Allosteric modulation in monomers and oligomers of a G protein-coupled receptor
- University of Toronto, Canada
- Hershey Medical Center, United States
Figures
Figure 1 with 2 supplements
Oligomeric and monomeric preparations of the M2 muscarinic receptor.
(A) Gels were loaded with samples of FLAG- and c-Myc-tagged receptor extracted from co-infected Sf9 cells (lane 1, 20–30 fmol of receptor per lane) or from the precipitate obtained upon treatment of …
-
Figure 1—source data 1
Panel B–Monomeric status of the purified M2 receptor after chemical cross-linking.
Panel C–Parametric values for the effect of gallamine on the rate of dissociation of [3H]QNB. Panel D–Parametric values for the effect of gallamine on the binding of [3H]NMS at equilibrium. Panels E–G–Parametric values for the effect of strychnine on the binding of [3H]NMS to oligomers and monomers.
- https://doi.org/10.7554/eLife.11685.004
-
Figure 1—source data 2
Figure 1–figure supplement 1–Parametric values for the effect of strychnine on the binding of [3H]NMS and [3H]QNB to membrane-bound M2 receptor.
- https://doi.org/10.7554/eLife.11685.005
-
Figure 1—source data 3
Figure 1-figure supplement 2–Rate constants for the simulated binding of strychnine and [3H]NMS according to Figure 6.
- https://doi.org/10.7554/eLife.11685.006
Figure 1—figure supplement 1
Effect of strychnine on the binding of [3H]NMS and [3H]QNB to membrane-bound M2receptor.
The binding of [3H]NMS (A) or [3H]QNB (B) to M2 receptor in membranes from CHO cells was measured at the concentrations of strychnine shown on the x-axes. Strychnine and the radioligand were added …
Figure 1—figure supplement 2
Simulated effect of strychnine on the binding of [3H]NMS according to Figure 6.
Total specific binding of the orthosteric ligand (RL + ARL) at graded concentrations of strychnine was simulated according to Figure 6 (Equations 13–17) with the parametric values listed in Figure …
Figure 2 with 3 supplements
FRET-based detection of conformational change at the allosteric site.
(A) A computed molecular model of the M2 receptor (red) fused at the N-terminus to mCherry (yellow) and labeled with FlAsH (green) at a tetracysteine motif inserted in ECL2 between Val166 and Gly167.…
-
Figure 2—source data 1
Panel A–Time-resolved fluorescence and fluorescence anistoropy of eGFP and eGFP-tagged M2 receptors.
Panel E–Levels of significance for ligand-dependent changes in the FRET efficiency of FlAsH-reacted mCh-M2-FCM.
- https://doi.org/10.7554/eLife.11685.010
Figure 2—figure supplement 1
Expression and localization of fluorophore-tagged M2 receptors.
CHO cells were transfected with plasmids coding for mCh-M2-FCM (A), mCh-M2(D103A)-FCM (B), M2-FCM (C), eGFP-M2 (D), mCh-M2 (E), and eGFP-M2-mCh (F). Images were recorded on a confocal microscope, as …
Figure 2—figure supplement 2
Modulatory effect of gallamine on the binding of [3H]NMS to mCh-M2-FCM.
Membranes were prepared from CHO cells expressing M2 receptor bearing mCherry at the N-terminus and the FlAsH-reactive sequence FCM in the second extracellular loop. The binding of [3H]NMS was …
Figure 2—figure supplement 3
FRET between eGFP fused at the N-terminus and mCherry inserted after Val166.
CHO cells expressing the eGFP- and mCherry-tagged M2 receptor were excited at 473 nm at a power of 0.37 µW. The emission spectrum was recorded from the region of the plasma membrane and analyzed to …
Figure 3 with 1 supplement
The nature of high-affinity allosteric interactions.
