Fig. 2.

Effect of the allosteric modulator HMA on the sodium pocket. a ²³Na NMR spectra as a function of Na⁺ concentration (10–1000 mM NaCl) in the presence of 50 μM apo A_2AR-V229C. b Line width-based binding isotherm for NaCl derived from (a), showing a dissociation constant of K_d, of 61 ± 27 mM. (The deviation was defined by three replicates and was applied into all ±values unless described otherwise.) c ²³Na NMR spectra of A_2AR-V229C, in the presence of 100 mM NaCl, as a function of stoichiometric equivalents of the allosteric modulator HMA. d Low-energy pose of HMA docked to A_2AR in the absence of sodium, showing sodium (purple) and water molecules (red) from PDB ID: 4EIY, overlaid with HMA, which was docked in the absence of sodium and internal water molecules. e ²³Na NMR CPMG relaxation series of apo A_2AR-V229C. Each spectrum was acquired using 256 scans with a constant T₂-refocusing period of 70 ms and a pulse refocusing frequency, Ѵ_CPMG, as described in the figure. Note that no discernible dispersion was observed with the identical experiment performed in the presence of MNG-3 detergent micelles without A_2AR