G proteins
11339383 · 2022-05-24
Assignee
Inventors
- Byron Carpenter (Swindon, GB)
- Andrew Leslie (Swindon, GB)
- Rony Nehmé (Swindon, GB)
- Christopher Gordon Tate (Swindon, GB)
- Antony Warne (Swindon, GB)
Cpc classification
C12Y306/05001
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The invention provides a mutant of a parent heterotrimeric G protein alpha (Gα) subunit, which mutant (i) lacks at least one helix of the helical domain of the parent Gα subunit; (ii) is capable of binding to a GPCR in the absence of a heterotrimeric G protein beta (Gβ) subunit and a heterotrimeric G protein gamma (Gγ) subunit; and (iii) has an amino acid sequence that contains one or more mutations compared to the amino acid sequence of the parent heterotrimeric Gα subunit, which mutations are selected from a deletion, a substitution and an insertion.
Claims
1. A mutant of a human parent heterotrimeric G protein alpha (Gα) subunit, which mutant: lacks the amino acid sequence of at least one of helix A, helix B, helix C, helix D, helix E, or helix F of the helical domain of the naturally-occurring parent Gα subunit, corresponding to amino acid residues 85-113, 123-136, 144-154, 157-168, 174-186 and 194-199, respectively, according to the numbering of the long isoform of human Gα-s subunit of SEQ ID NO:92; is capable of binding to a GPCR in the absence of a heterotrimeric G protein beta (Gβ) subunit and a heterotrimeric G protein gamma (Gγ) subunit; and has an amino acid sequence that contains one or more mutations compared to the amino acid sequence of the parent heterotrimeric Gα subunit, which mutations are selected from a deletion, a substitution and an insertion.
2. The mutant Gα subunit according to claim 1, wherein the mutant lacks a region of the helical domain of the parent heterotrimeric Gα subunit corresponding to amino acid residues 70-193, 71-193, 85-193, or 85-199 according to the numbering of the long isoform of human Gα-s subunit as set out in SEQ ID NO:92.
3. The mutant Gα subunit according to claim 1 wherein binding of the mutant Gα subunit to a GPCR increases the affinity of the GPCR for an agonist.
4. The mutant Gα subunit according to claim 1, wherein binding of the mutant Gα subunit to a GPCR activates the Gα subunit.
5. The mutant Gα subunit according to claim 4, wherein activation of the Gα subunit generates a Gα protein signal in a cell.
6. The mutant Gα subunit according to claim 1, wherein the mutant Gα subunit has increased stability under denaturing conditions compared to its parent Gα subunit or is expressed at a higher level than its parent Gα subunit, when expressed in a cell.
7. The mutant Gα subunit according to claim 1, wherein the mutant Gα subunit is able to stabilize a particular conformation of the GPCR upon binding to the GPCR.
8. The mutant Gα subunit according to claim 1, wherein the mutant Gα subunit is capable of binding to a nucleotide, such as a guanine nucleotide, or wherein the mutant Gα subunit is capable of binding to a Gβ and/or Gγ subunit of a heterotrimeric G protein.
9. The mutant Gα subunit according to claim 1, wherein the Gα subunit is any one of a Gα.sub.s, Gα.sub.i/o, Gα.sub.q/11, or Gα.sub.12/13 subunit.
10. The mutant Gα subunit according to claim 1, wherein the switch I region of the parent heterotrimeric G protein alpha subunit is not deleted, or wherein the switch I region of the parent heterotrimeric G protein alpha subunit is deleted or replaced by a switch I region of a small GTPase, or wherein the switch I region of the parent heterotrimeric G protein alpha subunit corresponds to amino acid residues 194-207 according to the numbering of the long isoform of human Gα-s subunit as set out in SEQ ID NO:92.
11. The mutant Gα subunit according to claim 1, wherein the helical domain or part thereof and/or the switch I region or part thereof, of the parent heterotrimeric G protein alpha subunit is replaced by a linker sequence, or wherein the region of the parent heterotrimeric Gα subunit that corresponds to amino acid residues 65 to 203 according to the numbering of the long isoform of human Gα-s subunit as set out in SEQ ID NO:92, is deleted or replaced by a linker sequence.
12. The mutant Gα subunit according to claim 1, which, when compared to the parent Gα subunit, has an N-terminally truncated amino acid sequence, or contains one or more mutations in the switch I region.
13. The mutant Gα subunit according to claim 1, wherein the switch III region of the parent heterotrimeric G protein alpha subunit is deleted.
14. The mutant Gα subunit according to claim 1, wherein the switch II region, or part thereof, of the parent heterotrimeric Gα subunit, is replaced by a linker sequence.
15. The mutant Gα subunit according to claim 1, which, when compared to the parent Gα subunit, has a different amino acid at a position which corresponds to any one or more of the following positions according to the numbering of the long isoform of human Gα-s subunit as set out in SEQ ID NO:92: Val 36, His 41, Ala 48, Gly 49, Glu 50, Met 60, Leu 63, Leu 197, Cys 200, Arg 201, Phe 208, Asn 218, Gly 226, Glu 230, Ala 249, Ser 252, Leu 272, Ile 372, Val 375.
16. The mutant Gα subunit according to claim 1, wherein the mutant Gα subunit has at least 20% sequence identity to the amino acid sequence of the long isoform of human Gα-s subunit as set out in SEQ ID NO:92, or to any of the amino acid sequences as set out in SEQ ID NO:95-140.
17. The mutant Gα subunit according to claim 1, which, when compared to the parent Gα subunit, comprises one or more dominant negative mutations.
18. The mutant Gα subunit according to claim 1, which, when compared to the parent Gα subunit, has one or more different amino acids within the NKXD motif.
19. The mutant Gα subunit according to claim 1, wherein the mutant lacks helices A to E or lacks helices A to F of the helical domain of the parent Gα subunit.
20. The mutant Gα subunit according to claim 1, which is a mutant Gαt subunit wherein the amino acid residue at a position that corresponds to Cys 347 according to the numbering of the Gαt subunit as set out in any one of SEQ ID NO:141-156, is chemically modified.
21. The mutant Gα subunit according to claim 1, wherein the mutant Gα subunit has increased stability under denaturing conditions compared to its parent Gα subunit and is expressed at a higher level than its parent Gα subunit, when expressed in a cell.
22. The mutant Gα subunit according to claim 1, wherein the mutant Gα subunit is capable of binding to a nucleotide, such as a guanine nucleotide, and wherein the mutant Gα subunit is capable of binding to a Gβ and/or Gγ subunit of a heterotrimeric G protein.
Description
(1) The invention will now be described by reference to the following figures and examples.
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(5) (a) For clarity, curves representing the binding reactions are described in order from the left hand side of the graph to the right hand side of the graph (Ki for isoprenaline binding is shown in parentheses, with number of independent experiments (n) also indicated): (i) β.sub.1AR-WT+Nb80 (Ki 5.8±0.8 nM, n=2); (ii) β.sub.1AR-WT+Gs-Nb35 (Ki 6.8±0.6 nM, n=2); (iii) β.sub.1AR-WT+Gs (Ki 17±2 nM, n=2); (iv) β.sub.1AR-WT (40±0 nM, n=2).
(6) (b) For clarity, curves representing the binding reactions are described in order from the left hand side of the graph to the right hand side of the graph (Ki for isoprenaline binding is shown in parentheses, with number of independent experiments (n) also indicated): (i) β.sub.1AR-84+Gs-Nb35 (Ki 16±4 nM, n=3); (ii) β.sub.1AR-84+Nb80 (Ki 28±1 nM, n=2); (iii) β.sub.1AR-84+Gs (Ki 271±54 nM, n=2); (iv) β.sub.1AR-84 (2.6±0.3 μM, n=2).
(7) (c) Experiments performed at 20° C. For clarity, curves representing the binding reactions are described in order from the left hand side of the graph to the right hand side of the graph (Ki for isoprenaline binding is shown in parentheses, with number of independent experiments (n) also indicated): (i) β.sub.1AR-84+Mini Gs.sub.77-βγ-Nb35 (Ki 3.6±0.8 nM, n=2); (ii) β.sub.1AR-84+Mini Gs.sub.77 (Ki 1.9±0.2 μM, n=3); (iii) β.sub.1AR-84 (Ki 2.6±0.3 μM, n=15).
(8) (d) Experiments performed at 4° C. For clarity, curves representing the binding reactions are described in order from the left hand side of the graph to the right hand side of the graph (Ki for isoprenaline binding is shown in parentheses, with number of independent experiments (n) also indicated): (i) β.sub.1AR-84+Mini Gs.sub.77-βγ-Nb35 (Ki 10 nM, n=2); (ii) β.sub.1AR-84+Mini Gs.sub.77 (Ki 117 nM, n=2); (iii) β.sub.1AR-84 (Ki 2.1±0.2 μM, n=12).
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(15) (b) Receptor in membranes. For clarity, curves representing the binding reactions are described in order from the left hand side of the graph to the right hand side of the graph (Ki for isoprenaline binding is shown in parentheses, with number of independent experiments (n) also indicated): (i) β.sub.1AR-84+mini Gs.sub.393 (Ki 3.6±0.0 nM, n=2); (ii) β.sub.1AR-84+Gs-Nb35 (Ki 16±4 nM, n=2); (iii) β.sub.1AR-84 (Ki 2.6±0.3 μM, n=15).
(16) (c) Receptor solubilised in DDM. For clarity, curves representing the binding reactions are described in order from the left hand side of the graph to the right hand side of the graph (Ki for isoprenaline binding is shown in parentheses, with number of independent experiments (n) also indicated): (i) β.sub.1AR-84+mini Gs.sub.393 (Ki 4.7±0.4 nM, n=2); (ii) β.sub.1AR-84+Gs-Nb35 (Ki 23±7 nM, n=2); (iii) β.sub.1AR-84 (Ki 2.8±0.2 μM, n=2).
(17) (d-f) Analytical gel filtration analysis of mini Gs complexes. (d) Mini Gs.sub.393 was purified with a yield of 100 mg per litre E. coli culture (inset), and resolved as a single peak with a retention volume of 17.2 ml by gel filtration. (e) Mini Gs.sub.399, a construct in which N-terminal residues 6-25 were replaced and the L272D mutation reversed retained its ability to bind Gβ.sub.1γ.sub.2. An equimolar mixture of mini Gs.sub.399 and Gβ.sub.1γ.sub.2 resolved as a single peak with a retention volume of 14.6 ml, compared with retention volumes of 15.8 ml or 16.4 ml for Gβ.sub.1γ.sub.2 or mini Gs.sub.399, respectively. (f) Mini Gs.sub.393 was able to bind purified β.sub.1AR-WT in LMNG detergent. An equimolar mixture of mini Gs.sub.393 and β.sub.1AR-WT resolved as a predominant peak with a retention volume of 13.2 ml, compared with retention volumes of 13.6 ml or 17.1 ml for β.sub.1AR-WT or mini Gs.sub.393, respectively.
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(19) (b) Thermostability in octylglucoside; uncoupled β.sub.1AR-WT did not survive solubilisation in OG detergent. For clarity, thermostability curves are described in order from the left hand side of the graph to the right hand side of the graph (apparent Tm is shown in parentheses, with number of independent experiments (n) also indicated): (i) β.sub.1AR-WT+Gs-Nb35 (Tm 13.6±0.2° C., n=3); (ii) β.sub.1AR-WT+Nb80 (Tm 14.3±0.2° C., n=3); (iii) β.sub.1AR-WT+mini Gs.sub.393 (Tm 19.7±0.5° C., n=3).
(20) (c-d) GTP-mediated dissociation of β.sub.1AR-84 complexes, measured by competitive binding assay in membranes. Mini Gs.sub.404, is an identical construct to mini Gs.sub.393, except the I372A and V375I mutations were reversed.
(21) (c) No GTPγS present in the assay. For clarity, curves representing the binding reactions are described in order from the left hand side of the graph to the right hand side of the graph (Ki for isoprenaline binding is shown in parentheses, with number of independent experiments (n) also indicated): (i) β.sub.1AR-84+mini Gs.sub.393 (Ki 3.6±0.0 nM, n=3); (ii) β.sub.1AR-84+mini Gs.sub.404 (Ki 18±2 nM, n=2); (iii) β.sub.1AR-84+Gs (Ki 271±54 nM, n=2); (iv) β.sub.1AR-84 (Ki 2.6±0.3 μM; n=15).
(22) (d) In the presence of 0.25 mM GTPγS in the assay. For clarity, curves representing the binding reactions are described in order from the left hand side of the graph to the right hand side of the graph (Ki for isoprenaline binding is shown in parentheses, with number of independent experiments (n) also indicated): (i) β.sub.1AR-84+mini Gs.sub.393 (Ki 5.2±0.7 nM, n=2); (ii) β.sub.1AR-84+mini Gs.sub.404 (Ki 700±60 nM, n=2); (iii) β.sub.1AR-84+Gs (Ki 2.7±0.1 μM, n=2); (iv) β.sub.1AR-84 (Ki 3.0±0.1 μM; n=2).
(23) There was no statistical difference in the isoprenaline affinity of the β.sub.1AR-WT-mini Gs.sub.393 in the presence or absence of GTPγS. Curves shown are from a representative experiment performed in duplicate.
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(25) (b) Thermostability in nonylglucoside; uncoupled β.sub.1AR-WT did not survive solubilisation in NG detergent. For clarity, thermostability curves are described in order from the left hand side of the graph to the right hand side of the graph (apparent Tm is shown in parentheses, with number of independent experiments (n) also indicated): (i) β.sub.1AR-WT+Nb80 (Tm 16.7±0.7° C., n=2); (ii) β.sub.1AR-WT+Gs-Nb35 (Tm 19.0±0.2° C., n=2); (iii) β.sub.1AR-WT+mini Gs.sub.393 (Tm 24.7±0.4° C., n=2). Data represent mean±SEM of the number of independent experiments (n) indicated in the legend. Curves shown are from a representative experiment performed in duplicate.
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(27) (a) Absence of GTPγS. For clarity, curves representing the binding reactions are described in order from the left hand side of the graph to the right hand side of the graph (Ki for isoprenaline binding is shown in parentheses, with number of independent experiments (n) also indicated): (i) β.sub.1AR-84+mini Gs.sub.391(Ki 3.0±0.4 nM, n=2); (ii) β.sub.1AR-84 (Ki 2.6±0.3 μM, n=15).
(28) (b) In the presence of GTPγS (0.25 mM). For clarity, curves representing the binding reactions are described in order from the left hand side of the graph to the right hand side of the graph (Ki for isoprenaline binding is shown in parentheses, with number of independent experiments (n) also indicated): (i) β.sub.1AR-84+mini Gs.sub.391 (Ki 4.7±0.1 nM, n=2); (ii) β.sub.1AR-84 (Ki 3.0±0.1 μM, n=2).
(29) There was no statistical difference in the isoprenaline affinity of the β.sub.1AR-WT-mini Gs.sub.391 in the presence or absence of GTPγS. Curves shown are from a representative experiment performed in duplicate.
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EXAMPLE 1: ENGINEERING A MINIMAL G PROTEIN TO FACILITATE CRYSTALLISATION OF G PROTEIN COUPLED RECEPTORS IN THEIR ACTIVE CONFORMATION
Introduction
(59) G protein coupled receptors (GPCRs) modulate cytoplasmic signalling pathways in response to stimuli such as hormones and neurotransmitters. Structure determination of GPCRs in all activation states is vital to elucidate the precise mechanism of signal transduction. However, due to their inherent instability, crystallisation of GPCR-G protein complexes has proved particularly challenging. Here, we describe the design of a minimal G protein, which is composed solely of the GTPase domain from the adenylate cyclase stimulating G protein (Gs). Mini Gs is a small, soluble protein, which efficiently couples GPCRs in the absence of Gβγ subunits. We engineered mini Gs to form a stable complex with the β1 adrenergic receptor (β1AR), even when solubilised in short chain detergents. Mini G proteins induce similar pharmacological and structural changes in GPCRs as heterotrimeric G proteins. They are therefore novel tools, which will facilitate high throughput structure determination of GPCRs in their active conformation.
(60) Results
(61) Developing a Sensitive Assay to Detect Gs Coupling to β1AR
(62) We developed a sensitive competitive binding assay, which could detect the interaction of different binding partners with β.sub.1AR, by measuring the response in agonist binding affinity. The binding partners used during this work were: Nb80.sup.38, a Nanobody that binds β.sub.2AR and induces a comparable shift in agonist affinity to lipidated Gs; Nb35.sup.40, a Nanobody that stabilises Gs in its GPCR-bound conformation; non-lipidated Gs (Gαsβ.sub.1γ.sub.2); and non-lipidated Gβγ (Gβ.sub.1γ.sub.2). The concentration of binding proteins used in the assays was standardised to 25 μM, which is approximately 30-fold above the equilibrium dissociation constant (K.sub.D) for Nb80 binding to β.sub.1AR.sup.96. No affinity data was available for Gs, however we anticipated this concentration to be at least 10-fold above K.sub.D.
(63) A heterologous competitive binding assay was used to measure competition between the antagonist .sup.3H-dihydroalprenolol (.sup.3H-DHA) and the agonist isoprenaline. Inhibition constant (K.sub.i) values were calculated using the dissociation constant (K.sub.d) of .sup.3H-DHA derived from saturation binding experiments (see
(64) Isolation of the Gαs GαGTPase Domain and Measuring Binding to β.sub.1AR-84
(65) The structure of the β.sub.2AR-Gs complex.sup.10 revealed that only the GαGTPase domain from Gs forms significant interactions with the receptor (see
(66) Thermostabilisation of the β.sub.1AR-Mini Gs Complex
(67) We thermostabilised mini Gs in complex with β.sub.1AR. Mutants were screened using our competitive binding assay at both 4° C. and 20° C. Due to the low, and variable expression level of the mutants, it was impossible to standardize the concentration used in the assays. Instead, the total mini Gs purified from 1 L of E. coli culture was used per competition curve (Table 3). Approximately 100 mutants were tested during the initial screen. Mutations that shifted the agonist affinity of β.sub.1AR-84 more than 2-fold compared to the parental mini Gs construct (mini Gs.sub.77) at either temperature were classed as positive. A total of 14 positive mutations, covering 11 unique positions were identified (Table 3).
