Mini-G proteins: Novel tools for studying GPCRs in their active conformation

Abstract

Mini-G proteins are the engineered GTPase domains of Gα subunits. They couple to GPCRs and recapitulate the increase in agonist affinity observed upon coupling of a native heterotrimeric G protein. Given the small size and stability of mini-G proteins, and their ease of expression and purification, they are ideal for biophysical studies of GPCRs in their fully active state. The first mini-G protein developed was mini-Gs. Here we extend the family of mini-G proteins to include mini-Golf, mini-Gi1, mini-Go1 and the chimeras mini-Gs/q and mini-Gs/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 GPCRs in a fully active state.

Fig 1. Phylogenetic relationship of human Gα subunits.

All the Gα subunits that have been highlighted in the family-specific colours were attempted to be converted into mini-G proteins. The phylogenetic relationships were determined using TreeDyn.

Fig 2. Alignment of Gα GTPase domain protein sequences.

The amino acid sequences aligned are of the wild type GTPase domains of the Gα subunits used in this study to create the initial mini-G proteins. The GαAH domain (not shown) was deleted and replaced by a linker (GGGGGGGG or GGSGGSGG in red). To construct mini-G proteins, the residues highlighted in grey were deleted and residues highlighted in magenta were mutated to the following (Gα_s residue number and the CGN in superscript): D49^S1H1.3, N50^S1H1.4, D249^S4.7, D252^S4H3.3, D272^H3.8, A372^H5.4, I375^H5.7. The glycine mutation (G217D, highlighted in cyan) was incorporated into G_i1 only, to improve expression (see Results & Discussion). Numbering above the sequences is for Gα_s and the CGN system below the sequence is used for reference [6].

Fig 3. The β₁AR−mini-G_s and A_2AR−mini-G_s complexes.

(a) FSEC traces of GFP-mini-G_s with β₁AR (retention volumes are given in parentheses): green, GFP-mini-G_s (15.1 ml); orange, GFP-mini-G_s with β₁AR bound to the inverse agonist ICI118551 (15.1 ml); black, GFP-mini-G_s with β₁AR bound to the agonist isoprenaline (8 ml, 12.1 ml and 15.1 ml). Representative chromatograms from at least two independent experiments are shown. (b) Measurement of GFP-mini-G_s affinity to DDM-solubilized β₁AR using a fluorescent saturation binding assay (FSBA); blue circles, β₁AR bound to the agonist isoprenaline (total binding); orange squares, β₁AR bound to the inverse agonist ICI118551 (non-specific binding); green triangles, specific binding, with an apparent K_D of 200 ± 1 nM (mean ± SEM, n = 2). Curves shown are from a representative experiment. (c) FSEC traces of GFP-mini-G_s with DDM-solubilised A_2AR (retention volumes are given in parentheses): green, GFP-mini-G_s (15.1 ml); orange, GFP-mini-G_s with A_2AR bound to the inverse agonist ZM241385 (15.1 ml); black, GFP-mini-G_s with A_2AR bound to the agonist NECA (12.5 ml and 15.1 ml). Representative chromatograms from at least two independent experiments are shown. (d) Measurement of mini-G_s affinity to DDM-solubilized A_2AR using FSBA: blue circles, A_2AR bound to the agonist NECA (total binding); orange squares, A_2AR bound to the inverse agonist ZM241385 (non-specific binding); green triangles, specific binding, with an apparent K_D of 430 ± 24 nM (mean ± SEM, n = 2). (e) Analytical size exclusion chromatography (SEC) of mini-G_s bound to purified A_2AR (retention volumes are given in parentheses): black, A_2AR–mini-G_s complex, 153 kDa (13 ml); blue, A_2AR, 133 kDa (13.3 ml); green, mini-G_s, 22 kDa (17.2 ml). Three panels to the right of the SEC traces are coomassie blue-stained SDS-PAGE gels of fractions from 3 separate SEC experiments: top panel, mini-G_s; middle panel, A_2AR; bottom panel, mini-G_s mixed with NECA-bound A_2AR (1.2:1 molar ratio).

Fig 4. The A_2AR−mini-G_olf complex.

(a) Analytical SEC of mini-G_olf bound to purified A_2AR (retention volumes are given in parentheses): black, A_2AR–mini-G_olf complex, 153 kDa (13 ml); blue, A_2AR, 133 kDa (13.3 ml); green, mini-G_olf, 23 kDa (17.1 ml). Three panels to the right of the SEC traces are coomassie blue-stained SDS-PAGE gels of fractions from 3 separate SEC experiments: top panel, mini-G_olf; middle panel, A_2AR; bottom panel, mini-G_olf mixed with NECA-bound A_2AR (1.2:1 molar ratio). (b) Thermostability assay (TSA) of unpurified DM-solubilized, ³H-NECA-bound A_2AR. Data were analysed by nonlinear regression and apparent T_m values were determined from analysis of the sigmoidal dose-response curves fitted. T_m values represent mean±SEM of two independent experiments, each performed in duplicate: blue circles, no mini-G_olf (27 ± 0.3°C); green squares, mini-G_olf (33 ± 1°C). Curves shown are from a representative experiment.

Fig 5. Thermostability assays of various complexes between mini-G_s/q chimeras and GPCRs.

