTICYPEYAG SNTYEEAAAY IQCQFEDLNK RKDTKEIYTH FTCSTDTKNV QFVFDAVTDV IIKNNLKDCG LF SEQ ID No. 23 DYKDDDDAL SEQ ID No. 24 NSTGHLQDAPNATSLHVPHSQEGNSTSLQEGLQDLIHTATLVTCTFLLAVIFCLGSYGNFIVFLSFFDPA FRKFRTNFDFMILNLSFCDLFICGVTAPMFTFVLFFSSASSIPDAFCFTFHLTSSGFIIMSLKTVAVIAL HRLRMVLGKQPNRTASFPCTVLLTLLLWATSFTLATLATLKTSKSHLCLPMSSLIAGKGKAILSLYVVDF TFCVAVVSVSYIMIAQTLRKNAQVRKCPNLSTAKDSKAVVTCVIIVLSVLVCCLPLGISLVQVVLSSNGS FILYQFELFGFTLIFFKSGLNPFIYSRNSAGLRRKVLWCLQYIGLGFFCCKQKTRLRAMGKGNLEVNRNK SS SEQ ID No. 25 MGCTLSAEERAALERSKAIEKNLKEDGISAAKDVKLLLLGAGESGKNTIVKQMKIIHEDGFSGEDVKQYK PVVYSNTIQSLAAIVRAMDTLGIEYGDKERKADAKMVCDVVSRMEDTEPFSAELLSAMMRLWGDSGIQEC FNRSREYQLNDSAKYYLDSLDRIGAADYQPTEQDILRTRVKTTGIVETHFTFKNLHFRLFDVGAQRSERK KWIHCFEDVTAIIFCVALSGYDQVLHEDETTNRMHASLMLFDSICNNKFFIDTSIILFLNKKDLFGEKIK KSPLTICFPEYTGPNTYEDAAAYIQAQFESKNRSPNKEIYCHMTCSTDTNNIQVVFDAVTDIIIANNLRG CGLYSEQ ID No. 26 MKTIIALSYI FCLVFADYKD DDDALNSTGH LQDAPNATSL HVPHSQEGNS TSLQEGLQDL IHTATLVTCT FLLAVIFCLG SYGNFIVFLS FFDPAFRKFR TNFDFMILNL SFCDLFICGV TAPMFTFVLF FSSASSIPDA FCFTFHLTSS GFIIMSLKTV AVIALHRLRM VLGKQPNRTA SFPCTVLLTL LLWATSFTLA TLATLKTSKS HLCLPMSSLI AGKGKAILSL YVVDFTFCVA VVSVSYIMIA QTLRKNAQVR KCPNLSTAKD SKAVVTCVII VLSVLVCCLP LGISLVQVVL SSNGSFILYQ FELFGFTLIF FKSGLNPFIY SRNSAGLRRK VLWCLQYIGL GFFCCKQKTR LRAMGKGNLE VNRNKSSMGC TLSAEERAAL ERSKAIEKNL KEDGISAAKD VKLLLLGAGE SGKNTIVKQM KIIHEDGFSG EDVKQYKPVV YSNTIQSLAA IVRAMDTLGI EYGDKERKAD AKMVCDVVSR MEDTEPFSAE LLSAMMRLWG DSGIQECFNR SREYQLNDSA KYYLDSLDRI GAADYQPTEQ DILRTRVKTT GIVETHFTFK NLHFRLFDVG AQRSERKKWI HCFEDVTAII FCVALSGYDQ VLHEDETTNR MHASLMLFDS ICNNKFFIDT SIILFLNKKD LFGEKIKKSP LTICFPEYTG PNTYEDAAAY IQAQFESKNR SPNKEIYCHM TCSTDTNNIQ VVFDAVTDII IANNLRGCGL Y SEQ. ID No. 27 Gαs long variant 1-394 with the following mutations: S54N, G226A, E268A, N271K, K274D, R280K, T284D, I285T, A366S. (protein sequence from: AAM12611.1) SEQ. ID No. 28 Gαq 1-359 with the following mutations: S53N, G208A, E250A, A253K, R256D, P262K, N266D, S267T, A331S. (protein sequence from; NP_002063.2)

Claims

CLAIMS 1. A G protein coupled receptor (GPCR)-G protein complex, wherein the G protein is a cognate heterotrimeric G protein comprising an alpha (Gα) subunit, a beta (Gβ) subunit and a gamma (Gγ) subunit; wherein the GPCR is fused to the Gα subunit of the G protein; wherein the Gα subunit comprises an amino acid sequence that contains one or more mutations compared to the amino acid sequence of the naturally-occurring parent heterotrimeric Gα subunit; wherein the mutations stabilize the GPCR-G protein complex in its nucleotide-free state; and wherein the GPCR maintains its stability in an active conformation without the presence of a GPCR ligand (apo-active state).