(68) A new parental construct (mini Gs.sub.161), which contained a modified N-terminus and linker region (see Table 2), was used to combine mutations. Positive mutations were combined with one of the best mutations from the first round of screening (A249D), and their stability in complex with β.sub.1AR-WT was tested using a thermostability assay in n-dodecyl-β-D-maltopyranoside (DDM) detergent. The agonist .sup.3H-norepinephrine (.sup.3H-NE) was used in the Tm assay, however due to the high background signal associated with this ligand, a maximum concentration of 200 nM could be used in the assay. This was approximately equal to the K.sub.i of uncoupled β.sub.1AR-WT, but approximately 250-fold above K.sub.i of β.sub.1AR-WT complexed with Nb80 or Gs-Nb35 (see
(69) The A249D mutant (mini Gs.sub.162) had an apparent Tm of 25.1° C. (Table 4), which was lower than that of uncoupled of β.sub.1AR-WT (25.9° C.). A double mutant (mini Gs.sub.164), containing the A249D mutation and switch III deletion, increased the apparent Tm of the complex to 28.6° C. Addition of G49D, E50N, S252D and L272D mutations produced similar apparent Tm values (within 0.2° C. of the double mutant). These six mutations were utilised in the final construct, because of their positive individual effect on the agonist affinity of membrane-embedded β.sub.1AR-84 (Table 3). None of the other positive mutations from the first round of screening further increased the Tm of the complex and so were rejected. All of the combinations, except L272D, also increased the Tm of the basal GDP-bound state (Table 4), as assessed by differential scanning fluorimetry (DSF).
(70) Five of the six mutations, which were successfully combined, were clustered around the nucleotide-binding pocket and phosphate-binding loop (P-loop) (
(71) Screening Mutations that Stabilise the β.sub.1AR-WT-Mini Gs Complex in Detergent
(72) Mini Gs.sub.183, which contained the six stabilising mutations from the first round of screening, was unable to fully stabilise detergent-solubilised β.sub.1AR-WT. Complexes of Nb80 or Gs-Nb35 had apparent Tm values 3.3° C. or 7.1° C. higher than mini Gs.sub.183, respectively (Table 4). Therefore, a second panel of approximately 150 mutants were designed based on the structure of Gs in its receptor-bound conformation.sup.10, using mutagenesis. These mutations were screened in a parental construct, which had a modified linker region (see Table 2). We identified four additional mutations that increased the stability of the complex in detergent (Table 4). The best mutant (I372A) increased the apparent Tm of the complex from 29.2° C. to 34.00° C. and combined additively with V375I giving a final apparent Tm of 35.0° C. This was 3.0° C. higher than Nb80 and only 0.8° C. lower than Gs-Nb35. All of the detergent stabilising mutations decreased the stability of mini Gs in the GDP-bound conformation (Table 4).
(73) The detergent stabilising mutations were located around the α1-α5 helix interface. Alignment of Gαs in the GPCR-bound conformation.sup.10 with the GTP-bound structure.sup.81 revealed a steric clash between Ile-372 (in the α5 helix) and residues Met-60 and His-64 (in the α1 helix) (
(74) Validation of Mini Gs
(75) The detergent stabilised construct (mini Gs.sub.345) was modified for crystallography applications by changing the linker and shortening the N-terminus (see Table 2). The final stabilised construct was named mini Gs.sub.393 (see
(76) Mini Gs.sub.393 was highly expressed in E. coli: it could be purified with a yield of 100 mg per litre culture, and concentrated to over 100 mg/ml (
(77) The stability of the β.sub.1AR-WT-mini Gs.sub.393 complex was tested in a number of different detergents. In longer chain detergents such as DDM the β.sub.1AR-WT-mini Gs.sub.393 complex had an apparent Tm of 34.1° C., which was 8.2° C. higher than uncoupled β.sub.1AR-WT. In DDM mini Gs.sub.393 was slightly less stabilising (1.7° C.) than Gs-Nb35, but slightly more stabilising (2.1° C.) than Nb80 (
(78) The nucleotide binding properties of the mutants were not extensively studied in this work, but one interesting observation was made: β.sub.1AR-84 complexes involving mini Gs.sub.393 were completely resistant to GTP-mediated dissociation (
(79) Treatment of the complex with GTPγS fully reversed the shift in agonist binding affinity induced by Gs from 271±54 nM to 2.7±0.1 μM (
Discussion
(80) Several novel approaches have been developed to stabilise and crystallise GPCRs in active-like conformations, these include complexation with: G protein-derived peptides.sup.36,37, G protein-mimicking nanobodies.sup.38,100,101, and nanobody-stabilised heterotrimeric G proteins.sup.10. However, these approaches are not ideal: the G protein-derived peptides do not appear to induce the same conformational changes in the receptor as the heterotrimeric G protein; the G protein-mimicking nanobodies cannot recreate the native GPCR-G protein interface; and the heterotrimeric G protein complexes are large, dynamic, and unstable in detergent, making them particularly challenging targets for crystallisation. Therefore, we designed a minimal G protein, which was amenable to high throughput crystallisation of native-like GPCR-G protein complexes. Recently, we solved the structure of mini Gs in complex with the wild type human adenosine A.sub.2a receptor at 3.4 Å resolution (see Example 4). The molecular organisation of the A.sub.2a complex is remarkably similar to that of the β.sub.2AR-Gs complex.sup.10, strongly suggesting that it is an accurate reflection of the native signalling complex.
(81) The G protein engineering work has also provided unique insight into the mechanism of G protein activation. We identified a steric clash between the α1 and α5 helices in the receptor-bound conformation, which appears to prevent close packing of the α1 helix against the core of the GαGTPase domain (
(82) Mini G proteins are novel tool, which have many potential applications, including: characterisation of receptor pharmacology, binding kinetic studies, thermostabilisation of GPCR in their active conformation, drug discovery, and crystallisation of native-like GPCR-G protein complexes. Furthermore, all of the mutations reported here are located within conserved regions of the Gα subunit. Therefore, the concept is believed to be transferable to all classes of heterotrimeric G protein, which would allow the production of a panel of mini G proteins capable of coupling any GPCR.
(83) Materials and Methods
(84) Cloning
(85) Details of all constructs used in this work are provided in Tables 1 and 2. Site directed mutagenesis was performed using the Quick Change protocol (Stratagene). Insertions and deletions were performed using a modified version of a previously described method.sup.102.
(86) Baculovirus Expression of G Proteins
(87) G protein genes were cloned into the transfer vector pBacPAK8 (Clontech), and baculoviruses were prepared using the flashBAC ULTRA system (Oxford Expression Technologies). Trichopulsia ni cells (Expression Systems) were grown in ESF921 serum-free media (Expression Systems) in 5 L Optimum growth flasks (Thompson Instrument Company). Immediately before infection, heat inactivated foetal bovine serum (Sigma) was added to a final concentration of 5%. Cells were infected with third passage virus at a final concentration of 3%. In the case of co-infection with multiple viruses (for heterotrimeric Gs or Gβγ) each virus was added to a final concentration of 3%. The final volume of culture was 3 L per flask and the final cell density was 3×10.sup.6 cells/ml. Cells were harvested 48 h post-infection by centrifugation at 5000 g for 5 mins, flash-frozen in liquid nitrogen and stored at −80° C.
(88) Purification of Non-Lipidated Gαs
(89) The cell pellet from 6 L of insect cell culture was resuspended to 400 ml in buffer A (30 mM TRIS, pH 8.0, 100 mM NaCl, 5 mM MgCl.sub.2, 5 mM imidazole, 50 μM GDP). PMSF (1 mM), Pepstatin-A (2.5 ELM), Leupeptin (10 μM), Complete protease tablets (Roche), DNase I (50 μg/ml), and DTT (100 μM) were added. Cells were broken by sonication (10 minutes at 70% amplitude) and clarified by centrifugation (38,000 g for 1 h). The supernatant was loaded onto a 5 ml Ni Sepharose FF column (GE Healthcare) at 5 ml/min. The column was washed sequentially with 25 ml buffer A, 50 ml buffer B (20 mM TRIS, pH 8.0, 300 mM NaCl, 10 mM imidazole, 10% glycerol, 1 mM MgCl.sub.2, 50 μM GDP), and 25 ml buffer C (20 mM TRIS, pH 8.0, 300 mM NaCl, 30 mM imidazole, 10% glycerol, 1 mM MgCl.sub.2, 50 μM GDP) at 5 ml/min. The column was eluted with 25 ml buffer D (20 mM TRIS, pH 9.0, 50 mM NaCl, 500 mM imidazole, 10% glycerol, 1 mM MgCl.sub.2, 50 μM GDP). The eluate was diluted to 250 ml in buffer E (20 mM TRIS, pH 9.0, 50 mM NaCl, 10% glycerol, 1 mM MgCl.sub.2, 50 μM GDP, 1 mM DTT) and loaded onto a 5 ml Q Sepharose HP column (GE Healthcare) at 5 ml/min. The column was washed with 50 ml buffer E and eluted with a linear gradient of 50-300 mM NaCl (in buffer E). Peak fractions were pooled and TEV protease was added to give a final ratio of 1:20 w/w (TEV: Gαs). The sample was dialysed overnight against 1 L buffer F (20 mM HEPES, pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM MgCl.sub.2, 10 μM GDP). Imidazole (20 mM) and Ni-NTA resin (4 ml) were added to the sample and mixed for 1 h. The mixture was poured onto a disposable column containing 1 m Ni-NTA resin, and the flow-through collected. The column was washed with 10 ml buffer F and this wash was pooled with the flow-through. The pooled sample was concentrated to 1.5 ml using a 10 KDa MWCO Amicon Ultra centrifugal filter (Millipore). The sample was loaded onto a Superdex-200 26/600 gel filtration column (GE Healthcare), equilibrated with buffer G (10 mM HEPES, pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM MgCl.sub.2, 1 μM GDP, 0.1 mM TCEP). Peak fractions were pooled and concentrated to 50 mg/ml. The pure protein was aliquoted, flash-frozen in liquid nitrogen, and stored at −80° C. A typical yield was 6.5 mg pure Gαs per litre culture.
(90) Purification of Non-Lipidated Gs Heterotrimer
(91) Purification of non-lipidated heterotrimeric Gs was performed essentially as described for non-lipidated Gαs, except: the Ni Sepharose column was washed with 50 ml buffer C, instead of 25 ml; buffer D contained 300 mM imidazole, instead of 500 mM; the pH of buffers D and E was 8.5, instead of 9.0; the Q Sepharose column was eluted with a linear gradient of 50-200 mM NaCl, instead of 50-300 mM; and no TEV cleavage step was performed, instead fractions from the Q Sepharose column were concentrated and loaded on the Superdex-200 column. A typical yield was 7 mg pure Gs per litre culture.
(92) Purification of Non-Lipidated Gβγ Dimer
(93) Purification of non-lipidated Gβγ dimer was performed essentially as described for non-lipidated Gαs, except: GDP was omitted from all buffers; MgCl.sub.2 was omitted from buffers B-G; buffers B and C contained 250 mM NaCl, instead of 300 mM; buffer D contained 25 mM NaCl, instead of 50 mM; buffer D contained 300 mM imidazole, instead of 500 mM; buffers E and F were supplemented with 1 mM EDTA; the Q Sepharose column was eluted with a linear gradient of 25-200 mM NaCl, instead of 50-300 mM; and no TEV cleavage step was performed, instead fractions from the Q Sepharose column were concentrated and loaded on the Superdex-200 column. A typical yield was 7.5 mg pure Gβγ per litre culture.
(94) Expression and Purification of Nanobodies
(95) Synthetic genes (Integrated DNA Technologies) for Nb80 and Nb35 were cloned into pET26b (Novagen) for periplasmic expression in E. coli strain BL21(DE3)RIL (Agilent Technologies). Cells were lysed by sonication (10 mins at 70% amplitude). Nb80 was purified by IMAC and gel filtration, with a typical yield of 12 mg pure protein per litre culture. Nb35 was purified by IMAC, cation exchange chromatography and gel filtration, with a typical yield of 26 mg pure protein per litre culture.
(96) Expression and Purification of Mini G Proteins (for Screening)
(97) Mini G protein mutants were cloned in pET15b (Novagen). Expression was performed in E. coli strain BL21(DE3)RIL. Cells were grown in 2TY media supplemented with glucose (0.1%). Cultures were induced with IPTG (100 μM) at 15° C. for 20 h. Cells were lysed by sonication (2 mins at 70% amplitude). Mutants were partially purified by IMAC. Imidazole was removed on a PD10 column (GE Healthcare), and samples were concentrated to 20 mg/ml. The partially pure protein was aliquoted, flash-frozen in liquid nitrogen, and stored at −80° C.
(98) Expression and Purification of Mini G Proteins (Final Protocol)
(99) BL21(DE3)RIL cells transformed with the mini G protein construct were grown in TB media supplemented with glucose (0.2%) and MgSO.sub.4 (5 mM) and antifoam (0.01%). Cells were cultured in 2 L baffled flasks (Simax), shaking at 140 rpm. Cultures were grown at 30° C. until an OD.sub.600 of 0.8 was reached. Expression was induced with IPTG (50 μM) and the temperature reduced to 25° C. Cells were harvested 20 h post-induction by centrifugation at 5000 g for 10 mins, flash-frozen in liquid nitrogen and stored at −80° C.
(100) The cell pellet from 1 L of culture was resuspended to 200 ml in buffer A (40 mM HEPES, pH 7.5, 100 mM NaCl, 10% glycerol, 10 mM imidazole, 5 mM MgCl.sub.2, 50 μM GDP). PMSF (1 mM), Pepstatin-A (2.5 μM), Leupeptin (10 μM), Complete protease tablets, DNase I (50 μg/ml), and DTT (100 μM) were added. Cells were broken by sonication (10 minutes at 70% amplitude) and clarified by centrifugation (38,000 g for 45 mins). The supernatant was loaded onto a 10 ml Ni Sepharose FF column at 5 ml/min. The column was washed with 100 ml buffer H (20 mM HEPES, pH 7.5, 500 mM NaCl, 40 mM imidazole, 10% glycerol, 1 mM MgCl.sub.2, 50 μM GDP) at 5 ml/min. The column was eluted with 30 ml buffer I (20 mM HEPES, pH 7.5, 100 mM NaCl, 500 mM imidazole, 10% glycerol, 1 mM MgCl.sub.2, 50 μM GDP). TEV protease was added to give a final ratio of 1:20 w/w (TEV: Gαs). DTT (1 mM) was added and the sample was dialysed overnight against 2 L buffer J (20 mM HEPES, pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM MgCl.sub.2, 10 μM GDP). Imidazole (20 mM) and Ni-NTA resin (4 ml) were added to the sample and mixed for 1 h. The mixture was poured onto a disposable column containing 1 m Ni-NTA resin, and the flow-through collected. The column was washed with 10 ml buffer F and this wash was pooled with the flow-through. The pooled sample was concentrated to 1.5 ml and loaded onto a Superdex-200 26/600 gel filtration column, equilibrated with buffer K (10 mM HEPES, pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM MgCl.sub.2, 1 μM GDP, 0.1 mM TCEP). Peak fractions were pooled and concentrated to 100 mg/ml. The pure protein was aliquoted, flash-frozen in liquid nitrogen, and stored at −80° C. A typical yield was 100 mg pure protein per litre culture.
(101) Saturation Binding Assay
(102) Insect cells expressing β.sub.1AR were resuspended in 1 ml of assay buffer (20 mM HEPES, pH 7.5, 100 mM NaCl), supplemented with Complete EDTA-free protease inhibitors (Roche). Cells were broken by 10 passages through a bent 26 G needle. Cell debris was removed by centrifugation (3000 g for 5 mins at 4° C.). The supernatant was diluted and 2×0.96 ml aliquots taken for each sample. Alprenolol (120 μl) was added to the negative sample (1 mM final concentration) and assay buffer (120 μl) was added to the positive sample. Samples were aliquoted (12×108 μl) into a PCR plate. [.sup.3H]-dihydroalprenolol (12 μl) was added to each well (to give final concentrations in the range: 2.5 nM-2.56 μM). Samples were mixed and incubated at 20° C. for 2 h. Samples (2×50 μl duplicates) were vacuum filtered through 96-well glass fibre filter plates (Merck Millipore), pre-soaked with PEI (0.1%). Each well was washed with assay buffer (3×200 μl). Filters were dried, punched into scintillation vials and 4 ml Ultima Gold scintillant (Perkin Elmer) was added. Radioactivity was quantified by scintillation counting (1 min per sample) using a Tri-Carb counter (Perkin Elmer). Data for negative samples were subtracted from positive samples. Data were plotted graphically (Prism) and K.sub.d values derived from one site saturation binding analysis.
(103) Competitive Binding Assay
(104) Insect cells expressing β.sub.1AR were resuspended in 1 ml of assay buffer (25 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl.sub.2, 1 mM ascorbate), supplemented with Complete EDTA-free protease inhibitors (Roche). Cells were broken by 10 passages through a bent 26 G needle. Cell debris was removed by centrifugation (3000 g for 5 mins at 4° C.). The supernatant was diluted and a single 1.68 ml aliquot taken for each sample. Binding partner (240 μl) was added (25 μM final concentration). The mixture was aliquoted (17×96 μl) into a 0.2 ml PCR plate at 20° C. Isoprenaline (12 μl), prepared in buffer containing 1 U/ml apyrase (Sigma-Aldrich), was added to each well (final concentrations in the range: 1 pM-10 mM). Alprenolol (12 μl) was added to the negative sample (100 μM final concentration). Samples were mixed and incubated at 20° C. for 1.5 h. [.sup.3H]-dihydroalprenolol (12 μl) was added to each well (5 nM or 20 nM final concentrations for β.sub.1AR-WT or β.sub.1AR-84, respectively). Samples were mixed and incubated at 20° C. for 1.5 h. Samples (2×50 μl duplicates) were vacuum filtered exactly as described in the saturation binding assay protocol. Data were plotted graphically and K.sub.i values derived from one site fit K.sub.i analysis.