(a) Thermostability of unpurified digitonin-solubilized, ¹²⁵I-AngII-bound AT₁R (T_m values in parentheses): blue circles, no mini-G_s/q (23 ± 0.4°C); green squares, mini-G_s/q57 (T_m not determined); magenta inverted triangles, mini-G_s/q70 (31 ± 1°C); red triangles, mini-G_s/q71 (30 ± 0.8°C). (b) Thermostability of unpurified DDM-solubilized, ³H-NTS-bound NTSR1: blue, no mini-G_s/q (25 ± 0.4°C); green squares, mini-G_s/q57 (27 ± 0.7°C); black circles, mini-G_s/q58 (25 ± 0.4°C); magenta inverted triangles, mini-G_s/q70 (32 ± 0.3°C); red diamonds, mini-G_s/q71 (29 ± 1.1°C). (c) Thermostability of unpurified DM-solubilized, ³H-NECA-bound A_2AR: blue circles, no mini-G_s/q (27 ± 0.3°C); green squares, mini-G_s/q57 (31 ± 0.3°C); black circles, mini-G_s/q58 (27 ± 0.5°C); magenta inverted triangles, mini-G_s/q70 (27 ± 0.2°C). In all panels, data (n = 3) were analysed by nonlinear regression and apparent T_m values were determined from analysis of the sigmoidal dose-response curves fitted with values shown as mean ± SEM. Curves shown are from a representative experiment.

Fig 6. The 5HT_1BR−mini-G_i1 complexes.

(a) Mini-G_i1 coupling increases agonist affinity to 5HT_1BR. Competition binding curves were performed in duplicate (n = 2) by measuring the displacement of the antagonist ³H-GR125743 with increasing concentration of the agonist sumatriptan (K_i values representing mean ± SEM in parentheses): blue circles, 5HT_1BR (K_i 280 ± 10 nM); green hexagons, 5HT_1BR and mini-G_i1 (K_i 80 ± 13 nM); brown squares, 5HT_1BR and mini-G_s/i1 (K_i 36 ± 2 nM); pale blue triangles, 5HT_1BR and mini-G_i1β₁γ₂ (K_i 15 ± 1 nM); red diamonds, 5HT_1BR and mini-G_s/i1β₁γ₂ (K_i 7.2 ± 0.8 nM). Error bars represent the SEM. (b) Measurement of mini-G_s/i1 chimera affinity to the DDM-solubilized, donitriptan-bound 5HT_1BR using FSBA: brown circles, 5HT_1BR and GFP-mini-G_s/i1 (total binding); purple squares, 5HT_1BR and GFP-mini-G_s (non-specific binding); green triangles, specific binding. The apparent K_D of 390 ± 47 nM represents the mean ± SEM of two independent experiments. Curves shown are from a representative experiment. (c) FSEC traces of GFP-mini-G_i1 with 5HT_1BR in DDM: black, GFP-mini-G_i1 and donitriptan-bound 5HT_1BR purified in DDM (13.5 ml); green GFP-mini-G_i1 (13.5 ml). (d) FSEC traces of GFP-mini-G_i1 with 5HT_1BR in LMNG: black, GFP-mini-G_i1 and donitriptan-bound 5HT_1BR purified in LMNG (12.2 ml and 14.3 ml); green, GFP-mini-G_i1 (14.3 ml). (e) FSEC traces of GFP-mini-G_s/i1 with 5HT_1BR: black, GFP-mini-G_s/i1 and donitriptan-bound 5HT_1BR purified in DDM (13.2 ml); brown, GFP-mini-G_s/i1 (15.1 ml). (f) FSEC traces of GFP-mini-G_i1β₁γ₂ with 5HT_1BR: black, GFP-mini-G_i1β₁γ₂ and donitriptan-bound 5HT_1BR purified in LMNG (11.8 ml); pale blue, GFP-mini-G_i1β₁γ₂ (14.3 ml). In panels c-f, retention volumes are given in parentheses.

Fig 7. The 5HT_1BR−mini-G_o1 complex.

(a) Competition binding curves were performed on membranes in duplicate (n = 2) by measuring the displacement of the antagonist ³H-GR125743 with increasing concentration of the agonist sumatriptan (apparent K_i values representing mean ± SEM are in parentheses): blue circles, 5HT_1BR (K_i 280 ± 10 nM); green squares, 5HT_1BR and mini-G_o1 (K_i 32 ± 3 nM). Error bars represent SEM. (b) Measurement of GFP-mini-G_o1 affinity to DDM-solubilized, donitriptan-bound 5HT_1BR using the FSBA: blue circles, 5HT_1BR and GFP-mini-G_o1 (total binding); purple squares, 5HT_1BR and GFP-mini-G_s (non-specific binding); green triangles, specific binding. The apparent K_D value (180 ± 24 nM) represents mean ± SEM of two independent experiments. Curves shown are from a representative experiment. (c) FSEC traces of GFP-mini-G_o1 with DDM-solubilized unpurified 5HT_1BR bound to the following (retention volumes are shown in parentheses): orange, the antagonist SB224289 (14.9 ml); black, the agonist donitriptan (11.3 ml and 14.9 ml). Free GFP-mini-G_o1 (green) resolved as a predominant peak with a retention volume of 14.9 ml (d) Mini-G_o1 forms a complex with purified 5HT_1BR. The three panels are coomassie blue-stained SDS-PAGE gels of fractions from 3 separate SEC experiments: top panel, mini-G_o1; middle panel, 5HT_1BR; bottom panel, mini-G_o1 mixed with donitriptan-bound 5HT_1BR (1:1 molar ratio). (e) FSEC traces of GFP-mini-G_o1 with purified 5HT_1BR: black, GFP-mini-G_o1 with 5HT_1BR purified in DDM (13 ml); green, GFP-mini-G_o1 (14.8 ml). (f) FSEC traces of GFP-mini-G_s with purified 5HT_1BR: black, GFP-mini-G_s with 5HT_1BR purified in DDM (negative control; 15.1 ml); purple, GFP-mini-G_s (15.1 ml). Retention volumes are shown in parentheses.