Volume 25, Issue 8, Pages 1275-1285.e4 (August 2017) Structures of Human A1 and A2A Adenosine Receptors with Xanthines Reveal Determinants of Selectivity  Robert K.Y. Cheng, Elena Segala, Nathan Robertson, Francesca Deflorian, Andrew S. Doré, James C. Errey, Cédric Fiez-Vandal, Fiona H. Marshall, Robert M. Cooke  Structure  Volume 25, Issue 8, Pages 1275-1285.e4 (August 2017) DOI: 10.1016/j.str.2017.06.012 Copyright © 2017 Elsevier Ltd Terms and Conditions

Structure 2017 25, 1275-1285.e4DOI: (10.1016/j.str.2017.06.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Ligands Used in This Study Xanthine derivatives are boxed, and atom numbers of the xanthine core are indicated on caffeine. Structure 2017 25, 1275-1285.e4DOI: (10.1016/j.str.2017.06.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Superposition of Backbone Ribbons of the A1R-StaR-Δ3-b562RIL Structure in Complex with PSB36, in Blue, and the A2AR-StaR2-b562RIL Structure in Complex with ZM241385, in Orange (A) Side view, with the approximate boundaries of the membrane indicated as blue bars (Out is the extracellular side, In is the intracellular side), (B) extracellular view, and (C) intracellular view. Structure 2017 25, 1275-1285.e4DOI: (10.1016/j.str.2017.06.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 PSB36 in the Binding Site of A1R A1R is shown as blue ribbons. Interacting residues are shown as sticks with carbon, nitrogen, and oxygen atoms colored gray, blue, and red, respectively. Hydrogen bonds with N2546.55 are shown as red dashes and the distances between heavy atoms indicated. PSB36 is represented in stick with carbon, nitrogen, and oxygen atoms colored green, blue, and red, respectively. Structure 2017 25, 1275-1285.e4DOI: (10.1016/j.str.2017.06.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Comparison of Xanthine Binding Modes (A–C) The binding sites of A2AR with (A) caffeine (with carbon atoms in yellow) in binding mode A and (B) caffeine (with carbon atoms in yellow) in binding mode B and (C) theophylline (with carbon atoms in blue). (D) Two views of ligands from the superposed structures of A2AR with theophylline (with carbon atoms in blue), A2AR with PSB36 (with carbon atoms in purple), and A1R with PSB36 (with carbon atoms in green). Structure 2017 25, 1275-1285.e4DOI: (10.1016/j.str.2017.06.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Comparison of Binding Modes of PSB36 in A1R and A2AR (A) Superposition of the structures of A1R (blue ribbon) with PSB36 (green carbon atoms) and A2AR (orange ribbon) with PSB36 (purple carbon atoms). (B) Section through A1R with PSB36 bound and the protein surface in blue. (C) Section through A2AR with PSB36 bound and the protein surface in orange. Structure 2017 25, 1275-1285.e4DOI: (10.1016/j.str.2017.06.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Competition Binding Experiments for Wild-Type and Mutated A2A and A1 Receptors Competition binding experiments for wild-type (WT) and mutated (residue at position 270) A2AR and A1R receptors for (A) ZM241385, (B) SCH442416, (D) PSB36, and (E) DPCPX. Each graph is from one of the three to seven separate experiments, each performed in duplicate or quadruplicate. Curves were normalized to competition binding values at a competitor concentration of 10−12 M. Calculated pKi values for (C) the A2A-selective ligands ZM241385 and SCH442416, and for (F) the A1-selective ligands PSB36 and DPCPX. pKi values are reported as means ± SEM of three to seven separate experiments each performed in duplicate or quadruplicate. Data have been analyzed with one-way ANOVA with Dunnett's test comparing the Ki values of the WT with the Ki values of the mutant at position 270 and the other three mutants analyzed in this study (see Table 2). Significant difference from WT: ∗∗p < 0.01, ∗∗∗p < 0.001. Structure 2017 25, 1275-1285.e4DOI: (10.1016/j.str.2017.06.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 Superposed Structures of A1R, Blue Ribbon, with PSB36, Green Carbon Atoms, and A2AR, Orange Ribbon, with PSB36, Purple Carbon Atoms, with the Side Chains of M2707.35 in A2AR and T2707.35 in A1R (A) Viewed approximately from the extracellular surface. (B) Viewed approximately from the plane of the membrane. Structure 2017 25, 1275-1285.e4DOI: (10.1016/j.str.2017.06.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 8 Superposed Structures of A1R, Blue Ribbon, with PSB36, Green Carbon Atoms, from This Study, and A1R, Pink Ribbon, with DU172, Orange Carbon Atoms, from Glukhova et al. (2017) (A) Side view, with the approximate boundaries of the membrane indicated as gray bars. (B) The ligand binding sites, with the side chain of Asn 2546.55 shown in each structure. The pocket surfaces are shown in gray mesh for the A1R structure with PSB36 and pink solid for A1R with DU172, having been defined using GRID with a CH3 methyl group probe contoured at 1 kcal/mol. Structure 2017 25, 1275-1285.e4DOI: (10.1016/j.str.2017.06.012) Copyright © 2017 Elsevier Ltd Terms and Conditions