(105) Competitive binding assays using detergent-solubilised β.sub.1AR-84 were performed using a similar protocol, except: all steps were performed at 4° C.; membranes were solubilised with DDM (0.1% final concentration) for 30 minutes, prior to addition of binding partner; separation of bound from free ligand (by gel filtration) was performed exactly as described in the thermostability assay protocol.
(106) Thermostability Measurement of β.sub.1AR-WT-Mini Gs Complexes
(107) Insect cells expressing wild type β.sub.1AR-WT were resuspended in 1 ml of assay buffer (25 mM HEPES, pH 7.5, 400 mM NaCl, 1 mM MgCl.sub.2, 1 mM ascorbate, 0.1% BSA, 0.004% bacitracin), supplemented with Complete EDTA-free protease inhibitors (Roche). Cells were broken by 10 passages through a bent 26 G needle. Cell debris was removed by centrifugation (3000 g for 5 mins at 4° C.). The supernatant was diluted and 2×0.78 ml aliquots taken for each sample. Norepinephrine (120 μl) was added to the negative sample (200 μM final concentration) and assay buffer (120 μl) was added to the positive sample. Binding partner (120 μl) was added to both samples (25 μM final concentration). .sup.3H-norepinephrine (120 μl), prepared in buffer containing 1 U/ml apyrase (Sigma-Aldrich), was added to both samples (200 nM final concentration). Samples were mixed and incubated at 4° C. for 1 h. Detergent (60 μl) was added to both samples (final concentration: DDM=0.1%; DM=0.13%; OG=0.8%). Samples were mixed and incubated on ice for 1 h. Insoluble material was removed by centrifugation (17000 g for 5 mins at 4° C.). The supernatant was aliquoted (9×120 μl) into 0.2 ml PCR tubes. Each sample was heated to the desired temperature (between 4 and 50° C.) for exactly 30 minutes, followed by quenching on ice for 30 minutes. Samples (2×50 μl duplicates) were applied to Toyopearl HW-40F resin, which was pre-equilibrated (25 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl.sub.2, 0.025% DDM) and packed (225 μl bed volume) in 96-well filter plates (Merck Millipore). Plates were centrifuged (1800 rpm for 5 mins at 4° C.). The filtrate was transferred to Isoplates (Perkin Elmer), and 200 μl Optiphase Supermix scintillant (Perkin Elmer) was added to each well. Radioactivity was quantified by scintillation counting (1 min per well) using a MicroBeta counter (Perkin Elmer). Data for negative samples were subtracted from positive samples. Data were plotted graphically and apparent melting temperature (Tm) values derived from sigmoidal dose-response (variable slope) analysis.
(108) Thermostability Measurement of GDP-Bound Mini Gs Mutants by Differential Scanning Fluorimetry (DSF)
(109) Mini Gs mutants (30 μg) were diluted to 135 μl with assay buffer (10 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl.sub.2, 1 mM GDP, 2 mM DTT). SYPRO-orange (15 μl) was added from a 20× stock solution to give a final concentration of 2×. Samples were mixed and 2×50 μl aliquots (duplicates) were transferred to 0.2 ml PCR tubes (Qiagen). Thermostability measurements were performed using a Rotor-Gene Q (Qiagen). Samples were equilibrated for 90 s at 25° C. before ramping from 25 to 99° C. at 4 s/° C. The melting temperature (Tm), corresponding to the inflection point of the curve, was derived from analysis using the Rotor-Gene Q software. Tm values were calculated as the mean±SEM from three independent experiments.
(110) Gel Filtration Analysis of Mini G Protein Complexes
(111) Mini Gs-βγ complexes were prepared using mini Gs.sub.399: a construct in which N-terminal residues 6-25 were replaced and the L272D mutation was reversed (see Table 2). Purified mini Gs.sub.399 was mixed with non-lipidated Gβ.sub.1γ.sub.2 subunits in an equimolar ratio (6.7 nmol each), diluted to 200 μl with buffer L (10 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl.sub.2, 1 μM GDP, 0.1 mM TCEP) and incubated on ice for 4 hours. The entire sample (200 μl) was loaded onto a Superdex-200 10/300 gel filtration column, equilibrated with buffer L.
(112) The β.sub.1AR-mini Gs complex was prepared using wild type β.sub.1AR-WT purified in LMNG detergent. Purified β.sub.1AR-WT and mini Gs were mixed in an equimolar ratio (3.3 nmol each), diluted to 200 μl with buffer M (10 mM HEPES, pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM MgCl.sub.2, 1 μM ascorbic acid, 1 μM isoprenaline, 0.002% LMNG) and incubated on ice for 4 hours. The entire sample (200 μl) was loaded onto a Superdex-200 10/300 gel filtration column, equilibrated with buffer M.
EXAMPLE 2: DEVELOPMENT OF AN ASSAY TO DETECT COUPLING OF NON-LIPIDATED GS TO THE β.SUB.1.AR
Introduction
(113) A myriad of structural and biophysical data has provided clues as to why obtaining a high-resolution structure of a G protein-GPCR complex has proved difficult: flexibility within the nucleotide-free G protein appears to be the main problem.sup.7, 24, 42, 61. We have engineered a minimal GPCR-binding protein that still couples to a GPCR but removes much of the flexibility that has made crystallisation of the complex so difficult. First, we developed an assay capable of detecting coupling of non-lipidated Gs to the β.sub.1AR. Next, we expressed the isolated GTPase domain from Gαs and demonstrated that it was able to couple β.sub.1AR even in the absence of the βγ dimer. However, production of the GTPase domain was difficult due to poor expression and severe thermal instability. Therefore, we performed mutagenic screens and identified mutations that improved both the expression and stability of the isolated GTPase domain, whilst retaining the basic guanine nucleotide binding properties and functionality of the protein. The mutations we discovered are well conserved amongst the heterotrimeric G proteins, and are anticipated to transfer to members of all four classes of α subunits. Therefore, this approach can be used to produce a repertoire of GTPase domains capable of coupling almost all GPCRs. Herein, we describe the design of Minimal, Engineered, G protein Alpha (MEGA) domains, which couple activated GPCRs and induce the core pharmacological and conformational changes associated with the high-affinity agonist-bound state.
(114) Results
(115) Development of an Assay to Detect Coupling of Non-Lipidated Gs to the β.sub.1AR
(116) We have developed a competitive binding assay capable of detecting coupling of purified, non-lipidated Gs (see Experimental Procedures) to cell membranes containing the β.sub.1AR receptor. Initially, we screened different β.sub.1AR constructs in order to identify receptors that displayed a large increase in agonist affinity in response to Nb80.sup.38 binding. A near wild-type β.sub.1AR construct (β6) had a relatively high affinity for isoprenaline (approximately 180 nM), which only increased 3.5 fold in response to Nb80 binding (
(117) Expression and Characterization of the Isolated GTPase Domain of Gαs
(118) A large number of constructs were tested to find the best method of isolating the GTPase domain from Gαs. This process essentially involved deletion of the helical domain from its position within the switch I region. The strategies evaluated were: (1) deletion of the helical domain and any associated regions (residues 57-207) that were disordered in the crystal structure of the β.sub.2AR-Gs complex.sup.7; (2) deletion of the helical domain (residues 70-193), and linking the resulting termini with a short glycine linker to retain a near native switch I region; (3) deletion of the helical domain and switch I (residues 65-203), and linking the resulting termini with a longer glycine linker; (4) deletion of the helical domain and switch I (residues 67-205), and insertion of the switch I region from structurally related small GTPases. A number of variations of each strategy were tested, thus residue ranges quoted are approximations. We found that strategy (1) removed regions of the GTPase domain vital for its stability in the absence of the receptor, and was therefore unsuitable. Strategies 2-4 were all successful, and resulted in expression of a small amount of isolated GTPase domain in E. coli (approximately 100 μg/L culture). The expression level between different constructs was variable, but difficult to quantify. Generally, complete removal of the helical domain and switch I (strategy 3) resulted the highest expression levels.
(119) The isolated GTPase domain was partially purified from E. coli, and its ability to couple the β.sub.1AR was determined using the agonist-shift assay described above. First, we tested coupling of the GTPase domain, but no increase in affinity was observed (
(120) Stabilization of the GTPase Domain Through Mutagenesis
(121) An initial screen of approximately 50 modifications (mutations, deletions and chimeras) was performed. The modifications were designed to: remove superfluous sequences (compared to the small GTPases), stabilise the nucleotide-binding site, constrain the conformationally dynamic switch regions, stabilise the inactive state of the G protein, or stabilise the active conformation of the G protein. No modifications were made in regions that directly interact with the receptor. The parental construct used for mutagenesis consisted of: a 20 amino acid deletion of the N-terminus, complete deletion of the helical domain, and retention of a slightly modified switch I region (see Experimental Procedures).
(122) Mutants were expressed in E. coli and partially purified by IMAC. In order to estimate the stabilising effect of each modification, agonist-shift assays were performed at 4 and 20° C.; a summary of the assay data is presented in Table 5. The expression levels of the mutants differed widely, thus the concentration of each mutant used in the assays could not be standardised. Instead the entire quantity of protein purified from one litre of culture was included in each assay. Importantly, the concentration of protein used in the assay does affect the magnitude of the shift in agonist affinity. Thus, the data must be interpreted as a combination of expression level and stabilising effect. Four key modifications (Δ switch III, A249D, L272D and H41I), which dramatically improved expression and/or stability of the isolated GTPase domain, were identified (Table 5). Both the A249D mutation and deletion of switch III resulted in significantly improved expression levels (approximately 1-2 mg/L culture); the L272D and H41I mutations did not significantly improve expression levels. All of the mutants induced a large shift in agonist affinity (10-60 nM final isoprenaline affinity) when assayed at 4° C. (Table 5). Furthermore, the A249D and Δ switch III mutants induced a large shift in agonist affinity (53 and 30 nM final isoprenaline affinity respectively) when assayed at 20° C. (Table 5). The L272D and H41I mutants also induced a shift in agonist affinity (464 and 589 nM final isoprenaline affinity respectively) when assayed at 20° C. (Table 5), albeit not as large as the A249D and Δ switch III mutants. However, it must be noted that the concentration of the A249D and Δ switch III mutants used in the assay was approximately 5-fold higher. Four additional mutations (G49D, E50N, G226A and S252D) that improved the stability of the GTPase domain were also identified (Table 5). However, the proximity of these mutations to the aforementioned sites indicates that their mechanism of action is likely to be similar, therefore, their additive properties must be determined empirically.
Discussion
(123) The solution of high-resolution structures of GPCRs in their fully active conformation is of major importance for the design of novel agonist compounds. We have developed a unique strategy to engineer the isolated GTPase domain of G protein α subunits to couple and conformationally activate GPCRs.
(124) Heterotrimeric G proteins can couple GPCRs efficiently in their lipidated, membrane associated state.sup.5. MEGA domains include non-lipidated mutant Gα subunits, whose mechanism of receptor binding is anticipated to be similar to that of the holoenzyme. Therefore, a prerequisite to the design of MEGA domains was the development of an assay capable of detecting coupling of non-lipidated G proteins to GPCRs. Initially, we found that non-lipidated Gs induced only a small shift in agonist affinity for a minimally thermostabilised β.sub.1AR. However, addition of Nb35, which was used to facilitate crystallisation of the β.sub.2AR-Gs complex.sup.7, produced a much larger response. Nb35 appears to inhibit dissociation of the G protein heterotrimer by conformationally constraining the switch II region, and stabilising the α/β subunit interface. Nb35 is therefore likely to reduce the conformational dynamics of the GPCR-G protein ternary complex, possibly mimicking the stabilising effect of membrane anchorage, albeit through a different mechanism.
(125) We assessed several different strategies to isolate the GTPase domain from Gs. We found that complete removal of the helical domain and switch I resulted in slightly better expression and stability. Initially, we found that the GTPase domain induced a significant shift in agonist affinity only in the presence of βγ dimer and Nb35, suggesting that βγ subunits were still required for efficient coupling. However, we hypothesised that the βγ dimer and Nb35 may simply act to stabilise the thermally labile GTPase domain.sup.28. Therefore, we repeated the assays at 4° C., and found that the GTPase domain was capable of efficiently coupling the receptor in a βγ-independent manner. GPCRs can catalyse low-level nucleotide exchange on Gα subunits.sup.4, however, the βγ dimer is required to facilitate rapid exchange and thus signal amplification.sup.56,57. Deletion of the helical domain allows efficient coupling of the GTPase domain to the receptor, in a βγ-independent manner. This is probably due to more rapid GDP dissociation from the GTPase domain, which results in more efficient coupling the receptor. Together, these data suggests that the mechanism of interaction between the receptor and the isolated GTPase domain is similar to that of the holoenzyme. Therefore, MEGA domains are likely to induce native-like conformational changes in the receptor, and thus represent a true mimetic of G protein coupling.
(126) We used mutagenesis to improve the stability and expression of the GTPase domain. A number of key mutations were identified that dramatically improved the expression level and/or stability of the GTPase domain, the mechanisms of which are discussed below. The A249D mutation improved both the expression and stability of the GTPase domain. In the small GTPases an aspartic acid is often found in this position, where it stabilises the lysine of the NKXD motif through a salt-bridge interaction. This lysine residue forms the base of the nucleotide-binding pocket and participates in a π-cation stacking interaction with the guanine ring.sup.92. This position is not exclusively occupied by an aspartic acid in the small GTPases, however within each class it is generally conserved or non-conserved, indicating that it may be inherent to the stability of certain GTPase families. In heterotrimeric G proteins this position is occupied by either an alanine or serine residue, except Gαz, where a glutamic acid residue is present. However, the lysine from the NKXD motif is stabilized through a salt-bridge interaction with an aspartic or glutamic acid from the helical domain (Asp-173 in Gαs). This interaction is broken when the domain interface separates during activation.sup.7. The A249D mutation is thought to stabilise the nucleotide-binding pocket and increase the GDP binding affinity, although this has not yet been tested.
(127) Deletion of switch III improved both the expression and stability of the GTPase domain. In heterotrimeric G proteins switch III is involved in mediating the conformational changes induced by GTP uptake, and is required for effector binding.sup.93. In the small GTPases switch III is absent and the corresponding region consists of distinct secondary structural elements: the β4 strand terminates in a type-I turn, which connects directly to a 3.sub.10 helical segment preceding the α3 helix (secondary structure assignments were performed using the STRIDE web-server.sup.94, 95). The improvements in stability achieved by deletion of switch III are likely to be a result of replacing a highly flexible loop with more ordered secondary structure elements. The increase in expression level is likely to result from a combination of the improved stability and a more energetically favourable folding pathway.
(128) The H41I mutation significantly improved the stability of the isolated GTPase domain. Histidine 41 has been reported to contribute significantly to the elevated levels of basal nucleotide exchange observed in Gαs compared with Gt.sup.96. It was previously reported that mutation of histidine 41 to valine, which is found in this position in Gt, halved the level of basal nucleotide exchange of Gαs.sup.96. We showed that the H41V mutation improved the stability of the MEGA domain (see Table 5), however, the H41I mutation was optimal in this position. This mutation improves the stability of the GTPase domain because it enhances the interactions between the α5 helix and the αN/β1 loop. Close packing in this region stabilises the α5 helix, thus reducing the rate of GDP dissociation.sup.96.
(129) The L272D mutation, which is located in the α2 helix (adjacent to switch II), significantly improved the stability of the isolated GTPase domain. Switch II changes structure dramatically between GDP and GTP-bound states.sup.97: in the GDP bound state switch II is more dynamic, and often disordered in crystal structures.sup.98; in the GTP-bound state switch II becomes highly ordered.sup.30,99, and in Gs this region forms the main effector-binding site.sup.100. The L272D mutation is likely to directly interact with switch II, and conformationally constrain the whole region. Intriguingly, it may form a salt bridge interaction with a highly-conserved arginine residue in switch II (Arg-231), which is ideally positioned for such an interaction in the GTP-bound state.sup.30. This is likely to improve the stability of the GTPase domain by limiting exposure of the hydrophobic residues beneath switch II to the aqueous environment.
(130) In summary, we have demonstrated that, despite being unstable and poorly expressed, the isolated GTPase domain from Gαs can efficiently couple the β.sub.1AR in a βγ-independent manner. We have performed an extensive mutagenesis screen and identified four key mutations, which dramatically increase the expression and/or stability of the domain.
(131) Methods and Materials
(132) β.sub.1AR Constructs
(133) The 384 construct, which was used for G protein binding assays, contained a number of modifications: an N-terminal MBP fusion protein; N-terminal truncation (residues 1-32); intracellular loop 3 deletion (residues 244-271); C-terminal truncation at residue 367; C-terminal hexa histidine-tag; a C116L mutation; an engineered disulphide bond (M40C-L103C); and four thermostabilising mutations (M90V, D322K, F327A and F338M). The receptor was expressed using the BaculoGold baculovirus expression system (BD Bioscience) in the Trichopulsia ni (High Five) cell line (Life Technologies).