The sensor (mCh-M2-FCM) (A), a mutant thereof that does not bind NMS [mCh-M2(D103A)-FCM] (B), and the mutant plus the wild-type M2 receptor (C) were expressed or co-expressed in CHO cells and …
-
Figure 3—source data 1
Panel D–Levels of significance for ligand-dependent changes in the FRET efficiency of FlAsH-reacted mCh-M2-FCM and mCh-M2(D103A)-FCM
- https://doi.org/10.7554/eLife.11685.015
Figure 3—figure supplement 1
Coexpression of the M2 receptor and a non-binding sensor.
(A) CHO cells were cotransfected with the plasmids coding for eGFP-M2 and mCh-M2(D103A)-FCM. The cells were treated with FlAsh, and those expressing both receptors were identified by the presence of …
Figure 4 with 1 supplement
Intramolecular interactions between orthosteric and allosteric ligands.
Models of the liganded receptor were simulated by molecular dynamics as described in Materials and Methods. The region of the ligand-binding sites is shown in the figure, with NMS and strychnine (A, …
Figure 4—figure supplement 1
Chemical structures of allosteric and orthosteric ligands to the M2 muscarinic receptor.
The ligands shown are: NMS, N-methylscopolamine; QNB, quinuclidinylbenzilate; Str, strychnine; and Gal, gallamine. Different liganded states of the receptor are identified as X_R_Y, where X and Y …
Figure 5 with 3 supplements
Effects of orthosteric and allsteric ligands on the aromatic cap and Trp422.
(A) The three residues of the aromatic cap (i.e., Tyr104, Tyr403, and Tyr426) and Trp422 are shown in an overlay of the results of three simulations: a vacant receptor (yellow), a QNB-bound receptor …
-
Figure 5—source data 1
Figure 5–figure supplement 2–Distance between the α-carbon atoms of Tyr177 and Asn419 in crystal structures of the M2 receptor
- https://doi.org/10.7554/eLife.11685.021
-
Figure 5—source data 2
Figure 5–figure supplement 3–Mean distances (Å) between the α-carbon atoms of Tyr177 and Asn419 in the M2 receptor with different combinations of allosteric and orthosteric ligands
- https://doi.org/10.7554/eLife.11685.022
Figure 5—figure supplement 1
Orientation of ligands at the orthosteric site.
Strychnine adopts a similar pose at the vacant receptor and in the presence of either NMS (A) or QNB at the orthosteric site. The position of gallamine at a vacant receptor (B) is consistent …
Figure 5—figure supplement 2
Width of the vestibule to the orthosteric site.
Changes in the distance between the α-carbon atoms of Tyr177 and Asn419 were taken as a measure of the effect of an orthosteric ligand on the conformation of the allosteric site. The structures of …
Figure 5—figure supplement 3
Distribution of distances between the α-carbon atoms of Tyr177 and Asn419.
The distances from all frames of the molecular dynamics simulations over the production period of 30 ns are shown in the figure for a receptor with NMS (A) or QNB (B) at the orthosteric site. In …
Figure 6
A receptor (R) binds an orthosteric ligand (L) and an allosteric ligand (A) to form a ternary complex (ARL).
Each ligand can bind separately to form AR or RL, but the ternary complex is accessible only via RL. The orthosteric site of the M2 receptor is located within the cluster of helical domains, with …
Tables
Table 1
Correlates of electrostatic repulsion between orthosteric and allosteric ligands. The inter-cationic distance was calculated as that between the cationic nitrogen atom of the orthosteric ligand and …
| Allosteric–orthosteric pair | ||||
|---|---|---|---|---|
| Str_R_NMSa | Str_R_QNB | Gal_R_NMS | Gal_R_QNBa | |
| Inter-cationic distance (Å) | 13.7 | 15.7 | 16.5 | 16.8 |
| Difference in electrostatic potential (kcal/mol) | 0.0085 | 0.063 | 0.096 | 0.41 |
-
a The distances are shown in Figures 4A and B.
Additional files
-
Source code 1
Analysis of binding.
- https://doi.org/10.7554/eLife.11685.027
-
Source code 2
Calculation of FRET by spectral unmixing.
- https://doi.org/10.7554/eLife.11685.028
-
Source code 3
Simulation of the capped allosteric ternary complex model.
- https://doi.org/10.7554/eLife.11685.029