2. The GPCR-G protein complex of claim 1, wherein the GPCR is a Class B1 GPCR, NPY2R or GPR75.

3. The GPCR-G protein complex of claim 2, wherein the Gα subunit comprises a mutant amino acid sequence that contains one or more dominant negative mutations.

4. The GPCR-G protein complex of claim 3, wherein the mutant Gα subunit comprises an amino acid sequence selected from: Gαs long variant: 1-394 of SEQ. ID No. 27 with the following mutations: S54N, G226A, E268A, N271K, K274D, R280K, T284D, I285T, A366S; Gαi: 1-354 of SEQ. ID No. 21 with the following mutations: S47N, G203A, E245A, A326S; Gαo: 1-354 of SEQ. ID No. 25 with the following mutations: S47N, G204A, E246A, A326S; and Gαq: 1-359 of SEQ. ID No. 28 with the following mutations: S53N, G208A, E250A, A253K, R256D, P262K, N266D, S267T, A331S.

5. The GPCR-G protein complex of claim 4, wherein the C-terminus of the GPCR is fused to the N-terminus of an amino acid of the cognate Gα subunit.

6. The GPCR-G protein complex of claim 5, wherein the amino acid of the Gα subunit that is fused to the GPCR corresponds to an amino acid residue between 20 and 25 in the C- terminal region past helix 8 of the GPCR where helix 8 is defined as the last helical structure of a Class A or Class B GPCR.

7. The GPCR-G protein complex of claim 6, wherein the GPCR maintains its stability after the complex is extracted from a cell membrane in a purified form.

8. The GPCR-G protein complex of claim 7, wherein the fused GPCR-Gα subunit, the Gβ subunit, and the Gγ subunit form a monodispersed complex.

9. The GPCR-G protein complex of claim 7, wherein the GPCR is capable of engaging with the trimeric G protein without the presence of a GPCR ligand.

10. The GPCR-G protein complex of claim 9, wherein the GPCR is a glucose-dependent insulinotropic polypeptide receptor (GIPR).

11. The GPCR-G protein complex of claim 9, wherein the GPCR is a glucagon receptor (GCGR).

12. The GPCR-G protein complex of claim 9, wherein the GPCR is a glucagon-like peptide-1 receptor (GLP-1R).

13. The GPCR-G protein complex of claim 9, wherein the GPCR is a calcitonin receptor (CTR) or its heterodimer complexes with the receptor-activity modifying protein 1, 2, and 3, which respectively form amylin receptor 1, 2 and 3.

14. The GPCR-G protein complex of claim 9, wherein the GPCR is a neuropeptide Y type 2 receptor (NPY2R).

15. The GPCR-G protein complex of claim 9, wherein the GPCR is GPR75.

16. The GPCR-G protein complex of any one of claims 1-15, wherein the complex is used for screening and discovering novel GPCR ligands, wherein the screening is selected from a biophysical screening and a computer-aided virtual screening.

17. The GPCR-G protein complex of claim 16, wherein the biophysical screening is selected from a surface plasmon resonance screening, an affinity selection mass spectrometry screening and a DNA-encoded library screening.

18. The GPCR-G protein complex of any one of claims 1-15, wherein the apo-active complex is leveraged in a structure-based drug design.

19. The GPCR-G protein complex of any one of claims 1-15, wherein the apo-active complex is further incubated (‘soaked’) with a GPCR ligand to form a ternary complex, and the atomic resolution structure of the ternary complex is then determined via cryo-electron microscopy (cryo-EM).

20. The liganded GPCR-G protein ternary complex of claim 19, wherein the atomic resolution structure is leveraged in a structure-based drug design, wherein the structure-based drug design is selected from a human intelligence based drug design and a computer-aided drug design.

21. The GPCR-G protein complex of claim 6, wherein the amino acid residue past helix 8 of the GPCR is partially or fully replaced with an affinity tag or with a generic linker sequence comprising any amino acids.

22. The GPCR-G protein complex of claim 21, wherein the amino acid residue past helix 8 of the GPCR is partially or fully replaced with an affinity tag selected from an AVI tag, a FLAG tag, and a his tag.

23. The GPCR-G protein complex of claim 21, wherein the amino acid residue past helix 8 of the GPCR is partially or fully replaced with a generic linker sequence comprising any amino acids.

24. The GPCR-G protein complex of any one of claims 1-15, wherein the extracellular region N-terminal before transmembrane helix 1 of the GPCR is replaced and fused with a foreign soluble protein to enhance protein expression where transmembrane helix 1 is defined as the first transmembrane helix of a Class A or Class B GPCR.

25. The GPCR-G protein complex of claim 24, wherein the foreign soluble protein is BRIL.

26. The GPCR-G protein complex of any one of claims 1-15 and 24-25, wherein the N- terminus, C-terminus, or any of the intracellular and extracellular loops of the GPCR comprises one or more alterations selected from a point mutation, a truncation to the N-terminus, a truncation to the C-terminus, and a truncation to the intracellular and extracellular loops of the GPCR.