(134) Gαs GTPase Domain Constructs
(135) The parental GTPase domain used for the initial mutagenesis screens is described below (all numbering refers to the long isoform of Gαs). The construct consisted of: an N-terminal hexa histidine-tag; N-terminal deletion (residues 1-20); helical domain deletion (residues 71-193), leaving a near native switch I intact, and linking the termini with a Gly.sub.2 linker; and two mutations in switch I region (L197A and C200S), to remove unfavorable surface residues exposed by removal of the helical domain.
(136) Expression and Purification of Heterotrimeric Gs
(137) Non-lipidated heterotrimeric Gs used in this study was composed of: human Gαs (long-form: including the variably spliced region in linker-1), which contained a four amino acid deletion of the N-terminus to remove all potential palmitoylation sites.sup.101; human Gβ1 (containing an N-terminal hexa histidine-tag); and human Gγ2 containing a C68S mutation to remove the prenylation site.sup.102. Baculovirus constructs encoding each individual subunit were constructed using the flashBAC ULTRA system (Oxford Expression Technologies). The Gs heterotrimer was expressed in Spodoptera frugiperda (SF9) cells grown in TNM-FH media (Sigma) containing 10% foetal calf serum (Gibco) and 1% lipids (BD Bioscience). Cells were infected using P3 virus at concentration of 2% for each subunit, in a 1:1:1 ratio. Cells were incubated for 48 hours at 27° C. Cells were harvested by centrifugation at 4000 g for 10 minutes and washed with of PBS (15% of culture volume). The cell pellet was flash frozen in liquid nitrogen and stored at −80° C.
(138) The cell pellet from three litres of culture was resuspended in 150 ml of lysis buffer (30 mM Tris, 100 mM NaCl, 10% glycerol, 5 mM MgCl.sub.2, 100 μM GDP, 0.5 mM PMSF, 2.5 μM Pepstatin-A, 10 μM Leupeptin, 50 μg/ml DNasel, 50 μg/ml RNaseA, pH 8.0) containing Complete EDTA-free protease inhibitors, and DTT was added to a final concentration of 0.1 mM. The cells were broken by sonication, and insoluble material removed by centrifugation at 38000 g for 40 minutes. The supernatant was filtered (0.45 μM) and loaded onto a 5 ml Ni-Sepharose fast flow HisTrap column (GE Healthcare) at 5 ml/min. The column was washed with ten column volumes of lysis buffer, followed by ten column volumes of wash buffer (20 mM Tris, 250 mM NaCl, 5 mM imidazole, 10% glycerol, 1 mM MgCl.sub.2, 50 μM GDP, pH 8.0) at 5 ml/min. The column was eluted with 25 ml elution buffer (20 mM Tris, 50 mM NaCl, 200 mM imidazole, 10% glycerol, 1 mM MgCl.sub.2, 50 μM GDP, pH 8.3) at 2 ml/min. The eluent was diluted with 225 ml of Q buffer (20 mM Tris, 50 mM NaCl, 10% glycerol, 0.5 mM MgCl.sub.2, 50 μM GDP, 1 mM DTT, pH 8.3). The mixture was loaded directly onto a 5 ml Q-Sepharose HP HiTrap column (GE Healthcare) at 5 ml/min. The column was washed with ten column volumes of Q buffer at 5 ml/min. Gs was eluted with a linear NaCl gradient from 50 mM to 250 mM (Q buffer containing 250 mM NaCl) over 40 column volumes at 2 ml/min. Fractions containing Gs were pooled and concentrated to 5-10 mg/ml using a 10 KDa cut off Amicon Ultra concentrator (Millipore). Concentrated Gs was loaded onto a Superdex-200 (16/60) gel filtration column (GE Healthcare) equilibrated with GF buffer (20 mM Tris, 100 mM NaCl, 10% glycerol, 0.2 mM MgCl.sub.2, 2 μM GDP, 0.1 mM TCEP, pH 8.0) at 1 ml/min. Fractions containing pure Gs were pooled, concentrated to 10 mg/ml, flash frozen in liquid nitrogen and stored at −80° C. The typical yield was 1-2 mg of pure Gs per litre of culture.
(139) Expression and Purification of Nb35
(140) The Nb35.sup.10 gene was synthesised (Integrated DNA Technologies) and cloned into the pET26b vector (Merck). Nb35 was expressed in BL21(DE3)-RIL cells (Merck). Cultures were grown in terrific broth media, supplemented with glucose (0.1%) and MgSO.sub.4 (2 mM), to an OD.sub.600 nm of 0.8 at 37° C. Expression was induced with IPTG (50 μM), at 28° C. for approximately 18 hours. Cells were harvested by centrifugation at 4000 g, and stored at −80° C.
(141) The cell pellet from six litres of culture was resuspended in 200 ml of lysis buffer (40 mM Hepes, 100 mM NaCl, 5 mM imidazole, 5 mM MgCl.sub.2, 1 mM PMSF, 100 μg/ml lysozyme, 50 μg/ml DNaseA, pH 7.5) containing Complete EDTA-free protease inhibitors. The cells were incubate on ice for 30 minutes, then broken by sonication, and insoluble material removed by centrifugation at 38000 g for 30 minutes. The supernatant was filtered (0.45 μM) and loaded onto a 5 ml Ni-Sepharose fast flow HisTrap column (GE Healthcare) at 5 ml/min. The column was washed with 15 column volumes of wash buffer (20 mM Hepes, 300 mM NaCl, 40 mM imidazole, pH 7.5) at 5 ml/min. The column was eluted with 25 ml elution buffer (20 mM Hepes, 500 mM imidazole, pH 7.0) at 2 ml/min. The eluent was diluted with 225 ml of SP buffer (20 mM Hepes, pH 7.0), and loaded directly onto a 5 ml SP-Sepharose HP HiTrap column (GE Healthcare) at 5 ml/min. The column was washed with ten column volumes of SP buffer at 5 ml/min. Nb35 was eluted with a linear NaCl gradient from 0 mM to 250 mM (SP buffer containing 250 mM NaCl) over 40 column volumes at 2 ml/min. Fractions containing Nb35 were pooled and dialysed against 500 ml of GF buffer (20 mM Tris, 100 mM NaCl, 10% glycerol, pH 7.5) overnight, with two external buffer changes. Nb35 was concentrated to 20 mg/ml using a 3 KDa cut off Amicon Ultra concentrator (Millipore). Concentrated Nb35 was loaded onto a Superdex-200 (16/60) gel filtration column (GE Healthcare) equilibrated with GF buffer at 1 ml/min. Fractions containing pure Nb35 were pooled, concentrated to 20 mg/ml, flash frozen in liquid nitrogen and stored at −80° C. The typical yield was 5 mg of pure Nb35 per litre of culture.
(142) Partial Purification of Gαs GTPase Domains for Use in Agonist-Shift Assays
(143) GTPase domains were expressed in BL21(DE3)-RIL cells. Cultures were grown in 2TY media, supplemented with glucose (0.1%), to an OD.sub.600 nm of 0.5-0.8 at 25° C. Expression was induced with IPTG (100 μM), at 15° C. for approximately 16 hours. Cells were harvested by centrifugation at 4000 g, and stored at −80° C.
(144) The cell pellet from two litres of culture was resuspended in 22 ml of lysis buffer (30 mM Tris, 100 mM NaCl, 10 mM imidazole, 20% glycerol, 5 mM MgCl.sub.2, 3 mM ATP, 100 μM GDP, 0.5 mM PMSF, 2.5 μM Pepstatin-A, 10 μM Leupeptin, 50 μg/ml lysozyme, 20 μg/ml DNasel, pH 7.5) containing Complete EDTA-free protease inhibitors. DTT (0.1 mM) was added and the cells were incubated on ice for 30 minutes. The cells were broken by sonication, and insoluble material removed by centrifugation at 50000 g for 40 minutes. The supernatant was filtered (0.45 μM), 1 ml Ni-Sepharose fast flow resin (GE Healthcare) was added, and the suspension was mixed at 4° C. for 1.5 hours. The mixture was poured into an empty PD10 column (GE Healthcare) and washed with 20 ml of wash buffer (20 mM Tris, 300 mM NaCl, 40 mM imidazole, 20% glycerol, 1 mM MgCl.sub.2, 50 μM GDP, pH 7.5). The column was eluted with 2.5 ml elution buffer (20 mM Tris, 100 mM NaCl, 400 mM imidazole, 20% glycerol, 1 mM MgCl.sub.2, 50 μM GDP, pH 7.5). The partially purified protein was desalted into GF buffer (20 mM Tris, 100 mM NaCl, 10% glycerol, 1 mM MgCl.sub.2, 50 μM GDP, 0.1 mM DTT, pH 7.5) using a PD10 column (GE Healthcare). The desalted protein was concentrated to a final volume of 400 μL using a 10 KDa cut off Amicon Ultra concentrator (Millipore). The concentrated protein was flash frozen in liquid nitrogen and stored at −80° C.
(145) Agonist-Shift Assay
(146) The cell pellet from approximately 2 ml of High Five culture expressing the 384 receptor construct was resuspended in 1 ml of lysis buffer (20 mM Tris, 100 mM NaCl, 1 mM MgCl.sub.2, 1 mM ascorbic acid, pH 7.5) containing Complete EDTA-free protease inhibitors (Roche). Cells were lysed by 10 passages through a bent 26 G needle, and insoluble material removed by centrifugation (5 mins at 3000 g). The supernatant, containing crude membrane fractions, was diluted to 8 ml in lysis buffer (0.8 ml per competition curve required). G protein, MEGA domain, Nb80 or buffer (200 μl) was added to the crude membranes (0.8 ml), and homogenised by 3 passages through a bent 26 G needle. The final concentration of G protein or Nb80 used in the assay was approximately 1 mg/ml; the final concentration of MEGA domains used depended on their expression levels. Nine aliquots (88 μl each) were transferred to a 96-well PCR plate (on ice). Isoprenaline (11 μl) was added to seven samples to give final competitive ligand concentration curve of 1×10.sup.−3-1×10.sup.−9 M; isoprenaline dilutions were prepared in lysis buffer containing 1 U/ml apyrase (Sigma). Buffer (11 μl) was added to one of the remaining samples to determine total signal, and alprenolol (11 μl) was added to the final sample to determine background signal (100 μM final concentration). Samples were incubated at 4° C. for 2 hours (or at 20° C. for 1 hour). .sup.3H-dihydroalprenolol (Perkin Elmer) was added to each well (11 μl) to give a final concentration of 10 nM (≤Kd of β84). Samples were incubated at 4° C. for 2 hours (or at 20° C. for 1 hour). Samples were filtered on 96-well GF/B filter plates (Millipore), pre-soaked in lysis buffer containing 0.1% PEI. Plates were washed three times (200 μl) with ice-cold wash buffer (20 mM Tris, 100 mM NaCl, 1 mM MgCl.sub.2, pH 7.5). Plates were dried and filters punched out into scintillation vials. Scintillant (4 ml) was added, samples were incubated overnight and then tritium was counted in a liquid scintillation counter (Beckmann Coulter). Data were analysed using the ‘one site—fit log IC50’ function of Prism (GraphPad).
EXAMPLE 3: APPLICATIONS
(147) MEGA domains have a wide range of applications in the design of therapeutics to modulate GPCR and G protein activity.
(148) Stabilisation of GPCRs During Purification
(149) GPCRs are conformationally dynamic, which contributes to their poor thermostability in detergent.sup.69. MEGA domains are likely to conformationally and thermally stabilise GPCR, and will therefore improve the efficiency of purification procedures.
(150) Thermostabilisation of GPCRs in their Fully Active Conformation
(151) MEGA domains are likely to significantly improve the thermostability of their bound GPCR. However, further dramatic improvements in stability may be achieved through mutagenic thermostabilisation of the receptor.sup.88 whilst in complex with the MEGA domain. The resulting MEGA-StaR complex will be highly stable, and suitable for even the most demanding applications. Furthermore, GPCRs thermostabilised in this manner may adopt a fully active conformation even in the absence of the MEGA domain or ligand, providing a unique opportunity for drug design.
(152) Structure Determination of GPCRs in their Fully Active Conformation
(153) The stabilising properties of MEGA domains will permit high-resolution structure determination of the high-affinity agonist-bound state of GPCRs, using both x-ray crystallography and NMR.
(154) Fragment Library Screening Against Activated GPCRs
(155) MEGA domains may also be a valuable tool for fragment library screening using both structural and non-structural methods. There is strong evidence to suggest that once the ternary G protein-GPCR complex is formed the ligand can be removed from the binding pocket without causing dissociation of the complex: hydroxylamine treatment of the nucleotide-free rhodopsin-transducin complex causes hydrolysis of the Schiff base bond between rhodopsin and retinal, resulting in the release of retinaloxime.sup.14. However, this causes neither dissociation of the complex, or decay of the Meta-II photochemical state into inactive opsin, furthermore the chromaphore site appears to remain in its open conformation.sup.14. Therefore it may be possible to produce a ligand-free MEGA-GPCR complex in which the empty ligand-binding pocket maintains the high-affinity agonist-bound conformation. Ligand-free complexes represent an ideal substrate for fragment library screening using biophysical methods or crystal soaking techniques. These complexes will also be of significant importance for the design of agonists to target orphan receptors.
(156) Screening Compounds that Block Specific G Protein-GPCR Interfaces
(157) The ligand-binding pocket and extracellular surface of GPCRs are the main targets exploited in drug design, however, downstream signalling proteins also have significant therapeutic potential. Several peptides and small molecules that modulate G protein a subunits have been reported.sup.70-72. Although these molecules generally target a single class of G protein, the promiscuous nature of the G protein signalling means they are unlikely to be suitable for therapeutic applications. Structures of MEGA-GPCR complexes will allow the design of small molecules that target a specific G protein-receptor interface. Thus, signalling through a specific G protein-receptor pair could be inhibited, whilst retaining the activity of both the receptor and G protein in other signalling cascades.
(158) Development of Cell-Based Assays
(159) Due to their monomeric nature, MEGA domains will be useful in the development of fluorescent assays to study receptor/G protein coupling in vivo.
(160) Understanding the Molecular Mechanisms of Receptor Specificity
(161) MEGA domains may also allow us to determine the molecular mechanisms of receptor specificity beyond the Gα-GPCR interface. MEGA domains can be reconstituted with different combinations of βγ subunits, these GPCR-MEGA-βγ complexes may be more amenable to crystallisation than the full G protein-GPCR complexes. Therefore, the interactions between C-terminus of the receptor and the βγ subunits can be studied. This may allow design of allosteric modulators that can target specific GPCR-G protein complexes, based on the βγ components of the G protein heterotrimer.
(162) MEGA Domains as Therapeutic Agents
(163) MEGA domains can be engineered to sequester GPCRs, βγ subunits or downstream effectors. These dominant negative mutants may themselves be valuable therapeutic agents, for example in cancer therapy.
EXAMPLE 4: STRUCTURE OF THE ADENOSINE A.SUB.2A .RECEPTOR BOUND TO AN ENGINEERED G PROTEIN
Introduction
(164) G protein-coupled receptors (GPCRs) are essential components of the chemical intercellular signalling network throughout the body. To understand the molecular mechanism of signalling, structures are necessary of receptors in both an inactive conformation and in an active conformation coupled to a heterotrimeric G protein. Here we report the first structure of the adenosine A.sub.2A receptor (A.sub.2AR) bound to a highly engineered G protein, mini-G.sub.s, to 3.4 Å resolution. Mini-G.sub.s binds to A.sub.2AR through an extensive interface (1048 Å.sup.105) that is similar, but not identical, to the interface between the β.sub.2-adrenergic receptor and G.sub.s. The structure of A.sub.2AR bound to mini-G.sub.s identifies key amino acid residues involved in the transition of the receptor from an agonist-bound active-intermediate state to the fully active G protein bound state. The structure highlights both the diversity and similarity in GPCR-G protein coupling and hints at the potential complexity of the molecular basis for G protein specificity.
(165) Adenosine is a signalling molecule that activates four different adenosine receptors in humans, A.sub.1, A.sub.2A, A.sub.2B and A.sub.3, and has been implicated in a wide range of physiological processes including angiogenesis, immune function and sleep regulation (reviewed in.sup.104,105). In addition, there is strong evidence that high concentrations of extracellular adenosine is deleterious to cell health and contributes to pathological effects observed in neurodegenerative diseases, inflammatory disorders, cancer and ischaemia-reperfusion injury (reviewed in.sup.106). There is thus considerable interest in the development of subtype specific agonists and antagonists to the adenosine receptors. Over the last 40 years a wide range of compounds have been developed by traditional medicinal chemistry.sup.105,107 and, more recently, structure based drug design has been implemented to develop novel antagonists of the adenosine A.sub.2A receptor (A.sub.2AR) for the potential treatment of Parkinson's disease.sup.108. An agonist targeting A.sub.2AR (regadenoson) is approved by the FDA for myocardial perfusion imaging.sup.107 and agonists specific for A.sub.3R are under development for their anti-cancer and anti-inflammatory properties.sup.109.
