Homo-oligomerization of the human adenosine A2A receptor is driven by the intrinsically disordered C-terminus
- Department of Chemistry and Biochemistry, University of California, Santa Barbara, United States
- Department of Chemical Engineering, University of California, Santa Barbara, United States
- C. Eugene Bennett Department of Chemistry, West Virginia University, United States
Figures
Figure 1 with 2 supplements
Method for collecting size-exclusion chromatography (SEC) data and assessing A2AR oligomerization.
The SEC data is recorded every second as absorbance at 280 nm. The baseline is corrected to ensure uniform fitting and integration across the peaks. The areas under the curve, resulting from a …
Figure 1—figure supplement 1
The purity and identity of A2AR are confirmed with total protein stain and western blot.
(A) Representative total protein stain (upper panel) and western blot (lower panel) of A2AR-WT during purification. Positive ([+] ctrl) and negative ([–] ctrl) controls consist of 5 OD cell lysate …
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Figure 1—figure supplement 1—source data 1
Raw representative total protein stain of A2AR-WT during purification.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig1-figsupp1-data1-v2.tif.zip
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Figure 1—figure supplement 1—source data 2
Labeled representative total protein stain of A2AR-WT during purification.
Positive ([+] ctrl) and negative ([–] ctrl) controls consist of 5 OD cell lysate of Saccharomyces cerevisiae BJ5464 cells expressing and not expressing A2AR WT, respectively. ‘IMAC FT’ indicates the flow-through from IMAC step. ‘XAC inactive’ and ‘XAC active’ indicate the fractions that do not and do bind to XAC during the ligand-affinity chromatography step. MagicMark protein ladder (LC5602) is used as the molecular weight standard.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig1-figsupp1-data2-v2.tif.zip
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Figure 1—figure supplement 1—source data 3
Raw representative western blot of A2AR-WT during purification.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig1-figsupp1-data3-v2.tif.zip
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Figure 1—figure supplement 1—source data 4
Labeled representative western blot of A2AR-WT during purification.
Positive ([+] ctrl) and negative ([–] ctrl) controls consist of 5 OD cell lysate of Saccharomyces cerevisiae BJ5464 cells expressing and not expressing A2AR WT, respectively. ‘IMAC FT’ indicates the flow-through from IMAC step. ‘XAC inactive’ and ‘XAC active’ indicate the fractions that do not and do bind to XAC during the ligand-affinity chromatography step. MagicMark protein ladder (LC5602) is used as the molecular weight standard.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig1-figsupp1-data4-v2.tif.zip
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Figure 1—figure supplement 1—source data 5
Raw representative western blot of A2AR-WT during size-exclusion chromatography separation.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig1-figsupp1-data5-v2.tif.zip
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Figure 1—figure supplement 1—source data 6
Labeled representative western blot of A2AR-WT during size-exclusion chromatography separation.
Each lane on the blot is from 0.5 mL fractions eluted from a Superdex 200 10/300 GL (GE Healthcare) column. MagicMark protein ladder (LC5602) is used as the molecular weight standard.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig1-figsupp1-data6-v2.tif.zip
Figure 1—figure supplement 2
Size-exclusion chromatographic traces and data distribution of all A2AR variants used in the main text of this study.
(A) Curve fitting using OriginLab of all A2AR variants used in the main text of this study, listed by the order they appear. By default, each oligomeric peak is fitted with one curve using Gaussian …
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Figure 1—figure supplement 2—source data 1
Raw size-exclusion chromatography data of five experimental replicates of A2AR-WT.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig1-figsupp2-data1-v2.xlsx
Figure 2
Residue C394 helps stabilize A2AR oligomerization via disulfide bonds.
(A) The effect of C394X substitutions on A2AR oligomerization. The levels of dimer (dark colors) and high-molecular-weight oligomer (light colors) are expressed relative to the monomeric population …
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Figure 2—source data 1
Raw western blot of size-exclusion chromatography-separated dimeric populations of A2AR-WT with and without 5 mM TCEP.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig2-data1-v2.tif.zip
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Figure 2—source data 2
Raw western blot of size-exclusion chromatography-separated dimeric populations of A2AR-WT with and without 5 mM TCEP.
