Fig. 3: Molecular dynamics simulations reveal that BnOCPA binding modes can uniquely drive both agonist- and antagonist-like intracellular conformations of the A₁R.

a Adenosine binding pose: N254^6.55 (Ballesteros-Weinstein superscript enumeration) is engaged in key hydrogen bonds, while important hydrophobic contacts are shown as cyan transparent surfaces (F171^ECL2 and I274^7.39). b On the basis of structural similarities and the dynamic docking (Supplementary Movie 2), HOCPA was predicted to bind with a geometry analogous to adenosine; the cyclopentyl group makes further hydrophobic contacts with L253^6.54, as shown by simulation. c The xanthine scaffold of the antagonist PSB36 makes hydrogen bonds with N254^6.55 side chains and hydrophobic contacts with F171^ECL2 and I274^7.39. d BnOCPA agonist-like binding Mode A (Supplementary Movie 1): the benzyl group orients towards the ECL2 and makes hydrophobic contacts with I175^ECL2 (and M177^5.35) side chains. e BnOCPA antagonist-like binding Mode B: the benzyl group orients towards the top of TM5/TM6 and makes hydrophobic contacts with L258^6.59 side chain. f BnOCPA agonist-like binding Mode C: the benzyl group orients towards the top of TM7 and makes hydrophobic contacts with Y271^7.36 side chain. g Binding orientation of BnOCPA in antagonist-like Mode D: the benzyl group orients under ECL3 and occupies the hydrophobic pocket defined by L253^6.54, T257^6.58, T270^7.35, and L269^7.34. Key hydrogen bonds with N254^6.55 and T277^7.42 are shown as dotted lines; main hydrophobic contacts are highlighted as cyan transparent surfaces. h Extracellular view of the A₁R showing the four BnOCPA binding Modes A (cyan), B (magenta), C (green), and D (red) as randomly extracted from the MD simulations. i, j Root-mean-square deviation (RMSD) distributions considering the inactive N^7.49PXXY^7.53 motif on the distal part of TM7 as reference. i HOCPA (blue broken line), BnOCPA Mode A (cyan curve), BnOCPA Mode C (green curve) and the apo receptor (dark green broken line) have a common distribution centring around the active confirmation of the A₁R (orange broken line; Supplementary Fig. 9) leading to A₁R signalling. In contrast, j PSB36 (black broken line), BnOCPA Mode B (magenta curve) and BnOCPA Mode D (red curve) RMSD values have the tendency to move closer to the inactive N^7.49PXXY^7.53 geometry (leftward shift of the curves towards broken grey line at x = 0) preventing A₁R signalling.