(166) Comparison of the structures of A.sub.2AR bound to either inverse agonists.sup.110-112 or agonists.sup.1,4,113 elucidated molecular determinants of subtype specificity and efficacy.sup.114. However, the mechanism of activation of the receptor to allow coupling to G proteins and the basis of G protein selectivity is not fully understood. Structures of A.sub.2AR in the inactive state have been determined bound either to the antagonists ZM241385.sup.110-112, XAC110, caffeine.sup.110 or 1,2,4-triazines.sup.108, and all the structures are very similar. An intramembrane Na.sup.+ ion that can act as an allosteric antagonist was identified in the highest resolution structure (1.8 Å).sup.115, and a homologous Na.sup.+ ion has been subsequently identified in other high-resolution structures of GPCRs.sup.96,116,117. Four agonist-bound structures of A.sub.2AR have also been determined after co-crystallisation with either adenosine.sup.1, NECA.sup.1, CGS21680.sup.113 or UK432097.sup.4. All the structures are very similar and are thought to represent an active-intermediate conformation of the receptor, but not the fully active receptor that binds a G protein.sup.1. Observations that support this conclusion include the presence of key rotamer changes of conserved amino acid residues associated with activation of other GPCRs, but the absence of a significant movement of the cytoplasmic end of transmembrane helix 6 (H6) away from the receptor core.sup.114. The G protein-coupled state of A.sub.2AR exhibits higher affinity binding of agonists compared to the uncoupled state.sup.18, but it is unclear whether the agonist bound structures determined so far depict the binding pocket in a high affinity or low affinity conformation. In contrast to A.sub.2AR, crystallisation of either β.sub.1AR or β.sub.2AR bound to agonists resulted in structures of the inactive conformation that differ only subtly from structures bound to antagonists.sup.2,3. It is now apparent that β.sub.2AR exists in an ensemble of conformations whether bound to antagonists, agonists or to no ligand at all, and the presence of agonists increases the probability of formation of the active state.sup.119. The activated state is then stabilised by the binding of a G protein.sup.10 or by a G protein mimetic (nanobody).sup.38. Therefore, in order to elucidate the structure of the activated state of A.sub.2AR, we have determined its structure bound to an engineered G protein.
(167) Results
(168) Structure of G Protein-Bound A.sub.2AR.
(169) There is a single reported structure of a GPCR bound to a heterotrimeric G protein, namely G.sub.s-bound β.sub.2AR.sup.10. The crystallisation was a real tour de force, requiring the development of a specific nanobody, Nb35, to stabilise the Gαβγ trimer by binding at the interface between Gα and Gβ, fusion of T4-lysozyme to the N-terminus of β.sub.2AR, the use of a novel mono-olein derivative MAG 7:7 for crystallisation in meso and a complex procedure for purification and crystallisation. The β.sub.2AR-G.sub.s structure.sup.10 showed that virtually all the contacts between the receptor and G protein were made to the Gα subunit and therefore, in theory, Gβγ was unnecessary for complex formation. We therefore adopted an alternative approach, engineering the Gα.sub.s subunit to make it more amenable for the formation of well-ordered crystals, which in principle should allow the crystallisation of any G.sub.s coupled receptor in the activated state.sup.120. We developed a minimal G protein, mini-G.sub.s, that comprised a truncated form of the GTPase domain of Gα.sub.s, including 8 point mutations to stabilise the protein in the absence of Gβγ and in the presence of detergents.sup.120. Truncations included the switch III region, 23 amino acids from the N-terminus and the α-helical domain, all of which would benefit crystal formation by decreasing the structural heterogeneity of the complex. Mini-G.sub.s reproduced the increase in agonist affinity that occurred upon incubation of the receptor in the presence of the heterotrimeric G protein G.sub.s and it also showed identical sensitivity to the presence of the allosteric antagonist Na.sup.+ (
(170) There are two mini-G.sub.s-A.sub.2AR complexes per crystallographic asymmetric unit composed of either chains A and C (complex AC) or chains B and D (complex BD). The best electron density was observed for complex AC (chain A, A.sub.2AR; chain C, mini-G.sub.s), and included density for the agonist NECA bound to A.sub.2AR and density for a molecule of GDP bound to mini-G.sub.s (
(171) The interface between A.sub.2AR and mini-G.sub.s in complex AC is formed between 20 amino acid residues from the receptor and 17 residues in mini-G.sub.s (
(172) Comparison of the Receptor-G Protein Interface in β.sub.2AR-Gs and A.sub.2AR-Mini-Gs Structures
(173) Superposition of the receptors in the A.sub.2AR-mini-G.sub.s complex and the β.sub.2AR-G.sub.s complex.sup.10 shows that the receptors have very similar architectures (rmsd 1.7 Å over 1239 atoms), with the majority of the differences occurring in the extracellular region where the amino acid sequences are the most divergent (
(174) Although there are significant differences in receptor-G protein contacts, there are also many similarities (
(175) Conformational Changes in A.sub.2AR Upon Mini-G.sub.s Binding
(176) Previously reported structures of A.sub.2AR bound to agonists are in an active-intermediate conformation.sup.1,4,113. This assignment is based on the similarities of rotamer changes in the receptor core and the movement of transmembrane helices that are also observed in the structures of the active state of β.sub.2AR bound to a nanobody.sup.36 and rhodopsin in an active state.sup.36,122. However, as the extent of H6 movement in agonist-bound A.sub.2AR is less than one half of that observed in the other receptors, there would be insufficient room in the cytoplasmic cleft to accommodate the C-terminal peptide of a G protein.sup.1. Comparison of the active-intermediate state of UK432097-bound A.sub.2AR.sup.12 with the structure of A.sub.2AR bound to mini-G.sub.s identified major re-arrangements in the cytoplasmic half of the receptor core to accommodate G protein binding (
(177) In contrast to the considerable re-arrangements of the cytoplasmic half of the receptor to allow mini-G.sub.s binding, there are no significant changes in the extracellular half of the receptor when the NECA-bound A.sub.2AR-mini-G.sub.s structure is compared to NECA-bound A.sub.2AR (
Conclusions
(178) A.sub.2AR is the first GPCR where both an active-intermediate and a fully active conformation have been defined structurally. However, the structure of the neurotensin receptor bound to the agonist peptide neurotensin.sup.1,4,110-114 shows very similar characteristics to adenosine-bound A.sub.2AR and therefore probably also represents an active-intermediate state.sup.23,124. Recently, it has been proposed based on extensive EPR data that β.sub.2AR also exists in two distinct states in the active conformation, although the structure of the second state has not yet been elucidated.sup.119. However, it is clear from this work that there can be distinct conformations with or without G protein bound that lie on the activation pathway of agonist bound receptors. Given the highly conserved nature of the mechanism of GPCR activation, it is likely that the active-intermediate of A.sub.2AR may represent a common intermediate for many Class A GPCRs, although it may exist only transiently depending on the energy landscape of the receptor.
(179) The similarities and differences between the G protein interfaces of β.sub.2AR and A.sub.2AR are a consequence of the different amino acid sequences of the receptors and result in a slightly different position and orientation of the G protein with respect to the receptor. Thus it is to be expected that G.sub.s will also interact slightly differently with other G.sub.s-coupled receptors and that other G proteins, such as G.sub.i and G.sub.q, will also show differences in the details of their interactions with receptors, due both to different amino acid sequences and the flexible nature of both receptors and G proteins. Thus the relatively ‘loose’ nature of the G protein binding interface may allow significant variations in G protein orientation. However, one of the beauties of the conserved mechanism of G protein activation by GPCRs.sup.103 is provided that helix α5 is displaced away from the nucleotide binding pocket, causing the order-to-disorder transition of α1 and nucleotide release, the exact mode of interaction with the receptor is largely superfluous.
(180) Methods and Materials
(181) Expression and Purification.
(182) Mini-G.sub.s (construct 414) was expressed in E. coli and purified by immobilised metal affinity chromatography (IMAC) and gel filtration chromatography (see further methods). The wild type human A.sub.2AR (residues 1-308), which contained the N154A mutation to remove a potential N-linked glycosylation site, was expressed in insect cells utilising the baculovirus expression system. A.sub.2AR was purified in the presence of the agonist NECA, in n-decyl-β-D-maltopyranoside (DM) detergent by IMAC and gel filtration chromatography (see further methods).
(183) Complexation and Crystallisation.
(184) Agonist-bound, purified A.sub.2AR (in DM) was mixed with a 1.2-fold molar excess of mini-G.sub.s, apyrase was added and the sample was incubated overnight on ice. The complex was exchanged into n-octyl-β-D-thioglucopyranoside (OTG) detergent, purified by gel filtration, and crystallised by vapour diffusion (see further methods).
(185) Data Collection, Structure Solution and Refinement.
(186) Diffraction data were collected from two cryo-cooled crystals (100K), using either standard or helical collection modes, at beamline ID23-2 (European Synchrotron Radiation Facility). The structure was solved by molecular replacement using thermostabilised A.sub.2AR (PDB code 2YDV) and the Gα.sub.s GTPase domain from the β.sub.2AR-Gs complex (3SN6) as search models (see further methods).
(187) Further Methods
(188) Expression and Purification of Mini-G.sub.s
(189) Mini-G.sub.s414, which incorporated an N-terminal histidine tag (His.sub.10) and TEV protease cleavage site was expressed in E. coli strain BL21(DE3)RIL. Expression was induced with IPTG (50 μM) for 20 h at 25° C. Cells were harvested by centrifugation and lysed by sonication in lysis buffer (40 mM HEPES pH 7.5, 100 mM NaCl, 10% glycerol, 10 mM imidazole, 5 mM MgCl.sub.2, 50 μM GDP, 1 mM PMSF, 2.5 μM Pepstatin-A, 10 μM Leupeptin, 50 μg/ml DNase I, 100 μg/ml lysozyme, 100 μM DTT), supplemented with Complete™ protease inhibitors (Roche). The lysate was clarified by centrifugation and loaded onto a 10 ml Ni.sup.2+ Sepharose FF column. The column was washed with wash buffer (20 mM HEPES pH 7.5, 500 mM NaCl, 40 mM imidazole, 10% glycerol, 1 mM MgCl.sub.2, 50 μM GDP) and eluted with elution buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 500 mM imidazole, 10% glycerol, 1 mM MgCl.sub.2, 50 μM GDP). TEV protease was added and the sample was dialysed overnight against dialysis buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM MgCl.sub.2, 10 μLM GDP). TEV protease was removed by negative purification on Ni.sup.2+-NTA resin (Qiagen). The sample was concentrated to 1.5 ml and loaded onto a Superdex-200 26/600 gel filtration column, equilibrated with gel filtration buffer (10 mM HEPES pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM MgCl.sub.2, 1 μM GDP, 0.1 mM TCEP). Peak fractions were pooled and concentrated to 100 mg/ml. The pure protein was aliquoted, flash-frozen in liquid nitrogen, and stored at −80° C. A typical yield was 100 mg of pure mini-G.sub.s414 per litre of culture.
(190) Expression and Purification of Adenosine A.sub.2AR
(191) Wild type human adenosine A.sub.2AR (residues 1-308) was modified to contain a C-terminal histidine tag (His.sub.10) and TEV protease cleavage site. The N154A mutation was introduced to remove a potential N-linked glycosylation site. Baculoviruses were prepared using the flashBAC ULTRA system (Oxford Expression Technologies). Trichopulsia ni cells were grown in ESF921 media (Expression Systems) to a density of 3×10.sup.6 cells/ml, infected with A.sub.2AR baculovirus and incubated for 72 h. Cells were harvested and membranes prepared by two ultracentrifugation steps in membrane buffer (20 mM HEPES pH7.5, 1 mM EDTA, 1 mM PMSF).
(192) NECA (100 μM), NaCl (300 mM), PMSF (1 mM) and Complete™ protease inhibitors (Roche) were added to the membranes, and the sample was mixed for 30 min at room temperature. Membranes were solubilised with 2% n-decyl-β-D-maltopyranoside (DM) on ice for 1 h. The sample was clarified by ultracentrifugation and loaded onto a 5 ml Ni-NTA column (Qiagen). The column was washed with wash buffer (20 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 80 mM imidazole, 100 μM NECA, 0.15% DM), and eluted with elution buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 10% glycerol, 300 mM imidazole, 100 μM NECA, 0.15% DM). The eluate was concentrated using a 50 kDa cutoff Amicon unit (Millipore), and exchanged in to desalting buffer (10 mM HEPES pH 7.5, 100 mM NaCl, 10% glycerol, 100 μM NECA, 0.15% DM) using a PD10 column (GE Healthcare). TEV protease was added, and the sample was incubated on ice overnight. The sample was concentrated to 0.2 ml and loaded onto a Superdex 200 column (GE Healthcare). Peak fractions were pooled and concentrated to approximately 20 mg/ml. A typical yield was 2 mg of pure A.sub.2AR per litre of culture.
(193) Complexation and Crystallisation
(194) Purified A.sub.2AR was mixed with a 1.2-fold molar excess of mini-G.sub.s414. MgCl.sub.2 (1 mM) and apyrase (0.1 U) were added, and the mixture was incubated on ice overnight. The sample was diluted 10-fold in gel filtration buffer (10 mM HEPES pH 7.5, 100 mM NaCl, 100 μM NECA, 0.35% n-octyl-β-D-thioglucopyranoside OTG), concentrated to 0.2 ml, and loaded on to a Superdex 200 column (pre-equilibrated in the same buffer). Peak fractions, containing the A.sub.2AR-mini-G.sub.s complex, were pooled and concentrated to 20 mg/ml. The A.sub.2AR-mini-G.sub.s complex was crystallised by vapour diffusion in OTG either in the presence or absence of cholesterol hemisuccinate (CHS). Crystallisation plates were set up at 4° C. using 120 nl sitting drops. Crystals used for structure solution were grown in two conditions, either: 0.1 M NaOAc pH 5.5, 10% PEG 2000 (in the presence of CHS); or 0.1 M NaOAc pH 5.7, 9.5% PEG 2000 MME (in the absence of CHS). Crystals were cryo-protected in mother liquor supplemented with 30% PEG 400 and flash frozen in liquid nitrogen.
(195) Data Collection, Processing and Refinement
(196) Diffraction data were collected at the European Synchrotron Radiation Facility on beamline ID23-2 with a Pilatus 2M detector, using a 10 μm microfocus beam (0.8729 Å wavelength). Data were collected using either standard or helical collection modes. Data from two crystals were used for structure solution. Data were processed using MOSFLM.sup.104 and AIMLESS.sup.105. The structure was solved by molecular replacement with PHASER.sup.106 using the structures of the thermostabilised A.sub.2AR (PDB code 2YDV).sup.107 and the Gα.sub.s GTPase domain (residues 40-59 and 205-394) from the β.sub.2AR-G.sub.s complex (PDB code 3SN6).sup.108 as search models. Model refinement and rebuilding were performed using REFMAC.sup.109 and COOT.sup.110.
(197) Competition Binding Assay
(198) FreeStyle HEK293-F cells transiently expressing wild type A.sub.2AR were resuspended in either assay buffer A (25 mM HEPES, pH 7.5, 100 mM KCl, 1 mM MgCl.sub.2), assay buffer B (25 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl.sub.2), or assay buffer C (25 mM HEPES, pH 7.5, 500 mM NaCl, 1 mM MgCl.sub.2), and were lysed by 10 passages through a 26 G needle. Purified binding partners were buffer-exchanged to the respective buffer before being added to the membranes at a final concentration of 25 μM. The mixture was aliquoted and NECA was added (0 to 1 mM final concentration, prepared in assay buffers containing 1 u/mL apyrase). The samples were incubated for 90 min at 22° C., .sup.3H-ZM241385 was added at its apparent K.sub.d (2.5 nM), and the samples were incubated for a further 90 min at 22° C. Non-specific binding was determined in the presence of 100 μM of ZM241385. Receptor-bound and free radioligand were separated by filtration through 96-well GF/B filter plates (pre-soaked with 0.1% polyethyleneimine), and washed 3 times with the appropriate buffer. Plates were dried and radioactivity was quantified by liquid scintillation counting using a Tri-Carb 2910 TR (Perkin Elmer). Data were analyzed by nonlinear regression using GraphPad Prism software. The K.sub.i for NECA binding was derived from one-site fit Ki analysis. Data from at least three independent experiments, each performed in duplicate, were analyzed using an unpaired two-tailed t-test for statistical significance.
(199) Thermostability Assay
(200) Membranes from Trichopulsia ni cells expressing wild type human A.sub.2AR were resuspended in Tm buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MgCl.sub.2) and homogenised by 10 passages through a 26-gauge needle. Binding partner was added at a final concentration of 25 μM. .sup.3H-NECA and unlabelled NECA were mixed in a ratio of 1:5 and added to the membranes to give a final concentration of 1 μM (approximately 10-fold above the apparent K.sub.d). The samples were incubated at room temperature for 1 h, then chilled on ice for 30 min. DDM, DM or OG were added to a final concentration of 0.1%, 0.13% or 0.8%, respectively, and samples were incubated on ice for 1 h. Cell debris was removed by centrifugation for 5 min at 20,000×g and the supernatant was aliquoted into PCR strips. Samples were heated to the desired temperature for exactly 30 min, then quenched on ice for 30 min. Samples (50 μl) were loaded onto gel filtration resin packed into a 96-well filter plate (Millipore), which was centrifuged to separate receptor-bound from free radioligand. Non-specific binding was determined in the presence of 200 μM unlabeled NECA. Radioactivity was quantified by liquid scintillation counting using a MicroBeta TriLux scintillation counter (PerkinElmer). Data were analyzed by nonlinear regression using GraphPad Prism software. Apparent T.sub.m values were derived from sigmoidal dose-response analysis. Results represent the mean±SEM of two independent experiments, performed in duplicate.