MagicMark protein ladder (LC5602) is used as the molecular weight standard.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig2-data2-v2.tif.zip
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Figure 2—source data 3
Raw western blot of size-exclusion chromatography-separated dimeric populations of A2AR-Q372ΔC with and without 5 mM TCEP.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig2-data3-v2.tif.zip
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Figure 2—source data 4
Raw western blot of size-exclusion chromatography-separated dimeric populations of A2AR-Q372ΔC with and without 5 mM TCEP.
MagicMark protein ladder (LC5602) is used as the molecular weight standard.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig2-data4-v2.tif.zip
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Figure 2—source data 5
Raw size-exclusion chromatography data of A2AR-WT and C394X variants.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig2-data5-v2.xlsx
Figure 3
Truncating the C-terminus systematically affects A2AR oligomerization.
(A) Depiction of where the truncation points are located on the C-terminus, with region 354–359 highlighted (in black) showing critical residues. (B) The levels of dimer and high-molecular-weight (HM…
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Figure 3—source data 1
Raw size-exclusion chromatography data of A2AR-WT and C-terminally truncated ΔC variants.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig3-data1-v2.xlsx
Figure 4
Non-bonded interactions of the extended C-terminus of A2AR play a critical role in stabilization of the dimeric interface.
(A) Dimer configurations from cluster analysis in GROMACS of the 394-residue variant identify two major clusters involving either (1) the C-terminus of one protomer and the C-terminus, ICL2, and …
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Figure 4—source data 1
Detailed data regarding the multiple interfaces of A2AR and the network of non-bonded interactions that stabilize these interfaces.
(A) Dimer configurations from cluster analysis in GROMACS of the 394-residue variant. (B) Average number of residues that form electrostatic contacts as a function of sequence length of A2AR. (C) Average number of residues that form hydrogen bonds as a function of sequence length of A2AR.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig4-data1-v2.xlsx
Figure 5 with 1 supplement
The effects of ionic strength on the oligomerization of various A2AR variants reveal the involvement of depletion interactions.
The levels of (A) dimer and (B) high-molecular-weight oligomer + dimer are expressed relative to the monomeric population as an arbitrary unit and plotted against ionic strength, with reported …
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Figure 5—source data 1
Raw size-exclusion chromatography data of various A2AR variants under different ionic strengths of 0.15, 0.45, and 0.95 M.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
The dimer/oligomerization of A2AR is a thermodynamic process where the dimer and high-molecular-weight oligomer once formed are kinetically trapped.
(A) Size-exclusion chromatography (SEC) chromatograms of the consecutive rounds of SEC performed on A2AR-WT and Q372ΔC. The first rounds of SEC are to separate the dimer/oligomer population and the …
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Figure 5—figure supplement 1—source data 1
Raw size-exclusion chromatography (SEC) data of the consecutive rounds of SEC performed on A2AR-WT and Q372ΔC.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig5-figsupp1-data1-v2.xlsx
Figure 6 with 1 supplement
The A2AR C-terminus is prone to aggregation.
(A) Absorbance at 450 nm of the A2AR C-terminus in solution, with NaCl and GdnHCl concentrations varied to achieve ionic strengths 0–4 M. Inset: the solution at ionic strength 4 M achieved with …
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Figure 6—source data 1
Detailed data showing the propensity of A2AR C-terminus to aggregate.
(A) Absorbance at 450 nm of the A2AR C-terminus in solution, with NaCl and GdnHCl concentrations varied to achieve ionic strengths 0–4 M. (B) SYPRO orange fluorescence of solutions containing the A2AR C-terminus as the temperature was varied from 20°C to 70°C (gray). The change in fluorescence, measured in relative fluorescence unit (RFU), was calculated by taking the first derivative of the fluorescence values.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
The C-terminus of A2AR can form non-polar contacts.
(A) Hydropathy plot against A2AR residue number showing the hydrophobicity of A2AR C-terminus, scored with ProtScale using method described by Kyte and Doolittle, window size of 3. Positive scores …
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Figure 6—figure supplement 1—source data 1
Detailed data showing the ability of A2AR C-terminus to form non-polar contacts.
(A) Hydropathy plot against A2AR residue number showing the hydrophobicity of A2AR. C-terminus, scored with ProtScale using method described by Kyte and Doolittle, window size of 3. Positive scores represent hydrophobicity and negative scores hydrophilicity. (C) Average number of residues that form non-polar contacts as a function of sequence length of A2AR.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig6-figsupp1-data1-v2.xlsx
Figure 7 with 1 supplement
Visualizing A2AR dimeric interface and observing conformational changes of the TM7 using MD simulations.
