Ligand modulation of sidechain dynamics in a wild-type human GPCR
- The University of Texas Southwestern Medical Center, United States
- CUNY Advanced Science Research Center, United States
- University of Copenhagen, Denmark
- Weill Cornell Medical College, United States
- City College of New York, United States
- City University of New York, United States
Figures
Figure 1 with 2 supplements
A2AR expression and Gs activation.
(A) Wild-type A2AR was expressed in P. pastoris as a fusion with an optimized version of the α-mating factor (MFα) signal sequence that contains mutations and a deletion that increase receptor …
Figure 1—figure supplement 1
A2AR processing and purification.
(A) Anti-His6 Western blots of wild-type GPCRs expressed in Pichia as a fusion with the wild-type (WT) and optimized (MFαOpt) α-mating factor secretion signal. Receptor that has not been fully …
Figure 1—figure supplement 2
Ligand binding assays on A2AR yeast membranes.
(A) Competition binding curve of WT A2AR membranes incubated with [3H]-ZM241385 and increasing amounts of unlabeled NECA in low ionic strength assay buffer (50 mM Tris pH 7.4, 10 mM MgCl2, 1 mM …
Figure 2 with 3 supplements
NMR spectra of A2AR in different liganded states with NaCl.
(A) 1H/13C HMQC spectrum of Ile δ1-labeled WT A2AR in DDM micelles and 150 mM NaCl with inverse agonist ZM241385 (black). Resonances assigned in this work (I923.40, I2386.40, I2747.39, I292, and the …
Figure 2—figure supplement 1
A2AR and Gs complex formation.
(A) Gel filtration trace of purified NECA-bound A2AR alone (black) and A2AR complexed with Gs heterotrimer (red) injected on a Superdex200 10/300 column. Total protein inputs were 360 μg and 833 μg, …
Figure 2—figure supplement 2
Assignments of select Ile-δ1 methyl resonances in A2AR.
top - 1H/13C HMQC spectra of Ile δ1-labeled mutants (blue) of A2AR in DDM micelles with inverse agonist ZM241385 collected to make the assignments shown in Figure 2A, compared to the WT spectrum (bla…
Figure 2—figure supplement 3
Lifetime of A2AR NMR sample.
(A) 1H/13C HMQC spectrum of Ile δ1-labeled WT A2AR in DDM micelles with agonist NECA collected immediately after final SEC column. (B) 1H/13C HMQC spectrum of the same Ile δ1-labeled WT A2AR in DDM …
Figure 3 with 1 supplement
Ligand- and cation-dependent chemical shift changes in A2AR.
(A) 1H/13C HMQC spectrum of Ile δ1-labeled WT A2AR in DDM micelles and 150 mM NaCl with inverse agonist ZM241385 (black). Residue numbers of assigned peaks and peak IDs of unassigned peaks are …
Figure 3—figure supplement 1
Overlays of ligand- and cation-dependent chemical shift changes in A2AR.
Overlays of pairs of spectra in Figure 3. (A) 1H/13C HMQC spectra of Ile δ1-labeled WT A2AR in DDM micelles and 150 mM NaCl with inverse agonist ZM241385 (black) and agonist NECA (red). (B) 1H/13C …
Figure 4
Solvent PRE analysis of A2AR.
(A) Solvent PRE 1H/13C HMQC spectra of Ile δ1-labeled WT A2AR in DDM micelles with inverse agonist ZM241385 in the presence (red) and absence (black) of paramagnetic Gd3+-DTPA. Disappearance of …
Figure 5 with 1 supplement
Modified 3Q relaxation for MBP.
(A) Crystal structure of MBP complexed with β-cyclodextrin (PDB 1DMB[Sharff et al., 1993]). The β-cyclodextrin ligand is shown as orange sticks, while protein isoleucine residues are shown as blue …
Figure 5—figure supplement 1
Adaptation of 3Q methyl relaxation experiment to A2AR samples.
(A) Traditional 3Q methyl relaxation experiments (Sun et al., 2011) involve collecting matched pairs of allowed (SQ) and forbidden (MQ) spectra at several relaxation delay times, calculating the …
Figure 6 with 1 supplement
Changes in dynamics of methyl groups of A2AR in the presence of different ligands.
(A) Plot of the 1H-1H dipolar cross-correlation rateη, which is proportional to the S2axis order parameter, for 20 peaks in the Ile δ1 region for A2AR in DDM micelles with agonist NECA (red) and …
Figure 6—figure supplement 1
Changes in dynamics of methyl groups of A2AR in the presence of different ligands from MD trajectories.
Plot of the S2axis order parameter for all 29 Ile δ1 methyl groups extracted from two ~ 100 ns MD simulations of A2AR in DDM micelles with agonist NECA (red) and inverse agonist ZM241385 (grey). A …
Figure 7
Structural contexts of isoleucine peak assignments.
A2AR ribbon diagrams are shown for structures solved in complex with inverse agonist ZM241385 (gray; PDB 4EIY[Liu et al., 2012b]), agonist NECA (red; PDB 2YDV[Lebon et al., 2011]), and agonist …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.28505.018