(201) TABLE-US-00001 TABLE 1 Turkey β1AR constructs used during this work. The β1AR-WT construct contained N- and C-terminal truncations, and the C116L mutation. These modifications were designed to prevent glycosylation or improve expression.sup.15. The β1AR-84 construct contained an additional deletion of cytoplasmic loop three. Thermostabilising mutations (M90V, D322K, F327A, and F388M).sup.2,3. A disulphide link between transmembrane helices 1 and 2, facilitated by the M400 and L1030 mutations. The C358A mutation, designed to prevent palmitoylation. An N-terminal MBP fusion, designed to facilitate crystallisation. Construct Deleted residues Mutations Other modifications β.sub.1AR-WT 1-32 C116L 6 His tag (C-terminus) 424-483 β.sub.1AR-84 1-32 M40C MBP fusion 244-271 M9OV (N-terminus) 368-483 L103C 6 His tag (C-terminus) C116L D322K F327A C358A F388M
(202) TABLE-US-00002 TABLE 2 Parental mini Gs constructs used during this work. Construct Deleted residues GαAH linker Mutations Other modifications Mini Gs.sub.77 1-21 Gly.sub.3 L197A N-terminal 6 His tag 67-193 C200S Mini Gs.sub.161 1-21 Gly.sub.5 None N-terminal 6 His tag 65-208 Mini Gs.sub.199 1-21 Gly.sub.8 G49D N-terminal 6 His tag 65-203 E50N 254-963 A249D (switch III) S252D L272D Mini Gs.sub.391 1-25 GGSGGSGG G49D N-terminal 6 His tag 65-203 E50N TEV protease site 254-263 A249D (switch III) S252D L272D I372A Mini Gs.sub.393 1-25 GGSGGSGG G49D N-terminal 6 His tag 65-203 E50N TEV protease site 254-263 A249D (switch III) S252D L272D I372A V375I Mini Gs.sub.399 1-5 GGSGGSGG G49D N-terminal 6 His tag 65-203 E50N TEV protease site 254-263 A249D (switch III) S252D I372A V375I Mini Gs.sub.404 1-25 GGSGGSGG G49D N-terminal 6 His tag 65-203 E50N TEV protease site 254-263 A249D (switch III) S252D L272D
(203) TABLE-US-00003 TABLE 3 Competitive binding assay data showing the isoprenaline K.sub.i of β.sub.1AR-84 in response to different binding partners. Data are from a single experiment performed in duplicate unless otherwise stated in the table, in these cases data represent mean ± SEM, from the number of independent experiments (n) indicated. The effect of mutations on the expression level of mini Gs was estimated from SDS-PAGE gels. Mutants that caused more than a 2-fold change in expression compared to the parental construct are shown simply as an increase (+) or decrease (−). .sup.a Common Gα numbering (CGN) system. .sup.b Not applicable..sup.c Not determined. .sup.d Substitution of switch II residues 227-230 with two glycine residues..sup.e Deletion of switch III residues 254-263. Binding CGN β.sub.1AR-WT Tm in Mini Gs basal partner Mutation code.sup.a complex DDM (° C.) Tm (° C.) None n.a..sup.b n.a. 25.9 ± 0.0 (n = 3) n.a. Nb80 n.a. n.a. 32.0 ± 0.0 (n = 3) n.a. Gs-Nb35 n.a. n.a. 35.8 ± 0.1 (n = 3) n.a. Gαs n.a. n.a. n.d..sup.c 50.1 ± 0.1 (n = 3) Mini Gs.sub.162 A249D 25.1 (n = 1) 60.6 ± 0.1 (n = 3) Mini Gs.sub.164 A249D-SIII.sup.d 28.6 (n = 1) 66.5 ± 0.0 (n = 3) Mini Gs.sub.165 A249D-S252D-SIII 28.5 ± 0.2 (n = 2) 68.7 ± 0.0 (n = 3) Mini Gs.sub.169 A249D-S252D-SIII- 28.8 (n = 1) 67.1 ± 0.0 (n = 3) Mini Gs.sub.183 G49D-E50N-A249D- 28.7 ± 0.2 (n = 4) 72.5 ± 0.0 (n = 3) S252D-SIII-L272D Mini Gs.sub.199.sup.e G49D-E50N-A249D- 29.2 ± 0.2 (n = 17) 72.5 ± 0.0 (n = 3) S252D-SIII-L272D Mini Gs.sub.254 M60A 60.sup.G.H1.8 31.5 ± 0.3 (n = 5) 70.3 ± 0.0 (n = 3) Mini Gs.sub.350 L63Y 63.sup.G.11.1 30.9 ± 0.4 (n = 2) 70.7 ± 0.0 (n = 3) Mini Gs.sub.340 I372A 372.sup.G.H5.4 34.0 (n = 1) 66.6 ± 0.1 (n = 3) Mini GS.sub.303 V375I 375.sup.G.H5.7 31.5 ± 0.6 (n = 3) 70.3 ± 0.0 (n = 3) Mini Gs.sub.352 L63Y-1372A 34.5 (n = 1) 64.7 ± 0.1 (n = 3) Mini Gs.sub.345 I372A-V375I 35.0 (n = 1) 65.4 ± 0.1 (n = 3)
(204) TABLE-US-00004 TABLE 4 Thermostability (Tm) measurements for either β.sub.1AR-WT complexes or mini Gs mutants in the basal GDP-bound state. Tm values represent the mean ± SEM from the number of independent experiments (n) indicated in the table. Some Tm values were determined from a single experiments performed in duplicate, with an assumed error of ± 0.5° C. Tm values for mini Gs in the GDP-bound state were determined by differential scanning fluorimetry..sup.a Common Gα numbering (CGN) system..sup.b Not applicable..sup.c Not determined..sup.d Deletion of switch III residues 254-263 is referred to as SIII..sup.e Mini Gs.sub.199 contains the same mutations as mini GS.sub.183, but has a redesigned linker region (see Table 2), and was used as the parental construct for screening detergent stabilising mutations. Binding CGN β.sub.1AR-84 isoprenaline Ki (nM) Effect on partner Mutation code.sup.a 4° C. 20° C. expression None n.a..sup.b n.a. 2080 ± 181 (n = 12) 2615 ± 273 (n = 15) n.a. Nb80 n.a. n.a. n.d..sup.c 28 ± 1 (n = 2) n.a. Gs n.a. n.a. 419 ± 80 (n = 2) 271 ± 54 (n = 2) n.a. Gs-Nb35 n.a. n.a. n.d. 16 ± 4 (n = 3) n.a. Mini Gs.sub.77 Parental n.a. 99 ± 12 (n = 4) 1867 ± 228 (n = 3) n.a. Mini Gs.sub.81 H41I 41.sup.G.S1.2 32 393 Mini Gs.sub.84 H41V 41.sup.G.S1.2 51 491 Mini Gs.sub.186 A48L 48.sup.G.s1h1.2 43 174 Mini Gs.sub.130 G49D 49.sup.G.s1h1.3 25 285 Mini Gs.sub.116 E50N 50.sup.G.s1h1.4 37 724 Mini Gs.sub.134 R201A 201.sup.G.hfa2.2 31 1479 − Mini Gs.sub.98 227-230 sub.sup.d 227.sup.G.s3h2.3 23 533 Mini Gs.sub.175 E230A 230.sup.G.h2.3 51 545 − Mini Gs.sub.92 A249D 249.sup.G.S4.7 10 35 + Mini Gs.sub.104 A249E 249.sup.G.S4.7 70 388 Mini Gs.sub.117 S252D 252.sup.G.sh4.3 14 94 + Mini Gs.sub.118 S252E 252.sup.G.s4h3.3 38 383 + Mini Gs.sub.105 254-263 del.sup.e 254.sup.G.s4h3.5 21 20 + Mini Ge.sub.94 L272D 272.sup.G.H3.8 7 310
(205) TABLE-US-00005 TABLE 5 Agonist-shift assay data for GTPase domain mutants. Assays were performed using membranes containing the 61AR reconstituted with partially purified GTPase domain mutants (total amount purified from 1 litre of E. coli culture). Assays were performed at 4 and 20° C. for each mutant. The table shows the final isoprenaline affinity of the receptor under each condition. The starting isoprenaline affinity of the receptor was 3.03 ± 0.81 μM (n = 16), with values in the range of 1.5-4.4 μM. Therefore, a shift in agonist affinity less than threefold cannot be considered significant. Quantification of expression levels was not possible due to the use of partially purified material. Therefore, a simplified scale was used to indicate the relative expression level compared to the parental construct. Note: some mutants that did not induce a shift in agonist affinity at 4° C. were not tested at 20° C. (denoted by n.d. in the table). Approximate isoprenaline IC50 (nM) Approximate Modification/mutation At 4° C. At 20° C. expression level Parental construct 136 2100 V36D/N218K 115 2100 - V36D/N218K/T40A 551 2400 -- V36D/N218K/T40D 427 3200 = Y37D 81 2000 = Y37R/R42D 2300 n.d. --- H41I 58 589 = H41L 285 3900 - H41M 307 1800 = H41V 77 737 = A48D 185 1800 --- G49D 37 428 = E5ON 55 1100 = G49D/E5ON 44 364 = S54N 462 2100 --- R199K 108 1900 + R199D 109 1800 = R201A 46 2200 = F208N 250 1300 = G226A 127 1200 = Δ227-230/GG linker 34 799 = W234A/F238A 584 4000 - C237E 93 1900 = D240G 510 4400 - A249S 146 1600 - A249D 15 53 ++ A249E 104 582 + S252D 21 142 + S252E 57 575 + L270N/I348N 4300 n.d. --- L272D 10 464 + L272E 42 110 = S275D 136 2000 = N279E/I235K 1200 2800 --- N279D/Q235K 102 2000 --- S286C/I382C 4200 7000 --- D295N 2400 n.d. --- R356S 229 2800 = R356D 161 3300 = Δ255-262/G linker 1300 n.d. -- Δ254-263 31 30 ++ Δ254-263/Y253P 23 142 ++ Δ266-340/Gαil chimera 4400 n.d. --- Δ266-341/ras chimera 2200 n.d. ---
(206) TABLE-US-00006 TABLE 6 Data collection and refinement statistics Data collection Space group P 2.sub.12.sub.12.sub.1 Cell dimensions a, b, c (Å) 90.6, 111.8, 161.3 Resolution (Å) 40.3-3.4 (3.49-3.40) R.sub.merge 0.173 (0.747) I/σl 3.6 (1.2) Completeness (cY0) 90.6 (78.5) Redundancy 2.6 (2.4) Refinement Resolution (Å) 40.3-3.4 No. reflections 19788 R.sub.work/R.sub.free (%) 28.4/31.5 No. atoms 7359 Protein 7248 Ligand/detergent/nucleotide 44/40/27 Water 0 B-factors (Å.sup.2) Protein 79.9 Ligand/detergent/nucleotide 67.9/98.6/69.0 R.M.S.D. Bond lengths (Å) 0.008 Bond angles (°) 1.15
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EXAMPLE 5: FURTHER MINI-G PROTEINS
Summary
(212) The first mini-G protein developed was mini-G.sub.s. Here we extend the family of mini-G proteins to include mini-G.sub.olf, mini-G.sub.i1, mini-G.sub.o1 and the chimeras mini-G.sub.s/q and mini-G.sub.s/i. The mini-G proteins were shown to couple to relevant GPCRs and to form stable complexes with purified receptors that could be purified by size exclusion chromatography. Agonist-bound GPCRs coupled to a mini-G protein showed higher thermal stability compared to the agonist-bound receptor alone. Fusion of GFP at the N-terminus of mini-G proteins allowed receptor coupling to be monitored by fluorescence-detection size exclusion chromatography (FSEC) and, in a separate assay, the affinity of mini-G protein binding to detergent-solubilised receptors was determined. This work provides the foundation for the development of any mini-G protein and, ultimately, for the structure determination of any GPCR in a fully active state.
Introduction
(213) The concept of mini-G proteins shows great promise for accelerating the rate of structure determination of GPCRs in their active states. However, there are four families of Gα subunits (
(214) Materials and Methods
(215) Ligands
(216) The β.sub.1-adrenergic receptor (β.sub.1AR) agonist isoproterenol hydrochloride and inverse agonist ICI118551 hydrochloride were from Sigma-Aldrich. The adenosine A.sub.2A receptor (A.sub.2AR) agonist NECA and antagonist ZM241385 were also from Sigma Aldrich. Serotonin 5HT.sub.1B receptor (5HT.sub.1BR) agonist donitriptan hydrochloride and selective antagonist SB224289 hydrochloride were from Santa Cruz Biotechnology; the agonist sumatriptan succinate was from Cayman chemical. Angiotensin II receptor (AT.sub.1R) agonist angiotensin II was from Tocris. All radioactive ligands were from PerkinElmer.
(217) GPCR Constructs, Expression and Purification
(218) Human adenosine A.sub.2A receptor (A.sub.2aR)
(219) Two different A.sub.2AR constructs were used during this work. For SEC experiments using purified receptor, an A.sub.2AR construct was used that contained an N-terminal thioredoxin fusion protein to increase the molecular weight of the receptor. Without this fusion protein, A.sub.2AR and the mini-G protein had identical mobility on SDS-PAGE, thus making it difficult to visualise the separate components when analyzing a complex. The thioredoxin-A.sub.2AR fusion protein consisted of an N-terminal cleavable leader sequence (gp67), His10 tag and TEV protease cleavage site, followed by thioredoxin, which was connected to wild-type human A.sub.2AR (residues 6-316) through an EAAAKA linker. A.sub.2AR contained the N154A mutation to remove a potential N-linked glycosylation site. For all other experiments, a C-terminally truncated human A.sub.2AR construct was used (residues 1-317), which contained a C-terminal His10 tag and TEV protease cleavage site and the N154A mutation to remove the potential N-linked glycosylation site. Both constructs were expressed using the baculovirus expression system as described previously [19] (see Example 4). Cells were harvested by centrifugation 72 hours post infection, resuspended in hypotonic buffer (20 mM HEPES pH7.5, 1 mM EDTA, 1 mM PMSF), flash-frozen in liquid nitrogen and stored at −80° C. until use. Purification of the receptor was performed in DDM using Ni.sup.2+-affinity chromatography followed by SEC essentially as described previously [19].
(220) Turkey β.sub.1-Adrenergic Receptor (β.sub.1AR)
(221) A truncated version of wild type turkey β.sub.1AR (construct βAR6; [25]) contained truncations at the N-terminus and the C-terminus and a C-terminal His6 tag for purification [25], and was expressed using the baculovirus expression system at 27° C. as described previously [26]. Cells were harvested by centrifugation 48 hours post infection, resuspended in hypotonic buffer (20 mM Tris HCl pH8, 1 mM EDTA, 1 mM PMSF), flash-frozen in liquid nitrogen and stored at −80° C. until use.
(222) Human Angiotensin Type II Receptor 1 (AT.sub.1R)
(223) Wild type AT.sub.1R (residues 1-359) had a C-terminal factor X cleavage site followed by GFP and a His10 tag for purification, and was expressed using the tetracycline-inducible mammalian expression system as a stable cell line in HEK293 cells [27]. Cells were grown in DMEM containing 5% tetracycline-free FBS until they were 80% confluent and then tetracycline was added to a final concentration of 1 μg/ml. Cells were grown for 24 hours and then harvested, and resuspended in PBS, flash frozen in liquid nitrogen and stored at −80° C. until use.
(224) Rat Neurotensin Receptor (NTSR1)
(225) NTSR1 was expressed as described previously [13]. The baculovirus construct NTSR1 consisted of the hemagglutinin signal peptide and the Flag tag, followed by the wild-type rat NTSR1 (residues 43-396) and a C-terminal His10 tag. Recombinant baculovirus was generated using a modified pFastBac1 transfer plasmid (Invitrogen). Trichoplusia ni cells were infected with recombinant virus, and the temperature was lowered from 27° C. to 21° C. Cells were harvested by centrifugation 48 hours post infection, resuspended in hypotonic buffer (10 mM HEPES pH 7.5, 10 mM MgCl.sub.2, 20 mM KCl), flash-frozen in liquid nitrogen and stored at −80° C. until use.
(226) Human Serotonin 5HT.sub.1B Receptor (5HT.sub.1BR)
(227) Wild-type 5HT.sub.1BR (residues 34-390) was modified to contain a C-terminal TEV cleavage site and a His10 tag, cloned into plasmid pBacPAK8 and recombinant baculoviruses were prepared using the FlashBAC ULTRA system (Oxford Expression Technologies). Trichoplusia ni cells were grown in ESF921 media (Expression Systems) to a density of 3×10.sup.6 cells/ml, infected with 5HT.sub.1BR baculovirus and incubated for 48 h at 27° C. for expression. Purification of the receptor was performed in either DDM or LMNG using Ni.sup.2+-affinity chromatography followed by SEC.
(228) Expression, Purification and Stability of G Protein Subunits
(229) For constructs see
(230) SEC of the A.sub.2AR-Mini-Gs Complex
(231) The thioredoxin fusion construct of A.sub.2AR was purified in DDM, mini-G protein was added in excess at a 1:1.2 molar ratio, incubated overnight on ice and then loaded onto a Superdex S200 10/300 size exclusion column (10 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MgCl.sub.2, 100 μM NECA, 0.02% DDM; 4° C., 0.5 ml/min). Peak fractions were analysed by SDS-PAGE.
(232) FSEC Assays
(233) (1) A.sub.2AR
(234) Insect cell membranes containing a total of 20 μg (560 pmol) wild-type A.sub.2aR (20×10.sup.6 cells) were solubilized for 30 min on ice in 40 mM HEPES pH7.5, 500 mM NaCl, 2 mM MgCl.sub.2, 2 U/mL apyrase (Sigma-Aldrich), and 0.5% (v/v) DDM in a final volume of 2 ml. Insoluble material was removed by ultracentrifugation (30 min, 4° C., 135,000×g). The supernatant was divided into aliquots for the subsequent assay. To 500 μl of the supernatant was added either the agonist NECA or the inverse agonist ZM241385 (negative control), both at a final concentration of 60 μM. GFP-mini-G.sub.s (6 μg; 110 pmol) was then added and allowed to bind for 90 min on ice before loading 200 μl onto a Superdex S200 10/300 size exclusion column (buffer 20 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl.sub.2, 1 μM NECA or ZM241385, 0.03% DDM, 4° C., flow rate 0.45 ml/min). The control sample contained 6 μg GFP-mini-G.sub.s only in 500 μl assay buffer. GFP fluorescence was detected by a Hitachi fluorometer (mV) set to an excitation of 488 nm and an emission of 525 nm.