(A) Representative snapshot of A2AR-C394ΔC dimers shows salt bridge formation between a sample trajectory. The insets are closeups of the salt bridges, which can be both intra- and intermolecular. …
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Figure 7—source data 1
MD simulations data used to visualize A2AR dimeric interface and observe the conformational changes of the TM7.
(A) List of all C-terminal residue pairs of A2AR-C394ΔC dimers engaging in electrostatic interactions. (B) Helical tilt angles for TM7 helix in A2AR as a function of protein length.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Helical tilt angles for TM1–6 helices in A2AR as a function of protein length.
Systematic truncations of the C-terminus lead to rearrangement of the heptahelical bundle, propagated to the entire receptor and is especially pronounced in helices proximal to the C-terminus, that i…
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Figure 7—figure supplement 1—source data 1
Helical tilt angles for TM1–6 helices in A2AR as a function of protein length.
- https://cdn.elifesciences.org/articles/66662/elife-66662-fig7-figsupp1-data1-v2.xlsx
Author response image 1
Decorrelation time calculated from representative trajectories of each A2AR truncated system.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Recombinant DNA reagent | pITy (plasmid) | Parekh et al., 1996 | ||
| Strain, strain background (Saccharomyces cerevisiae) | BJ5464 | Robinson Lab – Carnegie Mellon University | ||
| Strain, strain background (Escherichia coli) | BL21 (DE3) | Sigma, St. Louis, MO, USA | #CMC0014 | |
| Chemical compound, drug | DDM | Anatrace, Maumee, OH, USA | #D310 | |
| Chemical compound, drug | CHAPS | Anatrace, Maumee, OH, USA | #C216 | |
| Chemical compound, drug | CHS | Anatrace, Maumee, OH, USA | #CH210 | |
| Chemical compound, drug | Xanthine amine congener | Sigma, St. Louis, MO, USA | #X103 | |
| Chemical compound, drug | Theophylline | Sigma, St. Louis, MO, USA | #T1633 | |
| Commercial assay, kit | Affigel 10 resin | BioRad, Hercules, CA, USA | #1536099 | |
| Commercial assay, kit | Tricorn Superdex 200 10/300 GL column | GE Healthcare, Pittsburgh, PA, USA | #17-5175-01 | |
| Antibody | Anti-A2AR, clone 7F6-G5-A2 (Mouse monoclonal) | Millipore, Burlington, MA, USA | #05-717 | (1:500) dilution |
| Antibody | Anti-Mouse IgG H&L DyLight 550 (Goat monoclonal) | Abcam, Cambridge, MA, USA | #ab96880 | (1:600) dilution |
| Software, algorithm | MODELLER 9.23 | Eswar et al., 2006 | ||
| Software, algorithm | martinize.py script | de Jong et al., 2013 | ||
| Software, algorithm | ELNeDyn elastic network | Periole et al., 2009 | ||
| Software, algorithm | MARTINI coarse-grained force field v2.2 | Monticelli et al., 2008 | ||
| Software, algorithm | GROMACS 2016 | Abraham et al., 2015 | ||
| Software, algorithm | backward.py script | Wassenaar et al., 2014 | ||
| Software, algorithm | LINCS | Hess et al., 1997 | ||
| Software, algorithm | CHARMM36 and TIP3P force fields | Best et al., 2012; Jorgensen et al., 1983 | ||
| Software, algorithm | LOOS | Romo and Grossfield, 2009 | ||
| Software, algorithm | VMD | Humphrey et al., 1996 |
Additional files
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Supplementary file 1
Results from curve fitting using OriginLab and calculations of the high-molecular-weight (HMW) oligomer and dimer levels for all A2AR variants used in the main text of this study.
The variants are grouped by the order they appear and numbered corresponding to Figure 1—figure supplement 2. The levels of dimer and HMW oligomer are expressed relative to the monomeric population in arbitrary units as monomer-equivalent concentration ratios. The errors are calculated from the variance of the fit, not experimental variations, and are within 95% confidence interval. Only the WT replicates are represented with standard deviation as experimental variations (last row; n = 5; mean ± SD).
- https://cdn.elifesciences.org/articles/66662/elife-66662-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/66662/elife-66662-transrepform-v2.docx
