Article Figures & Data
Figures
- Fig. 1.
The model used for calculating the ligand bias in GPCRs. The allosteric communication pipelines starting from the residues in the extracellular loop region and passing through the residues in the agonist binding site (agonist shown as blue spheres) and terminating in the residues that couple to the G-protein (GPI shown as pink surfaces) are shown as pink sticks. The allosteric communication pipelines that connect to the β-arrestin interface (BAI shown as green surfaces) are shown in green sticks. The ratio of the strength of these two pipelines for a test ligand with respect to a reference ligand is defined as the ligand bias.
- Fig. 2.
Comparison of computational ligand bias (y axis) to the experimental ligand bias factors (x axis) for (A) the β-arrestin–biased agonists in β2AR. epi, epinephrine; iso, isoproterenol; sal, salbutamol; form, formoterol; BI, BI-167107; carm, carmoterol. (B) the G-protein–biased agonists in κOR. u69, u69593. (A) The bias values obtained using epinephrine as the reference agonist are shown as green triangles, and those obtained using isoproterenol as the reference agonist are shown as green circles. The respective reference agonists from both studies are marked at zero. The error bars for the bias factors of all β2AR agonists represent S.E. with 95% confidence limits except for BI-167107 which is 99% confidence limit. The number of experiments for these cases were n ≥ 3. (B) The bias values obtained for the G-protein–biased system κOR is shown using U69593 as the reference agonist. For the opioid receptor agonists, the experimental bias factor was calculated from at least three independent experiments. All computational ligand bias values are reported with S.E. and were obtained from five independent MD simulations.
- Fig. 3.
The shape of the agonist binding cavity in β2AR of (A) a neutral agonist epinephrine and (B) formoterol, a biased agonist. The allosteric communication pipelines to the G-protein and the β-arrestin coupling interfaces are also depicted. This figure shows β2AR surfaces sliced to reveal the shape of the ligand binding cavity.
- Fig. 4.
(A) The residues within 5Å of formoterol in β2AR. (B) The binding site residues within 5 Å of epinephrine in β2AR. The size of the circles is proportional to the percentage of snapshots from the MD simulations that show the contacts. Large circles: agonist-residue contacts present in more than 75% of the snapshots from MD simulations. Medium-size circles: between 60% and 75% of the snapshots. Small circles: between 40% and 60% of the snapshots. Residues shown in red text with yellow highlighting are the functional hotspot residues.
- Fig. 5.
(A) The residues within 5Å of the G-protein–biased agonist 1.1 in κOR. (B) The binding site residues within 5 Å of the balanced agonist U69593 in κOR. The size of the circle is proportionate to the percentage of snapshots from the MD simulations that show the contacts. Large circles: agonist-residue contacts present in more than 75% of the snapshots from MD simulations. Medium-size circles: between 60% and 75% of the snapshots. Small circles: between 40% and 60% of the snapshots. Residues shown in red text with yellow highlight are the functional hotspot residues.
- Fig. 6.
Heat map depicting the extent of bias in β2AR and in κOR. The thickness of the cartoon is directly proportional to the contribution of that residue to arrestin or G-protein bias. (A) β2AR in complex with formoterol (β-arrestin–biased). Regions of the receptor that are strongly β-arrestin–biased are represented by shades of blue to red. (B) κOR in complex with probe 1.1 (G-protein–biased). Regions of the receptor that are strongly G-protein–biased are represented by shades of blue to red.
Additional Files
Data Supplement
- Supplemental Data -
Materials and Methods
Supplemental Table 1 - List of all systems used in the current study, including the Computational Ligand Bias values and details about the studies from which the Experimental Bias Factors were obtained
Supplemental Table 2 - The list of residues in the GPCR β-arrestin interface and G-protein interface that we have used in this study for calculating the allosteric pipelines for the different GPCRs
Supplemental Table 3 - Experimental bias factors for agonists formeterol and carmeterol using reference agonist isoproterenol
Supplemental Figure 1 - Two-dimensional representation of the agonists studied here
Supplemental Figure 2 - Variation of mean error in allosteric pipeline strength as a function of the number of snapshots from MD simulations
Supplemental Figure 3 - Plot of the standard error in the ratio of the strength of the allosteric pipelines to the beta-arrestin to that of the G-protein PRlig in equation (1) in the main text, as a function of simulation time
Supplemental Figure 4 - Calculated Ligand Bias for the β2ARTYY mutant in complex with epinephrine compared to that of the WT β2AR in complex with epinephrine
Supplemental Figure 5 - Side-chain rotameric angle distribution of residues altering the binding site shape for formoterol vs. epinephrine
Supplemental Figure 6 - Three-dimensional orientation of the formeterol binding site in β2AR
Supplemental Figure 7 - Side-chain rotameric angle distribution of residues altering the binding site shape for U69593 vs. Probe 1.1
Supplemental Figure 8 - The residues in the probe 1.1 binding cavity in KOR that contact the agonist for over 75% of the MD simulation are shown
References
- Supplemental Data -