(235) (2) β.sub.1AR
(236) Insect cell membranes containing a total of 8 μg (178 pmol) wild-type β.sub.1AR (30×10.sup.6 cells) were solubilized for 30 min on ice in 20 mM Tris-HCl pH8, 500 mM NaCl, 5 mM MgCl.sub.2, 2 U/mL apyrase and 0.5% (v/v) DDM. Insoluble material was removed by ultracentrifugation (30 min, 4° C., 135,000×g). The supernatant was divided into aliquots for the subsequent assay. Isoprenaline (100 μM final concentration) or ICI118551 (10 μM final concentration) were added to 500 μl of the supernatant. GFP-mini-G.sub.s (6 μg) was then added and allowed to bind for 90 min on ice before loading 200 μl onto a Superdex S200 10/300 size exclusion column (buffer 20 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl.sub.2, 1 μM isoprenaline or ICI118551, 0.03% DDM, 4° C., flow rate 0.45 ml/min). The control sample contained 6 μg GFP-mini-G only in 500 μl assay buffer.
(237) (3) 5HT.sub.1BR
(238) When detergent-solubilized unpurified receptor was used, insect cells expressing 610 pmol 5HT.sub.1BR (40×10.sup.6 cells) were resuspended in 20 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl.sub.2 to a final cell density of 20×10.sup.6 cells/ml and solubilized with 0.5% DDM (45 min, 4° C.). Insoluble material was removed by ultracentrifugation (30 min, 4° C., 135,000×g). The supernatant was divided into 900 μl aliquots for the subsequent assay. GFP-mini-G.sub.o1 (5 μg) was added with either donitriptan or SB224289, each to a final concentration of 100 μM, and allowed to bind for 90 min on ice before loading 500 μl onto a Superdex S200 10/300 size exclusion column. The control sample contained 5 μg GFP-mini-G.sub.o1 in 500 μl assay buffer.
(239) In some FSEC experiments, purified 5HT.sub.1BR was used. Donitriptan-bound, purified receptor (120 μg; 3 nmol) in either LMNG or DDM was incubated for 90 min on ice with 4 μg (60-80 pmol) either of GFP-mini-G.sub.i1, GFP-mini-G.sub.o1 or GFP-mini-G.sub.s (negative control) in a final volume of 450 μl. Samples (200 μl) were then loaded onto Superdex S200 10/300 size exclusion column (buffer 20 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl.sub.2, 1 μM 0.03% DDM or 0.001% LMNG buffer, 4° C., flow rate 0.45 ml/min).
(240) Fluorescent Saturation Binding Assay (FSBA)
(241) (1) β.sub.1AR
(242) Membranes prepared from insect cells expressing β.sub.1AR (50×10.sup.6 cells) were solubilized in 20 mM Tris-HCl pH8, 500 mM NaCl, 3 mM imidazole, 0.5% DDM (1 hour, 4° C., final volume 8 ml). Insoluble material was removed by ultracentrifugation (30 min, 4° C., 135,000×g) and the supernatant was divided into two aliquots. The agonist isoprenaline was added to one sample (final concentration 10 μM) and the inverse agonist ICI118551 was added to the other (final concentration 1 μM). Samples were then aliquoted 200 μl per well into a black Ni.sup.2+-coated 96-well plate (Pierce; Thermo Fisher). The receptor was allowed to bind via its His tag for 1 h on ice. The supernatant was then aspirated and 200 μl GFP-mini-G.sub.s at varying concentrations (0 to 2.8 μM) were added and incubated for a further 90 min on ice. The supernatant was then removed by aspiration and each well washed 4 times with buffer A (10 μM isoprenaline (agonist), 20 mM Tris-HCl pH8, 100 mM NaCl, 1 mM MgCl.sub.2, 1 mg/mL BSA, 30 mM imidazole, 0.03% DDM,) or buffer B (1 μM ICI118551 (inverse agonist), 20 mM Tris-HCl pH8, 100 mM NaCl, 1 mM MgCl.sub.2, 1 mg/mL BSA, 30 mM imidazole, 0.03% DDM). Elution of the receptor-GFP-mini-G.sub.s complex from the sides of the well to make a homogeneous solution was performed with 200 μl of the respective wash buffers that contained 300 mM imidazole. Fluorescence was then measured using a Pherastar plate reader (BMG Labtech, Inc.) with excitation at 485 nm and emission at 520 nm. ΔF data (fluorescence agonist condition minus fluorescence antagonist condition) corresponding to specific binding were analysed by non-linear regression using GraphPad Prism version 5.0 (GraphPad Software, San Diego, Calif.) and apparent K.sub.D values derived from one site-specific binding analysis.
(243) (2) A.sub.2aR
(244) The assay was performed essentially as described above for β.sub.1AR, but the buffer conditions were different. Solubilisation of insect cell membranes (40×10.sup.6 cells) was performed in 10 ml of 20 mM Tris-HCl pH8, 500 mM NaCl, 10 mM imidazole and 0.5% DDM. After ultracentrifugation, the agonist NECA (10 μM final concentration) was added to one supernatant sample and the inverse agonist ZM241385 (10 μM final concentration) to the other. Washing buffers for A.sub.2aR were buffer C (10 uM NECA, 20 mM Tris-HCl pH8, 100 mM NaCl, 1 mM MgCl2, 1 mg/mL BSA, 50 mM imidazole, 0.03% DDM) or buffer D (10 μM ZM241385, 20 mM Tris-HCl pH8, 100 mM NaCl, 1 mM MgCl2, 1 mg/mL BSA, 50 mM imidazole, 0.03% DDM).
(245) (3) 5HT.sub.1BR
(246) Insect cells expressing 5HT.sub.1BR (50×10.sup.6 cells) were solubilized with buffer containing 10 μM Donitriptan, 20 mM Tris-HCl pH8; 500 mM NaCl; 10 mM imidazole, 0.5% DDM (1 h, 4° C., final volume 6 ml). Insoluble material was removed by ultracentrifugation (30 min, 4° C., 135,000×g) and 200 μl of supernatant was then aliquoted per well into a black Ni.sup.2+-coated 96-well plate. The receptor was allowed to bind via its His tag for 1 h on ice. The supernatant was then aspirated and 200 μl either of GFP-mini-G.sub.o1, GFP-mini-G.sub.s/i1 or GFP-mini-G.sub.s (negative control) at varying concentrations (from 0 to 5 μM) were added and incubated for a further 90 min on ice. The supernatant was then removed by aspiration and each well washed 4 times with buffer E (1 μM Donitriptan, 20 mM Tris-HCl pH8, 100 mM NaCl, 1 mM MgCl.sub.2, 1 mg/mL BSA, 50 mM imidazole, 0.03% DDM). Elution was carried out with 200 μL of buffer E containing 300 mM imidazole. ΔF data (fluorescence G.sub.o1 condition minus fluorescence G.sub.s condition) corresponding to specific binding were analysed by non-linear regression using GraphPad Prism version 5.0 (GraphPad Software, San Diego, Calif.) and apparent K.sub.D values derived from one site-specific binding analysis.
(247) Competition Binding Assay
(248) Insect cells expressing 5HT.sub.1BR were resuspended in 1 ml of assay buffer (20 mM HEPES pH7.5, 100 mM NaCl, 1 mM MgCl.sub.2, 1 mM ascorbate, 20 μM pargyline) at a final concentration of 2×10.sup.6 cells/ml. Cells were sheared by 10 passages through a bent 26G needle. The supernatant was diluted 10-fold in assay buffer and aliquots (900 μl) taken for each sample. Mini-G protein (100 μl, 25 μM final concentration) or buffer (negative control) was added. The mixture was aliquoted into a 0.2 ml PCR plate, 96 μl per well. Sumatriptan (12 μl), prepared in assay buffer also containing 2 U/ml apyrase, was added to each well (final concentrations in the range of 100 μM to 1 mM). Non-specific binding was determined in the presence of 100 μM donitriptan. Samples were mixed and incubated at 4° C. for 2 h. [.sup.3H]-GR125743 (12 μl) was added at its apparent K.sub.D (10 nM) concentration. Samples were mixed and incubated at 4° C. for 2 h before filtering through 96-well glass fibre GF/B filter plate (Merck Millipore) and washing with ice-cold assay buffer. Filters were dried, punched into scintillation vials and 4 ml Ultima Gold scintillant (Perkin Elmer) were added. Radioactivity was quantified by scintillation counting (1 min per sample) using a Tri-Carb counter (Perkin Elmer), and K.sub.i values were determined using GraphPad Prism version 5.0 (GraphPad Software, San Diego, Calif.).
(249) Thermostability Assay
(250) (1) A.sub.2AR
(251) Membranes from Trichoplusia ni cells expressing wild-type human A.sub.2AR were resuspended in T.sub.m buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MgCl.sub.2) and homogenized by ten passages through a 26G needle. Mini-G protein was added at a final concentration of 25 μM. .sup.3H-NECA and unlabeled NECA were mixed in a molar ratio of 1:5 and added to the membranes to give a final concentration of 1 μM (approximately ten-fold above the apparent K.sub.D). The samples were incubated at room temperature for 1 h, then chilled on ice for 30 min. Decylmaltoside (DM) was added to a final concentration of 0.13%, and samples were incubated on ice for 1 h. Cell debris and insoluble material were removed by centrifugation (5 min, 20,000×g, 4° C.) and the supernatant was aliquoted (120 μl) into PCR strips. Samples were heated to the desired temperature for exactly 30 min, then quenched on ice for 30 min. Samples (50 μl) were loaded onto gel-filtration resin (Toyopearl HW-40F) packed into a 96-well filter plate (Millipore), which was centrifuged to separate receptor-bound from free radioligand [28]. Nonspecific binding was determined in the presence of 200 μM unlabelled NECA. Radioactivity was quantified by liquid scintillation counting using a MicroBeta TriLux scintillation counter (PerkinElmer). Data were analysed by nonlinear regression using GraphPad Prism software. Apparent T.sub.m values were derived from sigmoidal dose-response analysis performed by non-liner regression. Results represent the mean±SEM of two independent experiments, performed in duplicate.
(252) (2) NTSR1
(253) Cell pellets from 10 ml of insect cell cultures were resuspended in 1.8 ml buffer containing DDM to give a final buffer composition of 50 mM TrisHCl pH 7.4, 100 mM NaCl, 1 mM MgCl2, 1% (w/v) DDM. The samples were placed on a rotating mixer at 4° C. for 1 hour. Cell debris and non-solubilized material were removed by ultracentrifugation (152,800×g, 4° C., 30 min), and the supernatant containing detergent-solubilized NTSR1 was used to test for thermal stability in the presence of NTS and mini-G proteins. For thermal denaturation curves, the supernatants were diluted 6.67-fold into assay buffer (50 mM TrisHCl pH 7.4, 100 mM NaCl, 1 mM MgCl.sub.2) containing 22.5 μM mini-G protein and 10 nM .sup.3H-NTS and incubated for 1 hour on ice. After addition of apyrase (0.25 units/ml, NEB), the sample was placed on ice for an additional 30 min. Samples (120 μl aliquots) were exposed to different temperatures between 0° C. and 60° C. for 30 min and placed on ice. Separation of receptor-ligand-mini-G protein complex from free .sup.3H-NTS (100 μl) was achieved by centrifugation-assisted gel filtration (spin assay) using Bio-Spin 30 Tris columns (BioRad), equilibrated with RDB buffer [50 mM TrisHCl pH7.4, 1 mM EDTA, 0.1% (w/v) DDM, 0.2% (w/v) CHAPS, 0.04% (w/v) CHS], essentially as described previously [29]. Control reactions on ice were recorded at the start and at the end of each denaturation experiment. The percentage of activity remaining after heat exposure was determined with respect to the unheated control. Data were analyzed by nonlinear regression using a Boltzmann sigmoidal equation in the Prism software (GraphPad).
(254) (3) AT.sub.1R
(255) HEK 293 cells expressing wild type AT.sub.1R were resuspended in a radioligand binding assay buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% BSA, 40 μg/ml bacitracin) and homogenized by sonication (4 sec pulse). Mini-G protein and apyrase were added at a final concentration of 25 μM and 0.1 units/ml, respectively. .sup.125I-Ang II and unlabeled Ang II were added at a concentration of 0.5 nM and 25 nM respectively (approximately 50 times the apparent K.sub.D value). The sample was incubated at room temperature (20° C.) for an hour, chilled on ice for 10 minutes and then digitonin was added to a final concentration of 1% and incubated on ice for an hour. Insoluble material was removed by centrifugation (2 min, 20,000×g, 4° C.). The reaction mix was split into a number of 115 μl aliquots and each was incubated at various temperatures for exactly 30 minutes. The reactions were then quenched on ice for 5 minutes. .sup.125I-Ang II bound to AT.sub.1R was separated from unbound .sup.125I-Ang II using centrifugation-assisted gel filtration column, essentially as described previously [27]. Non-specific binding was determined using a 500-fold excess of cold ligand. Radioactivity was measured using liquid scintillation counting. Data was analysed by non-linear regression using GraphPad prism software and apparent T.sub.m values were derived by non-linear regression of the sigmoidal dose-response curve.
(256) Results and Discussion
(257) Initial Development of New Mini-G Proteins
(258) The recently designed minimal G protein, mini-G.sub.s [23], comprises only the GαGTPase domain from G.sub.s and 3 deletions and 7 mutations to thermostabilise it (
(259) Archetypical members from each Gα family were selected and include the following: G.sub.olf from the G.sub.s family, G.sub.i1, G.sub.o1, G.sub.z and G.sub.t from the G.sub.i family, G.sub.q and G.sub.16 from the G.sub.q/11 family, and G.sub.12 from the G.sub.12/13 family. The mutations required to convert Gα.sub.s into mini-G.sub.s were transferred en bloc to the selected Gα proteins to produce a mini-G protein version of each (
(260) Four mini-G proteins were not expressed in E. coli, namely mini-G.sub.z, mini-G.sub.t, mini-G.sub.16 and mini-G.sub.q (amino acid sequences are given in
(261) Assay Development and Validation Using the Mini-G.sub.s System
(262) The ultimate goal of developing mini-G proteins is the structure determination of GPCRs in the fully active state bound to an agonist and a mini-G protein. In the simplest format, this necessitates the purification of the GPCR in detergents and forming the G protein-GPCR complex from the purified components in vitro. It was therefore essential to devise some simple assays that could assess whether a mini-G protein had coupled to a GPCR in detergent solution. This turned out to be not as straightforward as originally anticipated due to the potential instability of either the GPCR and/or mini-G protein in either their inactive and/or active conformations. These issues were not obvious when the original work on the development of mini-G.sub.s was performed, because mini-G.sub.s is one of the most stable mini-G proteins developed and also the thermostabilised β.sub.1-adrenergic receptor (β.sub.1AR) and the wild type adenosine A.sub.2A receptor (A.sub.2AR) were both much more stable than other GPCRs. We therefore developed five separate assays for assessing whether a mini-G protein coupled to a GPCR and/or formed a stable complex in detergent. These were all first tested using mini-G.sub.s coupling to β.sub.1AR and A.sub.2AR. Each assay has its own limitations, which are often apparent in the subsequent sections where they were used on less stable receptors and the newly developed mini-G proteins, and these are discussed below. The five different assays that were used are the following: (i) agonist affinity shift assay; (ii) thermostability assay (TSA); (iii) fluorescence-based saturation binding analysis (FSBA) of GFP-mini-G protein binding; (iv) fluorescence-detection size exclusion chromatography (FSEC); (v) size exclusion chromatography (SEC) of purified complex. A brief rationale for the use of each assay with their advantages and disadvantages are given below.
(263) (i) Agonist Affinity Shift Assay
(264) The development of mini-G.sub.s relied on the agonist affinity shift assay to identify those mutants that coupled to β.sub.1AR [23]. It is generally considered that the defining feature of G protein coupling is an increase in the affinity of an agonist for the G protein-GPCR complex compared to the GPCR alone. For example, wild type β.sub.2AR binds an agonist 100-fold more tightly when coupled to a G protein than the receptor alone [31]. However, the shift in agonist affinity in other receptors is often considerably smaller than that observed for β.sub.2AR, such as the 10-fold shift in agonist affinity observed in β.sub.1AR [31] and may be entirely absent eg NTSR1. However, the advantage of this assay is that it can be performed using standard pharmacological procedures in high-throughput, using receptors in either membrane preparations or solubilized in detergent. Assays may use either a radiolabelled agonist in saturation binding experiments or, more usually, a radiolabelled antagonist in competition binding experiments [19, 23 and Examples 1 and 4] (and see experiments below on the serotonin 5HT.sub.1B receptor). The advantage of this assay is that it is very sensitive and can be performed on membrane-bound receptors i.e. in a format where the receptor is most stable in all conformations. The disadvantage of this assay is that some receptors may not show a shift in agonist affinity when coupled to a G protein.
(265) (ii) Thermostability Assay
(266) The thermostability of a detergent-solubilised GPCR depends upon the type of detergent used and whether the receptor is either ligand-free, agonist-bound or antagonist-bound [32, 33]. In addition, the receptor stability tends to be increased by an increase in affinity and/or decrease in the off-rate of the ligand [34]. Often, the agonist bound state is one of the least stable conformations of a receptor, presumably because agonists increase the probability of transitions to a fully active state. In the inactive state there is close packing of the intracellular surface of the transmembrane α-helices. Upon activation, the outward movement of helices 5 and 6 disrupts this close packed structure and creates a crevice where the C-terminus of the G protein binds, thus allowing G protein coupling [35]. The structures of non-rhodopsin GPCRs in the fully active state have been determined only when they have been stabilized through binding of a heterotrimeric G protein [2], a conformation-specific nanobody [14, 17, 18] or a mini-G protein [19]. The interface between a GPCR and a G protein is over 1000 Å.sup.2 [2, 19], and is therefore predicted to increase the thermostability of the agonist-bound GPCR-G protein complex compared to the agonist-bound GPCR. This was observed for both β.sub.1AR and A.sub.2AR, which were consistently more stable in the agonist-bound state when coupled to mini-G.sub.s in a variety of different detergents compared to when mini-G.sub.s was absent [19, 23 and Examples 1 and 4].
(267) A typical thermostability assay measures how much of a radiolabelled agonist remains bound to a detergent-solubilised receptor after heating at different temperatures for 30 minutes [33]. The advantage of this assay is that it is fast and high-throughput and can be performed in any detergent of choice. Another advantage is that the agonist-GPCR-mini-G protein complex can be pre-formed in membranes, which may stabilise the receptor upon detergent solubilisation, allowing the assay to be performed. If there is a shift in thermostability in the presence of a mini-G protein, then this is strongly suggestive of binding or coupling.
(268) (iii) Fluorescence-Detection Size Exclusion Chromatography (FSEC)
(269) FSEC is a rapid methodology for assessing whether a membrane protein fused to GFP is stable in detergent by performing SEC on an unpurified detergent solubilisate and monitoring GFP fluorescence in the eluate [36]. A membrane protein stable in detergent gives a symmetrical peak at a size consistent with the molecular weight of the membrane protein plus the mass of specifically bound detergent and lipid. By fusing GFP to the N-terminus of mini-G proteins (
(270) The advantage of this assay is that it is a quick assessment of whether a GPCR forms a complex with a mini-G protein, because the receptor does not need to be purified and the SEC experiment takes under an hour. However, the major limitation is that only small amounts of GFP-mini-G.sub.s can be used per experiment to avoid saturation of the detector and producing a very broad peak that would obscure the presence of the complex between the GPCR and GFP-mini-G protein. Thus the concentration of the mini-G protein is below its K.sub.D for association with a receptor and therefore the assay is not quantitative. In addition, the receptor-mini-G protein complex must be detergent-stable for a peak to be observed. Many GPCR-G protein complexes are too unstable to be observed in DDM and therefore it is essential to assess milder detergents such as LMNG (see section on the serotonin 5HT.sub.1B receptor).
(271) (iv) Fluorescence-Based Saturation Binding Analysis of Mini-G Protein Binding
(272) To determine the affinity of mini-G protein binding to a receptor, the fluorescence-based saturation binding assay (FSBA) was developed. In this assay, the amount of the GFP-mini-G protein specifically bound to an immobilized receptor was determined using a fluorescent plate reader. As proof of principle, DDM-solubilized β.sub.1AR or A.sub.2AR were immobilized onto Ni.sup.2+-coated wells of a 96-well plate via their C-terminal poly-histidine tag, in the presence of either an agonist or inverse agonist. GFP-mini-G.sub.s was then added at increasing concentrations. After washing to remove any non-specifically bound GFP-mini-G.sub.s, the amount of GFP-mini-G.sub.s fluorescence was measured (
(273) The FSBA is a simple assay for determining the affinity of mini-G protein binding to a receptor in vitro. However, it must be appreciated that the apparent affinity determined may be specific only for the conditions in the assay. In particular, the type of detergent used may have a profound effect on the affinity, especially if it slightly destabilizes the active state of the receptor. The agonist may also affect the apparent affinity of the mini-G protein, depending on how effective the agonist is in stabilizing the active state of the receptor. However, the FSBA remains a useful tool for biophysical analyses of mini-G protein binding to a receptor.
(274) (v) Size Exclusion Chromatography (SEC)
(275) The ultimate biochemical assay for observing coupling of mini-G proteins to a receptor is combining the purified components in vitro and then observing the co-elution of the relevant proteins on SEC [23 and Example 1]. Purified A.sub.2AR and purified mini-G.sub.s were mixed at a molar ratio of 1:1.2 in the presence of the agonist NECA, the complex allowed to form and then separation was performed by SEC. The A.sub.2AR-mini-G.sub.s complex resolved as a predominant peak with an apparent molecular weight of 153 kDa compared with 133 kDa for the receptor alone and 22 kDa for mini-G.sub.s alone. SDS-PAGE analysis confirmed the presence of both A.sub.2AR and mini-G.sub.s in fractions from the 153 kDa complex (
(276) The advantage of using purified components and SEC for analyzing complex formation is that complex formation is observed unambiguously. The conditions for complex formation can be refined and the stability of the complex can be assessed readily after a period of days by repeating the SEC. These data are essential for successful determination of the structure of a GPCR-mini-G protein complex. The disadvantage of this assay is that sufficient quantities of purified receptor are required and this may be limiting in the initial stages of a project.
(277) Characterisation of Mini-G Proteins
(278) Mini-G.sub.olf Couples and Stabilizes A.sub.2AR
(279) The GTPase domains of G.sub.olf and G.sub.s share 87% sequence identity (80% for the full length α subunits) and both G proteins couple to A.sub.2AR [37]. Of the 17 amino acid residues in mini-G.sub.s that make direct contact to residues in A.sub.2AR in the crystal structure of the A.sub.2AR-mini-G.sub.s complex [19 and Example 4], all of these residues are identical except that two Arg residues in G.sub.s are replaced with two Lys residues in G.sub.olf. Despite the high degree of sequence homology between these two isoforms, Gα.sub.olf is far more difficult to overexpress than Gα.sub.s, in fact, the only method reported to produce functional Gα.sub.olf is co-expression with the molecular chaperone RIC8B in insect cells [38]. Therefore, we constructed mini-G.sub.olf to investigate whether the mini-G protein version would be better expressed that native α subunit. Mini-G.sub.olf was constructed by transferring the 7 point mutations and 3 deletions from mini-G.sub.s (
(280) The results with mini-G.sub.olf were very encouraging in terms of both the transferability of the mutations, the expression and stability of the mini-G.sub.olf and the stability of the A.sub.2AR-mini-G.sub.olf complex. Thus where there is a high degree of homology between G proteins, then there is good transferability of the mutations, as was previously observed for the transfer of thermostabilising mutations between GPCRs [39]. These data also suggested that even if the native α subunit is poorly expressed the mini-G protein version may be highly expressed and very stable.
(281) Development of Chimeric Mini-G.sub.s/g to Study G.sub.q-Coupled Receptors
(282) The expression of mini-G.sub.q in E. coli was unsuccessful. One possibility to explain this is that efficient folding of G.sub.q in vivo is dependent on the molecular chaperone Ric8 [30] and that mini-G.sub.q had a similar requirement. Indeed, co-expression of Ric8 with mini-G.sub.q in the baculovirus expression system led to the overproduction of mini-G.sub.q. However, upon purification of mini-G.sub.q it was not possible to dissociate Ric8 (results not shown), suggesting that the mini-G.sub.q was perhaps not correctly folded and/or was very unstable. Given the lack of success in transferring the mini-G protein mutations from G.sub.s to G.sub.q, another strategy was developed.
(283) The second strategy used to try and develop mini-G.sub.q was to transfer the specificity determinants of G.sub.q onto mini-G.sub.s. It is well established that the C-terminal region of a Gα subunit forms the main receptor binding site [40] and is one of the main determinants of coupling specificity [41, 42]. Mutating as few as 3-5 amino acids at the C-terminus of the G alpha subunit has been shown to switch the specificity of coupling to some GPCRs [41, 42]. However, the two GPCR-G protein structures published to date [2, 19] revealed an extensive interface between the receptors and Gα, suggesting that other regions of the G protein may also play a role in specificity. Recent in vivo FRET studies suggest that residues within the α5 helix, but distal to the five C-terminal residues, strongly influence specificity [43].
(284) Mini-G.sub.s did not couple to any of the G.sub.q-coupled receptors tested (results not shown). We then evaluated a number of mini-G.sub.s/q chimeras (
(285) Two other chimeras were also constructed to try and improve on mini-G.sub.s/q70. Mini-G.sub.s/q72 contained the additional mutation C379V.sup.H5.11 compared to mini-G.sub.s/q70 and, although the C379.sup.H5.11 side chain does not form direct contacts with either A.sub.2AR or β.sub.2AR, its mutation to Val is predicted to introduce a direct interaction between the Val γ2 carbon and Leu110 from A.sub.2AR. However, the AT.sub.1R-mini-G.sub.s/q72 complex did not have a higher thermostability than AT.sub.1R-mini-G.sub.s/q70 (results not shown). Finally, the chimera mini-G.sub.s/q71 was constructed in which residues from other regions of Gα that form direct contacts with either β.sub.2AR [2] or A.sub.2AR [19] were mutated to match those in Gα.sub.q. This included the seven mutations in mini-G.sub.s/q70 (R380K.sup.H5.12, Q384L.sup.H5.16, R385Q.sup.H5.17, H387N.sup.H5.19, Q390E.sup.H5.22, E392N.sup.H5.24 and L394V.sup.H5.26) and six additional mutations (A39R.sup.HNS1.3, H41 L.sup.S1.2, D343K.sup.H4.23, L346V.sup.H4.26, R347D.sup.H4.27 and Y358I.sup.H4S6.11). D343.sup.H4.23 was the only amino acid residue whose side chain did not interact with either A.sub.2AR or β.sub.2AR, but the mutation to Lys was included because the longer side chain could potentially interact with a receptor and the charge reversal may be important for specificity. Conversely, Thr350.sup.H4S6.3 was not mutated to Pro in mini-G.sub.s/q71 even though its side chain forms direct contacts with β.sub.2AR. Alignment of Gα.sub.s with two independently solved structures of Gα.sub.q [44, 45] showed that this region of the G proteins differ significantly and thus, in Gα.sub.q, this residue is unlikely to interact with the receptor. However, after all these considerations to make an improved version of mini-G.sub.s/q70, mini-G.sub.s/q71 did not improve the thermostability of agonist-bound G.sub.q-coupled receptors compared to mini-G.sub.s/q70 (
(286) Mini-G.sub.i1: Tackling Stability Issues
(287) Transfer of the 7 point mutations and 3 deletions from mini-G.sub.s into Gα.sub.i1 to make mini-G.sub.i1 was not successful, as the resultant protein was very poorly expressed and had low stability (results not shown). Whilst the work on developing chimeras of mini-G.sub.s/q was underway, we decided to first study the reasons why mini-G.sub.i1 appeared to be so unstable. Therefore, to improve expression, stability and to allow binding of the mini-G.sub.i1 to the βγ subunits, the N-terminus (residues 4-18) was re-inserted, Asp249.sup.H3.8 was mutated back to Leu, and the G217D.sup.H2S4.3 mutation introduced based on a sequence comparison between G.sub.i1 (poorly expressed) and G.sub.s/G.sub.o (highly expressed) (
(288) The serotonin 5-HT.sub.1B receptor (5HT.sub.1BR) was used as a model G.sub.i-coupled receptor for developing mini-G.sub.i1 because it could be expressed and purified in DDM using the baculovirus expression system and its structure determined in the inactive state [10]. Initially, GFP-mini-G.sub.i1 was tested using FSEC for binding to purified 5HT.sub.1BR (in DDM) and bound to the agonist donitriptan. However, the GFP-mini-G.sub.i1 (
(289) Although the mini-G.sub.i1β.sub.1γ.sub.2 trimer coupled successfully to LMNG-solubilised 5HT.sub.1BR, this is not as desirable for crystallography as a mini-G protein coupled receptor due to the large size of the heterotrimeric G protein. Therefore, following the successful strategy of changing the coupling of mini-G.sub.s to that of G.sub.q by making a mini-G.sub.s/q chimera, the same strategy was applied to engineer a mini-G.sub.s/i1 chimera (
(290) In order to compare all the mini-G.sub.i1 constructs and the role of β.sub.1γ.sub.2, agonist affinity shift assays were performed on 5HT.sub.1BR. The uncoupled receptor showed a K.sub.i for the agonist sumatriptan in this assay of 276±10 nM, which was shifted by mini-G.sub.i146 and mini-G.sub.s/i143 to 80±13 nM and 36±2 nM, respectively (
(291) Coupling of Mini-G.sub.o1 to 5HT.sub.1BR
(292) The GTPase domain of G.sub.o1 and G.sub.i1 are highly conserved (80% identity), but the mini-G proteins derived from them behaved very differently. Unlike the unstable mini-G.sub.i1, mini-G.sub.o1 expressed well (100 mg/L), had high stability comparable to mini-G.sub.s and it was largely insensitive to the presence of mild detergents. Since 5HT.sub.1BR couples to both G.sub.o and G.sub.i family members [46], we tested mini-G.sub.o1 coupling to 5HT.sub.1BR and compared the results to coupling with mini-G.sub.i1 (see above). On FSEC, GFP-mini-G.sub.o112 (
(293) The properties of mini-G.sub.o1 make this an ideal choice for structural studies of G.sub.o/G.sub.i coupled receptors, rather than using mini-G.sub.s/i, as it is more highly expressed and more tolerant of detergents.
Conclusions
(294) The aim of the work presented here was to generate a range of mini-G proteins that could be used as a basis for the structure determination of GPCRs in their fully active state. The original work in developing mini-G proteins was performed on G.sub.s [23], which turned out to be one of the best expressed and most stable of the mini-G proteins. Transfer of the relevant mutations to other G proteins was successful in deriving mini-G.sub.olf, mini-G.sub.o1 and mini-G.sub.12. Both mini-G.sub.olf and mini-G.sub.o1 coupled to relevant receptors only in the presence of an agonist and formed stable complexes that could be purified by SEC. Currently, we have not been able to demonstrate binding of mini-G.sub.12 to any receptor (results not shown), even though it is highly expressed in E. coli and has high thermal stability, suggesting that the protein is in a folded state. In contrast, initial trials to generate mini-G.sub.t1, mini-G.sub.z mini-G.sub.q and mini-G.sub.16 were unsuccessful due to no expression in E. coli. Mini-G.sub.i1 expressed very poorly, but was improved upon further mutagenesis, but was still not as stable as mini-G.sub.s and required binding of βγ subunits to attain a full agonist affinity shift in the 5HT.sub.1BR.
(295) The second approach to generate mini-G proteins for those that did not work initially was to make chimeras by converting the specificity of mini-G.sub.s to the specificity of the desired G protein. This was developed initially for G.sub.q by mutating in mini-G.sub.s only those residues in the α5 helix whose side chains make contact to either β.sub.2AR or A.sub.2AR in the crystal structures of the relevant complexes [2, 19], to match the equivalent residues in G.sub.q. The final mini-G.sub.s/q chimera was stable, overexpressed in E. coli and coupled to G.sub.q-coupled receptors but not to G.sub.s-coupled receptors. The process was also successful in generating a mini-G.sub.s/i1 amd mini-G.sub.s/o chimera. The α5 helix provides ˜70% of the buried surface area between the GTPase domain and the receptor in the two G protein-GPCR complexes crystallised to date. The work here shows that changing these contacts is sufficient to alter the specificity of coupling. However, this is not to say that the remaining 30% of the interface is not important, merely that a range of amino acid residues can be accommodated in this interface and therefore it plays a less important role in defining both specificity and the affinity of G protein binding.
(296) The mini-G proteins and their properties are shown in Table 7. On the whole, the expression levels are satisfactory in E. coli and the stability of the mini-G proteins in the absence of detergent is also good. However, their stability decreases in detergent, particularly in high concentrations, with the greatest decrease in stability observed at high detergent concentrations (greater than 0.5% w/v) and with detergents that are regarded as harsh for membrane protein purification [47]. Thus care must be exercised in the initial choice of detergent for forming receptor-mini-G protein complexes.
(297) In conclusion, the range of mini-G proteins developed here will lead to further knowledge on the active structures of receptors through the crystallisation of receptor-mini-G protein complexes. This will expand our understanding of the signaling of GPCRs as well as having useful applications for drug discovery.
(298) TABLE-US-00007 TABLE 7 Mutants of mini-G proteins and their characteristics. Yield Stability of pure meas- Stability in protein ured detergent measured per L of by by native DSF (° C) Mini-G Con- E. coli DSF No 0.1% 0.1% protein struct (mg) (° C) detergent LMNG DDM G.sub.s 393 100 65.3 ± 0.0 47.7 ± 0.2 44.9 ± 0.2 39.1 ± 0.0 G.sub.olf 6 80 64.8 ± 0.4 44.3 ± 0.1 41.9 ± 0.0 37.4 ± 0.2 G.sub.s/q 70 50 67.2 ± 0.4 47.2 ± 0.3 44.2 ± 0.2 36.2 ± 0.1 G.sub.s/i1 43 40 69.0 ± 0.1 44.8 ± 0.0 41.1 ± 0.1 35.9 ± 0.1 G.sub.o1 12 100 63.8 ± 0.1 43.6 ± 0.2 40.7 ± 0.1 32.6 ± 0.2 G.sub.l2 8 25 72.6 ± 0.3 50.3 ± 0.1 46.0 ± 0.1 41.2 ± 0.2 Mini-G Yield of pure Stability protein that Con- protein per L of measured bind βγ struct E. coli (mg) by DSF (° C) G.sub.s 399 100 71.6 ± 0.0 G.sub.olf 9 144 66.1 ± 0.1 G.sub.i1 76 30 70.7 ± 0.1 G.sub.s/i1 46 12 47.8 ± 0.3 G.sub.s/i1 48 10 72.1 ± 0.1 G.sub.s/o1 16 15 69.0 ± 0.1
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EXAMPLE 6: EFFECT OF MINI-Gs ON GLP1R
(300) To assess the effect of mini-Gs on receptor stability we measured GLP1R stability following detergent solubilisation. To this end, prior to solubilisation cells expressing human GLP1R receptor were incubated with tritiated peptide agonist and mini-Gs. The mixture was allowed to reach equilibrium at room temperature for 1 hour before solubilisation at 4° C. for 1 hour. Aliquots of the receptor/ligand/mini-Gs were incubated at different temperatures for 30 minutes. Following separation of excess unbound ligand from the receptor bound molecules, the levels of retained radioactivity were measured for each temperature which was plotted against temperature points. The results was compared with the control arm of the experiment with was identical but lacked mini-Gs. Presence of the mini-Gs significantly increased the stability of the agonist bound conformation (